Human claudin 18.2 binders and uses thereof

Abstract

Human claudin 18.2 (CLDN18.2) binders are provided, as well as related polynucleotides, vectors, host cells, methods of production, compositions (e.g., pharmaceutical compositions), uses, and methods of use, e.g., for treatment of cancer.

Description

HUMAN CLAUDIN 18.2 BINDERS AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 695,075, filed September 16, 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 13, 2024, is named 26055-WO-PCT_SL.xml and is 54,057 bytes in size.FIELD

[0003] This disclosure relates to anti-human claudin 18.2 (CLDN18.2) binders such as antibodies and antigen-binding fragments that can bind to human CLDN18.2.BACKGROUND

[0004] CLDN18.2 and claudin 18.1 (CLDN18.1) are alternative splicing isoforms of the same gene, CLDN18. CLDN18.2 is a transmembrane protein and an important component of tight junctions. In normal tissues, CLDN18.2 is expressed in differentiated epithelial cells of the gastric mucosa and not in any other healthy tissues under physiological conditions. CLDN18.2 is thought to be inaccessible and is restricted to tight junctions. However, upon malignant transformation, changes in cell polarity result in epitopes of CLDN 18.2 being exposed on the cancer cell surface, including in gastric cancer, pancreatic cancer, esophageal cancer, bile tract cancer, and colorectal cancer. CLDN18.2 is therefore a promising target for cancer therapy based on its limited expression and accessibility in normal tissues and high expression across several cancer indications.

[0005] Therefore, there is a need in the art for improved human CLDN18.2 binders and methods of making and using the same.SUMMARY

[0006] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1A-1C are a series of graphs showing results of cell binding experiments of anti- CLDN18.2 antibodies to CHO-K1 control cells (FIG. 1A), CHO-K1 CLDN18.1 engineered cells (FIG. IB), or CHO-K1 CLDN18.2 engineered cells (FIG. 1C). The Y axis shows Mean Fluorescence Intensity (MFI) and the X axis shows the antibody concentration.

[0008] FIG. 2 is a graph showing results of cell binding experiments of anti-CLDN18.2 antibodies to endogenous tumor cell line NUGC-4.

[0009] FIGS. 3A-3C are a series of graphs showing results of cell binding experiments of anti- CLDN18.2 scFv constructs to CHO-K1 control cells (FIG. 3 A), CHO-K1 CLDN18.1 engineered cells (FIG. 3B), or CHO-K1 Claudin-18.2 engineered cells (FIG. 3C). The Y axis shows Mean Fluorescence Intensity (MFI) and the X axis shows the antibody concentration.

[0010] FIG. 4 is a graph showing results of cell binding experiments of anti-CLDN18.2 scFv constructs to endogenous tumor cell line NUGC-4.

[0011] FIG. 5 is a schematic diagram ofthe structure of CLDN18.1 and CLDN18.2. There are only 8 amino acids in the ECL1 region that are different between human CLDN18.1 and CLDN18.2, and the ECL2 region is identical betw een human CLDN18. 1 and CLDN18.2.DETAILED DESCRIPTION

[0012] The present disclosure is directed to human CLDN18.2 binders, as well as related polynucleotides, vectors, host cells, methods of production, compositions (e.g., pharmaceutical compositions), uses, and methods of use, e.g., for treatment of cancer.

[0013] As is described further in the Examples, the human CLDN18.2 binders described herein have a number of surprising advantages compared to existing human CLDN18.2 binders, including in terms of cell binding ability and binding affinity to human CLDN18.2. Moreover, the human CLDN18.2 binders described herein can be successfully used in anumber of formats, including full-length antibodies and scFvs. Additionally, certain human CLDN18.2 binders disclosed herein bind to distinct epitopes compared to existing human CLDN18.2 binders.Definitions

[0014] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0015] 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. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0016] As used herein, the articles “a” and “an” refer to one or to more than one (i. e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0017] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0018] Ranges provided herein are understood to be shorthand for all of the values within the range, are inclusive of the endpoints, and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). For example, a range of 1 to 20 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, as well as all intervening decimal values between the aforementioned integers such as, for example. 1.1, 1.2, 1.3, 1.4. 1.5, 1.6, 1.7, 1.8. and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0019] As used herein, the term “comprising” includes the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ the enumerated components or “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0020] As used herein, the term “human CLDN18.2” refers to any form of human CLDN18.2, including soluble and / or membrane-bound human CLDN18.2. An exemplar}' amino acid sequence of human CLDN18.2 is show n in UniProt accession number P56856-2.26055

[0021] As used herein, the term “human CLDN18.2 binder’7refers to an antibody or antigen binding fragment that binds to soluble and / or membrane-bound human CLDN18.2. A human CLDN18.2 binder includes but is not limited to an antibody, a bivalent antibody tetramer (2H+2L), a monovalent antibody (H+L), a multispecific antibody (e.g., a bispecific antibody that binds to human CLDN18.2 and another target), a Fab fragment, a Fab’ fragment. a F(ab’)2 fragment, an Fv region, and an scFv. In some embodiments, the human CLDN18.2 binder is an antibody. In other embodiments, the human CLDN18.2 binder is an antigen binding fragment of an antibody.

[0022] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction betw een members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including kinetic exclusion assay (e.g., KINEXA®) and surface plasmon resonance (SPR; e.g., BlACORE®). Specific illustrative and exemplary embodiments for measuring binding affinity are described in this disclosure.

[0023] With regard to the binding of an antibody to a target molecule, the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a nontarget molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.

[0024] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. KD = kd / ka. In some embodiments, the affinity of an antibody is described in terms of the KD for an interaction between such antibody and its antigen. For clarity, as known in the art, a smaller KD value indicates a higher affinity interaction, while a larger KD value indicates a lower affinity interaction.

[0025] The term “administering” refers to the act of delivering a composition (e.g., a pharmaceutical composition) or combination described herein into a subject by such routes as intravenous, subcutaneous, parenteral, intraperitoneal, intramuscular oral, mucosal, topical,suppository, intralesional, intrathecal, intranasal or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration generally occurs after the onset of the disease, disorder, or condition, or its symptoms but, in certain instances, can occur before the onset of the disease, disorder, or condition, or its symptoms (e.g., administration for patients prone to such a disease, disorder, or condition).

[0026] As used herein, in some embodiments, the terms “administration in combination with” and “administered in combination” are used to represent a form of drug administration in which a plurality of active ingredients are contained or encapsulated in different preparations and administered simultaneously or separately or sequentially in any order at different times, or both contained or encapsulated in the same preparation and administered, unless the context otherwise requires and unless technically inconsistent. Simultaneous administration means that the plurality of active ingredients, or the pharmacological effects thereof, appear simultaneously (i.e., overlap for some period of time) in the patient.

[0027] As used herein, the term “amino acid” refers to a simple organic compound containing both a carboxyl ( — COOH) and an amino ( — NH2) group. Amino acids are the building blocks for proteins, polypeptides, and peptides. Amino acids occur in L-form and D-form, with the L- form in naturally occurring proteins, polypeptides, and peptides. Amino acids and their code names are listed in Table 1 below.Table 1: Amino acids and their codes

[0028] As used herein, the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of anon- polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity’ (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary' conservative substitutions are listed in Table 2 below.Table 2: Conservative amino acid substitutions

[0029] The term "‘immunoglobulin’' refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed.. Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region typically comprises three domains, abbreviated CHI, CH2, and CH3. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0030] The terms “antibody” or “antibodies” are meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific, etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1. CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl. IgA2, IgGl, IgG2, IgG3 and IgG4.26055Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. An antibody provided herein may include post-translational modifications thereof, e.g., C-terminal lysine clipping in the heavy chain or conversion of glutamine or glutamic acid to pyroglutamate or pyroglutamic acid, which may occur when recombinantly expressed in host cells (e g., CHO cells), or during purification / storage.

[0031] A “Complementary Determining Region (CDR)” refers to one of three hypervariable regions (Hl, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH p-sheet framework, or one of three hypervariable regions (LI, L2 or L3) within the nonframework region of the antibody VL P-sheet framework. CDRs are variable region sequences interspersed within the framework region sequences. CDRs are well recognized in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains. See Kabat et al.. J Biol Chem, 1977, 252:6609-6616 and Kabat, Adv Protein Chem. 1978, 32: 1-75. CDRs have also been defined structurally by Chothia as those residues that are not part of the conserved P-sheet framework, and thus are able to adapt different conformations. See Chothia and Lesk, J Mol Biol, 1987, 196:901-917. Both the Kabat and Chothia nomenclatures are well known in the art. AbM, Contact and IMGT also define CDRs. CDR positions within a canonical antibody variable domain have been determined by comparison of numerous structures. See Morea et al., Methods, 2000, 20:267-279 and Al- Lazikani et al., J Mol Biol, 1997, 273:927-48. Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a. b, c and so forth next to the residue number in the canonical variable domain numbering scheme. Such terminology is well known to those skilled in the art.

[0032] A number of hypen ariable region delineations are in use and are included herein. The Kabat CDRs are based on sequence variability and are the most commonly used. See Kabat et al. (1992) Sequences of Proteins of Immunological Interest, DIANE Publishing: 2719. Chothia refers instead to the location of the structural loops (Chothia and Lesk, supra)' . The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The Contact hypervariable regions are based on an analysis of the available complex crystal structures.

[0033] More recently, a universal numbering system ImMunoGeneTics (IMGT) Information System™ has been developed and widely adopted. See Lefranc et al., Dev Comp Immunol, 2003, 27:55-77. IMGT is an integrated information system specializing in immunoglobulins (IG), T26055 cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. The IMGT CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. Correspondence between the Kabat, Chothia and IMGT numbering is also well known in the art (Lefranc et al., supra). An exemplary system, shown herein, combines Kabat and Chothia CDR definitions. See Table 3 below for a summary of exemplary CDR numbering schemes that may be used in the context of the present disclosure.Table 3: Exemplary CDR numbering schemes

[0034] The entire nucleotide sequence of the heavy chain and light chain variable regions are commonly numbered according to Kabat while the three CDRs within the variable region may be defined according to any one of the aforementioned numbering schemes.

[0035] In general, the state of the art recognizes that in many cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on CDR3 of the heavy chain alone are known in the art (e.g., Beiboer et al., J. Mol. Biol. 296: 833-849 (2000); Klimka et al., British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al., Immunity 13: 37-45 (2000)).

[0036] The heavy chain from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also designated a, 8, s, y, and p, respectively. The IgG and IgA classes are further divided into subclasses on the basis of differences in sequence and function. Humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.26055

[0037] The light chain from any vertebrate species can be assigned to one of two types, called kappa (K) and lambda (X), based on the sequence of its constant domain.

[0038] The term ‘‘constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigenbinding site. The constant domain contains the CHI, CH2 and CH3 domains of the heavy chain and the CL domain of the light chain.

[0039] The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the EU numbering scheme. The EU numbering scheme is based upon the amino acid sequence of human IgGl (EU), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgGl described in Edelman et al., Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgGl, IgG2, IgG3, and IgG4 constant domains in Beranger et al., op. cit.

[0040] As used herein, the term “Fc domain” or “Fc” as used herein is the crystallizable fragment domain or region obtained from an antibody that comprises the CH2 and CH3 domains of an antibody. In an antibody, the two Fc domains are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The Fc domain may be obtained by digesting an antibody with the protease papain. Typically, amino acids in the Fc domain are numbered according to the EU numbering convention (See Edelmann et al., Biochem. 63: 78-85 (1969)).

[0041] As used herein, the term “antigen” as used herein refers to any foreign substance which induces an immune response in the body.

[0042] As used herein, the term “antigen binding fragment” refers to a polypeptide or polypeptides comprising a fragment of a full-length antibody, w hich retains the ability to specifically bind to the antigen (e.g., human CLDN18.2) bound by the full-length antibody, and / or to compete with the full-length antibody for specifically binding to the antigen (e.g., human CLDN18.2). Examples of antigen binding fragments include but are not limited to Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fv region, and scFv. In some embodiments, an26055 antigen binding fragment includes a complex molecule (e.g., a complex protein) that includes the antigen binding fragment, e g., an antibody-drug conjugate.

[0043] The term ‘‘human antibody" refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are “derived from'’ sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody'’ ty pically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J. Mol. Biol. 296:57-86, or a synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J. Mol. Biol. 397:385-396. and in Int. Patent Publ. No. W02009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of “human antibody”.

[0044] The term “humanized antibody” refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

[0045] As used herein, the term “Fab fragment” refers to an antigen binder comprising one antibody light chain and the CHI and VH of one antibody heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.26055

[0046] As used herein, the term “Fab’ fragment” refers to an antigen binder comprising one antibody light chain and a portion or fragment of one antibody heavy chain that contains the VH and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab’ fragments to form a F(ab’)2 fragment.

[0047] As used herein, the term “F(ab’)2 fragment” refers to an antigen binder comprising two antibody light chains and two heavy chains containing the VH and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab’)2 fragment thus is composed of two Fab’ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab’)2 fragment” can be the product of pepsin cleavage of an antibody.

[0048] As used herein, the term “Fv region” refers to an antigen binder comprising the variable regions from both the heavy and light chains of an antibody but lacks the constant regions.

[0049] As used herein, the term “scFv” or “single-chain variable fragment” refers to a fusion protein comprising a VH and VL fused or linked together by a short linker peptide typically of about 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C- terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0050] As used herein, the term “diabody” refers to an antigen binder comprising a small antibody fragment with two antigen-binding regions, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding regions. Diabodies are described more fully in, e.g., EP 404,097; WO 93 / 11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1 136.

[0051] These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility’ in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.26055

[0052] As used herein, the term “isolated” human CLDN18.2 binder (e.g., antibodies or antigen-binding fragments) are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

[0053] As used herein, the term “monoclonal antibody” refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991), and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. See also Presta, J. Allergy Clin. Immunol. 116: 731 (2005).

[0054] “Multispecific” refers to a molecule that binds two or more distinct antigens or two or more distinct epitopes within the same antigen. Multispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0055] “Single-domain antibodies” or “sdAb(s)” are antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single-domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit,26055 bovine. For example, in some embodiments, the single-domain antibodies of the disclosure are obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization'’ of a naturally occurring VHH domain or by expression of a nucleic acid encoding such a humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, and in particular from a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; (5) by “camelization’" of a “domain antibody” or “Dab,” or by expression of a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a single-domain antibody using techniques for nucleic acid synthesis known in the field, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of one or more of the foregoing. The term single-domain antibody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation.

[0056] The term “VHH” as used herein, refers to single chain antibody binding domain devoid of light chain. In some cases, a VHH is derived from an antibody of the type that can be found in Camelidae or cartilaginous fish which are naturally devoid of light chains or to a synthetic and non-immunized VHH which can be constructed accordingly. Each heavy chain comprises a variable region encoded by V-, D- and J exons. A VHH, in some cases, is a natural VHH, such as a Camelid derived VHH, or a recombinant protein comprising a heavy chain variable domain. In some embodiments, the VHH is derived from a species selected from the group consisting of camels, llamas, vicugnas, guanacos, and cartilaginous fish (such as. but not limited to, sharks). In another embodiment, the VHH is denved from an alpaca (such as, but not limited to. a Huacaya Alpaca or a Suri alpaca).

[0057] The term “antibody-drug conjugate” or “ADC” refers to a conjugate comprising an human CLDN18 2 binder conjugated to one or more cytotoxic agents, optionally through one or more linkers.

[0058] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. The cytotoxic agent can be an anti- angiogenic agent, a pro-apoptotic agent, an anti-mitotic agent, an anti-kinase agent, an alky lating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabohte, an antibiotic, an alkaloid, or a radioactive isotope. Exemplary cytotoxic agents include calicheamycin, camptothecin, carboplatin, irinotecan, SN- 38, carboplatin, camptothecan, cyclophosphamide, cytarabine, dacarbazine, docetaxel,26055 dactinomycin, daunorubicin. doxorubicin, doxorubicin, etoposide, idarubicin, topotecan. vinca alkaloid, maytansinoid, maytansinoid analog, pyrrolobenzodiazepine, taxoid, duocarmycin, dolastatin, auristatin, and derivatives thereof.

[0059] A “linker’ refers to a molecule that connects one composition to another, e.g., an antibody to an agent. Linkers described herein can conjugate an antibody to a cytotoxic agent. Exemplary linkers include a peptide linker, a labile linker, an acid labile linker, a photolabile linker, a charged linker, a disulfide-containing linker, a peptidase-sensitive linker, a |3- glucuronide-linker, a dimethyl linker, a thio-ether linker, and a hydrophilic linker. A linker can be cleavable or non-cleavable.

[0060] As used herein, the term “gene” is used broadly to refer to any segment of nucleic acid associated with a biological function. Thus, genes include coding sequences and / or the regulatory sequences required for their expression. For example, “gene” refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including regulatory sequences.“Genes” also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. “Genes” can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from know n or predicted sequence information, and may include sequences designed to have desired parameters. Genes include both naturally occurring nucleotide sequences encoding a molecule of interest and synthetically derived nucleotide sequences encoding a molecule of interest, for example, complementary DNA (cDNA) obtained from a messenger RNA (mRNA) nucleotide sequence.

[0061] As used herein, the term “germline” or “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER® germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al.. Nucleic Acids Res. 33: D256-D261 (2005).

[0062] As used herein, the term “library” as used herein is, typically, a collection of related but diverse polynucleotides that are, in general, in a common vector backbone. For example, a light chain or heavy chain immunoglobulin library may contain polynucleotides, in a common vector backbone, that encode light and / or heavy chain immunoglobulins, which are diverse but related in their nucleotide sequence; for example, which immunoglobulins are functionally diverse in their abilities to form complexes with other immunoglobulins, e.g., in an antibody display system of the present disclosure, and bind a particular antigen.26055

[0063] As used herein, the terms “polynucleotide;’ “polynucleic acid.” “nucleic acid.” or “nucleic acid molecule” refer to a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. A polynucleotide includes DNA (e.g., cDNA) and RNA, which may be single-stranded, double-stranded, or a mix of single- and double-stranded. In some embodiments, polynucleotide(s) as disclosed herein, e.g., polynucleotide(s) encoding a human CLDN18.2 binder disclosed herein or an immunoglobulin chain or component of a human CLDN18.2 binder of the present disclosure may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry’ sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5’- and 3 ’-non-coding regions, and the like. In some examples, a single polynucleotide encodes a human CLDN18.2 binder disclosed herein or an immunoglobulin chain or component of a human CLDN18.2 binder of the present disclosure. In other examples, a set of polynucleotides together encodes a human CLDN 18.2 binder disclosed herein or an immunoglobulin chain or component of a human CLDN18.2 binder of the present disclosure.

[0064] “Protein” or “polypeptide” are used interchangeably herein are refers to a molecule that comprises one or more polypeptides each comprised of at least two amino acid residues linked by a peptide bond. Protein may be a monomer, or may be a protein complex of two or more subunits, the subunits being identical or distinct. Small polypeptides of less than 50 amino acids may be referred to as “peptides.” Protein may' be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acety lation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation. ADP-ribosylation. pegylation or biotinylation.

[0065] “Recombinant” refers to polynucleotides, polypeptides, vectors, viruses and other macromolecules that are prepared, expressed, created or isolated by recombinant means.

[0066] As used herein, a “promoter” or “promoter sequence” is a DNA regulatory' region capable of binding an RNA polymerase in a cell (e.g.. directly or through other promoter-bound proteins or substances) and initiating transcription of a coding sequence. A promoter sequence is, in general, bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary' to initiate transcription at any level. Within the promoter sequence may be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. The promoter may be operably associated with other expression control sequences, including enhancer and26055 repressor sequences or with a nucleic acid of the disclosure. Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter (U.S. Patent Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist, et al., Nature 290: 304-310 (1981)), the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980)). the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors such as the p-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727- 3731 (1978)), or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)); and promoter elements from yeast or other fungi such as Gall promoter, Gal4 promoter, ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter or alkaline phosphatase promoter.

[0067] As used herein, the terms “vector,’" “cloning vector,"’ and “expression vector’" include a vehicle (e.g.. a plasmid) by which a DNA or RNA sequence can be introduced into a host cell so as to transform the host and, optionally, promote expression and / or replication of the introduced sequence. In some embodiments, polynucleotide(s) encoding an a human CLDN18.2 binder disclosed herein or an immunoglobulin chain or component of a human CLDN18.2 binder of the present disclosure may be in a vector or collectively in a set of vectors.

[0068] The terms “host cell,” “host cell line,” and "‘host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “engineered cells,” “transformants,” and “transformed cells,” which include the primary engineered (e.g., transformed) cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. As appropriate, the host cells can be stably or transiently transfected with a polynucleotide(s) encoding a human CLDN18.2 binder, as described herein.

[0069] As used herein, the term “control sequences” or “regulatory sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for expression in eukary otes, for example, include a promoter, operator or enhancer sequences, transcription termination sequences, and polyadenylation sequences for expression of a messenger RNA encoding a protein and a ribosome binding site for facilitating translation of the messenger RNA.26055

[0070] As used herein, a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence, e g., a regulatory sequence. For example, DNA for a pre-sequence or secretory' leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0071] As used herein, the term “encoding” refers to the inherent property' of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA. or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may' include introns.

[0072] As used herein, the term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0073] The term “treating” (and variations thereof such as “treat” or “treatment”) refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed during the course of clinical pathology. Desirable effects of treatment include preventing recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the term “treating” or any grammatical variation thereof refers to reducing and / or ameliorating the severity and / or duration26055 of a given disease, disorder or condition, and / or a symptom related thereto, such as (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, and / or (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions.

[0074] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e g., those described herein individually or in combination) that is sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition, and / or a symptom related thereto. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary for (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, and / or (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or condition. A “therapeutically effective amount” of an agent of the present disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule / agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of an agent effective to “treat” a disease, disorder, or condition, in a subject or mammal.

[0075] As used herein, the term “combination therapy” refers to treatment of a human or animal individual comprising administering a first therapeutic agent and a second therapeutic agent consecutively or concurrently to the individual. In general, the first and second therapeutic agents are administered to the individual separately and not as a mixture; however, there may be embodiments where the first and second therapeutic agents are mixed prior to administration.

[0076] As used herein, the term “individual,” “subject,” and “patient” are used interchangeably and mean a mammalian subject. Exemplary7subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, pigs and sheep. In preferred embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with an agent provided herein. In some embodiments, the disease or condition is a cancer.

[0077] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory7agency of the federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans. Additionally, or alternatively, the phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact26055 wi th the tissues of human beings and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problem or complication, commensurate with a reasonable benefit risk ratio.

[0078] The term ‘‘pharmaceutically acceptable carrier'’ includes, but is not limited to. any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / w-ater emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

[0079] As used herein, the term “concurrently” means at the same time. For example, if two treatment regimens for a single patient are being conducted concurrently, then they are being conducted at the same time. It ill be understood that tw o treatment regimens happening at the same time, does not necessarily mean that actual delivery of two drugs happens at the same time, as each regimen may call for a different dosing schedule and / or different delivery’ modes.

[0080] “Cancer” refers to any physiological condition in mammals characterized by unregulated cell growth; in particular, cellular-proliferative disease states. As used herein, “tumor” refers to any neoplastic cell growth or proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The cancer may be a cancer associated with expression of CLDN 18.2, such as gastrointestinal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bile tract cancer, or colorectal cancer.

[0081] As used herein, the term “percent (%) amino acid sequence identity"’ with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identify can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs or software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for26055 measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.Human CLDN18.2 binders

[0082] Provided herein are human CLDN 18.2 binders. The human CLDN 18.2 binders of the present disclosure may be chimeric or fully human antibodies or antigen binding fragments that specifically bind human CLDN18.2. In some embodiments, the human CLDN18.2 binders comprise a VH domain and a VL domain, each domain comprising three CDRs and four Frameworks (FR) in the following arrangement:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0083] In some embodiments, the human CLDN18.2 binder comprises six complementarity determining regions (CDRs) comprising a particular combination of three CDRs from a VH and three CDRs from the VL that pairs with the VH. The CDR sequences may be defined according to any numbering scheme useful for defining CDR sequences including but not limited to the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme. Guidance for defining the CDR sequences may be found in the general rules disclosed in bioinf.org.uk: Prof. Andrew C.R. Martin’s Group and in Table 3 above.

[0084] In a particular embodiment, the CDRs are defined by Kabat. The CDR amino acid sequences shown in Tables 4-8 are set forth according to the Kabat numbering scheme for identifying CDR amino acid sequences.26055

[0085] A particular CDR amino acid sequence determined using any one of the schemes for identifying CDR amino acid sequences (see Table 3) may have more or fewer amino acids than that of CDR amino acid sequences determined according to any other numbering scheme but the CDR amino acid sequences will typically overlap to some extent. Thus, the CDR amino acid sequences defined according to Kabat are not to be construed as limiting and any human CLDN18.2 binder in which the CDR amino acid sequences have been identified by another numbering scheme will fall within the scope of the human CLDN18.2 binders of the present disclosure provided the amino acid sequences for such human CLDN18.2 binders comprise the six CDR amino acid sequences as identified by Kabat. For all human CLDN18.2 binders disclosed herein, unless indicated otherwise, the amino acids comprising the variable domains as a whole are numbered according to the Kabat numbering scheme independently of how the amino acids comprising the CDR are defined. Unless indicated otherwise, heavy chain constant domains are numbered according to the EU numbering scheme.26055

[0086] In some embodiments, provided herein is a human CLDN18.2 binder that comprises one, two, three, four, five, or all six complementarity determining regions (CDRs) of any antibody disclosed herein.

[0087] In some embodiments, provided herein is a human CLDN18.2 binder that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3. The CDRs can be defined according to any numbering scheme known in the art. In some embodiments, the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 3.

[0088] In particular embodiments of the disclosure, the human CLDN18.2 binder comprises (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0089] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity' to the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising an amino acid sequence having about 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 90% sequence identity' to the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising an ammo acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 95%26055 sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, any of the human CLDN18.2 binders described in this paragraph comprise (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0090] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0091] In some embodiments, provided herein is a human CLDN18.2 binder that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11. The CDRs can be defined according to any numbering scheme known in the art. In some embodiments, the CDRs are defined using the Kabat. Chothia, AbM. ImMunoGeneTics (1MGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 3.

[0092] In particular embodiments of the disclosure, the human CLDN18.2 binder comprises (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17. wherein the CDR sequences are defined by the Kabat numbering scheme.

[0093] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80%, 85%,2605590%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising an amino acid sequence having about 90% sequence identity7to the amino acid sequence of SEQ ID NO: 11. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, provided herein is a human CLDN 18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of the human CLDN18.2 binders described in this paragraph comprise (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 12. a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17. wherein the CDR sequences are defined by the Kabat numbering scheme.

[0094] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0095] In some embodiments, provided herein is a human CLDN18.2 binder that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chainvariable domain (VL) comprising the ammo acid sequence of SEQ ID NO: 19. The CDRs can be defined according to any numbering scheme known in the art. In some embodiments, the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 3.

[0096] In particular embodiments of the disclosure, the human CLDN18.2 binder comprises (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 compnsing the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0097] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80%. 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. or 99% sequence identity’ to the amino acid sequence of SEQ ID NO: 19. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, provided herein is a human CLDN 18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 99% sequence identity’ to the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising an amino acid sequence having about99% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, any of the human CLDN18.2 binders described in this paragraph comprise (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0098] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0099] In some embodiments, provided herein is a human CLDN18.2 binder comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27. The CDRs can be defined according to any numbering scheme known in the art. In some embodiments, the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 3.

[0100] In particular embodiments of the disclosure, the human CLDN18.2 binder comprises (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 comprising the amino acid sequence of SEQ ID NO: 29. and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0101] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising an amino acid sequence having about 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to the ammo acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80% sequence identity to the amino acid26055 sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising an amino acid sequence having about 95% sequence identity7to the amino acid sequence of SEQ ID NO: 27. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 27. In some embodiments, any of the human CLDN18.2 binders described in this paragraph comprise (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 30; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0102] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0103] In some embodiments, provided herein is a human CLDN18.2 binder comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35. The CDRs can be defined according to any numbering scheme known in the art. In some embodiments, the CDRs are defined using the Kabat, Chothia, AbM. ImMunoGeneTics (IMGT), or Contact numbering scheme, or a combination thereof. In some embodiments, the CDRs are defined according to a numbering scheme in Table 3.26055

[0104] In particular embodiments of the disclosure, the human CLDN18.2 binder comprises (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 41, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0105] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80%. 85%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising an amino acid sequence having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising an amino acid sequence having about 80% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising an amino acid sequence having about 90% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, provided herein is a human CLDN18.2 binder that compnses a heavy chain variable domain (VH) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising an amino acid sequence having about 95% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, provided herein is a human CLDN 18.2 binder that comprises a heavy chain variable domain (VH) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising an amino acid sequence having about 99% sequence identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, any of the human CLDN18.2 binders described in this paragraph comprise (a) a VH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and (b) a VL domain comprising a CDR1 comprising the amino acid sequence26055 of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 41, wherein the CDR sequences are defined by the Kabat numbering scheme.

[0106] In some embodiments, provided herein is a human CLDN18.2 binder that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.

[0107] Any of the human CLDN18.2 binders disclosed herein may be used in an antibody drug conjugate. In some embodiments, the antibody is linked by a linker to a cytotoxic agent. Any suitable linker and cytotoxic agent may be used.

[0108] In another aspect, provided herein is an antibody that competes for binding to human CLDN18.2 with any of the antibodies disclosed herein. For example, provided herein is an antibody that competes for binding to human CLDN18.2 with any of the antibodies disclosed in any one of Tables 4-8.

[0109] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0110] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0111] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0112] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0113] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid26055 sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the ammo acid sequence of SEQ ID NO: 19.

[0114] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0115] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the ammo acid sequence of SEQ ID NO: 27.

[0116] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0117] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.

[0118] For example, provided herein is an antibody that competes for binding to human CLDN18.2 with an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.

[0119] In another aspect, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as any of the antibodies disclosed herein. For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as any of the antibodies disclosed in any one of Tables 4-8.

[0120] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity' determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.26055

[0121] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0122] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0123] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0124] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0125] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0126] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy’ chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0127] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0128] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising aheavy chain variable domain (VH) comprising the amino acid26055 sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the ammo acid sequence of SEQ ID NO: 35.

[0129] For example, provided herein is an antibody that binds to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.

[0130] Also provided herein are means for binding to the same epitope of human CLDN18.2 as any of the antibodies disclosed herein.

[0131] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0132] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3.

[0133] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0134] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 11.

[0135] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0136] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the26055 amino acid sequence of SEQ ID NO: 18 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 19.

[0137] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0138] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 26 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 27.

[0139] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises the six complementarity determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.

[0140] For example, provided herein is means for binding to the same epitope of human CLDN18.2 as an antibody that comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 34 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 35.Human CLDN18.2 binders comprising an effector-silent Fc domain

[0141] Effector-silent human CLDN 18.2 binders of the present disclosure (e.g., full-length antibodies) may comprise an HC constant domain or Fc domain thereof that has been modified such that the antibody displays no measurable binding to one or more Fc receptors (FcRs) or displays reduced binding to one or more FcRs compared to that of an unmodified antibody of the same IgG isotype. The effector-silent antibodies may in further embodiments display no measurable binding to each of FcyRIIIa, FcyRIIa, and FcyRI or display reduced binding to each of FcyRIIIa, FcyRIIa, and FcyRI compared to that of an unmodified antibody of the same IgG isotype. In particular embodiments, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

[0142] In particular embodiments, the effector-silent antibody comprises an Fc domain of an IgGl isotype that has been modified to lack V-glycosylation of the asparagine (Asn) residue at position 297 (EU numbering system) of the HC constant domain. The consensus sequence for N-26055 glycosylation is Asn-Xaa-Ser / Thr (wherein Xaa at position 298 is any amino acid except Pro); the A-glycosylation consensus sequence is Asn-Ser-Thr. The modification may be achieved by replacing the codon encoding the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, for example Ala, Asp, Gin, Gly, or Glu. e.g., N297A, N297Q, N297G, N297E. or N297D. Alternatively, the codon for Ser at position 298 may be replaced with the codon for Pro or the codon for Thr at position 299 may be replaced with any codon except the codon for Ser. In a further alternative, each of the amino acids comprising the A-glycosylation consensus sequence is replaced with another amino acid. Such modified IgG molecules have no measurable effector function. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations. In further embodiments, such IgGs modified to lack N- glycosylation at position 297 may further include one or more additional mutations disclosed herein for eliminating measurable effector function.

[0143] An exemplary IgGl HC constant domain mutated at position 297, which abolishes the A-glycosylation of the HC constant domain, is of SEQ ID NO: 49. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9. or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0144] In particular embodiments, the Fc domain of the IgGl HC constant domain comprising the effector-silent antibody is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A. N297D, D265S, and P331S (wherein the positions are identified according to EU numbering) and wherein said HC constant domain is effector- silent. In particular embodiments, the modified IgGl further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0145] In particular embodiments, the HC constant domain comprises L234A, L235A, and D265S substitutions (wherein the positions are identified according to EU numbering). In particular embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to EU numbering). These and other substitutions are disclosed in WO9428027;W02004099249; W020121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815; Sondermann et al. Nature 406, 267-273 (2000)).26055

[0146] In particular embodiments of the above, the HC constant domain comprises L234A / L235A / D265A; L234A / L235A / P329G; L235E; D265A; D265A / N297G; or V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions, wherein the positions are identified according to EU numbering. In particular embodiments, the HC molecules further comprises 1, 2. 3, 4, 5. 6, 7, 8, 9. or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

[0147] In particular embodiments, the effector-silent antibody comprises an IgGl isotype, in which the Fc domain of the HC constant domain has been modified to be effector-silent by substituting the amino acids from position 233 to position 236 of the IgGl with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgG4 HC, wherein the positions are identified according to EU numbering (Armour et al., Eur. J. Immunol. 29(8):2613- 24 (1999); Shields et al.. J. Biol. Chem. 276(9):6591-604(2001)). In particular embodiments, the modified IgGl further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and / or deletions, wherein said substitutions may be conservative mutations or nonconservative mutations.

[0148] In particular embodiments, the effector-silent antibody comprises a VH fused or linked to a hybrid human immunoglobulin HC constant domain, which includes a hinge region, a CH2 domain and a CH3 domain in an Abterminal to C-terminal direction, wherein the hinge region comprises an at least partial amino acid sequence of a human IgD hinge region or a human IgGl hinge region; and the CH2 domain is of a human IgG4 CH2 domain, a portion of which, at its N- terminal region, is replaced by 4-37 amino acid residues of an A'- terminal region of a human IgG2 CH2 or human IgD CH2 domain. Such hybrid human HC constant domain is disclosed in U.S. Pat. No. 7,867,491, which is incorporated herein by reference in its entirety.

[0149] Exemplary IgGl HC constant domains include HC constant domains comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 42-50.

[0150] In particular embodiments of the human CLDN18.2 binder, the human CLDN18.2 binder is an antibody comprising an IgGl Fc domain as disclosed herein, which further comprises a C-terminal lysine or lacks either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

[0151] In any one of the embodiments disclosed herein, the light chain may comprise a human kappa light chain constant domain comprising SEQ ID NO: 51.26055

[0152] Alternatively, or additionally, in another embodiment of the present disclosure, the human CLDN18.2 binders and other peptides provided herein may undergo post-translational modifications as know n in the art. Examples of post-translational modifications include, but are not limited to, chemical modifications, such as disulfide bonds, oligosaccharides, N-terminal pyroglutamate formation, N-terminal pyroglutamatic acid formation, C-terminal lysine processing (such that lysine is removed), deamidation, isomerization, oxidation, glycation, peptide bond cleavage, non-reducible cross-linking, truncation and others known in the art. See, e.g., Liu et al., J. Pharma. Sci. vol. 97, no. 7, pp. 2426-2447 (July 2008). Other types of modifications include noncovalent interaction, conformational heterogeneity, and aggregation.

[0153] In some embodiments, an N-terminal E or Q of a human CLDN 18.2 binder provided herein is substituted with pyroglutamate or pyroglutamic acid. In some embodiments, a C- terminal K of a human CLDN18.2 binder provided herein is removed. In other embodiments, an N-terminal E or Q of a human CLDN18.2 binder provided herein is substituted with pyroglutamate or pyroglutamic acid and a C-terminal K of the human CLDN18.2 binder (e.g.. heavy chain C terminal amino acid) is removed. The present disclosure includes any of the above-described post-translational modifications of any of the human CLDN18.2 binders and polypeptides provided herein. For example, provided herein is a human CLDN18.2 binder that comprises the same sequences as any of the antibodies disclosed herein, except that the first N- terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid; and / or the C-terminal amino acid of the heavy chain is removed. For example, in some embodiments, provided herein is a human CLDN18.2 binder that comprises the same sequences as NG034.5A9, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid: and / or the C-terminal amino acid of the heavy chain is removed. In some embodiments, provided herein is a human CLDN18.2 binder that comprises the same sequences as NG034.7A6, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid; the first N-terminal amino acid of the VL region is substituted with pyroglutamate or pyroglutamic acid; and / or the C-terminal amino acid of the heavy chain is removed. In some embodiments, provided herein is a human CLDN18.2 binder that comprises the same sequences as NG034.8A7, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid; the first N-terminal amino acid of the VL region is substituted with pyroglutamate or pyroglutamic acid; and / or the C-terminal amino acid of the heavy chain is removed. In some embodiments, provided herein is a human CLDN18.2 binder that comprises the same sequences as NG034.17H6, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic26055 acid; the first N-terminal amino acid of the VL region is substituted with pyroglutamate or pyroglutamic acid; and / or the C-terminal amino acid of the heavy chain is removed. In some embodiments, provided herein is a human CLDN18.2 binder that comprises the same sequences as NG034. 17H7. 1, except that the first N-terminal amino acid of the VH region is substituted with pyroglutamate or pyroglutamic acid; the first N-terminal amino acid of the VL region is substituted with pyroglutamate or pyroglutamic acid; and / or the C-terminal amino acid of the heavy chain is removed.

[0154] In some specific embodiments, provided herein is an antibody or antigen binding fragment (e.g., scFv) comprising an amino acid sequence of any one of SEQ ID NOs: 2, 10, 11,18, 19, and 26 to 28, except that the first amino acid at the N-terminus is substituted with pyroglutamate.

[0155] In some specific embodiments, provided herein is an antibody or fragment (e.g., scFv) or a polyprotein comprising an amino acid sequence of any one of SEQ ID NOs: 2, 10. 11. 18,19. and 26 to 28. except that the first amino acid at the N-terminus is substituted with pyroglutamic acid.

[0156] In other specific embodiments, provided herein is antibody or fragment comprising an amino acid sequence of any one of SEQ ID NOs: 42-50, except that the C-terminal lysine (K) is removed. scFv fusion proteins that bind human CLDN18.2

[0157] Any of the antibody sequences provided herein may be used in a scFv fusion protein that binds to human CLDN18.2. In particular embodiments, a VH and VL disclosed herein are expressed as an scFv fusion protein in which the VL and VH domains are linked together by a linker, e.g., a peptide linker. In some embodiments, the peptide linker joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH-VL paring and antigen-binding sites. Thus, the scFv may comprise a fusion protein in which the C-terminus of a VL is linked by a peptide linker to the N- terminus of a VH or a fusion protein in which the C-terminus of a VH is linked by a peptide linker to the N-terminus of a VL.

[0158] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and VH comprises an amino acid sequence having2605580%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 2.

[0159] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 3 and VH comprises the amino acid sequence of SEQ ID NO: 2.

[0160] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 10.

[0161] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 11 and VH comprises the amino acid sequence of SEQ ID NO: 10.

[0162] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 18.

[0163] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 19 and VH comprises the amino acid sequence of SEQ ID NO: 18.

[0164] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 27 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 26.

[0165] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 27 and VH comprises the amino acid sequence of SEQ ID NO: 27.

[0166] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises an amino acid sequence having2605580%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 35.

[0167] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-linker-VH or VH-linker-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 34 and VH comprises the amino acid sequence of SEQ ID NO: 35.

[0168] Any suitable linker may be used. In some embodiments, peptide linkers for linking the variable domains can vary from 10 to 25 amino acids in length and are typically, but not always, composed of hydrophilic amino acids such as glycine (G) and serine (S) having the structure G4S (SEQ ID NO: 52), for example, (G4S)n (SEQ ID NO: 53), wherein n is 1, 2, 3, 4, or 5. Peptide linkers of shorter lengths (0-4 amino acids) have also been used; however, scFvs bearing shorter linkers may form multimers. Generally, the (G4S)s peptide comprising three repeating G4S unitsC‘(G4S)s” disclosed as SEQ ID NO: 54) is used as an scFv peptide linker (See for example. Leath et al., Int. J. Oncol. 24:765-771 (2004); Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Iliades et al., FEBS Lett. 409:437-441 (1997)).

[0169] Exemplary scFv fusion proteins include the structure VL-(G4S)n-VH or VH-(G4S)n- VL wherein (a) VL comprises the amino acid sequence of SEQ ID NO: 3 and VH comprises the amino acid sequence of SEQ ID NO: 2; (b) VL comprises the amino acid sequence of SEQ ID NO: 3 and VH comprises the amino acid sequence set for in SEQ ID NO: 26; (c) VL comprises the amino acid sequence of SEQ ID NO: 3 and VH comprises the amino acid sequence set for in SEQ ID NO: 27; (d) VL comprises the amino acid sequence of SEQ ID NO: 3 and VH comprises the amino acid sequence set for in SEQ ID NO: 28; (e) VL comprises the amino acid sequence of SEQ ID NO: 11 and VH comprises the amino acid sequence set for in SEQ ID NO: 10; or (1) VL comprises the amino acid sequence of SEQ ID NO: 19 and VH comprises the amino acid sequence set for in SEQ ID NO: 18, wherein n is 1, 2, 3, 4, or 5.

[0170] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 and VH comprises an amino acid sequence having 80%, 85%. 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 2, wherein n is 1, 2, 3, 4, or 5.

[0171] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises the amino acid sequence26055 of SEQ ID NO: 3 and VH comprises the amino acid sequence of SEQ ID NO: 2. wherein n is L 2, 3, 4, or 5.

[0172] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 10, wherein n is 1, 2, 3, 4, or 5.

[0173] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 11 and VH comprises the amino acid sequence of SEQ ID NO: 10, wherein n is 1, 2, 3, 4, or 5.

[0174] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 18, wherein n is 1, 2. 3, 4, or 5.

[0175] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 19 and VH comprises the amino acid sequence of SEQ ID NO: 18, wherein n is 1, 2, 3, 4. or 5.

[0176] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and VH comprises an amino acid sequence having 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 27, wherein n is 1, 2, 3, 4, or 5.

[0177] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 27 and VH comprises the amino acid sequence of SEQ ID NO: 26, wherein n is 1, 2, 3, 4, or 5.

[0178] In another embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises an amino acid sequence26055 having 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and VH comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to comprises the amino acid sequence of SEQ ID NO: 35, wherein n is 1, 2, 3, 4, or 5.

[0179] For example, in one embodiment, provided herein is a scFv fusion protein comprising the structure VL-(G4S)n-VH or VH-(G4S)n-VL, wherein VL comprises the amino acid sequence of SEQ ID NO: 34 and VH comprises the amino acid sequence of SEQ ID NO: 35, wherein n is 1, 2, 3, 4, or 5.

[0180] The scFvs disclosed herein may be provided in a bispecific format comprising a CD3 binder (scFv) linked by a peptide linker to an scFv that binds CLDN18.2 as disclosed herein. When these molecules, called bispecific T-cell engagers, bind CD3 on T cells and CLDN18.2 expressed on the surface of a cell, it brings the T cells to a tumor site.

[0181] scFvs disclosed herein may also be fused or conjugated to cytotoxic agents, cellular toxins, radioisotopes, cytokines, and enzymes for cancer, autoimmune, and / or inflammatory therapeutic applications. In particular embodiments, the peptide linker may comprise 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G4S peptide units (SEQ ID NO: 55).

[0182] In further embodiments, the scFvs disclosed herein may be linked to or inserted in different locations of an intact IgG molecule to confer dual epitope binding. For example, a bispecific antibody may be provided comprising two heterodimeric heavy chain constant domains wherein the N-terminus of one heavy chain constant domain is fused to the C-terminus of an scFv disclosed herein and the N-terminus of the other heavy chain constant domain is fused to the C-terminus of an scFv that targets an antigen other than human CLDN18.2 or a Fab' that targets an antigen other than human CLDN18.2.Nucleic acids, vectors, host cells, and methods for making human CLDN18.2 binders

[0183] The present disclosure provides nucleic acids (e.g., isolated nucleic acids) encoding a human CLDN18.2 binder as disclosed herein. The present disclosure provides vectors comprising one or more nucleic acids individually or collectively encoding a human CLDN18.2 binder as disclosed herein. The present disclosure also provides host cells comprising one or more nucleic acids individually or collectively encoding a human CLDN18.2 binder or comprising a vector or set of vectors as disclosed herein. The present disclosure also includes methods for making human CLDN18.2 binders. Also provided herein are human CLDN18.2 binders produced by any of the methods disclosed herein.26055

[0184] Provided herein are polynucleotide molecules encoding a human CLDN 18.2 binder described herein. Also provided herein are sets of polynucleotides molecules together encoding a human CLDN18.2 binder described herein. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

[0185] In some examples, the methods comprise introducing into a host cell (i) an expression vector that encodes the VH and VL of a human CLDN 18.2 binder or the HC and LC of a human CLDN18.2 binder, or (ii) two expression vectors, one encoding the VH of a human CLDN18.2 binder or the HC of a human CLDN 18.2 binder and the other encoding the VL of a human CLDN18.2 binder or the LC of a human CLDN 18.2 binder. In some embodiments, nucleic acid molecules or polynucleotides encoding the VH, VL, HC, or LC are operably linked to a promoter and other transcription and translation regulatory sequences. In some examples, the methods comprise introducing into a host cell (i) an expression vector that encodes the VH and VL of a human CLDN18.2 binder or (ii) two expression vectors, one encoding the VH of a human CLDN18.2 binder and the other encoding the VL of a human CLDN18.2 binder. In some embodiments, nucleic acid molecules or polynucleotides encoding the VH, VL, HC, or LC are operably linked to a promoter and other transcription and translation regulatory sequences. The host cell is cultured under conditions and a time period suitable for expression of the nucleic acid molecules followed by isolating the human CLDN18.2 binder from the host cell and / or medium in which the host cell is grown. See e.g., W02004041862, WO2006122786, W02008020079, WO2008142164 or W02009068627. Any suitable expression vector may be used. For example, the expression vector may be a plasmid or viral vector. The disclosure also relates to host cells that contain such nucleic acid molecule encoding the human CLDN 18.2 binder or components thereof, e.g., solely the VH or HC or solely the VL or HC.

[0186] Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the human CLDN 18.2 binder, are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Thus, mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse, and hamster cells. Cell lines of particular preference may be selected through determining which cell lines have high expression levels, e.g., compared to other cell lines. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia m), amphibian cells,26055 bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Saccharomyces cerevisiae, and Trichoderma reesei. The present disclosure includes any host cell comprising a human CLDN18.2 binder of the present disclosure or comprising one or more nucleic acid molecules encoding such a human CLDN18.2 binder or comprising an expression vector that comprises one or more nucleic acid molecules encoding such human CLDN18.2 binder.

[0187] Further, expression of a human CLDN18.2 binder from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0216846B1, 0256055B1, 0323997B1, and 0338841B1. Thus, in an embodiment of the disclosure, the mammalian host cells lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium w herein, how ever, the nucleic acid molecule encoding the human CLDN18.2 binder comprises a glutamine synthetase gene which complements the lack of the gene in the host cell. Such host cells containing the human CLDN18.2 binder or nucleic acid(s) or expression vector(s) as discussed herein as well as expression methods, as discussed herein, for making the human CLDN18.2 binder using such a host cell are part of the present disclosure.

[0188] The present disclosure includes methods for purifying a human CLDN 18.2 binder comprising introducing a sample (e.g., culture medium, cell lysate or cell lysate fraction, e.g., a soluble fraction of the lysate) comprising the human CLDN18.2 binder to a purification medium (e.g., cation-exchange medium, anion-exchange medium and / or hydrophobic exchange medium) and either collecting purified human CLDN 18.2 binder from the flow-through fraction of said sample that does not bind to the medium; or, discarding the flow-through fraction and eluting bound human CLDN18.2 binder from the medium and collecting the eluate. In an embodiment of the disclosure, the medium is in a column to which the sample is applied. In an embodiment of the disclosure, the purification method is conducted following recombinant expression of the human CLDN18.2 binder in a host cell, e.g., wherein the host cell is first lysed and, optionally, the lysate is purified of insoluble materials prior to purification on a medium; or wherein the human CLDN18.2 binder is secreted into the culture medium by the host cell and the medium or a fraction thereof is applied to the purification medium.

[0189] In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of a human CLDN18.2 binder26055 will depend on the particular cell line or transgenic animal used to produce the human CLDN 18.2 binder. Human CLDN18.2 binders comprising only non-fucosylated N-glycans are part of the present disclosure and may be advantageous, because non-fucosylated antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See e.g., Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); and U.S. Patent Nos. 6,946,292 and 7,214,775). These human CLDN18.2 binders with non-fucosylated N- glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

[0190] The present disclosure includes human CLDN18.2 binders comprising N-linked glycans that are typically added to immunoglobulins produced in Chinese hamster ovary cells (CHO N- linked glycans) or to engineered yeast cells (engineered yeast N-linked glycans), such as, for example, Pichia pastoris. For example, in an embodiment of the disclosure, the human CLDN18.2 binder comprises one or more of the “engineered yeast N-linked glycans'’ or “CHO N-linked glycans” (e.g., GO. GO-F, Gl, Gl-F, G2-F, and / or Man5). In an embodiment of the disclosure, the human CLDN18.2 binder comprises the engineered yeast N-linked glycans, i.e., GO, Gl, and / or G2, optionally, further including Man5. In an embodiment of the disclosure, the human CLDN18.2 binders comprise the CHO N-linked glycans, i.e., GO-F, Gl-F and G2-F, optionally, further including GO. Gl, G2, and / or Man5. In an embodiment of the disclosure, about 80% to about 95% (e.g., about 80-90%, about 85%, about 90% or about 95%) of all N- linked glycans on the human CLDN18.2 binders are engineered yeast N-linked glycans or CHO N-linked glycans. See Nett et al. Yeast. 28: 237-252 (2011); Hamilton et al. Science. 313: 1441- 1443 (2006); Hamilton et al. Curr Opin Biotechnol. 18(5): 387-392 (2007). For example, in an embodiment of the disclosure, an engineered yeast cell is GF15.0 or YGLY8316 or strains of U.S. Patent No. 7,795,002 or Zha et al. Methods Mol Biol. 988: 31-43 (2013). See also WO2013066765.Administration and pharmaceutical compositions

[0191] The human CLDN18.2 binder may be provided in suitable pharmaceutical compositions comprising the human CLDN18.2 binder and a pharmaceutically acceptable carrier. Any suitable carrier may be used. The carrier may be a diluent, adjuvant, excipient, or vehicle with which the human CLDN18.2 binder is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by26055 conventional, well-known sterilization techniques (e.g.. filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, and the like. The concentration of the molecules or of the disclosure in such pharmaceutical formulation may vary widely, i.e.. from less than about 0.5%, usually to at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: 77re Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp. 691-1092, see especially pp. 958-989.

[0192] Any suitable mode of administration may be used. The mode of administration of the human CLDN18.2 binder may be parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal) or other means appreciated by the skilled artisan, as well known in the art. In some embodiments, the human CLDN18.2 binder is administered intravenously. In other embodiments, the human CLDN18.2 binder is administered subcutaneously. Subcutaneous administration of the human CLDN18.2 binder can be facilitated by agents such as hyaluronan degrading enzymes, including hyaluronidases, including soluble PH20 polypeptides, and variants thereof. For systemic administration, the facilitating agents can be modified to increase pharmacological properties, such as serum half-life, by modifying the agents, such as with polymers. See, e.g., U.S. Patent Nos.7, 767, 429, 8.431,380, 7,871,607, International Publication No. WO 2020 / 022791. U.S. Patent Publication No. US2006 / 0104968 and European Patent 1858926, and in numerous other patents and publications. Exemplar}7of such agents is the known agent PEGPH20 or rHuPH20. Accordingly, specific embodiments relate to pharmaceutical compositions comprising the human CLDN18.2 binder and any one of a hyaluronan degrading enzyme, hyaluronidase, soluble hyaluronidase, soluble PH20 polypeptide, or a variant of any of the foregoing. In particular embodiments, the pharmaceutical composition comprises the human CLDN18.2 binder and a soluble PH20 polypeptide or a variant thereof.

[0193] In some embodiments, the human CLDN18.2 binder may be administered to an individual (e.g., patient) parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously, or intraperitoneally. In some embodiments, i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.26055

[0194] The dose given to an individual having a cancer is sufficient to alleviate or at least partially arrest the disease being treated and may be, e.g., about 0.005 mg / kg to about 100 mg / kg, e.g., about 0.05 mg / kg to about 30 mg / kg or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg or about 24 mg / kg, or, e.g., about 1, 2, 3, 4, 5, 6, 7. 8, 9 or 10 mg / kg. but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 30. 40. 50, 60, 70, 80, 90 or 100 mg / kg.

[0195] In some examples, between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat cancer or malignancy, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.

[0196] The administration of the human CLDN 18.2 binder may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration. The repeated administration may be at the same dose or at a different dose. For example, the human CTDN18.2 binder in the methods of the disclosure may be administered at 8 mg / kg or at 16 mg / kg at weekly interval for 8 weeks, followed by administration at 8 mg / kg or at 16 mg / kg every two weeks for an additional 16 weeks, followed by administration at 8 mg / kg or at 16 mg / kg every four weeks by intravenous infusion.

[0197] The human CLDN18.2 binder may be administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more. For example, human CLDN18.2 binder in the methods of the disclosure may be provided as a daily dosage in an amount of about 0.1-100 mg / kg, such as 0.5, 0.9, 1.0, 1.1. 1.5, 2, 3, 4. 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18. 19, 20. 21. 22. 23, 24, 25, 26, 27, 28, 29, 30, 40, 45. 50. 60. 70. 80. 90 or 100 mg / kg. per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24. 12. 8, 6, 4, or 2 hours, or any combination thereof.

[0198] The human CLDN18.2 binder may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of the occurrence of an event in cancer progression, and / or reduce the risk of recurrence when a cancer is in remission. This may be especially useful in patients in whom it is difficult to locate a tumor that is known to be present due to other biological factors.26055Methods and compositions for use in treating cancer

[0199] In some embodiments, any of the human CLDN18.2 binders may be used for treatment of cancer.

[0200] In one example, provided herein is a method for treating a cancer in an individual in need thereof comprising administering to the individual a therapeutically effective amount of any one of the human CLDN18.2 binders disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising a therapeutically effective amount of any one of the human CLDN18.2 binders disclosed herein.

[0201] In another example, provided herein is a human CLDN18.2 binder for use in treatment of a cancer in an individual in need thereof. In another example, provided herein is a therapeutically effective amount of a human CLDN18.2 binder for use in treatment of a cancer in an individual in need thereof. Any of the human CLDN18.2 binders described herein may be used in treatment of a cancer in an individual in need thereof.

[0202] In another example, provided herein is a composition comprising a human CLDN 18.2 binder for use in treatment of a cancer in an individual in need thereof. In another example, provided herein is a therapeutically effective amount of a composition comprising a human CLDN18.2 binder for use in treatment of a cancer in an individual in need thereof.

[0203] In another example, provided herein is the use of any one of the human CLDN18.2 binders disclosed herein, or a composition (e.g., a pharmaceutical composition) comprising any one of the human CLDN18.2 binders disclosed herein, in the manufacture of a medicament for treating a cancer in an individual in need thereof.

[0204] Any suitable cancer may be treated. In particular embodiments, the cancer is gastrointestinal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bile tract cancer, colorectal cancer, melanoma, non-small cell lung cancer, head and neck cancer, bladder cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma (e.g., non-Hodgkin lymphoma or Hodgkin lymphoma), brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, renal cancer, mesothelioma, ovarian cancer, small cell lung cancer, anal cancer, cervical cancer, thyroid cancer, testicular cancer, salivary' cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

[0205] In another embodiment, the cancer is gastrointestinal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bile tract cancer, or colorectal cancer.26055Combination therapy treatments

[0206] In some embodiments, a combination therapy of the present disclosure comprises a human CLDN18 2 binder and one or more additional therapeutic agents (e.g., small molecules or antibodies) that may be used for the treatment of cancer.

[0207] In particular embodiments, a combination therapy of the present disclosure (including any combination described below) may be used to treat gastrointestinal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bile tract cancer, colorectal cancer, melanoma, non-small cell lung cancer, head and neck cancer, bladder cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, lymphoma (e.g., non-Hodgkin lymphoma or Hodgkin lymphoma), brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, renal cancer, mesothelioma, ovarian cancer, small cell lung cancer, anal cancer, cervical cancer, thyroid cancer, testicular cancer, salivary cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

[0208] In another embodiment, the combination therapy of the present disclosure may be used to treat gastric cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction cancer, bile tract cancer, or colorectal cancer.Combination therapy comprising a human CLDN18.2 binder and chemotherapy

[0209] A combination therapy of the present disclosure may be administered to an individual having a cancer in combination with chemotherapy. The individual may undergo the chemotherapy before, at the same time, or after the individual is undergoing the therapy with a human CLDN18.2 binder of the present disclosure. The combination therapy of the present disclosure may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapsed cancer and who is undergoing chemotherapy or who has completed chemotherapy.

[0210] Any suitable chemotherapy may be used. The chemotherapy may include one or more chemotherapy agents selected from the group consisting of:(i) alkylating agents, including but not limited to, bifunctional alkylators, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan;(ii) monofunctional alkylators, including but not limited to, dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine);26055(iii) anthracy clines, including but not limited to. daunorubicin, doxorubicin, epirubicin. idarubicin, mitoxantrone, and valrubicin;(iv) cytoskeletal disruptors (taxanes), including but not limited to, paclitaxel, docetaxel, nab-paclitaxel (ABRAXANE®), and taxotere;(v) epothilones, including but not limited to ixabepilone and utidelone;(vi) histone deacetylase inhibitors, including but not limited to vorinostat and romidepsin;(vii) inhibitors of topoisomerase I, including but not limited to irinotecan and topotecan;(viii) inhibitors of topoisomerase II, including but not limited to etoposide, teniposide, and tafluposide;(ix) kinase inhibitors, including but not limited to bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib;(x) nucleotide analogs and precursor analogs, including but not limited to azacytidine, azathioprine, fluoropyrimidines (e g., such as capecitabine, carmofur. doxifluridine, fluorouracil, and tegafur) cytarabine, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine (formerly thioguanine);(xi) peptide antibiotics, including but not limited to bleomycin and actinomycin;(xii) a platinum-based agent, including but not limited to, carboplatin, cisplatin, and oxaliplatin:(xiii) a retinoid, including but not limited to, tretinoin, alitretinoin, and bexarotene; and(xiv) vinca alkaloids and derivatives, including but not limited to, vinblastine, vincristine, vindesine, and vinorelbine.

[0211] Selecting a dose of the chemotherapy agent(s) for chemotherapy depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. The dose of the additional therapeutic agent(s) should be an amount that provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each additional therapeutic agent will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd. Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis. Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et26055 al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians’ Desk Reference 2003 (Physicians’ Desk Reference, 57thEd); Medical Economics Company; ISBN: 1563634457; 57thedition (November 2002). Determination of the appropriate dose regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the individual’s clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0212] Thus, the present disclosure contemplates embodiments of the combination therapy of the present disclosure that includes a chemotherapy step comprising platinum-containing chemotherapy, pemetrexed and platinum chemotherapy or carboplatin and either paclitaxel or nab-paclitaxel. In particular embodiments, the combination therapy with a chemotherapy step may be used for treating at least gastric cancer, esophageal cancer, gastroesophageal junction cancer, pancreatic cancer and any other gastrointestinal cancer.

[0213] In another example, the present disclosure contemplates embodiments of the combination therapy of the present disclosure that includes a chemotherapy such as 5- fluorouracil, leucovorin, and / or oxaliplatin (e.g., mFOLFOX6), e.g., for treatment of gastric cancer (e.g., a locally advanced or metastatic gastric cancer). In some embodiments, mFOLFOX6 is administered even' two weeks. In some embodiments, a human CLDN18.2 binder disclosed herein is administered in combination with 5 -fluorouracil. In some embodiments, a human CLDN18.2 binder disclosed herein is administered in combination with leucovorin. In some embodiments, a human CLDN18.2 binder disclosed herein is administered in combination with oxaliplatin. In some embodiments, a human CLDN18.2 binder disclosed herein is administered in combination with 5 -fluorouracil, leucovorin, and oxaliplatin (e.g., mFOLFOX6). In some embodiments, an mFOLFOX6 regimen may include administering to the individual oxaliplatin 85 mg / m2and leucovorin 400 mg / m2(or / -leucovorin 200 mg / m2), e.g.. as a 2-hour infusion, followed by 5 -fluorouracil 400 mg / m2as a bolus and 2,400 mg / m2as a 46-hour infusion) every 2 weeks.Combination Therapy Comprising A Human CLDN18.2 Binder And A Therapeutic Antibody

[0214] The human CLDN 18.2 binder of the present disclosure may be administered in combination with one or more therapeutic antibodies for treatment of cancer. The individual may undergo treatment with the therapeutic antibody before, at the same time, or after the individual is undergoing the therapy with the human CLDN18.2 binder of the present disclosure. The26055 combination therapy of the present disclosure may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapsed cancer and who is undergoing chemotherapy or who has completed chemotherapy. In particular embodiments, the therapeutic agent targets the programmed death 1 receptor or ligand, PD-1 and PD-L1, respectively.

[0215] In some aspects, a method provided herein may further comprise administering a therapeutically effective amount a PD-1 inhibitor or PD-L1 inhibitor to the subject. In some aspects, the method provided herein may further comprise administering a therapeutically effective amount an anti-PD-1 antibody or an anti-PD-Ll antibody to the subject. Exemplary' anti-PD-1 antibodies that may be used in a combination therapy with the human CLDN18.2 binder include any antibody that binds PD-1 and inhibits PD-1 from binding PD-L1. In various embodiments, the anti-PD-1 antibody is pembrolizumab, cemiplimab, dostarlimab, toripalimab, retifanlimab, tislelizumab, camrelizumab, sintilimab, vopratelimab, spartalizumab, INCMGA00012 (MGA012), AMP-514 (MEDI0680). acrixolimab, or nivolumab. In various embodiments, the anti-PD-Ll antibody is atezolizumab, durvalumab. KN035, cosibelimab, or avelumab. In some aspects, a method provided herein may further comprise administering a therapeutically effective amount of pembrolizumab (also know n as KEYTRUDA®) to the subject.

[0216] Exemplary antibodies include the following anti-PD-1 antibodies and compositions comprising an anti-PD-1 antibody and a pharmaceutically acceptable salt.

[0217] Pembrolizumab, also known as KEYTRUDA®, lambrolizumab, MK-3475 or SCH- 900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No. 8,354,509 and W02009 / 114335 and disclosed, e.g., in Hamid, et al.. New England J. Med. 369 (2): 134-144 (2013).

[0218] Nivolumab, also known as OPDIVO®, MDX- 1106-04, ONO-4538, or BMS-936558, is a fully human IgG4 anti-PD-1 antibody described in W02006 / 121168 and U.S. Pat. No. 8,008,449.

[0219] Cemiplimab-rwlc, also known as cemiplimab, LIBTAYO® or REGN2810, is a recombinant human IgG4 monoclonal antibody that is described in WO2015112800 and U.S. Pat. No. 9,987,500.

[0220] Dostarlimab-gxly, also known as dostarlimab or JEMPERLI®, is a humanized anti-PD- 1 antibody that is described in U.S. Patent Nos. 9,815,897, 10,738,117, and 11.155,624.

[0221] Retifanlimab-dlwr, also known as retifanlimab and ZYNYZ®, is a humanized anti-PD-1 antibody that is described in U.S. Patent No. 10,577,422.26055

[0222] Toripalimab-tpzi. also known as tonpalimab and LOQTORZ1®. is a humanized anti- PD-1 antibody that is described in U.S. Patent Nos. 10,066,013 and 10,915,302.

[0223] In particular embodiments, the anti-PD-1 antibody comprises (i) a VH comprising the three HC-CDRs of pembrolizumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of pembrolizumab fused or linked to a LC kappa or lambda constant domain.

[0224] In particular embodiments, the anti-PD-1 antibody comprises (i) a VH comprising the three HC-CDRs of nivolumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of nivolumab fused or linked to a LC kappa or lambda constant domain.

[0225] In particular embodiments, the anti-PD-1 antibody comprises (i) a VH comprising the three HC-CDRs of cemiplimab-rwlc fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of nivolumab fused or linked to a LC kappa or lambda constant domain.

[0226] In particular embodiments, the anti-PD-1 antibody VH may be fused or linked to an IgGl, IgG2, IgG3, or IgG4 HC constant domain that is not currently linked to the particular VH or is linked to an IgGl, IgG2, IgG3, or IgG4 HC constant domain has been modified to include one or more mutations in the Fc domain that render the resulting anti-PD-1 antibody effecter- silent.Injection device for administering a human CLDN18.2 binder

[0227] The present disclosure also provides an injection device comprising a human CLDN18.2 binder as set forth herein or a composition (e.g.. a pharmaceutical composition) thereof. An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscularly, subcutaneously or intravenously. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., comprising the human CLDN 18.2 binder or a pharmaceutical composition thereof), a needle for piecing skin and / or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the disclosure, an injection device that comprises a human CLDN18.2 binder or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the human CLDN 18.2 binder or a pharmaceutical composition thereof in a cannula or trocar / needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated Ringer26055 solution comprising NaCL sodium lactate. KCL CaCh and optionally including glucose) introduced into the body of the subject through the cannula or trocar / needle.

[0228] The human CLDN18.2 binder or a pharmaceutical composition thereof may, in an embodiment of the disclosure, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the disclosure, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the human CLDN18.2 binder or a pharmaceutical composition thereof to a patient’s body. External infusion pumps are medical devices that deliver the human CLDN18.2 binder or a pharmaceutical composition thereof into a patient’s body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery', an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.Kits comprising a human CLDN18.2 binder

[0229] Further provided are kits comprising one or more components that include, but are not limited to, a human CLDN18.2 binder, as discussed herein in association with one or more additional components including, but not limited to, a further therapeutic agent, as discussed herein. The human CLDN18.2 binder and / or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

[0230] In one embodiment, the kit includes a human CLDN 18.2 binder or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a further therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0231] In another embodiment, the kit comprises a combination of the disclosure, including a human CLDN18.2 binder or pharmaceutical composition thereof in combination with one ormore therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0232] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above. Thus, the present disclosure includes a kit comprising an injection device and the human CLDN18.2 binder, e.g., wherein the injection device includes human CLDN18.2 binder or wherein the human CLDN18.2 binder is in a separate vessel.

[0233] The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the disclosure may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.Methods and kits for detecting CLDN18.2

[0234] According to another embodiment of the disclosure, methods and kits for detecting expression of human CLDN18.2 in vitro or in vivo are provided. The kits may include any of the foregoing human CLDN18.2 binders (e.g., a delectably labeled human CLDN18.2 binder), and, optionally, one or more compounds for detecting the label. In some embodiments, the label is selected from the group consisting of a fluorescent label, an enzyme label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

[0235] In some cases, CLDN18.2 expression is detected in a biological sample. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies, and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid, or urine. A biological sample is typically obtained from a mammal, such as a human or non-human primate.

[0236] In one embodiment, provided is a method of determining if a subject has cancer (e.g.. a CLDN18.2+ cancer) by contacting a sample from the subject with a human CLDN18.2 binder as disclosed herein; and detecting binding of the human CLDN18.2 binder to the sample. Anincrease in binding of the human CLDN 18.2 binder to the sample as compared to binding of the human CLDN18.2 binder to a control sample identifies the subject as having cancer.

[0237] In another embodiment, provided is a method of confirming a diagnosis of cancer (e.g., a CLDN18.2+ cancer) in a subject by contacting a sample from a subject diagnosed with cancer with a human CLDN18.2 binder as disclosed herein; and detecting binding of the human CLDN18.2 binder to the sample. An increase in binding of the human CLDN18.2 binder to the sample as compared to binding of the human CLDN18.2 binder to a control sample confirms the diagnosis of cancer in the subject.

[0238] In some examples of the disclosed methods, the human CLDN18.2 binder is directly labeled. In some examples, the methods further include contacting a second antibody that specifically binds the human CLDN18.2 binder with the sample; and detecting the binding of the second antibody. An increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects cancer in the subject or confirms the diagnosis of cancer in the subject. In some cases, the cancer is a gastric cancer or any other type of cancer that expresses CLDN18.2. In some examples, the control sample is a sample from a subject without cancer. In particular examples, the sample is a blood or tissue sample.

[0239] In some cases, the human CLDN18.2 binder is directly labeled with a detectable label. In another embodiment, the human CLDN18.2 binder (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human CLDN18.2 binder is labeled. A second antibody is chosen such that it is able to specifically bind the specific species and class of the human CLDN18.2 binder. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the antibody or secondary antibody are described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Non-limiting examples of suitable fluorescent materials include umbelliferon, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include125I,1311,35S or3H.26055

[0240] In an alternative embodiment, CLDN18.2 can be assayed in a biological sample by a competition immunoassay utilizing CLDN18.2 standards labeled with a detectable substance and an unlabeled antibody that specifically binds CLDN18.2. In this assay, the biological sample, the labeled CLDN18.2 standards and the human CLDN18.2 binder are combined and the amount of labeled CLDN18.2 standard bound to the unlabeled antibody is determined. The amount of CLDN18.2 in the biological sample is inversely proportional to the amount of labeled CLDN18.2 standard bound to the human CLDN18.2 binder.

[0241] The immunoassays and method disclosed herein can be used for a number of purposes. In one embodiment, the human CLDN18.2 binder may be used to detect the production of CLDN18.2 in cells in cell culture. In another embodiment, the human CLDN18.2 binder can be used to detect the amount of CLDN18.2 in a biological sample, such as a tissue sample, or a blood or serum sample. In some examples, the CLDN18.2 is cell-surface CLDN18.2. In other examples, the CLDN18.2 is soluble CLDN18.2 (e.g., CLDN18.2 in a cell culture supernatant or soluble CLDN18.2 in a body fluid sample, such as a blood or serum sample).

[0242] In one embodiment, a kit is provided for detecting CLDN18.2 in a biological sample, such as a blood sample or tissue sample. For example, to confirm a cancer diagnosis in a subject, a biopsy can be performed to obtain a tissue sample for histological examination. Alternatively, a blood sample can be obtained to detect the presence of soluble CLDN18.2 protein or fragment. Kits for detecting a polypeptide will ty pically comprise a human CLDN18.2 binder, according to the present disclosure, that specifically binds CLDN18.2. In some embodiments, an antibody fragment, such as an scFv fragment, a VH domain, or a Fab is included in the kit. In a further embodiment, the human CLDN18.2 binder is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).

[0243] In one embodiment, a kit includes instructional materials disclosing means of use of a human CLDN18.2 binder. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk), may be visual (such as video files), or provided through an electronic network, for example, over the internet, World Wide Web, an intranet, or other network. The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzy matic labels, filter sets to detect fluorescent labels, appropriate secondary’ labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.26055

[0244] In one embodiment, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting CLDN18.2 in a biological sample generally includes the steps of contacting the biological sample with a human CLDN18.2 binder which specifically reacts, under immunologically reactive conditions, to a CLDN18.2 polypeptide. The human CLDN18.2 binder is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.

[0245] Methods of determining the presence or absence of a cell surface marker are well known in the art. For example, the human CLDN18.2 binder can be fused or conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds, or drugs. The human CLDN18.2 binders can also be utilized in immunoassays such as but not limited to radioimmunoassays (RIAs), ELISA, or immunohistochemical assays. The human CLDN18.2 binders can also be used for fluorescence activated cell sorting (FACS). FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells. See U.S. Patent No. 5,061,620. Any of the human CLDN18.2 binders as disclosed herein can be used in these assays. Thus, the human CLDN18.2 binder can be used in a conventional immunoassay, including, without limitation, an ELISA, an RIA, FACS, tissue immunohistochemistry, Western blot, or immunoprecipitation.EXAMPLES

[0246] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Materials and MethodsProtein enzvme-linked immunosorbent assays (ELISAs)

[0247] The wells of a 96-well plate were coated with 50 pL of purified human or rhesus CLDN18.2 in a carbonate coating buffer (5.3 g NaHCCh and 3.2 g Na2COs in 2L FLO) or HYCLONE™ Dulbecco’s phosphate buffered saline (DPBS; HYCLONE™. catalog (cat)# SH30028.02) at a concentration of 1-2 pg / mL and incubated overnight at 4 °C. Afterwards, the plate wells were washed with well wash solution (phosphate buffered saline (PBS) containing 0.05% poly sorbate 20). The plate wells were then blocked by adding Super Block Blocking26055Buffer T20 (Thermo Scientific, #37536) for 1-2 hours at room temperature followed by washing with well wash solution. Next, 50 pL / well hybridoma supernatant fraction (used neat), or purified antibody starting at 10 pg / mL and serially diluted 4- or 5-fold in ELISA buffer (DPBS + 0.1% bovine serum albumin (BSA) +0.05% polysorbate 20) was added to the wells. The plates were incubated for an hour at room temperature, then the wells were washed with well wash solution and 50 pL / well horseradish peroxide (HRP)-conjugated anti-species IgG diluted 1:3000- 5000 in ELISA buffer was added to the wells. HRP-conjugated anti-species IgG included goat anti-mouse IgG-HRP (Southern Biotech, cat# 1030-05), goat anti-rat IgG-HRP (Southern Biotech, cat# 3030-05), and goat anti-human IgG-HRP (Jackson Immunologies, cat# 109-036- 098). The plates were incubated for an hour at room temperature, then the wells washed with well wash and 50 pL / well ABTS Peroxidase Substrate (Kirkgaard & Perry Laboratories) was added to the wells. After 10 minutes, the optical density (OD) at 405 nm was measured using an ELISA plate reader.Cell ELISA on Chinese hamster ovary (CHO) cells

[0248] The wells of a 96-well plate were seeded with sufficient cells to provide 95-100% confluency on day 0 (the day of assay). On day 0 the culture medium was removed and 50 pL / well hybridoma supernatant fraction, or purified antibody starting at 10 pg / mL or equal molar concentration and serially diluted 4- or 5-fold in CHO medium (DMEM F12, 10% bovine serum). The plate was then incubated for 1 hour at room temperature or a temperature up to 37 °C. Next, about 50 pL of a pre-determined concentration of ligand was added to the wells of the plate and the plate was incubated for 30 minutes at room temperature or a temperature up to 37 °C. The plate wells were washed with ELISA wash (PBS containing 0.05% polysorbate 20). Next, 50 pL / well of HRP-conjugated anti-species IgG diluted 1 :2000 in CHO medium was added to the wells and the plate incubated for an hour at room temperature or a temperature up to 37 °C. HRP-conjugated anti-species IgG included goat anti -mouse IgG-HRP (Southern Biotech, cat# 1030-05), goat anti -rat IgG-HRP (Southern Biotech, cat# 3030-05). and goat anti-human IgG- HRP (Jackson Immunologies, cat# 109-036-098). The plate wells were washed with the ELISA wash. Next, 50 pL / well 3,3',5,5'-tetramethylbenzidine (TMB) substrate (1-STEP™ Ultra TMB- ELISA: Thermo Fisher, Cat# 34029) was added to the wells. When the blank started to turn blue, 50 pL / well TMB stop solution (KPL, Cat# 50-85-06) was added to the wells and A450 - A620 was measured on an ELISA reader.Immunizing Humanized Mice and Fusion

[0249] TRIANNI™ (1 .2.5) C57 BL / 6 female mice at 6-8 weeks old were immunized with human CLDN18.2 Full Length Protein-VLP (Aero Biosystems, Catalog CL2-H52P7) at 20 pg dosage. A second cohort of TRIANNI™ (1.2.5) C57 BL / 6 female mice at 6-8 weeks old were immunized with Human CLDN18.2-mRNA-LNP at 50 pg dosage. Cheek bleed samples were used for assessing titer using a FACS binding assay. Mice were euthanized and harvested for splenocytes and lymph nodes per IACUC standards. Splenocytes and lymph nodes were gently homogenized using glass mortar and pestle. Red blood cells were lysed using ACK lysing buffer for 5 minutes. B cells were counted and fused with myeloma cell line SP2 / 0-Agl4 using a Nepa Gene Bulldog electroporator, 2-pulse setting. Hybridoma cells w ere plated in tissue culture plates for two weeks and screened using a FACS binding assay as described below-. Candidates were chosen based on cell surface binding to human CLDN18.2-CHOK1 cells and negative binding to both human CLDN18. 1-CHOK1 and CHOK1 parental cells.Hybridoma Supernatant FACS Binding Assay

[0250] Human CLDN18.1-CHOK1 (BPS Bioscience, Catalog #78361) and Pooled Human CLDN18.2-CHOK1 were grown in 175 cm2flask using F-12K media (Kaighn's Modification of Ham's F-12 Medium) + 10% heat inactivated fetal bovine serum (FBS) + 1.0% Penicillin- Streptomycin + 1.0 mg / mL GENETICIN® selective antibiotic and DMEM / F12 (1: 1 mixture) + 10% heat inactivated FBS + 1.0% penicillin-streptomycin+1.0 mg / mL GENETICIN®, respectively. Parental CHOK1 cells were grown in DMEM / F12 (1: 1 mixture) + 10% heat inactivated FBS + 1.0% penicillin-streptomycin. All cells were harvested using 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) (CLDN18. 1-CHOK1) or trypsin and washed twice with PBS buffer. Cell trace dye (CELLTRACE™-Violet, Thermo Scientific, cat# C34557 and CELLTRACE™-Far Red, Thermo Scientific catalog # 34564) was diluted at optimized concentrations in PBS (1 ml staining volume / lOM cells) and used to resuspend cell pellets prepared from the different recombinant cell lines. Cells were incubated with the dyes at 37° C for 20 minutes in the dark under shaking. These staining reactions were stopped by adding warm DMEM / F12 complete medium with 10% FBS, using 5X the original staining volume, and incubated at 37° C for 10 minutes under shaking. Stained cells were spun down and washed once with 10 ml PBS and resuspended in FACS buffer (2.0% FBS in Hank’s Balanced Salt Solution (HBSS)). Cells were then checked to confirm both positive staining with the dyes and that each recombinant cell line demonstrated a separate fluorescent intensity using an INTELLICYT® iQue Screener PLUS instrument. The different recombinant cell lines w ere then mixed byresuspending in fluorescence activated cell sorting (FACS) buffer and aliquoted into 96-V-well plates (50 pL / well, 30xl04 / well). Cells were stained with either 50 pL of hybridoma supernatant or purified control antibodies (10 pg / mL) for 30 minutes and then spun down and washed once with FACS buffer. Finally, the cells were stained with a fluorescently-labeled secondary antibody (specific to the Fc domain of the supernatant or purified control antibody) for 15 minutes and spun down and washed twice with FACS buffer. Cells resuspended in 100 pL FACS buffer were then analyzed using an INTELLICYT® iQue Screener PLUS instrument.Next-Generation Sequencing of Lysed Hybridomas

[0251] Candidates of interest were lysed in 85 pL of Qiagen TCL buffer with 1% betamercaptoethanol and RNA was isolated using Qiagen TURBOCAPTURE™ tubes (Qiagen, cat# 72251). cDNA was generated using SUPERSCRIPT™ IV reverse transcriptase (Thermo, cat# 18090050) in the presence of a template switching oligo (TSO) for 5‘ rapid amplification of cDNA ends (5’RACE). A first round polymerase chain reaction (PCR) was then performed using GOTAQ® polymerase (Promega, cat# M7422), a forw ard R1 primer, and a reverse equimolar mixture of Tri IgK, AF IgGl / 2abc, and AF IgG3 nested primers. A second round of PCR, also using GOTAQ® polymerase, further amplified the PCR products and introduced ILLUMINA® MISEQ™ NGS adaptors and indices for high-throughput, multiplexed NGS sequencing. Mixed PCR2 pools of heavy, kappa, and lambda chain samples were bead purified at a ratio of 0.8x using AGENCOURT® AMPURE® XP purification system (Beckman Coulter, cat# A63881). Individual indexed PCR products were then pooled and gel purified using a Qiagen gel purification kit (Qiagen, cat# 28704). Sequences that showed unique variable regions (framework and CDR regions) were recombinantly expressed for further testing.Purified Antibodies EC50 FACS Binding Assay (CLND18.1 / 18.2-CHOK1 and NUGC-4)

[0252] Human CLDN18.1-CHOK1, Human CLDN18.2-CHOK1 Clone 1C5, and CHOK1 parental cells were grown in 175 cm2flasks using media recipes described above. Endogenous tumor cell line NUGC-4 was grown in RPMI 1640 medium + 10% heat inactivated FBS. All cells were harvested as described above. Cell trace dye was added in a similar manner as described above for the hybridoma supernatant FACS binding assay. The different recombinant cell lines were then mixed by resuspending in FACS buffer and aliquoted into 96-V-well plates (50 pl / well, 30xl04 / well). In separate 96-V-well plates, purified human CLDN18.2 antibodies and control antibodies were diluted in FACS buffer with starting concentration of 10 pg / mL and titered 1:3, for 8 total points. Diluted antibodies were added to cells for 30 minutes and then spun26055 down and washed once with FACS buffer. Finally, the cells were stained with a fluorescently labeled secondary antibody (specific to the Fc domain of the supernatant or purified control antibody) for 15 minutes and spun down and washed twice with FACS buffer. Cells were resuspended in 100 pL FACS buffer were then analyzed using aNovoCyte 3000 flow cytometer system.SPR binding affinity7

[0253] SPR measurements were performed on a BIACORE® 4000 (Cytiva, USA) at 25°C. Buffers (Teknova cat# H8022) were degassed and filter-sterilized through 0.2 pm filters prior to use. Anti-human Fc antibody (Cytiva cat# 29234600, lot# 10330472) was immobilized onto a CM5 sensor chip (lot# 10330431) using standard amine coupling chemistry'. All purified antibodies were diluted in assay buffer IX HBS-EP pH 7.6 (TeknoVa, cat# H8022) to 1 pg / mL then captured on chip for 60s. For kinetic analysis, a concentration series of CLDN18.2 (0-200 nM) (Genscript, cat# Z03504-1) were injected over the antibodies and reference surfaces at a flow rate of 30 pL / min. The association and dissociation were measured for 180 s and 300 s, respectively. After each sample injection, the surface was regenerated by a 30 s injection of 3M MgCh. Affinity' and kinetic constants were determined by fitting the sensorgrams with the 1 : 1 Langmuir model using BIACORE® 4000 evaluation software (Cytivia).Epitope binning

[0254] A biosensor surface containing an array of covalently coupled monoclonal antibodies (mAbs) was prepared on an HC30M sensor chip (Carterra) via amine-coupling using the Carterra LSA® platform’s surface array preparation protocol using the reagents summarized in Table 9 below. The running buffer for chip preparation was 25 mM MES (2-(N- morpholino)ethanesulfonic acid) pH 5.5, 150mM NaCl, and 0.05% (v / v) TWEEN®-20. The entire sensor surface was first activated with a mixture of 40 mM EDC (l-ethyl-3-(3- dimethylaminopropyl)carbodiimide) and 10 mM sulfo-NHS (N-hydroxysulfosuccinimide) in 100 mM MES buffer at pH 5.5 for 5 min. mAbs diluted to approximately 10 pg / mL in 10 mM sodium acetate pH 4.5, 0.05% TWEEN®-20 in duplicate were coupled to unique spots on the EDC-sulfo-NHS-modified surface for 10 min, followed by a 5 min injection of 1 M ethanolamine-HCl (pH 8.5) to quench any remaining reactive esters. The resulting array of covalently coupled mAbs on the sensor surface was used for subsequent epitope binning measurements.26055

[0255] Binning in this study was performed in a “classical sandwich format” at 25 °C in IxHBS-TE + 0.5 mg / ml BSA using the single flow cell mode of the Carterra LSA® instrument. Each binning experiment cycle involved first a 3 min injection of the antigen at 100 nM followed by a 3 min injection of the secondary' antibody at 50 pg / rnL. The array surface was regenerated with two 15 s pulses of 10 mM glycine pH 2.2 after each binning cycle. The raw high-throughput epitope binning results were processed using the LSA® epitope software to generate a heat map as yvell as other visualizations such as a combined dendrogram and netyvork / community plots.Table 9: Reagents for epitope binningEpitope mapping by SPR

[0256] Epitope mapping was carried out using single mutant variants of Claudinl 8.2 to conduct a comprehensive exploration of the eight amino acid differences between Claudinl 8. 1 and Claudinl 8.2 to pinpoint the key residues for binding using the reagents and instrument summarized in Table 10. The binding affinities of mutants to the set of Claudinl 8.2-directed mAbs yvere measured on SPR similar to wildtype (WT). Conclusions are supported by loss-of binding data.Table 10: Reagents for epitope mapping by SPR26055Media and passaging for CH0-K1 engineered cell lines and NUGC-4 endogenous tumor cell line

[0257] CHO-K1 adherent cell lines were grown in T75 flasks. Negative control wild-type (WT) CHO-K1 cells were grown in DMEM / F12 (Gibco cat# 11320033), 10% FBS (Coming cat# 35- 015-CV), and IX penicillin / streptomycin (Gibco V15140122). The CHO-K1 Claudinl8.1 counter screen cell line was grown in F12K (Gibco 21126022), 10% FBS, IX penicillin / streptomycin and 1 mg / mL GENETICIN® (Gibco 10131-027). The CHO-K1 Claudinl8.2 target cell line was grown in DMEM / F12, 10% FBS, IX penicillin / streptomycin and 1 mg / mL GENETICIN®.

[0258] The NUGC-4 endogenous tumor cell line was grown in suspension in T75 flasks with RPMI (Gibco cat# 11875093), 10% FBS, and IX penicillin / streptomycin.Cell Preparation for CHO-K1, CHO-K1 Claudinl8,l and CHO-K1 Claudinl8,2

[0259] Adherent cell lines were grown to confluence in T75 flasks. On the day of the assaygrowth medium was aspirated, cells were rinsed with DPBS, no Ca, no Mg (Gibco cat# 14190136) and detached using TRYPLE™ enzyme (Gibco cat# 12604013). Cells were centrifuged at 500 ref for 3 minutes and resuspended in 10 mL of PBS (Gibco cat# 20012027) and counted with the Invitrogen COUNTESS™ II cell counter. Cell concentration was adjusted to 1E6 cells per mL in PBS and stained with Near- Infrared LIVE / DEAD™ Stain (Invitrogen cat# L34975) at room temperature (RT) in the dark for 30 minutes. Cells were centrifuged and resuspended at 1E6 cells per mL of BD stain buffer (BD Pharmingen cat# 554657). 50 pL per well of each cell suspension was then added to a 96-well v-bottom plate (Costar cat# 3897) for target staining.26055Cell Preparation for Endogenous Tumor Cell Line NUGC-4

[0260] NUGC-4 suspension cells were grow n in T75 flasks. On the day of the assay, cells were collected in 50 mL FA CON® tubes and centrifuged at 500 ref for 3 minutes, growth medium was removed, and cells w ere resuspended in 10 mL of PBS and counted with the Invitrogen COUNTESS™ II cell counter. Cell concentration was adjusted to 1E6 cells per mL in PBS and stained with Near-Infrared LIVE / DEAD™ Stain at RT in the dark for 30 minutes. Cells were centrifuged and resuspended at 1E6 cells per mL of BD Stain buffer. 50 pL per well of each cell suspension w as added to a 96-well v-bottom plate for target staining. scFv-Fc Flow Cvtometrv Dose Titration protocol on CHO-K1. CHO-K.1 Claudinl8,l and CHO- K1 Claudinl8,2 and Endogenous Tumor Cell Line NUGC-4

[0261] scFv samples were prepared at 2x concentration in BD stain buffer in a 96-well polypropylene source plate. 50 pL of the scFv samples were added to each of the cell plates for a final top concentration of 200 nM and a decreasing 5-fold serial dilution for 7 points down the plate, with the 8throw left blank for a 0 nM point. Samples were allowed to incubate for 1 hour on ice. Following the incubation, cells w ere pelleted by centrifuging for 1 minute at 500 ref and washed 2 times with 150 pL of BD stain buffer. Cells w ere resuspended in 50 pL per well of 1:200 PE-conjugated anti-mouse Fc secondary’ (Jackson 115-115-205) and incubated in the dark on ice for 30 minutes. Following the incubation, cells w ere pelleted by centrifuging for 1 minute at 500 ref and washed 2 times w ith 150 pl of BD stain buffer. Cells were then washed and resuspended for a total of 150 pL per well and read on an ATTUNE™ NXT flow cytometer.Data Analysis

[0262] Viable, single cell populations were gated in FLOWJO™ V 10.9 software and the final readout for each sample w as median fluorescence intensity (MFI) of PE. Plots of MFI over nanomolar concentration were generated in GraphPad Prism 9 and EC50s were determined by nonlinear regression [Agonist] vs. response — Variable slope (three or four parameters) fit. Bmax w as determined by One site - Total binding fit in GraphPad Prism.Example 1 - Immunization and screening for human CLDN18.2 binders

[0263] TRIANNI™ mice (AbCellera, Vancouver. BC, Canada) were immunized using mRNA encoding the full-length human mature CLDN18.2. Splenocytes from the immunized animals were subsequently isolated and used for the fusion with the Sp2 / 0 myeloma partner to produce hybridomas. Hybridomas were then separately plated into 96-well plates to produce a hybridoma26055 library and antibody supernatant fractions from the plated hybridomas were screened for binding to human CLDN18.2, species cross-reactivity, affinity, binding to cells expressing full-length human CLDN18.2, and binding to the human CLDN18.2. Positive binding wells were selected for subcloning to ensure monocl onality. Monoclonal hybridomas that were obtained were then used for small scale antibody production and purification. Purified antibodies were subjected to another round of screening, including affinity, epitope binning, full length protein binding, human CLDN18.2 binding, protein, and cell binding, and competition assays.

[0264] The hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034. 17H7. 1 were selected for analysis.Example 2 - Antibody production

[0265] EXPICHO™ cells growing in suspension were transfected with antibody expression plasmids (HC+LC) using commercially available protocols and EXPIFECTAMINE™ CHO reagents (ThermoFisher) (Sivasubramanian et al., MABS 9: 29-42 (2017)). In brief, cells were transfected at day 0 using 1 pg total DNA (3:2 ratio LC: HC) per 1 mL cells at a density of 6 million cells per mL and a viability >95% measured using a Vi-Cell (Beckman-Coulter). On day one, EXPICHO™ feed and enhancer were added to the cell culture and the cell culture temperature was lowered to 32°C. On day five, a second EXPICHO™ feed was performed; cell viability was measured using a Vl-CELL™ cell viability analyzer (Beckman-Coulter). Cell cultures were harvested between day 8 and day 12 depending on a cell viability' greater than 80% and the harvested cell culture fluid was centrifuged to remove cells and debris to provide a clarified supernatant fraction.

[0266] Antibodies were punfied from clarified supernatant fraction using Protein A resin chromatography (MABSELECT SURE™ LX, GE Healthcare) as follows.

[0267] Protein A resin in a loading buffer was incubated with the clarified supernatant fraction overnight in 4°C on a roller mixer. Afterwards, the protein A resin was collected from the clarified supernatant fraction and transferred to a chromatography column and washed with 10 column volumes (CV) of phosphate buffered saline (PBS). Antibody w as then eluted from the chromatography column using an elution buffer comprising 20 mM sodium acetate, pH 3.5. One column volume (CV) fractions w ere collected and tested by Bradford assay to determine presence of protein. In some cases, Protein A purification was followed by anion exchange chromatography (CAPTO™ Q, GE Healthcare). Purified antibodies were buffer exchanged into a final formulation buffer comprising 20 mM sodium acetate, 9% sucrose, pH 5.5. Purified antibody was checked for purity by reduced and non-reduced capillary electrophoresis sodium26055 dodecyl sulfate (CE-SDS) (PerkinElmer). concentration was measured by A280. and aggregate content was analyzed by SEC-UPLC (size exclusion ultra-performance liquid chromatography) using a BEH200 UPLC- SEC analytical column (Waters Corporation). Endotoxin was quantified using ENDOSAFE® NEXGEN-MCS™ (Charles River). Intact mass was confirmed via SYNAPT® G2S QTOF or XEVO® G2 TOF (Waters).

[0268] The amino acid sequences for the antibodies produced by hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1 are shown in Tables 4-8 and 21.Example 3 - CLDN18.2 Antibody and scFv Characterization

[0269] Antibodies and scFv created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034. 17H6, and NG034.17H7.1 w ere analyzed in terms of cell binding, affinity, epitope binning, and epitope mapping.Cell binding - recombinant antibodies

[0270] Binding of antibodies created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1, isotype control, or comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab to CHO-K1 control cells or engineered CHO-K1 cells expressing Claudinl8.1 or Claudinl8.2 was assessed (FIGS. 1A-1C). A summary of the results for EC50 and maximal binding (Bmax) for binding to CHO-K1 Claudinl8.2 cells is shown in Table 11. Overall, in this experiment, the binding in terms of EC50 for NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7. 1 was superior compared to the comparator antibodies CARsgene8E5-2I and zolbetuximab. Similarly, the Bmax was superior or similar compared to the comparator antibodies.Table 11: EC50 and Bmax for antibody binding to engineered CHO-K1 cells26055

[0271] Binding of antibodies created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1, isotype control, or comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab to the NUGC4 endogenous tumor cell line was also assessed (FIG. 2). A summary' of the results for EC50 and maximal binding (Bmax) is shown in Table 12.Table 12: EC50 and Bmax for antibody binding to NUGC4 cellsCell binding - scFv

[0272] Binding of scFv constructs created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7. NG034.17H6, and NG034.17H7.1 to CHO-K1 control cells or engineered CHO-K1 cells expressing Claudinl8.1 or Claudinl8.2 was assessed (FIGS. 3A-3C). The binding was compared with scFv constructs generated from comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab. A summary of the results for EC50 and maximal binding (Bmax) is shown in Table 13. Overall, in this experiment. NG034.5A9. NG034.7A6.NG034.8A7, NG034.17H6, and NG034.17H7.1 showed comparable or lower EC50 of cell binding compared to 8E5-2I and zolbetuximab. The Bmax of NG034.5A9 and NG034.17H7. 1 was higher than 8E5-2I and zolbetuximab, suggesting better accessibility of their binding site and / or binding kinetics.Table 13: EC50 and Bmax for scFv binding to engineered CHO-K1 cells26055

[0273] Binding of scFv constructs created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1 to the NUGC4 endogenous tumor cell line was also assessed (FIG. 4). The binding was compared with scFv constructs generated from comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab. A summary of the results for EC50 and maximal binding (Bmax) is shown in Table 14. For the NUGC4 cell line, which expresses an endogenous level (much lower than CHO-K1 Claudinl8.2) of membranebound CLDN18.2, NG034.17H7.1 and NG034.5A9 showed superior binding in terms of EC50 and Bmax compared to 8E5-2I and zolbetuximab.Table 14: EC50 and Bmax for scFv binding to NUGC4 cellsAffinity - recombinant antibodies

[0274] The binding affinity of antibodies created from hybridoma clones NG034.5A9, NG034.7A6. NG034.8A7. NG034.17H6, and NG034.17H7.1 or comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab to CLDN18.2 was evaluated using SPR. The results are shown in Table 15. Overall, the binding of antibodies created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1 was significantly stronger than the comparator antibody zolbetuximab.26055Table 15: Binding affinity for antibodiesAffinity - scFv-Fc

[0275] The binding affinity of scFv constructs created from hybridoma clones NG034.5A9, NG034.7A6, NG034.8A7, NG034.17H6, and NG034.17H7.1 or comparator anti-CLDN18.2 antibodies CARsgene8E5-2I and zolbetuximab to CLDN18.2 was evaluated using SPR. The results are shown in Table 16.Table 16: Binding affinity for scFv constructs26055Epitope binning

[0276] Epitope binning for anti-CLDN18.2 antibodies as described herein was performed using an LSA® instrument. Clones with very low signals, poor regeneration or aggregation were excluded. An anti-His antibody was included as a control. After data curation, 5 communities were identified. The communities are also summarized below in Table 17. Community 1 included NG034.8A7, NG034.17H6, and NG034.5A9, as well as the comparator antibody CARsgene 8E5-2I. Community 2 included NG034.7A6, NG034.17H7.1, as well as comparator antibody zolbetuximab. Community 3 included 3 pan-claudin binders. Community 1 and community 2 mutually blocked each other. Community 2 blocked community 3 while community 1 did not.Table 17: Results of epitope binning26055Epitope mapping

[0277] CLDN18.1 and CLDN18.2 are tetra-span transmembrane proteins. CLDN18.1 is predominantly expressed in the lung, and CLDN18.2 is predominately expressed in the stomach. There is high sequence homology between human (hu)CLDN18.1 and huCLDN18.2; only 8 amino acids at ECL1 are different. ECL1 is only 53 amino acids in length, such that there are limited epitopes present (FIG. 5).

[0278] Epitope mapping was performed by measuring binding of the 8 single muteins to anti- CLDN18.2 antibodies (Table 18). For the 5 selected antibodies disclosed in these Examples, E56 is a critical binding site for both bin 1 and bin 2 antibodies. N37 is important in the binding for bin 2 antibodies, while Q29 plays a role in binding of some bin 1 antibodies. For the pan-claudin binder NG034.4H6, the single mutations impacted binding minimally.26055Table 18: Results of epitope mappingSummary

[0279] Table 19 shows a summary of results in terms of epitope bin, binding affinity (KD), and cell binding data for antibody constructs. Table 20 shows a summar\' of results in terms of epitope bin, binding affinity (KD), and cell binding data for scFv constructs.Table 19: Summary of results for antibody constructs26055Table 20: Summary of results for scFv constructs

[0280] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0281] All references (e.g.. publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) w as specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.2605526055260552605526055

Claims

WHAT IS CLAIMED IS:

1. A human claudin 18.2 (CLDN18.2) binder comprising:(a) the six complementarity' determining regions (CDRs) of an antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 3;(b) the six CDRs of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 11 ;(c) the six CDRs of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19;(d) the six CDRs of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 26 and a VL comprising the amino acid sequence of SEQ ID NO: 27; or(e) the six CDRs of an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 34 and a VL comprising the amino acid sequence of SEQ ID NO: 35; wherein in (a), (b), (c), (d), or (e), the CDRs are defined using the Kabat, Chothia, AbM, ImMunoGeneTics (IMGT), or Contact numbering scheme.

2. The human CLDN18.2 binder of claim 1, wherein the VH comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 6; and the VL comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO:

7. a CDR 2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 9.

3. The human CLDN18.2 binder of claim 1, wherein the VH comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 14; and the VL comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 17.

4. The human CLDN18.2 binder of claim 1, wherein the VH comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR 2 comprising the amino acid sequence ofSEQ ID NO:

21. and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 22; and the VL comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 25.

5. The human CLDN18.2 binder of claim 1, wherein the VH comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 30; and the VL comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 33.

6. The human CLDN18.2 binder of claim 1, wherein the VH comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 36, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 37, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 38; and the VL comprises a CDR 1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR 2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR 3 comprising the amino acid sequence of SEQ ID NO: 41.

7. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises:(a) a VH comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 wherein the 1st amino acid residue of SEQ ID NO: 2 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 wherein the 1st amino acid residue of SEQ ID NO: 3 is pyroglutamate or pyroglutamic acid; or(b) a VH comprising the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10 wherein the 1st amino acid residue of SEQ ID NO: 10 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 11 ; or(c) a VH comprising the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence of SEQ ID NO: 18 wherein the 1st amino acid residue of SEQ ID NO: 18 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acidsequence of SEQ ID NO: 19 or the amino acid sequence of SEQ ID NO: 19 wherein the 1st amino acid residue of SEQ ID NO: 19 is pyroglutamate or pyroglutamic acid; or(d) a VH comprising the amino acid sequence of SEQ ID NO: 26 or the amino acid sequence of SEQ ID NO: 26 wherein the 1st amino acid residue of SEQ ID NO: 26 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 27 or pyroglutamic acid; or(e) a VH comprising the amino acid sequence of SEQ ID NO: 34 or the amino acid sequence of SEQ ID NO: 34 wherein the 1st amino acid residue of SEQ ID NO: 34 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence of SEQ ID NO: 35 wherein the 1st amino acid residue of SEQ ID NO: 35 is pyroglutamate or pyroglutamic acid.

8. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 2 and a VL comprising the amino acid sequence of SEQ ID NO: 3.

9. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 2, and a VL comprising the amino acid sequence of SEQ ID NO: 3 wherein the 1st amino acid residue of SEQ ID NO: 3 is pyroglutamate or pyroglutamic acid.

10. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 2 wherein the 1st amino acid residue of SEQ ID NO: 2 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 3.

11. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 2 wherein the 1st amino acid residue of SEQ ID NO: 2 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 3 wherein the 1st amino acid residue of SEQ ID NO: 3 is pyroglutamate or pyroglutamic acid.2605512. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 and a VL comprising the amino acid sequence of SEQ ID NO: 11.

13. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 10 wherein the 1st amino acid residue of SEQ ID NO: 10 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 11.

14. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 19.

15. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, and a VL comprising the amino acid sequence of SEQ ID NO: 19, wherein the 1st amino acid residue of SEQ ID NO: 19 is pyroglutamate or pyroglutamic acid.

16. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, wherein the 1st amino acid residue of SEQ ID NO: 18 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 19.

17. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 18, wherein the 1st amino acid residue of SEQ ID NO: 18 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 19, wherein the 1st amino acid residue of SEQ ID NO: 19 is pyroglutamate or pyroglutamic acid.

18. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO:

26. and a VL comprising the amino acid sequence of SEQ ID NO: 27.

19. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 26, wherein the 1st amino acid residue of SEQ ID NO: 26 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 27.

20. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 35.

21. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, and a VL comprising the amino acid sequence of SEQ ID NO: 35, wherein the 1st amino acid residue of SEQ ID NO: 35 is pyroglutamate or pyroglutamic acid.

22. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, wherein the 1st amino acid residue of SEQ ID NO: 34 is pyroglutamate or pyroglutamic acid, and a VL comprising the amino acid sequence of SEQ ID NO: 35.

23. The human CLDN18.2 binder of claim 7, wherein the human CLDN18.2 binder comprises a VH comprising the amino acid sequence of SEQ ID NO: 34, wherein the 1st amino acid residue of SEQ ID NO: 34 is pyroglutamate or pyroglutamic acid, and a VL comprising or the amino acid sequence of SEQ ID NO: 35, wherein the 1st ammo acid residue of SEQ ID NO: 35 is pyroglutamate or pyroglutamic acid.

24. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises a VH comprising a variant of the amino acid sequence of SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a VL comprising a variant of the amino acid sequence of SEQ ID NO: 3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

25. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises a VH comprising a variant of the amino acid sequence of SEQ ID NO: 10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof,and a VL comprising a variant of the amino acid sequence of SEQ ID NO: 11 having 1, 2.

3. 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

26. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises a VH comprising a variant of the amino acid sequence of SEQ ID NO: 18 having 1. 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a VL comprising a variant of the amino acid sequence of SEQ ID NO: 19 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

27. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises a VH comprising a variant of the amino acid sequence of SEQ ID NO: 26 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a VL comprising a variant of the amino acid sequence of SEQ ID NO: 27 having 1, 2, 3. 4, 5, 6, 7, 8, 9. or 10 amino acid substitutions, additions, deletions, or combinations thereof.

28. The human CLDN18.2 binder of claim 1, wherein the human CLDN18.2 binder comprises a VH comprising a variant of the amino acid sequence of SEQ ID NO: 34 having 1, 2, 3, 4, 5, 6. 7, 8, 9. or 10 amino acid substitutions, additions, deletions, or combinations thereof, and a VL comprising a variant of the amino acid sequence of SEQ ID NO: 35 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, deletions, or combinations thereof.

29. The human CLDN18.2 binder of any one of claims 1-28, wherein the human CLDN18.2 binder comprises an antibody comprising a heavy chain constant domain of the IgGl isotype and a light chain constant domain of the human kappa or human lambda isotype.

30. The human CLDN18.2 binder of claim 29, wherein the heavy chain constant domain comprises the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 42.

31. The human CLDN18.2 binder of claim 29, wherein the heavy chain constant domain of the IgGl isotype comprises an Fc domain comprising one or more mutations that render the constant domain effector-silent.

32. The human CLDN18.2 binder of claim 31, wherein the effector-silent constant domain comprises(i) the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 43;(ii) the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 44;(iii) the amino acid sequence of SEQ ID NO: 45 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 45;(iv) the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 46;(v) the amino acid sequence of SEQ ID NO: 47 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 47;(vi) the amino acid sequence of SEQ ID NO: 48 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 48;(vii) the amino acid sequence of SEQ ID NO: 49 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 49; or(viii) the amino acid sequence of SEQ ID NO: 50 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 50.

33. The human CLDN18.2 binder of claim 32, wherein the effector-silent constant domain comprises the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having amino acids 1-329 of SEQ ID NO: 43.

34. The human CLDN18.2 binder of claim 29, wherein the light chain constant domain comprises a human kappa isotype light chain constant domain comprising the amino acid sequence of SEQ ID NO: 51.

35. The human CLDN18.2 binder of any one of claims 1-28, wherein the human CLDN18.2 binder comprises a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, an Fv region, or an scFv.

36. A composition comprising the human CLDN18.2 binder of any one of claims 1-35 and a pharmaceutically acceptable carrier or diluent.2605537. A method for treating a cancer in an individual in need thereof comprising administenng to the individual a therapeutically effective amount of a human CLDN18.2 binder of any one of claims 1-35 or the composition of claim 36.

38. A human CLDN18.2 binder of any one of claims 1-35 or the composition of claim 36 for use in treatment of a cancer in an individual in need thereof.

39. Use of a human CLDN18.2 binder of any one of claims 1-35 or the composition of claim 36 in the manufacture of a medicament for treating a cancer in an individual in need thereof.

40. A combination therapy for treating a cancer in an individual in need thereof comprising a human CLDN18.2 binder of any one of claims 1-35 or the composition of claim 36 and a therapeutic agent.

41. The combination therapy of claim 40, wherein the therapeutic agent is a chemotherapy agent or a therapeutic antibody.

42. The combination therapy of claim 41, wherein the therapeutic antibody is an anti-PD-1 antibody or an anti-PD-Ll antibody.

43. The combination therapy of claim 41, wherein the anti-PD-1 antibody is pembrolizumab.

44. A nucleic acid molecule encoding a human CLDN18.2 binder of any one of claims 1-35. or a set of nucleic acid molecules together encoding the human CDLN18.2 binder.

45. The nucleic acid molecule of claim 44, which is a set of nucleic acid molecules together encoding the human CDLN18.2 binder, wherein the human CLDN18.2 binder comprises a VH and a VL, wherein the set comprises a first nucleic acid molecule encoding the VH domain of human CDLN18.2 binder and a second nucleic acid molecule encoding the VL domain of human CDLN18.2 binder.

46. An expression vector comprising the nucleic acid molecule or set of nucleic acid molecules of claim 44 or 45; or a set of expression vectors, each expression vector encoding a nucleic acid molecule of the set of nucleic acid molecules.2605547. A host cell comprising the expression vector or set of expression vectors of claim 46.

48. A method for producing a human CLDN18.2 binder comprising (a) providing a host cell of claim 47; (b) cultivating the host cell in a medium under conditions suitable for expressing the human CLDN18.2 binder; and, optionally, (c) isolating the human CLDN18.2 binder from the medium.

49. A human CLDN18.2 binder of any one of claims 1-35 conjugated to a detectable moiety.

50. The human CLDN18.2 binder of claim 49, wherein the detectable moiety is detectable by magnetic resonance imaging (MRI) or by X-ray imaging.

51. A human CLDN18.2 binder, wherein the human CLDN18.2 binder comprises one or two of the modifications relative to the human CLDN18.2 binder of any one of claims 1-35 selected from:(i) a C-terminal amino acid residue K of a heavy chain being removed; and(ii) an N-terminal amino acid residue E or Q of a VH, a VL. a heavy chain or a light chain being substituted with pyroglutamate or pyroglutamic acid.

52. A human CLDN18.2 binder, wherein the human CLDN18.2 binder is obtainable by expressing the nucleic acid molecule or the set of nucleic acid molecules of claim 44 or 45 or the expression vector or the set of expression vectors of claim 46 in a host cell.

53. A method for detecting human CLDN18.2 on the surface of a cell in an individual comprising administering to the individual a human CLDN18.2 binder of claim 49 or 50 and detecting the cells in the individual bound to the human CLDN18.2 binder by detecting the label.

54. A kit comprising a human CLDN18.2 binder of any one of claims 1-35 and 51-52 and instructions to administer the human CLDN18.2 binder to treat cancer in an individual in need thereof.

55. The kit of claim 54, further comprising one or more additional therapeutic agents.2605556. The composition of claim 36 or the kit of claim 54 further comprising an hyaluronan degrading enzy me.

57. The composition or kit of claim 56, wherein the hyaluronan degrading enzy me is a soluble hyaluronidase.

58. The composition or kit of claim 56, wherein the hyaluronan degrading enzyme is a soluble PH20.

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