NZ738493B2 - Antibodies against claudin 18.2 useful in cancer diagnosis - Google Patents

Abstract

The invention relates to antibodies directed against an epitope located within the C-terminal portion of CLDN18.2 which are useful, for example, in diagnosing cancer and/or in determining whether cancer cells express CLDN18.2.

Description

ANTIBODIES AGAINST CLAUDIN 18.2 USEFUL IN CANCER DIAGNOSIS This application is a divisional of New Zealand Application No. 718280, in turn a divisional application of New Zealand Application No.700823, the entire contents of which are herein incorporated by reference.

Claudins are integral ne proteins located within the tight junctions of epithelia and endothelia. Claudins are predicted to have four transmembrane segments with two extracellular loops, and N- and C-termini located in the cytoplasm. The n (CLDN) family of transmembrane proteins plays a critical role in the maintenance of epithelial and elial tight junctions and might also play a role in the maintenance of the eleton and in cell signaling.

The claudin 18 (CLDN18) molecule is an integral transmembrane protein (tetraspanin) having four membrane spanning hydrophobic regions and two extracellular loops (loop1 embraced by hydrophobic region 1 and hydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4). CLDN18 exists in two different splice variants, which are described in mouse and in human (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). The splice variants (Genbank accession number: splice variant 1 (CLDN18.1): NP_057453, NM_016369, and splice t 2 (CLDN18.2): NM_001002026, 002026) have a molecular weight of approximately 27,9 / 27,72 kD. The splice ts CLDN18.1 and CLDN18.2 differ in the N- terminal n which comprises the first transmembrane (TM) region and loop1, whereas the primary protein sequence of the C-terminus is identical; see Figure 1.

CLDN18.1 is selectively expressed on cells of normal lung, whereas CLDN18.2 is expressed only on gastric cells. However, CLDN18.2 expression in normal stomach is restricted to the differentiated short-lived cells of stomach epithelium. CLDN18.2 expression has been identified in various tumor s. For example, .2 has been found to be expressed in pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is a valuable target for the prevention and/or ent of primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non small cell lung cancer (NSCLC), ovarian , colon cancer, hepatic cancer, head-neck , and cancers of the gallbladder, and metastases f, in ular c cancer metastasis such as Krukenberg tumors, peritoneal metastasis, and lymph node metastasis.

The differential expression of CLDN18.2 between cancer and normal cells, its membrane localization, its absence from the vast ty of toxicity relevant normal tissues, its restriction of sion to a dispensable cell population in stomach, differentiated c cells, which can be replenished by target-negative stem cells of the h, makes .2 an attractive target for cancer immunotherapy and the use of antibody-based therapeutics for targeting CLDN18.2 in cancer therapy promises a high level of therapeutic specificity.

The clinical application of CLDN18.2-targeting antibodies faces the obstacle that human CLDN18.2 is highly homologous to human CLDN18.1. CLDN18.2-specific antibodies targeting the N-terminal extracellular domain of CLDN18.2 displaying ce differences between human .2 and human CLDN18.1 could successfully be established. Attempts to produce antibodies targeting the N-terminal portion of CLDN18.2 and having ties making them clinically applicable for diagnostic purposes, e.g. for detection of CLDN18.2 expression in cells of cancer tissue sections, failed.

Surprisingly, the present inventors found that antibodies directed against a certain epitope located within the C—terminal portion of CLDN18.2 fulfill the criteria for the diagnostic applicability of antibodies, in particular for detecting and identifying cells expressing CLDN18.2. Most surprisingly, these antibodies gh directed against a sequence which is identical between CLDN18.1 and CLDN18.2 do not target non- ous lung cells.

The antibodies of the invention are useful, for example, in diagnosing cancer and/or in determining whether cancer cells express CLDN18.2. Preferably, a cancer disease or a cancer cell is characterized by surface expression of .2. Cancer cells expressing CLDN18.2 are suitable targets for therapies targeting .2 such as therapy with antibodies directed against CLDN18.2. In one embodiment, cancer cells express or aberrantly express .2 while the corresponding normal cells do not express CLDN18.2 or express CLDN18.2 at a lower level. The cells expressing CLDN18.2 are preferably ed from the group ting of tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon, hepatic, head-neck, and gallbladder cancer cells.

Summary of the invention The present invention relates to an antibody or antigen-binding fragment thereof which (i) binds to a peptide having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or SKHDYV (SEQ ID NO: 6) and/or (ii) binds to claudin 18.2 (CLDN18.2), wherein said antibody or antigen-binding nt thereof binds to CLDN18.2 by binding at least to an epitope within CLDN18.2 having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6).

In one embodiment, said CLDN18.2 is cell e membrane-bound CLDN18.2. In one embodiment, said CLDN18.2 is present on cancer cells, wherein said cancer cells are preferably CLDN18.2 sing cancer cells. In one embodiment, said cancer cells are selected from the group consisting of gastric, esophageal, pancreatic, lung, ovarian, colon, hepatic, eck, and gallbladder cancer cells. In one embodiment, an dy or antigen-binding fragment of the invention does not bind to non—cancerous cells except stomach epithelial cells. In one embodiment, an dy or antigen-binding fragment of the ion does not bind to non-cancerous lung cells. In one embodiment, an antibody of the invention is a chimeric, human or humanized antibody.

In one embodiment, an antibody of the ion is a monoclonal antibody.

In these and further aspects the present invention relates to an antibody comprising: (1) an antibody heavy chain comprising: (i) an antibody heavy chain sequence according to SEQ ID NO: 7 or a variant thereof, (ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence according to SEQ ID NO: 7 or a variant thereof, or (iii) a CDR3 sequence according to SEQ ID NO: 10 or a variant thereof and preferably further comprising a CDRl ce according to SEQ ID NO: 8 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 9 or a variant thereof, and/or (II) an dy light chain comprising: (i) an antibody light chain sequence according to SEQ ID NO: 11 or a variant thereof, (ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence according to SEQ ID NO: 11 or a variant thereof, or (iii) a CDR3 sequence according to SEQ ID NO: 14 or a variant thereof and preferably further comprising a CDRl sequence according to SEQ ID NO: .12 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 13 or a variant thereof.

In the above and further aspects the present invention also relates to an antibody comprising: (1) an antibody heavy chain comprising: (i) an antibody heavy chain sequence according to SEQ ID NO: 15 or a variant thereof, (ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence ing to SEQ ID NO: 15 or a variant thereof, or (iii) a CDR3 ce according to SEQ ID NO: 18 or a t thereof and preferably further comprising a CDRl sequence according to SEQ ID NO: 16 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 17 or a variant thereof, and/or (II) an antibody light chain comprising: (i) an antibody light chain sequence according to SEQ ID NO: 19 or a variant thereof, (ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence according to SEQ ID NO: 19 or a variant f, or (iii) a CDR3 sequence according to SEQ ID NO: 22 or a variant thereof and ably further comprising a CDRl sequence according to SEQ ID NO: 20 or a variant thereof and/or a CDR2 sequence ing to SEQ ID NO: 21 or a variant thereof.

In preferred embodiments, an antibody of the invention comprises an antibody heavy chain comprising a gamma-1 heavy chain constant region, preferably a human gamma-1 heavy chain constant region and/or comprises an antibody light chain comprising a kappa light chain constant region.

In the above and r aspects the present invention s to an antibody produced by or obtainable from a hybridoma cell deposited at the DSMZ fenstr. 7B, 38124 Braunschweig, Germany) and having one of the following designations and accession numbers: I. muAB 43-14A, sion no. DSM ACC3144, deposited on October 6, 2011; or 2. muAB 35-22A, sion no. DSM ACC3143, deposited on October 6, 2011.

Antibodies of the invention are designated herein by referring to the designation of the antibody and/or by referring to the clone producing the antibody, e. g. muAB 43-14A.

Further preferred antibodies are those having the specificity of the dies produced by and obtainable from the above-described hybridomas and, in particular, those comprising an antigen binding portion or antigen binding site, in particular a variable , identical or highly homologous to that of the antibodies produced by and obtainable from the described hybridomas. It is contemplated that preferred dies are those having CDR regions either cal or highly homologous to the CDR regions of antibodies produced by and obtainable from the above-described hybridomas. By "highly homologous" it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in each CDR region.

Particularly preferred antibodies are the ized and humanized forms of the dies produced by and obtainable from the above-described hybridomas.

Thus, an antibody of the invention may be selected from the group ting of (i) an antibody produced by or obtainable from a clone deposited under the accession no.

DSM ACC3144 (muAB 43-14A) or DSM ACC3143 (muAB 35-22A), (ii) an antibody which is a chimerized or humanized form of the antibody under (i), (iii) an antibody which has the specificity of the antibody under (i), and (iv) an antibody comprising the antigen g portion or antigen binding site of the antibody under (i). The antigen binding portion or antigen binding site of the antibody under (i) may comprise the variable region of the antibody under (i). Furthermore encompassed by the present invention are antigen-binding fragments of the antibodies described herein.

An antibody of the invention is preferably able to bind to CLDN18.2 in its native, i.e. naturally occurring or non-denatured state, or in its denatured state.

In one embodiment, an antibody of the invention is obtainable by a method sing the step of immunizing an animal with a peptide sing, ably consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or an immunologically equivalent peptide, or a nucleic acid or host cell expressing said peptide. Preferably said peptide comprises not more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 contiguous amino acids of CLDNl 8.2.

In one embodiment, an antibody of the ion is obtainable by a method comprising the step of immunizing an animal with a peptide comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or an immunologically equivalent peptide, or a nucleic acid or host cell expressing said peptide. Preferably said peptide ses not more than 110, 100, 90, 80, or 75 contiguous amino acids of CLDN18.2.

Antibodies or antigen-binding nts of the invention may be coupled, i.e. covalently or valently linked, to other moieties such as detectable labels.

The t invention also relates to a cell such as a hybridoma cell producing an dy as described herein.

Preferred hybridoma cells are those deposited at the DSMZ (Inhoffenstr. 7B, 38124 Braunschweig, Germany) and having one of the following designations and accession numbers: 1. muAB 43-14A, accesssion no. DSM ACC3144, deposited on October 6, 2011; or 2. muAB 35-22A, accesssion no. DSM ACC3143, deposited on r 6, 2011.

The t invention also relates to a peptide comprising, preferably consisting of the amino acid ce of SEQ ID NO: 5 or SEQ ID NO: 6, or an immunologically equivalent peptide. Preferably said peptide comprises not more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 contiguous amino acids of CLDN18.2.

The present invention also relates to a peptide comprising, preferably consisting of the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or an immunologically lent peptide, or a nucleic acid or host cell expressing said peptide. Preferably said peptide comprises not more than 110, 100, 90, 80, or 75 contiguous amino acids of CLDN18.2.

The present invention also relates to nucleic acids comprising genes or nucleic acid sequences encoding antibodies or parts thereof, e.g. an antibody chain, or antigen- binding fragments, or peptides as described herein. Preferably, the nucleic acid of the invention is operatively attached to expression control elements allowing expression in eukaryotic or prokaryotic cells. Control elements ensuring expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.

The c acids of the invention may be comprised in a vector, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering. The vector may comprise further genes such as marker genes which allow for the selection of the vector in a suitable host cell and under suitable conditions.

Furthermore, the vector may comprise expression control elements allowing proper expression of the coding regions in le hosts. Such l elements are known to the artisan and may include a promoter, a splice cassette, and a translation initiation codon. s for construction of nucleic acid molecules, for construction of vectors comprising nucleic acid les, for introduction of vectors into appropriately chosen host cells, or for causing or achieving expression of nucleic acid molecules are well- known in the art.

A further aspect of the present invention s to a host cell sing a nucleic acid or vector as sed herein.

A r aspect the present invention relates to the detection of CLDN18.2 or CLDN18.2-expressing cells or determination of the quantity of CLDN18.2 or CLDN18.2-expressing cells using an antibody or antigen-binding fragment of the invention. CLDN18.2 or CLDN18.2-expressing cells are detected or the quantity of CLDN18.2 or CLDN18.2-expressing cells is determined by detecting or determining the amount of a complex between CLDN18.2 and an antibody or antigen-binding nt of the invention. ion of a complex indicates the presence of CLDN18.2 or CLDN18.2-expressing cells. Such detection or determination of the amount may be 3O carried out in a number of ways, including but not d to immunodetection using an antibody or n-binding fragment of the invention. Methods for using antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, and the like. In general, such assays use an antibody or antibody fragment that specifically binds the target peptide or protein directly or indirectly bound to a label that provides for ion, e.g. indicator enzymes, abels, hores, or paramagnetic particles. The methods of the invention allow quantitative and/or qualitative evaluations, e.g., absolute and/or relative evaluations, of CLDN18.2 levels or of levels of CLDNl 8.2-expressing cells.

In one aspect, the present invention relates to a method for detecting CLDN18.2 or determining the quantity of .2 in a sample comprising the steps of: (i) contacting a sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention and (ii) ing the ion of a complex or determining the quantity of a complex between the antibody, the antigen—binding fragment or the conjugate and .2.

In one embodiment, the sample is a cellular sample, i.e. a sample comprising cells such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, the antigen-binding fragment or the conjugate and CLDN18.2 expressed by cells in said sample.

In one aspect, the present invention relates to a method for determining whether cells express CLDN18.2 comprising the steps of: 2O (i) ting a cellular sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention and (ii) detecting the formation of a complex between the antibody, the antigen-binding fragment or the ate and CLDN18.2 expressed by cells in said sample.

In one embodiment, the cells in the sample are cancer cells. The complex is preferably formed between the antibody, the antigen-binding fragment or the conjugate and CLDN18.2 expressed by cells in said sample.

Further aspects of the present invention relate to s of diagnosing or classifying diseases by targeting CLDN18.2 using an antibody or antigen-binding fragment of the invention. These methods provide for the selective detection of cells that express CLDN18.2 thereby entiating these cells from normal cells not expressing CLDN18.2 or diseased cells not expressing .2. Diseases characterized by diseased cells expressing CLDN18.2 are treatable by a therapy targeting CLDN18.2 such as therapy with therapeutic antibodies ed against CLDN18.2. red diseases for a therapy or diagnosis are those in which CLDN18.2 is expressed or aberrantly expressed, in particular cancer diseases, such as those described herein.

In one aspect the present invention relates to methods for diagnosis, detection or monitoring, i.e. ining the regression, progression, course and/or onset, of a cancer disease comprising the detection of CLDN18.2 or CLDN18.2-expressing cells and/or determination of the quantity of CLDN18.2 or CLDN18.2-expressing cells in a biological sample ed from a patient using an antibody or antigen—binding fragment of the invention. Such methods may be used to detect whether a subject has a cancer e or is at (increased) risk of developing a cancer disease or, for instance, r a treatment regimen is efficient.

Thus, in one aspect, the present invention relates to a method for diagnosis, detection or monitoring of cancer comprising the steps of: (i) contacting a biological sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention and (ii) detecting the formation of a complex and/or determining the quantity of a complex n the antibody, the n-binding fragment or the conjugate and .2.

In one embodiment, the biological sample is a cellular sample, i.e. a sample comprising cells such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, the antigen-binding fragment or the conjugate and CLDN18.2 sed by cells in said sample.

The methods of monitoring according to the invention preferably comprise a detection of and/or determination of the quantity of CLDN18.2 or CLDN18.2—expressing cells in a first sample at a first point in time and in a r sample at a second point in time, wherein the regression, progression, course and/or onset of a tumor disease may be determined by comparing the two samples.

Typically, the level of CLDN18.2 or level of CLDNl 8.2-expressing cells in a biological sample is compared to a reference level, wherein a deviation from said reference level is tive of the presence and/or stage of a cancer e in a subject. The reference level may be a level as determined in a control sample (e.g., from a healthy tissue or subject, in particular a patient without a cancer disease) or a median level from healthy subjects. A "deviation" from said reference level designates any significant change, such as an se by at least 10%, 20%, or 30%, Preferably by at least 40% or 50%, or even more.

Preferably, the presence of CLDN18.2 or CLDN18.2-expressing cells and/or a quantity of CLDNl8.2 or CLDN18.2-expressing cells which is increased compared to a reference level, e.g. compared to a patient without a cancer disease, indicates the presence of or risk for (i.e. a potential for a development of) a cancer disease in the patient.

A quantity of CLDN18.2 or CLDN18.2-expressing cells which is decreased compared to a biological sample taken earlier from a patient may indicate a regression, a positive , e. g. a successful treatment, or a reduced risk for an onset of a cancer disease in a patient.

A quantity of CLDNl 8.2 or CLDNl 8.2-expressing cells which is increased compared to a biological sample taken earlier from a patient may indicate a progression, a negative course, e.g. an essful treatment, ence or metastatic behaviour, an onset or a risk for an onset of a cancer disease in said patient.

In one aspect, the present invention s to a method for determining whether a cancer is treatable by a cancer therapy targeting CLDN18.2 comprising the steps of: (i) contacting a sample comprising cancer cells with an antibody or n-binding fragment of the invention or a ate of the invention and (ii) detecting the formation of a complex between the dy, the antigen-binding fragment or the conjugate and CLDN18.2.

The complex is preferably formed between the antibody, the antigen-binding nt or the conjugate and CLDN18.2 expressed by cancer cells in said sample.

Such methods may be used to detect whether a t is suitable for a therapy involving the targeting of cells expressing CLDN18.2 such as a therapy using antibodies exerting one or more immune effector functions such as cytotoxic .2 specific antibodies, e.g. antibodies labeled with a cytotoxic substance such as a toxin or a radiolabel or inducing a cell killing mechanism such as CDC or ADCC. Diseases characterized by diseased cells expressing CLDN18.2 are treatable by a therapy targeting CLDN18.2 such as cancer diseases, in particular those described herein.

In one embodiment of any of the above aspects, the sample, cellular sample or ical sample is from a t having a cancer disease, being suspected of having or falling ill with a cancer disease or having a potential for a cancer disease. In one embodiment, the sample, cellular sample or biological sample is from a tissue or organ wherein the cells when the tissue or organ is free of cancer do not substantially express CLDN18.2. Preferably said tissue is a tissue other than stomach tissue. Preferably, said tissue is tissue of lung, esophagus, pancreas or breast and the tissue or organ optionally has y been diagnosed as being affected by a cancer disease, e.g. by visual tion or culture g of cells of said tissue or organ. In this embodiment, the presence of .2 or CLDN18.2-expressing cells and/or a quantity of CLDN18.2 or CLDN18.2-expressing cells which is increased compared to a reference level, e.g. compared to a patient without a tumor disease, may indicate that a patient is suitable for a therapy ing the targeting of cells expressing CLDN18.2.

In one aspect, the invention provides compositions, e.g., diagnostic compositions, or kits, comprising an antibody or antigen-binding fragment or a combination of antibodies and/or or antigen-binding fragments described herein. Such diagnostic compositions or test kits are useful in the methods of the ion such as the methods for diagnosis, detection or ring of the invention. These kits may ally se a detectable label, e.g. indicator enzymes, radiolabels, fluorophores, or paramagnetic particles. Kits may include informative pamphlets, for example, pamphlets informing 3O one how to use reagents to practice a method disclosed herein.

Other features and advantages of the t invention will be apparent from the following detailed description and claims. ed description of the invention Although the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be d only by the ed claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as ly tood by one of ordinary skill in the art.

In the following, the ts of the present invention will be bed. These elements are listed with specific ments, however, it should be understood that they may be ed in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments.

This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. erger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, .

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA ques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless the context es otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other , integer or step or group of members, integers or steps gh in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e. the subject— matter consists in the inclusion of a stated member, integer or step or group of members, integers or steps. The terms "a" and "an" and "the" and r reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the , unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited . All methods described herein can be performed in any suitable order unless otherwise indicated herein or ise clearly contradicted by context. The use of any and all examples, or exemplary language (e. g., "such as"), provided herein is intended merely to better illustrate the ion and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should. be construed as indicating any non-claimed element essential to the practice of the invention.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, ific publications, manufacturer's cations, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their ty. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The term "recombinant" in the context of the t invention means "made through genetic engineering". ably, a "recombinant object" such as a recombinant cell in the t of the present invention is not occurring naturally.

The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally d by man in the laboratory is naturally occurring.

The term "antigen" relates to an agent comprising an epitope against which an immune response is directed and/or is to be generated. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen. The term "antigen" includes in particular ns, peptides, polysaccharides, nucleic acids, especially RNA and DNA, and nucleotides.

The term "epitope" refers to an antigenic determinant in a molecule, i.e., to the part in a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are the te, three-dimensional sites on an antigen, which are recognized by the immune . Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural teristics, as well as specific charge characteristics. Conformational and nformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein such as a CLDN preferably ses a uous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 8, 2O 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in .

The term "discontinuous epitope" as used herein, means a conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein.

In a preferred embodiment, an antigen is a tumor-associated antigen, such as CLDN18.2, i.e., a constituent of cancer cells which may be derived from the cytoplasm, the cell surface and the cell nucleus, in particular those antigens which are produced, preferably in large ty, intracellular or as surface antigens on cancer cells.

In the context of the t invention, the terms -associated antigen" or "tumor antigen" relate to proteins that are under normal conditions cally expressed in a d number of tissues and/or organs or in specific developmental stages, for example, the tumor-associated antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are sed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means not more than 3, more preferably not more than 2. The tumor-associated antigens in the context of the present invention include, for e, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain entiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific ns. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell e of a cancer cell and is preferably not or only rarely expressed in normal tissues. Preferably, the tumor-associated antigen or the aberrant expression of the associated antigen identifies cancer cells. In the context of the present invention, the tumor—associated antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is preferably a self-protein in said subject. In preferred embodiments, the tumor-associated antigen in the t of the present invention is expressed under normal ions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system. ably, the amino acid ce of the tumor-associated antigen is cal between the tumor-associated antigen which is expressed in normal tissues and the tumor-associated antigen which is expressed in cancer tissues.

Examples for entiation antigens which ideally fulfill the criteria for tumor- associated antigens as target structures in tumor immunotherapy, in particular, in tumor ation are the cell surface proteins of the claudin family, such as CLDN18.2.

Claudins are a family of proteins that are the most important components of tight junctions, where they establish the llular barrier that controls the flow of 3O molecules in the intercellular space between cells of an epithelium. Claudins are transmembrane proteins spanning the membrane 4 times with the N-terminal and the C- terminal end both located in the cytoplasm.

The term "claudin 18" or "CLDN18" preferably relates to human CLDN18 and includes any Splice variants such as CLDN18.1 and CLDN18.2 of CLDN18. CLDN18.1 and CLDN18.2 differ in the N-terminal portion which comprises the first transmembrane (TM) region and loop1, whereas the primary protein sequence of the C-terminus is cal.

The term "CLDN18.1" preferably relates to human CLDN18.1, and, in particular, to a protein comprising the amino acid sequence ing to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence.

The term "CLDN18.2" preferably relates to human CLDN18.2, and, in particular, to a protein comprising the amino acid sequence according to SEQ ID NO: 2 of the sequence listing or a variant of said amino acid sequence.

The term "variant" according to the ion refers, in particular, to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often r.

Complete gene sequencing often identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence.

The terms "CLDN", "CLDN18", "CLDN18.1" and 8.2" shall encompass any posttranslationally ed variants and conformation variants.

CLDN18.2 is selectively expressed in normal tissues in entiated epithelial cells of the gastric mucosa. CLDN18.2 is sed in cancers of various origins, and is particularly suited as target ure for the development of antibody-mediated cancer immunotherapy due to its selective sion (no expression in a ty relevant 3O normal tissue) and localization to the plasma membrane. For example, CLDN18.2 has been found to be expressed in pancreatic carcinoma, geal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is a valuable target for the prevention and/or treatment of primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non small cell lung cancer (NSCLC), ovarian cancer, colon cancer, hepatic cancer, eck cancer, and cancers of the gallbladder, and metastases thereof, in particular gastric cancer metastasis such as Krukenberg tumors, peritoneal metastasis, and lymph node metastasis. Cells expressing CLDN18.2 are preferably cancer cells and are, in particular, selected from the group consisting of tumorigenic gastric, esophageal, pancreatic, lung, ovarian, colon, hepatic, head-neck, and gallbladder cancer cells.

According to the invention, a cell expressing CLDN18.2 is preferably characterized by cell-surface membrane—bound CLDN18.2, i.e. CLDN18.2 is associated with the cell e. Furthermore, according to the invention, cellular CLDN18.2 is preferably cell- surface membrane-bound CLDN18.2. A cell expressing CLDN18.2 or a cell characterized by association of .2 with its cell surface preferably is a cancer cell, preferably a cancer cell from a cancer described herein.

The term "associated with the cell surface" means that a tumor-associated antigen such as CLDN18.2 is associated with and located at the plasma membrane of a cell, wherein at least a part of the tumor-associated antigen faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by dies located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or ct. For example, the association may be by one or more embrane s, one or more lipid s, or by the ction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For e, a tumor-associated antigen associated with the e of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane n.

"Cell e" or "surface of a cell" is used in accordance with its normal meaning in the 3O art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules.

According to the invention CLDN18.2 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression and association exceeds the level of expression and association in non-cancerous tissue other than stomach by no more than 2-fold, preferably 1,5—fold, and preferably does not exceed the level of expression and association in said non-cancerous . Preferably, .2 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression or association is below the detection limit and/or if the level of expression or association is too low to allow binding by CLDNl8.2-specific antibodies added to the cells.

According to the invention CLDN18.2 is expressed in a cell and is associated with a cell surface if the level of expression and association s the level of expression and association in non-cancerous tissue other than stomach, preferably by more than 2-fold, preferably 10-fold, lOO-fold, old, or IOOOO-fold. Preferably, CLDN18.2 is expressed in a cell and is associated with a cell surface if the level of expression and association is above the detection limit and/or if the level of sion and association is high enough to allow binding by CLDNl8.2-specific antibodies added to the cells.

The term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any le comprising an antigen binding portion thereof. The term "antibody" includes monoclonal antibodies and fragments or derivatives of antibodies, including, without limitation, human dies, humanized antibodies, chimeric dies, single chain antibodies, e.g., scFv’s and antigen-binding antibody nts such as Fab and Fab' fragments and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain le region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining s (CDR), interspersed with regions that are more conserved, termed framework 3O regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that cts with an antigen. The constant regions of the antibodies may mediate the g of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the cal complement .

The antibodies described herein may be human antibodies. The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline globulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

The term "humanized antibody" refers to a molecule having an antigen g site that is substantially derived from an immunoglobulin from a non-human species, wherein the ing immunoglobulin structure of the molecule is based upon the structure and/or sequence of a human immunoglobulin. The n g site may either se complete le domains fused onto nt domains or only the complementarity determining regions (CDR) grafted onto appropriate ork regions in the variable domains. Antigen binding sites may be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody.

The term "chimeric antibody" refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another. Typically, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of s, while 3O the constant portions are homologous to sequences of antibodies derived from another.

One clear advantage to such chimeric forms is that the variable region can conveniently be derived from presently known s using readily available B-cells or hybridomas from non-human host organisms in combination with nt regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human t when the antibodies are injected than would the constant region from a non human .

However the definition is not limited to this particular example.

The terms "antigen-binding portion" of an antibody (or simply "binding portion") or "antigen-binding fragment" of an antibody (or simply ng fragment") refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments ting of the VL and VH domains of a single arm of an dy, (v) dAb fragments (Ward et al., (1989) Nature 341: 6), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more ed CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by te genes, they can be , using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH s pair to form lent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et a1. (1988) Proc.

Natl. Acad. Sci. USA 85: 883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an globulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. 3O The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding-domain globulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The antibodies described herein may be monoclonal antibodies. The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A onal antibody displays a single binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell.

The antibodies described herein may be recombinant antibodies. The term "recombinant antibody", as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) dies isolated from an animal (e.g., a mouse) that is transgenic or hromosomal with respect to the globulin genes or a hybridoma prepared therefrom, (b) dies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

The term "transfectoma", as used herein, includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/O cells, HEK293 cells, T cells, plant cells, or fungi, including yeast cells.

As used herein, a "heterologous antibody" is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid ce corresponding to that found in an organism not consisting of the transgenic organism, and being generally derived from a species other than the transgenic organism.

As used herein, a "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origins. For e, an antibody having a human heavy chain ated with a murine light chain is a heterohybrid antibody.

The invention includes all dies and derivatives of antibodies as bed herein which for the purposes of the invention are encompassed by the term "antibody". The term "antibody derivatives" refers to any modified form of an antibody, e.g., a conjugate of the antibody and another agent or antibody, or an antibody fragment.

The antibodies described herein are preferably isolated. An "isolated antibody" as used herein, is intended to refer to an antibody which is substantially free of other dies having different antigenic specificities. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

According to the present invention, an antibody is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said ermined target in standard assays. "Affinity" or "binding y" is often measured by equilibrium dissociation constant (KD). Preferably, the term "significant affinity" refers to the binding to a predetermined target with a iation constant (KB) of 10'5 M or lower, 10'6 M or lower, 10'7 M or lower, 10'8 M or lower, 10'9 M or lower, 10'10 M or lower, 10'll M or lower, or 10'12 M or lower.

An antibody is not (substantially) capable of binding to a target if it has no significant y for said target and does not bind significantly, in particular does not bind detectably, to said target in standard assays. Preferably, the antibody does not detectably bind to said target if t in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 ug/ml or higher. Preferably, an antibody has no significant y for a target if it binds to said target with a KD that is at least 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, or 106-fold higher than the KB for binding to the predetermined target to which the antibody is capable of binding. For example, if the KB for binding of an antibody to the target to which the dy is capable of binding is '7 M, the KB for binding to a target for which the antibody has no significant y would be is at least 10'6 M, 10'5 M, 10'4 M, 10'3 M, 10'2 M, or 10" M.

An antibody is specific for a predetermined target if it is capable of g to said 3O predetermined target while it is not capable of binding to other targets, i.e. has no significant affinity for other targets and does not significantly bind to other targets in standard assays. According to the invention, an antibody is specific for CLDN18.2 if it is capable of binding to CLDN18.2 but is not (substantially) capable of binding to other targets, in ular proteins other than n proteins, preferably proteins other than , in particular proteins other than CLDN18.2. Preferably, an dy is specific for CLDN18.2 if the affinity for and the binding to such other targets does not significantly exceed the affinity for or binding to claudin-unrelated proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin transmembrane proteins such as MHC les or transferrin receptor or any other specified polypeptide. Preferably, an antibody is c for a predetermined target if it binds to said target with a KD that is at least 10-fold, 100—fold, 103-fold, 104-fold, 105- fold, or 106-fold lower than the K9 for binding to a target for which it is not specific.

For example, if the K0 for binding of an antibody to the target for which it is specific is 10'7 M, the K0 for binding to a target for which it is not specific would be at least 10'6 M, 10'5 M, 10'4 M, 10'3 M, 10'2 M, or 10'1 M.

Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In ental Immunology, Paul, W. 13., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the e 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled n. The affinity data may be analyzed, for example, by the method of Scatchard et al., Ann NY.

Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if ed under different conditions, e.g., salt tration, pH.

Thus, measurements of affinity and other n-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.

As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. Antibodies according to the invention include polyclonal and monoclonal antibodies and include IgG2a (e.g. IgGZa, K, 7»), IgG2b (e.g.

IgGZb, K, 7»), IgG3 (e.g. IgG3, K, 7») and IgM antibodiesfiHowever, other dy es are also encompassed by the invention, including IgGl, IgAl, IgA2, secretory IgA, IgD, and IgE antibodies.

As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

The term "rearranged" as used herein refers to a configuration of a heavy chain or light chain globulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to gerrnline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the ration n the V segment is not recombined so as to be ately nt to a D or J segment.

According to the invention, antibodies may be derived from different species, including but not limited to mouse, rat, rabbit, guinea pig and human. Antibodies also include chimeric molecules in which an dy constant region derived from one species, preferably human, is combined with the antigen binding site derived from another species. Moreover, antibodies include humanized les in which the antigen binding sites of an antibody derived from a non-human species are combined with constant and framework regions of human origin.

Antibodies can be produced by a variety of ques, including tional monoclonal antibody methodology, e.g., the standard c cell hybridization technique of Kohler and in, Nature 256: 495 (1975). gh somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B- lymphocytes or phage y techniques using libraries of antibody genes. 3O The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g. described in Spieker-Polet et al., Proc.

Natl. Acad. Sci. USA. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124: 295 ).

In yet another preferred ment, human monoclonal antibodies directed against CLDN18.2 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice known as HuMAb mice and KM mice, respectively, and are tively referred to herein as "transgenic mice." The production of human antibodies in such transgenic mice can be med as described in detail for CD20 in WO2004 035607 Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing dies of defined specificity; see e.g. Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For details of recombinant dy engineering see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN896038 and Benny K.C. Lo dy Engineering ISBN 1-58829—092-1.

To generate antibodies to CLDN18.2, mice can be immunized with carrier-conjugated peptides derived from the CLDN18.2 sequence, an ed ation of recombinantly expressed CLDN18.2 antigen or nts thereof and/or cells expressing CLDN18.2 or fragments thereof, as described. Alternatively, mice can be immunized with DNA encoding full length human CLDN18.2 or fragments thereof. In the event that immunizations using a purified or enriched preparation of the CLDN18.2 antigen do not result in dies, mice can also be immunized with cells expressing 3O CLDN18.2, e.g., a cell line, to promote immune responses.

The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being ed by tail vein or retroorbital bleeds. Mice with sufficient titers of anti-CLDN18.2 immunoglobulin can be used for fusions. Mice can be d intraperitonealy or intravenously with CLDN18.2 expressing cells 3-5 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas.

To generate omas ing monoclonal antibodies to CLDN18.2, cells from lymph nodes, spleens or bone marrow obtained from immunized mice can be isolated and fused to an riate immortalized cell line, such as a mouse myeloma cell line.

The resulting omas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By fluorescence and FACS analysis using CLDN18.2 expressing cells, antibodies with city for CLDN18.2 can be identified. The dy secreting hybridomas can be replated, screened again, and if still positive for anti- CLDN18.2 monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization.

Antibodies of the invention can also be produced in a host cell transfectoma using, for example, a combination of inant DNA techniques and gene transfection methods as are well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be d into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in WO 62, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS/O cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the 3O antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies.

Recombinant antibodies can be isolated and purified from these e supematants and/or cells.

Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g. E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. l. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Chimeric antibodies are dies, the different portions of which are derived from different animal species, such as those having a variable region d from a murine dy and a human immunoglobulin constant region. Chimerisation of antibodies is achieved by joining of the variable regions of the murine antibody heavy and light chain with the constant region of human heavy and light chain (e.g. as described by Kraus et al., in Methods in Molecular Biology , Recombinant antibodies for cancer therapy ISBN896038). In a preferred embodiment, chimeric antibodies are generated by joining human kappa-light chain constant region to murine light chain variable region.

In an also preferred embodiment, chimeric antibodies can be generated by g human lambda-light chain constant region to murine light chain variable region. The preferred heavy chain constant regions for generation of chimeric dies are IgGl, IgG3 and IgG4. Other red heavy chain constant regions for generation of chimeric antibodies are IgG2, IgA, IgD and IgM.

Antibodies interact with target antigens predominantly through amino acid residues that are d in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than ces e of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express inant antibodies that mimic the properties of specific lly occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a ent 3O antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323—327; Jones, P. et al. (1986) Nature 321: 522—525; and Queen, C. et al. (1989) Proc.

Natl. Acad. Sci. U. S. A. 86: 10029—10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual positions evenly across the variable region. For e, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the y- terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not ary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant dy having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequences spanning the CDR regions are typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The ne sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic ucleotides by ligation or PCR amplification. atively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic le region clone. This process has n advantages such as elimination or inclusion or ular restriction sites, or optimization of ular codons.

The tide sequences of heavy and light chain transcripts from hybridomas may be used to design an pping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate ucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266: 19867- 3O 19870); and HindIII sites are engineered upstream of the translation tion sites.

For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span ts of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These pping products are then combined by PCR amplification to form the complete variable region.

It may also be desirable to include an overlapping fragment of the heavy or light chain constant region in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs.

The reconstructed chimerized or zed heavy and light chain variable regions are then combined with cloned er, leader, translation initiation, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, nsfected, serially transfected, or tely transfected into host cells which are then fused to form a host cell expressing both chains. Plasmids for use in construction of expression vectors for human IgGK are described. The plasmids can be constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences can be used to reconstruct complete heavy and light chain minigenes. These ds can be used to express completely human, or chimeric IgGl, Kappa or IgG4, Kappa antibodies. Similar plasmids can be constructed for sion of other heavy chain isotypes, or for expression of dies comprising lambda light chains.

Thus, in another aspect of the ion, the structural features of the anti-CLDN18.2 antibodies bed herein, are used to create structurally related humanized anti- CLDN18.2 antibodies that retain at least one onal property of the antibodies of the invention, such as binding to CLDN18.2. More specifically, one or more CDR regions of mouse monoclonal antibodies can be combined recombinantly with known human 3O framework regions and CDRs to create additional, recombinantly-engineered, humanized anti-CLDN18.2 antibodies.

The ability of an antibody to bind CLDN18.2 can be determined using standard binding assays, e.g., ELISA, Western Blot, Immunofluorescence and Flow cytometric analysis.

ELISA can be used to demonstrate the presence of antibodies in sera of immunized mice or g of monoclonal antibodies to CLDN18.2 protein or peptides. Peptides or protein used for immunization may be used for determining the specificity of hybridoma supematants or analysing serum titers.

In order to trate ce of antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells, flow cytometry can be used. Cell lines expressing naturally or after transfection antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in oma tants or in PBS containing 1% FBS, and can be incubated at 4°C for 30 min. After washing, the APC— or 47-labeled anti IgG antibody can bind to antigen—bound onal antibody under the same conditions as the primary dy staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding antigen and a cent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, antigen-specific monoclonal antibodies bind preferentially to fluorescence marker sing cells, whereas ecific antibodies bind in a comparable ratio to non—transfected cells. An alternative assay using fluorescence copy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy.

In order to demonstrate presence of anti-CLDN18.2 antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing CLDN18.2, fluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection CLDN18.2 and negative controls lacking CLDN18.2 expression are grown in chamber slides under standard growth conditions in DMEM/F 12 medium, supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 ug/ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with onal antibodies against CLDN18.2 for 30 min. at 25°C. After washing, cells can be reacted with an Alexa555-labelled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy.

Total .2 levels in cells can be observed when cells are methanol fixed or paraformaldehyde fixed and permeabilized with Triton X-100. In living cells and non— permeabilized, paraformaldehyde fixed cells surface localization of CLDN18.2 can be examined. Additionally targeting of CLDN18.2 to tight junctions can be analyzed by co-staining with tight junction markers such as ZO—l. Furthermore, effects of antibody binding and .2 localization within the cell membrane can be examined.

Anti-CLDN18.2 IgG can be further tested for reactivity with CLDN18.2 antigen by Western Blotting. Briefly, cell extracts from cells expressing CLDN18.2 and appropriate ve controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel ophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG g can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.

Anti-CLDN18.2 mouse IgGs can be further tested for reactivity with CLDN18.2 antigen by Immunohistochemistry in a manner well known to the d person, e.g. using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue ns fixed with paraformaldehyde from non-cancer tissue or cancer tissue s obtained from patients during routine surgical procedures or from mice ng xenografted tumors inoculated with cell lines sing spontaneously or after transfection with CLDN18.2. For immunostaining, antibodies reactive to CLDN18.2 can be incubated ed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit 3O antibodies ing to the vendors instructions.

One particularly preferred methodology for assaying CLDN18.2 in the methods of the invention is Immunohistochemistry or IHC. Immunohistochemistry or IHC refers to the- process of detecting antigens (e.g., ns) in cells of a tissue section, e.g. cells of the tissues mentioned herein. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Visualising an antibody— antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine.

Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. This requires proper tissue collection, fixation and sectioning. Paraformaldehyde is usually used with n. Depending on the purpose and the ess of the mental sample, either thin (about 4-40 pm) sections are sliced from the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole. The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides.

The sample may require additional steps to make the es available for antibody binding, including deparaffinization and antigen retrieval. Detergents like Triton X-100 are generally used in Immunohistochemistry to reduce e tension, allowing less reagent to be used to achieve better and more even coverage of the sample.

The direct method of immunohistochemical staining uses one labelled antibody, which binds directly to the antigen being stained for. The ct method of immunohistochemical ng which is more common uses one dy against the antigen being probed for, and a second, labelled, antibody t the first.

To reduce ound staining in IHC, the samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Primary antibodies are raised t an antigen of interest and are typically unconjugated (unlabelled), while secondary antibodies are raised against 3O immunoglobulins of the primary dy species. The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody is ly bound to the reporter molecule . Common blocking buffers include normal serum, non-fat dry milk, BSA or gelatin, and commercial blocking buffers. er molecules vary based on the nature of the detection method, and the most popular methods of detection are with enzyme- and fluorophore-mediated chromogenic and fluorescence detection, respectively. With chromogenic reporters, an enzyme label is reacted with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, enic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT. Fluorescent ers are small, organic molecules used for IHC detection. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully-quantitative data, respectively, to correlate the level of reporter signal to the level of n expression or localization.

After immunohistochemical staining of the target n, a second stain is often applied to provide st that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design. Hematoxylin, Hoechst stain and DAPI are ly used.

Mapping of epitopes recognized by antibodies can be performed as described in detail in "Epitope g Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN3 75-9 and in "Epitope Mapping: A Practical ch" cal Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay.

The term "immune effector functions" in the context of the present invention includes any functions mediated by components of the immune system that result in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition 3O of tumor dissemination and metastasis. Preferably, immune effector functions result in g of cancer cells. Preferably, the immune effector functions in the context of the present invention are antibody-mediated effector functions. Such functions comprise complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity , antibody-dependent cell-mediated phagocytosis (ADCP), ion of apoptosis in the cells carrying the tumor-associated antigen, for example, by binding of the antibody to a surface antigen, tion of CD40L-mediated signal transduction, for example, by binding of the antibody to the CD40 receptor or CD40 ligand (CD40L), and/or inhibition of proliferation of the cells carrying the tumor- ated antigen, ably ADCC and/or CDC. Thus, antibodies that are capable of mediating one or more immune effector functions are ably able to mediate killing of cells by inducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and/or phagocytosis, preferably by inducing CDC-mediated lysis and/or ADCC-mediated lysis. Antibodies may also exert an effect simply by binding to tumor- associated antigens on the surface of a cancer cell. For example, antibodies may block the on of the tumor—associated n or induce apoptosis just by binding to the tumor-associated antigen on the surface of a cancer cell.

ADCC describes the cell-killing ability of effector cells, in particular lymphocytes, which preferably es the target cell being marked by an antibody. ADCC preferably occurs when antibodies bind to antigens on cancer cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. Several families of Fc receptors have been fied, and specific cell populations characteristically express defined Fc ors. ADCC can be viewed as a ism to directly induce a variable degree of immediate tumor destruction that also leads to n presentation and the induction of tumor-directed T-cell responses. Preferably, in vivo induction of ADCC will lead to tumor-directed T-cell responses and further host- derived antibody responses.

CDC is another illing method that can be directed by antibodies. lgM is the most effective isotype for complement tion. lgGl and lgG3 are also both very effective at directing CDC via the classical complement-activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the uncloaking of multiple Clq binding sites in close ity on the CH2 domains of participating 3O antibody molecules such as lgG molecules (Clq is one of three subcomponents of complement C1). Preferably these uncloaked Clq binding sites convert the previously low-affinity Clq—lgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector—cell chemotactic/activating agents C3a and C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell and may lead to apoptosis.

The term "immune effector cells" in the context of the t invention relates to cells which exert effector functions during an immune reaction. For example, such cells secrete cytokines and/or ines, kill microbes, secrete antibodies, recognize ous cells, and optionally eliminate such cells. For example, immune effector cells comprise T-cells (cytotoxic T-cells, helper T-cells, tumor infiltrating T-cells), B-cells, l killer cells, neutrophils, macrophages, and dendritic cells.

A nucleic acid is ing to the invention preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro ribed RNA (IVT RNA). Nucleic acids include according to the ion genomic DNA, cDNA, mRNA, recombinantly prepared and chemically sized molecules. A c acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic can be employed for introduction into, i.e. transfection of, cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, g, and enylation.

The nucleic acids described herein may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, Viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome Vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors.

Expression vectors comprise plasmids as well as Viral vectors and generally contain a d coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host sm (e.g., bacteria,_ yeast, plant, insect, or mammal) or in in vitro expression systems. g vectors are generally used to er and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

As the vector for expression of an antibody, either of a vector type in which the antibody chains are t in different s or a vector type in which the antibody chains are present in the same vector can be used.

As used herein, the term "RNA" means a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2'—position of a beta-D-ribo-furanose moiety. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally ing RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non- nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non- standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or ucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

According to the present invention, the term "RNA" includes and preferably s to "mRNA" which means "messenger RNA" and relates to a "transcript" which may be produced using DNA as template and encodes a peptide or protein. mRNA typically ses a 5’ non translated , a protein or peptide coding region and a 3’ non translated region. mRNA has a limited halftime in cells and in vitro.

In the context of the t invention, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA.

The nucleic acids described according to the invention have preferably been isolated.

The term ”isolated nucleic acid" means according to the invention that the nucleic acid 3O was (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel- electrophoretic onation, or (iv) synthesized, for example by chemical synthesis. An ed nucleic acid is a nucleic acid which is ble for manipulation by inant DNA techniques.

Nucleic acids may, according to the invention, be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In preferred embodiments, a nucleic acid is functionally linked to expression control ces which may be homologous or heterologous with respect to said nucleic acid. The term "homologous" means that the nucleic acids are also functionally linked naturally and the term "heterologous" means that the nucleic acids are not functionally linked naturally.

A nucleic acid and an expression l sequence are "functionally" linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said nucleic acid is under the control or under the influence of said sion control sequence. If the nucleic acid is to be translated into a functional protein, then, with an expression l sequence functionally linked to a coding. sequence, induction of said expression control ce results in transcription of said nucleic acid, without g a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide.

The term "expression control sequence" or "expression control element" comprises according to the invention promoters, me binding sites, ers and other control elements which regulate transcription of a gene or translation of a mRNA. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of expression control sequences may vary as a function of the s or cell type, but generally comprises 5’-untranscribed and 5’- and 3’- untranslated ces which are involved in initiation of transcription and translation, respectively, such as TATA box, g sequence, CAAT sequence, and the like.

More specifically, 5’-untranscribed expression control sequences comprise a er region which es a promoter sequence for transcriptional control of the functionally linked nucleic acid. Expression control sequences may also comprise er sequences or upstream activator sequences.

According to the invention the term "promoter" or "promoter region" relates to a nucleic acid ce which is located upstream (5’) to the nucleic acid sequence being expressed and controls expression of the sequence by providing a recognition and binding site for RNA-polymerase. The "promoter region" may include further recognition and binding sites for further factors which are involved in the regulation of transcription of a gene. A er may control the transcription of a prokaryotic or eukaryotic gene. Furthermore, a promoter may be "inducible" and may initiate transcription in response to an inducing agent or may be "constitutive" if transcription is not controlled by an inducing agent. A gene which is under the control of an inducible er is not expressed or only expressed to a small extent if an inducing agent is absent. In the presence of the inducing agent the gene is ed on or the level of transcription is sed. This is mediated, in general, by binding of a specific transcription factor.

Promoters which are preferred according to the invention include promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMV promoter, and artificial hybrid promoters thereof (e.g. CMV) where a part or parts are fused to a part or parts of promoters of genes of other cellular proteins such as e.g. human GAPDH (glyceraldehydephosphate dehydrogenase), and including or not including (an) additional (s).

The term "expression" is used herein in its broadest meaning and comprises the production of RNA or of RNA and n or peptide. With respect to RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. Expression may be transient or may be stable. According to the invention, the term expression also includes an "aberrant expression" or "abnormal expression".

"Aberrant expression" or "abnormal expression" means according to the ion that expression is altered, preferably sed, ed to a reference, e.g. a state in a subject not having a disease associated with aberrant or abnormal expression of a certain protein, e. g., a tumor-associated antigen. An increase in sion refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%, or more. In one embodiment, expression is only found in a diseased tissue, while 3O expression in a healthy tissue is repressed.

The term "specifically expressed" means that a protein is essentially only expressed in a c tissue or organ. For example, a tumor—associated antigen specifically expressed in gastric mucosa means that said protein is primarily expressed in gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other tissue or organ types. Thus, a protein that is ively sed in cells of the gastric mucosa and to a significantly lesser extent in any other tissue is specifically expressed in cells of the gastric mucosa. In some embodiments, a tumor-associated n may also be specifically expressed under normal conditions in more than one tissue type or organ, such as in 2 or 3 tissue types or organs, but preferably in not more than 3 different tissue or organ types. In this case, the tumor-associated antigen is then specifically expressed in these organs.

The term "translation" ing to the invention relates to the process in the ribosomes of a cell by which a strand of ger RNA directs the assembly of a sequence of amino acids to make a n or peptide.

According to the invention, the term "nucleic acid encoding" means that nucleic acid, if present in the appropriate environment, preferably within a cell, can be expressed to produce a protein or peptide it encodes.

The term "peptide" comprises oligo- and ptides and refers to substances comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids joined covalently by peptide bonds. The term in" refers to large peptides, preferably to peptides with more than 100 amino acid residues, but in general the terms "peptides" and "proteins" are synonyms and are used interchangeably herein.

Preferably, the proteins and peptides described according to the invention have been isolated. The terms "isolated protein" or "isolated e" mean that the protein or peptide has been separated from its natural environment. An isolated protein or e 3O may be in an essentially d state. The term "essentially purified" means that the protein or peptide is essentially free of other substances with which it is associated in nature or in viva.

The teaching given herein with respect to specific amino acid sequences, e.g. those shown in the sequence listing, is to be construed so as to also relate to modifications, i.e. variants, of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino acid sequences exhibiting properties identical or similar to those of the c amino acid sequences. One important property is to retain binding of an antibody to its target. Preferably, a sequence modified with respect to a specific sequence, when it es the specific sequence in an antibody retains binding of said antibody to the target.

It will be appreciated by those d in the art that in particular the ces of the CDR sequences, hypervariable and variable s can be modified without losing the ability to bind to a target. For example, CDR sequences will be either identical or highly homologous to the CDR sequences specified herein.

By "highly homologous" it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made.

The term "variant" according to the invention also includes mutants, splice variants, conformations, isoforms, allelic variants, species ts and species homologs, in particular those which are naturally present. An c variant s to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous c variants for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence.

For the purposes of the present ion, "variants" of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid tution variants. Amino acid deletion variants that se the deletion at the N-terminal and/or C-terminal end of the protein are 3O also called N-terminal and/or inal truncation variants.

Amino acid ion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with riate screening of the resulting product is also possible.

Amino acid addition ts comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of l, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein.

Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not ved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., tutions of similarly charged or uncharged amino acids. A conservative amino acid change es substitution of one of a family of amino acids which are related in their side chains. lly occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, ine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, phan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified y as aromatic amino acids.

Preferably the degree of similarity, preferably ty between a given amino acid ce and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of similarity or identity is given for the entire length of the nce amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, ably EMBOSS::needle, Matrix: 62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" indicates the tage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

The term ntage identity" is intended to denote a tage of amino acid residues which are identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences n the two sequences being distributed randomly and over their entire length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these ces after having aligned them optimally, said comparison being carried out by segment or by "window of comparison" in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local gy algorithm of man and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci.

USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The tage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences.

Homologous amino acid ces exhibit according to the invention at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

The amino acid sequence variants described herein may readily be ed by the skilled , for e, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing proteins and es having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.

Furthermore, the peptides and amino acid ts described herein may be readily prepared with the aid of known peptide synthesis ques such as, for example, by solid phase synthesis and similar methods.

The invention includes derivatives of the peptides or proteins described herein which are comprised by the terms "peptide" and "protein". According to the invention, "derivatives" of proteins and peptides are modified forms of proteins and peptides. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecules ated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, "derivatives" of proteins or peptides include those modified analogs resulting from glycosylation, acetylation, orylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term "derivative" also extends to all functional chemical equivalents of said proteins and peptides. Preferably, a modified peptide has increased stability and/or increased immunogenicity.

According to the ion, a variant, derivative, d form, fragment, part or portion of an amino acid sequence, peptide or protein ably has a functional property of the amino acid sequence, peptide or protein, respectively, from which it has been derived, i.e. it is onally equivalent. In one embodiment, a variant, derivative, modified form, fragment, part or portion of an amino acid ce, peptide or protein is logically equivalent to the amino acid sequence, peptide or protein, respectively, from which it has been derived. In one embodiment, the functional property is an logical property.

The term "derived" means ing to the invention that a particular entity, in particular a particular sequence, is t in the object from which it is derived, in particular an organism or le. In the case of amino acid sequences, especially particular sequence regions, "derived" in particular means that the relevant amino acid sequence is derived from an amino acid sequence in which it is present.

The term "cell" or "host cell" ably is an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as s, organelles, or genetic material. An intact cell preferably is a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions. The term "cell" includes according to the invention prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, and primates. The cells may be derived from a large number of tissue types and include y cells and cell lines. The term "cell" includes non-cancerous cells and cancer cells such as cells of the cancer types disclosed herein.

A cell which comprises a nucleic acid molecule preferably express the peptide or protein encoded by the nucleic acid.

"Target cell" shall mean a cell which is a target for an immune se such as an antibody. Target cells include any undesirable cell such as a cancer cell as described herein. In preferred embodiments, the target cell is a cell expressing CLDN18.2. Cells sing CLDN18.2 typically include cancer cells.

The terms "transgenic animal" refers to an animal having a genome comprising one or more transgenes, preferably antibody heavy and/or light chain enes, or transchromosomes (either integrated or non-integrated into the 's natural genomic DNA) and which is preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with CLDN18.2 antigen and/or cells expressing CLDN18.2. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, e.g., HuMAb mice, such as HCo7 or HC012 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as bed in WO 02/43478. Such transgenic and transchromosomal mice may be e of producing multiple isotypes of human monoclonal antibodies to CLDN18.2 (e. g., lgG, lgA and/or IgE) by oing V-D-J recombination and e ing.

The term "immunologically lent" means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect such as induction of a humoral immune reaction, the strength and/or duration of the induced immune reaction, or the specificity of the immune reaction. In the context of the present invention, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of a peptide or peptide variant used for zation or an antibody. A particular immunological 2O property is the ability to bind to antibodies and, where appropriate, generate an immune response, preferably by stimulating the generation of antibodies. For e, an amino acid sequence is immunologically equivalent to a nce amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction, preferably antibodies, having a specificity of reacting with the reference amino acid sequence, such as the reference amino acid sequence forming part of CLDN18.2.

The invention provides methods for detecting the ce of CLDN18.2 antigen in a sample, or measuring the amount of CLDN18.2 antigen, comprising contacting the sample, and optionally a control sample, with an antibody of the ion which binds to CLDN18.2, under conditions that allow for formation of a complex between the antibody and CLDN18.2. The formation of a complex is then detected, wherein a difference in x formation between the sample compared to a control sample is indicative for the presence of CLDNl 8.2 antigen in the sample.

Methods as described above are useful, in particular, for diagnosing CLDN18.2-related diseases such as cancer diseases. Preferably an amount of CLDN18.2 in a sample which is higher than the amount of CLDN18.2 in a reference or l sample is indicative for the presence of a CLDN18.2-related disease in a subject, in particular a human, from which the sample is derived.

When used in methods as described above, an antibody bed herein may be provided with a label that functions to: (i) provide a detectable ; (ii) ct with a second label to modify the detectable signal provided by the first or second label, e.g.

FRET (Fluorescence Resonance Energy Transfer); (iii) affect ty, e.g. electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture moiety, e.g., affinity, antibody/antigen, or ionic complexation. Suitable as label are structures, such as fluorescent labels, luminescent labels, chromophore labels, radioisotopic labels, isotopic labels, preferably stable ic labels, isobaric labels, enzyme labels, particle labels, in particular metal particle labels, magnetic particle labels, polymer le labels, small organic molecules such as , ligands of receptors or binding molecules such as cell adhesion ns or lectins, label-sequences comprising nucleic acids and/or amino acid residues which can be detected by use of binding agents, etc. Labels comprise, in a nonlimiting manner, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, amide, sodium tyropanoate and radio diagnostic, including positron emitters such as fluorine-1 8 and carbon-11, gamma emitters such as iodine—123, technetium-99m, iodine-131 and -1 1 l, nuclides for nuclear magnetic resonance, such as e and gadolinium.

According to the invention, a "reference" such as a reference sample or reference organism may be used to correlate and compare the results obtained in the s of the invention from a test sample or test organism. Typically the reference organism is a healthy organism, in particular an organism which does not suffer from a disease such as a cancer disease. A "reference value" or "reference level" can be determined from a reference empirically by measuring a sufficiently large number of references. Preferably the reference value is determined by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.

"Reduce" or "inhibit" as used herein means the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level. The term "inhibit" or similar s includes a te or essentially complete tion, i.e. a reduction to zero or essentially to zero.

Terms such as "increase" or "enhance" preferably relate to an increase or enhancement by about at least 10%, ably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

The agents, compositions and methods described herein can be used to diagnose a t with a disease. Diseases which can be diagnosed encompass all diseases expressing CLDN18.2. Particularly preferred diseases are cancer diseases such as cancer diseases described herein.

According to the invention, the term "disease" refers to any pathological state, ing cancer diseases, in particular those forms of cancer diseases described herein.

The term l" such as used in the terms "normal tissue" or "normal ions" refers to healthy tissue or the conditions in a healthy subject, i.e., non-pathological conditions, wherein "healthy" preferably means non-cancerous.

"Disease ing cells expressing CLDN18.2" means according to the invention that expression of CLDN18.2 in cells of a diseased tissue or organ is preferably sed compared to the state in a healthy tissue or organ. An increase refers to an se by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only found in a ed tissue, while expression in a healthy tissue is repressed.

According to the invention, diseases involving or being associated with cells expressing CLDN18.2 include cancer diseases, in particular those forms of cancer described herein.

According to the ion, the term "tumor" or "tumor disease" refers to a swelling or lesion formed by an al growth of cells (called neoplastic cells or tumor cells). By "tumor cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that ted the new growth cease.

Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre-malignant or malignant.

A benign tumor is a tumor that lacks all three of the malignant properties of a cancer.

Thus, by definition, a benign tumor does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not spread to non-adjacent tissues (metastasize). Common examples of benign tumors include moles and uterine fibroids.

The term "benign" implies a mild and nonprogressive disease, and indeed, many kinds of benign tumors are ss to the health. However, some neoplasms which are defined as "benign tumors" because they lack the invasive properties of a cancer, may still produce negative health effects. Examples of this include tumors which produce a "mass effect” (compression of vital organs such as blood vessels), or "functional" tumors of ine tissues, which may overproduce certain hormones (examples include thyroid adenomas, adrenocortical adenomas, and pituitary adenomas).

Benign tumors typically are surrounded by an outer surface that ts their ability to behave in a malignant manner. In some cases, certain n" tumors may later give rise to malignant cancers, which result from additional genetic changes in a subpopulation of the tumor's neoplastic cells. A prominent example of this phenomenon is the r adenoma, a common type of colon polyp which is an important sor to colon cancer. The cells in tubular adenomas, like most tumors which frequently progress to cancer, show certain abnormalities of cell maturation and appearance 3O collectively known as dysplasia. These cellular abnormalities are not seen in benign tumors that rarely or never turn cancerous, but are seen in other pre-cancerous tissue abnormalities which do not form discrete , such as pre-cancerous lesions of the e cervix. Some authorities prefer to refer to dysplastic tumors as "pre-malignant", and reserve the term n" for tumors which rarely or never give rise to cancer. sm is an abnormal mass of tissue as a result of neoplasia. Neoplasia (new growth in Greek) is the abnormal proliferation of cells. The growth of the cells exceeds, and is uncoordinated with that of the normal tissues around it. The growth persists in the same excessive manner even after cessation of the stimuli. It usually causes a lump or tumor. sms may be benign, pre-malignant or malignant.

"Growth of a tumor" or "tumor growth" according to the invention relates to the tendency of a tumor to increase its size and/or to the tendency of tumor cells to proliferate.

Cancer (medical term: ant neoplasm) is a class of diseases in which a group of cells display uncontrolled growth ion beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumors, which are self-limited, and do not invade or metastasize. Most cancers form a tumor but some, like leukemia, do not.

According to the invention, the terms "cancer" and "tumor" or "cancer disease" and "tumor disease" are lly used interchangeably herein to refer to diseases n cells display an uncontrolled growth and ally invasion and/or metastasis.

Preferably, a "cancer disease" ing to the invention is characterized by cells expressing CLDN18.2. A cell expressing CLDN18.2 preferably is a cancer cell, preferably of the tumors and cancers described herein. Preferably, such cell is a cell other than a stomach cell.

Cancers are classified by the type of cell that les the tumor and, therefore, the tissue presumed to be the origin of the tumor. These are the histology and the location, respectively.

The term r" according to the invention comprises leukemias, seminomas, melanomas, teratomas, lymphomas, lastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, al cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine , head and neck cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate , cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, al carcinomas, or metastases of the cancer types or tumors described above. The term cancer ing to the invention also comprises cancer metastases.

The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). There are three main sub-types of the non-small cell lung carcinomas: us cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas account for approximately 10% of lung cancers. This cancer usually is seen erally in the lungs, as opposed to small cell lung cancer and squamous cell lung cancer, which both tend to be more centrally located.

According to the invention, a "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common s, including the common foms of breast, prostate, lung and colon cancer.

By tasis" is meant the spread of cancer cells from its original site to another part of the body. The ion of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs.

Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the y tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention relates to nt metastasis" which relates to a metastasis which is remote 3O from the primary tumor and the regional lymph node system.

The cells of a secondary or metastatic tumor are like those in the original tumor. This means, for example, that, if ovarian cancer asizes to the liver, the secondary tumor is made up of abnormal ovarian cells, not of abnormal liver cells. The tumor in the liver is then called metastatic ovarian cancer, not liver cancer.

A relapse or recurrence occurs when a person is ed again by a condition that ed them in the past. For example, if a patient has suffered from a cancer disease, has ed a successful treatment of said disease and again develops said disease said newly developed disease may be considered as relapse or recurrence. However, according to the invention, a e or recurrence of a cancer disease may but does not necessarily occur at the site of the original cancer disease. Thus, for example, if a patient has suffered from ovarian tumor and has received a successful ent a relapse or recurrence may be the occurrence of an ovarian tumor or the ence of a tumor at a site different to ovary. A e or ence of a tumor also includes situations wherein a tumor occurs at a site different to the site of the original tumor as well as at the site of the original tumor. Preferably, the original tumor for which the patient has received a treatment is a y tumor and the tumor at a site different to the site of the original tumor is a secondary or metastatic tumor.

By "treat" is meant to administer a compound or composition to a subject in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the ncy or severity of symptoms and/or recurrences in a subject who currently has or who previously has had a disease; and/or prolong, i.e. increase the an of the subject.

In particular, the term "treatment of a e" includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting ssion or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

By "being at risk" is meant a subject, i.e. a patient, that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had, or who currently has, a disease, in particular cancer is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease. Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases.

The term "immunotherapy" s to a ent involving a specific immune reaction.

In the context of the present invention, terms such as "protect", "prevent", "prophylactic", "preventive", or "protective" relate to the prevention or treatment or both of the ence and/or the propagation of a disease in a subject and, in particular, to minimizing the chance that a subject will develop a disease or to delaying the development of a disease. For example, a person at risk for a tumor, as described above, would be a candidate for therapy to prevent a tumor. therapy may be performed using any of a variety of techniques, in which agents function to remove antigen- expressing cells from a patient.

Within certain ments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against diseased cells with the administration of immune response-modifying agents (such as reactive peptides and nucleic acids).

Within other embodiments, therapy may be passive immunotherapy, in which treatment involves the delivery of agents with ished tumor-immune reactivity (such as antibodies) that can directly or indirectly mediate antitumor effects and does not arily depend on an intact host immune system.

The term "in vivo" relates to the situation in a subject.

The terms "subjectH II , individual", "organism" or nt" are used interchangeably and relate to vertebrates, preferably mammals. For example, mammals in the context of the present ion are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc. as well as animals in captivity such as animals of zoos. The term "animal" as used herein also includes humans. The term "subject" may also include a 3O patient, i.e., an animal, ably a human having a disease, preferably a disease as described herein.

According to the invention, a "sample" may be any sample useful according to the present invention, in particular a biological sample such a tissue sample, including body ‘ fluids, and/or a cellular sample and may be obtained in the conventional manner such as by tissue biopsy, including punch biopsy, and by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the invention, the term "sample" also includes processed s such as ons or isolates of biological samples, e.g. nucleic acid and peptide/protein isolates. Preferably a sample contains cells or tissue of the organ which is to be examined, e.g. which is to be diagnosed for cancer. For example, if the cancer to be diagnosed is lung cancer a sample may contain cells or tissue ed from lung.

According to the invention a sample may be a sample such as a bodily sample derived from a patient containing or being expected of containing tumor or cancer cells. The bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases or any other sample containing tumor or cancer cells.

The present ion is described in detail by the figures and es below, which are used only for illustration purposes and are not meant to be limiting. Owing to the description and the examples, further embodiments which are se included in the invention are accessible to the skilled worker.

Figures: Figure 1: Sequence alignment of n 18 proteins (human/murine) The sequence alignment shows the high homology between human and mouse claudin 18.2 and human claudin 18.1 and claudin 18.2.

Figure 2: Recombinant protein including the C-terminal portion of CLDN18.2 (aa191-261) used for immunization of mice Figure 3: Sequence-analysis of 43-14A and 35-22A antibodies Examples The techniques and methods used herein are described herein or carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods including the use of kits and reagents are carried out according to the manufacturers’ information unless specifically indicated.

Example 1: Generation of monoclonal antibodies The aim of this project was to generate murine monoclonal CLDN18-specific antibodies capable of detecting CLDN18.2 expressing tumor cells in stomach CA, gus CA, pancreas CA and lung CA FFPE tissues.

To generate a highly specific, high affinity diagnostic CLDN18.2 antibody it was essential to start immunization protocols with a big variation of different immunogens and nts. During the project about 100 mice (C57Bl/6 and Balb/c) were inoculated, using various immunization strategies to trigger an 18 immune response.

To trigger the mouse immune system and to me the immune tolerance we used like-particles (VLP), peptide—conjugates and recombinant proteins coding for different parts of human CLDN18.2 expressed as recombinant fusion proteins with different expression rs (tags).

Out of 13 different immunization strategies the best results were achieved by treating mice with gged CLDN18 C-termal recombinant protein (see Figure 2; Immunization #20) in combination with various adjuvants (see Table 1, below, fusion ).

One candidate (35-22A) ed from a 4 step-immunization gy (30 days). A further candidate (43—l4A) was generated following a 7-step-immunization protocol (79 days)(see Table 1, below, fusion 43).

Two days before splenectomy, the mice were boosted to activate the targeted s.

On the day of fusion the mouse splenocytes were isolated and fused to a mouse myeloma cell line Ag8.653. For fusion of mouse cells to the myeloma we followed the standard protocol published by Kohler and Milstein 1975. After HAT selection supernatants were tested in ELISA for secretion of antibodies recognizing the antigen used for immunizations.

The oma cells of ELISA positive supernatants were subcloned to generate monoclonal hybridomas and supernatants of the subcloned hybridoma cells were rescreened in ELISA. Hybridoma cells of positive clones were expanded and atants analyzed further. e 2: Western Blot Screen of monoclonal hybridoma supernatants To answer the question if ELISA-positive dies in the supernatants are able to bind to either recombinant claudin 18 or protein lysates from stable transfected claudin l8 expressing HEK293 cells Western Blot—analysis was med. Antibodies which were able to bind specifically to claudin 18 in a Western Blot-analysis were expanded. Cells were cryoconserved and antibodies purified via MABselect (FPLC). The antibodies selected by the Western Blot screening were purified and evaluated for their ability to bind their antigen in formalin fixed n-embedded tissues (FFPE) by immunohistochemistry.

Example 3: Histological analysis - first screen of Western Blot ve antibodies The aim of this experiment was to check the CLDN18 specificity and sensitivity of the antibodies. This was done by using CLDN18 expressing FFPE normal stomach tissue. 3O In a first experiment the Western Blot tested purified antibodies were analyzed at a concentration of 0,5 ug/ml on human stomach FFPE sections. Antibodies which performed well and did not produce high amounts of background were further titrated to 0,2 & 0,1 ug/ml on various normal h tissues to test the sensitivity and specificity.

In later development stages the freshly generated dies were directly tested at a concentration of 0,2 ug/ml because the best antibody already performed very well at 0,2 ug/ml and did serve as a benchmark. Antibodies generating strong signals on the mucosa lium of the tested human stomach s and no background on the adjacent mucosa s were selected for further titration experiments and specificity analysis. Two antibodies did perform outstanding: 35-22A and 43-14A; see Tables 2 and 3, below.

Antibodies producing strong signals on the tested normal stomach tissue were further analyzed on cancer tissues. The corresponding hybridoma cells were adapted to serum free media. The signals produced using mumAb 43—14A were slightly er than the signals produced using mumAb 35-22A; see Table 4, below.

Example 4: Histological in depth analysis and dy characterization The serum free produced antibodies were used to stain stomach CA tissue microarrays (TMA). The amount of stained cases, the strength of the signal and the amount of positive tumor cells were ed.

The staining intensities of the mumAbs 35-22A and 43-14A were excellent. No significant differences in the staining pattern and only slight differences in the staining intensities between the tested antibodies 35-22A and 43-14A were detected.

Example 5: Analysis of dy specificity using a normal tissue panel The selected antibodies were tested on various, relevant normal tissues to ensure the high CLDN18 target specificity; see Tables 5A and SB, below.

No significant ences in the ng pattern and staining intensities of the antibodies 35-22A and 43-14A were visible in the previous experiments. Therefore the 3O antibodies were subjected to staining experiments with a more clinically oriented protocol. To simulate the staining processes applied in standard pathology labs a One- Day-Protocol with a short (1 hour) primary antibody incubation step was established.

In all analyzed cases mumAb 43-14A performed extremely good and even better compared to mumAb 35-22A; see Table 6, below.

Example 6: In depth analysis on relevant tissue - respiratory epithelium mumAb 43-14A was additionally analyzed on various relevant respiratory tissues to ensure its specificity, especially in target tissues of the lung/bronchial tract. For these tissues the expression of CLDN18.1 was reported. To analyze whether the diagnostic antibody cross reacts with the ronchial expressed isoform of CLDN18.1 all available lung/bronchial tissues were screened. No s were detected with lung and bronchial tissues; see Table 7, below. The CLDN18 isoform expressed in these respiratory tissues is not recognized by the antibody 43—14A.

Example 7: Epitope mapping of the mumAbs 43—14A and 35-22A Peptide ELISA was med to identify the antibody-binding epitopes on CLDN18.2.

Each purified antibody was tested on overlapping peptides covering the inal sequence of CLDN18.2. 35-22A and 43-14A both showed specific binding to an e mapping to the e TEDEVQSYPSKHDYV. The following sequence was determined as the ve sequence: EVQSYPSKHDYV.

Example 8: Sequence analysis of the mumAbs 43—14A and 35-22A An analysis of the sequence of the antibodies 43-14A and 35-22A is shown in Figure 3. e 9: Staining of different cancer tissues Immunohistochemistry (IHC) was performed on slides of 4% ed formalin fixed paraffin embedded samples. Paraffin embedding was performed according to standard protocols.

After ffinization, all slides were subjected to antigen retrieval by boiling in 10 mM citric acid supplemented with 0.05% Tween—20 (pH 6.0) at 120°C for 10 min, subsequently quenched (by 2% H202) blocked and incubated overnight at 4°C with 0.2 to 0.5 ug/ml diagnostic monoclonal mouse LNDN18.2 antibody 43-14A or 35— 22A. dy binding was visualized with horseradish-peroxidase-labeled secondary antibodies using the polymer-based Powervision antibody (Power Vision HRP goat-a- mouse; Immunologic, Duiven, The lands) and a substrate—chromogen solution (VectorRed; Vector Labs, game, USA). Sections were subsequently counter- stained with s haematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and subjected to tion by the raters.

Histological assessment All samples were analyzed regarding the relative proportion of positive stained tumor cells in relation to all visible tumor cells for each section. The intensity of the staining was classified as negative (-), weakly ve (1+), medium positive (2+) and strongly positive (3+). Only membranous ng was considered as positive. Human stomach tissue served as positive control for each staining. Since PanIN (pancreatic intraepithelial neoplasia) are frequently found strong positive, those areas were also considered as internal staining intensity reference for strong positivity (3+).

Strong, membranous signals were generated by both antibodies in pancreatic, esophageal and stomach cancerous tissues (Table 8) or with antibody 43-14A in lung cancerous tissues (Table 9). The number of positive tumor cells varied interindividually between the different tumor cases. The biggest part of the analysed samples was 2+ to 3+ positive. coHumuuchHEE oasHo> H1OON HOOON HOOON H1OON 350m . . .

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Tumor % positive cells cells H/2010/10869 IVG 43-14A pancreas CA Acinar cell carcinoma - 0 Neuroendocrine 0522 43-14A pancreas CA ++ , 70 carcmoma Neuroendocrine H/2006/2 2797* * IA 43-14A pancreas CA 0 carcinoma Neuroendocrine 308/6284-VC 35-22A pancreas CA 0 carcinoma, NET 61 ndocrine 808/8549-4 35-22A pancreas CA _ carcinoma, NET 61 805/8523-3 806/16136-NP2 807/14168 -—_-m 807/14935 pancreas CA _—-I 33-lll3 pancreas CA 807/7430 pancreas CA 808/5618—2 pancreas CA BlO/l4198—VC pancreas CA PDAC +++ BlO/706-VC3 pancreas CA PDAC +++ Bll/2059-D pancreas CA PDAC +++ 311/4084 35-22A pancreas CA PDAC +++ 112500530 43-14A as CA PDAC 112500527 43-14A pancreas CA PDAC ++ HO 112500524 43-14A pancreas CA PDAC ++ U1O 523 43-14A pancreas CA PDAC ++ l-| OO 112500525 43-14A pancreas CA PDAC +++ U) 0 112500528 43-14A pancreas CA PDAC +++ H/2008/13074 43-14A pancreas CA + In.++ /13194 43—14A pancreas CA /380 43-14A pancreas CA —-+++ 90 H/2009/11847 43-14A pancreas CA 15 H/2009/20336 43-14A pancreas CA 90 H/2009/23598 V” B 43-14A pancreas CA H/2010/11569 43'14A pancreas CA ' H/2011/17191 VA 43-14A as CA so H/2009/4917 43-14A pancreas CA 70 pancreas CA H/2010/6709 43-14A pancreas CA 112500519 esophagus CA so BOQ/l491-Ill-2 adenocarcinoma 06/14957-2 adenocarcinoma 1125005.17 arcinoma 10b06684-ll—3 adenocarcinoma +++ “ 112500310 adenocarcinoma 11250056 adenocarcinoma -“ PDAC = pancreatic ductal adenocarcinoma Table 9: Analysis of .2 expression in lung cancerous tissues, using monoclonal murine antibody 43-14A - _-cells mug/m._— 807/4771-3 0,2p.g/m| carcinoid - 307/5358 |l2 0,2ug/ml carcinoid — Boa/33824 awe/ml Boa/ssas-ne awe/ml l_809/12293ll2 0,2p.g/m| carcinoma,spinoce|lular — BOG/12562- 0,2p.g/ml Carcinoma, largecell - |||3 306/10820-ll2 0,2ug/ml carcinoma, adeno 806/10876-2 0,2p.g/m| carcinoma, adeno — - 0,2p.g/m| carcinoma, adeno +++ 80% 307/03255 0,2p.g/ml carcinoma, adeno +++ 5% 807/2296-3 0,2p.g/m| carcinoma, adeno _— 0,2ug/ml carcinoma, adeno ++ <5% BlO/lZ713—ll1 carcinoma, adeno ++ <5% 197-2 0,2ug/ml carcinoma, adeno _— BlO/16367-V2 0,2ug/ml carcinoma, adeno I1% 43 ||3 0,2ug/ml carcinoma, adeno, I squamous 916-1 ml carcinoma, large cell I BlO/lO814-3 0,2ug/ml oma, large cell + 1% neuro endocrine ———_ 306/3204—2 0,2ug/ml carcinoma,squamousce|l_ 808/292-2 0,2ug/ml carcinoma,squamouscel| ++ seam-w awe/ml—__ B09/45-2 _— ————— alo/lsm —_—— ale/1704s_— ale/lame_— BO7/6782—1 0,2ug/ml ++ <1% cell BO7/9741—5 0,2p.g/ml carcinoma;adeno,clear cell 808/16425—2 0,2ug/ml largecellneuro—endocrine ++ <5% 19-V3 0,2ug/ml non—small cell CA 99 V3 0,2ug/ml nt small cell long CA <1% BO8/12010-2 0,2ug/ml nt small cell long CA ++ <1% BIO/17662— 0,2p.g/m| small cell carcinoma Applicant's or agent‘s International applieationNo. “‘6Wm 342-70 PCT PCT/EP20 13/00 133 1 L INDICATIONS NG TO TED RGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13bis) A. The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 4 .Iine 32 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [2] Name ofdepositai'y institution DSMZ-Deutsche ng von Mikroorganismen und Zellkulturen GmbH Address of depositary institution (including postal code and country) Inhoffenstr. 7 B 38124 Braunschweig Date of deposit Accession Number October 6. 2011 DSM ACC3144 C. ADDITIONAL INDICATIONS (leave blank. lfnot applicable) This information is ued on an additional sheet I - Mouse (Mus musculus) myeloma AgB.653 fused with mouse (Mus musculus) splenocytes - Hybridoma secreting antibody against human CLDN18.2 D. ATED STATES FOR WHICH INDICATIONS ARE MADE lift/w indications are not/or all ated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifnnt applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature ofthe tions e.g., ’Viceersion Number ofDeputit”) For receiving Office use only For International Bureau use only This sheet was received with the international application I: This sheet was received by the lntemational Bureau on: Authorized officer Authorized officer Dobbelaere, Julie Form PCT/RO/l 34 (Julyl998; reprint January 2004) Additional Sheet for Biological Material Identification of further deposits: 1) The Name and Address of depositary institution for the deposits are: DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr. 7 B 38124 Braunschweig DE Date of desposit Accession Number The indications made below relate to the deposited microorganism in the ption on the following page(s) October 6, 2011 DSM ACC3143 age 4, line 33 Additional Indications for all above mentioned deposits: - Mouse (Mus musculus) myeloma Ag8.653 fused with mouse (Mus musculus) splenocytes - oma ing dy against human CLDN18.2 2) Depositor: All above mentioned depositions were made by: Ganymed Pharmaceuticals AG FreiligrathstraBe 12 55131 Mainz

Claims (14)

The claims defining the invention are as follows:

1. An antibody binding to CLDN18.2 or an antigen-binding fragment thereof, being selected from the group consisting of: (i) an dy produced by a clone deposited under the accession no. DSM 4 (muAB 43-14A) or DSM 3 (muAB 35-22A), (ii) an antibody which is a chimerized or humanized form of the antibody under (i), (iii) an antibody which has the specificity of the antibody under (i), wherein said antibody binds to a peptide consisting of the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6), and (iv) an antibody sing the antigen binding portion or antigen binding site of the dy under (i), or an antigen-binding fragment of the antibody under any one of (i) to (iv), wherein the CDR sequences of the antibody according to (ii)-(iv) are identical to the CDR sequences of the antibody according to (i).

2. The dy or an antigen-binding fragment of claim 1 thereof, wherein the dy comprises: an antibody heavy chain comprising: a CDR1 sequence according to SEQ ID NO:8, a CDR2 sequence according to SEQ ID NO:9, and a CDR3 ce according to SEQ ID NO:10, an antibody light chain comprising: a CDR1 sequence according to SEQ ID NO:12, a CDR2 sequence according to SEQ ID NO:13, and a CDR3 sequence according to SEQ ID NO:14, an antibody heavy chain comprising: a CDR1 sequence according to SEQ ID NO:16, a CDR2 sequence according to SEQ ID NO:17, and a CDR3 sequence according to SEQ ID NO:18, an dy light chain comprising: a CDR1 sequence according to SEQ ID NO:20, a CDR2 sequence according to SEQ ID NO:21, and a CDR3 sequence according to SEQ ID NO:22.

3. The antibody of claim 2, wherein the antibody (A) comprises the le region of SEQ ID NO:7 or 11, and the antibody (B) comprises the variable region of SEQ ID NO:15 or

4. The antibody of claims 2 or 3, wherein the antibody (A) comprises the amino acid sequence of SEQ ID NO:7 or 11, and the antibody (B) comprises the amino acid sequence of SEQ ID NO:15 or 19.

5. A conjugate comprising an dy or antigen-binding fragment of any one of claims 1 to 4 coupled to at least one detectable label.

6. An in vitro hybridoma cell line capable of producing the antibody of any one of claims 1 to 5.

7. A hybridoma ted under the accession no. DSM ACC3144 (muAB 43-14A) or DSM 3 (muAB 35-22A).

8. A method for detecting CLDN18.2 or determining the quantity of CLDN18.2 in a sample comprising the steps of: (i) contacting a sample with the antibody or n-binding fragment of any one of claims 1 to 4 or the conjugate of claim 5 and (ii) detecting the formation of a complex or determining the quantity of a complex between the antibody, the antigen-binding fragment or the conjugate and CLDN18.2.

9. A method for determining whether cells express CLDN18.2 comprising the steps of: (i) contacting a cellular sample with the dy or antigen-binding fragment of any one of claims 1 to 4 or the conjugate of claim 5 and (ii) detecting the formation of a complex between the antibody, the antigen-binding fragment or the conjugate and CLDN18.2 expressed by cells in said sample.

10. A method for diagnosis, detection or monitoring of cancer comprising the steps of: (i) contacting a biological sample with the antibody or antigen-binding nt of any one of claims 1 to 4 or the ate of claim 5 and (ii) detecting the formation of a complex and/or determining the quantity of a complex between the antibody, the antigen-binding fragment or the conjugate and .2.

11. A method for determining whether a cancer is treatable by a cancer therapy targeting CLDN18.2 comprising the steps of: (i) contacting a sample comprising cancer cells with the antibody or antigen-binding fragment of any one of claims 1 to 4 or the ate of claim 5 and (ii) detecting the formation of a complex between the dy, the antigen-binding fragment or the conjugate and CLDN18.2.

12. A diagnostic test kit which comprises the antibody or antigen-binding fragment of any one of claims 1 to 4 or the conjugate of claim 5.

13. The antibody or antigen-binding fragment of any one of claims 1 to 4, the conjugate of claim 5, the oma of claim 6 or 7, or the method of any one of claims 8 to 11, wherein said CLDN18.2 comprises the amino acid sequence according to SEQ ID NO: 2 of the sequence g or a variant of said amino acid sequence.

14. The antibody or antigen-binding fragment of any one of claims 1 to 4, the conjugate of claim 5 or 13, the hybridoma of claims 6, 7 or 13, or the method of any one of claims 8 to 11 or 13, substantially as herein described with nce to the figures and/or examples, excluding comparative examples.