Antibody drug conjugates targeting claudin 18.2

Abstract

Provided are antibody drug conjugates comprising a drug moiety linked 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.

Description

[0001] This application is a divisional application of the patent application with the application date of November 5, 2020, the application number of 202080053845.0, and the invention name of “Antibody drug conjugate targeting claudin 18.2”. BACKGROUND

[0002] Claudins, such as claudin 18.2, are considered promising targets for cancer immunotherapy. Claudins form a protein family that are important components of tight cell junctions. They establish a paracellular barrier that controls the flow of molecules between cells. The proteins have an N- and C-terminal end in the cytoplasm. Different claudins are expressed in different tissues and alterations of their function are associated with cancer formation in the respective tissue. Claudin-1 is expressed in colon cancer, claudin-18 in gastric cancer, claudin-10 in hepatocellular carcinoma.

[0003] 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. Claudin 18.2 expression in normal tissue is strictly confined to the differentiated epithelial cells of the gastric mucosa, but is absent in the gastric stem cell zone. Claudin 18.2 is retained upon malignant transformation and is expressed in a substantial fraction of primary gastric carcinomas and their metastases. Ectopic activation of claudin 18.2 is also frequently found in pancreatic, esophageal, ovarian and lung tumors. These data suggest that CLDN18.2 has a highly restricted expression pattern in normal tissue and is frequently ectopically activated in a variety of human cancers. SUMMARY

[0004] It was discovered herein that anti-claudin 18.2 antibodies selectively bind to wild-type claudin 18.2 and the common mutant M149L and do not bind to other claudin 18 isoforms, such as claudin 18.1. In a surprising and unexpected finding, the present disclosure demonstrates that these antibodies are very effective in inducing receptor-mediated antibody internalization, particularly in comparison to the lead anti-claudin 18.2 antibody IMAB362 (claudiximab) that is in clinical development. Thus, when conjugated to a drug moiety, these antibodies are able to efficiently deliver the drug into target cells, such as cancer cells that overexpress the claudin 18.2 protein.

[0005] The greatly enhanced ability of the antibodies of the present disclosure to induce receptor-mediated antibody internalization can be attributed to the way these antibodies bind to the claudin 18.2 protein. As demonstrated in Example 14 and Figure 20As illustrated, 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). More importantly, it is contemplated that the residues involved in binding to the antibody include N45, Y46, G48, V54, R55, E56, S58, F60, and E62 (located between the b3 strand and the b4 strand of the first extracellular loop), and Y169 and G172 (located in the b5 of the second extracellular loop). In contrast, the known anti-Claudin 18.2 antibodies are believed to only bind one of the extracellular loops.

[0006] According to one embodiment of the disclosure, there is provided an antibody drug conjugate comprising a drug moiety covalently linked to an antibody or fragment thereof having binding specificity for a wild-type human Claudin 18.2 (CLDN18.2) protein, wherein the antibody or fragment thereof binds to the b3-b4 loop and the b5 strand of CLDN18.2. The b3-b4 loop consists of residues 45-63 of SEQ ID NO: 30 (NYQGLWRSCVRESSGFTEC), and the b5 strand consists of residues 169-172 of SEQ ID NO: 30 (YTFG).

[0007] 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.

[0008] In some embodiments, the antibody or fragment thereof does not bind to b1 and b2, or binds to b1 or b2 with at least 10-fold lower affinity than to the b3-b4 loop or b5 strand. In some embodiments, the antibody or fragment thereof does not bind to a CLDN18.1 protein, or binds to CLDN18.1 with at least 10-fold lower affinity than to CLDN18.2.

[0009] In some embodiments, the antibody or fragment thereof binds to a CLDN18.2 M149L mutant with an affinity that is at least 1% of the affinity for the wild-type CLDN18.2 protein.

[0010] 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.

[0011] The drug moiety can be a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The drug moiety, once released in a cancer cell, can inhibit the proliferation of the cancer cell, or cause apoptosis of the cancer cell. Examples of drug moieties are selected from the group consisting of DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-l-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethyl auristatin-D or N-methyl-L-valyl-N-[(lS,2R)-2-methoxy-4-[(2S)-2-[(lR,2R)-l-methoxy-2-methyl-3-oxo-3-[[(lS)-2-phenyl-l-(2- thiazolyl)ethyl]amino]propyl]-l-pyrrolidinyl]-l-[(lS)-l-methylpropyl]-4-oxobutyl]-N- methyl-(9C1)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethyl auristatin F or N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(aR,βR,2S)-β-methoxy-a- methyl-2-pyrrolidinopropionyl-L-phenylalanine), and mc-Val-Cit-PABA-MMAE (6- maleimidocaproyl-ValcCit-(p-aminobenzyloxy carbonyl)-monomethyl auristatin E or N- [[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbonyl)- L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(lS,2R)-4-[(2S)-2-[(lR,2R)- 3-[[(lR,2S)-2-hydroxy-l-methyl-2-phenylethyl]amino]-l-methoxy-2-methyl-3-oxopropyl]-l- pyrrolidinyl]-2-methoxy-l-[(lS)-l-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide). DM1 is a derivative of the microtubulin inhibitor maytansine, while MMAD, MMAE, and MMAF are auristatin derivatives.

[0012] Methods and uses of 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 to the patient an antibody drug conjugate of the disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Flow cytometry shows that mouse sera from all mice reacted at high titer with HEK293 cells transfected with CLD 18A2 after DNA immunization, with CLD 18A1 as a negative control.

[0014] Figure 2 Cell ELISA or flow cytometry shows that hybridoma supernatants can bind to HEK293 cells transfected with human CLD18A2.

[0015] Figure 3 Flow cytometry shows that purified murine antibodies can bind to MKN45 cells transfected with human CLD18A2 at high EC50 compared to a positive reference antibody.

[0016] Figure 4 Flow cytometry shows that purified murine antibodies can bind to SU620 cells endogenously expressing human CLD18A2 with M149L mutation at high EC50, while reference antibodies cannot.

[0017] Figure 5 Flow cytometry shows that purified murine antibodies can bind to HEK293 cells transfected with mouse CLD18A2 at high EC50.

[0018] Figure 6 Flow cytometry shows that purified murine antibodies can bind to HEK293 cells transfected with cynomolgus CLD18A2 at high EC50.

[0019] Figure 7 Flow cytometry shows that purified murine antibodies can bind to HEK293 cells transfected with human CLD18A2 at high EC50.

[0020] Figure 8 Flow cytometry shows that chimeric antibodies can bind to MKN45 cells transfected with human CLD18A2 at high EC50 compared to a positive reference antibody.

[0021] Figure 9 Flow cytometry shows that chimeric antibodies can bind to MKN45 cells transfected with human CLD18A1.

[0022] Figure 10 Flow cytometry shows that humanized antibodies can bind to MKN45 cells transfected with human CLD18A2 at high EC50 compared to a positive reference antibody.

[0023] Figure 11 It was shown by flow cytometry that the humanized antibody could not bind to MKN45 cells transfected with human CLD18A1.

[0024] Figure 12 It was shown by flow cytometry that the humanized antibody with CDR mutations could bind to MKN45 cells transfected with human CLD18A2 with high EC50 compared to the positive reference antibody.

[0025] Figure 13 It was shown by flow cytometry that the humanized antibody with CDR mutations could not bind to MKN45 cells transfected with human CLD18A1.

[0026] Figure 14 It was shown that the de-risked variants have potent binding to cell surface CLD18A2.

[0027] Figure 15 It was shown that certain mutations of CLD18A2 have a significant impact on the binding of the indicated antibodies to HEK293 cells transfected with these mutants, indicating that these amino acid residues make up at least part of the epitope.

[0028] Figure 16 It was shown that antibodies 4F11E2, 72C1B6A3 and 120B7B2 have superior binding to both high and low tight junction protein 18.2 expressing CHO-K1 cells compared to 175D10.

[0029] Figure 17 It was shown that 4F11E2, 72C1B6A3 and 120B7B2 have potent ADCC test results using the 175D10 antibody as a reference.

[0030] Figure 18 It was shown that the S239D / I332E versions of 4F11E2, 72C1B6A3 and 120B7B2 outperform the 175D10 counterparts in ADCC tests.

[0031] Figure 19 It was shown that 4F11E2, 72C1B6A3 and 120B7B2 also have a better ADCP effect than 175D10.

[0032] Figure 20 The 3D structure and motif structure of tight junction proteins are illustrated.

[0033] Figure 21 It was shown that the internalization results of the test chimeric antibodies compared to the reference antibody IMAB362 on CHO cells expressing tight junction protein 18.2.

[0034] Figure 22 Shown are the internalization results of the test humanized antibodies compared to the reference antibody IMAB362 on Claudin 18.2-expressing CHO cells.

[0035] Figure 23 Shown are the internalization results of the test humanized antibodies compared to the reference antibody IMAB362 on MKN45 cells expressing Claudin 18.2.

[0036] Figure 24 The binding affinities of the antibody and its drug conjugate are shown.

[0037] Figure 25A -C shows the cytotoxicity of the test antibody-MMAE conjugate after internalization in DAN-G, NUGC, or SCG-7901 transfectants.

[0038] Figure 26 The cytotoxicity of the test antibody-MMAE conjugate after internalization in SNU620 cells endogenously expressing human claudin 18.2 is shown.

[0039] Figure 27 The reduction of tumor growth in test animals was compared between the antibody drug conjugate and the antibody alone.

[0040] Figure 28 The average or individual tumor reduction effects of the antibody drug conjugates are shown. DETAILED DESCRIPTION

[0041] definition

[0042] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "antibody" is understood to refer to one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0043] As used herein, the term "polypeptide" is intended to encompass both singular "polypeptide" as well as plural "polypeptides" and refers to a molecule that consists of a linear sequence of monomers (amino acids) joined by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, and not to a specific length of the product. Thus, 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 are included within the definition of "polypeptide," and the term "polypeptide" can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" also means the products of polypeptide expression, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / protecting groups, proteolytic cleavage or by non-naturally occurring amino acids. A polypeptide can be from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any manner, including by chemical synthesis.

[0044] The term "isolated," as used herein with respect to a cell, nucleic acid (e.g., DNA or RNA), refers to a molecule that is separated from other DNA or RNA present in the natural source of the macromolecule, respectively. The term "isolated," as used herein, also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that is separated from other cellular proteins or tissues.

[0045] The term "recombinant," as used herein with respect to a polypeptide or polynucleotide, means a form of the polypeptide or polynucleotide that does not occur in nature, with one non-limiting example being generated by joining together polynucleotides or polypeptides that would not otherwise be associated.

[0046] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in the sequences, which can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. Sequences that are "unrelated" or "non-homologous" have less than 40% identity, but preferably less than 25% identity, to one of the sequences of the disclosure.

[0047] A polynucleotide or region of a polynucleotide (or a polypeptide or region of a polypeptide) has a certain percent (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) "sequence identity" to another sequence if that percent of bases (or amino acids) are the same, when aligned for comparison. Such an alignment and percent homology or sequence identity can be determined using software known in the art, such as those described in Ausubel et al. eds., Current Protocols in Molecular Biology (2007). Preferably, the alignment is performed using default parameters. One alignment program that uses default parameters is BLAST. 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 those having a certain percent homology described above and encoding a polypeptide having the same or similar biological activity.

[0048] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology or sequence identity to the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a certain degree of homology to the nucleotide sequence or its complement. In one aspect, a homolog of a nucleic acid is capable of hybridizing to the nucleic acid or its complement. Likewise, an "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 aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the 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 aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0049] Hybridization reactions can be performed under different "stringency" conditions. Typically, low stringency hybridization reactions are performed in about 10 x SSC or equivalent ionic strength / solvent at about 40°C. Medium stringency hybridization reactions are typically performed in about 6 x SSC at about 50°C, and high stringency hybridization reactions are typically performed in about 1 x SSC at about 60°C. Hybridization reactions can also be performed under "physiological conditions" known to those of skill in the art. One non-limiting example of physiological conditions is the temperature, ionic strength, pH, and Mg2 + concentrations.

[0050] A polynucleotide consists of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into databases in computers having central processing units and used for 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 portion thereof. At least two different forms, i.e., two different nucleotide sequences, in a portion of a gene are referred to as a "polymorphic region of the gene." A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0051] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any sequence of nucleotides which is of any length and can be deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of a polynucleotide can be interrupted by non-nucleotide components. A polynucleotide can be further modified, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise indicated, a polynucleotide in any embodiment of the present disclosure encompasses both the double-stranded form and each single-stranded form of a polynucleotide known or predicted to be capable of forming the double-stranded form.

[0052] The term "encodes" applied to a polynucleotide means that if the polynucleotide, in its native state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA and / or a polypeptide and / or fragments thereof, then the polynucleotide is said to "encode" the polypeptide. The antisense strand is the complement of such a nucleic acid from which the coding sequence can be deduced.

[0053] 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 an intact 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 that binds to an antigen with biological

[0054] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of structure, the antibody fragment binds to 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 functions in a complex similar to an antibody by binding to a specific antigen.

[0055] "Single-chain variable fragment" or "scFv" refers to a fusion protein of an immunoglobulin heavy chain variable domain (V H ) and light chain variable domain (V L ) linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N-terminus of the V H to the C-terminus of the V L , or vice versa. Despite the removal of constant regions and the introduction of the linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art, for example described in U.S. Patent 5,892,019.

[0056] The term antibody includes a wide variety of polypeptides that can be biochemically distinguished. Those of skill will appreciate that the heavy chain is classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that some of these subclasses are further divided into subclasses or isotypes (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA, IgG, or IgE. The subclasses of immunoglobulins (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to impart particular functionality. Modified versions of each of these classes and isotypes are readily discernible to one of skill in light of the present disclosure and thus are within the scope of the present disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule includes two identical light chains polypeptides of approximately 23,000 daltons in molecular weight, and two identical heavy chains polypeptides of 53,000-70,000 in molecular weight. These four chains are typically linked together by disulfide bonds into a “Y” configuration, with the light chains bracketing the heavy chains from the mouth of the “Y” and continuing through the variable region.

[0057] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the present disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, 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-linked Fv (sdFv), fragments comprising a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to the LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0058] Light chains are classified as kappa or lambda (κ, λ). Each class of heavy chain can associate with either kappa or lambda light chains. Generally, light chains covalently associate with heavy chains, and the “tails” of the two heavy chains covalently associate with each other through disulfide bonds or non-covalently associate when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0059] Both the light and heavy chains are divided into structural and functional domains. The terms "constant" and "variable" are used in the functional sense. In this regard, it is understood that the variable domains of the light chain portion (VK) and the variable domains of the heavy chain portion (VH) determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CK) and the constant domains of the heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases as they move away from the antigen binding site or the amino terminus of the antibody. The N-terminal portion is the variable region and at the C-terminal portion is the constant region; the CH3 and CK domains actually comprise the carboxy terminus of the heavy and light chains, respectively.

[0060] As noted above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK and VH domains or a subset of the complementarity determining regions (CDRs) of the antibody combine to form the variable region, which defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule can consist of only heavy chains, without light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0061] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous sequences of amino acids that, due to the antibody's three-dimensional configuration when it is presented in an aqueous environment, are specifically positioned to form the antigen binding domain. The remaining amino acids in the antigen binding domain, referred to as "framework" regions, show less inter-molecular variability. The framework regions primarily adopt a beta-sheet conformation and the CDRs form loops that connect, and in some cases form part of, the beta-sheet structure. Thus, framework regions act to form a scaffold for the CDRs, positioning them in the correct orientation for antigen binding. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to the antigen's epitope on the immunologically active antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDRs and the framework regions, respectively, as they have been precisely defined (see "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)).

[0062] If a term used in this disclosure and / or accepted in the art has two or more definitions, the definition of the term used in this disclosure is intended to encompass all such meanings, unless expressly stated otherwise. One specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found in the variable region of heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Department 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 incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. However, the use of either definition to refer to the CDRs of an antibody or variant thereof is intended to fall within the scope of the term as defined and used herein. Suitable amino acid residues comprising the CDRs as defined by each of the above-cited references are listed in the table below for comparison. The exact number of residues comprising 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 comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0063] Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0064] Kabat et al. also defined a numbering system for variable domain sequences of any antibody. This "Kabat numbering" system can be assigned to any variable domain sequence by one of ordinary skill in the art without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983).

[0065] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts about 9 residues after the first cysteine residue (i.e., about the 31st amino acid), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts 15 residues after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts about 33 amino acid residues after the end of CDR-H2, includes 3-25 amino acids, and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 starts about the 24th residue (i.e., after the cysteine residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 starts about 16 residues after the end of CDR-L1, includes about 7 residues. CDR-L3 starts about 33 residues after the end of CDR-L2 (i.e., after the cysteine residue), includes about 7-11 residues, and ends at the sequence F or W-G-X-G, where X is any amino acid.

[0066] The antibodies disclosed herein can be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from a shark).

[0067] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge domain (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present disclosure can include a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure includes a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure can lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, one of ordinary skill in the art will appreciate that heavy chain constant regions can be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0068] 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 comprise 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 comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion can comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0069] 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 comprises at least one of a kappa constant domain or a lambda constant domain.

[0070] A "light chain-heavy chain pair" refers to the collection of a light chain and a heavy chain, which can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0071] As previously noted, 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 domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is located amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0072] The term "CH2 domain" as used herein includes the portion of a heavy chain molecule, for example, extending from about residue 244 to residue 360 of an antibody (residues 244 to 360, Kabat numbering system; and residues 231 to 340, EU numbering system; see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983). The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also noted that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0073] The term "hinge region" as used herein includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises about 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161 :4083 (1998)).

[0074] The term "disulfide bond" as used herein 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, the CH1 and CK regions are connected by one disulfide bond, and the two heavy chains are connected by two disulfide bonds corresponding to positions 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0075] The term "chimeric antibody" as used herein shall be taken to mean any antibody in which the immunologically active region or site is obtained or derived from a first species and the constant region (which can be intact, partial, or modified in accordance with the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate), and the constant region is human.

[0076] As used herein, "percent humanized" is calculated by determining the number of framework amino acid differences (i.e., non-CDR differences) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, then dividing by the total number of amino acids and multiplying by 100.

[0077] “Specifically binds” or “has specificity for” generally refers to the binding of an antibody by its antigen binding domain to an epitope, and binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope by its antigen binding domain more readily than it binds to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity of a certain antibody to a certain epitope. For example, an antibody “A” can be considered to have a higher specificity for a given epitope than an antibody “B”, or to say that the binding of antibody “A” to an epitope “C” is more specific than to a related epitope “D”.

[0078] As used herein, the term “treat” or “treatment” refers to therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) 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, lessening of disease extent, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented.

[0079] “Subject” or “individual” or “animal” or “patient” or “mammal” refers to any subject, particularly a mammalian subject, in need of diagnosis, prognosis, or treatment. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.

[0080] As used herein, phrases such as “a patient in need of treatment” or “a subject in need of treatment” include a subject, such as a mammalian subject, who would benefit from administration of an antibody or composition of the present disclosure, e.g., for detection, diagnostic procedures, and / or treatment.

[0081] Anti-claudin 18.2 antibodies and fragments

[0082] The present disclosure provides anti-claudin 18.2 antibodies with high affinity to both wild-type claudin 18.2 and the common mutant M149L (SU620 cells endogenously express this mutation). To the inventors' knowledge, none of the currently known anti-claudin 18.2 antibodies bind to this mutant. Thus, the antibodies of the present disclosure have the unique advantage of being able to target both wild-type and M149L mutant claudin 18.2 proteins. This advantage is important because a significant fraction of cancer patients carry this common mutation. It is also noteworthy that the antibodies of the present disclosure do not bind to (or bind to claudin 18.1 with much lower affinity) other claudin 18 isoforms, i.e., claudin 18.1.

[0083] The antibodies and fragments of the present disclosure exhibit superior properties even when using a clinical candidate as a reference. 175D10 (IMAB362; clivatuzumab) is currently undergoing phase III clinical trials for the treatment of gastric and gastroesophageal junction adenocarcinoma. In comparison to 175D10, the antibodies and fragments of the present disclosure not only exhibit stronger binding activity, but also exhibit higher ADCC and ADCP activity under a variety of different conditions.

[0084] Also importantly, the present disclosure demonstrates that these antibodies are very effective at inducing receptor-mediated antibody internalization, even in comparison to IMAB362. The ability of the antibodies of the present disclosure to induce receptor-mediated antibody internalization is greatly increased, which is thought to be attributable to how these antibodies bind to the claudin 18.2 protein. As demonstrated in Example 14 and Figure 20 As demonstrated, the amino acid residues on the claudin 18.2 protein that are important for binding to antibodies include those that are important for stabilizing the conformation of the extracellular loop (e.g., W30, L49, W50, C53, C63, and R80). W30, L49, and W50 are part of the W-LW-C-C consensus motif, which helps to stabilize the conformation of loop 1. C53 and C63 form an inter-beta-strand disulfide bond. R80 can be important for maintaining interactions between claudin 18.2 molecules on the cell surface in parallel, or for stabilizing the conformation of loop 1.

[0085] Also important for antibody binding are 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 the b3 strand, or across C63 in the b4 strand. This region consists of residues 45-63 of SEQ ID NO: 30 (NYQGLWRSCVRESSGFTEC), referred to herein as the "b3 to b4 loop", which is part of the first extracellular loop (loop 1) of claudin 18.2. In contrast, Y169 and G172 are part of the b5 strand of the second extracellular loop (loop 2) (residues 169-172 of SEQ ID NO: 30; YTFG).

[0086] The antibodies of the present disclosure induce greatly increased receptor-mediated antibody internalization, presumably due to their ability to bind to residues of the b3 to b4 loop and the b5 strand. In this case, it is believed that known anti-claudin 18.2 antibodies bind to only one of these loops.

[0087] Experimental data also indicate that the antibodies of the present disclosure have higher binding specificity and improved ADCC and ADCP compared to known antibodies.

[0088] Human claudin 18.2 sequence

[0089]

[0090] According to one embodiment of the present disclosure, there is provided an antibody or fragment thereof having binding specificity for a wild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody or fragment thereof binds to both the first extracellular loop and the second extracellular loop of the CLDN18.2. In some embodiments, the antibody or fragment thereof binds to both the b3-b4 loop and the b5 strand of the CLDN18.2.

[0091] According to another embodiment of the present disclosure, there is provided an antibody or fragment thereof having binding specificity for a wild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody or fragment thereof further binds to a M149L mutant of the CLDN18.2 protein. In some embodiments, the antibody or fragment thereof does not bind to a human wild-type claudin 18.1 (CLDN18.1) protein, or does not bind to CLDN18.1 with an affinity that is greater than about 1% of the affinity for the wild-type CLDN18.2 protein.

[0092] The binding affinity of an antibody or fragment to a protein can be measured using a number of methods known in the art. For example, cell-free assays can be performed using isolated CLDN18.1 protein or CLDN18.2 protein. However, preferably, the measurement is performed using CLDN18.1 protein or CLDN18.2 protein on the surface of a cell that mimics the actual binding environment. Such binding assays are well exemplified in the experimental examples.

[0093] In some embodiments, the antibody or fragment thereof has a binding affinity for the M149L mutant that is at least 1%, or 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.

[0094] In some embodiments, the antibody or fragment thereof does not bind to human CLDN18.1. In some embodiments, the antibody or fragment thereof binds to human CLDN18.1 much less, for example but not limited to, no more than 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% as compared to binding to CLDN18.2.

[0095] As mentioned above, the antibodies and fragments thereof of the present disclosure bind to claudin 18.2 at an epitope that is different from the known antibodies (see Figure 4 ; at least the reference antibodies interact with M149, whereas the antibodies of the present disclosure do not). Thus, in one embodiment, an antibody or fragment thereof having binding specificity for a wild-type human claudin 18.2 (CLDN18.2) protein is provided, wherein the binding between the antibody or fragment thereof and the wild-type CLDN18.2 protein involves amino acid residues comprising at least one amino acid residue of the wild-type CLDN18.2 protein 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.

[0096] 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.

[0097] In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from the group consisting of 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 the group consisting of 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 the group consisting of 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 the group consisting of N45, Y46, G48, V54, R55, E56, S58, F60, and E62.

[0098] 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 the group consisting of Y169 and G172 of CLDN18.2.

[0099] In some embodiments, the amino acid residues involved in the binding comprise W30 of the wild-type CLDN18.2 protein; two, three, four, five or more amino acid residues 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 the wild-type CLDN18.2 protein. In some embodiments, the amino acid residues involved in the binding comprise W30, N45, Y46, G48, V54, R55, E56, S58, F60, E62, and Y169 of the wild-type CLDN18.2 protein.

[0100] The binding to these amino acids on CLDN18.2 is weaker 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 acid as 175D10. For example, the antibody or fragment of the disclosure binds at least one, two, three, four, five, or all of D28, Q33, N38, V43, G59, and V79 weaker than 175D10 (IMGT / 2D structure-DB accession number: 10473).

[0101] 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 a M149L mutant of the CLDN18.2 protein.

[0102] According to one embodiment of the disclosure, there is provided an antibody or fragment thereof, comprising heavy and light chain variable domains, the CDR regions of which are as shown in the CDR combinations of Table A.

[0103] Table A. CDR combinations of tested antibodies (Kabat numbering)

[0104]

[0105]

[0106] Table B. CDRs of 120B7B2 (Kabat numbering)

[0107]

[0108] Table C. CDRs of 72C1B6A3 (Kabat numbering)

[0109]

[0110] Table D. CDRs of 4F11E2 (Kabat numbering)

[0111]

[0112] Antibodies containing these CDR regions, whether mouse, humanized, or chimeric, have potent Claudin 18.2 binding and inhibitory activity. As shown in Examples 11 and 12, certain residues within the CDRs can be modified to retain or improve this property or to reduce the likelihood that they have post-translational modifications (PTMs). Such modified CDRs can be referred to as affinity matured CDRs or de-risked CDRs.

[0113] The third column of Tables B-D provides non-limiting examples of de-risked CDRs. Affinity matured CDRs can include those with one, two, or three additions, deletions, and / or substitutions of amino acids. In some embodiments, the substitutions can be conservative substitutions.

[0114] A "conservative amino acid substitution" refers to the replacement of a amino acid residue by another amino acid residue having similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including 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 nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In a further embodiment, strings of amino acid can be replaced with a string of similar structure that differs in the order and / or composition of members of the side chain family.

[0115] The following table provides non-limiting examples of conservative amino acid substitutions, where a similarity score of 0 or higher indicates a conservative substitution between two amino acids.

[0116] Table E. Amino acid similarity matrix

[0117] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0118] Table F. Conservative amino acid substitutions

[0119]

[0120]

[0121] Accordingly, in one embodiment, there is provided an antibody or fragment thereof having binding specificity to a wild-type human Claudin 18.2 (CLDN18.2) protein, wherein 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, and wherein the CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 are selected from combinations 1-33 or each of combinations 1-33 of Table A, wherein one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 comprises one, two, or three amino acid additions, deletions, conserved amino acid substitutions, or combinations thereof, respectively.

[0122] In some embodiments, provided is an anti-CLDN18.2 antibody or fragment comprising a CDRL1, a CDRL2, a CDRL3, a CDRH1, a CDRH2, and a CDRH3, each of which is selected from Table A or Tables B-D. For example, provided is an antibody or fragment thereof having binding specificity for wild-type human Claudin 18.2 (CLDN18.2) protein, wherein 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, and wherein: the CDRL1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 208-226, or 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; the CDRL2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 227-233, or an amino acid sequence derived from any one of SEQ ID NOs: 227-233 by addition, deletion, or amino acid substitution of an amino acid; the CDRL3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 3, 8, 13, 19, and 42-58, or 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; the CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 234-254, or 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; the CDRH2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 255-280, or 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 the CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 281-303, or 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.

[0123] In some embodiments, the CDRL1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 208-226, 304-305, and 308-309; the CDRL2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 227-233; the CDRL3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 3, 8, 13, 19, 20, and 42-58; the CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 234-254; the CDRH2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 255-280, 306, 310, and 311; and the CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 281-303, 307, and 312-314.

[0124] Antibody 120B7B2 has been shown to be an effective inhibitor of Claudin 18.2. Table B provides its CDR sequences as well as some de-risked versions. In one embodiment, the present disclosure provides an antibody or fragment thereof having binding specificity to wild-type human Claudin 18.2 (CLDN18.2) protein, wherein 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, and wherein: the CDRL1 comprises the amino acid sequence of 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, the CDRL2 comprises the amino acid sequence of WAS (SEQ ID NO: 2), or an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 2, the CDRL3 comprises the amino acid sequence of 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, the CDRH1 comprises the amino acid sequence of GYTFTGYI (SEQ ID NO: 4), or an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 4, the CDRH2 comprises the amino acid sequence of 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 the CDRH3 comprises the amino acid sequence of 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.

[0125] It is interesting to note that the CDRs from different antibodies have a high degree of homology (see Table A). It is then expected that each respective CDR can be interchanged without greatly 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.

[0126] In some embodiments, an antibody or fragment thereof is provided having binding specificity to a wild-type human Claudin 18.2 (CLDN18.2) protein. 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, and wherein: the 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, the 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; the 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; the 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; the 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 the 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.

[0127] In some embodiments, 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: 227, the CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, 19, or 20, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, the CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or 306, and the CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or 307.

[0128] Non-limiting examples of light chain variable regions comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 192-195, and 206-207, 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: 141, 192-195, and 206-207.

[0129] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NOs: 171, 188-191, and 205, 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: 171, 188-191, and 205.

[0130] In some embodiments, 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 CDRH3 comprises the amino acid sequence of SEQ ID NO: 307. One non-limiting example of the antibody or fragment includes 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.

[0131] Similarly, 72C1B6A3 has proven to be a good antibody. Accordingly, in another embodiment, there is provided an antibody or fragment thereof having binding specificity to wild-type human Claudin 18.2 (CLDN18.2) protein, wherein 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, and wherein: the 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, the 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, the 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, the 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, the 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 the 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.

[0132] In some embodiments, 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.

[0133] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 185-187, and 203-204, 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: 124, 185-187, and 203-204.

[0134] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NOs: 153 and 181-184, 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: 153 and 181-184.

[0135] In some embodiments, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, 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. One non-limiting example of an antibody or fragment thereof includes 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.

[0136] In addition, 4F11E2 has proven to be a good antibody. Accordingly, in another embodiment, an antibody or fragment thereof having binding specificity to wild-type human Claudin 18.2 (CLDN18.2) protein is provided, wherein 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, and wherein: the 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, the 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, the 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, the 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, the 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 the 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.

[0137] In some embodiments, the CDRL1 comprises the amino acid sequence of SEQ ID NO: 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 NO: 268, 310, or 311, and the CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, 312, 313, or 314.

[0138] Non-limiting examples of a 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.

[0139] Non-limiting examples of a 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.

[0140] In some embodiments, 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. One non-limiting example of the antibody or fragment includes 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.

[0141] In some embodiments, the antibody is a humanized antibody. As shown in Example 9, a humanized antibody can include one or more back mutations of the mouse counterpart. Examples of such back mutations are shown in Table 3. In some embodiments, the antibody or fragment can include one, two, three, four, five, or more of the back mutations.

[0142] In some embodiments, the anti-claudin 18.2 antibodies of the present disclosure comprise a VL of any one of SEQ ID NOs: 117-144, 178-180, 185-187, 192-195, 201-202, 203-204, or 206-207, and a VH of any one of SEQ ID NOs: 145-174, 175-177, 181-184, 188-191, 196-200, or 205, or a biological equivalent of each thereof. A biological equivalent of a VH or VL is a sequence that includes the specified amino acids and has an overall 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. Thus, a biological equivalent of SEQ ID NO: 145 can be a VH that has an overall 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 145, but retains the CDRs, and optionally one or more or all of the back mutations.

[0143] One of ordinary skill in the art will also appreciate that the antibodies disclosed herein can be modified such that their amino acid sequence is not identical to the naturally occurring binding polypeptide from which they are derived. For example, a polypeptide or amino acid sequence derived from a specified protein can be similar to the starting sequence, e.g., have a certain percentage of identity, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0144] In certain embodiments, the antibodies comprise an amino acid sequence or one or more moieties not normally associated with antibodies. Exemplary modifications are described in more detail below. For example, the antibodies of the present disclosure can comprise a flexible linker sequence, or can be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0145] The antibodies, variants, or derivatives thereof of the present disclosure include modified derivatives, i.e., by covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not hinder the antibody from binding to the epitope. For example, and not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to a cell ligand or other protein, and the like. Any of numerous chemical modifications can be introduced into the antibody by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of glycosylation, and the like. Moreover, the antibodies can comprise one or more non-canonical amino acids.

[0146] Antibody drug conjugates

[0147] In some embodiments, the antibody or fragment may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0148] In one embodiment, the antibody or fragment of the present disclosure is covalently linked to a drug moiety. The drug moiety can be, or be modified to include a group that reacts with a coupling site on the antibody. For example, the drug moiety can be linked by alkylation (e.g., at the ε-amino lysine or N-terminus of the antibody), reductive amination of oxidized carbohydrates, transesterification between a hydroxyl and carboxyl group, amidation of an amino or carboxyl group, and coupling with a thiol.

[0149] In some embodiments, the number of drug moieties conjugated per antibody molecule, p, is between 1 and 8; an average of 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is 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 is 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 is between 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.

[0150] For example, when chemical activation of a protein results in the formation of free thiol groups, the protein can be coupled with a sulfhydryl-reactive agent. In one aspect, the agent is a reagent that is substantially specific for free thiol groups. For example, such agents include maleimides, haloacetamides (e.g., iodo, bromo, or chloro), haloesters (e.g., iodo, bromo, or chloro), halomethylketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodide, bromide, or chloride), vinyl sulfones, and pyridyl sulfides.

[0151] The drug can be linked to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal proteases or endosomal proteases. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl] cyclohexane-l-carboxylate (smcc). Sulfosmcc conjugation occurs via the maleimido group's reaction with a sulfhydryl group (thiol, -SH), while its sulfos-NHS ester is reactive with primary amines (as found in lysines and at the N-terminus of proteins or peptides). Still another linker is a maleimidocaproyl (mc) linker. Other suitable linkers include linkers that hydrolyze at a particular pH or pH range, such as a hydrazone linker. Other suitable cleavable linkers include disulfide linkers. The linker can be covalently linked to the antibody to the extent that the antibody must be degraded intracellularly for the drug to be released, e.g., a mc linker or the like.

[0152] The linker can comprise a group for linking to the antibody. For example, the linker can comprise an amino, hydroxyl, carboxyl, or sulfhydryl reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinyl sulfone, and pyridylthio).

[0153] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The conjugate can be used to inhibit the proliferation of tumor or cancer cells, to cause apoptosis of tumor or cancer cells, or to treat cancer in a patient. The conjugate can be used accordingly in various contexts for treating cancer in an animal. The conjugate can be used to deliver a drug to a tumor or cancer cell. Without being bound by theory, in some embodiments, the conjugate binds or associates with a cancer cell that expresses claudin 18.2, and the conjugate and / or drug can be taken up into the tumor or cancer cell by receptor-mediated endocytosis.

[0154] Once inside the cell, one or more specific peptide sequences in the conjugate (e.g., in the linker) are hydrolytically cleaved by one or more tumor cell or cancer cell associated proteases, causing release of the drug. The released drug then freely migrates within the cell and induces cytotoxicity or cytostasis or other activity. In some embodiments, the drug is cleaved from the antibody outside the tumor cell or cancer cell, and the drug then penetrates the cell, or exerts its effect at the cell surface.

[0155] Examples of drug moieties or payloads 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-monomethyl auristatin-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-(9C1)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethyl auristatin 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-(aR, bR,2S)-b-methoxy-a-methyl-2-pyrrolidinepropionyl-L-phenylalanine), and mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-ValcCit-(p- aminobenzyloxy carbonyl)-monomethyl auristatin 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 microtubulin 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.

[0156] The antibody or fragment can be conjugated or fused to a therapeutic agent, which 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 (which can be a drug or a toxin), an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0157] The antibody can be detectably labeled by coupling to a chemiluminescent compound. The presence of the chemiluminescent tag on the antigen-binding polypeptide is then determined by detecting the light produced from the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, an acridinium ester, an imidazole, an oxalate ester, and an oxalate.

[0158] The antibody can also be detectably labeled using a metal that emits fluorescence upon 152Eu or other metals of the lanthanide series are detectably labeled. These metals can be attached to the antibody using metal-chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known, see, e.g., 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.), Marcel 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).

[0159] Polynucleotides encoding antibodies and methods of making antibodies

[0160] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy and light chain variable regions of the antigen binding polypeptides, variants, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Further, the polynucleotides of the present disclosure can encode portions of the heavy and light chain variable regions of the antigen binding polypeptides, variants, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0161] 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 can be made using techniques described in the art and as described herein. For example, fully human antibodies to a particular antigen can be made by administering the antigen to a transgenic animal that has been altered to produce such antibodies when challenged with the antigen, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. Patents 6,150,584, 6,458,592, 6,420,140, the entire contents of which are incorporated by reference.

[0162] Methods of treatment

[0163] As described herein, the antibodies, variants, or derivatives of the present disclosure can be used in certain methods of treatment and diagnosis.

[0164] The present disclosure further relates to antibody-based therapies involving administration of the antibodies, fragments, or antibody drug conjugates of the present disclosure to a patient (e.g., an animal, a mammal, and a human) 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 as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0165] The antibodies of the present disclosure can also be used to treat or inhibit cancer. As provided previously, Claudin 18.2 can be overexpressed in tumor cells, particularly gastric, pancreatic, esophageal, ovarian, and lung tumors. Inhibition of Claudin 18.2 has been shown to be useful in treating tumors.

[0166] Accordingly, in some embodiments, methods of treating cancer in a patient in need thereof are provided. In one embodiment, the method entails 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 cancer cell (e.g., stromal cell) in the patient overexpresses Claudin 18.2.

[0167] The present disclosure also provides cell therapies, such as chimeric antigen receptor (CAR) T cell therapies. Suitable cells can be used that are contacted with (or alternatively engineered to express) an anti-claudin 18.2 antibody of the present disclosure. Through such contacting or engineering, the cells can then be introduced into a cancer patient in need of treatment. The cancer patient can have any type 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.

[0168] In some embodiments, the cells are isolated from the cancer patient themselves. In some embodiments, the cells are provided by a donor or from a cell bank. When the cells are isolated from the cancer patient, an unwanted immune reaction can be minimized.

[0169] Non-limiting examples of cancers 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.

[0170] Other 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, but are not limited to, the progression and / or metastasis of malignancies and related diseases, such as leukemias (including acute leukemias (e.g., acute lymphatic, acute myeloid (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) and chronic lymphocytic leukemias)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, hemangiosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, 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, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0171] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody, variant or derivative thereof used, the age, body weight, general health, sex, and diet of the patient, the time of administration, the rate of excretion, the combination with other drugs, and the severity of the particular disease being treated. The judgment of the medical practitioner is a relevant consideration in these matters. The amount will also depend on the individual patient to be treated, the route of administration, the formulation of the drug, the nature of the compound used, the severity of the disease and the effect desired. The amount can be determined by principles known in pharmacology and pharmacokinetics.

[0172] Methods of administering the antibodies, fragments, or antibody drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen binding polypeptides or compositions can be administered by any convenient route, for example, by infusion or bolus injection, either singly or in combination, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal tract linings, etc.), and can be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen binding polypeptides of the present disclosure can be orally administered, rectally administered, parenterally administered, intracisternally administered, intravaginally administered, intraperitoneally administered, topically administered (e.g., as by powders, ointments, drops, or transdermal patches), nasally administered, or as an oral or nasal spray.

[0173] The term "parenterally" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous and intraarticular injection and infusion.

[0174] Administration can be systemic or local. In addition, it can be desirable to introduce the antibodies of the present disclosure into the central nervous system by any means acceptable in the art, including intercerebroventricular and intrathecal injection; intercerebroventricular injection can be facilitated by an intercerebroventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., through the use of an inhaler or insufflator, and formulation with an aerosol.

[0175] It can be desirable to administer the antigen binding polypeptides or compositions of the present disclosure locally to the area in need of treatment; this can be achieved by, for example, and without limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the present disclosure, care must be taken to use a material that does not absorb the protein.

[0176] The amount of the antibodies, fragments, or antibody drug conjugates of the present disclosure that can be effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder, or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0177] As a general proposition, the dosage of the antibody, fragment, or antibody drug conjugate of the present disclosure administered to a patient is generally 0.1 mg / kg to 100 mg / kg of the patient's body weight, between 0.1 mg / kg to 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. Typically, human antibodies have a longer half-life in humans than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration are generally possible. In addition, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by modifying (e.g., lipidation) to enhance absorption and tissue penetration (e.g., into the brain) of the antibody.

[0178] In another embodiment, the compositions of the present disclosure are administered in combination with a cytokine. Cytokines that can be administered with the compositions 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-alpha.

[0179] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

[0180] Compositions

[0181] The present disclosure also provides pharmaceutical compositions. The pharmaceutical compositions comprise an effective amount of the antibody, fragment, or antibody drug conjugate, and an acceptable carrier. In some embodiments, the compositions further include a second anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0182] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Further, a "pharmaceutically acceptable carrier" would typically be any type of nontoxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of whatever means of introduction is used.

[0183] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. The pharmaceutical carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. 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, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetate, citrate or phosphate. Antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity, such as sodium chloride or dextrose, are also envisioned. 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 with traditional binders and carriers such as, for example, triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, The

[0184] In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0185] The compounds of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc. DETAILED DESCRIPTION

[0187] Example 1: Generation of murine monoclonal antibodies against human claudin 18 subtype 2 (CLD 18A2)

[0188] a. Immunization

[0189] Balb / c and C57 / BL6 mice were immunized with a eukaryotic expression vector encoding a fragment of human claudin 18.2 (CLD 18A2). 50 μg of plasmid DNA was injected into the quadriceps muscle (intramuscular injection, i.m.) on day 1 and day 10. On day 20 the presence of antibodies against human CLD 18A2 in the mouse serum was monitored by flow cytometry using HEK293 cells transiently transfected with a nucleic acid encoding human CLD 18A2. Mice with a detectable immune response 7 were boosted three days and two days prior to fusion by intraperitoneal injection of 5 x 10 Figure 1 ) were boosted three days and two days prior to fusion by intraperitoneal injection of 5 x 10

[0190] b. Generation of hybridomas producing human monoclonal antibodies against CLD18A2

[0191] Mouse spleen cells were isolated according to standard protocols and fused to a mouse myeloma cell line with PEG. The resulting hybridomas were then screened by cell ELISA using HEK293 cells transiently transfected with a nucleic acid encoding human CLD18 to generate immunoglobulins with specificity for CLD18A2.

[0192] Single cell suspensions of spleen lymphocytes from immunized mice were fused with P3X63Ag8U.1 non-secreting mouse myeloma cells (ATCC, CRL 1597) at a ratio of 2:1 using 50% PEG (Roche Diagnostics, CRL 738641). Cells were plated at approximately 3 x 10 4Individuals / well were plated in flat bottom microtiter plates and then incubated in selective medium containing 10% fetal calf serum, 2% hybridoma fusion and cloning supplement (HFCS, Roche Diagnostics, CRL 1 363 735) plus 10 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 μg / ml gentamicin and 1 x HAT (Sigma, CRL H0262) for approximately two weeks. After 10 to 14 days, individual wells were screened for anti-CLD 18A2 monoclonal antibody production by cell ELISA Figure 2 ). Antibody secreting hybridomas were re-plated and again screened by FACS with HEK293 expressing CLD18A2 or CLD18A1 and if still CLD18A2 positive and CLD18A1 negative, subcloned by limiting dilution. Stable subclones were then cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization. At least one clone was selected from each hybridoma that retained the reactivity of the parent cell (by FACS). Each clone produced three vials of cell bank and were stored in liquid nitrogen.

[0193] c. Selection of monoclonal antibodies that bind to CLD 18A2 and not to CLD18A1

[0194] To determine the isotype of the antibodies, an isotype ELISA was performed. The Mouse monoAB ID kit (Zymed, CRL 90-6550) was used to determine the Ig subclass of the identified CLD18A2 reactive monoclonal antibodies. 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:

[0195] 64G11B4, mouse monoclonal IgG1, kappa antibody

[0196] 65G8B8, mouse monoclonal IgG1, kappa antibody

[0197] 56E8F10F4, mouse monoclonal IgG1, kappa antibody

[0198] 54A2C4, mouse monoclonal IgGl, kappa antibody

[0199] 44F6B11, mouse monoclonal IgGl, kappa antibody

[0200] 15C2B7, mouse monoclonal IgGl, kappa antibody

[0201] 20F1E10, mouse monoclonal IgGl, kappa antibody

[0202] 72C1B6A3, mouse monoclonal IgGl, kappa antibody

[0203] 58G2C2, mouse monoclonal IgG2a, kappa antibody

[0204] 101C4F12, mouse monoclonal IgG2b, kappa antibody

[0205] 103A10B2, mouse monoclonal IgG2b, kappa antibody

[0206] 40C10E3, mouse monoclonal IgGl, lambda antibody

[0207] 78E8G9G6, mouse monoclonal IgGl, kappa antibody

[0208] 4F11E2, mouse monoclonal IgGl, kappa antibody

[0209] 10G7G11, mouse monoclonal IgGl, kappa antibody

[0210] 12F1F4, mouse monoclonal IgGl, kappa antibody

[0211] 78C10B6G4, mouse monoclonal IgGl, kappa antibody

[0212] 119G11D9, mouse monoclonal IgGl, kappa antibody

[0213] 113G12E5E6, mouse monoclonal IgGl, kappa antibody

[0214] 116A8B7, mouse monoclonal IgGl, kappa antibody

[0215] 105F7G12, mouse monoclonal IgGl, kappa antibody

[0216] 84E9E12, mouse monoclonal IgGl, kappa antibody

[0217] 103F4D4, mouse monoclonal IgGl, kappa antibody

[0218] 110C12B6, mouse monoclonal IgGl, kappa antibody

[0219] 85H12E8, mouse monoclonal IgGl, kappa antibody

[0220] 103H2B4, mouse monoclonal IgGl, kappa antibody

[0221] 103F6D3, mouse monoclonal IgGl, kappa antibody

[0222] 113E12F7, mouse monoclonal IgG2a, kappa antibody

[0223] 120B7B2, mouse monoclonal IgG2a, kappa antibody

[0224] 111B12D11, mouse monoclonal IgG2a, kappa antibody

[0225] 111E7E2, mouse monoclonal IgG2a, kappa antibody

[0226] 100F4G12, mouse monoclonal IgG3, kappa antibody.

[0227] Example 2. Hybridoma Sequencing

[0228] Hybridoma cells (1 x 10 7 ) were harvested and total RNA was extracted using the Tri Reagent for spleen tissue described above. cDNA was prepared using the Superscript III kit according to the manufacturer's instructions described above. The resulting cDNA product was used as a template for PCR with primers VhRevU and VhForU, and the resulting 300 bp PCR product was cleaned up using a PCR cleanup kit and sequenced with the same primers. PCR reactions were also performed using light chain V region specific primers VkRev7 and VkFor (for variable region only) or KappaFor primers (for entire kappa light chain). Sequencing reactions were performed on the cleaned up PCR products to obtain the DNA sequences for the following 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. The variable sequences (VH and VL) are shown in Table 1 below.

[0229] Table 1. Sequences of antibody variable regions

[0230]

[0231]

[0232]

[0233]

[0234] Example 3. Production and purification of monoclonal antibodies reactive with CLD18A2

[0235] To generate milligram quantities of antibodies for functional characterization, hybridoma cells were plated at 2 x 10 6 Cells / ml were seeded in a dialysis-based bioreactor (CELLine CL1000, Integra, Chur, Switzerland). Antibody-containing supernatants were harvested weekly. 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.

[0236] Example 4. Binding of murine monoclonal antibodies reactive with CLD18A2

[0237] Harvest MKN45 cells overexpressing CLD18A2 from the flask. 6 cells / ml of cells and Figure 3 The indicated primary antibodies were incubated on ice for 30 minutes in a 3-fold serial dilution from 100 nM to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated with the secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μl of FACS buffer, then transferred to a BD Falcon 5ml tube and analyzed by FACS. Flow cytometry revealed that the purified murine antibodies bound to MKN45 cells transfected with human CLD18A2 with a high EC50 compared to a positive reference antibody.

[0238] Example 5. Binding of murine monoclonal antibodies reactive with CLD18A2 mutants

[0239] SU620 cells endogenously expressing CLD18A2 with the M149L mutation were harvested from the flask. 100 μl, 1×10 6 cells / ml of cells and Figure 4The primary antibodies were incubated on ice for 30 minutes with 3-fold serial dilutions ranging from 100 nM to 0.003 nM as indicated. After washing twice with 200 μl of FACS buffer, the cells were incubated with the secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μl of FACS buffer, then transferred to BD Falcon 5 ml tubes and analyzed by FACS. Flow cytometry showed that the purified mouse antibodies could bind to SU620 cells endogenously expressing human CLD18A2 with the M149L mutation with a high EC50, while the reference antibody could not ( Figure 4 ).

[0240] Example 6. Binding of murine monoclonal antibodies reactive with mouse and macaque CLD18A2

[0241] To evaluate the cross-reactivity of these antibodies with mouse and macaque CLD18A2, HEK293 cells overexpressing mouse, macaque, or human CLD18A2 were harvested from flasks. 6 cells / ml of cells and Figure 3 The primary antibodies were incubated on ice for 30 minutes with 3-fold serial dilutions from 100 nM to 0.003 nM as indicated. After washing twice with 200 μl of FACS buffer, the cells were incubated with the secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μl of FACS buffer, then transferred to BD Falcon 5 ml tubes and analyzed by FACS. The results of the flow cytometry study showed that the purified murine antibodies could bind to mouse and macaque CLD18A2 with high EC50, at least similar to the reference antibody ( Figure 5 、 6 and 7).

[0242] Example 7. Binding of chimeric antibodies reactive with CLD18A2

[0243] Murine VH and VK genes are synthetically generated and then cloned into vectors containing human γ1 and human κ constant domains, respectively. Purified chimeric antibodies are produced by transfected CHO cells.

[0244] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. 6 cells / ml of cells and Figure 4The primary chimeric antibodies shown at 3-fold serial dilutions from 100 nM to 0.003 nM were incubated on ice for 30 minutes. After washing twice with 200 μΐ of FACS buffer, the cells were incubated with secondary antibody for 30 minutes on ice. The cells were washed twice with 200 μΐ of FACS buffer, then transferred to BD Falcon 5 ml tubes and analyzed by FACS. The results of the study showed that the chimeric antibodies can bind to human CLD18A2 with high EC50, but not to CLD18A1 Figure 8 and 9 ).

[0245] Example 8. Antibody-dependent cellular cytotoxicity (ADCC) of chimeric antibodies

[0246] The ADCC reporter bioassay uses another readout at an early stage of ADCC MOA pathway activation: gene transcription by the NFAT (nuclear factor of activated T cells) pathway activation in effector cells. In addition, the ADCC reporter bioassay uses engineered Jurkat cells stably expressing the FcyRIIIa receptor (V158 (high affinity) variant) and the NFAT response element driving the expression of firefly luciferase as effector cells. Antibody bioactivity in the ADCC MOA is quantified by luciferase produced after NFAT pathway activation; luciferase activity in effector cells is quantified by luminescence readout Figure 1 ). The signal is high, and the assay background is low.

[0247] Serially diluted Claudin 18.2 chimeric monoclonal antibodies or reference antibodies were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) with or without ADCC bioassay target cells (expressing Claudin 18.2) at 37°C for 6 hours. Bio-Glo® TM reagent was used to quantify luciferase activity (Table 2). The results show that these chimeric antibodies have very strong ADCC activity.

[0248] Table 2. EC50 of tested antibodies

[0249] Antibody EC50 (pM) 4F11E2 22.18 12F1F4 36.77 64G11B4 125.7 72C186A3 46.32 78E8G9G6 15.86 103F6D3 79.53 120B7B2 5.806 Reference Antibody 458.5

[0250] Example 9. Humanization of 4F11E2, 72C1B6A3 and 120B7B2 mouse mAbs

[0251] The variable region genes of mAbs 4F11E2, 72C1B6A3 and 120B7B2 were used to create humanized mAbs. In the first step of the process, the amino acid sequences of the VH and VL of the mAbs were compared to existing databases of human Ig gene sequences to find the best overall matching human germline Ig gene sequences.

[0252] The amino acid sequences of the humanized antibodies are listed in Table 3 below.

[0253] Table 3. Humanized sequences

[0254]

[0255]

[0256]

[0257] The humanized VH and VL genes were generated synthetically and then cloned into vectors containing human γ1 and human κ constant domains, respectively. The pairing of human VH and human VL produced the humanized antibodies (see Table 4).

[0258] Table 4. Humanized antibody 4F11E2 with VH and VL regions

[0259]

[0260] 72C1B6A3

[0261]

[0262] 120B7B2

[0263]

[0264] Example 10. Binding of humanized antibodies reactive with CLD18A2

[0265] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. 6 cells / ml of cells and Figure 4 The primary humanized antibodies shown were incubated on ice for 30 minutes with 3-fold serial dilutions from 100 nM to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated with the secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μl of FACS buffer, then transferred to BD Falcon 5 ml tubes and analyzed by FACS. The results showed that the humanized antibodies shown could bind to human CLD18A2 with high EC50, but not to CLD18A1 ( Figure 10 and 11 ).

[0266] Example 11. Binding of PTM (post-transcriptional translation) risk-reducing humanized antibodies reactive with CLD18A2

[0267] Post-translational modifications (PTMs) can cause problems during the development of therapeutic proteins, such as increased heterogeneity, reduced bioactivity, decreased stability, immunogenicity, fragmentation, and aggregation. The potential impact of a PTM depends on its location and, in some cases, solvent exposure. The CDRs of the sequences were analyzed for the following potential PTMs: asparagine deamidation, aspartate isomerization, free cysteine ​​thiol groups, N-glycosylation, oxidation, and fragmentation at potential hydrolysis sites.

[0268] To reduce the risk of PTMs in 4F11E2, 72C1B6A3, and 120B7B2, some relevant amino acids in VH and VL were mutated. Nine antibodies were then generated:

[0269]

[0270] *Amino acid positions (e.g., N55) are numbered according to the amino acid residues in the corresponding VH or VL amino acid sequence, not Kabat or Chothia.

[0271]

[0272]

[0273] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. 6 cells / ml of cells and Figure 4 The primary mutant humanized antibodies shown were incubated on ice for 30 minutes with 3-fold serial dilutions from 100 nM to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated with the secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μl of FACS buffer, then transferred to a BD Falcon 5 ml tube and analyzed by FACS. The results showed that the antibodies shown could bind to human CLD18A2 with a high EC50, but not to CLD18A1 ( Figure 12 and 13 ).

[0274] To evaluate the antigen binding potency of the de-risked variants of 4F11E2d (HC N55E / LC S32A) and 4F11E2d (H N55E N104Q / LC S32A), the variants were tested in a cell-based binding assay. Serial dilutions of anti-CLDN18.2 antibodies starting at 100 nM were combined with 10 5The cells were incubated on ice for 30 minutes. After washing with FACS buffer, the cells were incubated with APC-labeled secondary antibody for another 30 minutes on ice. The cells bound to the antibody were analyzed by FACS. The variants showed efficient binding to the cell surface tight junction protein 18.2 Figure 14 ).

[0275] Example 12. Antibody-dependent cellular cytotoxicity (ADCC) of PTM- de-risked humanized antibodies

[0276] Serially diluted tight junction protein 18.2 PTM-de-risked humanized antibodies or reference antibodies were induced to incubate with engineered Jurkat effector cells (ADCC bioassay effector cells) at 37°C for 6 hours with or without ADCC bioassay target cells (expressing tight junction protein 18.2). Bio-Glo TM Reagents were used to quantify luciferase activity (Table 5). The results showed that these humanized antibodies have very strong ADCC activity.

[0277] Table 5. ADCC

[0278] No. Test Antibody EC50 (pM) 1 4F11E2-HC N55Q-LC N31E 238.1 2 4F11E2-HC N55Q-LC S32A 413.9 3 4F11E2-HC N55E-LCS32A 148.1 4 72C1B6A3-HC WT-LC N31E 1651 5 72C1B6A3-HC WT-LCS32A 190.5 6 120B7B2-HC G57D&104A-LC-N96E&N31E 492.6 7 120B7B2-HC G57D&l04A-LC-S32A&G97A 113.9 Reference Antibody Reference Antibody 158.3

[0279] Example 13. Epitope mapping

[0280] All amino acids of the extracellular domain of tight junction protein 18.2 were individually mutated to A. Each mutated or wild-type tight junction protein 18.2 was transfected into Hek293 cells. The expression of tight junction protein 18.2 was evaluated by the indicated antibodies. The results are shown in Figure 15 (only amino acid residues with reduced binding after mutation are shown).

[0281] As shown in Figure 15 , 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 tested antibodies 4F11E2 (H4F), 72C186A3 (H72C1) and 120B7B2 (120) or the reference antibody 175D10 (IMAB362). W30 seems to form a first cluster of residues in the first extracellular domain of tight junction protein 18.2. N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62 and C63 seem to be a second cluster of residues within the same extracellular domain. On the other hand, Y169 and G172 are located in or close to the second extracellular domain.

[0282] Crystal structures of various tight junction proteins have been resolved. As shown inFigure 20 As shown in Figure 1 (adapted from Suzuki et al., Ann. NY Acad. Sci., 1397: 25-34), tight junction proteins contain four transmembrane segments, a short intracellular N-terminus, a large first extracellular loop (loop 1, or ECS1) containing a consensus W-LW-CC motif, a shorter second extracellular loop (loop 2, or ECS2), and an intracellular C-terminal tail. Loop 1 contains four β strands β1, β2, β3, and β4, and the loop contains one β strand, β5.

[0283] Mutations of W30, L49, and W50 to alanine may destabilize the conformation of loop 1. Mutations of C53 or C63 may disrupt the disulfide bond between β3 and β4. R80 may be important for maintaining interactions 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 (in the β3 to β4 loop), and Y169 and G172 (in β5), may provide an interface for the binding of the antibodies tested here.

[0284] Example 14. Comparison of humanized 4F11E2, 72C1B6A3, and 120B7B2 antibodies with the benchmark 175D10 Claudin 18.2 antibody

[0285] Cell-based binding

[0286] To compare the humanized anti-claudin 18.2 antibodies: 4F11E2 (HC N55E / LC S32A), 72C1B6A3 (HC WT / LC S32A), and 120B7B2 (HC G57D S104A / LC S32A G97A) with the benchmark antibody 175D10 (IMAB362), this example determined cell-based binding in cells expressing human claudin 18.2. CHO-K1 cells stably expressing human CLD18A2 were divided into high and low expressers based on the expression level of human CLDN18.2. Serial dilutions of anti-CLDN18.2 antibodies starting at 100 nM were added to 10 cells. 5 The cells were incubated on ice for 30 minutes. After washing with FACS buffer, the cells were incubated with APC-labeled secondary antibody for another 30 minutes on ice. Cells bound to the antibody were analyzed by FACS.

[0287] like Figure 16 As shown, 4F11E2, 72C1B6A3, and 120B7B2 all showed better binding ability than 175D10 in CHO-K1 cells that highly expressed or lowly expressed Claudin 18.2.

[0288] ADCC assay

[0289] To further compare the ADCC potency of humanized anti-claudin 18.2 antibodies: 4F11E2 (HC N55E / LC S32A), 72C1B6A3 (HC WT / LC S32A), and 120B7B2 (HC G57D S104A / LC S32A G97A) to the benchmark antibody 175D10 (IMAB362), a cell-based ADCC assay was performed in this example. Briefly, NK92 cells were co-cultured with 293 cells overexpressing claudin 18.2 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 to the 175D10 antibody.

[0290] For certain therapeutic antibodies, enhanced ADCC can increase the therapeutic window of antibody-based targeted therapy. Enhanced ADCC can be achieved by engineering the Fc region, such as with S239D / I332E mutations. In the NK92 cell-based ADCC assay, antibodies 4H11E2, 72C1B6A3, and 120B7B2 with S239D / I332E mutations in the Fc region had stronger NK92-mediated cell killing of 293 cells overexpressing claudin 18.2 than control antibody 175D10 with the same S239D / I332E mutations. Figure 18

[0291] Antibody-dependent cellular phagocytosis (ADCP)

[0292] The effect of anti-CLDN 18.2 mAbs on macrophage phagocytosis of tumor cells 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 mAbs. 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 collected and reseeded in 24-well culture dishes overnight as effector cells. NUG-C4 cells expressing CLDN18.2-eGFP were added to the MDMs at a ratio of 5 tumor cells per phagocyte in the presence of different concentrations of anti-CLDN18.2 mAbs. After 3 hours of incubation, unphagocytosed target cells were washed away with PBS, and the remaining phagocytic cells were collected and stained with the macrophage marker CD14 before flow cytometry analysis. The phagocytosis index was calculated by quantifying the percentage of GFP+ cells among CD14+ cells, normalized to the IgG control. ​

[0293] As Figure 19 shown, all C18.2 mAbs significantly enhanced phagocytosis of NUG-C4 cells in a concentration-dependent manner. In both wild-type IgGl and S239D / I332E mutated IgGl formats, 4F11E2, 72C1B6A3 and 120B7B2 antibodies exhibited stronger ADCP effect than the reference antibody 175D10.

[0294] In summary, this example demonstrates that the newly developed 4F11E2, 72C1B6A3 and 120B7B2 antibodies have stronger cell-based binding and ADCC / ADCP potency than the reference antibody 175D10. It is expected that the improved properties of these new antibodies can be attributed to the higher binding specificity of these antibodies compared to the reference antibody 175D10. For example, Figure 15 In particular, the interaction of 175D10 with claudin 18.2 was strong across the spectrum, including strong binding to D28, Q33, N38 and V43, followed by strong binding to G59 and V79. In contrast, the new antibodies 4F11E2, 72C1B6A3 and 120B7B2 had higher specificity to W30 in the first half of the first extracellular domain and to G48 to E56 in the second half of the first extracellular domain. The new antibodies also had slightly stronger binding to Y46, which is also located in the second half. Their binding to D28, Q33, N38, V43, G59 and V79 was considerably weaker, which can have contributed to the improved ADCC and ADCP of the new antibodies.

[0295] Example 15. Coupling of pHAb to Claudin 18.2 antibodies

[0296] An internalization assay based on pHAb reactive dye was used to determine the internalization of CLDN18.2 binding anti-claudin 18.2 antibodies. pHAb dye is a pH sensor dye that has very low fluorescence at pH > 7 and the fluorescence sharply increases when the pH of the solution becomes acidic. pHAb dye has an excitation maximum (Ex) at 532 nm and an emission maximum (Em) at 560 nm. pHAb dye conjugated antibodies 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 enters endosomal and lysosomal vesicles with acidic pH, resulting in fluorescence emission by the pHAb dye. This fluorescence can be detected using various techniques, including cell imaging, flow cytometry and fluorescence plate-based readers with appropriate filters.

[0297] Experimental protocol:

[0298] A. Antibody Production

[0299] Twenty-seven chimeric antibodies, three humanized antibodies, and one control IgG1 were produced by transient transfection of ExpiCHO cells and purified by protein A affinity chromatography.

[0300] B. Coupling of Antibodies to Magnetic Beads Using pHAb Thiol-Reactive Dyes

[0301] 1. Gently shake or use an upright drum mixer to evenly resuspend Protein A Magnetic Beads (LC00695). When preparing equal amounts of beads, keep the suspension homogeneous.

[0302] 2. Add 50 μl of magnetic bead slurry to a 1.5 ml microcentrifuge tube. Place the tube on a magnetic stand for 10 seconds.

[0303] 3. Remove and discard the storage buffer.

[0304] 4. Add 250 μl of PBS (pH 7.4). Mix and place the tube on a magnetic stand for 10 seconds. Remove and discard the buffer.

[0305] 5. Add 1.0 ml of sample containing 100 μg of antibody to the magnetic beads.

[0306] 6. Mix the sample at room temperature for 60 minutes. Keep the beads in suspension by stirring continuously.

[0307] 7. Place the tube on a magnetic stand for 10 seconds. Remove the supernatant.

[0308] 8. Add 250 μl of thiol coupling buffer (10 mM phosphate buffer, pH 7.0, containing 1 mM EDTA) and mix. Place the tube on a magnetic stand for 10 seconds. Remove and discard the buffer. Repeat this step for a total of two washes.

[0309] 9. Add 100 μl of thiol coupling buffer.

[0310] 10. Add DTT to a final concentration of 2.5 mM.

[0311] 11. Mix the sample at room temperature for 60 minutes. Keep the beads suspended by continuous stirring.

[0312] 12. Place the tube on the magnetic stand for 10 seconds and discard the buffer.

[0313] 13. Add 250 μl of thiol coupling buffer and mix. Place the tube on a magnetic stand for 10 seconds. Remove and discard the buffer. Repeat this step for a total of two washes.

[0314] 14. Add 100 μl of thiol coupling buffer.

[0315] 15. Spin the pHAb Thiol Reactive Dye (G9835) quickly (i.e., 5-10 seconds at 14,000 x g in a table top centrifuge) and dissolve to 10 mg / ml by adding 25 μΐ of a 1 : 1 DMSO-water mixture to 0.25 mg of dye. Mix by vortexing. The dye can take 1-3 minutes to completely dissolve. Make this solution just prior to use.

[0316] 16. Add 1.2 μΐ of pHAb Thiol Reactive Dye to 100 μg of antibody to make a 20 molar excess of dye.

[0317] 17. Mix for 60 minutes. Keep the magnetic beads in suspension by constant agitation.

[0318] 18. Place the tube on a magnetic stand for 10 seconds. Remove and discard the supernatant. 19. Add 250 μΐ of Thiol Coupling Buffer and mix. Place on magnetic stand for 10 seconds. Remove and discard the binding / washing buffer (PBS, pH 7.4).

[0319] 20. Repeat step 19 for a total of two washes.

[0320] 21. Add 100 μΐ of Elution Buffer (0.1 M Glycine, pH 3.0) to the magnetic beads.

[0321] 22. Mix for 5 minutes at room temperature.

[0322] 23. Place the tube on a magnetic stand for 10 seconds. Remove the eluted sample and transfer to a new microfuge tube containing 5 μΐ of Neutralization Buffer (1 M Tris-HCl, pH 9.0).

[0323] The antibody concentration and dye-to-antibody ratio (DAR) of the test antibodies are shown in Table 6.

[0324] Table 6. Dye-to-antibody ratio (DAR)

[0325]

[0326] Example 16. Screening of CLDN18.2 antibody internalization

[0327] Stably transfected human CLDN18.2 MKN45 cells were harvested with 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded at a density of 90 μΐ per well of 20K in 96 well black plates (Thermo Scientific #165305). The plates were incubated for 20-24 hours prior to treatment with pHAb labeled antibodies.

[0328] For internalization, pHAb-conjugated tight junction protein 18.2 antibodies were added to the cells at two concentrations (20 nM and 100 nM), mixed gently on a plate mixer for 1-2 minutes, and then incubated overnight to allow internalization (which can be detected after a few hours). Plates were read on a Tecan Infinity M1000 Pro fluorescence plate reader at Ex / Em: 532 nm / 560 nm. To achieve higher sensitivity, PBS was used instead of culture medium before reading the plates.

[0329] Results normalized by DAR are shown in Table 7. The tested antibodies had a higher internalization efficiency than the reference antibody IMAB362.

[0330] Table 7. Internalization results

[0331]

[0332] Example 17. EC50 of chimeric tight junction protein 18.2 antibodies for internalization on CHO-tight junction protein 18.2 cells

[0333] Stably transfected human CLDN18.2 CHO cells were harvested with 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded in 96-well black plates (Thermo Scientific #165305) at a density of 90 μΐ per well of 10K. Plates were incubated for 20-24 hours before treatment with pHAb-labeled antibodies.

[0334] For internalization, pHAb-conjugated chimeric tight junction protein 18.2 antibodies were added to the cells at different concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM, and 0.01 nM), mixed gently on a plate mixer for 1-2 minutes, and then incubated overnight to allow internalization (which can be detected after a few hours). Plates were read on a Tecan Infinity M1000 Pro fluorescence plate reader at Ex / Em: 532 nm / 560 nm. To achieve higher sensitivity, PBS was used instead of culture medium before reading the plates.

[0335] Results normalized by DAR are shown in Figure 21 . The tested antibodies again had a higher internalization efficiency than the reference antibody IMAB362.

[0336] Example 18. EC50 of humanized tight junction protein 18.2 antibodies for internalization on CHO-tight junction protein 18.2 cells

[0337] Stably transfected human CLDN18.2 CHO cells were harvested with 0.05% Trypsin / EDTA (Gibco, 25300-054) and seeded in 96-well black plates (Thermo Scientific #165305) at a density of 10K in 90 μΐ per well. Plates were incubated for 20-24 hours before treatment with pHAb-labeled antibodies.

[0338] For internalization, pHAb-conjugated humanized Claudin 18.2 antibodies were added to the cells at different concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM and 0.01 nM), mixed gently on a plate mixer for 1-2 minutes and then incubated overnight to allow internalization (which can be detected after a few hours). Plates were read on a Tecan Infinity M1000 Pro fluorescence plate reader at Ex / Em: 532 nm / 560 nm. To achieve higher sensitivity, plates were read with PBS instead of culture medium before reading.

[0339] Results normalized to DAR are shown in Figure 22 which shows that the tested antibodies have a higher internalization efficiency than the reference antibody IMAB362.

[0340] Example 19. EC50 of humanized Claudin 18.2 antibodies internalization on MKN45-Claudin 18.2 cells

[0341] Stably transfected human CLDN18.2 MKN45 cells were harvested with 0.05% Trypsin / EDTA (Gibco, 25300-054) and seeded in 96-well black plates (Thermo Scientific #165305) at a density of 10K in 90 μΐ per well. Plates were incubated for 20-24 hours before treatment with pHAb-labeled antibodies.

[0342] For internalization, pHAb-conjugated humanized Claudin 18.2 antibodies were added to the cells at different concentrations (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM and 0.01 nM), mixed gently on a plate mixer for 1-2 minutes and then incubated overnight to allow internalization (which can be detected after a few hours). Plates were read on a Tecan Infinity M1000 Pro fluorescence plate reader at Ex / Em: 532 nm / 560 nm. To achieve higher sensitivity, plates were read with PBS instead of culture medium before reading.

[0343] Results normalized to DAR are shown in Figure 23 which shows that the tested antibodies have a higher internalization efficiency than the reference antibody IMAB362.

[0344] Example 20. Antibody drug conjugates

[0345] Each antibody was mixed with approximately three-fold excess TCEP and stirred at 37°C for 2 hours. The reaction was quickly pipetted onto more than eight-fold excess VC-MMAE and incubated on ice for 1 hour, and a 20-fold excess of cysteine was added to the drug linker to stop the reaction. Finally, the ADC product was purified by elution through Sephadex G-25 equilibrated in PBS and concentrated by centrifugal ultrafiltration. The conjugates were filtered through a 0.2 pm filter under sterile conditions and stored at -80°C for analysis and testing. The drug to antibody ratio was analyzed by UV spectrophotometry, the monomer content by SEC-HPLC, and the free drug content by RP-HPLC. The DAR of vcMMAE conjugated antibodies is shown in Table 8.

[0346] Table 8. DAR of vcMMAE conjugated antibodies

[0347] Antibody DAR 4F11E2 HC N55E-LC S32A 3.76 72C1B6A3 HC WT-LC S32A 3.93 IMAB362 (Reference Antibody) 4.00 IgG1 (Control) 3.81

[0348] Example 21. Relative binding affinity and specificity of anti-CLDN18.2 naked antibodies and antibody drug conjugates

[0349] This example determined the relative binding affinity and specificity of anti-CLDN18.2 naked antibodies and antibody drug conjugates by flow cytometry using CLDN18.2 positive and negative cell lines.

[0350] Cells were harvested from exponentially growing cultures with 0.05% trypsin / EDTA (Gibco, 25300-054) and counted using a Neubauer counting chamber. Cells were centrifuged for 5 min at 1,500 rpm (468 x g), the supernatant was discarded, and the cells were resuspended in FACS buffer (PBS with 2% FCS (Gibco, 10270-106) for analysis of toxin-conjugated antibodies, PBS with 2% FCS and 2 mM EDTA for screening of CLDN18.2 reactive naked antibodies) to 2 x 10 6 cells / ml. 100 pl of the cell suspension per well (equivalent to 2 x 10 5Cells were harvested with 0.05% trypsin / EDTA (Gibco, 25300-054), resuspended in cell culture medium and 50 μl of the cell suspension was seeded in a 96-well cell culture plate with the corresponding number of cells per well. After 24 hours, the appropriate concentration of toxin-conjugated IMAB362 or control antibody diluted in 50 μl of culture medium was added and the cells were incubated for another 72 hours. The effect of the toxin-conjugated humanized Claudin 18.2 antibody on cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572).

[0351] It should be noted that the toxin-conjugated antibodies and the naked antibodies were applied at equal concentrations. The results are shown in Figure 24 .

[0352] Example 22. Cytotoxicity of the toxin-conjugated humanized Claudin 18.2 antibody with MMAE is more efficient than the toxin-conjugated IMAB362 in DAN-G, NUGC or SCG-7901 transfectants

[0353] The cells overexpressing human Claudin 18.2 (DAN-G, NUGC or SCG-7901 transfectants) were harvested with 0.05% trypsin / EDTA (Gibco, 25300-054), resuspended in cell culture medium and 50 μl of the cell suspension was seeded in a 96-well cell culture plate with the corresponding number of cells per well. After 24 hours, the appropriate concentration of toxin-conjugated IMAB362 or control antibody diluted in 50 μl of culture medium was added and the cells were incubated for another 72 hours. The effect of the toxin-conjugated humanized Claudin 18.2 antibody with MMAE on cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572). The CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) was used to determine the effect of the toxin-conjugated humanized Claudin 18.2 antibody with MMAE on cell viability.

[0354] The protocol for the use of the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572) was as follows:

[0355] 1. Prepare the opaque-walled multiwell plates with mammalian cells in culture medium, 100 μΐ per well for 96-well plates or 25 μΐ per well for 384-well plates. The multiwell plates must be compatible with the luminescence photometer used.

[0356] 2. Prepare control wells containing cell-free culture medium to obtain background luminescence values.

[0357] 3. Add test compounds to the experimental wells and incubate according to the culture protocol.

[0358] 5. Add an equal volume of reagent to the volume of cell culture medium present in each well (e.g. for 96-well plates, add 100 μΐ of reagent to 100 μΐ of cell-containing medium, or for 384-well plates, add 25 μΐ of reagent to 25 μΐ of cell-containing medium).

[0359] 6. Mix the contents on an orbital shaker for 2 minutes to induce cell lysis.

[0360] 7. Incubate the plates at room temperature for 10 minutes to stabilize the luminescence signal. Note: Non-uniform luminescence signals within a standard plate can be caused by temperature gradients, non-uniform cell seeding or edge effects of the multiwell plate.

[0361] 8. Record the luminescence.

[0362] The results of the test are shown in Figure 25A -C. BG2001-C and BG2001-D, when conjugated to MMAE, have a much increased cytotoxicity in all the cells tested compared to the reference IMAB362-NMAE conjugate. These results thus demonstrate the improved ability of the antibodies of the disclosure to internalize the conjugated drug.

[0363] Example 23. Cytotoxicity of claudin 18.2 humanized antibodies with MMAE is more efficient than IMAB362 with MMAE in SNU620, which endogenously expresses human claudin 18.2

[0364] SNU620 cells were resuspended in cell culture medium and 50 μΐ of cell suspension was seeded with the corresponding number of cells per well in a 96-well cell culture plate. After 24 hours, the appropriate concentration of toxin-conjugated antibodies (including the reference antibody IMAB362) diluted in 50 μΐ of culture medium was added and the cells were incubated for 72 hours. The effect of claudin 18.2 humanized antibodies with MMAE on cell viability was determined using a luminescent cell viability test (G7572) as described in the manufacturer's instructions. Figure 26 ​​As shown, both BG2001-C and BG2001-D were more effective in delivering conjugated MMAE to SNU620 cells compared to the reference antibody IMAB362, a lead anti-claudin 18.2 antibody in clinical development.

[0365] Example 24. In vivo efficacy of antibody drug conjugates

[0366] This example tests the efficacy of one of the antibody drug conjugates (ADCs) compared to the antibody alone (monoclonal antibody) in reducing tumor growth in nude mice implanted with human tumor cells.

[0367] 0.1 mL (5 × 10 5 Human patient-derived cells (mixed with Matrigel at a ratio of 1:1) were subcutaneously inoculated into the right back of each mouse. 3 At the same time, 30 mice were selected for treatment experiments.

[0368] 18 days after inoculation, 5 mice with tumors ranging in size from 330 to 520 mm were selected. 3 Mice within the range were treated with each treatment for three weeks (1 mg / kg, 3 mg / mk, 10 mg / kg or 20 mg / kg ADC once a week). For comparison, antibody only (mAb) treatment was 10 mg / kg (twice a week).

[0369] The results are shown in Figure 27 Both 10 mg / kg and 20 mg / kg of ADC completely inhibited tumor growth without reducing the body weight of the animals. Figure 28 The mean tumor reduction effect and the individual tumor reduction effect for each animal are shown. Thus, the tumor reduction effect of the ADC was much greater than that of the antibody alone.

[0370] The scope of the present disclosure is not limited by the specific embodiments described, which are intended to be single illustrations of individual aspects of the present disclosure, and any compositions or methods that are functionally equivalent are 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. Therefore, the present disclosure is intended to encompass modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0371] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An antibody-drug conjugate comprising a drug moiety covalently linked to an antibody or fragment thereof, wherein the antibody or fragment thereof has binding specificity for human claudin 18.2 (CLDN18.2) protein, wherein the antibody or fragment thereof is linked to 2 to 10 drug moieties, wherein the antibody or fragment thereof comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises light chain complementary determining regions CDRL1, CDRL2, and CDRL3, and the heavy chain variable region comprises heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein: The amino acid sequence of CDRL1 is shown in SEQ ID NO: 304; The amino acid sequence of CDRL2 is shown in SEQ ID NO: 229; The amino acid sequence of CDRL3 is shown in SEQ ID NO: 8; The amino acid sequence of the CDRH1 is shown in SEQ ID NO: 242; The amino acid sequence of the CDRH2 is shown in SEQ ID NO: 263; and The amino acid sequence of the CDRH3 is shown in SEQ ID NO:

289.

2. The antibody-drug conjugate according to claim 1, wherein the antibody or fragment thereof comprises a light chain variable region and a heavy chain variable region, the light chain variable region having the amino acid sequence of SEQ ID NO: 203, and the heavy chain variable region having the amino acid sequence of SEQ ID NO:

181.

3. The antibody drug conjugate of claim 1, wherein the drug moiety is a cytotoxic agent or a cytostatic agent.

4. The antibody drug conjugate of claim 3, wherein the drug moiety is maytansinoid or auristatin. The antibody drug conjugate of claim 4 , wherein the drug moiety comprises DM1 or DM4.

6. The antibody drug conjugate of claim 4, wherein the drug moiety comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

7. The antibody drug conjugate according to any one of claims 1 to 6, wherein the drug moiety is linked to the antibody or fragment thereof via a linker. The antibody drug conjugate according to claim 7 , wherein the linker is hydrolyzable under acidic conditions.

9. Use of the antibody-drug conjugate according to any one of claims 1 to 8 in the preparation of a drug for treating cancer; wherein the cancer is selected from the group consisting of pancreatic cancer, gastric cancer and esophageal cancer.

10. The use according to claim 9, wherein the cancer is gastric cancer.

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