Antibody-drug conjugate targeting claudin 18.2

Abstract

To provide an anti-claudin 18.2 antibody having higher affinity for both wild-type claudin 18.2 and a general variant M149L (SU620 cell which endogenously expresses the variation).SOLUTION: Provided is an antibody having binding specificity to a wild-type human claudin 18.2 (CLDN18.2) protein, or an antibody-drug conjugate including a drug moiety bound to a fragment thereof. The antibody or a fragment thereof binds to β3-β4 loop (residues 45 63 of sequence number 30, NYQGLWRSCVRESSGFTEC) and β5 chain (residues 169 to 172 of sequence number 30, YTFG) of CLDN18.2. In some embodiment, a ratio between the number of drug moieties and the number of antibodies or fragments is 1:1 to 20:1.SELECTED DRAWING: None

Description

[Background technology]

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

[0002] Claudin 18 has two isoforms, isoform 1 and isoform 2. Isoform 2 (claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Claudin 18.2 expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa but is absent from gastric stem cell regions. Claudin 18.2 is retained during malignant transformation and is expressed in a significant proportion of primary gastric cancers and their metastases. Frequent ectopic activation of claudin 18.2 was also observed in pancreatic, esophageal, ovarian, and lung tumors. These data suggest that CLDN18.2 has a highly restricted expression pattern in normal tissues, with frequent ectopic activation in a variety of human cancers. Summary of the Invention [Means for solving the problem]

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

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

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

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

[0007] In some embodiments, the antibody or fragment thereof does not bind to β1 and β2, or binds to β1 or β2 with an affinity that is at least 10-fold lower than the affinity of its binding to the β3-β4 loop or β5 strand. In some embodiments, the antibody or fragment thereof does not bind to the CLDN18.1 protein, or binds to CLDN18.1 with an affinity that is at least 10-fold lower than the affinity of its binding to CLDN18.2.

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

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

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

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

[0012] [Figure 1] FIG. 1 shows that by flow cytometry, mouse sera from all mice after DNA immunization reacted with high titer with HEK293 cells transfected with CLD18A2, with CLD18A1 as a negative control.

[0013] [Figure 2] FIG. 2 shows that hybridoma supernatants can bind to HEK293 cells transfected with human CLD18A2 by cell ELISA or flow cytometry.

[0014] [Figure 3] FIG. 3 shows by flow cytometry that purified mouse antibodies can bind to human CLD18A2-transfected MKN45 cells with a higher EC50 compared to a positive reference antibody.

[0015] [Figure 4] FIG. 4 shows that by flow cytometry, the purified murine antibody can bind to SU620 cells endogenously expressing human CLD18A2 with the M149L mutation with a high EC50, while the reference antibody did not.

[0016] [Figure 5] FIG. 5 shows by flow cytometry that purified mouse antibodies can bind to HEK293 cells transfected with mouse CLD18A2 with high EC50.

[0017] [Figure 6] FIG. 6 shows by flow cytometry that purified mouse antibodies can bind to HEK293 cells transfected with cynomolgus CLD18A2 with high EC50.

[0018] [Figure 7] FIG. 7 shows by flow cytometry that purified mouse antibodies can bind to human CLD18A2-transfected HEK293 cells with high EC50.

[0019] [Figure 8] FIG. 8 shows by flow cytometry that the chimeric antibody can bind to MKN45 cells transfected with human CLD18A2 with a higher EC50 compared to the positive reference antibody.

[0020] [Figure 9] FIG. 9 shows by flow cytometry that the chimeric antibody is unable to bind to MKN45 cells transfected with human CLD18A1.

[0021] [Figure 10] FIG. 10 shows by flow cytometry that the humanized antibodies can bind to MKN45 cells transfected with human CLD18A2 with a higher EC50 compared to the positive reference antibody.

[0022] [Figure 11] FIG. 11 shows by flow cytometry that the humanized antibodies are unable to bind to MKN45 cells transfected with human CLD18A1.

[0023] [Figure 12]FIG. 12 shows by flow cytometry that humanized antibodies with CDR mutations can bind to human CLD18A2-transfected MKN45 cells with a higher EC50 compared to the positive reference antibody.

[0024] [Figure 13] FIG. 13 shows by flow cytometry that humanized antibodies with CDR mutations are unable to bind to MKN45 cells transfected with human CLD18A1.

[0025] [Figure 14] FIG. 14 shows that the risk-depleted variants had strong binding to cell surface CLD18A2.

[0026] [Figure 15] Figure 15 shows that specific mutations in CLD18A2 have a significant effect on the indicated antibody binding to HEK293 cells transfected with these mutants, suggesting that these amino acid residues constitute at least part of the epitope.

[0027] [Figure 16] FIG. 16 shows that antibodies 4F11E2, 72C1B6A3 and 120B7B2 had superior binding to both claudin-18.2 high and low CHO-K1 cells compared to 175D10.

[0028] [Figure 17] FIG. 17 shows the results of a potent ADCC test of 4F11E2, 72C1B6A3 and 120B7B2 using the 175D10 antibody as a reference.

[0029] [Figure 18] FIG. 18 shows that the S239D / I332E versions of 4F11E2, 72C1B6A3 and 120B7B2 outperformed their 175D10 counterparts in the ADCC assay.

[0030] [Figure 19] FIG. 19 shows that 4F11E2, 72C1B6A3 and 120B7B2 also had better ADCP effects than 175D10.

[0031] [Figure 20] FIG. 20 shows the 3D and motif structures of claudin proteins.

[0032] [Figure 21] FIG. 21 shows the internalization results of the test chimeric antibodies compared to the reference antibody IMAB362 on CHO cells expressing claudin 18.2.

[0033] [Figure 22] FIG. 22 shows the internalization results of the test humanized antibodies compared to the reference antibody IMAB362 on CHO cells expressing claudin 18.2.

[0034] [Figure 23] FIG. 23 shows the internalization results of the test humanized antibodies compared to the reference antibody IMAB362 on MKN45 cells expressing claudin 18.2.

[0035] [Figure 24] FIG. 24 shows the binding affinities of antibodies and their drug conjugates.

[0036] [Figure 25-1] Figure 25A shows the cytotoxicity of test antibody-MMAE conjugates upon internalization into DAN-G transfectants, and Figure 25B shows the cytotoxicity of test antibody-MMAE conjugates upon internalization into NUGC transfectants. [Figure 25-2] FIG. 25C shows the cytotoxicity of test antibody-MMAE conjugates upon internalization into SCG-7901 transfectants.

[0037] [Figure 26] FIG. 26 shows the cytotoxicity of test antibody-MMAE conjugates upon internalization into SNU620 cells that endogenously express human claudin 18.2.

[0038] [Figure 27] Figure 27 compares antibody-drug conjugates to antibody alone in reducing tumor growth in test animals.

[0039] [Figure 28] FIG. 28 shows the mean and individual tumor reduction efficacy of antibody-drug conjugates. DETAILED DESCRIPTION OF THE INVENTION

[0040] definition It should be noted that terms prefixed with "a" or "an" refer to one or more of that entity. For example, "an antibody" is understood to refer to one or more antibodies. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0041] As used herein, the term "polypeptide" is intended to encompass both a single "polypeptide" and multiple "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term "polypeptide" also refers to molecules that have undergone, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or other modifications. is intended to refer to the product of post-expression modification of a polypeptide, including modifications with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including chemical synthesis.

[0042] The term "isolated," as used herein with respect to cells, nucleic acids, e.g., DNA or RNA, refers to a molecule separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated," as used herein, also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. Furthermore, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that have been isolated from other cellular proteins or tissues. Isolated polypeptides are meant to encompass both purified and recombinant polypeptides.

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

[0044] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, 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 "non-homologous" sequence shares less than 40% identity with one of the sequences of the present disclosure, but preferably less than 25% identity.

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

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

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

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

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

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

[0051] 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 or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such include, but are not limited to, at least a portion of a heavy or light chain complementarity-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 a binding protein.

[0052] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, or scFv. Regardless of 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.

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

[0054] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of an antibody, as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in light of the present disclosure and, therefore, are within the scope of the present disclosure. While all immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. For IgG, a standard immunoglobulin molecule contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically held together by disulfide bonds in a "Y" configuration, with the light chains supporting the heavy chains, which begin at the mouth of the "Y" and continue through the variable region.

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

[0056] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can associate with either kappa or lambda light chains. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the forked ends of the Y to the C-terminus at the bottom of each chain.

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

[0058] As described above, the variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VK domain and VH domain of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of each of the VH and VK chains. In some instances, for example, in certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule may consist only of a heavy chain, without including a light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0059] In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids within the antigen-binding domain, called the "framework" regions, exhibit less inter-molecular variability. The framework regions primarily adopt a β-sheet structure, while the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act to form a scaffold that orients the CDRs through interchain noncovalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to its cognate epitope. The amino acids comprising each of the CDRs and framework regions are precisely defined ("Sequences of Proteins of Immunological Interes”, Kabat, E. et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)), can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art.

[0060] There are two or more definitions of a term that are used and / or accepted within the art. Where a term is used herein, its definition is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the discontinuous antigen-binding site found within the variable regions of both heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. MoI. Biol. 196:901-917 (1987), both of which are incorporated herein by reference in their entireties. The CDR definitions by Kabat and Chothia include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues encompassing the CDRs defined by each of the above-cited references are provided in the following table for comparison. The exact residue numbers encompassing a particular CDR will vary depending on 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. [Table 9]

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

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

[0063] The antibodies disclosed herein can be of any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken. In another embodiment, the variable region can be chondricthoid in origin (e.g., from sharks).

[0064] 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 a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and / or lower hinge region) domain, and / or a C1 domain. , a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise 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 comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As described above, it will be understood by those skilled in the art that the heavy chain constant region may be modified so that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

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

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

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

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

[0069] As used herein, the term "CH2 domain" includes, for example, the portion of an antibody heavy chain molecule extending from about residue 244 to residue 360, using the conventional numbering scheme (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., US 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. Rather, two N-linked branched carbohydrate chains are sandwiched between the two CH2 domains in intact, native IgG molecules. It is also well established that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

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

[0071] As used herein, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond, 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).

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

[0073] 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 it by the total number of amino acids and multiplying by 100.

[0074] "Specifically bind" or "having specificity" generally means that an antibody binds to an epitope via its antigen-binding domain and that the binding involves considerable complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope 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 a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B," or it can be said that antibody "A" binds to epitope "C" with greater specificity than it has for the related epitope "D."

[0075] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., not worsening) disease, delayed or slowed disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the condition or disorder is to be prevented.

[0076] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0077] As used herein, phrases such as "in a patient in need of treatment" or "subject in need of treatment" refer to the use of the present invention for, e.g., detection, diagnostic procedures and / or treatment. This includes subjects, eg, mammalian subjects, who would benefit from the administration of the indicated antibodies or compositions. Anti-claudin 18.2 antibodies and fragments

[0078] The present disclosure provides anti-claudin 18.2 antibodies with high affinity for both wild-type claudin 18.2 and the common M149L mutant (SU620 cells, which endogenously express this mutation). To the best of our 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 significant proportion of cancer patients have this common mutation. It is also noteworthy that the antibodies of the present disclosure do not bind to (or bind to with much lower affinity) another claudin 18 isoform, claudin 18.1.

[0079] The antibodies and fragments of the present disclosure have shown excellent properties even when used as a reference for clinical candidates. Currently, 175D10 (IMAB362; clodiximab) is undergoing Phase III clinical trials for treating gastric and gastroesophageal junction adenocarcinoma. Compared with 175D10, the present antibodies and fragments not only exhibit stronger binding activity, but also show higher ADCC and ADCP activity under various different conditions.

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

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

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

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

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

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

[0086] The binding affinity of antibody or fragment to protein can be measured by many methods known in the art.For example, it can be measured by cell-free assay using independent CLDN18.1 or CLDN18.2 protein.However, preferably, the measurement is carried out using CLDN18.1 or CLDN18.2 protein on the cell surface, which mimics the actual binding environment.Such binding assay is fully illustrated in the experimental examples.

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

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

[0089] As described above, the antibodies and fragments thereof of the present disclosure bind to claudin 18.2 protein at an epitope different from that of known antibodies (see FIG. 4; at least the reference antibody interacts with M149, while the antibodies of the present disclosure do not). Thus, 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 thereof and the wild-type CLDN18.2 protein involves amino acid residues including at least one amino acid residue selected from the group consisting of N45, Y46, G48, V54, R55, E56, S58, F60, and E62 of the wild-type CLDN18.2 protein, and at least one amino acid residue selected from the group consisting of Y169 and G172.

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

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

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

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

[0094] The weaker binding to these amino acids on CLDN18.2 may be compared to other amino acids, such as G48, L49, W50, C53, V54, R55, E56, etc. In some embodiments, the comparison is to the binding at the same amino acids for 175D10. For example, the binding of an antibody or fragment of the present disclosure is weaker than the binding of 175D10 (IMGT / 2Dstructure-DB Card No.: 10473) to at least one, two, three, four, five, or all of D28, Q33, N38, V43, G59, and V79.

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

[0096] According to one embodiment of the present disclosure, there is provided an antibody or fragment thereof comprising a heavy chain variable domain and a light chain variable domain having the CDR regions shown in the CDR combinations of Table A. [Table A-1] [Table A-2] [Table B] [Table C] [Table D]

[0097] Antibodies containing these CDR regions, whether murine, humanized, or chimeric, had potent claudin 18.2 binding and inhibitory activity. As shown in Examples 11 and 12, specific 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.

[0098] Non-limiting examples of at-risk CDRs are provided in column 3 of Tables B-D. Affinity matured CDRs include those with one, two, or three amino acid additions, deletions, and / or substitutions. In some embodiments, the substitutions may be conservative substitutions.

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

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

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

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

[0103] In some embodiments, CDRL1 comprises an amino acid sequence selected from the group of SEQ ID NOs: 208-226, 304-305, and 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, 8, 13, 19, 20, and 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, 306, 310, and 311, and CDRH3 comprises an amino acid sequence selected from the group of SEQ ID NOs: 281-303, 307, and 312-314.

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

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

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

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

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

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

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

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

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

[0113] Non-limiting examples of light chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 185-187, and 203-204, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 185-187, and 203-204.

[0114] Non-limiting examples of heavy chain variable regions include an amino acid sequence selected from the group consisting of SEQ ID NOs: 153 and 181-184, or a biological equivalent, for example, a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 153 and 181-184.

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

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

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

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

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

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

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

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

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

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

[0125] Antibodies, variants, or derivatives thereof of the present disclosure include derivatives that have been modified such that the covalent attachment does not prevent the antibody from binding to its epitope, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not limited to, antibodies may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or by binding to a cellular ligand or other protein. The antibody can be modified by conjugation to a protein, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids. Antibody-drug conjugates

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

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

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

[0129] For example, when chemical activation of the protein results in the formation of a free thiol group, the protein can be conjugated with a sulfhydryl-reactive agent. In one embodiment, the agent is substantially specific to the free thiol group. Such agents include, for example, maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzylhalide (e.g., iodide, bromide, or chloride), vinylsulfone, and pyridylthio.

[0130] The drug can be attached to the antibody or fragment by a linker. Suitable linkers include, for example, cleavable linkers and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker is a valine-citrulline (val-cit), phenylalanine-lysine (phe-lys) linker, or a maleimidocaproic acid-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PABA) linker. The linker may be a dipeptide linker such as a hydroxyl group (NHS) linker. Another linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group that reacts with sulfhydryls (thiols, -SH), while its sulfo-NHS ester is reactive with primary amines (such as those found in lysines and the N-terminus of proteins or peptides). Yet another linker is maleimidocaproyl (mc). Other suitable linkers include linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently attached to the antibody to the extent that the antibody must be degraded intracellularly to release the drug (e.g., an mc linker).

[0131] The linker may contain a group for binding to an antibody. For example, the linker may include an amino-, hydroxyl-, carboxyl-, or sulfhydryl-reactive group (e.g., maleimide, haloacetamide (e.g., iodo, bromo, or chloro), haloester (e.g., iodo, bromo, or chloro), halomethylketone (e.g., iodo, bromo, or chloro), benzyl halide (e.g., iodide, bromide, or chloride), vinylsulfone, and pyridylthio).

[0132] In some embodiments, the drug moiety is a cytotoxic or cytostatic agent, an immunosuppressant, a radioisotope, a toxin, or the like. The conjugate can be used to inhibit tumor or cancer cell growth, induce apoptosis in tumor or cancer cells, or treat cancer in a patient. Thus, the conjugate can be used in a variety of situations for the treatment of animal cancer. The conjugate can be used to deliver a drug to tumor or cancer cells. Without being bound by theory, in some embodiments, the conjugate binds to or associates with cancer cells that express claudin 18.2, and the conjugate and / or drug bind to or associate with cancer cells that express claudin 18.2, and the conjugate and / or drug bind to or associate with cancer cells that express claudin 18.2, and / or the drug binds to or associates ... claudin 18.2, and / or the drug binds to or associates with claudin 18.2, and / or the drug binds to or associates with claudin 18.2, and / or the drug binds to or associates with claudin 18.2, and / or the drug binds to or associates with claudin 18.2, and / or the drug binds to or associates with claudin 18.2, and / or the drug binds to It can be internalized by tumor or cancer cells through cytosis.

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

[0134] Examples of drug moieties or payloads are DM1 (maytansine, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)- or N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD (6-maleimidocaproyl-monomethylauristatin D or N-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S )-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide), mc-MMAF (maleimidocaproyl-monomethylauristatin F or N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl mc-Val-Cit-PABA-MMAE (6-maleimidocaproyl-Val-Cit-(p-aminobenzyloxycarbonyl)-monomethylauristatin E or N-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(a In some embodiments, the drug moiety is selected from the group consisting of mc-MMAF and mc-Val-Cit-PABA-MMAE.In some embodiments, the drug moiety is a maytansinoid or an auristatin.

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

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

[0137] The antibody also 152 Fluorescent metals such as Eu, or others of the lanthanide series, can be used to detectably label the antibody. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to antibodies are well known and are described, for example, in 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 Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985); and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev. 52:119-58 (1982). Polynucleotides encoding antibodies and methods for preparing antibodies

[0138] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Furthermore, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of an antigen-binding polypeptide, variant, or derivative thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

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

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

[0141] The present disclosure is further directed to antibody-based therapies that involve administering the antibodies, fragments, or antibody-drug conjugates of the present disclosure to patients, e.g., animals, mammals, and humans, to treat one or more of the disorders or conditions described herein. Therapeutic compounds of the present disclosure include, but are not limited to, the antibodies of the present disclosure (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding the antibodies of the present disclosure (including their variants and derivatives as described herein).

[0142] The antibody of the present disclosure can also be used to treat or inhibit cancer.As mentioned above, claudin18.2 can be overexpressed in tumor cells, particularly in stomach, pancreas, esophagus, ovary and lung tumors.Inhibition of claudin18.2 has been shown to be useful for treating tumors.

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

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

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

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

[0147] Antibodies or variants, or derivatives thereof, of the present disclosure can be used for treatment, prevention, diagnosis and / or treatment of Additional diseases or conditions associated with increased cell viability that may be or may be prognosed include malignancies and related disorders, such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (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 sarcomas and cancers, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphomas (e.g. ... These include, but are not limited to, progression and / or metastasis of malignant tumors, including, but not limited to, pulmonary sarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0148] The specific dosage and treatment regimen for any particular patient depends on various factors, including the specific antibody, its variant or derivative used, the patient's age, weight, general health, sex, and diet, as well as the administration time, excretion rate, drug combination, and the severity of the specific disease being treated.The judgment of such factors by a medical caregiver is within the ordinary skill of a person skilled in the art.The amount also depends on the individual patient being treated, the administration route, 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 well known in the art.

[0149] Methods of administration of antibodies, fragments, or antibody-drug conjugates include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops, or It may be administered bucally (as by a transdermal patch), or as an oral or nasal spray.

[0150] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[0151] Administration can be systemic or local.In addition, it may be desirable to introduce the antibody of the present disclosure into central nervous system by any suitable route, including intraventricular injection and intrathecal injection, and intraventricular injection can be facilitated by an intraventricular catheter attached to a reservoir such as an Ommaya reservoir.Pulmonary administration can also be used, for example, by using an inhaler or nebulizer, and by formulating with aerosolizing agent.

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

[0153] The amount of the antibody, fragment, or antibody-drug conjugate of the present disclosure that is effective in treating, inhibiting, and preventing inflammatory, immune, or malignant diseases, disorders, or conditions can be determined by standard clinical techniques. Furthermore, in vitro assays can be used, if necessary, to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

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

[0155] In further embodiments, the compositions of the present disclosure are administered in combination with cytokines, including, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0156] In further embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy. composition

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

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

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

[0160] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active ingredient. When the composition is administered by injection, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

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

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

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

[0164] Single-cell suspensions of splenic lymphocytes from immunized mice were fused with P3X63AG8U.1 non-secreting mouse myeloma cells (ATCC, CRL1597) at a 2:1 ratio using 50% PEG (Roche Diagnostics, CRL738641). The cells were plated in flat-bottom microtiter plates at approximately 3 × 10 4 Cells were seeded at 1000 x 1000 cells / well and subsequently incubated for approximately 2 weeks in selection medium containing 10% fetal bovine serum, 2% hybridoma fusion and cloning supplement (HFCS, Roche Diagnostics, CRL1363735) plus 10 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 μg / ml gentamicin, and 1x HAT (Sigma, CRLH0262). After 10-14 days, individual wells were cultured for anti-CLD18A2 monoclonal antibody using Cell Antibody-secreting hybridomas were replated and screened again by FACS on HEK293 cells expressing CLD18A2 or CLD18A1. If they were still positive for CLD18A2 and negative for CLD18A1, they were subcloned by limiting dilution. Stable subclones were then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. At least one clone from each hybridoma that retained the reactivity of the parental cells (by FACS) was selected. A cell bank of three vials was generated for each clone and stored in liquid nitrogen. c. Selection of monoclonal antibodies that bind to CLD18A2 but not CLD18A1:

[0165] To determine the antibody isotype, an isotype ELISA was performed. The Ig subclass of the identified CLD18A2-reactive monoclonal antibodies was determined using the mouse monoAB ID kit (Zymed, CRL90-6550). Thirty-two hybridoma cell lines were generated: 64G11B4, 65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, and 12F1F. 4, 78C10B6G4, 119G11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and 100F4G12. More details below: 64G11B4, mouse monoclonal IgG1, κ antibody 65G8B8, mouse monoclonal IgG1, κ antibody 56E8F10F4, mouse monoclonal IgG1, κ antibody 54A2C4, mouse monoclonal IgG1, κ antibody 44F6B11, mouse monoclonal IgG1, κ antibody 15C2B7, mouse monoclonal IgG1, κ antibody 20F1E10, mouse monoclonal IgG1, κ antibody 72C1B6A3, mouse monoclonal IgG1, κ antibody 58G2C2, mouse monoclonal IgG2a, kappa antibody 101C4F12, mouse monoclonal IgG2b, kappa antibody 103A10B2, mouse monoclonal IgG2b, kappa antibody 40C10E3, mouse monoclonal IgG1, lambda antibody 78E8G9G6, mouse monoclonal IgG1, κ antibody 4F11E2, mouse monoclonal IgG1, κ antibody 10G7G11, mouse monoclonal IgG1, κ antibody 12F1F4, mouse monoclonal IgG1, κ antibody 78C10B6G4, mouse monoclonal IgG1, κ antibody 119G11D9, mouse monoclonal IgG1, κ antibody 113G12E5E6, mouse monoclonal IgG1, κ antibody 116A8B7, mouse monoclonal IgG1, κ antibody 105F7G12, mouse monoclonal IgG1, κ antibody 84E9E12, mouse monoclonal IgG1, κ antibody 103F4D4, mouse monoclonal IgG1, κ antibody 110C12B6, mouse monoclonal IgG1, κ antibody 85H12E8, mouse monoclonal IgG1, κ antibody 103H2B4, mouse monoclonal IgG1, κ antibody 103F6D3, mouse monoclonal IgG1, κ antibody 113E12F7, mouse monoclonal IgG2a, kappa antibody 120B7B2, mouse monoclonal IgG2a, kappa antibody 111B12D11, mouse monoclonal IgG2a, kappa antibody 111E7E2, mouse monoclonal IgG2a, κ antibody 100F4G12, mouse monoclonal IgG3,κ antibody. Example 2. Hybridoma sequencing

[0166] Hybridoma cells (1 × 10 7 (1000 individuals) were collected, and total RNA was extracted using the Tri Reagent described above for spleen tissue. cDNA was prepared using the SuperScript III kit according to the manufacturer's instructions. The resulting cDNA product was used as a template for PCR using primers VhRevU and VhForU. The resulting 300-bp PCR product was cleaned up using a PCR cleanup kit and sequenced with the same primers. PCR reactions were also performed using light chain V region-specific primers VkReV7 and VkFor (variable region only) or KappaFor primers (entire kappa light chain). Sequencing reactions were performed on the purified PCR products to identify antibodies, 6 4G11B4, 65G8B8, 56E8F10F4, 54A2C4, 44F6B11, 15C2B7, 20F1E10, 72C1B6A3, 58G2C2, 101C4F12, 103A10B2, 40C10E3, 78E8G9G6, 4F11E2, 10G7G11, 12F1F4, 78C10B6G4, 119G DNA sequences were obtained for 11D9, 113G12E5E6, 116A8B7, 105F7G12, 84E9E12, 103F4D4, 110C12B6, 85H12E8, 103H2B4, 103F6D3, 113E12F7, 120B7B2, 111B12D11, 111E7E2, and 100F4G12, and their variable (VH and VL) sequences are shown in Table 1 below. Table 1: Antibody variable region sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] Example 3. Production and purification of monoclonal antibodies reactive with CLD18A2

[0167] To produce mg amounts of antibody for functional characterization, hybridoma cells were cultured at 2 x 10 6 Cells / ml were seeded into a dialysis-based bioreactor (CELLine CL1000, Integra, Chur, Switzerland). Antibody-containing supernatants were collected weekly. Each mouse monoclonal antibody was purified using Melon Gel (Pierce, Rockford, USA) and concentrated by ammonium sulfate precipitation. Antibody concentration and purity were estimated by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. Example 4. Binding of mouse monoclonal antibodies reactive with CLD18A2

[0168] MKN45 cells overexpressing CLD18A2 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ... Example 5. Binding of mouse monoclonal antibodies reactive with CLD18A2 mutants

[0169] SU620 cells that endogenously expressed CLD18A2 with the M149L mutation were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ... Example 6. Binding of mouse monoclonal antibodies reactive to mouse and cynomolgus monkey CLD18A2

[0170] To assess the cross-reactivity of these antibodies with mouse and cynomolgus monkey CLD18A2, HEK293 cells overexpressing mouse, cynomolgus monkey, or human CLD18A2 were harvested from flasks. 100 μl of 1 × 10 6 Cells at 10 ... Example 7. Binding of chimeric antibodies reactive with CLD18A2

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

[0172] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ... Example 8. Antibody-dependent cellular cytotoxicity (ADCC) of chimeric antibodies

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

[0174] Serial dilutions of claudin 18.2 chimeric monoclonal antibodies or reference antibodies were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) in the presence or absence of ADCC bioassay target cells (expressing claudin 18.2) for 6 hours at 37°C for induction. Luciferase activity was quantified using Bio-Glo™ reagent (Table 2). The results show that these chimeric antibodies have very strong ADCC activity. Table 2. EC50 of tested antibodies [Table 2] Example 9. Humanization of 4F11E2, 72C1B6A3, and 120B7B2 Murine mAbs

[0175] Humanized MAbs were generated using the mAB 4F11E2, 72C1B6A3, and 120B7B2 variable region genes. In the first step of this process, the VH and VL amino acid sequences of the MAbs were compared with available databases of human Ig gene sequences to find the best overall match to the human germline Ig gene sequence.

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

[0177] Humanized VH and VL genes were synthetically produced and then cloned into vectors containing the human γ1 and human kappa constant domains, respectively. Humanized antibodies were generated by pairing the human VH with the human VL (see Table 4). Table 4. Humanized antibodies with VH and VL regions [Table 4-1] [Table 4-2] Example 10. Binding of humanized antibodies reactive with CLD18A2

[0178] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ... Example 11. Binding of PTM (post-translational modification)-defective humanized antibodies reactive with CLD18A2

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

[0180] To reduce the risk of PTM in 4F11E2, 72C1B6A3, and 120B7B2, several relevant amino acids in VH and VL were mutated. Nine antibodies were then generated. [Table 11-1] [Table 11-2] * Amino acid positions (eg, N55) are according to the number of the amino acid residue in the corresponding VH or VL amino acid sequence, rather than Kabat or Chothia. [Table 12]

[0181] MKN45 cells stably expressing human CLD18A2 or CLD18A1 were harvested from a flask. 100 μl of 1 × 10 6 Cells at 10 ...

[0182] To evaluate the antigen-binding ability of the risk-deprived variants of 4F11E2d (HCN55E / LCS32A) and 4F11E2d (HN55EN104Q / LCS32A), the variants were tested in a cell-based binding assay. Serial dilutions of anti-CLDN18.2 antibodies, starting at 100 nM, were applied to 10 5 The variants were incubated with 1000 cells on ice for 30 minutes. After washing with FACS buffer, the cells were then incubated with APC-labeled secondary antibody on ice for another 30 minutes. Antibody-bound cells were analyzed by FACS. The variants showed strong binding to cell surface claudin 18.2 (Figure 14). Example 12. Antibody-dependent cellular cytotoxicity (ADCC) of PTM-derisked humanized antibodies

[0183] Serial dilutions of claudin 18.2 PTM-derisked humanized antibodies or reference antibodies were incubated with engineered Jurkat effector cells (ADCC bioassay effector cells) in the presence or absence of ADCC bioassay target cells (expressing claudin 18.2) for 6 hours of induction at 37°C. Luciferase activity was quantified using Bio-Glo™ reagent (Table 5). The results show that these humanized antibodies have very strong ADCC activity. Table 5.ADCC [Table 5] Example 13. Epitope mapping

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

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

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

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

[0188] To compare the humanized anti-claudin18.2 antibodies 4F11E2 (HCN55E / LCS32A), 72C1B6A3 (HCWT / LCS32A), and 120B7B2 (HCG57DS104A / LCS32AG97A) with the benchmark antibody 175D10 (IMAB362), in this example, cell-based binding in human claudin18.2-expressing cells was determined. Based on the level of human CLDN18.2 expression, CHO-K1 cells stably expressing human CLD18A2 were sorted into high- and low-expressing cells. Serially diluted anti-CLDN18.2 antibodies, starting at 100 nM, were added to 10 5The cells were incubated with 1000 cells on ice for 30 minutes. After washing with FACS buffer, the cells were then incubated with APC-labeled secondary antibody on ice for an additional 30 minutes. Antibody-bound cells were analyzed by FACS.

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

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

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

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

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

[0194] In summary, this example demonstrates that the newly developed antibodies 4F11E2, 72C1B6A3, and 120B7B2 possessed stronger cell-based binding and ADCC / ADCP potency than the reference antibody 175D10. It is hypothesized that the improved properties of these new antibodies may be due to their higher binding specificity compared to that of the reference antibody 175D10. For example, the interaction of 175D10 with claudin-18.2 is strong across the range shown in Figure 15, including strong binding to D28, Q33, N38, and V43, followed by 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 for G48-E56 within the second half of the first extracellular domain. The new antibodies also have slightly stronger binding to Y46, which is also located in the second half. Their binding to D28, Q33, N38, V43, G59 and V79 was significantly weaker, which likely contributed to the improved ADCC and ADCP of the new antibodies. Example 15: pHAb conjugation to claudin 18.2 antibody

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

[0196] Experimental Protocol:

[0197] A. Antibody production

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

[0199] B. On-bead antibody conjugation using pHAb thiol-reactive dye

[0200] 1. Resuspend the AmMag® Protein A beads (LC00695) uniformly by gentle shaking or using an end-over-end mixer. Keep the suspension uniform when making aliquots of beads.

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

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

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

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

[0205] 6. Mix the sample for 60 minutes at room temperature. Keep the beads suspended by continuous mixing.

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

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

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

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

[0210] 11. Mix the combined sample for 60 minutes at room temperature. Keep the beads suspended by continuous mixing.

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

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

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

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

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

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

[0217] 18. Place the tube in the magnetic stand for 10 seconds. Remove and discard the supernatant. 19. Add 250 μl of Thiol Conjugation Buffer and mix. Place in the magnetic stand for 10 seconds. Remove and discard the Binding / Wash Buffer (PBS, pH 7.4).

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

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

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

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

[0222] The antibody concentrations and dye-to-antibody ratios (DAR) of the tested antibodies are shown in Table 6. Table 6. Dye-to-Antibody Ratio (DAR) [Table 6] Example 16: Screening for internalization of claudin 18.2 antibodies

[0223] Stably transfected human CLDN18.2MKN45 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded into 96-well black plates (Thermo Scientific, catalog number 165305) at a density of 20K per well in 90 μl. The plates were incubated for 20–24 hours and then treated with pHAb-conjugated antibodies.

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

[0225] The results normalized by DAR are shown in Table 7. The internalization efficiency of the test antibodies was higher than that of the reference antibody IMAB362. Table 7. Internalization results [Table 7] Example 17: Internalization EC50 of chimeric claudin 18.2 antibodies to CHO-claudin 18.2 cells

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

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

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

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

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

[0231] The results, normalized by DAR, are shown in Figure 22 and show that the internalization efficiency of the test antibodies was greater than that of the reference antibody IMAB362. Example 19: Internalization EC50 of humanized claudin18.2 antibodies to MKN45-claudin18.2 cells

[0232] Stably transfected human CLDN18.2MKN45 cells were harvested using 0.05% trypsin / EDTA (Gibco, 25300-054) and seeded into 96-well black plates (Thermo Scientific, catalog number 165305) at a density of 10K per well in 90 μl. The plates were incubated for 20–24 hours and then treated with pHAb-conjugated antibodies.

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

[0234] The results, normalized by DAR, are shown in Figure 23 and show that the internalization efficiency of the test antibodies was greater than that of the reference antibody IMAB362. Example 20: Antibody drug conjugates

[0235] Each antibody was mixed with approximately 3x TCEP and stirred at 37°C for 2 hours. The reaction mixture was rapidly added dropwise to 8x VC-MMAE, incubated on ice for 1 hour, and quenched by adding a 20x excess of cysteine ​​onto the drug linker. Finally, the ADC product was purified by elution with Sephadex G-25 equilibrated in PBS and concentrated by centrifugal ultrafiltration. The conjugate was filtered through a 0.2 μm filter under sterile conditions and stored at -80°C for analysis and testing. The drug-antibody ratio was analyzed by UV spectroscopy, the monomer content was analyzed by SEC-HPLC, and the free drug content was analyzed by RP-HPLC. The DAR of the vcMMAE-conjugated antibodies is shown in Table 8. Table 8. DAR of vcMMAE-conjugated antibodies [Table 8-1] [Table 8-2] Example 21: Relative binding affinity and specificity of anti-CLDN18.2 naked antibodies and antibody-drug conjugates

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

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

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

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

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

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

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

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

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

[0245] 6. Mix contents on an orbital shaker for 2 minutes to induce cell lysis.

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

[0247] 8. Record the luminescence.

[0248] The test results are shown in Figures 25A-C. Both BG2001-C and BG2001-D, when conjugated with MMAE, showed significantly increased cytotoxicity in all cells tested compared to the reference IMAB362-NMAE conjugate. Thus, these results demonstrate the improved ability of the antibodies of the present disclosure to internalize conjugated drugs. Example 23: Cytotoxicity of claudin 18.2 humanized antibody with MMAE is more potent than that of IMAB362 with MMAE in SNU620 cells that endogenously express human claudin 18.2

[0249] SNU620 cells were resuspended in cell culture medium, and 50 μL of the cell suspension containing the corresponding amount of cells was seeded per well into a 96-well cell culture plate. After 24 hours, toxin-conjugated antibodies, including the reference antibody IMAB362, diluted in 50 μL of medium at the appropriate concentration were added, and the cells were cultured for an additional 72 hours. The effect of claudin 18.2 humanized antibodies bearing MMAE on cell viability was determined using the CellTiter-Glo® Luminescent Cell Viability Assay (G7572). As shown in Figure 26, both BG2001-C and BG2001-D were significantly more effective at delivering conjugated MMAE to SNU620 cells than the reference antibody IMAB362, the lead anti-claudin 18.2 antibody in clinical development. Example 24: In vivo efficacy of antibody drug conjugates

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

[0251] Inject 0.1 mL (5 × 10) of human patient-derived cells (mixed 1:1 with Matrigel) into the right dorsal region of each mouse. 5 The average tumor volume was 60-80 mm. 3 When the number of mice reached 100, 30 mice were selected for treatment experiments.

[0252] 18 days after inoculation, 330-520 mm 3 Five mice with tumor sizes ranging from 1 mg / kg to 20 mg / kg ADC were selected for each 3-week treatment (1 mg / kg, 3 mg / mk, 10 mg / kg, or 20 mg / kg ADC, QW). For comparison, antibody (mAb) alone was administered at 10 mg / kg BIW.

[0253] The results are shown in Figure 27. Both 10 mg / kg and 20 mg / kg ADC completely inhibited tumor growth without reducing the animal's body weight. Figure 28 shows the average and individual tumor reduction effects in each animal. Therefore, the tumor reduction effect of the ADC is significantly greater than that of the antibody alone.

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

[0255] 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

[Claim 1] The invention described in this specification.

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