RU2815883C1 - Antibodies useful in diagnosing cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are antibodies and their antigen-binding fragments, which bind to claudin 6 (CLDN6), as well as conjugates of these antibodies for CLDN6 detection. Hybridomas producing said antibodies are also provided. Anti-CLDN6 antibodies are used to diagnose cancer and to determine expression of CLDN6 cancer cells.

EFFECT: invention provides binding to CLDN6 in FFPE tissues affected by cancer, with high specificity and low background activity.

28 cl, 6 dwg, 4 tbl, 9 ex

Description

Claudins are integral membrane proteins located at the tight junctions of the epithelium and endothelium. Claudins are proposed to have four transmembrane segments with two extracellular loops, and N- and C-termini located in the cytoplasm. The claudin family of transmembrane proteins (CLDN) plays a critical role in maintaining epithelial-endothelial tight junctions and may also play a role in cytoskeletal maintenance and cell signaling.

Claudin 6 (CLDN6) is an oncofetal gene expressed in mouse and human stem cells and embryoid bodies committed to the epithelial cell lineage (Turksen, K. et al. (2001) Dev Dyn 222, 292-300; Anderson WJ et al. (2008) Dev Dyn 237, 504-12; Turksen K. et al. (2002) Development, 129, 1775-84; Assou S. et al. (2007) Stem Cells 25, 961-73). As a tumor-associated antigen, it can be classified as a differentiation antigen due to its expression during the early stage of epidermal morphogenesis, where it is determinant for epidermal differentiation and barrier formation. In addition, expression has been observed in epithelial tissues or neonatal normal epithelial tissues of the tongue, skin, stomach and mammary gland (Abuazza G. et al. (2006), Am J Physiol Renal Physiol 291, 1132-1141; Troy T.C. et al. (2007) ), Molecular Biotechnology 36, 166-74; Zhao L. et al. (2008), Am J Physiol Regul Integr Comp Physiol 294, 1856-1862). In addition, our data show low or very low expression of CLDN6 in human placenta, bladder, endometrium, prostate, and peripheral nerve and frequent overexpression of CLDN6 in various cancers. CLDN6 has been demonstrated to be overexpressed in tumors, including pediatric brain tumors, gastric adenocarcinomas and germ cell tumors, as well as visceral carcinomas such as ovarian carcinomas. It has also been demonstrated that overexpression of CLDN6 in gastric cancer cells results in increased invasiveness, migration and proliferation, indicating that CLDN6 is a marker of poor prognosis and may play a potential role in the maintenance of the malignant phenotype. Additionally, CLDN6 has been shown to act as a cancer suppressor through inhibition of cell proliferation and induction of apoptosis in breast cancer cell lines.

The sequence alignment of CLDN3, CLDN4, CLDN6 and CLDN9 shown in Fig. 1B illustrates that there is a high degree of conservation of CLDN6 relative to other claudin proteins. The high homology of CLDN6 to other claudin proteins, in particular CLDN9 and CLDN4, makes it difficult to obtain antibodies to CLDN6 that have properties such as specificity and affinity suitable for diagnostic purposes. The present inventors have found that antibodies directed against a specific epitope located in the C-terminal region of CLDN6 satisfy the criteria for the diagnostic utility of antibodies, in particular for the detection and identification of cells expressing CLDN6.

The antibodies of the present disclosure are useful, for example, in diagnosing cancer and/or in determining whether cancer cells express CLDN6. Preferably, the cancer or cancer cell is characterized by surface expression of CLDN6. Cancer cells expressing CLDN6 are suitable targets for therapies that specifically target CLDN6, such as antibody therapy directed against CLDN6. In accordance with one embodiment, cancer cells express or aberrantly express CLDN6, while corresponding normal cells do not express CLDN6 or express CLDN6 at a lower level.

Brief Disclosure of the Present Invention

In accordance with one aspect, the present invention provides an antibody or antigen binding fragment thereof that binds:

(i) with a peptide having the amino acid sequence EYPTKNY (SEQ ID NO: 38), and/or

(ii) claudin 6 (CLDN6), wherein said antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence EYPTKNY (SEQ ID NO: 38), and/or

(iii) with a peptide having the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and/or

(iv) claudin 6 (CLDN6), wherein said antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and/or

(v) with a peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), where the antibody or antigen binding fragment thereof does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14), and/or

(vi) with claudin 6 (CLDN6), wherein the antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), wherein the antibody or antigen binding fragment thereof does not bind with a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14).

In accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides a monoclonal antibody or antigen binding fragment thereof that

(i) binds to a peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), and/or

(ii) binds to claudin 6 (CLDN6), wherein the antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15).

In accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides an antibody or antigen binding fragment thereof that binds one or more, preferably all, of the following peptides:

PAISRGPSEYPTKNY (SEQ ID NO: 22), AISRGPSEYPTKNYV (SEQ ID NO: 15), ISRGPSEYPTKNYV (SEQ ID NO: 23), SRGPSEYPTKNYV (SEQ ID NO: 24), RGPSEYPTKNYV (SEQ ID NO: 25), GPSEYPTKNYV (SEQ ID NO : 26), PSEYPTKNYV (SEQ ID NO: 27), SEYPTKNYV (SEQ ID NO: 28) and EYPTKNYV (SEQ ID NO: 29), where the antibody or antigen binding fragment thereof does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO : 14).

In one embodiment, the difference in binding affinity for the peptide to which the antibody or antigen-binding fragment thereof binds with the lowest affinity and the peptide to which the antibody or antigen-binding fragment thereof binds with the greatest affinity is 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.

Antibodies or antigen binding fragments described herein preferably do not bind to a peptide containing the amino acid sequence EYPTK (SEQ ID NO: 59) and/or the amino acid sequence EYPTKN (SEQ ID NO: 60), but not containing the amino acid sequence EYPTKNY (SEQ ID NO: 38). In other words, an antibody or antigen binding fragment described herein that binds to a peptide having the amino acid sequence EYPTKNY (SEQ ID NO: 38) and/or a peptide having the amino acid sequence EYPTKNYV (SEQ ID NO: 29) binds to the specified peptide(s) due to the presence of a second tyrosine molecule that is not present in the amino acid sequence EYPTK (SEQ ID NO: 59) and/or the amino acid sequence EYPTKN (SEQ ID NO: 60). As shown herein, preferred antibodies or antigen binding fragments do not bind to a peptide containing the amino acid sequence YPTKNY (SEQ ID NO: 61) and/or EYPTKN (SEQ ID NO: 60) but not containing the amino acid sequence EYPTKNY (SEQ ID NO: 38), indicating that the amino acid sequence EYPTKNY (SEQ ID NO: 38) is a sequence that can be considered as a minimal epitope for binding of these antibodies.

In accordance with the aspects described herein, and in accordance with additional aspects, the present invention relates to an antibody produced by or derived from a hybridoma deposited in the DSMZ (Inhoffenstr. 7B, 38124, Braunschweig, Germany), and having one of the following designations and access numbers:

1. 58-4B-2, DSM accession number ACC3311, deposited on November 29, 2016;

2. 58-3A, accession number DSM ACC3312, deposited on November 29, 2016; or

3. 58-1B, DSM accession number ACC3313, deposited on November 29, 2016.

Antibodies of the present invention are referred to herein by reference to the antibody designation and/or by reference to the clone producing said antibody.

The present invention also provides an antibody that competes for CLDN6 binding with an antibody produced by or derived from the above-described hybridomas and/or has specificity for an anti-CLDN6 antibody produced or derived from the above-described hybridomas. In accordance with these and another embodiment, the present invention also provides an antibody comprising an antigen binding region or antigen binding site, in particular a variable region identical or highly identical to the variable region of antibodies produced by or derived from the above-described hybridomas. Preferred antibodies are believed to be those having CDR regions that are either identical or highly homologous to the CDR regions of antibodies produced by or derived from the above-described hybridomas. By “highly homologous” is meant that 1 to 5, preferably 1 to 4, for example 1 to 3 or 1 or 2 substitutions can be made in each CDR region. Particularly preferred antibodies are chimeric or humanized forms of antibodies produced by or derived from the hybridomas described above.

Accordingly, in accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides an antibody selected from the group consisting of:

(i) an antibody produced by or derived from a clone deposited under accession number DSM ACC3313 (58-1B), DSM ACC3312 (58-3A) or DSM ACC3311 (58-4B-2),

(ii) an antibody that is a chimeric or humanized form of an antibody according to (i),

(iii) an antibody that competes for binding of CLDN6 with the antibody in accordance with (i),

(iv) an antibody that has an antibody specificity in accordance with (i), and

(v) an antibody comprising an antigen binding site or an antibody antigen binding site in accordance with (i), or

an antigen-binding fragment of an antibody according to any of (i) to (v).

In accordance with one embodiment, the antigen binding region or antigen binding site of the antibody according to (i) comprises a variable region of the antibody according to (i).

In accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

an antibody heavy chain containing:

(i) an antibody heavy chain sequence containing the sequence according to SEQ ID NO: 40, 42 or 44 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence comprising the sequence of SEQ ID NO: 40, 42 or 44 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 53 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 51 or 57 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 52 or 58 or a variant thereof, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

an antibody light chain containing:

(i) an antibody light chain sequence comprising a sequence according to SEQ ID NO: 41, 43 or 45 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence comprising the sequence of SEQ ID NO: 41, 43 or 45 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 55 or a variant thereof, And

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with embodiments of the aspects described herein, and in accordance with additional aspects, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

(I) an antibody heavy chain containing:

(i) an antibody heavy chain sequence containing the sequence according to SEQ ID NO: 40, 42 or 44 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence comprising the sequence of SEQ ID NO: 40, 42 or 44 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 53 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 51 or 57 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 52 or 58 or a variant thereof, and/or

(II) an antibody light chain containing:

(i) an antibody light chain sequence comprising a sequence according to SEQ ID NO: 41, 43 or 45 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence comprising the sequence of SEQ ID NO: 41, 43 or 45 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 55 or a variant thereof, And

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

(I) an antibody heavy chain containing:

(i) an antibody heavy chain sequence containing the sequence according to SEQ ID NO: 40 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence comprising the sequence of SEQ ID NO: 40 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 53 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 51 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 52 or a variant thereof, And

(II) an antibody light chain containing:

(i) an antibody light chain sequence containing the sequence according to SEQ ID NO: 41 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence comprising the sequence of SEQ ID NO: 41 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 55 or a variant thereof, And

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

(I) an antibody heavy chain containing:

(i) an antibody heavy chain sequence containing the sequence according to SEQ ID NO: 42 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence comprising the sequence of SEQ ID NO: 42 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 53 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 57 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 58 or a variant thereof, And

(II) an antibody light chain containing:

(i) an antibody light chain sequence comprising the sequence according to SEQ ID NO: 43 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence comprising the sequence of SEQ ID NO: 43 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 55 or a variant thereof, And

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) antibodies containing:

(I) an antibody heavy chain containing:

(i) an antibody heavy chain sequence containing the sequence according to SEQ ID NO: 44 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody heavy chain sequence comprising the sequence of SEQ ID NO: 44 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 53 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 57 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 58 or a variant thereof, And

(II) an antibody light chain containing:

(i) an antibody light chain sequence comprising the sequence according to SEQ ID NO: 45 or a variant thereof,

(ii) at least one, preferably two, more preferably all three of the CDR sequences of an antibody light chain sequence comprising the sequence of SEQ ID NO: 45 or a variant thereof, or

(iii) a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and preferably further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and/or a CDR2 sequence according to SEQ ID NO: 55 or a variant thereof, And

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody comprising an antibody heavy chain containing the CDR3 sequence of SEQ ID NO: 53 or a variant thereof, and an antibody light chain containing the CDR3 sequence of SEQ ID NO: 56 or a variant thereof, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody comprising an antibody heavy chain comprising a CDR3 sequence of SEQ ID NO: 53 or a variant thereof and further comprising a CDR1 sequence of SEQ ID NO: 51 or a variant thereof and a CDR2 sequence of SEQ ID NO: 52 or a variant thereof, and an antibody light chain comprising a CDR3 sequence in accordance with SEQ ID NO: 56 or a variant thereof and further comprising a CDR1 sequence in accordance with SEQ ID NO: 54 or a variant thereof and a CDR2 sequence in accordance with SEQ ID NO: 55 or its variant, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody comprising an antibody heavy chain comprising a CDR3 sequence of SEQ ID NO: 53 or a variant thereof and further comprising a CDR1 sequence of SEQ ID NO: 57 or a variant thereof and a CDR2 sequence of SEQ ID NO: 58 or a variant thereof, and an antibody light chain comprising a CDR3 sequence according to SEQ ID NO: 56 or a variant thereof and further comprising a CDR1 sequence according to SEQ ID NO: 54 or a variant thereof and a CDR2 sequence according to SEQ ID NO: 55 or its variant, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody containing a heavy chain variable region (VH) containing the amino acid sequence presented under SEQ ID NO: 40 or a variant thereof, and a light chain variable region (VL) containing the amino acid sequence presented under SEQ ID NO: 41 or her option, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody containing a heavy chain variable region (VH) containing the amino acid sequence represented by SEQ ID NO: 42 or a variant thereof, and a light chain variable region (VL) containing the amino acid sequence presented by SEQ ID NO: 43 or her option, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, the present invention provides an antibody selected from the group consisting of:

(a) an antibody containing a heavy chain variable region (VH) containing the amino acid sequence presented under SEQ ID NO: 44 or a variant thereof, and a light chain variable region (VL) containing the amino acid sequence presented under SEQ ID NO: 45 or her option, and

(b) an antibody that competes for binding of CLDN6 to an antibody according to (a) and/or has specificity for the CLDN6 antibody according to (a), or an antigen binding fragment of said antibody.

In accordance with preferred embodiments, an antibody of the present invention comprises an antibody heavy chain containing a gamma-2a heavy chain constant region, preferably a human gamma-2a heavy chain constant region, and/or contains an antibody light chain containing a kappa light chain constant region.

The antibodies or antigen binding fragments described herein bind to CLDN6. The antibody or antigen binding fragment of the present invention is preferably capable of binding to CLDN6 in its native, ie, naturally occurring or undenatured state, or in its denatured state. In accordance with one embodiment, an antibody or antigen binding fragment thereof of the present invention binds to CLDN6, but not CLDN9, and preferably does not bind to CLDN4 and/or CLDN3. Preferably, the antibody or antigen binding fragment thereof does not substantially bind to a CLDN protein other than CLDN6. Preferably, the antibody or antigen binding fragment thereof of the present invention is specific for CLDN6.

In accordance with one embodiment, CLDN6 is cell surface membrane-bound CLDN6. In accordance with one embodiment, CLDN6 is present on cancer cells, wherein said cancer cells are preferably cancer cells expressing CLDN6. In one embodiment, said cancer cells are cells from a cancer selected from the group consisting of ovarian cancer, in particular ovarian adenocarcinoma and ovarian tetracarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell carcinoma and adenocarcinoma of the lung, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer , in particular, malignant pleomorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular small intestinal adenocarcinoma and ileal adenocarcinoma, embryonal testicular carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer , in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumors, and metastatic forms thereof.

In accordance with one embodiment, the antibody of the present invention is a chimeric, human, or humanized antibody. In accordance with one embodiment, the antibody of the present invention is a monoclonal antibody.

In one embodiment, an antibody of the present invention is produced by a method comprising the step of immunizing an animal peptide comprising the amino acid sequence of SEQ ID NO: 49, or an equivalent peptide, or a nucleic acid or host cell expressing said peptide, preferably consisting of them. Preferably, said peptide contains no more than 110, 100, 90, 80, 70, 60, 50 or 40 contiguous CLDN6 amino acids.

Antibodies or antigen-binding fragments of the present invention can be linked, ie, covalently or non-covalently linked, to other fragments, such as detectable labels.

In accordance with a further aspect, the present invention provides a conjugate comprising an antibody or antigen binding fragment described herein associated with at least one detectable label.

The present invention also relates to a cell, such as a hybridoma, producing an antibody described herein.

Preferred hybridomas are those deposited in the DSMZ (Inhoffenstr. 7B, 38124, Braunschweig, Germany) and having one of the following designations and accession numbers:

1. 58-4В-2, DSM accession number ACC3311, deposited on November 29, 2016;

2. 58-3A, DSM accession number ACC3312, deposited November 29, 2016; or

3. 58-1B, DSM accession number ACC3313, deposited November 29, 2016.

The present invention also relates to a peptide containing the amino acid sequence of SEQ ID NO: 49 or an immunologically equivalent peptide, preferably consisting of them. Preferably, said peptide contains no more than 110, 100, 90, 80, 70, 60, 50 or 40 contiguous CLDN6 amino acids.

The present invention also relates to nucleic acids encoding antibodies or parts thereof, such as antibody chains or antigen binding fragments, or peptides described herein. Preferably, the nucleic acid is operably linked to one or more expression control elements allowing expression in eukaryotic or prokaryotic cells. Control elements that ensure expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.

The nucleic acid of the present invention may be contained in a vector, for example, a plasmid, cosmid, virus, bacteriophage or other vector used, for example, routinely in genetic engineering. The vector may contain additional genes, such as marker genes, which enable selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector may contain expression control elements to ensure appropriate expression of the coding regions in suitable hosts. Such control elements are known to one of ordinary skill in the art and may include a promoter, a splice cassette, and a translation initiation codon.

Methods for constructing nucleic acid molecules, constructing vectors containing nucleic acid molecules, introducing vectors into suitably selected host cells, or initiating or achieving expression of nucleic acid molecules are well known in the art.

An additional aspect of the present invention relates to a host cell containing a nucleic acid or vector disclosed herein.

An additional aspect of the present invention relates to the detection of CLDN6 or cells expressing CLDN6, or determination of the amount of CLDN6 or cells expressing CLDN6, using an antibody or antigen binding fragment of the present invention. CLDN6 or cells expressing CLDN6 are detected, or the amount of CLDN6 or cells expressing CLDN6 is determined by detecting or quantifying the complex between CLDN6 and the antibody or antigen binding fragment of the present invention. Formation of the complex indicates the presence of CLDN6 or cells expressing CLDN6. Such detection or quantification may be accomplished by a number of methods, including, but not limited to, immunodetection using an antibody or antigen binding fragment of the present invention. Methods for using antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, etc. Typically, such assays use an antibody or antibody fragment that binds a target peptide or protein, directly or indirectly coupled to a label that provides detection, such as indicator enzymes, radiolabels, fluorophores or paramagnetic particles. The methods of the present invention provide quantitative and/or qualitative estimates, for example, absolute and/or relative estimates of CLDN6 levels or levels of cells expressing CLDN6.

In accordance with one aspect, the present invention provides a method for detecting CLDN6 or determining the amount of CLDN6 in a sample, comprising the steps of:

(i) contacting the sample with an antibody or antigen binding fragment thereof of the present invention or a conjugate of the present invention, and

(ii) detecting the formation of a complex or determining the amount of a complex between the antibody, antigen binding fragment or conjugate and CLDN6.

In accordance with one embodiment, the sample is a cellular sample, ie, a sample containing cells, such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, the antigen binding fragment and CLDN6 expressed by the cells in the sample.

In accordance with one aspect, the present invention provides a method for determining whether cells express CLDN6, comprising the steps of:

(i) contacting the cell sample with an antibody or antigen binding fragment thereof of the present invention or a conjugate of the present invention, and

(ii) detecting the formation of a complex between the antibody, the antigen binding fragment and CLDN6 expressed by the cells in the sample.

In accordance with one embodiment, the cells in the sample are cancer cells. The complex is preferably formed between the antibody, the antigen binding fragment and CLDN6 expressed by the cells in the sample.

Additional aspects of the present invention relate to methods for diagnosing or classifying diseases by targeting CLDN6 using an antibody or antigen binding fragment of the present invention. These methods provide selective detection of cells that express CLDN6, thereby differentiating these cells from normal cells that do not express CLDN6 or pathological cells that do not express CLDN6. Diseases characterized by abnormal cells expressing CLDN6 are treated with therapies that specifically target CLDN6, such as therapeutic antibody therapy directed against CLDN6. Preferred diseases for therapy or diagnosis are diseases in which CLDN6 is expressed or aberrantly expressed, in particular cancers such as those described herein.

In accordance with one aspect, the present invention relates to methods for diagnosing, detecting or monitoring, i.e., determining the regression, progress, course and/or onset of cancer, comprising detecting CLDN6 or cells expressing CLDN6 and/or determining the amount of CLDN6 or cells expressing CLDN6 in a biological sample isolated from a patient using an antibody or antigen binding fragment of the present invention. Such methods can be used to detect whether a subject has cancer or is at an (increased) risk of developing cancer, or, for example, whether a treatment regimen is effective.

Thus, in one aspect, the present invention relates to a method for diagnosing, detecting or monitoring cancer, comprising the steps of:

(i) contacting the biological sample with an antibody or antigen binding fragment thereof of the present invention or a conjugate of the present invention, and

(ii) detecting the formation of a complex and/or determining the amount of a complex between the antibody, antigen binding fragment or conjugate and CLDN6.

In accordance with one embodiment, the biological sample is a cellular sample, ie, a sample containing cells, such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, the antigen binding fragment and CLDN6 expressed by the cells in the sample.

The monitoring methods of the present invention preferably comprise detecting and/or quantifying CLDN6 or CLDN6 expressing cells in a first sample at a first time point and in an additional sample at a second time point, with regression, progression, progression and/or onset tumor disease can be determined by comparing two specified samples.

Typically, the level of CLDN6 or the level of cells expressing CLDN6 in a biological sample is compared to a reference level, with a deviation from said reference level indicating the presence and/or stage of cancer in the subject. The reference level may be a level determined in a control sample (eg, from healthy tissue or from a subject, particularly a non-cancer patient) or a median level from healthy subjects. "Deviation" from said reference level means any significant change, such as an increase of at least 10%, 20% or 30%, preferably at least 40% or 50% or even more.

Preferably, the presence of CLDN6 or cells expressing CLDN6, and/or an amount of CLDN6 or cells expressing CLDN6 that is increased compared to a reference level, for example, compared to a patient without cancer, indicates the presence or risk of development (i.e. , the possibility of developing) cancer in a patient.

A number of CLDN6 or cells expressing CLDN6 that is reduced compared to a biological sample previously recovered from a patient may indicate regression, improvement, such as effective treatment, or a reduced risk of cancer onset in the patient.

The number of CLDN6 or cells expressing CLDN6 that is increased compared to a biological sample previously recovered from a patient may indicate progression, decline, such as treatment failure, recurrence or metastasis, onset, or risk of onset of cancer in the patient. the specified patient.

In accordance with one aspect, the present invention provides a method for determining whether a cancer is treatable with cancer therapy targeting CLDN6, comprising the steps of:

(i) contacting a sample containing cancer cells with an antibody or antigen binding fragment thereof of the present invention or a conjugate of the present invention, and

(ii) detecting the formation of a complex between the antibody, the antigen binding fragment and CLDN6.

The complex is preferably formed between the antibody, the antigen binding fragment and CLDN6 expressed by the cancer cells in the sample.

Such methods can be used to detect whether a patient is suitable for therapy that targets cells expressing CLDN6, such as therapy using antibodies exhibiting one or more immune effector functions, such as cytotoxic CLDN6-specific antibodies, for example, antibodies labeled with a cytotoxic substance, such as a toxin or radiolabel, or inducing a cell killing mechanism, such as CDC or ADCC. Diseases characterized by abnormal cells expressing CLDN6 are treated with CLDN6-targeting therapies, such as cancers, particularly the diseases described herein.

In one embodiment of any of the above aspects, the sample, cellular sample, or biological sample comes from a patient who has, is suspected of having, or is likely to have cancer. In accordance with one embodiment, the sample, cellular sample, or biological sample is from a tissue or organ in which the cells, in the case where the tissue or organ does not have cancer, do not substantially express CLDN6. Preferably, said tissue is tissue other than placental tissue. Preferably, said tissue has already been diagnosed as cancerous, for example by visual inspection or cell culture testing of said tissue or organ. In accordance with this embodiment, the presence of CLDN6 or cells expressing CLDN6, and/or an amount of CLDN6 or cells expressing CLDN6 that is increased compared to a reference level, for example, compared to a patient without a tumor disease, may indicate that the patient is eligible for therapy that targets cells expressing CLDN6.

In accordance with one aspect, the present invention provides compositions, for example, diagnostic compositions or kits, containing an antibody or antigen binding fragment or a combination of antibodies and/or antigen binding fragments described herein. Such diagnostic compositions or test kits are useful in the methods of the present invention, such as the diagnostic, detection or monitoring methods of the present invention. These kits may optionally contain a detectable label, for example, indicator enzymes, radiolabels, fluorophores or paramagnetic particles. The kits may contain information leaflets, for example, leaflets containing information on how to use the reagents to practice the method described herein.

Other characteristics and advantages of the present invention will be apparent from the following detailed description and claims.

Detailed Disclosure of the Present Invention

Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is intended for descriptive purposes only of specific embodiments, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art.

Next, elements of the present invention will be described. These elements are shown in certain embodiments, but it is understood that they can be combined in any manner and in any quantity to provide additional embodiments. The variously described examples and preferred embodiments are not intended to limit the present invention to only those embodiments expressly described. It should be understood that the present invention supports and covers all embodiments that combine the expressly described embodiments with any number of disclosed and/or preferred elements. In addition, any permutations and combinations of all described elements in this application should be considered disclosed by the description of this application, unless the context indicates otherwise.

Preferably, terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

In the practice of the present invention, unless otherwise indicated, standard methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques will be used as set forth in the literature in the art (for comparison, for example, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” or “comprising” will be intended to include the specified representative, integer or stage or group of representatives, integers or stages but not to the exclusion of any other representative, integer or stage or group of representatives, integers or stages, although in some embodiments such other representative, integer or stage or group of representatives, integers or stages may be excluded, those. the subject matter is the inclusion of a specified representative, integer or stage or group of representatives, integers or stages. Terms in the singular and similar references used in the context of the description of the present invention (especially in the context of the following claims) are intended to include the singular and plural unless otherwise indicated herein or clearly contrary to the context. Quoting a range of values in this document is intended only as a shorthand way of referring to each individual value within the range. Unless otherwise specified herein, each individual value is included in the description as if it were cited individually herein. All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise inconsistent with the context. The use of any and all examples or illustrative style (eg, “such as”) provided herein is intended only to more effectively describe the present invention and does not limit the scope of the present invention unless otherwise indicated. No style herein should be construed as indicating any unclaimed element as being necessary for the purposes of the practice of the present invention.

Several documents are cited throughout this description. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's descriptions, instructions, etc.), without regard to above or below, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to claim such an invention by virtue of a prior invention.

The term "recombinant" in the context of the present invention means "created by genetic engineering." Preferably, the "recombinant entity", such as a recombinant cell in the context of the present invention, does not occur in nature.

The term "naturally occurring" when used in relation to an object refers to the fact that the object can be found in nature. For example, a peptide or nucleic acid that is present in the body (including viruses) and can be isolated from a natural source and that has not been intentionally modified by humans in a laboratory occurs in nature.

The term "antigen" refers to an agent containing an epitope against which an immune response is directed and/or intended to be generated. Preferably, an antigen in the context of the present invention is a molecule that, upon processing, optionally induces an immune response that is preferably specific to the antigen. The term "antigen" includes certain proteins, peptides, polysaccharides, nucleic acids, especially RNA and DNA, and nucleotides.

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

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

Antigens include tumor-associated antigens such as CLDN6, i.e., components of cancer cells that can be derived from the cytoplasm, cell surface and cell nucleus, in particular those antigens that are produced, preferably in large quantities, intracellularly or in the form of surface antigens on cancer cells.

In the context of the present invention, the terms “tumor-associated antigen” or “tumor antigen” refer to proteins that are normally specifically expressed in a limited number of tissues and/or organs or at specific stages of development, for example, a tumor-associated antigen may normally be specifically expressed in gastric tissue, preferably in the gastric mucosa, in reproductive organs, for example, in the testes, in trophoblast tissue, for example, in the placenta or in germline cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "limited amount" preferably means no more than 3, more preferably no more than 2. Tumor-associated antigens in the context of the present invention include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e. i.e., proteins that are normally specifically expressed in a certain cell type at a particular stage of differentiation, cancer antigens/testis antigens, i.e., proteins that are normally specifically expressed in the testes and sometimes in the placenta, and antigens , specific to the germ line. In the context of the present invention, the tumor-associated antigen of the present invention is preferably associated with the cell surface of a cancer cell and is preferably not expressed or only rarely expressed in normal tissues. Preferably, tumor-associated antigen or aberrant antigen expression is determined by cancer cells. In the context of the present invention, a tumor-associated antigen that is expressed by a cancer cell in a subject, for example a patient suffering from cancer, is preferably a self-protein in the body of said patient. In accordance with preferred embodiments, the tumor-associated antigen in the context of the present invention is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs that, when damaged by the immune system, do not cause death of the subject, or in organs or structures of the body that are inaccessible or only difficult to access by the immune system. Preferably, the amino acid sequence of the tumor-associated antigen is identical to a tumor-associated antigen that is expressed in normal tissues and a tumor-associated antigen that is expressed in cancer tissues.

Examples of differentiation antigens that ideally fit the criteria of tumor-associated antigens as targets for tumor immunotherapy, particularly in tumor vaccination, are cell surface proteins of the claudin family, such as CLDN6. Claudins are a family of proteins that are the most important components of tight junctions, where they form a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells.

Claudins are transmembrane proteins spanning the membrane 4 times with an N-terminal and a C-terminal end, each located in the cytoplasm.

The term “claudin 6” or “CLDN6” preferably refers to human CLDN6, and, in particular, to a protein containing an amino acid sequence in accordance with SEQ ID NO: 1 of the Sequence List or a variant of the specified amino acid sequence. With respect to CLDN6, the term “variant” specifically refers to a protein containing an amino acid sequence according to SEQ ID NO: 1 of the Sequence Listing, wherein Ile at position 143 is replaced by Val. The term "CLDN6" includes any variants of CLDN6, such as post-translationally modified variants and conformational variants.

The term “CLDN9” preferably refers to human CLDN9, and, in particular, to a protein containing an amino acid sequence in accordance with SEQ ID NO: 2 of the Sequence Listing or a variant of the specified amino acid sequence.

The term “CLDN4” preferably refers to human CLDN4, and, in particular, to a protein containing an amino acid sequence in accordance with SEQ ID NO: 4 of the Sequence Listing or a variant of the specified amino acid sequence.

The term “CLDN3” preferably refers to human CLDN3, and, in particular, to a protein containing an amino acid sequence in accordance with SEQ ID NO: 3 of the Sequence Listing or a variant of the specified amino acid sequence.

CLDN6 was found to be expressed in, for example, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanomas, head and neck cancer, sarcomas, bile duct cancer, renal cell cancer and bladder cancer. CLDN6 is detected and can be targeted in ovarian cancer, particularly ovarian adenocarcinoma and ovarian tetracarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell carcinoma and lung adenocarcinoma, cancer stomach, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in private, synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer , cancer of the small intestine, including ileal cancer, in particular, adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors such as teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumors, and metastatic forms thereof. In one embodiment, the cancer associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer, and metastatic lung cancer. Preferably, the ovarian cancer is a carcinoma or adenocarcinoma. Preferably the lung cancer is a carcinoma or adenocarcinoma, and preferably a bronchiolar cancer such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

In accordance with the present invention, a cell expressing CLDN6 is preferably characterized by CLDN6 associated with a cell surface membrane, ie, CLDN6 is associated with the cell surface. Moreover, in accordance with the present invention, cellular CLDN6 is preferably CLDN6 associated with a cell surface membrane. The cell expressing CLDN6, or a cell characterized by the association of CLDN6 with its cell surface, is preferably a cancer cell, preferably a cancer cell of a type of cancer described herein.

The term “cell surface associated” means that a tumor-associated antigen, such as CLDN6, is associated with or located on the plasma membrane of a cell, wherein at least a portion of the tumor-associated antigen faces the extracellular space of the cell and is accessible from the outside side of said cell, for example, to antibodies located outside the cell. In this context, the portion contains 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 indirect. For example, association may be accomplished through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, saccharide, or other structure that may be found on the outer portion of the cell's plasma membrane. For example, a tumor-associated antigen associated with a cell surface may be a transmembrane protein having an extracellular portion, or may be a protein associated with a cell surface by interaction with another protein that is a transmembrane protein.

The term "cell surface" or "cell surface" is used in accordance with its usual meaning in the art, and thus includes the outer surface of a cell that is accessible to binding by proteins and other molecules.

In accordance with the present invention, CLDN6 is essentially not expressed in a cell if the level of expression is lower than that of placental cells or placental tissue. Preferably, the level of expression is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of expression in placental cells or placental tissue, or even lower. Preferably, CLDN6 is essentially not expressed in the cell if the expression level is no more than 2-fold, preferably 1.5-fold, greater than the expression level in a non-cancerous tissue other than the placenta, and preferably is not more than the expression level in said tissue , not related to cancer. Preferably, CLDN6 is essentially not expressed in the cell if the level of expression is below the limit of detection and/or if the level of expression is too low to allow binding by CLDN6-specific antibodies added to the cells.

In accordance with the present invention, CLDN6 is expressed in a cell if the level of expression exceeds the level of expression in a non-cancerous tissue other than the placenta, preferably more than 2-fold, preferably more than 10-fold, more than 100-fold, more than 1000 times or more than 10000 times. Preferably, CLDN6 is expressed in a cell if the level of expression is above the limit of detection and/or if the level of expression is high enough to allow binding by CLDN6-specific antibodies added to the cells. Preferably, CLDN6 expressed in a cell is expressed or exposed to the surface of said cell.

The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any molecule containing its antigen-binding site. The term "antibody" includes monoclonal antibodies and antibody fragments or derivatives, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, such as scFv, and antigen binding antibody fragments, such as Fab and Fab' fragments, and also includes all recombinant forms of antibodies, for example, antibodies expressed in prokaryotes, non-glycosylated antibodies and antigen binding fragments and derivatives of antibodies described herein. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), located between regions that are more conserved, called framework regions (FR). Each of VH and VL consists of three CDRs and four FRs, ordered from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

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 present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).

The term “humanized antibody” refers to a molecule having an antigen binding site that is essentially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may contain either complete variable domains fused to constant domains or only complementarity determining regions (CDRs) grafted onto appropriate framework regions within the variable domains. Antigen binding sites may be wild type or modified with one or more amino acid substitutions, for example modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies retain all CDR sequences (for example, a humanized mouse antibody, which contains all six CDRs from a mouse antibody). Other forms have one or more CDRs that change relative to the original antibody.

The term "chimeric antibody" refers to those antibodies in which one segment of each of the amino acid sequences of the heavy and light chains is homologous with respect to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous in relative to corresponding sequences in another segment. Typically, the variable region of both the light and heavy chains mimics the variable regions of antibodies derived from one mammalian species, while the constant regions are homologous to antibody sequences derived from another species. One obvious advantage of such chimeric forms is that the variable region can conveniently be derived from currently known sources using readily available B cells or hybridomas from non-human organisms in combination with constant regions derived from, for example, human cell preparations . Although the variable region has the advantage of being easy to obtain and the specificity is not influenced by the source, the constant region, being human, is less likely to elicit an immune response from a human subject upon injection of an antibody than a constant region from a non-human source would be. . At the same time, the definition is not limited to this specific example.

The terms "antigen binding site" of an antibody (or simply "binding site") or "antigen binding fragment" of an antibody (or simply "binding fragment") refer to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be carried out using fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding region” of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F(ab') ₂ fragments, bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments, consisting of the VL and VH domains of one arm of the antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, using a synthetic linker, which allows them to be obtained as a single protein chain in which The VL and VH regions combine to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl Acad Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included within the term “antigen binding fragment” of an antibody. A further example are binding domain-immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) a CH3 constant region. immunoglobulin heavy chain fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Binding domain-immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are prepared using standard techniques known to those skilled in the art, and the fragments are tested for suitability in the same manner as 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 a single molecule composition. A monoclonal antibody is characterized by a single specificity and binding affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma that contains a B cell derived from a non-human animal, such as a 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 produced, expressed, created or isolated by recombinant means, for example, (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transchromosomal to immunoglobulin genes, or a hybridoma derived from them, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectoma, (c) antibodies isolated from a library of recombinant combinatorial antibodies, and (d ) antibodies produced, expressed, created or isolated by any other means that involve splicing immunoglobulin gene sequences with other DNA sequences.

The term "transfectome" as used herein includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant or fungal cells, including yeast cells .

As used herein, the term “heterologous antibody” is defined in relation to the transgenic organism producing such antibody. This term refers to an antibody having an amino acid sequence, or a sequence encoding a nucleic acid corresponding thereto, that occurs in an organism other than the transgenic organism, and is typically derived from a species other than the transgenic organism.

As used herein, the term “heterohybrid antibody” refers to an antibody having light and heavy chains originating from different organisms. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

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

The antibodies described herein are preferably isolated. The term “isolated antibody” as used herein refers to an antibody that is substantially free of other antibodies having different antigen specificities. Moreover, the isolated antibody may essentially be free of other cellular material and/or chemicals.

In accordance with the present invention, an antibody is capable of binding to a predetermined target if it has significant affinity for the predetermined target and binds to the predetermined target in standard assays. "Affinity" or "binding affinity" is often measured using the equilibrium dissociation constant ( _KD ). Preferably, the term "significant affinity" refers to binding to a predetermined target with a dissociation constant (K _D ) 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 below, 10 ^-10 M or below, 10 ^-11 M or below or 10 ^-12 M or below.

An antibody is (essentially) incapable of binding to a target unless it has significant affinity for said target and binds significantly, in particular does not bind detectably to said target in standard assays. Preferably, the antibody does not bind detectably to said target if present at a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 μg/ml or higher. Preferably, an antibody does not have significant affinity for a target if it binds to said target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times or 10 ⁶ times higher than K _D in the case of binding to a predetermined target to which the antibody is capable of binding. For example, if the K _D for an antibody binding to a target to which the antibody is capable of binding is 10 ^-7 M, then the K _D for binding to a target for which the antibody does not have significant affinity will be at least 10 ^{- 6} M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

An antibody is specific for a predetermined target if it is capable of binding to said predetermined target while it is not capable of binding to other targets, i.e., does not have significant affinity for other targets and does not bind significantly with other targets in standard assays. In accordance with the present invention, an antibody is specific for CLDN6 if it is able to bind to CLDN6, but is (essentially) unable to bind to other targets, in particular other CLDN proteins such as CLDN9, CLDN4 and/or CLDN3 and/or or proteins other than claudin proteins, preferably proteins other than CLDN6. Preferably, the antibody is specific for CLDN6 if the affinity for and binding to such other targets is not significantly greater than the affinity for and binding to non-claudin proteins such as bovine serum albumin (BSA), casein, human whey albumin (HSA) or non-claudin transmembrane proteins such as MHC molecules or transferrin receptor or any other specific polypeptide. Preferably, an antibody is specific for a predetermined target if it binds to said target with a K _D that is at least 10-fold, 100-fold, ^103- fold, ¹⁰⁴ -fold, ¹⁰⁵ -fold, or 106 ^- fold lower than K _D in case of binding to a target for which it is not specific. For example, if the K _D for an antibody binding to a target for which it is specific is 10 ^-7 M, then the K _D for binding to a target for which it is not specific will be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

The binding of an antibody to its target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, NY (1992), as well as the methods described herein. Affinity values can be easily determined using standard techniques such as equilibrium dialysis; using the BIAcore 2000 tool, using the general procedures described by the manufacturer; using a radioimmunoassay using a radiolabeled target antigen; or by other method known to those skilled in the art. Affinity data can be analyzed, for example, using the method of Scatchard et al., Ann NY Acad. ScL 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, eg salt concentration, pH. Thus, measurements of affinity and other antigen binding parameters, eg _KD , _IC50 , are preferably performed using standardized antibody and antigen solutions and a standardized buffer.

The term "compete" refers to the competition between two antibodies for a target antigen. If two antibodies do not block each other from binding to the target antigen, then such antibodies are non-competitive, and this is an indication that the antibodies do not bind to the same part, ie, epitope, of the target antigen. One skilled in the art is well aware of how to examine antibody competition for binding to a target antigen. An example of such a method is the so-called cross-competition assay, which, for example, can be carried out in the form of an ELISA or by flow cytometry. For example, an ELISA-based assay may be performed by coating the wells of an ELISA plate with one of the antibodies; adding a competing antibody and a His-tagged antigen/target and detecting whether the added antibody inhibited the binding of the His-tagged antigen to the coated antibody, for example by adding a biotinylated anti-His antibody, followed by the addition of streptavidin-poly-HRP, and further reacting with ABTS and absorbance measurements at 405 nm. For example, flow cytometry can be performed by incubating cells expressing the antigen/target with an excess of unlabeled antibody, incubating the cells with a suboptimal concentration of antibody, followed by incubation with fluorescently labeled streptavidin and analysis by flow cytometry.

Two antibodies have the same specificity if they bind to the same antigen and the same epitope. Regardless of whether the antibody to be tested recognizes the same epitope as a particular antigen-binding antibody, i.e., the antibodies bind to the same epitope, it can be tested using various methods known to one skilled in the art. , for example, based on competition of antibodies for the same epitope. Competition between antibodies can be detected using cross-blocking assays. For example, a competitive ELISA can be used as a cross-blocking assay. For example, the target antigen can be applied to the wells of a microtiter plate, and an antigen binding antibody and a candidate competing antibody of interest can be added. The amount of antigen-binding antibody bound to an antigen in a well indirectly correlates with the binding ability of a candidate competing antibody of interest that competes with it for binding to the same epitope. In particular, the greater the affinity of a candidate competing test antibody for the same epitope, the less is the amount of antigen-binding antibody bound to the antigen-coated well. The amount of antigen binding antibody bound to the well can be measured by labeling the antibody with detectable or measurable labeling agents.

An antibody that competes for binding to an antigen with another antibody, such as an antibody containing heavy and light chain variable regions described herein, or an antibody having specificity for an antigen of another antibody, such as an antibody containing heavy and light chain variable regions chains described herein may be an antibody containing variants of said heavy and/or light chain variable regions described herein, for example, modifications in the CDR and/or a certain degree of identity described herein.

As used herein, the term “isotope” refers to a class of antibodies (eg, IgM or IgG1) that are encoded by heavy chain constant region genes. Antibodies of the present invention include polyclonal and monoclonal antibodies and include IgG2a (e.g., IgG2a, κ, λ), IgG2b (e.g., IgG2b, κ, λ), IgG3 (e.g., IgG3, κ, λ) antibodies and IgM antibodies. However, other antibody isotypes are also covered by the present invention, including IgG1, IgA1, IgA2 antibodies, IgA, IgD and IgE secretory antibodies.

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

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

The term “unarranged” or “germline configuration” as used herein in relation to a V segment refers to a configuration where the V segment does not recombine so as to be directly adjacent to a D or J segment.

In accordance with the present invention, antibodies can be derived from a variety of species, including, but not limited to, mouse, rat, rabbit, guinea pig, and human. Antibodies also include chimeric molecules in which the constant region of an antibody derived from one species, preferably human, is combined with an antigen binding site derived from another species. Moreover, antibodies include humanized molecules 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 using a number of techniques, including standard techniques for preparing monoclonal antibodies, for example, standard somatic cell hybridization techniques by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B cells or phage display techniques using antibody gene libraries, can in principle be used.

The preferred animal system for producing hybridomas that secrete monoclonal antibodies is the murine system. Obtaining hybridomas from mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion procedures are also known.

Other preferred animal systems for producing hybridomas that secrete monoclonal antibodies are the mouse and rabbit systems (eg, those described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am J Clin Pathol 124: 295 (2005).

In another preferred embodiment, human monoclonal antibodies directed against CLDN6 can be produced using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and collectively referred to herein as “transgenic mice.” The production of human antibodies in such transgenic mice can be carried out as detailed in the case of CD20 in WO 2004 035607.

Another strategy for producing monoclonal antibodies is the direct isolation of genes encoding antibodies from lymphocytes that produce antibodies of a certain specificity; see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For a detailed description of the development of recombinant antibodies, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

To obtain antibodies to CLDN6, mice can be immunized with carrier-conjugated peptides derived from the CLDN6 sequence, an enriched preparation of recombinantly expressed CLDN6 antigen or fragments thereof, and/or cells expressing CLDN6 or fragments thereof, as described. Alternatively, mice can be immunized with DNA encoding full-length human CLDN6 or fragments thereof. In the event that immunization with a purified or enriched CLDN6 antigen preparation does not result in the formation of antibodies, mice can also be immunized with cells expressing CLDN6, for example, to activate immune responses.

The immune response can be monitored during the immunization protocol using blood plasma and serum samples obtained from the tail vein or using retro-orbital phlebotomies. Mice with sufficient anti-CLDN6 immunoglobulin titers can be used for fusions. Mice can be boosted intraperitoneally or intravenously with CLDN6-expressing cells 3 to 5 days before sacrifice and spleen removal to increase levels of specific antibody-secreting hybridomas.

To produce hybridomas producing monoclonal antibodies to CLDN6, cells from lymph nodes, spleen or bone marrow obtained from immunized mice can be isolated and fused with a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody-secreting hybridomas. By immunofluorescence analysis and FACS using cells expressing CLDN6, antibodies with specificity for CLDN6 can be detected. Antibody-secreting hybridomas can be subcultured, rescreened, and if still positive for anti-CLDN6 monoclonal antibodies, subcloned by limiting dilution. Stable subclones can then be cultured in vitro to produce antibody for characterization in tissue culture medium.

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

For example, in accordance with one embodiment, the gene(s) of interest, such as antibody genes, may be ligated into an expression vector, such as a eukaryotic expression plasmid such as those used the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841, or other expression systems well known in the art. The purified plasmid with cloned antibody genes can be introduced into eukaryotic host cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells such as plant cells, fungal cells or yeast cells. The method used to introduce these genes may be a method described in the art, such as electroporation, lipofection using lipofectamine, or the like. Once these antibody genes are introduced into host cells, cells expressing the antibody can be identified and selected. These cells represent transfectomes that can then be amplified for their expression levels and scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture and/or cell supernatants.

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

Chimeric antibodies are antibodies whose different parts are derived from different animal species, such as antibodies having a murine antibody-derived variable region and a human immunoglobulin constant region. Chimerization of antibodies is achieved by linking the variable regions of the heavy and light chain of a mouse antibody to the constant region of the heavy and light chain of a human (for example, as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603- 918-8). In a preferred embodiment, chimeric antibodies are produced by linking a human kappa light chain constant region to a murine light chain variable region. Also in a preferred embodiment, chimeric antibodies can be produced by linking the human lambda light chain constant region to the murine light chain variable region. Preferred heavy chain constant regions for the production of chimeric antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain constant regions for producing chimeric antibodies are IgG2, IgA, IgD and IgM.

Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, amino acid sequences within the CDR are more variable between individual antibodies than sequences outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from a specific naturally occurring antibody grafted onto framework sequences from another antibody with different properties (see, for example, 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 the fully assembled variable genes that are produced by V(D)J binding during B cell maturation. The germline gene sequences will also differ from the high affinity secondary repertoire antibody sequences at discrete positions uniformly within the variable region. For example, somatic mutations are relatively infrequent in the amino-terminal region of framework region 1 and in the carboxy-terminal region of framework region 4. In addition, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact antibody 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 binding gene segments contributed to the formation of the recombined antibody variable genes. The germline sequences are then used to fill in the missing portions of the variable regions. The leader sequences of the heavy and light chains 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 oligonucleotides by linkage or PCR amplification. Alternatively, the entire variable region can be synthesized as a collection of short overlapping oligonucleotides and combined by PCR amplification to create a fully synthetic variable region clone. This process is characterized by certain advantages, such as the elimination or inclusion of certain restriction sites, or codon optimization.

The nucleotide sequences of heavy and light chain transcripts from hybridomas can be used to design overlapping arrays of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capabilities as natural sequences. Synthetic heavy chain and kappa sequences can differ from natural sequences in three ways: strands of repeating nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are included according to Kozak rules (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and HindIII sites are developed upstream from translation initiation sites.

For both heavy and light chain variable regions, the optimized coding and corresponding non-coding strand sequences are separated into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each strand, the oligonucleotides can be assembled into overlapping double-stranded assemblies that span segments of 150-400 nucleotides. The pools are then used as templates to generate PCR amplification products of 150-400 nucleotides. Typically, a set of oligonucleotides with a single variable region will be divided into two pools, which are separately amplified to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping heavy and light chain constant region fragment in the PCR amplification to produce fragments that can be easily cloned into expression vector constructs.

The reconstructed chimeric or humanized heavy and light chain regions are then combined with cloned promoter sequences, leader sequences, translation initiation sequences, constant region sequences, 3' untranslated sequences, polyadenylation sequences and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, cotransfected, sequentially transfected, or separately transfected into host cells, which are then fused to produce a host cell expressing both chains. Plasmids are described for use in the construction of expression vectors for human IgGK. Plasmids can be constructed such that PCR-amplified heavy chain V region and kappa light chain V region cDNA sequences can be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express fully human or chimeric IgG1 kappa or IgG4 kappa antibodies. Similar plasmids can be designed to express other heavy chain isotypes or to express antibodies containing lambda light chains.

Thus, in accordance with another aspect of the present invention, the structural characteristics of the anti-CLDN6 antibodies described herein are used to create structurally related humanized anti-CLDN6 antibodies that retain at least one functional property of the antibodies of the present invention, such as binding to CLDN6. More specifically, one or more murine monoclonal antibody CDR regions can be combined with known human framework regions and CDRs to form additional recombinantly engineered humanized anti-CLDN6 antibodies.

The ability of an antibody to bind CLDN6 can be determined using standard assays such as ELISA, Western blotting, immunofluorescence assays and flow cytometry.

ELISA can be used to demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to the CLDN6 protein or peptides. The peptides or protein used for immunization can be used to determine the specificity of hybridoma supernatants or to analyze serum titers.

Flow cytometry can be used to demonstrate the presence of antibodies in the sera of immunized mice or the binding of monoclonal antibodies to living cells. Cell lines naturally or transfectedly expressing antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with varying concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and can be incubated at 4°C for 30 minutes. After washing, the APC- or Alexa647-labeled anti-IgG antibody can bind to the antigen-bound monoclonal antibody under the same conditions under which the primary antibody is stained. Samples can be analyzed by flow cytometry using a FACS instrument using light scattering and side scattering properties to gate single living cells. In order to distinguish antigen-specific monoclonal antibodies from non-specific binders in one dimension, a co-transfection method can be used. Cells transiently transfected with plasmids encoding the antigen and fluorescent marker can be stained as described above. Transfected cells may be detected in a different fluorescent channel than antibody-stained cells. Because most transfected cells express both transgenes, antigen-specific monoclonal antibodies bind preferentially to cells expressing the fluorescent marker, while nonspecific antibodies bind at a comparable rate to untransfected cells. An alternative analysis using fluorescence microscopy can be used in addition to or instead of flow cytometry. Cells can be stained exactly as described above and examined using fluorescence microscopy.

Immunofluorescence microscopy can be used to demonstrate the presence of antibodies to CLDN6 in the sera of immunized mice or the binding of monoclonal antibodies to living cells expressing CLDN6. For example, cell lines expressing either spontaneously or post-transfection CLDN6 and negative controls not expressing CLDN6 are grown in slide chambers under standard growth conditions in DMEM/F12 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. The cells can then be mixed with methanol or paraformaldehyde or left untreated. The cells can then react with anti-CLDN6 monoclonal antibodies for 30 minutes at 25°C. After washing, cells can be reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined using fluorescence microscopy.

Total CLDN6 levels in cells can be observed when cells are fixed with methanol or fixed with paraformaldehyde or permeabilized with Triton X-100. In living cells and non-permeabilized paraformaldehyde-fixed cells, the surface localization of CLDN6 can be examined. In addition, targeting of CLDN6 at tight junctions can be analyzed by co-staining with tight junction markers such as ZO-1. In addition, the effects of antibody binding and localization of CLDN6 to the cell membrane can be examined.

Anti-CLDN6 IgG antibody can be further tested for reactivity to CLDN6 antigen by Western blotting. Briefly, cell extracts from cells expressing CLDN6 and corresponding negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked and probed with monoclonal antibodies to be examined. IgG binding can be detected using a peroxidase-conjugated anti-mouse IgG antibody and visualized using ECL substrate.

Mouse anti-CLDN6 IgG antibody can be further examined for reactivity to the CLDN6 antigen by immunohistochemical analysis in a manner well known to one skilled in the art, for example, using paraformaldehyde- or acetone-fixed frozen sections or paraformaldehyde-embedded tissue sections from non-CLDN6 samples. being cancerous tissue or cancerous tissue samples obtained from patients during standard surgical procedures, or from mice bearing xenografted tumors inoculated with cell lines expressing spontaneously or after transfection of CLDN6. For immunostaining, CLDN6-reactive antibodies can be incubated followed by horseradish peroxidase-conjugated goat anti-mouse antibodies or goat anti-rabbit antibodies according to the suppliers' instructions.

One particularly preferred technique for analyzing CLDN6 in the methods of the present invention is immunohistochemical analysis or IHC. Immunohistochemical analysis or IHC refers to the process of detecting antigens (eg, proteins) in the cells of a tissue section, such as the tissue cells mentioned herein. Immunohistochemical staining is widely used in the diagnosis of abnormal cells, such as those found in cancerous tumors. Visualization of antibody-antigen interactions can be accomplished in a number of ways. In the most common case, the antibody is conjugated to an enzyme, such as peroxidase, which can catalyze the color-producing reaction. Alternatively, the antibody can also be labeled with a fluorophore such as fluorescein or rhodamine.

Sample preparation is critical to maintaining cellular morphology, tissue architecture, and antigenicity of target epitopes. This requires appropriate collection, fixation and preparation of tissue sections. Paraformaldehyde is usually used for fixation. Depending on the purpose and thickness of the experimental sample, either thin (approximately 4-40 µm) sections are cut from the tissue of interest, or if the tissue is not very dense and is permeable, then it is used entirely. Section preparation is usually done using a microtome, and the sections are placed on glass slides.

The sample may require additional steps to make epitopes available for antibody binding, including deparaffinization and antigen unmasking. Detergents such as Triton X-100 are widely used in immunohistochemical analysis to reduce surface tension, allowing less reagent to be used to achieve more efficient and more uniform sample coverage.

The direct immunohistochemical staining method uses a single labeled antibody that binds directly to the antigen to be stained. The indirect immunohistochemical staining method, which is more common, uses one antibody against the antigen to be probed and a second labeled antibody against the first antibody.

To reduce background staining in IHC, samples are incubated with a buffer that blocks reactive sites to which primary or secondary antibodies might otherwise bind. Primary antibodies are raised against the antigen of interest and are typically unconjugated (unlabeled), while secondary antibodies are raised against immunoglobulins from the primary antibody molecules. The secondary antibody is typically conjugated to a linker molecule, such as biotin, which then recruits reporter molecules, or the secondary antibody is directly coupled to the reporter molecule itself. Common blocking buffers include normal whey, nonfat dry milk, BSA, or gelatin, as well as commercial blocking buffers.

Reporter molecules vary based on the nature of the detection method, and the most common detection methods are chromogenic and fluorescent detection mediated by enzymes and fluorophores. In the case of chromogenic reporters, the enzymatic tag reacts with the substrate to produce an intensely colored product that can be analyzed using a conventional light microscope. Although the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes most widely used as protein detection tags. An array of chromogenic, fluorogenic and chemiluminescent substrates are available for use with any enzyme, including DAB or BCIP/NBT. Fluorescent reporters are small organic molecules used for IHC detection. For chromogenic and fluorescent detection methods, densitometric signal analysis can provide semi- and fully quantitative data, respectively, to correlate the level of the reporter signal with the level of protein expression or localization.

After immunohistochemical staining of the target antigen, a second dye is often used to provide contrast that helps shade the primary dye. Many of these dyes are specific for discrete cellular compartments or antigens, while other dyes will stain an entire cell. Both chromogenic and fluorescent dyes are available for IHC to provide a wide variety of reagents to suit each experimental design. Hematoxylin, Hoechst stain and DAPI are widely used.

Mapping of epitopes recognized by antibodies can be done as described in detail in "Epitope Mapping Protocols (Methods in Molecular Biology) Glenn E. Morris ISBN-089603-375-9 and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 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 inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, immune effector functions lead to the destruction of cancer cells. Preferably, the immune effector functions in the context of the present invention are antibody-mediated effector functions. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in tumor-associated antigen-bearing cells, e.g. by antibody binding to surface antigen, inhibition of CD40L-mediated signaling, for example by binding an antibody to the CD40 receptor or CD40 ligand (CD40L), and/or inhibiting the proliferation of cells bearing a tumor-associated antigen, preferably ADCC and/or CDC. Thus, antibodies that are capable of mediating one or more immune effector functions are preferably capable of mediating cell killing 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 have an effect simply by binding to tumor-associated antigens on the surface of the cancer cell. For example, antibodies can block the function of a tumor-associated antigen or induce apoptosis directly by binding to a tumor-associated antigen on the surface of a cancer cell.

ADCC describes the ability of effector cells to kill cells, particularly lymphocytes, which preferably requires that the target cell be labeled with an antibody. ADCC preferentially occurs when antibodies bind to antigens on cancer cells and the Fc domains of the antibody hijack Fc receptors (FcRs) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations express specific Fc receptors in a characteristic manner. ADCC can be considered as a mechanism for directly inducing varying degrees of immediate tumor destruction, which also leads to antigen presentation and induction of tumor-directed T cell responses. Preferably, in vivo induction of ADCC will result in tumor-directed T cell responses and additional host antibody responses.

CDC is another method of killing cells that can be directly directed by antibodies. IgM is the most effective epitope for complement activation. Both IgG1 and IgG3 are very effective in promoting CDC through the classical complement activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the unmasking of numerous Clq binding sites in close proximity to the _CH 2 domains of participating antibody molecules, such as IgG molecules (Clq is one of the three subcomponents of complement C1). Preferably, these unmasked Clq binding sites result in the conversion of a previously low-affinity Clq-IgG interaction into a high-avidity interaction that activates a cascade of events involving a series of other complement proteins and results in the proteolytic release of the C3a and C5a effector cell chemotactic/activating agents. Preferably, the complement cascade ends with the formation of a membrane attack complex, which forms pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell and can lead to apoptosis.

The term "immune effector cells" as used herein refers to cells that perform effector functions during an immune response. For example, such cells secrete cytokines and/or chemokines, kill microorganisms, secrete antibodies, recognize cancer cells, and optionally kill such cells. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells.

The nucleic acid of the present invention is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA). Nucleic acids include, in accordance with the present invention, genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid of the present invention may be in the form of a molecule that is single-stranded, double-stranded, or covalently closed to form a ring. The nucleic acid can be used for introduction, for example, transfection, into cells, in the form of RNA, which can be obtained by in vitro transcription from a DNA template. Moreover, RNA can be modified before use through sequence stabilization, capping, and polyadenylation.

The nucleic acids described herein may be contained in the vector. The term “vector” as used herein includes all vectors known to one skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage vectors, viral vectors such as adenoviral or baculovirus vectors, or vectors based on artificial chromosomes, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). These vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and typically contain the necessary coding sequence and corresponding DNA sequences necessary for expression of the operably linked coding sequences in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal ) or in in vitro expression systems. Cloning vectors are typically used to design and amplify a specific DNA fragment of interest and may lack the functional sequences necessary to express the desired DNA fragments.

As the vector for expressing an antibody, either a vector type in which antibody chains are present in different vectors or a vector type in which antibody chains are present in the same vector can be used.

As used herein, the term “RNA” means a molecule containing ribonucleotide residues. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of the beta-D-ribo-furanose moiety. The term also includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, especially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA due to addition, deletion, substitution and/or changes to one or more nucleotides. Such changes may include the addition of non-nucleotide material, for example, to the end(s) of the RNA or within, for example, near one or more RNA nucleotides. Nucleotides in RNA molecules may be non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be similar or similar to naturally occurring RNAs.

In accordance with the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA" and refers to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. The mRNA typically contains a 5' untranslated region, a protein or peptide coding region, and a 3' untranslated region. mRNA has a limited half-life in cells and in vitro.

In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA.

The nucleic acids described in accordance with the present invention have preferably been isolated. The term “isolated nucleic acid” means, in accordance with the present invention, that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example , by digestion and gel electrophoretic fractionation, or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for processing using recombinant DNA techniques.

Nucleic acids in accordance with the present invention may be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In accordance with preferred embodiments, the nucleic acid is operably linked to express control sequences that may be homologous or heterologous to the nucleic acid. The term “homologous” means that the nucleic acids are also functionally linked in nature, and the term “heterologous” means that the nucleic acids are not functionally linked in nature.

A nucleic acid and an expression control sequence are also “operably” linked to each other if they are covalently linked to each other such that the expression or transcription of the nucleic acid is controlled or influenced by the expression control sequence. Thus, if a nucleic acid to be translated into a functional protein is operably linked to a coding sequence by an expression control sequence, then induction of said expression control sequence results in transcription of said nucleic acid without causing a frameshift of the coding sequence, or said coding sequence is unable to be translated into the required protein or peptide.

The term "expression control sequence" or "expression control element" includes, in accordance with the present invention, promoters, ribosome binding sites, enhancers and other control elements that regulate gene transcription or mRNA translation. In accordance with specific embodiments of the present invention, expression control sequences can be adjusted. The exact structure of the expression control sequences may vary depending on the function of the molecule or the cell type, but typically contains 5'-untranscribed and 5'- and 3'-untranslated sequences that are involved in the initiation of transcription and translation, respectively, such as TATA- box, capping sequence, CAAT sequence, etc. More specifically, the 5' untranscribed expression control sequences contain a promoter region that contains a promoter sequence to control the transcription of an operably linked nucleic acid. Expression control sequences may also contain enhancer sequences or upstream activating sequences.

In accordance with the present invention, the term "promoter" or "promoter region" refers to a nucleic acid sequence that is located upstream (5') of the nucleic acid sequence to be expressed and controls the expression of the sequence by providing an RNA recognition and binding site polymerases. The term "promoter region" may further include recognition and binding sites for additional factors that are involved in the regulation of gene transcription. A promoter can control the transcription of a prokaryotic or eukaryotic gene. In addition, a promoter may be "inducible" and may result in initiation of transcription in response to an inducing antigen, or may be "constitutive" if transcription is not controlled by the inducing agent. A gene that is under the control of an inducible promoter is not expressed or only expressed to a small extent if the inducing agent is absent. In the presence of the inducing agent, the gene is turned on or the level of transcription is increased. This is usually mediated by the binding of a specific transcription factor.

Promoters that are preferred in accordance with the present invention include the SP6, T3 and T7 polymerase promoters, the U6 RNA promoter, the CMV promoter, and artificial hybrid promoters thereof (e.g., CMV) in which a portion or portions are fused to a portion or portions of the promoters genes for other cellular proteins, such as, for example, human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and also contain or do not contain additional intron(s).

The term "expression" is used herein in its broadest sense and includes the formation of RNA or RNA and protein or peptide. In relation to RNA, the term "expression" or "translation" refers specifically to the formation of peptides or proteins. Expression may be transient or may be stable. In accordance with the present invention, the term expression also includes “aberrant expression” or “atypical expression”.

The term “aberrant expression” or “atypical expression” means, in accordance with the present invention, that the expression is altered, preferably increased, compared to a reference, for example, a condition in which the subject does not have a disease associated with aberrant or atypical expression of a particular protein, e.g. tumor-associated antigen. An increase in expression refers to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100% or more. According to one embodiment, expression occurs only in diseased tissue, while expression in healthy tissue is suppressed.

The term "specifically expressed" means that the protein is essentially expressed only in a specific tissue or organ. For example, a tumor-associated antigen specifically expressed in the gastric mucosa means that the protein is primarily expressed in the gastric mucosa and is not expressed in other tissues or is not expressed to a significant extent in other types of tissues or organs. Thus, a protein that is exclusively expressed in gastric mucosal cells and to a much lesser extent in any other tissue is specifically expressed in gastric mucosal cells. In some embodiments, the tumor-associated antigen may also be specifically expressed under normal conditions in more than one tissue type or organ, such as 2 or 3 tissue types or organs, but preferably in no more than 3 different tissue types or organs. In this case, the tumor-associated antigen is thus specifically expressed in these organs.

The term "translation" as used herein refers to the process in a cell's ribosomes by which a strand of messenger RNA directs the assembly of a sequence of amino acids to form a protein or peptide.

In accordance with the present invention, the term "nucleic acid encoding" means that the nucleic acid, when present in an appropriate environment, preferably in a cell, can be expressed to form the protein or peptide that it encodes.

The term "peptide" includes oligo- and polypeptides and refers to substances containing 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 or more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids linked covalently by peptide bonds. The term “protein” refers to large peptides, preferably those with more than 100 amino acid residues, however, in general, the terms “peptides” and “proteins” are synonymous and are used interchangeably herein.

Preferably, the proteins and peptides described in accordance with the present invention have been isolated. The terms "isolated protein" or "isolated peptide" mean that the protein or peptide has been separated from its natural environment. The isolated protein or peptide may be in a substantially purified state. The term “substantially purified” means that the protein or peptide is essentially free of other substances with which it is naturally associated or in vivo.

The description given herein with respect to certain amino acid sequences, e.g., amino acid sequences in a sequence listing, is to be interpreted to also apply to modifications, e.g., variants, of said specific sequences resulting in sequences that are functionally equivalent to said specific sequences. sequences, for example, amino acid sequences, exhibiting properties identical or similar to those of certain amino acid sequences. One important property is the retention of the antibody's binding to its target. Preferably, the sequence modified from a specific sequence by replacing a specific sequence in an antibody preserves the binding of said antibody to its target.

Those skilled in the art will appreciate that, in particular, the sequences of the CDR, hypervariable and variable region sequences can be modified without losing the ability to bind the target. For example, the CDR sequences will be either identical or highly homologous to the CDR sequences described herein.

By “highly homologous” it is meant that 1 to 5, preferably 1 to 4, for example 1 to 3 or 1 or 2 substitutions can be made.

The term “variant” in accordance with the present invention also includes mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those that are present in nature. An allelic variant refers to a change in the normal sequence of a gene, the significance of which is often unclear. Whole gene sequencing often identifies multiple variants of a particular gene. A species homolog is a nucleic acid sequence or amino acid sequence with origin from a different species than a specific nucleic acid sequence or amino acid sequence.

For purposes of the present invention, amino acid sequence “variants” include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid deletion variants that contain a deletion at the N-terminal and/or C-terminal end of the protein are also referred to as N-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise insertions of one or two or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are included at a specific site in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product.

Amino acid addition variants include amino- and/or carboxy-terminal fusions of one or more amino acids, for example, 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 a sequence, such as by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can occur at any position of the protein.

Amino acid substitution variants are characterized by at least one residue in the sequence to be deleted and another residue to be inserted at that location. Preference is given to modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and/or to substitution of amino acids with other amino acids having similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, ie, substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves a substitution with an amino acid from a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a specific amino acid sequence and an amino acid sequence that is a variant of said specific 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 preferably given for an amino acid region that 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, then the degree of similarity or identity is given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least 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 reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using means known in the art, preferably using a sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, penalty for opening a gap is 10.0, the penalty for extending the gap is 0.5.

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

The term "percentage identity" is intended to indicate the percentage of amino acid residues that are identical between two sequences to be compared, obtained after an optimal alignment, wherein the percentage is purely statistical and the differences between the two sequences are distributed randomly and throughout their length. Sequence comparisons between two amino acid sequences are routinely performed by comparing the sequences after they have been optimally aligned, wherein the comparison is performed segment by segment or using a comparison window to identify and compare local areas of sequence similarity. Optimal alignment of sequences for comparison can be performed, in addition to the manual method, using the local homology algorithm Smith and Waterman, 1981, Ads App.Math. 2, 482, using the local homology algorithm Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search method Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, WI).

Percent identity is calculated by determining the number of identical positions between two sequences to be compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

Homologous amino acid sequences are characterized according to the present invention by at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably by at least 95%, at least 98, or at least 99% amino acid residue identity.

The amino acid sequence variants described herein can be readily produced by one skilled in the art, for example, through the use of recombinant DNA. The use of DNA sequences to obtain proteins and peptides having substitutions, additions, insertions or deletions is described, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as, for example, solid phase synthesis and similar methods.

The present invention includes derivatives of the peptides or proteins described herein, which are encompassed by the terms "peptide" and "protein". In accordance with the present invention, "derivatives" of proteins and peptides are modified forms of proteins and peptides. Such modifications include any chemical modification and contain one or more substitutions, deletions and/or additions of any protein or peptide associated molecules, 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, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, protecting/blocking groups, proteolytic cleavage or binding to an antibody or other cellular ligand. The term "derivative" also covers all functional chemical elements of the specified proteins and peptides. Preferably, the modified peptide has increased stability and/or increased or decreased immunogenicity.

In accordance with the present invention, a variant, derivative, modified form, fragment, part or region of an amino acid sequence, peptide or protein is preferably characterized by a functional property of the amino acid sequence, peptide or protein, respectively, from which it is derived, i.e., it is functionally equivalent. In accordance with one embodiment, a variant, derivative, modified form, fragment, portion or region of an amino acid sequence, peptide or protein is immunologically equivalent to the amino acid sequence, peptide or protein, respectively, from which it is derived. According to one embodiment, the functional property is an immunological property.

The term "derived" means in accordance with the present invention that a certain structure, in particular a certain sequence, is present in the object from which it originates, in particular an organism or molecule. In the case of amino acid sequences, especially certain sequence regions, "derived" specifically means that the corresponding amino acid sequence is derived from the amino acid sequence in which it is present.

The term "cell" or "host cell" preferably represents an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components, such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, i.e. a cell capable of performing its normal metabolic functions. The term “cell” includes, in accordance with the present invention, prokaryotic cells (eg, E. coli) or eukaryotic cells (eg, dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeasts and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats and primates. Cells can come from a wide variety of tissue types and include primarily cells and cell lines. The term “cell” includes non-cancer cells and cancer cells, such as cells of the cancer types disclosed herein.

A cell that contains a nucleic acid molecule preferably expresses a peptide or protein encoded by said nucleic acid.

"Target cell" will refer to a cell that is the target of an immune response, such as an antibody. Target cells include any unwanted cell, such as a cancer cell described herein. In preferred embodiments, the target cell is a cell expressing CLDN6. Cells expressing CLDN6 typically include cancer cells.

The term "transgenic animal" refers to an animal having a genome containing one or more transgenes, preferably heavy and/or light chain transgenes, or transchromosomes (either integrated or not integrated into the animal's natural genomic DNA), and which is preferably capable of expressing the transgenes. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome such that the mouse produces antibodies to human CLDN6 when immunized with CLDN6 antigen and/or cells expressing CLDN6. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as is the case with transgenic mice, such as HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene can be maintained extrachromosomal, as is the case with transchromosomal (eg, KM) mice , described in WO 02/43478. Such transgenic and transchromosomal mice may be capable of producing multiple isotypes of human monoclonal antibodies to CLDN6 (eg, IgG, IgA, and/or IgE) via V-D-J recombination and isotype switching.

The term “immunologically equivalent” means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid, has the same or substantially the same immunological properties and/or exerts the same or substantially the same immunological effects, e.g. to the type of immunological effect, such as the induction of a humoral immune response, the strength and/or duration of the induced immune response, or the specificity of the immune response. In the context of the present invention, the term "immunologically equivalent" is preferably used in relation to the immunological effects or properties of the peptide or peptide variant used for immunization or antibody. A certain immunological property is the ability to bind to antibodies and, if necessary, lead to the formation of an immune response, preferably by stimulating the formation of antibodies. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence, when exposed to the immune system of a subject, results in the induction of an immune response, preferably antibodies having specificity for reacting with the reference amino acid sequence, such as the reference amino acid sequence, forming part of CLDN6.

The present invention provides methods for detecting the presence of CLDN6 antigen in a sample, or measuring the amount of CLDN6 antigen, comprising contacting the sample, and optionally a control sample, with an antibody of the present invention that binds to CLDN6 under conditions that allow formation of a complex between the antibody and CLDN6 . Complex formation is then detected, with the difference in complex formation between the sample compared to the control sample indicating the presence of CLDN6 antigen in the sample.

The methods described above are suitable, in particular, for the diagnosis of CLDN6-related diseases, such as cancers, for example, the cancers described herein. Preferably, an amount of CLDN6 in a sample that is higher than an amount of CLDN6 in a reference or control sample indicates the presence of a CLDN6-related disease in the subject, particularly the person from which the sample originates.

When used in the methods described above, the antibody described herein may be provided with a label that functions to: (i) provide a detectable signal; (ii) interacting with a second label to modify the detectable signal generated by the first or second label, for example, using FRET (fluorescence resonance energy transfer); (iii) affecting mobility, eg, electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) providing a capture moiety, eg, affinity, antibody/antigen, or ionic complexation. Suitable labels include structures such as fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzyme labels, particulate labels, in particular metal particle labels, magnetic particle tags, polymer particle tags, small organic molecules such as biotin, receptor ligands or binding molecules such as cell adhesion proteins or lectins tagging sequences containing nucleic acids and/or amino acid residues that can be detected through the use of binding agents, etc. Labels include, but are not limited to, barium sulfate, yocetamic acid, iopanoic acid, calcium ipodate, sodium ditriazoate, meglumine diatriazoate, metrizamide, sodium tyropanoate and x-ray, including positron emitters such as fluorine-18 and carbon-11, gamma probes , such as iodine-123, technetium-99t, iodine-131 and indium-111, nuclear magnetic resonance nuclides such as fluorine and gadolinium.

In accordance with the present invention, a "reference object", such as a reference sample or a reference organism, can be used to correlate and compare the results obtained in the methods of the present invention with the test sample or test organism. Typically, the reference organism is a healthy organism, in particular an organism that is not suffering from a disease such as cancer. The "reference value" or "reference level" can be determined from a reference object empirically by measuring a sufficiently large number of reference objects. 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 at least 50 or preferably at least 100 reference objects.

The term "reduce" or "inhibit" as used herein means the ability to cause an overall reduction in level of preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. more. The term "inhibit" or similar expressions include complete or substantially complete inhibition, ie, reduction to zero or substantially zero.

Terms such as “increase” or “enhance” preferably refer to an increase or increase by about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably by 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 subject with a disease. The diseases that can be diagnosed include all diseases that express CLDN6. Particularly preferred diseases are cancers, such as the cancers described herein.

In accordance with the present invention, the term “disease” refers to any pathological condition, including cancer, in particular the forms of cancer described herein.

The term "normal", as used in terms of "normal tissue" or "normal conditions", refers to healthy tissue or conditions in the body of a healthy subject, i.e., non-pathological conditions, in which "healthy" preferably means not related to cancerous

The term “disease involving cells expressing CLDN6” means, in accordance with the present invention, that CLDN6 is expressed in cells of the affected tissue or organ. In accordance with one embodiment, the expression of CLDN6 is increased compared to the state in healthy tissue or organ. An increase refers to an increase of 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. According to one embodiment, expression occurs only in diseased tissue, while expression in healthy tissue is suppressed. In accordance with the present invention, diseases involving or associated with cells expressing CLDN6 include cancers, in particular the forms of cancer described herein.

In accordance with the present invention, the term "tumor" or "neoplastic disease" refers to a swelling or lesion formed as a result of atypical growth of cells (called neoplastic or tumor cells). By "tumor cell" is meant an atypical cell that grows as a result of rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the neoplasm cease. Tumors are characterized by a partial or complete lack of structural organization and functional coordination compared to normal tissue, and usually form a distinct mass of tissue that can be either benign, precancerous, or malignant.

A benign tumor is a tumor that lacks all three malignant properties of cancer. Thus, by definition, a benign tumor does not grow in an unrestricted aggressive manner, invade surrounding tissue, or spread to non-contiguous tissues (metastasize). Common examples of benign tumors include moles and uterine fibroids.

The term "benign" implies mild and non-progressive disease, and in fact many types of benign tumors are harmless. However, some neoplasms that are defined as “benign tumors” because they lack the invasive properties of cancer may still have negative health effects. Examples of this include tumors that result in "mass impact" formation (compression of vital organs such as blood vessels), or "functional" tumors of endocrine tissues that can overproduce certain hormones (examples include thyroid adenomas, adrenocortical adenomas and pituitary adenomas).

Benign tumors are typically surrounded by an outer membrane that inhibits their ability to function in a malignant manner. In some cases, certain "benign" tumors may later lead to the formation of malignant cancers that arise from additional genetic changes in a subpopulation of neoplastic cells in the tumor. A prominent example of this phenomenon is tubular adenoma, a common type of colon polyp that represents an important precursor to colon cancer. The cells of tubular adenomas, like most tumors that often progress to cancer, are characterized by certain abnormalities in cell maturation and appearance, collectively known as dysplasia. These cellular abnormalities are not noticeable in benign tumors, which rarely or never become cancerous, but are noticeable in other precancerous tissue lesions that do not form distinct masses, such as precancerous lesions of the cervix. Some experts prefer to label dysplastic tumors as "precancerous" and reserve the term "benign" for tumors that rarely or never lead to cancer.

The neoplasm is an atypical mass of tissue as a result of neoplasia. Neoplasia (neoplasm translated from Greek) is an atypical proliferation of cells. Cell growth is greater and uncoordinated than that of normal tissue cells around it. Growth continues in the same excessive manner even after the stimuli cease. It usually results in the formation of a lump or tumor. Neoplasms can be benign, precancerous or malignant.

“Tumor growth” or “tumor growth” in accordance with the present invention refers to the property of a tumor to increase in size and/or the property of tumor cells to proliferate.

Cancer (medical term: malignancy) is a class of diseases in which a group of cells is characterized by uncontrolled growth (dividing beyond normal limits), invasion (invading and destroying adjacent tissues), and sometimes metastasis (spreading to other sites in the body through lymph or blood). These three malignant properties of various cancers distinguish them from benign tumors, which are self-limiting and do not invade or metastasize. Most different cancers form a tumor, but sometimes, like leukemia, do not. In accordance with the present invention, the terms “cancer” and “tumor” or “cancer disease” and “tumor disease” are generally used interchangeably herein to refer to diseases in which cells are characterized by uncontrolled growth and, optionally, invasion and/or metastasis.

Preferably, the “cancer” according to the present invention is characterized by cells expressing CLDN6. The cell expressing CLDN6 is preferably a cancer cell, preferably tumors or various types of cancer described herein. Preferably, such a cell is a cell other than a placental cell.

Different types of cancer are classified by the type of cell that resembles the tumor, and thus the tissue that is thought to be the source of the tumor. They are characterized by histological structure and position, respectively.

The term "cancer" in accordance with the present invention includes leukemia, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, pancreatic cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, colon cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, cancer ear, nose and throat (ENT), breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, as well as their metastases. Examples include lung carcinomas, breast carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the types of cancer or tumor described herein. The term cancer in accordance with the present invention also includes 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 subtypes of non-small cell lung carcinomas: squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas account for approximately 10% of various types of lung cancer. This type of cancer is usually seen peripherally in the lungs, as opposed to small cell lung cancer and squamous cell lung cancer, both of which tend to be more centrally located.

In accordance with the present invention, "carcinoma" is a malignant tumor originating from epithelial cells. This group represents the most common types of cancer, including common forms of breast cancer, prostate cancer, and colon cancer.

"Adenocarcinoma" is a cancer that originates from glandular tissue. This tissue also represents a larger category of tissue known as epithelial tissue. Epithelial tissue includes the skin, glands, and a number of other tissues that line the cavities and organs of the body. The epithelium is embryologically derived from ectoderm, endoderm and mesoderm. To be classified as adenocarcinoma, the cells do not have to be part of the gland, provided that they are characterized by secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas tend to resemble the glandular tissue from which they originate, while poorly differentiated adenocarcinomas may not. By staining the cells obtained from the biopsy, the pathologist will determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can form in many tissues of the body due to the universal nature of glands in the body. Although each gland may not secrete the same substance, provided that there is an exocrine function in the cell, it is considered glandular, and its malignant form is thus called adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize if given enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma and endometrioid adenocarcinoma.

“Metastasis” refers to the spread of cancer cells from their original site to another part of the body. The formation of metastases is a very complex process and depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of endothelial basement membranes into the cavities and vessels of the body and then, after movement into the blood, infiltration of target organs. Ultimately, the growth of a new tumor, i.e., a secondary tumor or a metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may persist and exhibit metastatic potential. In accordance with one embodiment, the term "metastasis" in accordance with the present invention refers to "distant metastasis", which refers to a metastasis that is distant from the primary tumor and the regional lymph node system.

The cells of a secondary or metastatic tumor are similar to the cells in the original tumor. This means, for example, that if ovarian cancer metastasizes to the liver, the secondary tumor will form from atypical ovarian cells rather than from atypical liver cells. A tumor in the liver is therefore called metastatic ovarian cancer rather than liver cancer.

Relapse or relapse occurs when an individual is re-affected by a condition that affected them in the past. For example, if a patient has suffered from cancer, has received effective treatment for said disease, and develops said disease again, then said newly developed disease may be considered a relapse or relapse. At the same time, in accordance with the present invention, relapse or re-development of cancer may occur, but does not necessarily occur, at the site of the original cancer. Thus, for example, if a patient suffered from an ovarian tumor and received effective treatment, relapse or recurrence may represent the occurrence of an ovarian tumor or the occurrence of a tumor in a site other than the ovary. Recurrence or re-development of a tumor also includes situations in which a tumor forms in a site different from the site of the original tumor, as well as in a site of the original tumor. Preferably, the original tumor for which the patient was treated is a primary tumor, and the tumor at a site different from the original tumor is a secondary or metastatic tumor.

By “treat” is meant the administration of a compound or composition to a subject for the purpose of preventing or eliminating a disease, including reducing the size of a tumor or the number of tumors in the subject; stopping or slowing down the disease in the subject; suppressing or slowing the development of a new disease in the subject; reducing the frequency or severity of symptoms and/or recurrence in a subject who currently has or has previously had the disease; and/or prolongation, i.e., increasing the life expectancy of the subject.

In particular, the term “treating a disease” includes curing, reducing the duration of, alleviating, preventing, slowing or suppressing the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms.

The term "at risk" refers to a subject, i.e., a patient, who is defined as having a higher than normal likelihood of developing a disease, particularly cancer, compared to the general population. In addition, a subject who has had or currently has a disease, in particular cancer, is a subject who has an increased risk of developing the disease, since such a subject may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of developing cancer metastases.

The term "immunotherapy" refers to treatment that involves a specific immune response.

In the context of the present invention, terms such as “protect,” “prevent,” “prophylactic,” “prophylactic,” or “protective” refer to preventing or treating, or both, the occurrence and/or spread of a disease in a subject, particularly with for the purpose of minimizing the likelihood that a subject will contract a disease or for the purpose of slowing the progression of a disease. For example, an individual at risk of developing a tumor described herein would be a candidate for tumor prevention therapy. Immunotherapy can be carried out through a number of techniques in which the agents function to eliminate antigen-expressing cells from the patient's body.

In certain embodiments, the immunotherapy may be an active immunotherapy in which treatment is based on in vivo stimulation of the host's endogenous immune system to mount a reaction against diseased cells upon administration of immune response-modifying agents (such as immunoreactive peptides and nucleic acids).

In other embodiments, the immunotherapy may be a passive immunotherapy, in which the treatment involves the delivery of agents with established tumor immune reactivity (such as antibodies) that can directly or indirectly mediate antitumor effects, and is not necessarily dependent on an intact host immune system .

The term "in vivo" refers to the situation in the subject's body.

The terms "subject", "individual", "organism" or "patient" are used interchangeably and refer to vertebrates, preferably mammals. For example, mammals in the context of the present invention include humans, non-human primates, domestic animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as captive animals such as zoo animals. The term "animal" as used herein also includes humans. The term "subject" may also include a patient, i.e., an animal, preferably a human, having a disease, preferably a disease described herein.

In accordance with the present invention, a "sample" may be any sample suitable in accordance with the present invention, in particular a biological sample, for example a tissue sample, including biological fluids, and/or a cellular sample, and may be prepared in a standard manner such as tissue biopsy, including punch biopsy, and by drawing blood, bronchial aspiration, sputum, urine, stool, or other body fluids. In accordance with the present invention, the term “sample” also includes processed samples, such as fractions or isolates of biological samples, for example, nucleic acid and peptide/protein isolates. Preferably, the sample contains cells or tissue of an organ that are to be examined, for example, to be diagnosed for cancer. For example, if the cancer to be diagnosed is lung cancer, then the sample may contain cells or tissue obtained from the lung.

In accordance with the present invention, the sample may be a sample, such as a biological sample, originating from a patient, containing or suspected of containing tumor or cancer cells. The biological sample may be any tissue sample, such as blood, a tissue sample obtained from a primary tumor or from tumor metastases, or any other sample containing tumor or cancer cells.

The present invention is described in detail by means of the figures and examples below, which are used for illustrative purposes only and are not intended to be limiting. By way of description and examples, additional embodiments that are likewise included in the present invention will be readily available to one skilled in the art.

Description of drawings

Fig. 1A: Sequence alignment of claudin 6 and claudin 9 proteins (human/mouse)

Sequence alignment shows high homology between human and mouse claudin 6 and human claudin 9.

Fig. 1B: Sequence alignment of claudin 6 and claudin 3, 4 and 9 proteins (human)

Sequence alignment shows high homology in the multigene claudin family.

Fig. 2A and B: Specificity of antibodies tested in Western blotting

Cell lysates of HEK293 cells transfected with mock control, CLDN3, 4, 6, or 9, and CLDN6 positive human tumor cells (PA-1 SC12 and NEC-8) were blotted and bound antibodies (rabbit anti-CLDN3 antibody (hwitrogen), 0.5 µg/ml, mouse anti-CLDN4 (Invitrogen), 1 µg/ml, rabbit anti-CLDN6 (IBL), 0.2 µg/ml, goat anti-CLDN9 (Santa Cruz), 0.4 µg/ml, or monoclonal mouse antibodies (5 μg/ml) were detected using peroxidase-conjugated secondary antibodies.

Fig. 3: Histological analysis of Western blot positive antibodies

Binding of priority mumABs 58-1B, 58-3A, and 58-4B compared to rabbit anti-CLDN6 IBL antiserum to ovarian cancer FFPE sections.

Fig. 4A: Epitope mapping using synthetic overlapping peptides

Overlapping peptides were immobilized on microtiter plates and antibodies were added. Bound antibodies were visualized using appropriate peroxidase-conjugated secondary reagents.

Fig. 4B: Epitope mapping using biotinylated synthetic overlapping peptides

Highly overlapping peptides, N-terminally biotinylated with a flexible hydrophilic linker, were synthesized and loaded onto streptavidin-coupled microtiter plates. Antibodies (1 μg/ml) were applied and bound antibodies were detected and analyzed.

To compare the signal intensity of mumAB 58-4B-2 with rabbit serum (IBL), the maximum binding of antibodies to the C-terminal peptide 19 was determined as 100%. The intensity of binding of each antibody to a single peptide was calculated relative to the maximum binding for each system tested. mumAB binding was analyzed in 3 independent experiments in triplicate. Serum IBL binding was analyzed in 2 independent experiments in triplicate.

Fig. 4C: Binding site of monoclonal priority 58-4B-2 and polyclonal rabbit serum to CLDN6 (IBL)

Fig. 5: Sequences of antibodies 58-1B, 58-3A and 58-4B

Fig. 6: Background signals of various antibodies in relation to normal ovarian tissue

Comparison of priority antibodies mumAB 58-1B, 58-3A and 58-4B compared with commercial antibody IBL against normal ovarian tissue (antibody concentration 5 μg/ml; clinical protocol).

Examples

The techniques and methods used herein are described herein and are carried out as are well known 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, N.Y. All methods, including the use of kits and reagents, are carried out in accordance with the manufacturer's information unless otherwise stated.

Example 1. Materials and methods

Antibody binding site mapping (Figure 4A)

Mapping of epitopes recognized by antibodies can be done as described in detail in "Epitope Mapping Protocols (Methods in Molecular Biology) Glenn E. Morris ISBN-089603-375-9 and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 Olwyn M. R. Westwood, Frank C. Hay.

Epitope mapping using biotinylated peptides (Figure 4B)

Peptide ELISA was performed to identify the antigen binding site of monoclonal mouse priority antibodies and rabbit polyclonal serum from IBL. Biotinylated overlapping peptides spanning the C-terminal sequence of CLDN6 were coupled to SA-coated plates. Purified mumAB (1 μg/ml) or rabbit serum IBL (1 μg/ml) was applied to antigen-coated ELISA plates and unbound antibodies were washed away. Bound antibodies were detected using an appropriate enzyme-labeled secondary antibody (alkaline phosphatase-linked goat anti-mouse IgG(1+2a+2b+3) or alkaline phosphatase-linked goat anti-rabbit IgG F(ab)2) and ABTS enzyme substrate and signal intensities were analyzed. To compare the signal intensity of mumAB 58-4B-2 with rabbit serum (IBL), the maximum binding of antibodies to the C-terminal peptide 19 was determined as 100%. The intensity of binding of each antibody to a single peptide was calculated relative to the maximum binding for each system tested (mouse or rabbit) independently.

mumAB binding was analyzed in 3 independent experiments in triplicate. Serum IBL binding was analyzed in 2 independent experiments in triplicate.

Determination of isotypes

To determine the isotypes of purified antibodies, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, cat. no. 1493027) was used as described by the manufacturer.

Western blotting

The newly obtained anti-CLDN6 IgG antibody could be further examined for specific binding to the CLDN6 antigen by Western blotting. Briefly, cell extracts from cells expressing CLDN3, 4, 6, or 9 and corresponding negative controls could be prepared and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens were transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies of interest. IgG binding was detected using horseradish peroxidase-conjugated anti-mouse IgG antibody and visualized using ECL substrate.

Histological examination

Immunohistochemical analysis (IHC) was performed on glass slides containing tissue samples fixed in 4% buffered formalin and embedded in paraffin. Paraffin embedding was carried out according to standard protocols.

After deparaffinization and rehydration, all microscopic slides were subjected to antigen unmasking by boiling in 0.1 M Tris/0.01 M EDTA buffer with the addition of 15 mM sodium azide (pH 9.0) at 95-98°C for 30 minutes, and endogenous peroxidases were then quenched by adding 3% H ₂ O ₂ supplemented with 15 mM NaN ₃ . After washing with 150 mM sodium chloride buffer (pH 7.6) supplemented with 0.05% (w/v) Tween-20 and 0.005% (w/v) Proclin, microscope slides were incubated with 1.0 or 5.0 μg/ml diagnostic mouse monoclonal antibody to CLDN6 58-4B at room temperature for one hour. Antibody binding was visualized using a ready-to-use solution containing horseradish peroxidase-labeled polymer-based secondary antibody (Histofine MAX PO(M)/UIO MAX PO(M), Nichirei, Japan). The sections were then counterstained with Mayer's hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and assessed by experts.

Histological evaluation

All samples were analyzed based on the relative proportion of positive stained cells relative to all visible tumor cells for each section. Staining intensity was classified as negative (−), weakly positive (1+), moderately positive (2+), and highly positive (3+). Only membrane staining was considered positive. Ovarian cancer tissue served as a positive control for each stain. In addition, it was found that embryonic kidney tissue from New Zealand white rabbits on the 29th day of the gestational period was characterized by highly positive staining intensity. It was used as an internal standard for staining intensity for a positive staining result (2+ - 3+).

Example 2. Preparation of monoclonal antibodies

The goal of this work was to generate murine monoclonal CLDN6-specific antibodies capable of detecting tumor cells expressing CLDN6 in tissue affected by ovarian cancer or any other cancerous tissue of any histological structure, including tissues of primary peritoneal tumors or FFPE fallopian tube tumors.

To obtain a highly specific, high-affinity diagnostic antibody against CLDN6, it was necessary to initiate immunization protocols using various immunogens (Table 1) and adjuvants. During the study, approximately 130 mice (C57BL/6 and BALB/c) were exposed to different immunization strategies to activate an immune response against α-CLDN6 (Table 2).

To activate the mouse immune system and overcome immune tolerance, we used peptide conjugates and recombinant proteins encoding various parts of human CLDN6, expressed as recombinant fusion proteins with different epitopes to facilitate affinity purification (polyhistidine (HIS)- and glutathione-S-transferase-( GST)-tag) (Table 1).

Of the 20 different immunization strategies, the best results were achieved by treating mice with GST-tagged C-terminal recombinant CLDN6 protein (Table 2) in combination with various adjuvants (Table 2).

On the day of fusion, mouse splenocytes were isolated and fused with the murine myeloma cell line P3X63Ag8.653 (ATCC). To fuse mouse cells with a myeloma cell line, the standard protocol published by Köhler and Milstein 1975 was followed. After selection using hypoxanthine-aminopterin-thymidine (HAT), supernatants were tested in ELISA for the secretion of antibodies recognizing the antigen used for immunizations.

69 fusions were performed and more than 25,000 hybridoma cell clones were screened. Hybridoma cells from ELISA positive supernatants (402) were subcloned to produce monoclonal hybridomas. Supernatants of subcloned hybridoma cells were rescreened for antigen binding to claudin 6. Hybridoma cells from 88 positive clones (binding to antigen but not scaffold or tag) were expanded and further analyzed by Western blotting for their specificity.

Three priority candidates (58-4B, 58-1B and 58-3A) were obtained through an 11-stage immunization strategy (immunization #10 for 123 days). Three days before splenectomy, mice were boosted to activate target B cells followed by fusion with SEQ ID NO:58 (Table 3).

Example 3: Screening of monoclonal antibodies using Western blotting

To investigate whether the ELISA-positive monoclonal antibodies in the supernatants were able to specifically bind to recombinant claudin 6, they were analyzed by Western blotting. Antibodies that bound to CLDN6, but not to any other tagged protein, were purified, and the cells were expanded and cryopreserved. Antibodies were purified by FPLC using MABselect protein A affinity chromatography resin. Purified antibodies selected by Western blot screening were reanalyzed by Western blot to assess binding to CLDN6 positive tumor cell lines (PA-1, NEC-8) and to HEK293 cells transfected with CLDN3, 4, 6 or 9 (Fig. 2). In addition, antibodies were evaluated for their ability to bind their antigen in formalin-fixed paraffin-embedded (FFPE) tissues using immunohistochemical analysis. Supernatants from 33 hybridomas that specifically bind claudin 6 were purified and further analyzed in an immunohistochemical assay.

Example 4 Histological Analysis of Western Blot Positive Antibodies

The purpose of this experiment was to study the specificity and sensitivity of antibodies to CLDN6. This was accomplished using FFPE ovarian cancer tissues expressing CLDN6 (Figure 3).

In the first experiment, purified antibodies that were tested positive in the 1st Western blot screen were analyzed at concentrations of 1 μg/ml and 5 μg/ml on FFPE ovarian cancer sections. A laboratory standard and overnight staining protocol using nitrate unmasking buffer and a unmasking temperature of 120°C were used. Antibodies exhibiting tissue-specific CLDN6 staining without producing high levels of nonspecific background staining were further titrated to 0.2, 1, 2, and 5 μg/ml on various ovarian cancer tissues to further evaluate the sensitivity and specificity of these antibodies. In subsequent stages of development, the first antibodies obtained were tested directly at high concentrations of 1 and 5 μg/ml, since differences in staining intensities could not be clearly assessed at a concentration of 0.2 μg/ml. Antibodies exhibiting strong signals against ovarian tumor cell membranes of selected tissues and little or no background activity against adjacent healthy tissues were selected for additional dose determination experiments and specificity analyses. The following three antibodies met these criteria and were selected as priority candidates for additional study: 58-1B, 58-3A, and 58-4B.

Example 5: MumAb Epitope Mapping (Figure 4)

Peptide ELISA was performed to identify antibody-binding epitopes on CLDN6. Each purified antibody was screened against peptides spanning the 11 amino acids spanning the C-terminal sequence of CLDN6. Antibodies 46-5A, 58-1B, 58-3A, 58-4B, and 58-9B had specific binding to the epitope covered by the AISRGPSEYPTKNYV peptide (peptide 7; Fig. 4A). The binding site of these antibodies was further characterized using biotinylated peptides overlapping 14 of the 15 amino acids. Surprisingly, antibodies 58-1B, 58-3A and 58-4B and subclone 58-4B-2 binding to the EYPTKNY sequence (Fig. 4B) were able to bind CLDN6 in FFPE cancer tissues very specifically with low background activity. In contrast, antibody 46-5A was able to bind to peptides that did not contain the EYPTKNY sequence but contained part of it, i.e., the EYPTK sequence (Fig. 4B). A commercially available polyclonal rabbit antiserum to CLDN6 (IBL) bound to peptides containing the RGPS sequence (Fig. 4C).

Example 6 Antibody Specificity Analysis Using a Normal Tissue Panel

Antibodies with strong signals against ovarian cancer tissue (58-1B, 58-3A and 58-4B) were further analyzed using a laboratory standard and established staining protocol against various corresponding normal tissues, namely healthy tissues. donors (eg, lung, testis, uterus, and ovary) to confirm high target specificity for CLDN6. Staining of these selected tissues was performed using a laboratory standard and established staining protocol (overnight; unmasking temperature 120°C, citrate unmasking buffer). None of the three selected antibodies exhibited nonspecific staining signals relative to the panel of normal tissues studied, except for weak nonspecific signals in one peripheral region of one ovarian tissue sample. This weak signal was noticeable at higher concentrations of both tested (1 and 5 μg/ml), not representing ovarian substructure.

Example 7: Comparison of Antibody Sensitivity Using Ovarian Cancer Tissue Samples and Normal Tissue Panel Samples

Minor differences in the staining patterns and staining intensities of antibodies 58-3A, 58-1B, and 58-4B were noticeable in previous experiments. Thus, the antibodies were subjected to staining experiments using a more clinically oriented, namely faster, staining protocol. To mimic the staining processes used in standard pathology laboratories, a one-day protocol was introduced using a 1-hour primary antibody incubation step and a water bath (98°C) unmasking step. In addition, two different unmasking buffers were compared to examine whether different unmasking conditions could influence the sensitivity of the test antibodies for detection of CLDN6. For all antibodies, more intense signals and higher numbers of positively stained tumor cells were obtained using an adapted one-day protocol using Tris unmasking buffer compared with standard citrate unmasking buffer. More intense staining signals were obtained with 58-4B, reaching 80% of 3+ stained tumor cells, while candidate 58-1B staining intensities were the least pronounced, reaching only 30% of 3+ stained tumor cells.

The Tris unmasking buffer staining protocol resulting in the most sensitive staining signals was subsequently used to stain a panel of normal tissues to examine the specificity of selected antibodies under standard clinical laboratory conditions.

The most effective results regarding specificity and sensitivity for CLDN6 in FFPE tissues were obtained with the diagnostic antibody candidate 58-4B. No signals were detected in relation to normal tissues. All other antibodies showed signals against the Reinke crystals of the testis sample. In addition, antibody 58-3A showed weak staining for blood vessels. Antibody 58-1B was characterized by more intense signals (1+) in relation to the testes and tissues of normal ovaries.

In all samples analyzed, mumAb 58-4B was superior in demonstrating absence of background staining and strong binding to the corresponding tumor tissue, while mumAb candidates 58-3A and 58-1B were characterized by intense staining to the corresponding tumor tissue , but with higher background signals. In particular, the signals produced by mumAb 58-1B were high in intensity, but lacked high background staining.

For subsequent use in clinical practice, the corresponding hybridoma was adapted to serum-free media to obtain diagnostic antibody candidates. Unfortunately, adaptation of clone 58-3A to serum-free conditions was not possible. Thus, the antibody candidate 58-3A was also excluded from histological studies, leaving 58-4B as the final priority candidate.

Example 8. Histological in-depth analysis and characterization of antibodies

In the first step, a batch of antibody 58-4B produced under serum-free conditions was compared to a batch of antibody produced under serum-free conditions using a TMA normal tissue panel (MNO961) to identify differences in target sensitivity and target specificity between batches. Staining was performed using a similar clinical 1-day protocol (water bath unmasking, Tris unmasking buffer) using royalty-free components.

In both batches of antibody, weak staining was detected in some normal tissue samples (see Table 4). Compared to the antibody produced under serum conditions, the batch of antibody produced under serum-free conditions was purer, with fewer artificial contaminants in the samples analyzed. In relation to the testes, staining signals were prominent in relation to Reinke crystals for both batches of antibody.

In a second step, the antibody produced under serum-free conditions was tested against the TMA ovarian cancer panel and compared with the commercially available IBL antibody. This commercial antibody has already been used in staining in previous studies. Polyclonal IBL antibody was used according to the study protocol (overnight, unmasking temperature 120°C, citrate unmasking buffer). Antibody 58-4B was used with a standard clinical-like protocol (one-day protocol, water bath unmasking, Tris unmasking buffer).

Of the 72 TMA ovarian cancer cases detected, only one sample was positive for the IBL antibody. In contrast, 14/72 specimens tested were positive for CLDN6 when stained with antibody 58-4B.

Unfortunately, the quality of commercial TMA ovarian cancer samples was poor, resulting in weak staining signals relative to positive tumor samples. Therefore, the comparison was repeated using biopsy samples from Dr. Dhaene, representing larger areas of tumors with high-quality preservation of tumor samples of the lung, uterus and testes.

58-4B staining resulted in more sensitive and specific detection of CLDN6 in tumor samples compared to stainings performed with the polyclonal antibody IBL, meaning that more tumor cells were positive and a greater intensity of tumor cell staining was detected.

Example 9 Priority Antibody Sequence Analysis

Sequence analysis of antibodies 58-1B, 58-3A and 58-4B is presented in Fig. 5 and below

Additional protocol for biological material

Identification of additional deposits

1) Name and address of the escrow institution for making deposits:

Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

Inhoffenstr. 7V

38124 Braunschweig

DE

Additional designations for all deposits mentioned

- Mouse myeloma cells (Mus musculus) Ag8.653 fused with mouse splenocytes (Mus musculus)

- Hybridoma secreting antibody against human CLDN6

2) Depositor

All the above deposits were made:

Ganymed Pharmaceuticals AG

An der Goldgrube 12

55131 Mainz

DE

--->

LIST OF SEQUENCES

<110> ASTELLAS PHARMA INC.etc.

<120> ANTIBODIES SUITABLE IN CANCER DIAGNOSIS

<130> 342-99 PCT2

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<151> 2017-09-06

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Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp

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Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser

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Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

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Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu

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Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala

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Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu

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Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

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Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

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Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu

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Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser

100 105 110

Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala

115 120 125

Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile

130 135 140

Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met

145 150 155 160

Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

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Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro

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20 25 30

Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Met Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Val Thr Leu Leu Ile Val Leu Leu Gly Leu Leu

85 90 95

Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Asp Arg Asn

100 105 110

Ser Lys Ser Arg Leu Val Leu Ile Ser Gly Ile Ile Phe Val Ile Ser

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Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ser Ile Ile

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Gln Asp Phe Tyr Asn Pro Leu Val Ala Asp Ala Gln Lys Arg Glu Leu

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Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Ala Cys Ser Ser Gly Gly Thr Gln Gly

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Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

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Ser Gln Gly Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala

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Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg Pro Arg Gly Pro

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Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

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20 25 30

Trp Ile His Trp Val Lys Glu Arg Pro Gly Gln Gly Leu Val Trp Ile

35 40 45

Gly Ala Ile Phe Pro Gly Asn Ser Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Ser Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Asp Leu Ser Ser Leu Thr Asp Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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100 105 110

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Asp Ile Val Leu Thr Gln Asn Pro Leu Thr Leu Ser Val Thr Ile Gly

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Gln Thr Ala Ser Ile Ser Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Met Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Asn Thr

100 105 110

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Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

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20 25 30

Trp Met His Trp Val Arg Glu Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Glu Asn Ser Asp Ala Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ser Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Thr Asp Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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100 105 110

Ser Ser

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Asp Val Val Met Thr Gln Asn Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Thr Ala Ser Ile Ser Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Met Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105 110

<210> 44

<211> 114

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Antibody chain

<400> 44

Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Arg Thr Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Glu Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Glu Asn Ser Asp Ala Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ser Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Glu Leu Ser Ser Leu Thr Asp Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Phe Tyr Ala Thr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser

<210> 45

<211> 112

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Antibody chain

<400> 45

Asp Ile Val Leu Thr Gln Asn Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Thr Ala Ser Ile Ser Cys Lys Ser Ser Gln Asn Leu Leu Tyr Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Leu Met Ser Lys Leu Glu Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ser

100 105 110

<210> 46

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 46

Cys Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu

1 5 10 15

Leu

<210> 47

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 47

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

1 5 10 15

Pro Ser His Tyr

20

<210> 48

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 48

Cys Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser Arg Gly Pro Ser

1 5 10 15

Glu Tyr Pro Thr Lys

20

<210> 49

<211> 39

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 49

Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly Pro Ser His Tyr Met

1 5 10 15

Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser Arg Gly Pro Ser Glu

20 25 30

Tyr Pro Thr Lys Asn Tyr Val

35

<210> 50

<211> 61

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 50

Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp Ser Lys Ala Arg Leu

1 5 10 15

Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser Gly Val Leu Thr Leu

20 25 30

Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn

35 40 45

Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu Gly Ala

50 55 60

<210> 51

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable region CDR

<400> 51

Gly Tyr Ile Phe Thr Thr Tyr Trp

15

<210> 52

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable region CDR

<400> 52

Ile Phe Pro Gly Asn Ser Asp Thr

15

<210> 53

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable region CDR

<400> 53

Thr Arg Glu Phe Tyr Ala Thr

15

<210> 54

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain variable region CDR

<400> 54

Gln Asn Leu Leu Tyr Ser Asp Gly Lys Thr Tyr

1 5 10

<210> 55

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain variable region CDR

<400> 55

Leu Met Ser

1

<210> 56

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain variable region CDR

<400> 56

Trp Gln Gly Thr His Phe Pro Trp Thr

15

<210> 57

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable region CDR

<400> 57

Gly Tyr Thr Phe Thr Thr Tyr Trp

15

<210> 58

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Heavy chain variable region CDR

<400> 58

Ile Tyr Pro Glu Asn Ser Asp Ala

15

<210> 59

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 59

Glu Tyr Pro Thr Lys

15

<210> 60

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 60

Glu Tyr Pro Thr Lys Asn

15

<210> 61

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Peptide

<400> 61

Tyr Pro Thr Lys Asn Tyr

15

<---

Claims (103)

1. An antibody or antigen binding fragment thereof that binds claudin 6 (CLDN6), wherein the antibody or antigen binding fragment thereof comprises: the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having the amino acid sequence of SEQ ID NO: 51 or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one substitution, wherein the substitution is an Ile at position 3 substituted with an uncharged amino acid; VHCDR2 having the amino acid sequence of SEQ ID NO: 52, or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one to three substitutions selected from the group consisting of Phe at position 2 substituted with an aromatic amino acid, Gly at position 4 substituted with an acidic or uncharged polar amino acid, Thr at position 8 substituted with an uncharged amino acid, and combinations thereof; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56; And which are connected: (i) with a peptide having the amino acid sequence EYPTKNY (SEQ ID NO: 38), and/or (ii) claudin 6 (CLDN6), wherein said antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence EYPTKNY (SEQ ID NO: 38), and/or (iii) with a peptide having the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and/or (iv) with claudin 6 (CLDN6), wherein said antibody or its the antigen binding fragment binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and/or (v) with a peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), where the antibody or antigen binding fragment thereof does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14), and/or (vi) with claudin 6 (CLDN6), wherein the antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), wherein the antibody or antigen binding fragment thereof does not bind with a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14). 2. The antibody or antigen-binding fragment thereof according to claim 1, wherein said CLDN6 is cell surface membrane-bound CLDN6. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said CLDN6 is present on cancer cells. 4. The antibody or antigen-binding fragment thereof according to claim 3, wherein said cancer cells are cancer cells expressing CLDN6. 5. The antibody or antigen-binding fragment thereof according to claim 3 or 4, wherein said cancer cells are selected from the group consisting of ovarian cancer, testicular cancer, gastric cancer, breast cancer, pancreatic cancer, lung cancer, uterine cancer and urinary cancer bubble 6. The antibody or antigen-binding fragment thereof according to claim 3 or 4, wherein said cancer cells are metastatic cancer cells. 7. Antibody according to any one of paragraphs. 1-5, which is a chimeric, human or humanized antibody. 8. Antibody according to any one of paragraphs. 1-7, which is a monoclonal antibody. 9. A monoclonal antibody or antigen-binding fragment thereof that binds claudin 6 (CLDN6), wherein the antibody or antigen-binding fragment thereof contains: the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having the amino acid sequence of SEQ ID NO: 51 or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one substitution, wherein the substitution is an Ile at position 3 substituted with an uncharged amino acid; VHCDR2 having the amino acid sequence of SEQ ID NO: 52, or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one to three substitutions selected from the group consisting of Phe at position 2 substituted with an aromatic amino acid, Gly at position 4 substituted with an acidic or uncharged polar amino acid, Thr at position 8 substituted with an uncharged amino acid, and combinations thereof; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56; And which (i) bind to a peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), and/or (ii) bind to claudin 6 (CLDN6), wherein the antibody or antigen binding fragment thereof binds to CLDN6 by binding to at least an epitope in CLDN6 having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15). 10. An antibody or antigen-binding fragment thereof that binds claudin 6 (CLDN6), wherein the antibody or antigen-binding fragment thereof comprises: the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having the amino acid sequence of SEQ ID NO: 51 or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one substitution, wherein the substitution is an Ile at position 3 substituted with an uncharged amino acid; VHCDR2 having the amino acid sequence of SEQ ID NO: 52, or an amino acid substitution variant thereof, wherein the amino acid substitution variant comprises one to three substitutions selected from the group consisting of Phe at position 2 substituted with an aromatic amino acid, Gly at position 4 substituted with an acidic or uncharged polar amino acid, Thr at position 8 substituted with an uncharged amino acid, and combinations thereof; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56; And which bind to one or more, preferably all, of the following peptides: PAISRGPSEYPTKNY (SEQ ID NO: 22), AISRGPSEYPTKNYV (SEQ ID NO: 15), ISRGPSEYPTKNYV (SEQ ID NO: 23), SRGPSEYPTKNYV (SEQ ID NO: 24), RGPSEYPTKNYV (SEQ ID NO: 25), GPSEYPTKNYV (SEQ ID NO : 26), PSEYPTKNYV (SEQ ID NO: 27), SEYPTKNYV (SEQ ID NO: 28) and EYPTKNYV (SEQ ID NO: 29), where the antibody or antigen binding fragment thereof does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO : 14). 11. The antibody or antigen-binding fragment thereof according to claim 10, where the difference in binding affinity to the peptide to which the antibody or antigen-binding fragment thereof binds with the lowest affinity and to the peptide to which the antibody or antigen-binding fragment thereof binds with the greatest affinity is 50 % or less. 12. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-11, wherein the antibody or antigen-binding fragment contains: the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having an amino acid sequence according to SEQ ID NO: 51 or an amino acid sequence according to SEQ ID NO: 57; VHCDR2 having an amino acid sequence according to SEQ ID NO: 52 or an amino acid sequence according to SEQ ID NO: 58; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56. 13. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-12, wherein the antibody or antigen-binding fragment contains: the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having the amino acid sequence according to SEQ ID NO: 51; VHCDR2 having the amino acid sequence according to SEQ ID NO: 52; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56; or the variable region of the heavy chain (VH) of an antibody, including: VHCDR1 having the amino acid sequence according to SEQ ID NO: 57; VHCDR2 having the amino acid sequence according to SEQ ID NO: 58; And VHCDR3 having the amino acid sequence according to SEQ ID NO: 53; And the antibody light chain variable region (VL), including: VLCDR1 having the amino acid sequence according to SEQ ID NO: 54; VLCDR2 having the amino acid sequence according to SEQ ID NO: 55; And VLCDR3 having the amino acid sequence according to SEQ ID NO: 56. 14. Antibody or antigen-binding fragment thereof according to any one of paragraphs. 1-13, wherein the antibody or antigen-binding fragment contains: an antibody heavy chain sequence comprising an amino acid sequence according to SEQ ID NO: 40 and an antibody light chain sequence comprising an amino acid sequence according to SEQ ID NO: 41; or an antibody heavy chain sequence comprising an amino acid sequence according to SEQ ID NO: 42 and an antibody light chain sequence comprising an amino acid sequence according to SEQ ID NO: 43; or an antibody heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 44 and an antibody light chain sequence comprising the amino acid sequence of SEQ ID NO: 45. 15. Conjugate for detection of CLDN6 containing an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 associated with at least one detectable label. 16. Hybridoma capable of producing an antibody according to any one of paragraphs. 1-14, with the hybridoma deposited under accession number DSM ACC3313 (58-1B), DSM ACC3312 (58-3A) or DSM ACC3311 (58-4B). 17. A method for detecting CLDN6 or determining the amount of CLDN6 in a sample, comprising the steps of: (i) contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims. 1-14 or the conjugate according to claim 15, and (ii) detecting the formation of a complex or determining the amount of a complex between the antibody, antigen binding fragment or conjugate and CLDN6. 18. A method for determining whether cells express CLDN6, comprising the steps of: (i) contacting the cell sample with an antibody or antigen-binding fragment thereof according to any one of claims. 1-14 or the conjugate according to claim 15 and (ii) detecting the formation of a complex between the antibody, antigen binding fragment or conjugate and CLDN6 expressed by cells in the sample. 19. A method for diagnosing, detecting or monitoring cancer, including cancer cells expressing CLDN6, comprising the steps of: (i) contacting the biological sample with an antibody or antigen-binding fragment thereof according to any one of claims. 1-14 or the conjugate according to claim 15, and (ii) detecting the formation of a complex and/or determining the amount of a complex between the antibody, antigen binding fragment or conjugate and CLDN6. 20. A method for determining whether a cancer is treatable using cancer therapy that targets CLDN6, comprising the steps of: (i) contacting a sample containing cancer cells with an antibody or an antigen-binding fragment thereof according to any one of claims. 1-14 or the conjugate according to claim 15, and (ii) detecting the formation of a complex between the antibody, antigen binding fragment or conjugate and CLDN6. 21. A method for determining the prognosis of a patient with cancer, including stages: (i) contacting a sample containing cancer cells with an antibody or an antigen-binding fragment thereof according to any one of claims. 1-14 or the conjugate according to claim 15, and (ii) detecting the formation of a complex between the antibody, antigen binding fragment or conjugate and CLDN6. 22. An antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-14, a conjugate according to claim 15, a hybridoma according to claim 16, or a method according to any one of claims. 17-21, where the specified CLDN6 contains the amino acid sequence in accordance with SEQ ID NO: 1 or a variant of the specified amino acid sequence. 23. A diagnostic test kit for targeted action on CLDN6, which contains an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or the conjugate according to claim 15, and instructions. 24. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or the conjugate of claim 15 for detecting CLDN6 or determining the amount of CLDN6 in a sample. 25. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or the conjugate of claim 15 to determine whether the cells express CLDN6. 26. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or a conjugate according to claim 15 for diagnosing, detecting or monitoring cancer, including cancer cells expressing CLDN6. 27. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or the conjugate of claim 15 to determine whether the cancer is treatable by cancer therapy targeting CLDN6. 28. The use of an antibody or an antigen-binding fragment according to any one of paragraphs. 1-14 or the conjugate according to claim 15 to determine the prognosis of a patient with cancer.