Anti-claudin 18.2 antibodies and uses thereof

Abstract

Provided are antibodies or fragments thereof that have binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein and are also capable of binding to the M149L mutant. In contrast, such antibodies and fragments do not bind or weakly bind to claudin 18.1 (CLDN18.1).

Description

[0001] This application is a divisional application of the invention patent application with application date of May 20, 2019, application number 201980014037.0, and invention name “Anti-claudin 18.2 antibodies and their uses”. Background Art

[0002] Claudins are a family of proteins that are important components of tight cell junctions. They form a paracellular barrier that controls the flow of molecules between cells. These proteins have N-termini and C-termini in the cytoplasm. Different claudins are expressed in different tissues, and alterations in their function are associated with the development of cancer in these tissues. Claudin-1 is expressed in colon cancer, claudin-18 in gastric cancer, and 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 tissues is strictly limited to differentiated epithelial cells of the gastric mucosa but is absent in the gastric stem cell compartment. Claudin 18.2 is retained during malignant transformation and is expressed in most primary gastric cancers and their metastases. Ectopic activation of claudin 18.2 is also frequently found in pancreatic, esophageal, ovarian, and lung tumors. These data indicate that CLDN18.2 has a highly restricted expression pattern in normal tissues and is frequently ectopically activated in a variety of human cancers. Summary of the Invention

[0004] Antibodies against claudin 18.2 have been discovered that bind with high affinity to both wild-type claudin 18.2 and the common mutant M149L. Thus, these antibodies have the unique advantage of being able to target both wild-type and M149L mutant claudin 18.2 proteins. This advantage is significant, as a significant proportion of cancer patients harbor this common mutation. Furthermore, the antibodies disclosed herein do not bind to the other claudin 18 isoform, claudin 18.1, and are therefore highly selective.

[0005] According to one embodiment of the present disclosure, an antibody or fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein is provided, wherein the antibody or fragment also binds to the M149L mutant of the CLDN18.2 protein with an affinity of at least about 1% of the affinity for the wild-type CLDN18.2 protein, and wherein the antibody or fragment also binds to wild-type human claudin 18.1 (CLDN18.1) protein with an affinity greater than about 1% of the affinity for the wild-type CLDN18.2 protein. In some embodiments, the antibody or fragment thereof does not bind to the CLDN18.1 protein.

[0006] In one embodiment, an antibody or fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein is also provided, wherein the binding between the antibody or fragment thereof and wild-type CLDN18.2 protein involves at least one amino acid residue selected from the group consisting of Y46, G48, L49, W50, C53, V54, R55, E56, and S58 of wild-type CLDN18.2 protein; and at least one amino acid residue selected from the group consisting of Y169 and G172 of wild-type CLDN18.2 protein. In some embodiments, binding also involves W30 of the CLDN18.2 protein.

[0007] In some embodiments, binding involves amino acid residues comprising: W30 of the wild-type CLDN18.2 protein; two or more amino acid residues selected from the group consisting of G48, L49, W50, C53, and E56 of the wild-type CLDN18.2 protein; 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, binding involves amino acid residues comprising W30, G48, L49, W50, C53, E56, and Y169 of the wild-type CLDN18.2 protein.

[0008] Also provided are methods and uses for treating diseases and conditions. In one embodiment, a method of treating cancer in a patient in need of cancer treatment is provided, comprising administering to the patient an antibody or fragment of the antibody disclosed herein. In another embodiment, a method of treating cancer in a patient in need of cancer treatment is provided, comprising: (a) treating cells in vitro with an antibody or fragment of the antibody disclosed herein; and (b) administering the treated cells to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Flow cytometry showed that mouse sera from all mice after DNA immunization had high titer reactions with HEK293 cells transfected with CLD18A2, with CLD18A1 serving as a negative control.

[0010] Figure 2 It was shown that hybridoma supernatants could bind to HEK293 cells transfected with human CLD18A2 by cell ELISA or flow cytometry.

[0011] Figure 3 It was shown by flow cytometry that the purified murine antibody could bind to MKN45 cells transfected with human CLD18A2 with a high EC50 compared to a positive reference antibody.

[0012] Figure 4 It was shown by flow cytometry that the purified murine antibody can bind with high EC50 to SU620 cells endogenously expressing human CLD18A2 carrying the M149L mutation, whereas the reference antibody cannot.

[0013] Figure 5 It was shown by flow cytometry that purified murine antibodies can bind to HEK293 cells transfected with mouse CLD18A2 with a high EC50.

[0014] Figure 6 It was shown by flow cytometry that the purified murine antibody can bind to HEK293 cells transfected with macaque CLD18A2 with a high EC50.

[0015] Figure 7 It was shown by flow cytometry that the purified murine antibody can bind to HEK293 cells transfected with human CLD18A2 with a high EC50.

[0016] Figure 8 It was shown by flow cytometry that the chimeric antibody could bind to MKN45 cells transfected with human CLD18A2 with a high EC50 compared to a positive reference antibody.

[0017] Figure 9 It was shown by flow cytometry that the chimeric antibody was unable to bind to MKN45 cells transfected with human CLD18A1.

[0018] Figure 10 It was shown by flow cytometry that the humanized antibody could bind to MKN45 cells transfected with human CLD18A2 with a high EC50 compared to a positive reference antibody.

[0019] Figure 11 It was shown by flow cytometry that the humanized antibody was unable to bind to MKN45 cells transfected with human CLD18A1.

[0020] Figure 12 It was shown by flow cytometry that humanized antibodies with CDR mutations could bind to MKN45 cells transfected with human CLD18A2 with high EC50 compared to a positive reference antibody.

[0021] Figure 13 It was shown by flow cytometry that humanized antibodies with CDR mutations were unable to bind to MKN45 cells transfected with human CLD18A1.

[0022] Figure 14 The de-risked variants were shown to have efficient binding to cell surface CLD18A2.

[0023] Figure 15Certain mutations in CLD18A2 were shown to have a significant effect on the binding of the indicated antibodies to HEK293 cells transfected with these mutants, suggesting that these amino acid residues constitute at least part of the epitope.

[0024] Figure 16 It was shown that antibody 4F11E2, antibody 72C1B6A3, and antibody 120B7B2 all had superior binding in both claudin 18.2 high and low CHO-K1 cells compared to 175D10.

[0025] Figure 17 Shown are the results of effective ADCC tests using the 175D10 antibody as a reference, 4F11E2, 72C1B6A3, and 120B7B2.

[0026] Figure 18 It was shown that the S239D / I332E versions of 4F11E2, 72C1B6A3, and 120B7B2 outperformed the 175D10 counterpart in the ADCC assay.

[0027] Figure 19 It was shown that 4F11E2, 72C1B6A3 and 120B7B2 also had better ADCP effects than 175D10. DETAILED DESCRIPTION

[0028] definition

[0029] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" should be understood to mean one or more antibodies. Thus, the terms "a" (or "one"), "one or more" and "at least one" are used interchangeably herein.

[0030] As used herein, the term "polypeptide" is intended to encompass both singular "polypeptide" and plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any one chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms for referring to one or more chains of two or more amino acids, and the term "polypeptide" can replace any of these terms or be used interchangeably with any of these terms. The term "polypeptide" also refers to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, and are not necessarily translated from a specified nucleic acid sequence. Polypeptides can be produced in any way (including by chemical synthesis).

[0031] As used herein, the term "isolated" with respect to cells, nucleic acids such as DNA or RNA refers to molecules separated from other DNA or RNA present in the natural source of the macromolecule. As used herein, the term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology, or that are substantially free of chemical precursors or other chemicals when chemically synthesized. In addition, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and that do not exist under natural conditions. The term "isolated" is also used herein to refer to cells or polypeptides separated from other cellular proteins or tissues. An isolated polypeptide is meant to encompass purified and recombinant polypeptides.

[0032] As used herein, the term "recombinant" in reference to a polypeptide or polynucleotide means a form of the polypeptide or polynucleotide that does not occur in nature, a non-limiting example of which can be produced by combining polynucleotides or polypeptides that do not normally occur together.

[0033] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for the purpose 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. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "nonhomologous" sequence shares less than 40% identity, but preferably less than 25% identity, with one of the sequences of the present disclosure.

[0034] "Sequence identity" refers to the percentage of bases (or amino acids) that are identical when two sequences are compared, after alignment. Such alignment and homology or sequence identity percentage can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, the alignment is performed using default parameters. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Biologically equivalent polynucleotides are polynucleotides having the above specified percent homology and encoding polypeptides having the same or similar biological activity.

[0035] 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 with the nucleotide sequence of a nucleic acid or its complement. The homologue of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a certain degree of homology with its complement. On the one hand, the homologue of a nucleic acid can hybridize with a nucleic acid or its complement. Similarly, an "equivalent polypeptide" refers to a polypeptide having a certain degree of homology or sequence identity with 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, compared with a reference polypeptide or polynucleotide, an equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletions, substitutions and combinations thereof. In some aspects, an equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of a reference sequence.

[0036] Hybridization reactions can be carried out under conditions of varying "stringency." Typically, low stringency hybridization reactions are carried out at about 40°C in about 10×SSC or a solution of equal ionic strength / temperature. Moderate stringency hybridizations are typically carried out at about 50°C in about 6×SSC, and high stringency hybridization reactions are typically carried out at about 60°C in about 1×SSC. Hybridization reactions can also be carried out under "physiological conditions," which are well known to those skilled in the art. Non-limiting examples of physiological conditions are the temperature, ionic strength, pH, and Mg2+ levels typically found in cells. 2+ concentration.

[0037] Polynucleotide is made up of the specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Therefore, the term "polynucleotide sequence" is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be input into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches. The term "polymorphism" refers to the coexistence of more than one form of a gene or its part. A part of a gene with at least two different forms, that is, two different nucleotide sequences, is referred to as a "polymorphic region of a gene." A polymorphic region can be a single nucleotide whose identity is different in different alleles.

[0038] The terms "polynucleotide" and "oligonucleotide" can be used interchangeably and refer to a polymeric form of nucleotides of any length, i.e., deoxyribonucleotides or ribonucleotides or their analogs. A polynucleotide can have any three-dimensional structure and can perform any known or unknown function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polynucleotide assembly. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugating it to a labeling component. The term also refers to double-stranded molecules and single-stranded molecules. Unless otherwise stated or required, any embodiment of the disclosure that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0039] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA that produces a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0040] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule that has biological activity that binds to an antigen. Examples of such include, but are not limited to, a heavy or light chain complementary determining region (CDR) or ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0041] 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, etc. Regardless of the structure, an 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 acts like an antibody by binding to a specific antigen to form a complex.

[0042] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L In some aspects, the region is connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to improve flexibility and rich in serine or threonine to improve solubility, and V H The N-terminus of V L The C-terminus of the constant region is connected to the C-terminus of the constant region and vice versa. Despite the removal of the constant region and the introduction of the linker, the protein still retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0043] The term antibody includes various broad classes of polypeptides that can be biochemically distinguished. It will be understood by those skilled in the art that heavy chains are classified as γ, μ, α, δ or ε, with some subclasses (e.g., γ1-γ4). The nature of this chain determines that the "class" of the antibody is IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been well characterized and are known to confer functional specificity. In view of the present disclosure, modified forms of each of these classes and isotypes are easily distinguishable for those skilled in the art, and are therefore within the scope of the present disclosure. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically linked by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, beginning at the mouth of the "Y" and continuing through the variable region.

[0044] The 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, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (e.g., including anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present disclosure can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0045] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with either a kappa or lambda light chain. Typically, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide linkages or non-covalent linkages. 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 base of each chain.

[0046] The light chain and the heavy chain are both divided into regions with structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains (VK) of the light chain portion and the variable domains (VH) of the heavy chain portion determine antigen recognition and specificity. In contrast, the constant domains (CK) of the light chain and the constant domains (CH1, CH2 or CH3) of the heavy chain confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. By convention, the numbering of the constant region domains increases as they become more away from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region, while the C-terminal portion is the constant region; the CH3 and CK domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0047] As mentioned above, variable region allows antibody to selectively recognize and specifically bind epitopes on antigens. That is, the subset of the VK domain and VH domain or complementary determining region (CDR) of the antibody is combined to form a variable region that limits three-dimensional antigen binding site. This four-level antibody structure forms the antigen binding site at the end of each arm of Y. More specifically, the antigen binding site is limited by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each of the VH chain and the VK chain. In some cases, for example, some immunoglobulin molecules derived from camelid species or based on camelid immunoglobulin design, complete immunoglobulin molecules can be composed only of heavy chain, without light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0048] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-continuous sequences of amino acids that are specifically positioned to form the antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain (referred to as "framework" regions) show less intermolecular variability. The framework regions largely adopt a β-pleated sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-pleated sheet structure. Thus, the framework regions serve to form a scaffold that positions the CDRs in the correct orientation through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. One of ordinary skill in the art can readily identify, for any given heavy or light chain variable region, the amino acids comprising the CDRs and framework regions, respectively, since 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)).

[0049] In the case of two or more definitions of terms used and / or accepted in the art, unless clearly indicated to the contrary, the definitions of the terms used herein are intended to include all of these meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of heavy and light chain polypeptides. This specific region has been 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.MoI.Biol.196:901-917 (1987), the entire contents of which are incorporated herein by reference. When compared to each other, the CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues. However, the application of any one definition refers to the CDR of an antibody or its variants and is intended to be within the scope of the terms as defined and used herein. The appropriate amino acid residues for the CDRs defined by each reference in the references cited above are listed in the table below as a comparison. The exact number of residues encompassing a specific CDR will vary according to the sequence and size of the CDR. One skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of an antibody.

[0050] 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

[0051] Kabat et al. have also defined a numbering system for the variable domain sequences of any antibody. Those of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983).

[0052] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at approximately the 31st amino acid (i.e., approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at approximately the 15th residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at approximately the 33rd amino acid residue after the end of CDR-H2, includes 3-25 amino acids, and ends with the sequence WGXG, where X is any amino acid. CDR-L1 starts at approximately the 24th residue (i.e., after the cysteine ​​residue), includes approximately 10-17 residues, and ends at the next tryptophan residue. CDR-L2 starts at approximately the 16th residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue), comprises approximately 7-11 residues, and ends with the sequence F or WGXG, where X is any amino acid.

[0053] Antibodies disclosed herein can be from any animal origin, including birds and mammals. Preferably, the antibody is an antibody of a human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In another embodiment, the variable region can be derived from a condricthoid (e.g., from a shark).

[0054] 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 includes at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present disclosure may include: a polypeptide chain 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 containing a CH3 domain. In addition, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0055] The heavy chain constant region of the antibodies disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide can include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region can include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion can include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0056] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0057] A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0058] As previously mentioned, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0059] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule, for example, extending from approximately residue 244 to residue 360 ​​of an antibody using conventional numbering (residues 244-360, Kabat numbering system; and residues 231-340, 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 closely paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0060] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 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 different domains: an upper hinge domain, a middle hinge domain, and a lower hinge domain (Roux et al., J. Immunol 161:4083 (1998)).

[0061] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a sulfhydryl group that can form a disulfide bond with a second sulfhydryl group or bridged with a second sulfhydryl group. In most naturally occurring IgG molecules, the CH1 region and the CK region are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (positions 226 or 229, EU numbering system).

[0062] As used herein, the term "chimeric antibody" will be retained to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial, or modified according to 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), while the constant region is human.

[0063] As used herein, "percent humanization" 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 that number by the total number of amino acids and multiplying by 100.

[0064] The term "specific binding" or "specific for..." generally refers to the binding of an antibody to an epitope via its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an epitope when it binds to that epitope via 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 with which an antibody binds to an epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with a higher specificity than it does to a related epitope "D."

[0065] As used herein, the term "treat" or "treatment" refers to therapeutic treatment and preventive or prophylactic measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or illnesses, such as the progress of cancer. Useful or desired clinical outcomes include, but are not limited to, symptom relief, disease severity relief, stable disease state (i.e., no worsening), disease progression delay or slowing down, disease state improvement or alleviation, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an extension of survival compared to an untreated expected survival period. Those objects in need of treatment include those objects already suffering from the disease or illness and those objects prone to suffering from the disease or illness, or those objects wherein the disease or illness will be prevented.

[0066] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0067] As used herein, phrases such as "administered to a patient in need of treatment" or "a subject in need of treatment" include subjects (such as mammalian subjects) who would benefit from the administration of an antibody or composition of the present disclosure, e.g., for detection, diagnostic procedures, and / or therapy.

[0068] Anti-claudin 18.2 antibody

[0069] The present disclosure provides anti-claudin 18.2 antibodies that have high affinity for both wild-type claudin 18.2 and the common mutant M149L (SU620 cells that endogenously express this mutation). To the best of the inventors' knowledge, all currently known anti-claudin 18.2 proteins do not bind to this mutant. Therefore, 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 large proportion of cancer patients carry this common mutation. It is also worth noting that the antibodies of the present disclosure do not bind to another claudin 18 isoform, claudin 18.1 (or bind to claudin 18.1 with much lower affinity).

[0070] Experiments in the Examples show that certain amino acids in the first and second extracellular segments of the claudin 18.2 protein participate in or otherwise influence the binding of the presently disclosed antibodies to the protein. More specifically, three clusters of amino acid residues are shown representing the epitopes of the antibodies. The first cluster includes W30, which is in the first part of the first extracellular domain of the claudin 18.2 protein. The second cluster, including N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, and C63, is also located in the first extracellular domain. On the other hand, the third cluster, including Y169 and G172, is located at or near the second extracellular domain.

[0071] Even when using a clinical candidate as a reference, the antibodies and fragments of the present disclosure exhibit superior properties. 175D10 (IMAB362) is currently undergoing Phase III clinical trials for the treatment of gastric and gastroesophageal junction adenocarcinoma. Compared to 175D10, the antibodies and fragments of the present disclosure not only exhibited stronger binding activity but also exhibited higher ADCC and ADCP activity under various conditions.

[0072] The improved properties of these new antibodies and fragments are expected to be due to their higher binding specificity compared to those currently under development (such as 175D10). For example, the interaction of 175D10 with claudin 18.2 is Figure 15The spectrum of the new antibodies is strong, including strong binding to D28, Q33, N38 and V43 and to G59 and V79. In contrast, the new antibodies 4F11E2, 72C1B6A3 and 120B7B2 have higher specificity for W30 in the first half of the first extracellular domain and higher specificity for G48 to E56 in the second half of the first extracellular domain. The new antibodies also have slightly stronger binding to Y46 and S58, which are also in the second half. Their binding to D28, Q33, N38, V43, G59 and V79 is quite weak (or non-functional), which may contribute to the improved ADCC and ADCP of the new antibodies.

[0073] Human claudin 18.2 sequence

[0074]

[0075]

[0076] According to one embodiment of the present disclosure, an antibody or fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein is provided, wherein the antibody or fragment further binds to the M149L mutant of the CLDN18.2 protein. In some embodiments, the antibody or fragment does not bind to wild-type human claudin 18.1 (CLDN18.1) protein, or does not bind to CLDN18.1 with an affinity greater than about 1% of the affinity for wild-type CLDN18.2 protein.

[0077] The binding affinity of an antibody or fragment for a protein can be measured by a variety of methods known in the art. For example, it can be measured using a cell-free assay using isolated CLDN18.1 or CLDN18.2 protein. However, it is preferred that the measurement be performed using CLDN18.1 or CLDN18.2 protein on the surface of a cell that simulates the actual binding environment. Such binding assays are fully exemplified in the experimental examples.

[0078] 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 wild-type CLDN18.2 protein.

[0079] In some embodiments, the antibody or fragment thereof does not bind to human CLDN18.1. In some embodiments, the antibody or fragment thereof binds much weaker to human CLDN18.1 than to CLDN18.2, for example, no more than 10%, 5%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001%, but is not limited thereto.

[0080] As described above, the antibodies and fragments thereof disclosed herein bind to the claudin 18.2 protein at a different epitope than known antibodies (see Figure 4 , at least the reference antibody interacts with M149, while the currently disclosed antibodies do not interact with M149). Therefore, in one embodiment, an antibody or fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein is provided, wherein the binding between the antibody or fragment and the wild-type CLDN18.2 protein involves amino acid residues comprising at least one amino acid residue selected from the group consisting of Y46, G48, L49, W50, C53, V54, R55, E56, and S58 of the wild-type CLDN18.2 protein; 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, binding also involves one or more W30 residues of the CLDN18.2 protein.

[0081] 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.

[0082] In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from G48, L49, W50, C53, V54, R55, and E56. In some embodiments, the antibody or fragment thereof binds to at least three amino acid residues selected from G48, L49, W50, C53, V54, R55, and E56. In some embodiments, the antibody or fragment thereof binds to at least four amino acid residues selected from G48, L49, W50, C53, V54, R55, and E56. In some embodiments, the antibody or fragment thereof binds to at least five amino acid residues selected from G48, L49, W50, C53, V54, R55, and E56.

[0083] In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from Y46, G48, L49, W50, C53, V54, R55, E56, and S58. In some embodiments, the antibody or fragment thereof binds to at least three amino acid residues selected from Y46, G48, L49, W50, C53, V54, R55, E56, and S58. In some embodiments, the antibody or fragment thereof binds to at least four amino acid residues selected from Y46, G48, L49, W50, C53, V54, R55, E56, and S58. In some embodiments, the antibody or fragment thereof binds to at least five amino acid residues selected from Y46, G48, L49, W50, C53, V54, R55, E56, and S58.

[0084] In some embodiments, the antibody or fragment thereof binds to at least Y169 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to at least G172 of CLDN18.2. In some embodiments, the antibody or fragment thereof binds to at least two amino acid residues selected from Y169 and G172 of CLDN18.2.

[0085] In some embodiments, binding involves an amino acid residue comprising W30 in the wild-type CLDN18.2 protein; two, three, four, five or more amino acid residues selected from the group consisting of G48, L49, W50, C53 and E56 in the wild-type CLDN18.2 protein; 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, binding involves an amino acid residue comprising W30, G48, L49, W50, C53, E56 and Y169 of the wild-type CLDN18.2 protein.

[0086] In some embodiments, the binding between the antibody or fragment thereof and the wild-type CLDN18.2 protein involves at least Y46 and / or S58. In some embodiments, the binding between the antibody or fragment thereof and the wild-type CLDN18.2 protein does not involve N38 or V43. In some embodiments, the binding between the antibody or fragment thereof and the wild-type CLDN18.2 protein does not involve D28, Q33, N38, V43, G59, and V79, or has weaker binding to one, two, three, four, or all of D28, Q33, N38, V43, G59, and V79.

[0087] Compared to other amino acids such as G48, L49, W50, C53, V54, R55, and E56, the binding to these amino acids on CLDN18.2 is weaker. In some embodiments, the comparison is to binding to 175D10 at the same amino acid. For example, the antibodies or fragments of the present disclosure bind weaker to at least one, two, three, four, five, or all of D28, Q33, N38, V43, G59, and V79 than to 175D10 (IMGT / 2D Structure-DB Card Number: 10473).

[0088] In some embodiments, the antibody or fragment thereof does not bind to M149L of the CLDN18.2 protein. In some embodiments, the antibody or fragment thereof binds to the M149L mutant of the CLDN18.2 protein.

[0089] According to one embodiment of the present disclosure, an antibody or a fragment thereof is provided, comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain and the light chain variable domain have CDR regions as shown in the CDR combinations of Table A and Table A'.

[0090] Table A. CDR combinations of tested antibodies

[0091]

[0092]

[0093]

[0094] Table A'. CDR combinations of the tested antibodies (Kabat numbering)

[0095]

[0096]

[0097]

[0098] Table B.120B7B2 CDR

[0099]

[0100] Table C.72C1B6A3 CDR

[0101]

[0102] Table D. CDRs of 4F11E2

[0103]

[0104]

[0105] Table B'. CDRs of 120B7B2 (Kabat numbering)

[0106]

[0107] Table C'.72C1B6A3 CDRs (Kabat numbering)

[0108]

[0109]

[0110] Table D'.4F11E2 CDR (Kabat numbering)

[0111]

[0112] Antibodies containing these CDR regions, whether mouse, humanized, or chimeric, all 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 properties or to reduce the likelihood of post-translational modifications (PTMs). Such modified CDRs can be referred to as affinity matured or de-risked CDRs.

[0113] Non-limiting examples of risk-reducing CDRs are provided in the third column of Tables B-D and B'-D'. Affinity-matured CDRs may include CDRs with one, two, or three amino acid additions, deletions, and / or substitutions. In some embodiments, the substitutions may be conservative substitutions.

[0114] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family. In another embodiment, an amino acid string can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0115] Non-limiting examples of conservative amino acid substitutions are provided in the table below, 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] Therefore, in one embodiment, an antibody or fragment thereof having binding specificity to a 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 complementary determining regions CDRL1, CDRL2 and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and wherein CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 are selected from combinations 1-33 of Table A or Table A', or selected from each of combinations 1-33 in which one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 each comprise one, two or three amino acid additions, deletions, conservative amino acid substitutions or a combination thereof.

[0122] In some embodiments, an anti-CLDN18.2 antibody or fragment is provided, which comprises CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3, each of which is selected from Table A or Table B-Table D. For example, 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 complementary determining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein CDRL1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NOs: 31-38, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 1, SEQ ID NO: 12, and SEQ ID NOs: 31-38 by addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRL2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NOs: 39-41, or comprises an amino acid sequence derived from any one of SEQ ID NOs: 2, SEQ ID NO: 7, and SEQ ID NOs: 39-41 by addition, deletion, or amino acid substitution; and CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 12, and SEQ ID NOs: 31-38. NO:3, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:19 and SEQ ID NO:42-58, or an amino acid sequence derived from any one of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:19 and SEQ ID NO:42-58 by addition, deletion or amino acid substitution of one, two or three amino acids; CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14 and SEQ ID NO:59-76, or an amino acid sequence derived from any one of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14 and SEQ ID NO:59-76 by addition, deletion or amino acid substitution of one, two or three amino acids;CDRH2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NOs: 77-95, or an amino acid sequence derived from any one of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NOs: 77-95 by addition, deletion, or amino acid substitution of one, two, or three amino acids; and CDRH3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, and SEQ ID NOs: 96-116, or an amino acid sequence derived from any one of SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, and SEQ ID NOs: 96-116 by addition, deletion, or amino acid substitution of one, two, or three amino acids.

[0123] In some embodiments, CDRL1 comprises an amino acid sequence selected from the group of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 17-18, SEQ ID NO: 23-24, and SEQ ID NO: 31-38; CDRL2 comprises an amino acid sequence selected from the group of SEQ ID NO: 2, SEQ ID NO: 7, and SEQ ID NO: 39-41; CDRL3 comprises an amino acid sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19-20, and SEQ ID NO: 42-58; CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 59-76; CDRH2 comprises an amino acid sequence selected from the group of SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 25-26, and SEQ ID NO: 77-95; and CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NO: 6, SEQ ID NO: 7 The amino acid sequences of the group consisting of SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NOs: 27-29 and SEQ ID NOs: 96-116.

[0124] In some embodiments, an anti-CLDN18.2 antibody or fragment is provided, which comprises CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3, each of which is selected from Table A' or Table B'-Table D'. For example, an antibody or fragment thereof having binding specificity to a 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 complementary determining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein CDRL1 comprises an amino acid sequence selected from the group consisting 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; CDRL2 comprises an amino acid sequence selected from the group consisting 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; and CDRL3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, and SEQ ID NO: 20. NO: 42-58, or comprises an amino acid sequence derived from any one of SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, and SEQ ID NO: 42-58 by the addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NO: 234-254, or comprises an amino acid sequence derived from any one of SEQ ID NO: 234-254 by the addition, deletion, or amino acid substitution of one, two, or three amino acids; CDRH2 comprises an amino acid sequence selected from the group of SEQ ID NO: 255-280, or comprises an amino acid sequence derived from any one of SEQ ID NO: 255-280 by the addition, deletion, or amino acid substitution of one, two, or three amino acids; and CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NO: 281-303, or comprises an amino acid sequence derived from any one of SEQ ID NO: 281-303 by the addition, deletion, or amino acid substitution of one, two, or three amino acids.

[0125] In some embodiments, CDRL1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 208-226, SEQ ID NOs: 304-305, and SEQ ID NOs: 308-309; CDRL2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 227-233; CDRL3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NOs: 42-58; CDRH1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 234-254; CDRH2 comprises an amino acid sequence selected from the group of SEQ ID NOs: 255-280, SEQ ID NO: 306, SEQ ID NO: 310, and SEQ ID NO: 311; and CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 281-303, SEQ ID NO: 307, and SEQ ID NOs: 312-314.

[0126] Antibody 120B7B2 has been shown to be a potent inhibitor of claudin 18.2. Its CDR sequences are provided in Table B, along with 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 complementary determining regions CDRL1, CDRL2 and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and wherein CDRL1 comprises the amino acid sequence of QSLLNSGNQKNY (SEQ ID NO: 1), QSLLNAGNQKNY (SEQ ID NO: 17) or QSLLESGNQKNY (SEQ ID NO: 18), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 1, SEQ ID NO: 17, or SEQ ID NO: 18; CDRL2 comprises the amino acid sequence of WAS (SEQ ID NO: 2), or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 2; CDRL3 comprises CQNGYYFPFT (SEQ ID NO: 3), QNAYYFPFT (SEQ ID NO: 4), or QQNGYYFPFT (SEQ ID NO: 5). NO: 19) or QEGYYFPFT (SEQ ID NO: 20), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 3, SEQ ID NO: 19 or SEQ ID NO: 20; CDRH1 comprises an amino acid sequence of GYTFTGYI (SEQ ID NO: 4), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 4; CDRH2 comprises an amino acid sequence of INPYNDGT (SEQ ID NO: 5) or INPYNDDT (SEQ ID NO: 21), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 5 or SEQ ID NO: 21; and CDRH3 comprises an amino acid sequence of ARAYFGNSFAY (SEQ ID NO: 6) or ARAYFGNAFAY (SEQ ID NO: 22), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 6 or SEQ ID NO: 22.

[0127] It is interesting to note (see Table A) that CDRs from different antibodies have great homology. It is then considered that each corresponding 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 replaced by another amino acid present in the corresponding CDR from a different antibody. Therefore, in some embodiments, the following table summarizes some possible substitutions based on sequence alignment.

[0128] Table G1. Example substitutions of amino acid residues in SEQ ID NO: 1

[0129] Kabat numbering residue replace 27 Q M 29 L V 30 L F 30B S G 30E Q L or R 30F K R 31 N K or S

[0130] Table G2. Example substitutions of amino acid residues in SEQ ID NO: 2

[0131] Kabat numbering residue replace 50 W G or R 51 A S 52 S F

[0132] Table G3. Example substitutions of amino acid residues in SEQ ID NO: 3

[0133] Kabat numbering residue replace 89 Q H 91 G A, D, N, S, or V 92 Y F or L 93 Y F, I, or S 94 F Y 96 F C, L, W, or Y 97 T A

[0134] Table G4. Example substitutions of amino acid residues in SEQ ID NO: 4

[0135]

[0136] Table G5. Example substitutions of amino acid residues in SEQ ID NO: 5

[0137]

[0138] Table G6. Example substitutions of amino acid residues in SEQ ID NO: 6

[0139]

[0140] In some embodiments of the antibody or fragment thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:17, or SEQ ID NO:18, CDRL2 comprises the amino acid sequence of SEQ ID NO:2, CDRL3 comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:19, or SEQ ID NO:20, CDRH1 comprises the amino acid sequence of SEQ ID NO:4, CDRH2 comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:21, and CDRH3 comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:22.

[0141] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 141, SEQ ID NOs: 192-195, and SEQ ID NOs: 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 NO: 141, SEQ ID NOs: 192-195, and SEQ ID NOs: 206-207.

[0142] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NOs: 188-191, and SEQ ID NO: 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 NO: 171, SEQ ID NOs: 188-191, and SEQ ID NO: 205.

[0143] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO:17, CDRL2 comprises the amino acid sequence of SEQ ID NO:2, CDRL3 comprises the amino acid sequence of SEQ ID NO:19, CDRH1 comprises the amino acid sequence of SEQ ID NO:4, CDRH2 comprises the amino acid sequence of SEQ ID NO:21, and CDRH3 comprises the amino acid sequence of SEQ ID NO:22.

[0144] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 206 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 205, or a biological equivalent thereof.

[0145] Table A' - Table D' provide the CDR sequences according to Kabat numbering. It will be apparent to the skilled person that the substitutions disclosed above can be readily translated into CDRs according to a different numbering scheme.

[0146] In some embodiments, antibodies or fragments thereof that have binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein are provided. In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising light chain complementary determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, SEQ ID NO: 304, or SEQ ID NO: 305, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 210, SEQ ID NO: 304, or SEQ ID NO: 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 19, or SEQ ID NO: 20, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 3, SEQ ID NO: 19, or SEQ ID NO: 20, and CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 254. NO: 253, CDRH2 comprises the amino acid sequence of SEQ ID NO: 278 or SEQ ID NO: 306, or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 278 or SEQ ID NO: 306, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 303 or SEQ ID NO: 307, or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 303 or SEQ ID NO: 307.

[0147] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO:210, SEQ ID NO:304 or SEQ ID NO:305, CDRL2 comprises the amino acid sequence of SEQ ID NO:227, CDRL3 comprises the amino acid sequence of SEQ ID NO:3, SEQ ID NO:19 or SEQ ID NO:20, CDRH1 comprises the amino acid sequence of SEQ ID NO:253, CDRH2 comprises the amino acid sequence of SEQ ID NO:278 or SEQ ID NO:306, and CDRH3 comprises the amino acid sequence of SEQ ID NO:303 or SEQ ID NO:307.

[0148] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 141, SEQ ID NOs: 192-195, and SEQ ID NOs: 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 NO: 141, SEQ ID NOs: 192-195, and SEQ ID NOs: 206-207.

[0149] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 171, SEQ ID NOs: 188-191, and SEQ ID NO: 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 NO: 171, SEQ ID NOs: 188-191, and SEQ ID NO: 205.

[0150] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 19, CDRH1 comprises the amino acid sequence of SEQ ID NO: 253, CDRH2 comprises the amino acid sequence of SEQ ID NO: 306, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 307. Non-limiting examples of antibodies or fragments comprise 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.

[0151] Likewise, 72C1B6A3 has been shown to be a good antibody. Thus, 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 complementary determining regions CDRL1, CDRL2 and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and wherein CDRL1 comprises the amino acid sequence of QSLLNSGNQKNY (SEQ ID NO: 1), QSLLNAGNQKNY (SEQ ID NO: 17) or QSLLESGNQKNY (SEQ ID NO: 18), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 1, SEQ ID NO: 17 or SEQ ID NO: 18, CDRL2 comprises the amino acid sequence of RAS (SEQ ID NO: 7), or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 7, and CDRL3 comprises the amino acid sequence of QNDYIYPYT (SEQ ID NO: 8), or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 8. NO:8, CDRH1 comprises the amino acid sequence of GYTFTTYP (SEQ ID NO:9), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:9, CDRH2 comprises the amino acid sequence of FHPYNDDT (SEQ ID NO:10), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:10, and CDRH3 comprises the amino acid sequence of ARRAYGYPYAMDY (SEQ ID NO:11), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:11.

[0152] Likewise, each specific amino acid in a CDR can be substituted with another amino acid present in the corresponding CDR from a different antibody.Thus, in some embodiments, the following table summarizes some possible substitutions based on sequence alignment.

[0153] Table H1. Example substitutions of amino acid residues in SEQ ID NO: 1

[0154] Kabat numbering residue replace 27 Q M 29 L V 30 L F 30B S G 30E Q L or R 30F K R 31 N K or S

[0155] Table H2. Example substitutions of amino acid residues in SEQ ID NO: 7

[0156] Kabat numbering residue replace 50 R G or W 51 A S 52 S F

[0157] Table H3. Example substitutions of amino acid residues in SEQ ID NO: 8

[0158] Kabat numbering residue replace 89 Q H 91 D A, G, N, S, or V 92 Y F or L 93 I F, Y, or S 94 Y F 96 Y C, L, W, or F 97 T A

[0159] Table H4. Example substitutions of amino acid residues in SEQ ID NO: 9

[0160]

[0161]

[0162] Table H5. Example substitutions of amino acid residues in SEQ ID NO: 10

[0163]

[0164] Table H6. Example substitutions of amino acid residues in SEQ ID NO: 11

[0165]

[0166] In some embodiments of the antibody or fragment thereof of 23, CDRL1 comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:17 or SEQ ID NO:18, CDRL2 comprises the amino acid sequence of SEQ ID NO:7, CDRL3 comprises the amino acid sequence of SEQ ID NO:8, CDRH1 comprises the amino acid sequence of SEQ ID NO:9, CDRH2 comprises the amino acid sequence of SEQ ID NO:10, and CDRH3 comprises the amino acid sequence of SEQ ID NO:11.

[0167] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NOs: 185-187, and SEQ ID NOs: 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 NO: 124, SEQ ID NOs: 185-187, and SEQ ID NOs: 203-204.

[0168] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 153 and SEQ ID NOs: 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 NO: 153 and SEQ ID NOs: 181-184.

[0169] In some embodiments, in the antibody or fragment thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO: 17, CDRL2 comprises the amino acid sequence of SEQ ID NO: 7, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, CDRH1 comprises the amino acid sequence of SEQ ID NO: 9, CDRH2 comprises the amino acid sequence of SEQ ID NO: 10, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 11.

[0170] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 203 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 181, or their corresponding biological equivalents.

[0171] Table A' - Table D' provide the CDR sequences according to Kabat numbering. It will be apparent to the skilled person that the substitutions disclosed above can be readily translated into CDRs according to a different numbering scheme.

[0172] In some embodiments, 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 complementary determining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 210, SEQ ID NO: 304, or SEQ ID NO: 305, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 210, SEQ ID NO: 304, or SEQ ID NO: 305, CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 229, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 8, and CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: NO: 242, CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 263, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 289, or an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 289.

[0173] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO:210, SEQ ID NO:304, or SEQ ID NO:305, CDRL2 comprises the amino acid sequence of SEQ ID NO:229, CDRL3 comprises the amino acid sequence of SEQ ID NO:8, CDRH1 comprises the amino acid sequence of SEQ ID NO:242, CDRH2 comprises the amino acid sequence of SEQ ID NO:263, and CDRH3 comprises the amino acid sequence of SEQ ID NO:289.

[0174] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 124, SEQ ID NOs: 185-187, and SEQ ID NOs: 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 NO: 124, SEQ ID NOs: 185-187, and SEQ ID NOs: 203-204.

[0175] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 153 and SEQ ID NOs: 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 NO: 153 and SEQ ID NOs: 181-184.

[0176] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 304, CDRL2 comprises the amino acid sequence of SEQ ID NO: 229, CDRL3 comprises the amino acid sequence of SEQ ID NO: 8, CDRH1 comprises the amino acid sequence of SEQ ID NO: 242, CDRH2 comprises the amino acid sequence of SEQ ID NO: 263, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 289. Non-limiting example antibodies or fragments thereof comprise 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.

[0177] Furthermore, 4F11E2 has been shown to be a good antibody. Thus, 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 complementary determining regions CDRL1, CDRL2 and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2 and CDRH3, and wherein CDRL1 comprises the amino acid sequence of QSLLNSGNRKNY (SEQ ID NO: 12), QSLLESGNRKNY (SEQ ID NO: 23) or QSLLNAGNRKNY (SEQ ID NO: 24), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO: 12, SEQ ID NO: 23 or SEQ ID NO: 24, CDRL2 comprises the amino acid sequence of WAS (SEQ ID NO: 2), or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 2, and CDRL3 comprises the amino acid sequence of QNAYSYPFT (SEQ ID NO: 13), or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 14. NO:13, CDRH1 comprises the amino acid sequence of GFTFSTFG (SEQ ID NO:14), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:14, CDRH2 comprises the amino acid sequence of ITSGNSPI (SEQ ID NO:15), ITSGQSPI (SEQ ID NO:25), or ITSGESPI (SEQ ID NO:26), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:15, SEQ ID NO:25, or SEQ ID NO:26, and CDRH3 comprises the amino acid sequence of ARSSYYGNSMDY (SEQ ID NO:16), ARSSYYGQSMDY (SEQ ID NO:27), ARSSYYGESMDYDY (SEQ ID NO:28), or ARSSYYGNAMDY (SEQ ID NO:29), or comprises an amino acid sequence having one, two or three amino acid substitutions from SEQ ID NO:16, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO: An amino acid sequence having one, two or three amino acid substitutions of NO:29.

[0178] Likewise, each specific amino acid in a CDR can be substituted with another amino acid present in the corresponding CDR from a different antibody.Thus, in some embodiments, the following table summarizes some possible substitutions based on sequence alignment.

[0179] Table 11. Example substitutions of amino acid residues in SEQ ID NO: 12

[0180] Kabat numbering residue replace 27 Q M 29 L V 30 L F 30B S G 30E R L or Q 30F K R 31 N K or S

[0181] Table 12. Example substitutions of amino acid residues in SEQ ID NO: 2

[0182] Kabat numbering residue replace 50 W G or R 51 A S 52 S F

[0183] Table 13. Example substitutions of amino acid residues in SEQ ID NO: 13

[0184]

[0185]

[0186] Table 14. Example substitutions of amino acid residues in SEQ ID NO: 14

[0187]

[0188] Table 15. Example substitutions of amino acid residues in SEQ ID NO: 15

[0189]

[0190] Table 16. Example substitutions of amino acid residues in SEQ ID NO: 16

[0191]

[0192]

[0193] In some embodiments, in the antibody or fragment thereof, CDRL1 comprises the amino acid sequence of SEQ ID NO:12, SEQ ID NO:23 or SEQ ID NO:24, CDRL2 comprises the amino acid sequence of SEQ ID NO:2, CDRL3 comprises the amino acid sequence of SEQ ID NO:13, CDRH1 comprises the amino acid sequence of SEQ ID NO:14, CDRH2 comprises the amino acid sequence of SEQ ID NO:15, SEQ ID NO:25 or SEQ ID NO:26, and CDRH3 comprises the amino acid sequence of SEQ ID NO:16, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29.

[0194] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NOs: 178-180, and SEQ ID NOs: 201-202, or a biological equivalent thereof, such as a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NOs: 178-180, and SEQ ID NOs: 201-202.

[0195] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NOs: 175-177, and SEQ ID NOs: 196-200, or a biological equivalent thereof, such as a peptide having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NOs: 175-177, and SEQ ID NOs: 196-200.

[0196] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO:24, CDRL2 comprises the amino acid sequence of SEQ ID NO:2, CDRL3 comprises the amino acid sequence of SEQ ID NO:13, CDRH1 comprises the amino acid sequence of SEQ ID NO:14, CDRH2 comprises the amino acid sequence of SEQ ID NO:26, and CDRH3 comprises the amino acid sequence of SEQ ID NO:16.

[0197] In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 202 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 197, or their corresponding biological equivalents.

[0198] Table A' - Table D' provide the CDR sequences according to Kabat numbering. It will be apparent to the skilled person that the substitutions disclosed above can be readily translated into CDRs according to a different numbering scheme.

[0199] Some embodiments of the present disclosure provide antibodies or fragments thereof that have 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 complementary determining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein CDRL1 comprises the amino acid sequence of SEQ ID NO: 216, SEQ ID NO: 308, or SEQ ID NO: 309, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 216, SEQ ID NO: 308, or SEQ ID NO: 309, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, or comprises an amino acid sequence having one or two amino acid substitutions from SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, or comprises an amino acid sequence having one, two, or three amino acid substitutions from SEQ ID NO: 13, and CDRH1 comprises the amino acid sequence of SEQ ID NO: 216, SEQ ID NO: 308, or SEQ ID NO: 309. NO: 246, or an amino acid sequence comprising one, two or three amino acid substitutions from SEQ ID NO: 246, CDRH2 comprises the amino acid sequence of SEQ ID NO: 268, SEQ ID NO: 310 or SEQ ID NO: 311, or an amino acid sequence comprising one, two or three amino acid substitutions from SEQ ID NO: 268, SEQ ID NO: 310 or SEQ ID NO: 311, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 294, SEQ ID NO: 312, SEQ ID NO: 313 or SEQ ID NO: 314, or an amino acid sequence comprising one, two or three amino acid substitutions from SEQ ID NO: 294, SEQ ID NO: 312, SEQ ID NO: 313 or SEQ ID NO: 314.

[0200] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO:216, SEQ ID NO:308, or SEQ ID NO:309, CDRL2 comprises the amino acid sequence of SEQ ID NO:227, CDRL3 comprises the amino acid sequence of SEQ ID NO:13, CDRH1 comprises the amino acid sequence of SEQ ID NO:246, CDRH2 comprises the amino acid sequence of SEQ ID NO:268, SEQ ID NO:310, or SEQ ID NO:311, and CDRH3 comprises the amino acid sequence of SEQ ID NO:294, SEQ ID NO:312, SEQ ID NO:313, or SEQ ID NO:314.

[0201] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NOs: 178-180, and SEQ ID NOs: 201-202, or a biological equivalent, such as a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 129, SEQ ID NOs: 178-180, and SEQ ID NOs: 201-202.

[0202] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NOs: 175-177, and SEQ ID NOs: 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 NO: 159, SEQ ID NOs: 175-177, and SEQ ID NOs: 196-200.

[0203] In some embodiments, CDRL1 comprises the amino acid sequence of SEQ ID NO: 309, CDRL2 comprises the amino acid sequence of SEQ ID NO: 227, CDRL3 comprises the amino acid sequence of SEQ ID NO: 13, CDRH1 comprises the amino acid sequence of SEQ ID NO: 246, CDRH2 comprises the amino acid sequence of SEQ ID NO: 311, and CDRH3 comprises the amino acid sequence of SEQ ID NO: 294. Non-limiting examples of antibodies or fragments comprise 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.

[0204] In some embodiments, the antibody is a humanized antibody. As shown in Example 9, a humanized antibody can include one or more back mutations to a 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 back mutations.

[0205] In some embodiments, the anti-claudin 18.2 antibodies of the present disclosure include a VL of any one of SEQ ID NOs: 117-144, SEQ ID NOs: 178-180, SEQ ID NOs: 185-187, SEQ ID NOs: 192-195, SEQ ID NOs: 201-202, SEQ ID NOs: 203-204, or SEQ ID NOs: 206-207 and a VH of any one of SEQ ID NOs: 145-174, SEQ ID NOs: 175-177, SEQ ID NOs: 181-184, SEQ ID NOs: 188-191, SEQ ID NOs: 196-200, or SEQ ID NO: 205, or their corresponding bioequivalents. Bioequivalents of VH or VL are sequences comprising the specified amino acids while having an overall sequence identity of 80%, 85%, 90%, 95%, 98%, or 99%. Thus, a biological equivalent of SEQ ID NO: 145 may be a VH having overall 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 145 but retaining the CDRs, and optionally retaining one or more or all back mutations.

[0206] It will also be understood by those of ordinary skill in the art that an antibody as disclosed herein can be modified so that its amino acid sequence differs from the naturally occurring binding polypeptide from which it is derived. For example, a polypeptide or amino acid sequence derived from a given protein can be similar, such as having a certain percentage of identity to the starting sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the starting sequence.

[0207] In certain embodiments, the antibodies comprise an amino acid sequence or one or more moieties that are not typically bound to an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the present disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0208] The antibodies, variants thereof, or derivatives thereof disclosed herein include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibody can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody can contain one or more non-classical amino acids.

[0209] In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

[0210] The antibodies may be conjugated or fused to therapeutic agents, which may include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic or diagnostic agents, cytotoxic agents, which may be "drugs" or "toxins," ultrasound enhancing agents, non-radioactive labels, combinations thereof, and other such agents known in the art.

[0211] Antibodies can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide can then be determined by detecting the presence of luminescence generated during the chemical reaction process. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0212] The antibodies may also be made with fluorescent metals such as those in the lanthanide series. 152Eu or other metals can be detectably labeled. These metals can be attached to antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known, see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), 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)).

[0213] Bifunctional molecules

[0214] As tumor antigen targeting molecules, an antibody or antigen-binding fragment specific for claudin 18.2 can be combined with a second fragment specific for immune cells to generate a bispecific antibody.

[0215] In some embodiments, the immune cell is selected from the group consisting of T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils, and mast cells.

[0216] In some embodiments, the second specificity is for CD3, CD47, PD1, PD-L1, LAG3, TIM3, CTLA4, VISTA, CSFR1, A2AR, CD73, CD39, CD40, CEA, HER2, CMET, 4-1BB, OX40, SIRPA CD16, CD28, ICOS, CTLA4, BTLA, TIGIT, HVEM, CD27, VEGFR, or VEGF.

[0217] Also provided are different forms of bispecific antibodies. In some embodiments, the anti-claudin 18.2 fragment and the second fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment.

[0218] Also provided are bifunctional molecules that include not only antibodies or antigen binding fragments. As tumor antigen targeting molecules, antibodies or antigen binding fragments specific for claudin 18.2, such as those described herein, can optionally be combined with immune cytokines or ligands via a peptide linker. The immune cytokines or ligands connected 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, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT, and GITRL. This bifunctional molecule can combine immune checkpoint blockade with local immune regulation at the tumor site.

[0219] Polynucleotide encoding antibody and method for preparing antibody

[0220] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants thereof, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variants thereof, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy chain variable region and light chain variable region of an antigen-binding polypeptide, variants thereof, or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0221] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable region and constant region of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and described herein. For example, fully human antibodies against specific antigens can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigenic attack, but whose endogenous loci have lost their ability. Exemplary techniques for preparing such antibodies are described in U.S. Patents 6,150,584, 6,458,592, and 6,420,140, ​​the entire contents of which are incorporated herein by reference.

[0222] Treatment

[0223] As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0224] The present disclosure also relates to antibody-based therapies, which involve administering the antibodies of the present disclosure to patients, such as animals, mammals, and humans, to treat one or more disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0225] The antibodies disclosed herein can also be used to treat or inhibit cancer. As described above, 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.

[0226] Thus, in some embodiments, methods for treating cancer in a patient in need of such treatment are provided. In one embodiment, the methods entail administering to the patient an effective amount of an antibody of the present disclosure. In some embodiments, at least one of the cancer cells in the patient (e.g., a stromal cell) overexpresses claudin 18.2.

[0227] Also provided in the present disclosure is cell therapy, such as chimeric antigen receptor (CAR) T cell therapy. Suitable cells can be used to contact the suitable cells with the anti-claudin 18.2 antibodies disclosed herein (or alternatively engineered to express the anti-claudin 18.2 antibodies disclosed herein). After such contact or engineering, the cells can then be introduced into cancer patients in need of treatment. Cancer patients can suffer from any type of cancer disclosed herein. Cells (e.g., T cells) can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0228] In some embodiments, the cells are isolated from a cancer patient. In some embodiments, the cells are provided by a donor or a cell bank. When the cells are isolated from a cancer patient, undesirable immune responses can be minimized.

[0229] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the cancer is one or more of gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer.

[0230] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related diseases such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemias)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), 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, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

[0231] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or variant or derivative thereof used; the patient's age, weight, general health, sex, and diet; the time of administration; the rate of excretion; the drug combination; and the severity of the specific disease being treated. The judgment of the medical care provider regarding these factors is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles known in the art.

[0232] The method of administration of antibody, variant includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.Antigen-binding polypeptide or composition can be applied by any convenient approach, for example, by infusion or bolus injection, by epithelial or mucocutaneous lining (for example, oral mucosa, rectum and intestinal mucosa etc.) absorption, and can be applied together with other bioactivators.Therefore, the pharmaceutical composition containing antigen-binding polypeptide of the present disclosure can be oral, per rectum, parenteral, intracisternal, intravaginal, intraperitoneal, local (such as by powder, ointment, drops or transdermal patch), oral (bucally) or as oral or nasal spray administration.

[0233] As used herein, the term "parenteral" refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

[0234] Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir such as an Omayor reservoir. Pulmonary administration can also be performed, for example, by using an inhaler or nebulizer and formulated with an aerosolizing agent.

[0235] It may 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, for example, but not limited to, by local infusion during surgery, local administration, such as in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, wherein the implant is a porous, non-porous, or gel-like material, including a membrane (e.g., a silicone rubber membrane) or fiber. Preferably, when administering proteins (including antibodies) of the present disclosure, care must be taken to use materials that the protein does not absorb.

[0236] The amount of the antibodies disclosed herein that are effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be employed to help identify the optimal dosage range. The precise dosage used in the formulation also depends on the route of administration and the severity of the disease, disorder, or condition, and should be determined based on the practitioner's judgment and the circumstances of each patient. The effective dose can be inferred from a dose-response curve derived from an in vitro or animal model test system.

[0237] As a general suggestion, the dosage of the antigen-binding polypeptides of the present invention 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 and 20 mg / kg of the patient's body weight, or between 1 mg / kg and 10 mg / kg of the patient's body weight. Typically, due to the immune response to exogenous polypeptides, the half-life of human antibodies in the human body is longer than that of antibodies from other species. Therefore, lower doses of human antibodies and lower frequency of administration are generally possible. In addition, the dosage and frequency of administration of the antibodies of the present invention can be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibodies through modification (e.g., lipidation).

[0238] In another embodiment, the compositions of the present disclosure are administered in combination with cytokines. 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-α.

[0239] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or preventive regimens, such as, for example, radiation therapy.

[0240] Combination therapies and antibody-drug conjugates

[0241] Also provided are combination therapies comprising the use of one or more anti-claudin 18.2 antibodies or fragments thereof disclosed herein and a second anti-cancer agent (chemotherapeutic agent). In some embodiments, the chemotherapeutic agent is conjugated to the antibody or fragment.

[0242] Chemotherapeutic agents can be classified according to their mechanism of action into, for example, the following groups:

[0243] -Antimetabolites / anticancerosine agents, such as the pyrimidine analogs floxuridine, capecitabine, and cytarabine;

[0244] -Purine analogs, folic acid antagonists, and related inhibitors;

[0245] - Antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine) and microtubule-modulating agents such as taxanes (paclitaxel, docetaxel), vinblastine, nocodazole, epothilones, vinorelbine and epipodophyllotoxins (etoposide, teniposide);

[0246] DNA-damaging agents, such as actinomycin, acridine, busulfan, carboplatin, chlorambucil, cisplatin, and cyclophosphamide Actinomycin D, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, dichloromethane, mitomycin, mitoxantrone, nitrosoureas, procarbazine, paclitaxel, taxotere, teniposide, etoposide, and triethylenethiophosphoramide;

[0247] -Antibiotics, such as dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin;

[0248] - enzymes, such as L-asparaginase, which metabolize L-asparagine systemically and deprive cells that do not have the ability to synthesize their own asparagine;

[0249] - Antiplatelet agents;

[0250] - Antiproliferative / antimiotic alkylating agents, such as the nitrogen mustards cyclophosphamide and analogs (melphalan, chlorambucil, altretamine, and thiotepa), alkylnitrosoureas (carmustine) and analogs, streptozotocin, and triazene (dacarbazine);

[0251] -Antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate);

[0252] - Platinum coordination complexes (cisplatin, oxiloplatinim, and carboplatin), procarbazine, hydroxyurea, mitotane, and aminoglutethimide;

[0253] - Hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, and nilutamide), and aromatase inhibitors (letrozole and anastrozole);

[0254] -Anticoagulants, such as heparin, synthetic heparin salts, and other thrombin inhibitors;

[0255] -Fibrinolytics, such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel;

[0256] - anti-migration agents;

[0257] - Antisecretory agents (breveldin);

[0258] -The immunosuppressants tacrolimus, sirolimus, azathioprine, and mycophenolate mofetil;

[0259] - compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors);

[0260] -Angiotensin receptor blockers, nitric oxide donors;

[0261] - antisense oligonucleotides;

[0262] - Antibodies, such as trastuzumab and rituximab;

[0263] -Cell cycle inhibitors and differentiation inducers such as retinoic acid;

[0264] - inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, actinomycin D, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan, and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone);

[0265] - Growth factor signaling kinase inhibitors;

[0266] - Dysfunction-inducing agents;

[0267] - toxins, such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase activators; and

[0268] -chromatin.

[0269] Other examples of chemotherapeutic agents include:

[0270] -Alkylating agents, such as thiotepa and cyclophosphamide

[0271] -Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan;

[0272] -aziridines, such as benzodepa, carbaquinone, metodepa, and uredepa;

[0273] -emylerumine and memylamelamine, including alfretamine, triemylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemylolomelamine;

[0274] - annonaceous lactones, especially bratacin and bratacinone;

[0275] - Camptothecins, including the synthetic analog topotecan;

[0276] -Bryostatin;

[0277] - callystatin;

[0278] -CC-1065, including its synthetic analogs of adolesine, carzelesine, and biszelesine;

[0279] - nostocs, especially nostoc-1 and nostoc-8;

[0280] - Aplysia toxin;

[0281] -duocarmycins, including the synthetic analogs KW-2189 and CBI-TMI;

[0282] - Acanthopanax;

[0283] -Hydroxycandin;

[0284] -Coleocanthal;

[0285] -Spongostatin;

[0286] - Nitrogen mustards, such as chlorambucil, naphthyl mustard, cyclophosphamide, estramustine, ifosfamide, dichloromethane, dichloromethane hydrochloride, melphalan, nembicevic, phenylephrine, prednimustine, trofosamide, and uracil mustard;

[0287] -Nitrosoureas, such as carmustine, chlorozotocin, foremustine, lomustine, nimustine, and ranimustine;

[0288] - Antibiotics, such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma II and calicheamicin phi II), danemycins including danemycin A, bisphosphonates such as clodronate, esperamicins, neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calabikine, carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, dimethoate, daunorubicin ... torubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrro-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mesilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, oliveromycins, peplomycin, porphyromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, neocarzinostatin and daurubicin,

[0289] -Antimetabolites, such as methotrexate and 5-fluorouracil (5-FU);

[0290] -Folic acid analogs, such as demopterin, methotrexate, pteropterin, and trimetrexate;

[0291] -Purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, and thioguanine;

[0292] -pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxefuridine, enocitabine, and floxuridine;

[0293] -Androgens, such as caprotestosterone, drostanolone propionate, cyclopentolone, melastosterone, and testolactone;

[0294] -Antiadreners, such as aminoglutethimide, mitotane, and trilostane;

[0295] -Folic acid supplements, such as folic acid;

[0296] - trichothecenes, in particular T-2 toxin, vilakulin A, myclobutanil A and anguidine;

[0297] -Taxoids, such as paclitaxel and docetaxel

[0298] -Platinum analogs, such as cisplatin and carboplatin;

[0299] -acetylglucosyl ester, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, hestrabucil, bisantrene, edatrexate, fosfamide, colcemid, serquinone, elformthine, elformthine, epothilone, epothilone, etoglucose, gallium nitrate, hydroxyurea, lentinan, leucovorin, lonidamine, maytansines such as maytansine and ansamitocin, mitoxantrone, mitoxantrone, monoperitone, nitracrine, pentostatin, methambucil, pyridamole Rubicin, losoxantrone, fluoropyrimidine, folinic acid, podophyllic acid, 2-ethylhydrazide, procarbazine, polysaccharide-K (PSK), razoxane, rhizoxin, sizolan, spirogermanamine, tricorotriemylamine, triazoline, 2,2',2"-tricUorotriemylamine, urethane, vindesine, dacarbazine, mannomustine, dibromomannitol, dibromodulcitol, pipobroman, garcitocin, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, chlorambucil, gemcitabine 6-thioguanine, mercaptopurine, methotrexate, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vancristine, vinorelbine Mitoxantrone, teniposide, edatrexate, daunorubicin, aminopterin, xeoloda, ibandronate, CPT-11, the topoisomerase inhibitor RFS2000, difluoromethylornithine (DFMO), retinoids such as retinoic acid, capecitabine, FOLFIRI (fluorouracil, leucovorin, and irinotecan); and

[0300] - Any pharmaceutically acceptable salt, acid or derivative of the above.

[0301] Also included within the definition of "chemotherapeutic agent" are antihormonal agents such as antiestrogens and selective estrogen receptor modulators (SERMs), aromatase inhibitors, antiandrogens, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing used to modulate or inhibit the effects of hormones on tumors.

[0302] Examples of antiestrogens and SERMs include, for example, tamoxifen (including NOLVADEX TM ), raloxifene, droloxifene, 4-hydroxytamoxifen, troloxifene, raloxifene, LY117018, onapristone, and toremifene

[0303] Aromatase inhibitors regulate estrogen production in the adrenal glands. Examples include 4(5)-imidazole, aminoglutethimide, megestrol acetate Exemestane, formestane, fadrozole, vorozole Letrozole and anastrozole

[0304] Examples of antiandrogens include flutamide, nilutamide, bicalutamide, leuprohde, and goserelin.

[0305] Examples of chemotherapeutic agents also include anti-angiogenic agents, including but not limited to retinoic acid and its derivatives, 2-methoxyestradiol, Suramin, squalamine, tissue inhibitor of metalloproteinase-1, tissue inhibitor of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, cartilage-derived inhibitors, albumin-bound paclitaxel (nab-paclitaxel), platelet factor 4, protamine sulfate (herring protamine), sulfated chitin derivatives (prepared from crab shells), sulfated polysaccharide peptidoglycan complex (sp-pg), staurosporine, matrix metabolism regulators including proline analogs ((1-azetidine-2-carboxylic acid (LACA))), hydroxyproline, d,1-3,4-dehydroproline, thioproline , α,α'-bipyridyl, β-fumaric acid aminopropionitrile, 4-propyl-5-(4-acetylpyridyl)-2(3h)-oxazolone, methotrexate, mitoxantrone, heparin, interferon, 2-macroglobulin serum, chicken metalloproteinase-3 inhibitor (ChIMP-3), chymotrypsin inhibitor, β-cyclodextrin tetradecyl sulfate, eponemycin, fumagillin, gold sodium thiomalate, d-penicillamine, β-1-anticollagenase-serum, α-2-antiplasmin, bisantrene, lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthronilic acid (chloroanthronilic acid) Anti-angiogenic agents include, but are not limited to, disodium thalidomide, angiostatic steroids, carboxyaminoimidazoles, and metalloproteinase inhibitors such as BB-94. Other anti-angiogenic agents include antibodies, preferably monoclonal antibodies, to the angiogenic growth factors β-FGF, α-FGF, FGF-5, VEGF isoforms, VEGF-C, HGF / SF, and Ang-1 / Ang-2.

[0306] Examples of chemotherapeutic agents also include anti-fibrotic agents, including but not limited to compounds such as β-aminopropionitrile (BAPN), and compounds disclosed in U.S. Pat. No. 4,965,288 (Palfreyman et al.), which relates to lysyl oxidase inhibitors and their use in treating diseases and conditions associated with abnormal collagen deposition, and U.S. Pat. No. 4,997,854 (Kagan et al.), which relates to compounds that inhibit LOX for the treatment of various pathological fibrotic diseases, both of which are incorporated herein by reference. Other exemplary inhibitors are described in U.S. Pat. No. 4,943,593 (Palfreyman et al.), directed to compounds such as 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine; U.S. Pat. Nos. 5,021,456 (Palfreyman et al.), 5,059,714 (Palfreyman et al.), 5,120,764 (Mccarthy et al.), 5,182,297 (Palfreyman et al.), 5,252,608 (Palfreyman et al.), and U.S. Publication No. 2004 / 0248871 (Farjanel et al.), directed to 2-(1-naphthyloxymethyl)-3-fluoroallylamine, which are incorporated herein by reference.

[0307] Exemplary anti-fibrotic agents also include primary amines that react with the carbonyl group of the active site of lysyl oxidase, more particularly those that produce a product stabilized by resonance after binding to the carbonyl group, such as the following primary amines: emylenemamine, hydrazine, phenylhydrazine and its derivatives; semicarbazide and urea derivatives; aminonitriles such as BAPN or 2-nitroethylamine; unsaturated or saturated halamines such as 2-bromoethylamine, 2-chloroethylamine, 2-trifluoroethylamine, 3-bromopropylamine and p-halobenzylamine; and selenohomocysteine ​​lactone.

[0308] Other anti-fibrotic agents are copper chelators that penetrate or do not penetrate cells.Exemplary compounds include indirect inhibitors, which prevent the aldehyde derivatives produced by the oxidative deamination of lysyl and hydroxylysyl residues by lysyl oxidase.Example includes thiolamine (thiolamine), particularly D-penicillamine, and analogs such as 2-amino-5-mercapto-5-methylhexanoic acid, D-2-amino-3-methyl-3-((2-acetamidoethyl) disulfide) butyric acid, p-2-amino-3-methyl-3-((2-aminoethyl) disulfide) butyric acid, 4-((p-1-dimethyl-2-amino-2-carboxylethyl) disulfide) butane sodium sulfonate, 2-acetamidoethyl-2-acetamidoethanethiol sulfate and 4-mercaptobutanesulfinate sodium trihydrate.

[0309] Examples of chemotherapeutic agents also include immunotherapeutic agents, including but not limited to therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include simtuzumab, abavotumab, adecatumumab, aftuzumab, alemtuzumab, atumomab, amatuximab, anatumomab, acetimumab, bavituximab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cilivatuzumab, konam ... Conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, demuximab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, ertumaxomab, farletuzumab, ficlatuzumab, figi tumumab), flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab tiuxetan, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab monoclonal antibody, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab,onartuzumab, oportuzumab, ogovuzumab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, protumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumumab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, vorsetuzumab, zalutumumab, CC49, and 3F8. Rituximab can be used to treat indolent B-cell cancers, including marginal zone lymphoma, WM, CLL, and small lymphocytic lymphoma. Combinations of rituximab and chemotherapy agents are particularly effective.

[0310] Exemplary therapeutic antibodies can be further labeled or combined with radioactive isotope particles such as indium 111, yttrium 90, or iodine 131.

[0311] In one embodiment, the additional therapeutic agent is a nitrogen mustard alkylating agent. Non-limiting examples of nitrogen mustard alkylating agents include chlorambucil.

[0312] In one embodiment, the compounds and compositions described herein can be used or combined with one or more additional therapeutic agents. The one or more therapeutic agents include, but are not limited to, inhibitors of Abl, activated CDC kinases (ACK), adenosine A2B receptors (A2B), apoptosis signal-regulating kinases (ASK), aurora kinases, Bruton's tyrosine kinases (BTK), BET bromodomains (BRD) such as BRD4, c-Kit, c-Met, CDK-activating kinases (CAK), calmodulin-dependent protein kinases (CaMK), cyclin-dependent kinases (CDK), casein kinases (CK), discoidin domain receptors (DDR), epidermal growth factor receptor (EGFR), focal adhesion kinases (FAK), Flt-3, FYN, glycogen synthase kinases (GSK), HCK, histone deacetylase (HDAC), IKK such as IKKβε, isocitrate dehydrogenases (IDH) such as IDH1, Janus kinases (JAK), KDR, lymphoid tissue remodeling agents (LMTs), mitochondria sclerotin (MS / MTs), mitochondria sclerotin (MTS ... cell-specific protein tyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein (LOXL), LYN, matrix metalloproteinase (MMP), MEK, mitogen-activated protein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derived growth factor (PDGF), phosphorylase kinase (PK), polo-like kinase (PLK), phosphatidylinositol 3-kinase (PI3K), protein kinase (PK) such as protein kinase A, B and / or C, PYK, spleen tyrosine kinase (SYK), serine / threonine kinase TPL2, serine / threonine kinase STK, signal transduction and transcription (STAT), SRC, serine / threonine protein kinase (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascular endothelial growth factor receptor (VEGFR), YES, or any combination thereof.

[0313] ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitors include, but are not limited to, those described in WO 2011 / 008709 (Gilead Sciences) and WO 2013 / 112741 (Gilead Sciences).

[0314] Examples of BTK inhibitors include, but are not limited to, ibrutinib, HM71224, ONO-4059, and CC-292.

[0315] DDR inhibitors include inhibitors of DDR1 and / or inhibitors of DDR2. Examples of DDR inhibitors include, but are not limited to, those disclosed in WO 2014 / 047624 (Gilead Sciences), US 2009 / 0142345 (Takeda Pharmaceutical), US 2011 / 0287011 (Oncomed Pharmaceuticals), WO 2013 / 027802 (Chugai Pharmaceutical), and WO 2013 / 034933 (Imperial Innovations).

[0316] Examples of HDAC inhibitors include, but are not limited to, pracinostat and panobinostat.

[0317] JAK inhibitors inhibit JAK1, JAK2 and / or JAK3. Examples of JAK inhibitors include, but are not limited to, filgotinib, ruxolitinib, fedratinib, tofacitinib, baricitinib, lestaurtinib, pacritinib, XL019, AZD1480, INCB039110, LY2784544, BMS911543, and NS018.

[0318] LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4 and / or LOXL5. Examples of LOXL inhibitors include, but are not limited to, the antibodies described in WO 2009 / 017833 (Arresto Biosciences).

[0319] Examples of LOXL2 inhibitors include, but are not limited to, the antibodies described in WO 2009 / 017833 (Arresto Biosciences), WO 2009 / 035791 (Arresto Biosciences), and WO 2011 / 097513 (Gilead Biologics).

[0320] MMP inhibitors include inhibitors of MMP 1 to MMP 10. Examples of MMP9 inhibitors include, but are not limited to, marimastat (BB-2516), cipemastat (Ro 32-3555), and those described in WO 2012 / 027721 (Gilead Biologics).

[0321] PI3K inhibitors include inhibitors of PI3Kγ, PI3Kδ, PI3Kβ, PI3Kα and / or pan-PI3K. Examples of PI3K inhibitors include, but are not limited to, wortmannin, BKM120, CH5132799, XL756, and GDC-0980.

[0322] Examples of PI3Kγ inhibitors include, but are not limited to, ZSTK474, AS252424, LY294002, and TG100115.

[0323] Examples of PI3Kδ inhibitors include, but are not limited to, PI3K II, TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443, and compounds described in WO 2005 / 113556 (ICOS), WO 2013 / 052699 (Gilead Calistoga), WO 2013 / 116562 (Gilead Calistoga), WO 2014 / 100765 (Gilead Calistoga), WO 2014 / 100767 (Gilead Calistoga), and WO 2014 / 201409 (Gilead Sciences).

[0324] Examples of PI3Kβ inhibitors include, but are not limited to, GSK2636771, BAY 10824391, and TGX221.

[0325] Examples of PI3Kα inhibitors include, but are not limited to, buparix, BAY 80-6946, BYL719, PX-866, RG7604, MLN1117, WX-037, AEZA-129, and PA799.

[0326] Examples of pan-PI3K inhibitors include, but are not limited to, LY294002, BEZ235, XL147 (SAR245408), and GDC-0941.

[0327] Examples of SYK inhibitors include, but are not limited to, tamatinib (R406), fostamatinib (R788), PRT062607, BAY-61-3606, NVP-QAB 205AA, R112, R343, and inhibitors described in U.S. Pat. No. 8,450,321 (Gilead Connecticut).

[0328] TKIs can target epidermal growth factor receptor (EGFR) and receptors for fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIs targeting EGFR include, but are not limited to, gefitinib and erlotinib. Sunitinib is a non-limiting example of a TKI targeting FGF, PDGF, and VEGF receptors.

[0329] Diagnostic methods

[0330] Overexpression of claudin 18.2 is observed in certain tumor samples, and patients with cells that overexpress claudin 18.2 may respond to treatment with the anti-claudin 18.2 antibodies of the present disclosure. Thus, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

[0331] Preferably, the sample comprising cells can be obtained from a patient, which can be a cancer patient or a patient for whom diagnosis is desired. The cells are cells of tumor tissue or tumor mass, a blood sample, a urine sample, or any sample from a patient. After optional pretreatment of the sample, the sample can be incubated with the antibody of the present disclosure under conditions that allow the antibody to interact with the claudin 18.2 protein that may be present in the sample. The presence of claudin 18.2 protein in the sample can be detected using methods such as ELISA using anti-claudin 18.2 antibodies.

[0332] The presence of claudin 18.2 protein in a sample (optionally in terms of amount or concentration) can be used to diagnose cancer, as an indicator that the patient is suitable for antibody treatment, or as an indicator that the patient has (or has not) responded to cancer treatment. For prognostic methods, detection can be performed once, twice, or more times at certain stages after the start of cancer treatment to indicate the progress of treatment.

[0333] Composition

[0334] The present disclosure also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0335] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and particularly humans. Additionally, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0336] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle used together with the therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid such as water and oil, and the pharmaceutical carrier includes pharmaceutical carriers from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and aqueous glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier or pH buffer such as acetate, citrate or phosphate. Antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for regulating tonicity such as sodium chloride or dextrose are also contemplated. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can be formulated as suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will comprise a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, and an appropriate amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0337] In an embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site. Typically, the composition is provided in unit dosage form alone or mixed together, for example, as a lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a sachet, to indicate the amount of the active agent. When the composition is administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, sterile water for injection or saline in an ampoule can be provided so that the composition can be mixed before administration.

[0338] The compounds of the present disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0339] Example

[0340] Example 1: Generation of murine monoclonal antibodies against human claudin 18 isoform 2 (CLD 18A2)

[0341] a. Immunity:

[0342] Balb / c and C57 / BL6 mice were immunized with a eukaryotic expression vector encoding a fragment of human claudin 18.2 (CLD 18A2). On days 1 and 10, 50 μg of plasmid DNA was injected into the quadriceps muscle (intramuscularly, im). On day 20, the presence of antibodies against human CLD 18A2 in mouse sera was monitored by flow cytometry using HEK 293 cells transiently transfected with nucleic acid encoding human CLD 18A2. 5 × 10 mice transiently transfected with nucleic acid encoding human CLD 18A2 were injected intraperitoneally. 7 HEK293 cells, mice with detectable immune responses were boosted three days before and two days after fusion ( Figure 1 ).

[0343] b. Generation of Hybridomas Producing Human Monoclonal Antibodies Against CLD18A2:

[0344] Mouse splenocytes were isolated and fused to a mouse myeloma cell line using PEG according to standard protocols. The resulting hybridomas were then screened for the production of immunoglobulins specific for CLD18A2 by cell-based ELISA using HEK293 cells transfected with nucleic acid encoding human CLD18.

[0345] 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). 4The cells were plated / well in a flat-bottom microtiter plate and then incubated for approximately two weeks in selective medium containing 10% fetal bovine 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× HAT (Sigma, CRL H0262). After 10 to 14 days, each well was screened for anti-CLD 18A2 monoclonal antibodies ( Figure 2 ). Antibody-secreting hybridomas were re-plated and screened again by FACS with HEK293 expressing CLD18A2 or CLD18A1, and if still positive for CLD18A2 and negative for CLD18A1, 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 that retained parental cell reactivity was selected from each hybridoma (by FACS). Three vials of cell banks were generated for each clone and stored in liquid nitrogen.

[0346] c. Selecting monoclonal antibodies that bind to CLD18A2 but not CLD18A1:

[0347] To determine the subtype of the antibodies, a subtype 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, 78C1 0B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and 100F4G12, with the following details:

[0348] 64G11B4, mouse monoclonal IgG1, kappa antibody

[0349] 65G8B8, mouse monoclonal IgG1, kappa antibody

[0350] 56E8F10F4, mouse monoclonal IgG1, kappa antibody

[0351] 54A2C4, mouse monoclonal IgG1, kappa antibody

[0352] 44F6B11, mouse monoclonal IgG1, kappa antibody

[0353] 15C2B7, mouse monoclonal IgG1, kappa antibody

[0354] 20F1E10, mouse monoclonal IgG1, kappa antibody

[0355] 72C1B6A3, mouse monoclonal IgG1, kappa antibody

[0356] 58G2C2, mouse monoclonal IgG2a, kappa antibody

[0357] 101C4F12, mouse monoclonal IgG2b, kappa antibody

[0358] 103A10B2, mouse monoclonal IgG2b, kappa antibody

[0359] 40C10E3, mouse monoclonal IgG1, lambda antibody

[0360] 78E8G9G6, mouse monoclonal IgG1, kappa antibody

[0361] 4F11E2, mouse monoclonal IgG1, kappa antibody

[0362] 10G7G11, mouse monoclonal IgG1, kappa antibody

[0363] 12F1F4, mouse monoclonal IgG1, kappa antibody

[0364] 78C10B6G4, mouse monoclonal IgG1, kappa antibody

[0365] 119G11D9, mouse monoclonal IgG1, kappa antibody

[0366] 113G12E5E6, mouse monoclonal IgG1, κ antibody

[0367] 116A8B7, mouse monoclonal IgG1, kappa antibody

[0368] 105F7G12, mouse monoclonal IgG1, kappa antibody

[0369] 84E9E12, mouse monoclonal IgG1, kappa antibody

[0370] 103F4D4, mouse monoclonal IgG1, kappa antibody

[0371] 110C12B6, mouse monoclonal IgG1, kappa antibody

[0372] 85H12E8, mouse monoclonal IgG1, kappa antibody

[0373] 103H2B4, mouse monoclonal IgG1, kappa antibody

[0374] 103F6D3, mouse monoclonal IgG1, kappa antibody

[0375] 113E12F7, mouse monoclonal IgG2a, kappa antibody

[0376] 120B7B2, mouse monoclonal IgG2a, kappa antibody

[0377] 111B12D11, mouse monoclonal IgG2a, kappa antibody

[0378] 111E7E2, mouse monoclonal IgG2a, kappa antibody

[0379] 100F4G12, mouse monoclonal IgG3, kappa antibody.

[0380] Example 2. Hybridoma Sequencing

[0381] Harvest hybridoma cells (1×10 7 ), and total RNA was extracted using the Tri reagent described above for spleen tissue. As described above, cDNA was prepared using the SuperScript III kit according to the manufacturer's instructions. The resulting cDNA product was used as a PCR template with VhRevU and VhForU as primers, and the resulting 300 bp PCR product was purified using a PCR purification kit and sequenced using the same primers. PCR reactions were also performed using light chain V region specific primers VkRev7 and VkFor (for the variable region only) or KappaFor primers (for the entire kappa light chain). The purified PCR products were sequenced to obtain the DNA sequences of the following antibodies: 64G11B4, 65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G1 1, 12F1F4, 78C10B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2 and 100F4G12. Their variable sequences (VH and VL) are shown in Table 1 below.

[0382] Table 1. Sequences of variable regions of antibodies

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Example 3. Production and purification of monoclonal antibodies reactive with CLD18A2

[0391] To produce mg of antibody for functional characterization, hybridoma cells were plated at 2 × 10 6 The amount of cells / ml was inoculated in a dialysis-based bioreactor (CELLine CL1000, Integra, Chur, CH). The supernatant containing the antibody was collected weekly. Mouse monoclonal antibodies were 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 brilliant blue staining.

[0392] Example 4. Binding of murine monoclonal antibodies reactive with CLD18A2

[0393] Harvest MKN45 cells overexpressing CLD18A2 from the flask. Add 100 μl of 1 × 10 6 cells / ml of cells and Figure 3 The indicated primary antibodies were incubated on ice for 30 minutes at 3-fold serial dilutions ranging from 100 nM to 0.003 nM. After washing twice with 200 μl of FACS buffer, cells were incubated with secondary antibodies on ice for 30 minutes. 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 purified murine antibodies could bind to human CLD18A2-transfected MKN45 cells with a high EC50 as measured by flow cytometry, compared to a positive reference antibody.

[0394] Example 5. Binding of murine monoclonal antibodies reactive with CLD18A2 mutants

[0395] Harvest SU620 cells endogenously expressing CLD18A2 carrying the M149L mutation from a flask. Add 100 μl of 1 × 10 6 cells / ml of cells and Figure 4 The indicated primary antibodies were incubated on ice for 30 minutes at 3-fold serial dilutions ranging 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 purified mouse antibodies could bind to SU620 cells endogenously expressing human CLD18A2 carrying the M149L mutation with a high EC50 by flow cytometry, while the reference antibody could not bind to SU620 cells ( Figure 4 ).

[0396] Example 6. Binding of murine monoclonal antibodies reactive with mouse and macaque CLD18A2

[0397] To assess the cross-reactivity of these antibodies with mouse and macaque CLD18A2, HEK293 cells overexpressing mouse, macaque, or human CLD18A2 were harvested from flasks. 100 μl of 1 × 10 6 cells / ml of cells and Figure 3 The indicated primary antibodies were incubated on ice for 30 minutes at 3-fold serial dilutions ranging from 100 nM to 0.003 nM. After washing twice with 200 μl of FACS buffer, the cells were incubated with 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 purified murine antibodies could bind to mouse and macaque CLD18A2 with high EC50 by flow cytometry, at least similar to the reference antibody ( Figure 5 、 Figure 6 and Figure 7 ).

[0398] Example 7. Binding of chimeric antibodies reactive with CLD18A2

[0399] 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.

[0400] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. Add 100 μl of 1 × 10 6 cells / ml of cells and Figure 4The indicated primary chimeric antibodies were incubated on ice for 30 minutes at 3-fold serial dilutions ranging 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 chimeric antibodies could bind to human CLD18A2 with a high EC50, but not to CLD18A1 ( Figure 8 and Figure 9 ).

[0401] Example 8. Antibody-dependent cell-mediated cytotoxicity (ADCC) of chimeric antibodies

[0402] ADCC reporter gene bioassays use an alternative readout at an early point in ADCC MOA pathway activation: activation of gene transcription through the NFAT (nuclear factor of activated T cells) pathway in effector cells. Additionally, the ADCC reporter gene bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor, the V158 (high affinity) variant, and an NFAT response element driving firefly luciferase expression as effector cells. Antibody bioactivity in the ADCC MOA is quantified by luciferase produced by NFAT pathway activation; luciferase activity in effector cells is quantified by luminescence readout ( Figure 1 ). High signal and low measurement background.

[0403] Serial dilutions of claudin 18.2 chimeric mAb or Ref.Ab were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) for 6 hours at 37°C in the presence or absence of ADCC bioassay target cells (expressing claudin 18.2). TM The luciferase activity was quantified by ELISA (Table 2). The results showed that these chimeric antibodies had very strong ADCC activity.

[0404] Table 2. EC50 of the tested antibodies

[0405]

[0406]

[0407] Example 9. Humanization of 4F11E2, 72C1B6A3, and 120B7B2 Mouse Monoclonal Antibodies

[0408] The variable region genes of monoclonal antibodies 4F11E2, 72C1B6A3, and 120B7B2 were used to create humanized monoclonal antibodies. In the first step of the process, the amino acid sequences of the VH and VL of the monoclonal antibodies were compared with available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

[0409] The amino acid sequences of the humanized antibodies are listed in Table 3 below.

[0410] Table 3. Humanized sequences

[0411]

[0412]

[0413]

[0414] Humanized VH and VL genes were generated synthetically and then cloned into vectors containing human γ1 and human κ constant domains, respectively. Pairing of human VH and human VL produced humanized antibodies (see Table 4).

[0415] Table 4. Humanized antibody 4F11E2 with its VH and VL regions

[0416]

[0417] 72C1B6A3

[0418]

[0419] 120B7B2

[0420]

[0421] Example 10. Binding of humanized antibodies reactive with CLD18A2

[0422] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. Add 100 μl of 1 × 10 6 cells / ml of cells and Figure 4 The indicated primary antibodies were incubated on ice for 30 minutes at 3-fold serial dilutions ranging 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 indicated humanized antibodies could bind to human CLD18A2 with a high EC50, but could not bind to CLD18A1 ( Figure 10 and Figure 11).

[0423] Example 11. Binding of PTM (post-translational modification) risk-reducing humanized antibodies reactive with CLD18A2

[0424] Post-translational modifications (PTMs) can cause problems during the development of therapeutic proteins, such as increased heterogeneity, reduced biological activity, reduced stability, immunogenicity, fragmentation, and aggregation. The potential impact of PTMs depends on their position and, in some cases, on solvent exposure. The following potential PTMs were analyzed for the CDRs of the sequences: asparagine deamidation, aspartic acid isomerization, free cysteine ​​sulfhydryl groups, N-glycosylation, oxidation, fragmentation of potential hydrolysis sites, etc.

[0425] To reduce the risk of PTM in 4F11E2, 72C1B6A3, and 120B7B2, some relevant amino acids in VH and VL were mutated. Nine antibodies were then generated:

[0426]

[0427] *Amino acid positions (eg, N55) are numbered according to the amino acid residue in the corresponding VH or VL amino acid sequence (not Kabat or Chothia).

[0428]

[0429]

[0430] Harvest MKN45 cells stably expressing human CLD18A2 or CLD18A1 from flasks. Add 100 μl of 1 × 10 6 cells / ml of cells and Figure 4 The indicated primary mutant humanized antibodies were incubated on ice for 30 minutes with 3-fold serial dilutions ranging 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 indicated antibodies could bind to human CLD18A2 with a high EC50, but not to CLD18A1 ( Figure 12 and Figure 13 ).

[0431] To evaluate the antigen binding potency of the de-risked variants of 4F11E2d (HC N55E / LC S32A) and 4F11E2d (H N55E N104Q / LC S32A), these variants were tested in a cell-based binding assay. Serial dilutions of anti-CLDN18.2 antibodies starting at 100 nM were combined with 10 5 The cells were incubated on ice for 30 minutes. After washing with FACS buffer, the cells were incubated with APC-labeled secondary antibodies for another 30 minutes on ice. The cells bound to the antibodies were analyzed by FACS. The variants showed efficient binding to cell surface claudin 18.2 ( Figure 14 ).

[0432] Example 12. Antibody-dependent cell-mediated cytotoxicity (ADCC) of PTM-derisked humanized antibodies

[0433] Serial dilutions of the Claudin 18.2PTM de-risked humanized antibody or Ref.Ab were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) at 37°C for 6 hours in the presence or absence of ADCC bioassay target cells (expressing Claudin 18.2). Luciferase activity was quantified using Bio-Glo™ reagent (Table 5). The results demonstrated that these humanized antibodies possessed very potent ADCC activity.

[0434] No. Detected antibodies 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&l0 4A-LC-S32A&G97A 113.9 Ref.Ab Ref.Ab 158.3

[0435] Example 13. Epitope Mapping

[0436] All amino acids in the extracellular domain of claudin 18.2 were mutated to A. Each mutant or wild-type claudin 18.2 was transfected into HEK293 cells. The expression of claudin 18.2 was assessed by the indicated antibodies. The results are shown in Figure 14 (Only amino acid residues whose mutations reduce binding are shown).

[0437] like Figure 15As shown, 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 three tested antibodies, namely, 4F11E2 (H4F), 72C186A3 (H72C1), and 120B7B2 (120), or the reference antibody 175D10 (IMAB362). W30 appears to form a residue cluster in the first half of the first extracellular domain of the claudin 18.2 protein. N45, Y46, G48, L49, W50, C53, V54, R55, E56, S58, F60, E62, and C63 appear to be a second residue cluster within the same extracellular domain. On the other hand, Y169 and G172 are located at or near the second extracellular domain.

[0438] Example 14. Comparison of humanized 4F11E2, 72C1B6A3, and 120B7B2 antibodies with the benchmark 175D10 claudin 18.2 antibody

[0439] Cell-based binding

[0440] To compare the humanized anti-claudin 18.2 antibodies: 4F11E2 (HC N55E / LC S32A), 72C1B6A3 (HCWT / 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 sorted into high and low expressers based on human CLDN18.2 expression levels. Serial dilutions of anti-CLDN18.2 antibodies starting at 100 nM were mixed with 10 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 that bound to the antibody were analyzed by FACS.

[0441] like Figure 16 As shown, 4F11E2, 72C1B6A3, and 120B7B2 all showed superior binding to 175D10 in both claudin 18.2 high and low CHO-K1 cells.

[0442] ADCC assay

[0443] To further compare the ADCC effects 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) with the benchmark antibody 175D10 (IMAB362), this example performed a cell-based ADCC assay. 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. Figure 17 As shown, 4F11E2, 72C1B6A3, and 120B7B2 showed superior ADCC potency to that of the 175D10 antibody.

[0444] For certain therapeutic antibodies, enhanced ADCC can increase the therapeutic window of antibody-based target therapy. Enhanced ADCC can be achieved by engineering the Fc region, for example, with S239D / I332E mutations. In an NK92 cell-based ADCC assay, 4H11E2, 72C1B6A3, and 120B7B2 antibodies with S239D / I332E mutations in the Fc region mediated stronger NK92-mediated cell killing of 293 cells overexpressing claudin 18.2 compared to the control antibody 175D10 with the same S239D / I332E mutations ( Figure 18 ).

[0445] Antibody-dependent cell-mediated phagocytosis (ADCP)

[0446] The effect of anti-CLDN 18.2 monoclonal antibodies 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 varying concentrations of anti-CLDN 18.2 monoclonal antibodies. 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 re-plated in 24-well culture dishes overnight as effector cells. In the presence of varying concentrations of anti-CLDN18.2 monoclonal antibodies, NUG-C4 cells expressing CLDN18.2-eGFP were added as target cells to the MDMs at a ratio of 5 tumor cells per phagocyte. After incubation for 3 hours, unphagocytic target cells were rinsed with PBS, and the remaining phagocytes were collected and stained with the macrophage marker CD14, followed by flow cytometric analysis. The phagocytic index was calculated by quantifying the percentage of GFP+ cells among CD14+ cells and normalized to the IgG control.

[0447] like Figure 19 As shown, all C18.2 mAbs significantly enhanced phagocytosis of NUG-C4 cells in a concentration-dependent manner. In both wild-type IgG1 and S239D / I332E mutant IgG1 formats, 4H11E2, 72C1B6A3, and 120B7B2 antibodies exhibited stronger ADCP effects than the reference antibody 175D10.

[0448] 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, the interaction of 175D10 with claudin 18.2 is Figure 15 The spectrum of binding is strong, including strong binding to D28, Q33, N38, and V43, as well as G59 and V79. In contrast, the new antibodies 4F11E2, 72C1B6A3, and 120B7B2 have higher specificity for W30 within the first half of the first extracellular domain and higher specificity for G48 to E56 within the second half of the first extracellular domain. The new antibodies also bind slightly more strongly to Y46 in the second half. Their binding to D28, Q33, N38, V43, G59, and V79 is quite weak, which may contribute to the improved ADCC and ADCP of the new antibodies.

[0449] ***

[0450] The scope of the present disclosure is not limited to the specific embodiments described, which are intended to be a single illustration of various 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 may 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 cover modifications and variations of the present disclosure as long as these modifications and variations fall within the scope of the appended claims and their equivalents.

[0451] 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 or antigen-binding fragment thereof having binding specificity for wild-type human claudin 18.2 (CLDN18.2) protein, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising light chain complementary determining regions CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising heavy chain complementary determining regions CDRH1, CDRH2, and CDRH3, and wherein: (a) the CDRL1 consists of the amino acid sequence of SEQ ID NO: 210, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 3, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 278, and The CDRH3 consists of the amino acid sequence of SEQ ID NO: 303; (b) the CDRL1 consists of the amino acid sequence of SEQ ID NO: 304, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 19, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 306, and The CDRH3 consists of the amino acid sequence of SEQ ID NO: 307; or (c) the CDRL1 consists of the amino acid sequence of SEQ ID NO: 305, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 20, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 306, and The CDRH3 consists of the amino acid sequence of SEQ ID NO:

307.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The CDRL1 consists of the amino acid sequence of SEQ ID NO: 210, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 3, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 278, and The CDRH3 consists of the amino acid sequence of SEQ ID NO:

303.

3. The antibody or antigen-binding fragment thereof according to claim 2, comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 141 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:

171.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The CDRL1 consists of the amino acid sequence of SEQ ID NO: 304, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 19, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 306, and The CDRH3 consists of the amino acid sequence of SEQ ID NO:

307.

5. The antibody or antigen-binding fragment thereof according to claim 4, comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 206 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:

205.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The CDRL1 consists of the amino acid sequence of SEQ ID NO: 305, The CDRL2 consists of the amino acid sequence of SEQ ID NO: 227, The CDRL3 consists of the amino acid sequence of SEQ ID NO: 20, The CDRH1 consists of the amino acid sequence of SEQ ID NO: 253, The CDRH2 consists of the amino acid sequence of SEQ ID NO: 306, and The CDRH3 consists of the amino acid sequence of SEQ ID NO:

307.

7. The antibody or antigen-binding fragment thereof according to claim 6, comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 207 and a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:

205.

8. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. An isolated cell comprising a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 in the preparation of a medicament for treating a patient's cancer expressing wild-type human claudin 18.2 (CLDN18.2) protein or its M149L mutant, wherein the cancer is selected from gastric cancer, esophageal cancer and pancreatic cancer. The use according to claim 10 , wherein the cancer is gastric cancer.

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