Antibody-drug conjugate targeting Claudin 18.2

Abstract

Antibody-drug conjugates are provided, comprising a drug moiety conjugated to an antibody or fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein. The antibody or fragment thereof binds to the β3-β4 loop (residues 45-63 of SEQ ID NO: 30, NYQGLWRSCVRESSGFTEC) and the β5 strand (residues 169-172 of SEQ ID NO: 30, YTFG) of CLDN18.2. In some embodiments, the ratio of the number of drug moieties to the number of antibodies or fragments is 1:1 to 20:1.

Description

Background Art

[0001] Background Claudin, such as claudin 18.2, is considered a promising target for cancer immunotherapy. Claudin is a family of proteins that form important components of tight cell junctions. They establish a paracellular barrier that controls the flow of molecules between cells. The proteins have an N-terminus and a C-terminus in the cytoplasm. Different claudins are expressed in different tissues, and changes in their functions are associated with the formation of cancer in their respective tissues. Claudin-1 is expressed in colorectal cancer, claudin-18 is expressed in gastric cancer, and claudin-10 is expressed in hepatocellular carcinoma.

[0002] Claudin 18 has two isoforms, isoform 1 and isoform 2. Isoform 2 (claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. The expression of claudin 18.2 in normal tissues was strictly limited to the differentiated epithelial cells of the gastric mucosa and was not present in the gastric stem cell region. Claudin 18.2 was retained in malignant transformation and was expressed in a significant proportion of primary gastric cancers and their metastases. The frequent ectopic activation of claudin 18.2 was also seen in tumors of the pancreas, esophagus, ovary, and lung. These data suggest that CLDN18.2 has a highly restricted expression pattern in normal tissues with frequent ectopic activation in the diversity of human cancers.

Summary of the Invention

Means for Solving the Problems

[0003] Summary An anti-claudin 18.1 antibody is discovered herein that selectively binds to wild-type claudin 18.2 and a common mutant M149L, and does not bind to other claudin 18 isoforms such as claudin 18.1. In a surprising and unexpected discovery, the present disclosure demonstrates that these antibodies are highly effective in inducing receptor-mediated antibody internalization, particularly when compared to IMAB362 (claudiximab), a lead anti-claudin 18.2 antibody in clinical development. Thus, when conjugated to a drug moiety, these antibodies can efficiently deliver the drug to target cells such as cancer cells that overexpress the claudin 18.2 protein.

[0004] The substantial increase in the ability of the antibodies of the present disclosure to induce receptor-mediated antibody internalization may be due to how these antibodies bind to the claudin 18.2 protein. As demonstrated in Example 14 and shown in FIG. 20, the amino acid residues on the claudin 18.2 protein that are important for antibody binding include those that are important for stabilizing the conformation of the extracellular loops (e.g., W30, L49, W50, C53, C63, and R80). More importantly, the residues involved in antibody binding are thought to include N45, Y46, G48, V54, R55, E56, S58, F60, and E62, which are located between the β3 and β4 strands of the first extracellular loop, and Y169 and G172 in β5 of the second extracellular loop. In contrast, known anti-claudin 18.2 antibodies are thought to bind to only one of the extracellular loops.

[0005] According to one embodiment of the present disclosure, there is provided an antibody-drug conjugate comprising a drug moiety covalently attached to an antibody or a fragment thereof having binding specificity for the wild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody or the fragment thereof binds to the β3-β4 loop and the β5 strand of CLDN18.2. The β3-β4 loop consists of residues 45-63 (NYQGLWRSCVRESSGFTEC) of SEQ ID NO: 30, and the β5 strand consists of residues 169-172 (YTFG) of SEQ ID NO: 30.

[0006] In some embodiments, the ratio of the number of drug moieties to the number of antibodies or fragments is from 1:1 to 20:1. In some embodiments, the ratio is from 2:1 to 10:1. In some embodiments, the ratio is from 2:1 to 6:1. In some embodiments, the ratio is about 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.

[0007] In some embodiments, the antibody or fragment thereof does not bind to β1 and β2, or binds to β1 or β2 with an affinity that is at least one-tenth of the affinity for binding to the β3-β4 loop or β5 strand. In some embodiments, the antibody or fragment thereof does not bind to the CLDN18.1 protein, or binds to CLDN18.1 with an affinity that is at least one-tenth of the affinity for binding to CLDN18.2.

[0008] In some embodiments, the antibody or fragment thereof binds to the CLDN18.2 M149L variant with an affinity that is at least 1% of the affinity for the wild-type CLDN18.2 protein.

[0009] In some embodiments, the antibody or fragment thereof binds to at least one amino acid residue selected from the group consisting of N45, Y46, G48, V54, R55, E56, S58, F60, and E62, and at least one amino acid residue selected from the group consisting of Y169 and G172 of SEQ ID NO: 30.

[0010] The drug moiety can be a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, etc. When released into cancer cells, the drug moiety can inhibit the growth of cancer cells or cause apoptosis in cancer cells. Examples of the drug moiety are selected from the group consisting of DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide).DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE, and MMAF are auristatin derivatives.

[0011] Methods and uses for treating diseases and conditions are also provided. In one embodiment, a method of treating cancer in a patient in need thereof is provided, comprising administering an antibody-drug conjugate of the present disclosure to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1

[0013]

Figure 2

[0014]

Figure 3

[0015]

Figure 4

[0016]

Figure 5

[0017]

Figure 6

[0018]

Figure 7

[0019]

Figure 8

[0020]

Figure 9

[0021]

Figure 10

[0022]

Figure 11

[0023]

Figure 12

[0024]

Figure 13

[0025]

Figure 14

[0026]

Figure 15

[0027]

Figure 16

[0028]

Figure 17

[0029]

Figure 18

[0030]

Figure 19

[0031]

Figure 20

[0032]

Figure 21

[0033]

Figure 22

[0034]

Figure 23

[0035]

Figure 24

[0036]

Figure 25-1

Figure 25-2

[0037]

Figure 26

[0038]

Figure 27

[0039]

Figure 28

Mode for Carrying Out the Invention

[0040] Definitions Note that the use of a substantive term preceded by "a" or "an" refers to one or more than one of that entity. For example, "an antibody" is understood to represent one or more than one antibody. Thus, the terms "a" (or "an"), "one or more than one", and "at least one" can be used interchangeably herein.

[0041] As used herein, the term "polypeptide" is intended to include both a single "polypeptide" and a plurality of "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, the term "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to one or more chains of two or more amino acids, and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0042] As used herein, the term "isolated" with respect to a cell, nucleic acid, e.g., DNA or RNA, refers to a molecule separated from other DNA or RNA present in the natural source of the macromolecule. The term "isolated" as used herein also refers to a nucleic acid or peptide that, when produced by recombinant DNA techniques, substantially lacks cellular material, viral material, or culture medium, or, when chemically synthesized, substantially lacks chemical precursors or other chemicals. Further, "isolated nucleic acid" means including nucleic acid fragments that do not exist naturally as fragments and are not found in their natural state. The term "isolated" is also used herein to refer to a cell or polypeptide isolated from other cellular proteins or tissue. Isolated polypeptides are meant to include both purified polypeptides and recombinant polypeptides.

[0043] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide is intended to mean a form of polypeptide or polynucleotide that does not occur in nature, and non-limiting examples thereof can be made by combining polynucleotides or polypeptides that do not normally occur together.

[0044] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. The molecules are homologous at that position if the positions in the sequences being compared are occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, preferably less than 25% identity, with one of the sequences of the present disclosure.

[0045] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when aligned, that percentage of bases (or amino acids) is the same when comparing the two sequences. This alignment and percent homology or percent sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. (eds.), (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for the alignment. One alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides having the specified percent homology above and encoding polypeptides having the same or similar biological activity.

[0046] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence with a certain degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence with a certain degree of homology to its complement. In one embodiment, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Similarly, "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity to the amino acid sequence of a reference polypeptide. In some embodiments, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, an equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof compared to the reference polypeptide or polynucleotide. In some embodiments, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0047] Hybridization reactions can be carried out under conditions of different "stringencies". Generally, low stringency hybridization reactions are carried out at about 40 °C in a solution of about 10× SSC or equivalent ionic strength / temperature. Typically, moderate stringency hybridization is carried out at about 50 °C in about 6× SSC, and generally, high stringency hybridization reactions are carried out at about 60 °C in about 1× SSC. Hybridization reactions can also be carried out "under physiological conditions" well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg 2+ concentration typically found within a cell.

[0048] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T). When the polynucleotide is RNA, uracil (U) replaces thymine. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into a database within a computer having a central processing unit and can be used in bioinformatics applications such as functional genomics and homology searching. The term "polymorphism" refers to the coexistence of more than one form of a gene or a part thereof. A part of a gene that has at least two different forms, i.e., two different nucleotide sequences, is called a "polymorphic region of the gene". The polymorphic region can be a single nucleotide that is different in different alleles with different identities.

[0049] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, ESTs or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can include modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide, if present. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0050] The term "encoding," when applied to a polynucleotide, refers to a polynucleotide that, when in its native state or when manipulated by methods well known to those of skill in the art, can be transcribed and / or translated to produce mRNA for a polypeptide and / or a fragment thereof, and thus is said to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom.

[0051] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a full antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule having biological activity to bind an antigen. Examples of such include, but are not limited to, the complementarity determining regions (CDRs) of the heavy or light chain or the ligand-binding portion thereof, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0052] As used herein, the terms "antibody fragment" or "antigen-binding fragment" refer to a part of an antibody such as F(ab’)2, F(ab)2, Fab’, Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0053] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (V H ) and light chain (V L ) of an immunoglobulin. In some embodiments, the regions are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and rich in serine or threonine for solubility, and can connect the N-terminus of V H to the C-terminus of V L , or vice versa. This protein retains the specificity of the original immunoglobulin despite removal of the constant regions and introduction of the linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0054] The term antibody encompasses a variety of broad classes of polypeptides that can be biochemically distinguished. Those skilled in the art understand that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are several subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well-characterized and are known to confer functional specialization. The respective modified versions of these classes and isotypes are readily distinguishable to those skilled in the art in view of the present disclosure and are thus within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion generally targets the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000 - 70,000. The four chains are typically joined by "Y"-shaped disulfide bonds, and the light chains support the heavy chains starting from the mouth of the "Y" and continuing through the variable regions.

[0055] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present disclosure include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab’ and F(ab’)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-bonded Fv (sdFv), fragments containing either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulins or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0056] The light chain is classified as either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or lambda light chain. Generally, when an immunoglobulin is produced by any of a hybridoma, B cell or genetically engineered host cell, the light and heavy chains are covalently bonded to each other and the "tail" portions of the two heavy chains are bonded to each other by a covalent disulfide bond or non-covalent bond. In the heavy chain, the amino acid sequence extends from the N-terminus at the fork-shaped end of the Y-shape to the C-terminus at the bottom of each chain.

[0057] Both the light and heavy chains are divided into regions that are structurally and functionally homologous. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain portion (VK) and the heavy chain portion (VH) determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as, for example, secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region. The CH3 domain and the CK domain actually include the carboxy termini of the heavy and light chains, respectively.

[0058] As described above, the variable region enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK and VH domains of the antibody, or a subset of the complementarity-determining regions (CDRs), combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites that are present at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of each of the VH and VK chains. In some examples, for instance, in certain immunoglobulin molecules derived from camelids or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule can consist of only heavy chains and no light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0059] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short discontinuous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody adopts its three-dimensional conformation in an aqueous environment. The remaining portions of the amino acids within the antigen-binding domain, called the "framework" regions, have less intermolecular variability. The framework regions predominantly adopt a β-sheet structure, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, the framework regions act to form a scaffold that brings the CDRs into the correct orientation by non-covalent intermolecular interactions. The antigen-binding domain formed by the arranged CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. The amino acids that comprise the CDRs and framework regions, respectively, can be readily identified by one of ordinary skill in the art for any given heavy-chain variable region or light-chain variable region because they are precisely defined ("Sequences of Proteins of Immunological Interest", Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0060] Where two or more definitions of terms used and / or permitted in the art exist, the definition of the terms used herein is intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementary determining region" ("CDR") to describe the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are hereby incorporated by reference in their entirety. The definitions of CDR by Kabat and Chothia, when compared to each other, include overlapping or subsets of amino acid residues. Nevertheless, the application of either definition for referring to the CDR of an antibody or its variant is intended to be within the scope of the terms defined and used herein. The appropriate amino acid residues encompassing the CDR defined by each of the references cited above are shown in the following table for comparison. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residues are included in a particular CDR given the amino acid sequence of the variable region of the antibody.

Table 9

[0061] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0062] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins approximately at amino acid 31 (i.e., approximately 9 residues downstream from the first cysteine residue), contains approximately 5 - 7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the 15th residue from the end of CDR-H1, contains approximately 16 - 19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins approximately at the 33rd amino acid residue from the end of CDR-H2, contains 3 - 25 amino acids, and ends with the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins approximately at residue 24 (i.e., after the cysteine residue) and contains approximately 10 - 17 residues, and ends at the next tryptophan residue. CDR-L2 begins at the approximately 16th residue from the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins approximately at the 33rd residue from the end of CDR-L2 (i.e., after the cysteine residue), contains approximately 7 - 11 residues, and ends with the sequence F or W-G-X-G, where X is any amino acid.

[0063] The antibodies disclosed herein can be of any animal origin, including avian and mammalian. Preferably, the antibody is an antibody from human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, the variable region may be of condricthoid origin (e.g., from shark).

[0064] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include a polypeptide chain containing a CH1 domain; a polypeptide chain containing a CH1 domain, at least a part of a hinge domain, and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain containing a CH1 domain, at least a part of a hinge domain, and a CH3 domain, or a polypeptide chain containing a CH1 domain, at least a part of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. Further, an antibody for use in the present disclosure may lack at least a part of a CH2 domain (e.g., all or part of the CH2 domain). As described above, it will be understood by those skilled in the art that the heavy chain constant region can be modified to have an amino acid sequence different from that of a naturally occurring immunoglobulin molecule.

[0065] The heavy chain constant region of an antibody disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0066] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0067] The "light chain - heavy chain pair" refers to an aggregate of a light chain and a heavy chain that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0068] As described above, the subunit structures and three - dimensional configurations of the constant regions of various immunoglobulin classes are well - known. As used herein, the term "VH domain" includes the amino - terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino - terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino - terminal with respect to the hinge region of the immunoglobulin heavy - chain molecule.

[0069] As used herein, the term "CH2 domain" includes, for example, the portion of the heavy - chain molecule that extends from approximately residue 244 to residue 360 of an antibody, using conventional numbering schemes (residues 244 - 360, Kabat numbering system; and residues 231 - 340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N - linked branched carbohydrate chains are sandwiched between the two CH2 domains of an intact native IgG molecule. It has also been well - established that the CH3 domain extends from the CH2 domain to the C - terminus of the IgG molecule and contains approximately 108 residues.

[0070] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues, is flexible, and thus allows the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains, namely the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0071] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, using the Kabat numbering system (positions 226 or 229, EU numbering system), the CH1 and CK regions are joined by disulfide bonds, and the two heavy chains are joined by two disulfide bonds at positions corresponding to 239 and 242.

[0072] As used herein, the term "chimeric antibody" means any antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (which may be intact, partial, or modified according to the present disclosure) is obtained from a second species. In certain embodiments, the target-binding region or site is derived from a non-human source (e.g., mouse or primate) and the constant region is human.

[0073] As used herein, "percent humanized" is calculated by determining the number of amino acid differences in the framework between the humanized domain and the germline domain (i.e., non-CDR differences), subtracting that number from the total number of amino acids, then dividing that by the total number of amino acids and multiplying by 100.

[0074] As used herein, the terms "specifically binds" or "has specificity" generally mean that an antibody binds to an epitope via its antigen-binding domain and that the binding involves a significant complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope more readily via its antigen-binding domain than to a random irrelevant epitope. The term "specificity" is used herein to qualify the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with a higher specificity than it has for a related epitope "D".

[0075] As used herein, the terms "treating" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, with the goal of preventing or slowing (mitigating) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of the disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Persons in need of treatment include those already having a condition or disorder, those having a tendency to have a condition or disorder, or those in whom a condition or disorder should be prevented.

[0076] As used herein, "subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and animals for zoos, sports or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, female cows, and the like.

[0077] As used herein, phrases such as "in a patient in need of treatment" or "a subject in need of treatment" include, for example, a subject, such as a mammalian subject, who would benefit from administration of an antibody or composition of the present disclosure used for detection, diagnostic procedures and / or treatment. Anti-Claudin 18.2 antibodies and fragments

[0078] The present disclosure provides anti-Claudin 18.2 antibodies that have high affinity for both wild-type Claudin 18.2 and the common variant M149L (SU620 cells that endogenously express this mutation). To the best of the inventors' knowledge, all currently known anti-Claudin 18.2 proteins do not bind to this variant. Thus, the antibodies of the present disclosure have the unique advantage of being able to target both wild-type and M149L variant Claudin 18.2 proteins. This advantage is important since a significant portion of cancer patients have this common mutation. It is also noteworthy that the antibodies of the present disclosure do not bind (or bind to Claudin 18.1 with much lower affinity) to Claudin 18.1, which is another Claudin 18 isoform.

[0079] The antibodies and fragments of the present disclosure also demonstrated excellent properties when used with clinical candidates as a reference. Currently, 175D10 (IMAB362; zolbetuximab) is undergoing a Phase III clinical trial for the treatment of gastric and gastroesophageal junction adenocarcinoma. The present antibodies and fragments not only showed stronger binding activity compared to 175D10, but also showed higher ADCC and ADCP activities under various different conditions.

[0080] Also importantly, the present disclosure demonstrates that these antibodies are highly effective in inducing receptor-mediated antibody internalization even when compared to IMAB362. The substantial increase in the ability of the antibodies of the present disclosure to induce receptor-mediated antibody internalization may be attributed to how these antibodies bind to the claudin 18.2 protein. As demonstrated in Example 14 and shown in Figure 20, the amino acid residues on the claudin 18.2 protein that are important for binding to the antibody include those that are important for stabilizing the conformation of the extracellular loops (e.g., W30, L49, W50, C53, C63, and R80). W30, L49, and W50 are part of the W-LW-C-C consensus motif that helps to stabilize the conformation of loop 1. C53 and C63 form an inter-β strand disulfide bond. R80 may be important for maintaining the interaction between parallel claudin 18.2 molecules on the cell surface or for stabilizing the conformation of loop 1.

[0081] Also important for antibody binding are the residues N45, Y46, G48, V54, R55, E56, S58, F60, E62, Y169, and G172. Of these, N45, Y46, G48, V54, R55, E56, S58, F60, and E62 are located within β3 strand or are located via C63 of β4 strand. This region consisting of residues 45-63 (NYQGLWRSCVRESSGFTEC) of SEQ ID NO: 30 is referred to herein as the "β3-β4 loop" and is part of the first extracellular loop (loop 1) of claudin 18.2. In contrast, Y169 and G172 are part of the β5 strand (residues 169-172; YTFG) of the second extracellular loop (loop 2).

[0082] The highly increased activity of the antibodies of the present disclosure for inducing receptor-mediated antibody internalization is hypothesized to be due to their ability to bind to residues in both the β3-β4 loop and the β5 strand. In this regard, known anti-claudin 18.2 antibodies are thought to bind to only one of the loops.

[0083] The experimental data also show that the antibodies of the present disclosure have higher binding specificity and improved ADCC and ADCP compared to known antibodies. Human Claudin 18.2 sequence [Table 10-1] [Table 10-2]

[0084] According to one embodiment of the present disclosure, there is provided an antibody or a fragment thereof having binding specificity for a wild-type human Claudin 18.2 (CLDN18.2) protein, which binds to both the first extracellular loop and the second extracellular loop of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to both the β3-β4 loop and the β5 strand of CLDN18.2.

[0085] According to another embodiment of the present disclosure, there is provided an antibody or a fragment thereof having binding specificity for a wild-type human Claudin 18.2 (CLDN18.2) protein, which further binds to the M149L mutant of the CLDN18.2 protein. In some embodiments, the antibody or fragment does not bind to the human wild-type Claudin 18.1 (CLDN18.1) protein or binds to CLDN18.1 with an affinity that does not exceed about 1% of the affinity for the wild-type CLDN18.2 protein.

[0086] The binding affinity of an antibody or fragment for a protein can be measured by many methods known in the art. For example, a cell-free assay using the secreted form of CLDN18.1 or CLDN18.2 protein can be measured. However, preferably, the measurement is performed using CLDN18.1 or CLDN18.2 protein on the cell surface that mimics the actual binding environment. Such binding assays are well illustrated in the experimental examples.

[0087] In some embodiments, the antibody or fragment thereof has a binding affinity for the M149L variant that is at least 1%, or alternatively at least 0.001%, 0.01%, 0.1%, 0.5%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the affinity for the wild-type CLDN18.2 protein.

[0088] In some embodiments, the antibody or fragment thereof does not bind to human CLDN18.1. In some embodiments, the binding to human CLDN18.1 is much weaker compared to the binding to CLDN18.2, for example, 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% or less, but is not limited thereto.

[0089] As described above, the antibodies and fragments thereof of the present disclosure bind to the claudin 18.2 protein at an epitope different from that of known antibodies (see FIG. 4; at least the reference antibody interacts with M149, but the antibodies of the present disclosure do not interact with M149). Thus, in one embodiment, provided is an antibody or fragment thereof having binding specificity for the wild-type human claudin 18.2 (CLDN18.2) protein, wherein the binding between the antibody or fragment thereof and the wild-type CLDN18.2 protein involves at least one amino acid residue selected from the group consisting of N45, Y46, G48, V54, R55, E56, S58, F60 and E62 of the wild-type CLDN18.2 protein, and at least one amino acid residue selected from the group consisting of Y169 and G172.

[0090] In some embodiments, the antibody or fragment thereof binds to N45 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to Y46 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to G48 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to L49 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to W50 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to C53 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to V54 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to R55 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to E56 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to E58 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to F60 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to E62 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to C63 of CLDN18.2.

[0091] In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from N45, Y46, G48, V54, R55, E56, S58, F60, and E62. In some embodiments, the antibody or fragment thereof binds to at least three amino acid residues selected from N45, Y46, G48, V54, R55, E56, S58, F60, and E62. In some embodiments, the antibody or fragment thereof binds to at least four amino acid residues selected from N45, Y46, G48, V54, R55, E56, S58, F60, and E62. In some embodiments, the antibody or fragment thereof binds to at least five amino acid residues selected from N45, Y46, G48, V54, R55, E56, S58, F60, and E62.

[0092] In some embodiments, the antibody or fragment thereof binds to at least Y169 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to at least G172 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from Y169 and G172 of CLDN18.2.

[0093] In some embodiments, the binding involves 2, 3, 4, 5 or more amino acid residues selected from the group consisting of amino acid residues including W30, N45, Y46, G48, V54, R55, E56, S58, F60 and E62, and at least one amino acid residue selected from the group consisting of Y169 and G172 of the wild-type CLDN18.2 protein. In some embodiments, the binding involves amino acid residues including W30, N45, Y46, G48, V54, R55, E56, S58, F60, E62 and Y169 of the wild-type CLDN18.2 protein.

[0094] Weaker binding to these amino acids on CLDN18.2 may be compared to other amino acids such as G48, L49, W50, C53, V54, R55, E56. In some embodiments, the comparison is to the binding at the same amino acids to 175D10. For example, the binding of the antibody or fragment of the present disclosure is weaker than the binding of 175D10 (IMGT / 2Dstructure-DB accession number: 10473) to at least 1, 2, 3, 4, 5 or all of D28, Q33, N38, V43, G59 and V79.

[0095] In some embodiments, the antibody or fragment thereof does not bind to M149L of the CLDN18.2 protein. In some embodiments, the antibody or fragment thereof binds to the M149L mutant of the CLDN18.2 protein.

[0096] According to one embodiment of the present disclosure, there is provided an antibody or fragment thereof comprising a heavy chain variable domain and a light chain variable domain having CDR regions shown in the combinations of CDRs in Table A.

Table A-1

Table A-2

Table B

Table C

Table D

[0097] Antibodies containing these CDR regions had strong Claudin 18.2 binding and inhibitory activity, regardless of whether they were murine, humanized or chimeric. As shown in Examples 11 and 12, specific residues within the CDR can be modified to retain or improve properties, or to reduce the likelihood of undergoing post-translational modification (PTM). Such modified CDRs can be referred to as affinity matured CDRs or risk-reduced CDRs.

[0098] Non-limiting examples of risk-reduced CDRs are provided in the third column of Tables B - D. Affinity matured ones can include those having one, two or three amino acid additions, deletions and / or substitutions. In some embodiments, the substitutions can be conservative substitutions.

[0099] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a series of amino acids can be replaced with a structurally similar string in which the order and / or composition of the side chain family members is different.

[0100] Non-limiting examples of conservative amino acid substitutions are provided in the following table, where a similarity score of 0 or greater indicates a conservative substitution between two amino acids.

Table E

Table F

[0101] Accordingly, in one embodiment, there is provided an antibody or a fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, which comprises a light chain variable region containing light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and a heavy chain variable region containing heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 are selected from combinations 1 to 33 in Table A or an antibody or a fragment thereof is provided, wherein one or more than one of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 each contains one, two or three amino acid additions, deletions, conservative amino acid substitutions or combinations thereof selected from each of combinations 1 to 33.

[0102] In some embodiments, provided are anti-CLDN18.2 antibodies or fragments thereof, each of which is selected from Table A or Tables B - D, and which comprise CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3. For example, an antibody or fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, comprising a light chain variable region comprising light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 208 - 226, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 208 - 226 by addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227 - 233, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 227 - 233 by addition, deletion, or amino acid substitution; CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 8, 13, 19, and 42 - 58, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 3, 8, 13, 19, and 42 - 58 by addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 234 - 254, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 234 - 254 by addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 255 - 280, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 255 - 280 by addition, deletion, or amino acid substitution of one, two, or three amino acids; and CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 281 - 303, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 281 - 303 by addition, deletion, or amino acid substitution of one, two, or three amino acids, is provided.

[0103] In some embodiments, CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 208-226, 304-305, and 308-309, CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 227-233, CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 8, 13, 19, 20, and 42-58, CDRH1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 234-254, CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 255-280, 306, 310, and 311, and CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 281-303, 307, and 312-314.

[0104] Antibody 120B7B2 has been demonstrated to be a potent inhibitor of Claudin 18.2. Its CDR sequences are provided in Table B along with several de-risked versions. In one embodiment, the present disclosure provides an antibody or a fragment thereof having binding specificity for the wild-type human Claudin 18.2 (CLDN18.2) protein, comprising a light chain variable region containing light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region containing heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence QSLLNSGNQKNY (SEQ ID NO: 1), QSLLNAGNQKNY (SEQ ID NO: 17), or QSLLESGNQKNY (SEQ ID NO: 18), or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 1, 17, or 18, CDRL2 comprises the amino acid sequence WAS (SEQ ID NO: 2) or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 2, CDRL3 comprises the amino acid sequence CQNGYYFPFT (SEQ ID NO: 3), QNAYYFPFT (SEQ ID NO: 19), or QEGYYFPFT (SEQ ID NO: 20), or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 3, 19, or 20, CDRH1 comprises the amino acid sequence GYTFTGYI (SEQ ID NO: 4) or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 4, CDRH2 comprises the amino acid sequence INPYNDGT (SEQ ID NO: 5) or INPYNDDT (SEQ ID NO: 21), or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 5 or 21, and CDRH3 comprises the amino acid sequence ARAYFGNSFAY (SEQ ID NO: 6) or ARAYFGNAFAY (SEQ ID NO: 22), or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 6 or 22.

[0105] It is interesting to note that CDRs from different antibodies share significant homology (see Table A). In this case, it is assumed that each corresponding CDR can be exchanged without significantly affecting the binding affinity or activity of the antibody or fragment. Alternatively, each specific amino acid in a CDR can be substituted with another amino acid present in the corresponding CDR from a different antibody.

[0106] In some embodiments, an antibody or fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein is provided. In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304, or 305, or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 210, 304, or 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, 19, or 20, or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 3, 19, or 20, CDRH1 comprises the amino acid sequence of SEQ ID NO: 253 or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 253, CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or 306 or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 278 or 306, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or 307 or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 303 or 307.

[0107] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304, or 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, 19, or 20, CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or 306, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or 307.

[0108] Non-limiting examples of the light chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 192-195, and 206-207, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 192-195, and 206-207.

[0109] Non-limiting examples of the heavy chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 188-191, and 205, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 188-191, and 205.

[0110] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 19, CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, CDRH2 comprises the amino acid sequence of SEQ ID NO: 306, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 307. Non-limiting examples of the antibody or fragment include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 206 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 205.

[0111] Similarly, 72C1B6A3 has been shown to be a good antibody. Thus, in another embodiment, an antibody or a fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, comprising a light chain variable region containing light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and a heavy chain variable region containing heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304 or 305 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 210, 304 or 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 229 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 229, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 8, CDRH1 comprises the amino acid sequence of SEQ ID NO: 242 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 242, CDRH2 comprises the amino acid sequence of SEQ ID NO: 263 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 263, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 289 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 289 is provided.

[0112] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304 or 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 289.

[0113] Non-limiting examples of the light chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NO: 124, 185-187, and 203-204, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 124, 185-187, and 203-204.

[0114] Non-limiting examples of the heavy chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NO: 153 and 181-184, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 153 and 181-184.

[0115] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 289. Non-limiting examples of the antibody or fragment thereof include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 203 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 181.

[0116] In addition, 4F11E2 has been shown to be a good antibody. Thus, in another embodiment, an antibody or a fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, comprising a light chain variable region containing light chain complementarity determining regions CDRL1, CDRL2 and CDRL3, and a heavy chain variable region containing heavy chain complementarity determining regions CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 216, 308 or 309 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 216, 308 or 309, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227 or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 13, CDRH1 comprises the amino acid sequence of SEQ ID NO: 246 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 246, CDRH2 comprises the amino acid sequence of SEQ ID NO: 268, 310 or 311 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 268, 310 or 311, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, 312, 313 or 314 or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 294, 312, 313 or 314 is provided.

[0117] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 216, 308 or 309, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, CDRH2 comprises the amino acid sequence of SEQ ID NO: 268, 310 or 311, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, 312, 313 or 314.

[0118] Non-limiting examples of the light chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NOs: 129, 178-180, and 201-202, or a biological equivalent, such as a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 129, 178-180, and 201-202.

[0119] Non-limiting examples of the heavy chain variable region include an amino acid sequence selected from the group consisting of SEQ ID NOs: 159, 175-177, and 196-200, or a biological equivalent, such as a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 159, 175-177, and 196-200.

[0120] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 309, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, CDRH2 comprises the amino acid sequence of SEQ ID NO: 311, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 294. Non-limiting examples of the antibody or fragment include a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 197.

[0121] In some embodiments, the antibody is a humanized antibody. As shown in Example 9, the humanized antibody may contain one or more back mutations to the murine counterpart. Examples of such back mutations are shown in Table 3. In some embodiments, the antibody or fragment may contain one, two, three, four, five, or more back mutations.

[0122] In some embodiments, the anti-Claudin 18.2 antibodies of the present disclosure include any one of VL of SEQ ID NOs: 117-144, 178-180, 185-187, 192-195, 201-202, 203-204 or 206-207, and any one of VH of SEQ ID NOs: 145-174, 175-177, 181-184, 188-191, 196-200 or 205, or their respective biological equivalents. A biological equivalent of VH or VL is a sequence that contains the specified amino acids but has 80%, 85%, 90%, 95%, 98% or 99% sequence identity overall. Thus, a biological equivalent of SEQ ID NO: 145 can be a VH that has 80%, 85%, 90%, 95%, 98% or 99% sequence identity overall with SEQ ID NO: 145, retains the CDRs, and optionally retains one or more or all of the revertant mutations.

[0123] It is also understood by those skilled in the art that the antibodies disclosed herein can be modified such that their amino acid sequences are different from the naturally occurring binding polypeptides from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar, e.g., may have a certain percent identity with the starting sequence, e.g., may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

[0124] In certain embodiments, the antibody includes an amino acid sequence or one or more moieties not normally associated with the antibody. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can include a flexible linker sequence or can be modified to add a functional moiety (e.g., PEG, drug, toxin, or label).

[0125] Antibodies, variants or derivatives of the present disclosure include derivatives modified such that the covalent bond does not interfere with the binding of the antibody to the epitope, i.e., derivatives modified by covalent bonding of any kind of molecule to the antibody. For example, without limitation, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, proteolytic cleavage, binding to cell ligands or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicaamycin, etc. Further, the antibody may contain one or more non-classical amino acids. Antibody-drug conjugate

[0126] In some embodiments, the antibody or fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical, or PEG.

[0127] In one embodiment, the antibody or fragment of the present disclosure is covalently bound to a drug moiety. The drug moiety can be or can be modified to be a group reactive with the conjugation point on the antibody. For example, the drug moiety can be bound by alkylation (e.g., at the ε-amino group lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between hydroxyl and carboxyl groups, amidation at amino or carboxyl groups, and conjugation to thiols.

[0128] In some embodiments, the number of drug moieties p conjugated per antibody molecule ranges from an average of 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is an average of 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p ranges from an average of about 1 to about 20, about 1 to about 10, about 2 to about 10, about 2 to about 9, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 1 to about 2. In some embodiments, p ranges from about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3.

[0129] For example, if chemical activation of a protein results in the formation of free thiol groups, the protein can be conjugated to a sulfhydryl-reactive agent. In one aspect, the agent is substantially specific for free thiol groups. Such agents include, for example, maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethyl ketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinyl sulfone, and pyridylthio.

[0130] The drug can be conjugated to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases such as lysosomal protease or endosomal protease. In an exemplary embodiment, the linker can be a dipeptide linker such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or maleimidocaproic acid-valine-citrulline (citruline)-p-aminobenzyl oxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc). Sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-1-carboxylate (smcc) conjugation occurs via a maleimide group that reacts with sulfhydryl (thiol, -SH), and its sulfosuccinimidyl ester is reactive towards primary amines (as found at the N-terminus of lysine and proteins or peptides). Yet another linker is maleimidocaproy (mc). Other suitable linkers include linkers hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers. The linker can be covalently conjugated to the antibody to such an extent that the antibody must be degraded intracellularly for the drug to be released (e.g., such as mc linker).

[0131] The linker can contain a group for binding to the antibody. For example, linkers can include amino, hydroxyl, carboxyl, or sulfhydryl reactive groups (e.g., maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethyl ketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinyl sulfone, and pyridylthio).

[0132] In some embodiments, the drug moiety is a cytotoxic agent or cytostatic agent, immunosuppressant, radioisotope, toxin, etc. The conjugate can be used to inhibit the growth of tumor cells or cancer cells, to cause apoptosis in tumor or cancer cells, or to treat a patient's cancer. Thus, the conjugate can be used in various situations for the treatment of animal cancers. The conjugate can be used to deliver the drug to tumor cells or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds or associates with cancer cells expressing Claudin 18.2, and the conjugate and / or the drug can be taken up into the interior of the tumor cells or cancer cells by receptor-mediated endocytosis.

[0133] Once inside the cell, one or more specific peptide sequences within the conjugate (e.g., in the linker) are hydrolytically cleaved by one or more tumor cell or cancer cell-associated proteases, resulting in the release of the drug. The released drug then freely moves within the cell and induces cytotoxic or cytostatic activity or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug subsequently penetrates the cell or acts on the cell surface.

[0134] Examples of drug moieties or payloads include DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine) and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide), and are selected from the group consisting thereof. DM1 is a derivative of the tubulin inhibitor maytansine, while MMAD, MMAE and MMAF are auristatin derivatives. In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.In some embodiments, the drug moiety is a maytansinoid or an auristatin.

[0135] The antibody or fragment can be conjugated or fused to a therapeutic agent that can include a detectable label such as a radiolabel, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent that can be a drug or toxin, an ultrasonic enhancer, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0136] The antibody can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalate esters.

[0137] The antibody can also 152It can be detectably labeled using a fluorescent metal such as Eu, or other members of the lanthanide series. These metals can be conjugated to an antibody using a metal chelate group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to an antibody are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985)); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcell Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies’84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (52:119-58 (1982)). Polynucleotides encoding antibodies and methods of preparing antibodies

[0138] The present disclosure also provides an isolated polynucleotide or nucleic acid molecule encoding an antibody, variant or derivative thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Further, the polynucleotides of the present disclosure can encode a portion of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variant or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0139] Methods of making antibodies are well known in the art and are described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies are described in the art and can be made using the techniques described herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to the antigen challenge, but whose endogenous locus has been inactivated. Exemplary techniques that can be used to make such antibodies are described in U.S. Pat. Nos. 6,150,584, 6,458,592, 6,420,140, which are incorporated herein by reference in their entirety. Methods of treatment

[0140] As described herein, the antibodies, variants, derivatives or antibody-drug conjugates of the present disclosure can be used in certain methods of treatment and diagnosis.

[0141] The present disclosure further relates to antibody-based therapies involving administering to a patient, such as an animal, mammal, and human, an antibody, fragment, or antibody-drug conjugate of the present disclosure to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including variants and derivatives thereof described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof described herein).

[0142] The antibodies of the present disclosure can also be used to treat or inhibit cancer. As described above, Claudin 18.2 can be overexpressed in tumor cells, particularly in tumors of the stomach, pancreas, esophagus, ovary, and lung. Inhibition of Claudin 18.2 has been shown to be useful for treating tumors.

[0143] Accordingly, in some embodiments, provided is a method of treating cancer in a patient in need thereof. The method involves, in one embodiment, administering to the patient an effective amount of an antibody, fragment, or antibody-drug conjugate of the present disclosure. In some embodiments, at least one of the patient's cancer cells (e.g., stromal cells) overexpresses Claudin 18.2.

[0144] Also provided by the present disclosure are cell therapies such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used that are contacted with (alternatively, engineered to express) the anti-Claudin 18.2 antibody of the present disclosure. Once such contact or engineering has been performed, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any of the types of cancer disclosed herein. The cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof, but are not limited thereto.

[0145] In some embodiments, the cells are isolated from the cancer patient himself. In some embodiments, the cells are provided by a donor or from a cell bank. When the cells are isolated from the cancer patient, undesirable immune responses can be minimized.

[0146] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, and lung cancer.

[0147] Additional diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies or variants, or derivatives thereof, of the present disclosure include malignant tumors and related disorders such as leukemia (including acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, as well as sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, angioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma, including but not limited to the progression and / or metastasis of malignant tumors.

[0148] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors including the specific antibody, its variant or derivative being used, the patient's age, weight, general health, sex and diet, as well as the administration time, excretion rate, drug combination, and the severity of the particular disease being treated. The determination of such factors by a healthcare provider is within the ordinary skill of one in the art. The amount also depends on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound being used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0149] Methods of administering the antibody, fragment or antibody-drug conjugate include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antigen-binding polypeptide or composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or skin mucosal linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Accordingly, the pharmaceutical composition containing the antigen-binding polypeptide of the present disclosure can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (such as in the case of powders, ointments, drops or transdermal patches), buccally, or as an oral or nasal spray.

[0150] As used herein, the term "parenteral" refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injections and infusions.

[0151] Administration can be systemic or local. Further, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route including intracerebroventricular injection and intrathecal injection, and intracerebroventricular injection can be facilitated, for example, by an intracerebroventricular catheter attached to a reservoir such as an Ommaya reservoir. Lung administration can also be used, for example, by the use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0152] It may be desirable to locally administer the antigen-binding polypeptide or composition of the present disclosure to the area in need of treatment, which can be achieved, for example, but not limited to, local injection during surgery, such as topical application in combination with a wound dressing after surgery, by injection, by catheter, by suppository, or by implant, and the implant is a membrane such as a sialastic membrane, or a porous, non-porous, or gelatinous material containing fibers. Preferably, when administering a protein containing an antibody of the present disclosure, care must be taken to use a material that the protein does not absorb.

[0153] The amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure that is effective for the treatment, inhibition, and prevention of inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Further, in vitro assays can be used, if necessary, to assist in identifying the optimal dosage range. The exact dosage used in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The effective dosage can be extrapolated from the dose-response curve obtained from in vitro or animal model test systems.

[0154] As a general proposal, the dosage administered to a patient of the antibody, fragment or antibody-drug conjugate of the present disclosure is typically 0.1 mg to 100 mg per kg of the patient's body weight, 0.1 mg to 20 mg per kg of the patient's body weight, or 1 mg to 10 mg per kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to foreign polypeptides. Therefore, lower dosages of human antibodies and less frequent administrations are often possible. Furthermore, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibody by modifications such as lipidation.

[0155] In a further embodiment, the composition of the present disclosure is administered in combination with a cytokine. Cytokines that can be administered together with the composition of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L and TNF-α.

[0156] In a further embodiment, the composition of the present disclosure is administered in combination with other treatment or prophylactic regimens such as, for example, radiation therapy. Composition

[0157] The present disclosure also provides a pharmaceutical composition. Such a composition comprises an effective amount of an antibody, fragment, or antibody-drug conjugate, and an acceptable carrier. In some embodiments, the composition further comprises a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0158] In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, more specifically in humans. Furthermore, a "pharmaceutically acceptable carrier" is generally any kind of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.

[0159] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When administering the pharmaceutical composition intravenously, water is a preferred carrier. Saline solutions as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable formulations. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain, if desired, minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as benzyl alcohol and methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting isotonicity, such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated as a suppository with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences by E.W. Martin, which is incorporated herein by reference. Such compositions contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in purified form, together with a suitable amount of carrier to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0160] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. Optionally, the composition may also contain solubilizing agents and local anesthetics such as lignocaine to relieve pain at the injection site. Generally, the components are supplied separately in unit dosage forms or mixed together as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the active agent. When the composition is administered by infusion, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0161] The compounds of the present disclosure can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Examples

[0162] Example 1: Preparation of a mouse monoclonal antibody against human Claudin 18 isoform 2 (CLD18A2) a. Immunization: Balb / c and C57 / BL6 mice were immunized with a eukaryotic expression vector encoding a fragment of human Claudin 18.2 (CLD18A2). 50 μg of plasmid DNA was injected intramuscularly (i.m.) into the quadriceps muscle on days 1 and 10. The presence of antibodies against human CLD18A2 in the sera of the mice was monitored on day 20 by flow cytometry using HEK293 cells transiently transfected with the nucleic acid encoding human CLD18A2. Mice with a detectable immune response (Figure 1) were boost-immunized 3 and 2 days before fusion by intraperitoneal injection of 5 × 10 7 cells of HEK293 transiently transfected with the nucleic acid encoding human CLD18A2. b. Generation of hybridomas producing human monoclonal antibodies against CLD18A2:

[0163] Mouse spleen cells were isolated and fused to the mouse myeloma cell line with PEG based on a standard protocol. The resulting hybridomas were then screened for the production of immunoglobulins with CLD18A2 specificity by cell ELISA using HEK293 cells transfected with the nucleic acid encoding human CLD18.

[0164] A single cell suspension of spleen lymphocytes from immunized mice was fused with P3X63AG8U.1 non-secreting mouse myeloma cells (ATCC, CRL1597) at a ratio of 2:1 using 50% PEG (Roche Diagnostics, CRL738641). The cells were seeded at approximately 3 × 10 4Seed in wells and then incubate for about 2 weeks in a selection medium containing 10% fetal bovine serum, 2% hybridoma fusogen and cloning supplement (HFCS, Roche Diagnostics, CRL1363735) + 10 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 μg / ml gentamicin and 1×HAT (Sigma, CRLH0262). After 10 - 14 days, individual wells were screened by Cell ELISA for anti-CLD18A2 monoclonal antibody (Figure 2). Antibody-secreting hybridomas were re-seeded and re-screened with HEK293 expressing CLD18A2 or CLD18A1 by FACS. If CLD18A2 was still positive and CLD18A1 was negative, they were subcloned by limiting dilution. Then, stable subclones were cultured in vitro to produce small amounts of antibody in tissue culture medium. At least one clone from each hybridoma that retained the reactivity of the parental cells was selected (by FACS). Three vials of cell bank were generated for each clone and stored in liquid nitrogen. Selection of monoclonal antibodies that bind to CLD18A2 but not CLD18A1:

[0165] To determine the isotype of the antibodies, isotype ELISA was performed. The Ig subclass of the identified CLD18A2-reactive monoclonal antibodies was determined using a mouse monoAB ID kit (Zymed, CRL90-6550). Thirty-two hybridoma cell lines were generated: 64G11B4, 65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, 12F1F4, 78C10B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and 100F4G12. Further details are shown below: 64G11B4, mouse monoclonal IgG1, κ antibody 65G8B8, mouse monoclonal IgG1, κ antibody 56E8F10F4, mouse monoclonal IgG1, κ antibody 54A2C4, mouse monoclonal IgG1, κ antibody 44F6B11, mouse monoclonal IgG1, κ antibody 15C2B7, mouse monoclonal IgG1, κ antibody 20F1E10, mouse monoclonal IgG1, κ antibody 72C1B6A3, mouse monoclonal IgG1, κ antibody 58G2C2, mouse monoclonal IgG2a, κ antibody 101C4F12, mouse monoclonal IgG2b, κ antibody 103A10B2, mouse monoclonal IgG2b, κ antibody 40C10E3, mouse monoclonal IgG1, λ antibody 78E8G9G6, mouse monoclonal IgG1, κ antibody 4F11E2, mouse monoclonal IgG1, κ antibody 10G7G11, Mouse monoclonal IgG1, κ antibody 12F1F4, Mouse monoclonal IgG1, κ antibody 78C10B6G4, Mouse monoclonal IgG1, κ antibody 119G11D9, Mouse monoclonal IgG1, κ antibody 113G12E5E6, Mouse monoclonal IgG1, κ antibody 116A8B7, Mouse monoclonal IgG1, κ antibody 105F7G12, Mouse monoclonal IgG1, κ antibody 84E9E12, Mouse monoclonal IgG1, κ antibody 103F4D4, Mouse monoclonal IgG1, κ antibody 110C12B6, Mouse monoclonal IgG1, κ antibody 85H12E8, Mouse monoclonal IgG1, κ antibody 103H2B4, Mouse monoclonal IgG1, κ antibody 103F6D3, Mouse monoclonal IgG1, κ antibody 113E12F7, Mouse monoclonal IgG2a, κ antibody 120B7B2, Mouse monoclonal IgG2a, κ antibody 111B12D11, Mouse monoclonal IgG2a, κ antibody 111E7E2, Mouse monoclonal IgG2a, κ antibody 100F4G12, Mouse monoclonal IgG3, κ antibody. Example 2. Hybridoma Sequencing

[0166] Hybridoma cells (1×10 7Individuals) were collected, and total RNA was extracted from the spleen tissue using the above Tri reagent. cDNA was prepared using the SuperScript III kit according to the instructions of the above manufacturer. The obtained cDNA product was used as a template for PCR with primers VhRevU and VhForU, and the obtained 300 bp PCR product was cleaned up using a PCR clean-up kit and sequenced with the same primers. PCR reactions were also performed using light chain V region-specific primers VkReV7 and VkFor (variable region only) or KappaFor primer (entire κ light chain). Sequencing reactions were performed on the purified PCR product (cleaned PCR product) to obtain the DNA sequences for antibodies 64G11B4, 65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, 12F1F4, 78C10B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and 100F4G12. Their variable (VH and VL) sequences are shown in Table 1 below. Table 1: Sequences of the Variable Regions of Antibodies

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0167] For functional characterization, hybridoma cells were seeded at 2×10 6 cells / ml in a dialysis-based bioreactor (CELLine CL1000, Integra, Küttigen, Switzerland) to produce mg amounts of antibody. The antibody-containing supernatant was harvested once a week. Each mouse monoclonal antibody was purified using Melon Gel (Pierce, Rockford, USA) and concentrated by ammonium sulfate precipitation. Antibody concentration and purity were estimated by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. Example 4. Binding of mouse monoclonal antibodies reactive with CLD18A2

[0168] MKN45 cells overexpressing CLD18A2 were harvested from flasks. 100 μl of 1×10 6 cells / ml were incubated on ice for 30 minutes with the primary antibodies shown in Figure 3, starting at 100 nM and serially diluted 3-fold to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of this study showed by flow cytometry that the purified mouse antibodies could bind to MKN45 cells transfected with human CLD18A2 with a high EC50 compared to the positive reference antibody. Example 5. Binding of mouse monoclonal antibodies reactive with CLD18A2 variants

[0169] SU620 cells endogenously expressing CLD18A2 with the M149L mutation were harvested from flasks. 100 μl of 1×10 6Cells / ml of cells were incubated on ice for 30 minutes with the primary antibody shown in Figure 4, starting from 100 nM and serially diluted 3-fold down to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of this study showed by flow cytometry that the purified mouse antibody could bind to SU620 cells endogenously expressing human CLD18A2 with the M149L mutation at a high EC50, while the reference antibody did not bind (Figure 4). Example 6. Binding of Mouse Monoclonal Antibodies Reactive with Mouse and Cynomolgus CLD18A2

[0170] To evaluate the cross-reactivity of these antibodies with mouse and cynomolgus CLD18A2, HEK293 cells overexpressing mouse, cynomolgus or human CLD18A2 were harvested from flasks. 100 μl of 1×10 6 Cells / ml of cells were incubated on ice for 30 minutes with the primary antibody shown in Figure 3, starting from 100 nM and serially diluted 3-fold down to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of this study showed by flow cytometry that the purified mouse antibody could bind to mouse and cynomolgus CLD18A2 at a high EC50, at least as well as the reference antibody (Figures 5, 6 and 7). Example 7. Binding of Chimeric Antibodies Reactive with CLD18A2

[0171] Mouse VH and VK genes were synthesized and produced, and then cloned into vectors containing human γ1 and human κ constant domains, respectively. The purified chimeric antibodies were produced from transfected CHO cells.

[0172] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from the flask. 100 μl of cells at 1×10 6 cells / ml were incubated on ice for 30 minutes with the primary chimeric antibodies shown in Figure 4, starting from 100 nM and serially diluted 3-fold down to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of the study showed that the chimeric antibody could bind to human CLD18A2 with a high EC50 but did not bind to CLD18A1 (Figures 8 and 9). Example 8. Antibody-Dependent Cellular Cytotoxicity (ADCC) of Chimeric Antibodies

[0173] The ADCC reporter bioassay uses an alternative readout at an earlier time point in the activation of gene transcription via the NFAT (nuclear factor of activated T cells) pathway in the effector cells for the activation of the ADCC MOA pathway. Furthermore, the ADCC reporter bioassay uses engineered Jurkat cells that stably express the FcγRIIIa receptor, the V158 (high affinity) variant, and the NFAT response element driving the expression of firefly luciferase as effector cells. The biological activity of the antibody in the ADCC MOA was quantified by luciferase produced as a result of NFAT pathway activation, and the luciferase activity in the effector cells was quantified by a luminescence readout (Figure 1). The signal was high and the background of the assay was low.

[0174] Serial dilutions of the claudin 18.2 chimeric monoclonal antibody or a reference antibody were incubated with the engineered Jurkat effector cells (ADCC bioassay effector cells) for 6 hours of induction at 37°C, with and without the ADCC bioassay target cells (expressing claudin 18.2). Luciferase activity was quantified using the Bio-Glo™ reagent (Table 2). The results indicate that these chimeric antibodies have very strong ADCC activity. Table 2. EC50 of Test Antibodies [Table 2] Example 9. Humanization of 4F11E2, 72C1B6A3, and 120B7B2 Mouse mAbs

[0175] Using the variable region genes of mABs 4F11E2, 72C1B6A3, and 120B7B2, humanized MAbs were prepared. In the first step of this process, the amino acid sequences of the VH and VL of the MAb were compared with the available database of human Ig gene sequences to find the human germline Ig gene sequences that best matched overall.

[0176] The amino acid sequences of the humanized antibodies are listed in Table 3 below. Table 3. Humanized Sequences [Table 3-1] [Table 3-2]

[0177] The humanized VH and VL genes were synthesized and produced, and then cloned into vectors containing the human γ1 and human κ constant domains, respectively. Pairing of the human VH and human VL produced humanized antibodies (see Table 4). Table 4. Humanized Antibodies with VH and VL Regions [Table 4-1] [Table 4-2] Example 10. Binding of Humanized Antibodies Reactive with CLD18A2

[0178] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from the flask. 100 μl of 1×10 6Cells / ml were incubated on ice for 30 minutes with the primary humanized antibody shown in Figure 4, starting from 100 nM and serially diluted 3-fold to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of the study showed that the humanized antibody shown could bind to human CLD18A2 with a high EC50, rather than CLD18A1 (Figures 10 and 11). Example 11. Binding of PTM (Post-Translational Modification) De-Risked Humanized Antibodies Reactive with CLD18A2

[0179] Post-translational modifications (PTMs) can cause problems such as increased heterogeneity, decreased biological activity, decreased stability, immunogenicity, fragmentation, and aggregation during the development of therapeutic proteins. The potential impact of PTMs depends on their location and, in some cases, solvent exposure. The CDRs of the sequences were analyzed for the following potential PTMs: asparagine deamidation, aspartic acid isomerization, free cysteine thiol groups, N-glycosylation, oxidation, fragmentation by potential hydrolysis sites, etc.

[0180] To reduce the risk of PTMs occurring in 4F11E2, 72C1B6A3, and 120B7B2, several relevant amino acids in VH and VL were mutated. Nine antibodies were then generated. [Table 11-1] [Table 11-2] * The amino acid position (e.g., N55) follows the number of amino acid residues in the corresponding VH or VL amino acid sequence, rather than Kabat or Chothia. [Table 12]

[0181] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from the flask. 100 μl of cells at 1×10 6 cells / ml were incubated on ice for 30 minutes with the primary mutant antibodies shown in Fig. 4, starting from 100 nM and serially diluted 3-fold down to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated on ice for 30 minutes with the secondary antibody. The cells were washed twice with 200 μl of FACS buffer, transferred to a BD Falcon 5 ml tube, and analyzed by FACS. The results of the study showed that the antibodies shown could bind to human CLD18A2 but not CLD18A1 with high EC50 (Figs. 12 and 13).

[0182] To evaluate the antigen-binding ability of the risk-deleted variants of 4F11E2d (HCN55E / LCS32A) and 4F11E2d (HN55EN104Q / LCS32A), the variants were tested in a cell-based binding assay. Anti-CLDN18.2 antibodies serially diluted starting from 100 nM were incubated with 10 5 cells on ice for 30 minutes. After washing with FACS buffer, the cells were then incubated on ice for an additional 30 minutes with an APC-labeled secondary antibody. Cells bound to the antibody were analyzed by FACS. The variants showed strong binding to cell surface claudin 18.2 (Fig. 14). Example 12. Antibody-Dependent Cellular Cytotoxicity (ADCC) of PTM Risk-Deleted Humanized Antibodies

[0183] Serial dilutions of claudin 18.2 PTM risk-deleted humanized antibodies or reference antibodies were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) with and without ADCC bioassay target cells (expressing claudin 18.2) for 6 hours of induction at 37°C. Luciferase activity was quantified using Bio-Glo™ reagent (Table 5). The results show that these humanized antibodies have very strong ADCC activity. Table 5. ADCC

Table 5

[0184] All amino acids in the extracellular domain of Claudin 18.2 were individually mutated to A. Each mutant Claudin 18.2 or wild-type Claudin 18.2 was transfected into Hek293 cells. The expression of Claudin 18.2 was evaluated by the indicated antibodies. The results are shown in Fig. 15 (only amino acid residues where the mutation reduced binding are shown).

[0185] As shown in Fig. 15, the amino acids W30, N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, C63, R80, Y169 and G172 are involved in the binding of the three test antibodies, 4F11E2 (H4F), 72C186A3 (H72C1) and 120B7B2 (120), or the reference antibody 175D10 (IMAB362). W30 appeared to form a cluster of residues in the first half of the first extracellular domain of the Claudin 18.2 protein. N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62 and C63 appeared to be a second cluster of residues within the same extracellular domain. On the other hand, Y169 and G172 are located in or near the second extracellular domain.

[0186] The crystal structures of various Claudin proteins have been elucidated. As shown in Fig. 20 (Suzuki et al., Ann. N.Y. Acad. Sci., 1397:25 - 34), the Claudin protein contains four transmembrane segments, a short intracellular N-terminus, a large first extracellular loop (Loop 1, or ECS1) containing a consensus W-LW-C-C motif, a shorter second extracellular loop (Loop 2, or ECS2), and an intracellular C-terminal tail portion. Loop 1 contains four β-strands β1, β2, β3, and β4, and Loop 2 contains one β-strand β5.

[0187] The mutations to alanine at W30, L49, and W50 may have destabilized the conformation of loop 1. The mutations at C53 or C63 may have disrupted the disulfide bond between β3 and β4. R80 may be important for maintaining the interaction between parallel claudin 18.2 molecules on the cell surface or for stabilizing the conformation of loop 1. The remaining residues, including N45, Y46, G48, V54, R55, E56, S58, F60, and E62 (within the β3-β4 loop), as well as Y169 and G172 (within β5), may present an interface for binding to the antibodies tested here. Example 14. Comparison of Humanized 4F11E2, 72C1B6A3, and 120B7B2 Antibodies with Benchmark 175D10 Claudin 18.2 Antibody Cell-based Binding

[0188] To compare the humanized anti-claudin 18.2 antibodies: 4F11E2 (HCN55E / LCS32A), 72C1B6A3 (HCWT / LCS32A), and 120B7B2 (HCG57DS104A / LCS32AG97A) with the benchmark antibody 175D10 (IMAB362), in this example, cell-based binding in human claudin 18.2-expressing cells was determined. Based on the level of human CLDN18.2 expression, CHO-K1 cells stably expressing human CLD18A2 were sorted into high-expressing and low-expressing cells. Anti-CLDN18.2 antibodies serially diluted starting from 100 nM were incubated with 10 5 cells on ice for 30 minutes. After washing with FACS buffer, the cells were then incubated with an APC-labeled secondary antibody on ice for an additional 30 minutes. Cells bound to the antibody were analyzed by FACS.

[0189] As shown in Figure 16, 4F11E2, 72C1B6A3, and 120B7B2 showed better binding than 175D10 in both high and low CHO-K1 cells expressing claudin 18.2. ADCC Assay

[0190] To further compare the ADCC effects of humanized anti-Claudin 18.2 antibodies: 4F11E2 (HCN55E / LCS32A), 72C1B6A3 (HCWT / LCS32A) and 120B7B2 (HCG57DS104A / LCS32AG97A) with the benchmark antibody 175D10 (IMAB362), a cell-based ADCC assay was performed in this example. Briefly, NK92 cells were co-cultured with Claudin 18.2 overexpressing 293 cells in the presence of different doses of anti-Claudin 18.2 antibodies. As shown in Figure 17, 4F11E2, 72C1B6A3 and 120B7B2 showed superior ADCC potency compared to the 175D10 antibody.

[0191] For certain therapeutic antibodies, enhanced ADCC can increase the therapeutic window for antibody-based targeted therapies. Enhanced ADCC can be achieved by engineering the Fc region using mutations such as S239D / I332E. In an NK92 cell-based ADCC assay, the 4H11E2, 72C1B6A3 and 120B7B2 antibodies with the S239D / I332E mutation in the Fc region mediated stronger NK92-mediated cell killing of Claudin 18.2 overexpressing 293 cells compared to the control antibody 175D10 with the same S239D / I332E mutation (Figure 18). Antibody-dependent cell phagocytosis (ADCP)

[0192] The effect of anti-CLDN18.2 mAb on tumor cell phagocytosis by macrophages was evaluated in an in vitro assay in which CLDN18.2-positive NUG-C4 cells were co-cultured with human differentiated macrophages in the presence of different concentrations of anti-CLDN18.2 mAb. Briefly, CD14+ monocytes were purified from human peripheral blood mononuclear cells (PBMCs) and differentiated into mature macrophages in vitro for 6 days. Monocyte-derived macrophages (MDMs) were harvested and re-seeded in 24-well dishes as effector cells overnight. NUG-C4 expressing CLDN18.2-eGFP as target cells was added to MDMs at a ratio of 5 tumor cells per phagocytic cell in the presence of different concentrations of anti-CLDN18.2 mAb. After 3 hours of incubation, non-phagocytosed target cells were washed away with PBS, and the remaining phagocytic cells were harvested, stained with the macrophage marker CD14, and then subjected to flow cytometry analysis. The phagocytosis index was calculated by quantifying the percentage of GFP+ cells in CD14+ cells and normalizing it to that of the IgG control.

[0193] As shown in Figure 19, all C18.2 mAbs significantly enhanced the phagocytosis of NUG-C4 cells in a concentration-dependent manner. In both wild-type IgG1 and S239D / I332E mutant IgG1 formats, the 4H11E2, 72C1B6A3, and 120B7B2 antibodies showed a stronger ADCP effect than the reference antibody 175D10.

[0194] In summary, this example demonstrates that the newly developed 4F11E2, 72C1B6A3, and 120B7B2 antibodies had stronger cell-based binding and ADCC / ADCP potencies than the reference antibody 175D10. The improved properties of these new antibodies are presumably due to their higher binding specificities compared to the binding specificity of the reference antibody 175D10. For example, the interaction of 175D10 with claudin 18.2 is strong over a series of ranges in Figure 15 that include strong binding to D28, Q33, N38, and V43, then G59 and V79. In contrast, the new antibodies 4F11E2, 72C1B6A3, and 120B7B2 have higher specificity for W30 within the first half of the first extracellular domain and higher specificity for G48 - E56 within the second half of the first extracellular domain. The new antibodies also have slightly stronger binding to Y46, which is also in the second half. Their binding to D28, Q33, N38, V43, G59, and V79 is considerably weaker, which likely contributed to the improved ADCC and ADCP of the new antibodies. Example 15: pHAb Conjugation to Claudin 18.2 Antibodies

[0195] The internalization of anti-claudin 18.2 antibodies conjugated to CLDN18.2 was determined using a pHAb-reactive dye-based internalization assay. The pHAb dye is a pH sensor dye that has very low fluorescence at pH > 7 and whose fluorescence increases dramatically as the pH of the solution becomes acidic. The pHAb dye has an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm. The pHAb dye-conjugated antibody can be used to monitor receptor-mediated antibody internalization. When the antibody-pHAb dye conjugate binds to its receptor on the cell membrane, it exhibits minimal fluorescence. However, upon receptor-mediated internalization, the antibody-pHAb dye conjugate is transported to endosomal and lysosomal vesicles where the pH is acidic, causing the pHAb dye to fluoresce. This fluorescence can be detected using a variety of techniques including cell imaging, flow cytometry, and a fluorescence plate-based reader equipped with appropriate filters.

[0196] Experimental protocol:

[0197] A. Antibody production

[0198] Twenty-seven chimeric antibodies, three humanized antibodies, and control IgG1 were produced by transient transfection of ExpiCHO cells and purified by protein A affinity chromatography.

[0199] B. Antibody conjugation on beads using pHAb thiol-reactive dye

[0200] 1. Gently shake or use an end-over-end mixer to resuspend AmMag® Protein A beads (LC00695) uniformly. Keep the suspension uniform when making an aliquot of the beads.

[0201] 2. Add 50 μl of the bead slurry to a 1.5 ml microcentrifuge tube. Place the tube on a magnetic stand for 10 seconds.

[0202] 3. Remove and discard the storage buffer.

[0203] 4. Add 250 μl of PBS (pH 7.4). Mix the tube and place it on the magnetic stand for 10 seconds. Remove and discard the buffer.

[0204] 5. Add 1.0 ml of the sample containing 100 μg of the antibody to the beads.

[0205] 6. Mix the sample at room temperature for 60 minutes. Keep the beads in suspension by mixing continuously.

[0206] 7. Place the tube in the magnetic stand for 10 seconds. Remove the supernatant.

[0207] 8. Add 250 μl of the thiol conjugation buffer (10 mM phosphate buffer containing 1 mM EDTA, pH 7.0) and mix. Place the tube in the magnetic stand for 10 seconds. Remove and discard the buffer. Repeat this step a total of 2 times.

[0208] 9. Add 100 μl of the thiol conjugation buffer.

[0209] 10. Add DTT to a final concentration of 2.5 mM.

[0210] 11. Mix the combined sample at room temperature for 60 minutes. Keep the beads in suspension by mixing continuously.

[0211] 12. Place the tube in the magnetic stand for 10 seconds and discard the buffer.

[0212] 13. Add 250 μl of the thiol conjugation buffer and mix. Place the tube in the magnetic stand for 10 seconds. Remove and discard the buffer. Repeat this step a total of 2 times.

[0213] 14. Add 100 μl of thiol conjugation buffer.

[0214] 15. Quickly centrifuge the pHAb thiol-reactive dye (G9835) (i.e., 14,000 × g for 5 - 10 seconds in a benchtop centrifuge) and dissolve it at 10 mg / ml by adding 25 μl of a 1:1 DMSO - water mixture to 0.25 mg of the dye. Mix by vortexing. It may take 1 - 3 minutes for the dye to completely dissolve. Prepare this solution immediately before use.

[0215] 16. Add 1.2 μl of the pHAb thiol-reactive dye to 100 μg of the antibody to create a 20 - molar excess of the dye.

[0216] 17. Mix for 60 minutes. Keep the beads in suspension by continuous mixing.

[0217] 18. Place the tube in a magnetic stand for 10 seconds. Remove and discard the supernatant. 19. Add 250 μl of thiol conjugation buffer and mix. Place in the magnetic stand for 10 seconds. Remove and discard the binding / washing buffer (PBS, pH 7.4).

[0218] 20. Repeat step 19 for a total of 2 washes.

[0219] 21. Add 100 μl of elution buffer (0.1 M glycine, pH 3.0) to the beads.

[0220] 22. Mix at room temperature for 5 minutes.

[0221] 23. Place the tube in a magnetic stand for 10 seconds. Remove the elution sample and transfer it to a new microcentrifuge tube containing 5 μl of neutralization buffer (1 M Tris - HCl, pH 9.0).

[0222] The antibody concentration and the dye - to - antibody ratio (DAR) of the test antibody are shown in Table 6. Table 6. Dye - to - antibody ratio (DAR)

Table 6

[0223] Stably transfected human CLDN18.2 MKN45 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded into a 96-well black plate (Thermo Scientific catalog number 165305) at a density of 20K per 90 μl / well. After incubating the plate for 20-24 hours, it was treated with pHAb-labeled antibody.

[0224] For internalization, the pHAb-conjugated Claudin 18.2 antibody was added to the cells at two concentrations (20 nM and 100 nM), gently mixed with a plate mixer for 1-2 minutes, and then incubated overnight to allow internalization (internalization can be detected within a few hours). The plate was read with a fluorescence plate reader at Ex / Em: 532 nm / 560 nm on a Tecan Infinity M1000 Pro. To achieve higher sensitivity, the medium was replaced with PBS before reading the plate.

[0225] The results normalized by DAR are shown in Table 7. The internalization efficiency of the test antibody was higher than that of the reference antibody IMAB362. Table 7. Results of internalization

Table 7

[0226] Stably transfected human CLDN18.2 CHO cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded into 96-well black plates (Thermo Scientific catalog number 165305) at a density of 10K per 90 μl / well. After incubating the plates for 20 - 24 hours, they were treated with pHAb-labeled antibodies.

[0227] For internalization, pHAb-conjugated chimeric Claudin 18.2 antibodies were added to the cells at various concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM, and 0.01 nM), gently mixed for 1 - 2 minutes using a plate mixer, and then incubated overnight to allow internalization (internalization can be detected within a few hours). The plates were read on a fluorescence plate reader at Ex / Em: 532 nm / 560 nm on a Tecan Infinity M1000 Pro. To achieve higher sensitivity, the medium was replaced with PBS before reading the plates.

[0228] The results normalized by DAR are shown in Figure 21. Again, the internalization efficiency of the test antibody was higher than that of the reference antibody IMAB362. Example 18: EC50 of internalization of humanized Claudin 18.2 antibody against CHO-Claudin 18.2 cells

[0229] Stably transfected human CLDN18.2 CHO cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded into 96-well black plates (Thermo Scientific catalog number 165305) at a density of 10K per 90 μl / well. After incubating the plates for 20 - 24 hours, they were treated with pHAb-labeled antibodies.

[0230] For internalization, the pHAb-conjugated humanized Claudin 18.2 antibody was added to the cells at various concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM, and 0.01 nM), gently mixed with a plate mixer for 1 - 2 minutes, and then incubated overnight to allow internalization (internalization can be detected within a few hours). The plates were read with a fluorescence plate reader at Ex / Em: 532 nm / 560 nm on a Tecan Infinity M1000 Pro. To achieve higher sensitivity, the medium was replaced with PBS before reading the plates.

[0231] The results normalized by DAR are shown in Figure 22, which indicates that the internalization efficiency of the test antibody was greater than that of the reference antibody IMAB362. Example 19: EC50 of internalization of humanized Claudin 18.2 antibody against MKN45-Claudin 18.2 cells

[0232] Stably transfected human CLDN18.2 MKN45 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300 - 054) and seeded into a 96-well black plate (Thermo Scientific catalog number 165305) at a density of 10K per 90 μl / well. After incubating the plates for 20 - 24 hours, they were treated with the pHAb-labeled antibody.

[0233] For internalization, the pHAb-conjugated humanized Claudin 18.2 antibody was added to cells at various concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM, and 0.01 nM), gently mixed with a plate mixer for 1 - 2 minutes, and then incubated overnight to allow internalization (internalization can be detected within several hours). The plates were read with a fluorescence plate reader at Ex / Em: 532 nm / 560 nm on a Tecan Infinity M1000 Pro. To achieve higher sensitivity, the medium was replaced with PBS before reading the plates.

[0234] The results normalized by DAR are shown in Figure 23, which indicates that the internalization efficiency of the test antibody was greater than that of the reference antibody IMAB362. Example 20: Antibody - Drug Conjugate

[0235] Each antibody was mixed with approximately 3 - fold TCEP and stirred at 37 °C for 2 hours. The reaction system was rapidly added dropwise 8 - fold with respect to VC - MMAE, incubated on ice for 1 hour, and 20 - fold excess cysteine was added onto the drug linker to stop the reaction. Finally, the ADC product was purified by elution with Sephadex G - 25 equilibrated in PBS and concentrated by centrifugal ultrafiltration. The conjugate was filtered through a 0.2 μm filter under sterile conditions and stored at - 80 °C for analysis and testing. The drug - antibody ratio was analyzed by UV spectroscopy, the monomer content was analyzed by SEC - HPLC, and the free drug content was analyzed by RP - HPLC. The (DAR) of the vcMMAE - conjugated antibody is shown in Table 8. Table 8. DAR of vcMMAE - conjugated antibody

Table 8 - 1

Table 8 - 2

[0236] In this example, the relative binding affinity and specificity of anti-CLDN18.2 naked antibodies and antibody-drug conjugates were determined by flow cytometry using CLDN18.2 positive and negative cell lines.

[0237] Cells from exponentially growing cultures were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and counted using a Neubauer counting chamber. The cells were centrifuged at 1,500 rpm (468×g) for 5 minutes, the supernatant was discarded, and the cells were resuspended in FACS buffer (PBS containing 2% FCS (Gibco, 10270-106) for analysis using toxin-conjugated antibodies, PBS containing 2% FCS and 2 mM EDTA for screening of CLDN18.2-reactive naked antibodies) at 2×10 6 cells / ml. 100 μl of the cell suspension (2×10 5(corresponding to the number of cells per well) were transferred to a round-bottom 96-well microtiter plate. After centrifugation at 1500 rpm for 1 minute, the supernatant was discarded, and the cells were resuspended in FACS buffer containing the toxin-conjugated antibody or naked antibody at an appropriate concentration (up to 20 μg / ml for relative affinity measurement or up to 50 μg / ml for expression control) and incubated at 4°C for 30 - 45 minutes. (Table 8). The cells were centrifuged at 1500 rpm for 1 minute, and the supernatant was discarded. After washing the cells three times with FACS buffer, the cells were resuspended in FACS buffer containing APC-conjugated anti-human IgG (Jackson Immuno Research, 109 - 136 - 170) or APC-conjugated goat anti-mouse IgG (Jackson Immuno Research, 115 - 136 - 146) or Protein L-FITC (1 μg / ml, for analysis of chim mAB294) and incubated at 4°C for 30 minutes. (Table 3). After incubation, 100 μl of FACS buffer was added to each sample, the cells were centrifuged at 1500 rpm for 1 minute, and the supernatant was discarded. The washing step with FACS buffer was repeated twice. Finally, the cells were resuspended in 100 μl of FACS buffer, and the binding was determined using a BD FACS Array bioanalyzer.

[0238] It should be noted that the toxin-conjugated antibody and the naked antibody were applied at equal concentrations. The results are shown in Figure 24. Example 22: The cytotoxicity of the Claudin 18.2 humanized antibody using MMAE is more potent than that of IMAB362 using MMAE in DAN-G, NUGC, or SCG-7901 transfectants.

[0239] Cells overexpressing human Claudin 18.2 (DAN-G, NUGC or SCG-7901 transfectants) were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054), resuspended in cell culture medium, and 50 μl of cell suspension containing the corresponding amount of cells was seeded per well of a 96-well cell culture plate. After 24 hours, the toxin conjugate IMAB362 or control antibody diluted in 50 μl of medium at an appropriate concentration was added, and the cells were cultured for an additional 72 hours. The effect of the Claudin 18.2 humanized antibody with MMAE on cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (G7572).

[0240] The CellTiter-Glo® Luminescent Cell Viability Assay (G7572) protocol used was as follows:

[0241] 1. Prepare an opaque-walled multi-well plate containing mammalian cells in culture medium at 100 μl / well for a 96-well plate or 25 μl / well for a 384-well plate. The multi-well plate must be compatible with the luminometer used.

[0242] 2. Prepare control wells containing medium without cells to obtain the background luminescence value.

[0243] 3. Add the test compound to the experimental wells and incubate according to the culture protocol. 4. Equilibrate the plate and its contents at room temperature for approximately 30 minutes

[0244] 5. Add an equal volume of CellTiter-Glo® reagent to the volume of cell culture medium present in each well (e.g., add 100 μl of reagent to 100 μl of medium containing cells for a 96-well plate or 25 μl of reagent to 25 μl of medium containing cells for a 384-well plate).

[0245] 6. Induce cell lysis by mixing the contents with an orbital shaker for 2 minutes.

[0246] 7. Incubate the plates at room temperature for 10 minutes to stabilize the luminescence signal. Note: Heterogeneous luminescence signals within standard plates can be caused by temperature gradients, non-uniform seeding of cells, or edge effects in multi-well plates.

[0247] 8. Record the luminescence.

[0248] The test results are shown in FIGS. 25A - C. Both BG2001 - C and BG2001 - D, when conjugated with MMAE, showed a significant increase in cytotoxicity compared to the reference IMAB362 - NMAE conjugate in all the cells tested. Thus, these results demonstrate the improved ability of the antibodies of the present disclosure in the internalization of conjugate drugs. Example 23: The cytotoxicity of the Claudin 18.2 humanized antibody having MMAE is more potent than that of IMAB362 having MMAE in SNU620 that endogenously expresses human Claudin 18.2

[0249] SNU620 cells were resuspended in cell culture medium, and 50 μl of cell suspension containing the corresponding amount of cells was seeded per well in a 96 - well cell culture plate. After 24 hours, a toxin - conjugated antibody containing the reference antibody IMAB362 diluted in 50 μl of medium at an appropriate concentration was added, and the cells were cultured for an additional 72 hours. The effect of the Claudin 18.2 humanized antibody having MMAE on cell viability was determined using the CellTiter - Glo® Luminescent Cell Viability Assay (G7572). As shown in FIG. 26, both BG2001 - C and BG2001 - D were much more effective in delivering conjugated MMAE to SNU620 cells compared to the reference antibody IMAB362, which is a lead anti - Claudin 18.2 antibody in clinical development. Example 24: In Vivo Efficacy of Antibody-Drug Conjugates

[0250] In this example, the efficacy of one of the antibody-drug conjugates (ADCs) was tested for the reduction of tumor growth in nude mice implanted with human tumor cells, compared to the antibody alone (mAb).

[0251] 0.1 mL (5×10 5 cells) of cells derived from human patients (mixed 1:1 with Matrigel) were subcutaneously inoculated into the right dorsal part of each mouse. 3 When the average tumor volume reached 60 - 80 mm

[0252] 30 mice were selected for the treatment experiment when the tumor size reached 330 - 520 mm 3 18 days after inoculation, five mice with tumor sizes in the range of 330 - 520 mm

[0253] were selected for each 3-week treatment (1 mg / kg, 3 mg / mk, 10 mg / kg or 20 mg / kg ADC, QW). For comparison, the treatment with the antibody (mAb) alone was 10 mg / kg (BIW).

[0254] The present disclosure should not be limited in scope by the specific embodiments described as single examples of individual aspects of the present disclosure, and any composition or method that is functionally equivalent is within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they come within the scope of the appended claims and their equivalents.

[0255] All publications and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference.

Claims

**Claim 1**: An antibody-drug conjugate comprising a drug moiety covalently attached to an antibody or an antigen-binding fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising light chain complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising heavy chain complementarity determining regions CDRH1, CDRH2, and CDRH3, (a) said CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304, or 305, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, said CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, 19, or 20, said CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or 306, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or 307; (b) said CDRL1 comprises the amino acid sequence of SEQ ID NO: 216, 308, or 309, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, said CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, said CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 268, 310, or 311, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, 312, 313, or 314; or (c) said CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304, or 305, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, said CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, said CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 289, an antibody-drug conjugate. **Claim 2** said CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304 or 305, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, said CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, 19 or 20, said CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or 306, and The antibody-drug conjugate according to claim 1, wherein the CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or 307.

3. The antibody-drug conjugate according to claim 2, wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 192-195 and 206-207, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 192-195 and 206-207.

4. The antibody-drug conjugate according to claim 1 or 2, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 188-191 and 205, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 188-191 and 205.

5. The CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, The CDRL3 comprises the amino acid sequence of SEQ ID NO: 19, The CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 306, and The antibody-drug conjugate according to claim 2, wherein the CDRH3 comprises the amino acid sequence of SEQ ID NO:

307.

6. The antibody-drug conjugate according to claim 5, wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 206 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

205.

7. The CDRL1 comprises the amino acid sequence of SEQ ID NOs: 216, 308 or 309, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, The CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, The CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, The CDRH2 comprises the amino acid sequence of SEQ ID NOs: 268, 310 or 311, and The antibody-drug conjugate according to claim 1, wherein the CDRH3 comprises the amino acid sequence of SEQ ID NOs: 294, 312, 313 or 314.

8. The antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129, 178 to 180, and 201 to 202, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 129, 178 to 180, and 201 to 202, the antibody-drug conjugate according to claim 7.

9. The antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 159, 175 to 177, and 196 to 200, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 159, 175 to 177, and 196 to 200, the antibody-drug conjugate according to claim 7 or 8.

10. The CDRL1 comprises the amino acid sequence of SEQ ID NO: 309, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, The CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, The CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 311, and The CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, the antibody-drug conjugate according to claim 7.

11. The antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 197, the antibody-drug conjugate according to claim 10.

12. The CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, 304, or 305, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, The CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, The CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and The CDRH3 comprises the amino acid sequence of SEQ ID NO: 289, the antibody-drug conjugate according to claim 1.

13. The CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, The CDRL2 comprises the amino acid sequence of SEQ ID NO: 2297, The CDRL3 comprises the amino acid sequence of SEQ ID NO: 819, The CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, The CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and The antibody-drug conjugate according to claim 12, wherein the CDRH3 comprises the amino acid sequence of SEQ ID NO:

289.

14. The antibody-drug conjugate according to claim 13, wherein the antibody or the antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 203 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

181.

15. The antibody-drug conjugate according to any one of claims 1 to 14, wherein the drug moiety is a cytotoxic agent or a cytostatic agent.

16. The antibody-drug conjugate according to claim 15, wherein the drug moiety is a maytansinoid or an auristatin.

17. The antibody-drug conjugate according to claim 16, wherein the drug moiety comprises DM1 or DM4.

18. The antibody-drug conjugate according to claim 16, wherein the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

19. The antibody-drug conjugate according to any one of claims 1 to 18, wherein the drug moiety is bound to the antibody or the antigen-binding fragment thereof via a linker.

20. The antibody-drug conjugate according to claim 19, wherein the linker is hydrolysable under acidic conditions.

21. A pharmaceutical composition for treating cancer in a patient in need thereof, the pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 20.

22. The pharmaceutical composition according to claim 21, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer and thyroid cancer.

23. The pharmaceutical composition according to claim 21, wherein the cancer is stomach cancer.

24. The pharmaceutical composition according to claim 21, wherein the patient has the M149L variant of the CLDN18.2 protein.

Images