Antibodies useful for diagnosing cancer

Abstract

To provide antibodies directed against an epitope located within the C-terminal portion of CLDN6 which are useful for diagnosing cancer and / or determining whether cancer cells express CLDN6.SOLUTION: The present invention provides an antibody or an antigen-binding fragment thereof, which binds to a peptide having a specific amino acid sequence, and / or to claudin 6 (CLDN6), wherein the antibody or antigen-binding fragment thereof binds to CLDN6 by binding at least to an epitope within CLDN6 having a specific amino acid sequence.SELECTED DRAWING: None

Description

[Background technology]

[0001] Claudins are endogenous membrane proteins located within the tight junctions of the epithelium and endothelium. Claudins are predicted to have four transmembrane segments, along with two extracellular loops and cytoplasmic N-terminus and C-terminus. The transmembrane protein claudin (CLDN) family plays a crucial role in maintaining the tight junctions of the epithelium and endothelium and may also play a role in maintaining the cytoskeleton and in cellular signaling.

[0002] Claudin 6 (CLDN6) is a carcinoembryonic gene expressed in mouse and human stem cells and embryoid bodies involved in epithelial cell fate (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, CLDN6 can be classified as a differentiation antigen due to its expression in the early stages of epithelial morphogenesis, which is important for epithelial differentiation and barrier formation. Furthermore, expression has been observed in the epithelial tissues of the tongue, skin, stomach, and breast, as well as in the normal epithelial tissue of neonates (Abuazza G. et al. (2006) Am J Physiol Renal Physiol 291, 1132-1141; Troy TC 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 reveal low or very low expression of CLDN6 in the human placenta, bladder, endometrium, prostate, and peripheral nerves, as well as frequent overexpression of CLDN6 in various cancers. CLDN6 has been demonstrated to be overexpressed in tumors including pediatric brain tumors, gastric adenocarcinoma and germ cell tumors, as well as visceral cancers such as ovarian cancer. Overexpression of CLDN6 in gastric cancer cells has been shown to lead to increased aggression, migration, and proliferation, suggesting that CLDN6 may be a marker of poor prognosis and potentially play a role in maintaining a malignant phenotype. Furthermore, CLDN6 has been shown to function as a tumor suppressor in breast cancer cell lines through inhibition of cell proliferation and induction of apoptosis. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Turksen, K. et al. (2001) Dev Dyn 222, 292-300 [Non-Patent Document 2] Anderson WJ.et al.(2008)Dev Dyn 237,504-12 [Non-Patent Document 3] Turksen K. et al.(2002)Development,129,1775-84 [Non-Patent Document 4] Assou S.et al.(2007)Stem Cells 25,961-73 [Non-Patent Document 5] Abuazza G.et al.(2006)Am J Physiol Renal Physiol 291,1132-1141 [Non-Patent Document 6] Troy TCet al.(2007)Molecular Biotechnology 36,166-74 [Non-Patent Document 7] Zhao L.et al.(2008)Am J Physiol Regul Integr Comp Physiol 294,1856-1862 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] The sequence alignments of CLDN3, CLDN4, CLDN6, and CLDN9 shown in Figure 1B demonstrate that CLDN6 is highly conserved with respect to other claudin proteins. This high homology between CLDN6 and other claudin proteins, particularly CLDN9 and CLDN4, makes it difficult to provide CLDN6 antibodies with properties such as specificity and affinity suitable for diagnostic purposes. We have found that antibodies against specific epitopes located within the C-terminal region of CLDN6 meet the criteria for the diagnostic applicability of the antibodies, particularly for the detection and identification of cells expressing CLDN6.

[0005] The antibody of the present invention is useful, for example, for diagnosing cancer and / or determining whether cancer cells express CLDN6. Preferably, the cancer disease or cancer cells are characterized by surface expression of CLDN6. Cancer cells expressing CLDN6 are suitable targets for treatments targeting CLDN6, such as treatments using an antibody against CLDN6. In one embodiment, the cancer cells express or aberrantly express CLDN6, while the corresponding normal cells do not express CLDN6 or express CLDN6 at a lower level.

Means for Solving the Problems

[0006] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to the following. (i) A peptide having the amino acid sequence EYPTKNY (SEQ ID NO: 38), and / or (ii) Claudin 6 (CLDN6), wherein the antibody or its antigen-binding fragment binds to CLDN6 by binding to an epitope within CLDN6 having at least the amino acid sequence EYPTKNY (SEQ ID NO: 38), and / or (iii) A peptide having the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and / or (iv) Claudin 6 (CLDN6), wherein the antibody or its antigen-binding fragment binds to CLDN6 by binding to an epitope within CLDN6 having at least the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and / or (v) A peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), wherein the antibody or its antigen-binding fragment does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14), and / or (vi) Claudin 6 (CLDN6), wherein the antibody or antigen-binding fragment thereof binds to CLDN6 by binding to an epitope within CLDN6 having at least the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15) and does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14).

[0007] In embodiments and further aspects of the aspects described herein, the present invention (i) binds to a peptide having the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15) and / or (ii) binds to Claudin 6 (CLDN6) and binds to CLDN6 by binding to an epitope within CLDN6 having at least the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15). relates to a monoclonal antibody or antigen-binding fragment thereof.

[0008] In embodiments and further aspects of the aspects described herein, the present invention relates to 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) relates to an antibody or antigen-binding fragment thereof that binds to one or more, preferably all, of the above, wherein the antibody or antigen-binding fragment thereof does not bind to a peptide having the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14).

[0009] In one embodiment, the difference in binding affinity between the peptide to which the antibody or antigen-binding fragment binds with the lowest affinity and the peptide to which the antibody or antigen-binding fragment binds with the highest affinity is 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.

[0010] The antibodies or antigen-binding fragments described herein preferably do not bind to peptides containing the amino acid sequence EYPTK (SEQ ID NO: 59) and / or EYPTKN (SEQ ID NO: 60) but not EYPTKNY (SEQ ID NO: 38). In other words, the antibodies or antigen-binding fragments described herein that bind to peptides having the amino acid sequence EYPTKNY (SEQ ID NO: 38) and / or peptides having the amino acid sequence EYPTKNYV (SEQ ID NO: 29) bind to the peptide(s) said by the presence of the second tyrosine missing from the amino acid sequence EYPTK (SEQ ID NO: 59) and / or EYPTKN (SEQ ID NO: 60). As shown herein, preferred antibodies or antigen-binding fragments do not bind to peptides containing the amino acid sequence YPTKNY (SEQ ID NO: 61) and / or EYPTKN (SEQ ID NO: 60) but not EYPTKNY (SEQ ID NO: 38), suggesting that the amino acid sequence EYPTKNY (SEQ ID NO: 38) is a sequence that is considered to be the minimal epitope for binding of these antibodies.

[0011] In embodiments of the aspects described herein and in further embodiments, the present invention is deposited with DSMZ (Inhoffenstr. 7B, 38124 Braunschweig, Germany) and is named and accession number below: 1.58-4B-2, accession number DSM ACC3311, deposited on November 29, 2016; 2.58-3A, accession number DSM ACC3312, deposited on November 29, 2016; or 3.58-1B, accession number DSM ACC3313, deposited on November 29, 2016. This relates to antibodies produced by or obtained from hybridomas having any of the following characteristics.

[0012] The antibodies of the present invention are specified herein by reference to the name of the antibody and / or by reference to the clone that produces the antibody.

[0013] The present invention also relates to antibodies that, with respect to CLDN6 binding, compete with antibodies produced by and obtained from the above hybridoma, and / or have specificity for CLDN6 of antibodies produced by or obtained from the above hybridoma. In these and other embodiments, the present invention also relates to antibodies that include an antigen-binding moiety or antigen-binding site, particularly a variable region, that is identical or highly homologous to that of antibodies produced by or obtained from the above hybridoma. Preferred antibodies are intended to have a CDR region that is identical or highly homologous to the CDR region of antibodies produced by or obtained from the above hybridoma. "Highly homologous" is intended to mean that each CDR region can have 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2 substitutions. Particularly preferred antibodies are chimeric and humanized forms of antibodies produced by or obtained from the above hybridoma.

[0014] Therefore, in embodiments of the features described herein and in further embodiments, the present invention is (i) Antibodies produced by or obtained from clones deposited under accession number DSM ACC3313(58-1B), DSM ACC3312(58-3A), or DSM ACC3311(58-4B-2), (ii)Antibodies that are chimeric or humanized forms of antibodies under (i), (iii)Antibodies that compete with the antibody under (i) for CLDN6 binding, (iv)Antibodies having the specificity of antibodies under (i), and (v)(i) Antibodies containing the antigen-binding portion or antigen-binding site of the antibody below An antibody selected from the group consisting of, Antibody antigen-binding fragment of any one of (i) through (v) below Regarding.

[0015] In one embodiment, the antigen-binding portion or site of the antibody below (i) includes the variable region of the antibody below (i).

[0016] In embodiments and further embodiments described herein, the present invention (a) Antibodies containing the following: Antibody heavy chains including the following: (i) Antibody heavy chain sequences containing the sequences described in Sequence ID No. 40, 42, or 44, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody heavy chain sequence containing the sequence described in SEQ ID NO: 40, 42, or 44, or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 53, preferably a sequence or variant thereof further comprising a CDR1 sequence or variant thereof described in SEQ ID NO: 51 or 57, and / or a CDR2 sequence or variant thereof described in SEQ ID NO: 52 or 58. Furthermore (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0017] In embodiments and further embodiments described herein, the present invention (a) Antibodies containing the following: Antibody light chains containing the following: (i) Antibody light chain sequences containing the sequences described in Sequence ID No. 41, 43, or 45, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody light chain sequence containing the sequence described in SEQ ID NO: 41, 43, or 45, or a variant thereof, or (iii) The CDR3 sequence described in SEQ ID NO: 56 or a variant thereof, preferably the CDR1 sequence described in SEQ ID NO: 54 or a variant thereof, and / or SEQ ID NO: 55 (Amino acid sequence: LMS) The CDR2 sequence described above, or a sequence further comprising a variant thereof, or a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0018] In embodiments and further embodiments described herein, the present invention (a) Antibodies containing the following: (I) Antibody heavy chains including the following: (i) Antibody heavy chain sequences containing the sequences described in Sequence ID No. 40, 42, or 44, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody heavy chain sequence containing the sequence described in SEQ ID NO: 40, 42, or 44, or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 53, preferably a sequence further comprising a CDR1 sequence or variant thereof described in SEQ ID NO: 51 or 57, and / or a CDR2 sequence or variant thereof described in SEQ ID NO: 52 or 58, and / or (II) Antibody light chains containing the following: (i) Antibody light chain sequences containing the sequences described in Sequence ID No. 41, 43, or 45, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody light chain sequence containing the sequence described in SEQ ID NO: 41, 43, or 45, or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 56, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 54, and / or the CDR2 sequence or variant thereof described in SEQ ID NO: 55, and variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0019] In certain preferred embodiments, the present invention is (a) Antibodies containing the following: (I) Antibody heavy chains including the following: (i) an antibody heavy chain sequence containing the sequence described in Sequence ID No. 40, or a variant thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody heavy chain sequence containing the sequence described in Sequence ID No. 40 or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 53, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 51, and / or the CDR2 sequence or variant thereof described in SEQ ID NO: 52, and a variant thereof, and (II) Antibody light chains containing the following: (i) Antibody light chain sequences containing the sequence described in Sequence ID No. 41, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody light chain sequence containing the sequence described in Sequence ID No. 41 or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 56, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 54, and / or the CDR2 sequence or variant thereof described in SEQ ID NO: 55, and variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0020] In certain preferred embodiments, the present invention is (a) Antibodies containing the following: (I) Antibody heavy chains including the following: (i) Antibody heavy chain sequences containing the sequence described in Sequence ID No. 42, or a variant thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody heavy chain sequence containing the sequence described in Sequence ID No. 42, or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 53, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 57 and / or the CDR2 sequence described in SEQ ID NO: 58, and a variant thereof, and (II) Antibody light chains containing the following: (i) Antibody light chain sequences containing the sequence described in Sequence ID No. 43, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody light chain sequence containing the sequence described in Sequence ID No. 43 or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 56, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 54 and / or the CDR2 sequence or variant thereof described in SEQ ID NO: 55, and a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0021] In certain preferred embodiments, the present invention is (a) Antibodies containing the following: (I) Antibody heavy chains including the following: (i) Antibody heavy chain sequences containing the sequence described in Sequence ID No. 44, or a variant thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody heavy chain sequence containing the sequence described in Sequence ID No. 44 or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 53, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 57 and / or the CDR2 sequence described in SEQ ID NO: 58, and a variant thereof, and (II) Antibody light chains containing the following: (i) Antibody light chain sequences containing the sequence described in Sequence ID No. 45, or variants thereof, (ii) At least one, preferably two, more preferably all three, CDR sequences of an antibody light chain sequence containing the sequence described in Sequence ID No. 45 or a variant thereof, or (iii) A CDR3 sequence or variant thereof described in SEQ ID NO: 56, preferably a sequence further comprising the CDR1 sequence or variant thereof described in SEQ ID NO: 54 and / or the CDR2 sequence or variant thereof described in SEQ ID NO: 55, and a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0022] In certain preferred embodiments, the present invention is (a) an antibody comprising an antibody heavy chain containing the CDR3 sequence or a variant thereof described in SEQ ID NO: 53, and an antibody light chain containing the CDR3 sequence or a variant thereof described in SEQ ID NO: 56, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0023] In certain preferred embodiments, the present invention is (a) an antibody heavy chain comprising the CDR3 sequence or a variant thereof described in SEQ ID NO: 53, and further comprising the CDR1 sequence or a variant thereof described in SEQ ID NO: 51 and the CDR2 sequence or a variant thereof described in SEQ ID NO: 52, and an antibody light chain comprising the CDR3 sequence or a variant thereof described in SEQ ID NO: 56, and further comprising the CDR1 sequence or a variant thereof described in SEQ ID NO: 54 and the CDR2 sequence or a variant thereof described in SEQ ID NO: 55, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0024] In certain preferred embodiments, the present invention is (a) an antibody heavy chain comprising the CDR3 sequence or a variant thereof described in SEQ ID NO: 53, and further comprising the CDR1 sequence or a variant thereof described in SEQ ID NO: 57 and the CDR2 sequence or a variant thereof described in SEQ ID NO: 58, and an antibody light chain comprising the CDR3 sequence or a variant thereof described in SEQ ID NO: 56, and further comprising the CDR1 sequence or a variant thereof described in SEQ ID NO: 54 and the CDR2 sequence or a variant thereof described in SEQ ID NO: 55, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0025] In certain preferred embodiments, the present invention is (a) an antibody comprising a heavy chain variable region (VH) containing the amino acid sequence represented by SEQ ID NO: 40 or a variant thereof, and a light chain variable region (VL) containing the amino acid sequence represented by SEQ ID NO: 41 or a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0026] In certain preferred embodiments, the present invention is (a) an antibody comprising 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 represented by SEQ ID NO: 43 or a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0027] In certain preferred embodiments, the present invention is (a) an antibody comprising a heavy chain variable region (VH) containing the amino acid sequence represented by SEQ ID NO: 44 or a variant thereof, and a light chain variable region (VL) containing the amino acid sequence represented by SEQ ID NO: 45 or a variant thereof, and (b) An antibody that competes with the antibody of (a) for CLDN6 binding and / or has the specificity of the antibody of (a) for CLDN6. This relates to an antibody selected from the group consisting of the following, or to an antigen-binding fragment of the antibody.

[0028] In preferred embodiments, the antibody of the present invention comprises an antibody heavy chain including a γ2a heavy chain constant region, preferably a human γ2a heavy chain constant region, and / or an antibody light chain including a κ light chain constant region.

[0029] The antibodies or antigen-binding fragments described herein bind to CLDN6. The antibodies or antigen-binding fragments of the present invention can preferably bind to CLDN6 in their native state, i.e., in their naturally occurring state or in an undenatured state, or in a denatured state. In one embodiment, the antibody or antigen-binding fragment of the present invention binds to CLDN6 but not to CLDN9, and preferably not to CLDN4 and / or CLDN3. Preferably, the antibody or antigen-binding fragment of the present invention does not substantially bind to CLDN proteins other than CLDN6. Preferably, the antibody or antigen-binding fragment of the present invention is specific to CLDN6.

[0030] In one embodiment, CLDN6 is cell surface membrane bound CLDN6. In one embodiment, CLDN6 is present on cancer cells, and the cancer cells are preferably cancer cells that express CLDN6. In one embodiment, the cancer cells are ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma; gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, particularly basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, particularly malignant pleomorphic adenoma; sarcoma, particularly synovial sarcoma and carcinosarcoma; bile duct cancer; bladder cancer, particularly transitional cell carcinoma and papillary carcinoma. Cancer-derived cells selected from the group consisting of renal cell carcinoma, particularly including clear cell carcinoma and papillary renal cell carcinoma; colon cancer; small intestinal cancer, particularly small intestinal adenocarcinoma and ileal adenocarcinoma; embryonic testicular carcinoma; placental choriocarcinoma; cervical cancer; testicular cancer, particularly testicular seminoma, testicular teratoma and testicular embryonic carcinoma; germ cell tumors, particularly testicular germ cell tumors, including uterine cancer, teratomas, or embryonic cancers; and metastatic forms thereof.

[0031] In one embodiment, the antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. In another embodiment, the antibody of the present invention is a monoclonal antibody.

[0032] In one embodiment, the antibody of the present invention is obtained by a method comprising the step of immunizing an animal with a peptide containing, preferably comprising, the amino acid sequence of SEQ ID NO: 49, or an immunologically equivalent peptide, or a nucleic acid or host cell nucleic acid expressing the peptide. Preferably, the peptide contains 110, 100, 90, 80, 70, 60, 50, or 40 or fewer consecutive amino acids of CLDN6.

[0033] The antibody or antigen-binding fragment of the present invention can be bound to other parts, such as a detectable label, i.e., it can be covalently or noncovalently linked.

[0034] In a further embodiment, the present invention relates to a conjugate comprising an antibody or antigen-binding fragment described herein, conjugated to at least one detectable label.

[0035] The present invention also relates to cells such as hybridomas that produce antibodies as described herein.

[0036] The preferred hybridoma is deposited with DSMZ (Inhoffenstr. 7B, 38124 Braunschweig, Germany) and has the following name and accession number: 1.58-4B-2, accession number DSM ACC3311, deposited on November 29, 2016; 2.58-3A, accession number DSM ACC3312, deposited on November 29, 2016; or 3.58-1B, accession number DSM ACC3313, deposited on November 29, 2016. It possesses one of the following characteristics:

[0037] The present invention also relates to a peptide, preferably comprising, the amino acid sequence of SEQ ID NO: 49, or an immunologically equivalent peptide. Preferably, the peptide comprises 110, 100, 90, 80, 70, 60, 50, or 40 or fewer consecutive amino acids of CLDN6.

[0038] The present invention also relates to nucleic acids encoding antibodies or parts thereof, such as antibody chains, or antigen-binding fragments, or peptides as described herein. Preferably, the nucleic acids of the present invention are operably bound to one or more expression regulatory elements that enable expression in eukaryotic or prokaryotic cells. Regulatory elements that ensure expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.

[0039] The nucleic acids of the present invention may be contained within a vector, such as a plasmid, cosmid, virus, bacteriophage, or another vector conventionally used in genetic engineering. The vector may contain further genes, such as marker genes, that enable selection of the vector in a suitable host cell and under suitable conditions. Furthermore, the vector may contain expression regulatory elements that enable proper expression of the coding region in a suitable host. Such regulatory elements are known to those skilled in the art and may include promoters, splice cassettes, and translation start codons.

[0040] Methods for constructing nucleic acid molecules, constructing vectors containing nucleic acid molecules, introducing vectors into appropriately selected host cells, or inducing or achieving the expression of nucleic acid molecules are well known in the art.

[0041] Further aspects of the present invention relate to host cells comprising nucleic acids or vectors disclosed herein.

[0042] Further aspects of the present invention relate to the detection of CLDN6 or CLDN6-expressing cells or the determination of the amount of CLDN6 or CLDN6-expressing cells using the antibody or antigen-binding fragment of the present invention. CLDN6 or CLDN6-expressing cells are detected by detecting a complex of CLDN6 with the antibody or antigen-binding fragment of the present invention, or the amount of CLDN6 or CLDN6-expressing cells is determined by determining the amount of the complex. The formation of the complex indicates the presence of CLDN6 or CLDN6-expressing cells. Such detection or determination of quantity can be carried out by many methods, including but not limited to immunodetection using the antibody or antigen-binding fragment of the present invention. Methods using antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, and the like. Generally, such assays use antibodies or antibody fragments that specifically bind to a target peptide or protein directly or indirectly bound to a label that provides detection, e.g., an indicator enzyme, radiolabeling, a fluorophore, or a paramagnetic particle. The methods of the present invention allow for quantitative and / or qualitative assessment, e.g., absolute and / or relative assessment, of CLDN6 levels or levels of CLDN6-expressing cells.

[0043] In one aspect, the present invention relates to a method for detecting CLDN6 in a sample or determining the amount of CLDN6, (i) A step of contacting a sample with the antibody or antigen-binding fragment of the present invention or the conjugate of the present invention, and (ii) A step of detecting the formation of a complex between an antibody, antigen-binding fragment, or conjugate and CLDN6, or determining the amount of the complex. Regarding methods including

[0044] In one embodiment, the sample is a cell sample, i.e., a sample containing cells such as cancer cells. In this embodiment, the complex is formed between an antibody, antigen-binding fragment, or conjugate and CLDN6 expressed by the cells in the sample.

[0045] In one aspect, the present invention relates to a method for determining whether or not a cell expresses CLDN6, (i) A step of contacting a cell sample with the antibody or antigen-binding fragment of the present invention or the conjugate of the present invention, (ii) A step of detecting the formation of a complex between an antibody, antigen-binding fragment, or conjugate and CLDN6 expressed by cells in the sample. Regarding methods including

[0046] In one embodiment, the cells in the sample are cancer cells. The complex is formed between an antibody, antigen-binding fragment, or conjugate and CLDN6 expressed by the cells in the sample.

[0047] Further aspects of the present invention relate to methods for diagnosing or classifying diseases by targeting CLDN6 using antibodies or antigen-binding fragments of the present invention. These methods provide selective detection of cells expressing CLDN6, thereby distinguishing these cells from normal cells that do not express CLDN6 or diseased cells that do not express CLDN6. Diseases characterized by diseased cells expressing CLDN6 are treatable by CLDN6-targeted therapies, such as therapeutic therapies with therapeutic antibodies against CLDN6. Preferred diseases for treatment or diagnosis are diseases in which CLDN6 is expressed or abnormally expressed, particularly cancerous diseases such as those described herein.

[0048] In one embodiment, the present invention relates to a method for diagnosing, detecting, or monitoring cancer, i.e., determining regression, progression, course, and / or onset, including the detection of CLDN6 or CLDN6-expressing cells in a biological sample isolated from a patient and / or the determination of the amount of CLDN6 or CLDN6-expressing cells, using the antibody or antigen-binding fragment of the present invention. Such a method may be used to detect whether a subject has cancer, whether they are at risk (high risk) of developing cancer, or, for example, whether a treatment regimen is effective.

[0049] Therefore, in one embodiment, the present invention is a method for diagnosing, detecting, or monitoring cancer, (i) A step of contacting a biological sample with the antibody or antigen-binding fragment of the present invention or the conjugate of the present invention, and (ii) A step of detecting the formation of a complex between an antibody, antigen-binding fragment or conjugate and CLDN6 and / or determining the amount of the complex. Regarding methods including

[0050] In one embodiment, the biological sample is a cellular sample, i.e., a sample containing cells such as cancer cells. In this embodiment, the complex is formed between an antibody, antigen-binding fragment, or conjugate and CLDN6 expressed by the cells in the sample.

[0051] The monitoring method according to the present invention preferably includes the detection and / or determination of the amount of CLDN6 or CLDN6-expressing cells in a first sample at a first time point and in a further sample at a second time point, and the regression, progression, course and / or onset of the tumor disease can be determined by comparing the two samples.

[0052] Typically, the level of CLDN6 or the level of CLDN6-expressing cells in a biological sample is compared to a reference level, and the deviation from the reference level is an indicator of the presence and / or stage of cancer in the subject. The reference level may be a level determined in a control sample (e.g., healthy tissue or healthy subjects, particularly from patients without cancer) or an average level from healthy subjects. The “deviation” from the reference level represents a significant change, such as an increase of at least 10%, 20%, or 30%, preferably at least 40%, or 50%, or more.

[0053] Preferably, the presence of CLDN6 or CLDN6-expressing cells and / or an increased amount of CLDN6 or CLDN6-expressing cells compared to a reference level, for example, compared to a patient without cancer, indicates the presence or risk (i.e., likelihood of developing) cancer in the patient.

[0054] A decrease in the amount of CLDN6 or CLDN6-expressing cells compared to previously collected biological samples from the patient may indicate a regression of cancer disease, a positive course, such as successful treatment, or a reduced risk of developing cancer in the patient.

[0055] An increased amount of CLDN6 or CLDN6-expressing cells compared to previously collected biological samples from the patient may indicate progression of cancer, a negative course, such as treatment failure, recurrence or metastatic behavior, or the risk of developing or developing cancer in the patient.

[0056] In one aspect, the present invention relates to a method for determining whether cancer is treatable by cancer therapy targeting CLDN6, (i) A step of contacting a sample containing cancer cells with the antibody or antigen-binding fragment of the present invention or the conjugate of the present invention, (ii) A step to detect the formation of a complex between an antibody, antigen-binding fragment, or conjugate and CLDN6. Regarding methods including

[0057] The complex is preferably formed between an antibody, antigen-binding fragment, or conjugate and CLDN6 expressed by cancer cells in the sample.

[0058] Such methods may be used to determine whether a patient is suitable for therapies that involve targeting CLDN6-expressing cells, such as therapies using one or more immunoeffector antibodies, such as cytotoxic CLDN6-specific antibodies, antibodies labeled with cytotoxic substances such as toxins or radiolabeling, or antibodies that induce cell death mechanisms such as CDC or ADCC. Diseases characterized by disease cells expressing CLDN6, such as cancerous diseases, particularly those described herein, are treatable by CLDN6-targeted therapies.

[0059] In any one embodiment of the above configuration, the sample, cell sample, or biological sample is derived from a patient who has, is suspected of having, or is likely to have a cancerous disease. In one embodiment, the sample, cell sample, or biological sample is derived from a tissue or organ, and if cancer is not present in the tissue or organ, the cells do not substantially express CLDN6. Preferably, the tissue is a tissue other than placental tissue. Preferably, the tissue has already been diagnosed as having a cancerous disease, for example by visual examination of the tissue or organ or by a cell culture test. In this embodiment, the presence of CLDN6 or CLDN6-expressing cells, and / or an increased amount of CLDN6 or CLDN6-expressing cells compared to a reference level, for example compared to a patient without a tumor, may indicate that the patient is suitable for a treatment that includes targeting cells expressing CLDN6.

[0060] In one embodiment, the present invention provides compositions, such as diagnostic compositions, or kits, comprising antibodies or antigen-binding fragments or combinations of antibodies and / or antigen-binding fragments as described herein. Such diagnostic compositions or test kits are useful in the methods of the present invention, such as methods for diagnosis, detection, or monitoring. These kits may optionally include detectable labels, such as indicator enzymes, radiolabels, fluorophores, or paramagnetic particles. The kits may include informational brochures, such as brochures informing the use of reagents for carrying out the methods disclosed herein.

[0061] Other features and advantages of the present invention will become apparent from the following detailed description and claims. [Brief explanation of the drawing]

[0062] [Figure 1A] Sequence alignment of claudin 6 and claudin 9 proteins (human / mouse): The sequence alignment shows high homology between human and mouse claudin 6 and human claudin 9. [Figure 1B]Sequence alignment of claudin 6 and claudin 3, 4, and 9 proteins (human). Sequence alignment shows high homology of the claudin multiple gene family. [Figure 2A] Antibody specificity tested by Western blot analysis [Figure 2B] Antibody specificity tested by Western blot analysis: Cell lysates of mock, human CLDN3, 4, 6, or 9-transfected HEK293 cells, and CLDN6-positive tumor cells (PA-1 SC12 and NEC-8) were blotted, and conjugated antibodies (rabbit anti-CLDN3 (Invitrogen) 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 antibody (5 μg / mL)) were detected by peroxidase-conjugated secondary antibodies. [Figure 3] Histological analysis of Western blot-positive antibodies: Conjugation of mumAB leads 58-1B, 58-3A, and 58-4B to FFPE sections of ovarian cancer compared with rabbit anti-CLDN6 IBL antiserum. [Figure 4A] Epitope mapping using synthetic duplicate peptides: Duplicate peptides were immobilized on microtiter plates, and antibodies were added. The bound antibodies were then developed using an appropriate peroxidase-conjugated secondary reagent. [Figure 4B] Epitope Mapping Using Biotinylated Synthetic Duplication Peptides: Highly duplication peptides with the N-terminus biotinylated via a flexible hydrophilic linker were synthesized and packed into streptavidin-conjugated microtiter plates. Antibodies (1 μg / mL) were applied, and the bound antibodies were detected and analyzed. To compare the signal intensity of mumAB 58-4B-2 with rabbit serum (IBL), the maximum binding of the antibody to the C-terminal peptide 19 was defined as 100%. The binding intensity of each antibody to a single peptide was calculated by comparing it with the maximum binding for each test system. Binding of mumAB was triplicated in three independent experiments. Binding of IBL serum was triplicated in two independent experiments. [Figure 4C]Binding sites of monoclonal lead 58-4B-2 and polyclonal rabbit anti-CLDN6 serum (IBL). [Figure 5A] Sequences of antibodies 58-1B, 58-3A, and 58-4B [Figure 5B] Sequences of antibodies 58-1B, 58-3A, and 58-4B [Figure 6] Background signals of various antibodies on normal ovarian tissue. Comparison of lead antibodies mumAB 58-1B, 58-3A, and 58-4B on normal ovarian tissue with commercially available IBL antibodies (antibody concentration 5 μg / ml; clinic protocol). [Modes for carrying out the invention]

[0063] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and that these may vary. Furthermore, it should be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, and that the scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0064] The elements of the present invention are described below. These elements are listed together with specific embodiments, but it should be understood that they may be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to only the embodiments expressly describing the invention. This description should be understood as supporting and encompassing embodiments in which the expressly described embodiments are combined with any number of disclosed elements and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by this description unless specifically indicated in the context.

[0065] Preferably, the terms used herein are defined as those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G. W. Heuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0066] The implementation of this invention is permitted in accordance with the literature of the art (e.g., Molecular Cloning: A Laboratory Manual, 2) unless otherwise specified. nd Conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology are used, as described in Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0067] Throughout this specification and the following claims, unless otherwise specifically required by context, the word “includes” and variations such as “includes” shall be understood to mean the inclusion of the member, integer or process or group of members, integers or processes described, but not the exclusion of any other member, integer or process or group of members, integers or processes; however, in some embodiments, such other members, integers or processes or groups of members, integers or processes may be excluded, i.e., the subject matter lies in the inclusion of the member, integer or process or group of members, integers or processes described. The terms “one” and “it” and similar references used in connection with the description of the invention (particularly in connection with the claims) shall be interpreted as including both singular and plural unless otherwise specifically indicated herein or unless otherwise clearly inconsistent with the context. Enumerations of ranges of values ​​herein are intended to serve merely as a way of concisely referring to each separate value belonging to that range individually. Unless otherwise specifically indicated herein, each individual value is incorporated herein as if it were individually enumerated herein. All methods described herein may be carried out in any suitable order, unless otherwise specifically indicated herein or unless it is clearly inconsistent with the context. The use of any examples or illustrative language provided herein (e.g., "etc.") is intended solely to better illustrate the invention and not to limit the scope of the claimed invention. No language herein should be construed as referring to any unclaimed element essential to the practice of the invention.

[0068] Throughout this specification, several sources are referenced. Each of the sources referenced herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) is incorporated herein by reference in its entirety, either above or below. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such disclosures for the sake of prior art.

[0069] In relation to the present invention, the term "recombinant" means "produced through genetic manipulation." Preferably, in relation to the present invention, "recombinant objects," such as recombinant cells, do not exist in nature.

[0070] As used herein, the term “naturally occurring” refers to the fact that a substance can be found in nature. For example, a peptide or nucleic acid that is present in living organisms (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is considered naturally occurring.

[0071] The term “antigen” refers to an active substance comprising an epitope to which an immune response is directed and / or to which an immune response should be generated. Preferably, in relation to the present invention, the antigen is a molecule that induces an immune response, preferably antigen-specific, after processing. The term “antigen” includes proteins, peptides, polysaccharides, nucleic acids, particularly RNA and DNA, and nucleotides.

[0072] The term "epitope" refers to an antigenic determinant within a molecule, such as an antigen, that is, a portion of a molecule recognized by the immune system, for example, by an antibody. For example, an epitope is a distinct three-dimensional site on an antigen that is recognized by the immune system. Epitopes typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural properties and specific charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that the former lose their binding to a denaturing solvent, while the latter retains their binding. The epitopes of proteins such as CLDN preferably comprise continuous or discontinuous portions of the protein and are preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, and most preferably 10 to 25 amino acid lengths. For example, the epitopes may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid lengths.

[0073] As used herein, the term “discontinuous epitope” means a conformational epitope on a protein antigen that is formed from at least two distinct regions in the primary sequence of a protein.

[0074] Antigens include tumor-associated antigens such as CLDN6, i.e., components of cancer cells that may originate from the cytoplasm, cell surface, and cell nucleus, and are preferably produced in large quantities as surface antigens within or on cancer cells.

[0075] In relation to the present invention, the term “tumor-associated antigen” or “tumor antigen” refers to a protein that is specifically expressed in a limited number of tissues and / or organs or at a specific developmental stage under normal conditions. For example, a tumor-associated antigen may be specifically expressed in gastric tissue, preferably gastric mucosa, reproductive organs, such as the testes, trophoblast tissue, such as the placenta, or germline cells under normal conditions, and may be expressed or abnormally expressed in one or more tumor or cancerous tissues. In this context, “limited number” means preferably three or fewer, more preferably two or fewer. In relation to the present invention, tumor-associated antigens include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins that are specifically expressed in a specific cell type at a specific differentiation stage under normal conditions, cancer / testicular antigens, i.e., proteins that are specifically expressed in the testes and sometimes in the placenta under normal conditions, and germline-specific antigens. In relation to the present invention, tumor-associated antigens are preferably bound to the cell surface of cancer cells and are preferably not expressed or rarely expressed in normal tissues. Preferably, tumor-associated antigens or abnormal expression of tumor-associated antigens identify cancer cells. In connection with the present invention, the tumor-associated antigen expressed by a subject, for example, cancer cells of a patient suffering from a cancerous disease, is preferably an autoprotein of the subject. In a preferred embodiment, the tumor-associated antigen in connection with the present invention is specifically expressed in non-essential tissues or organs under normal conditions, i.e., tissues or organs that, if damaged by the immune system, do not result in the death of the subject, or in organs or body structures that are inaccessible or have little access to the immune system. Preferably, the amino acid sequence of the tumor-associated antigen is identical between the tumor-associated antigen expressed in normal tissue and the tumor-associated antigen expressed in cancerous tissue.

[0076] Examples of differentiation antigens that ideally meet the criteria for tumor-associated antigens as target structures in tumor immunotherapy, particularly 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, establishing the intercellular space barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that traverse the membrane four times, with both their N-terminus and C-terminus located in the cytoplasm.

[0077] The terms “Claudin 6” or “CLDN6” preferably refer to human CLDN6, in particular to proteins containing the amino acid sequence described in Sequence ID No. 1 of the sequence listing, or to variants of said amino acid sequence. With respect to CLDN6, the term “variant” specifically refers to proteins containing the amino acid sequence described in Sequence ID No. 1 of the sequence listing, in which Ile at position 143 is replaced with Val. The term “CLDN6” includes any CLDN6 variants, such as post-translational modification variants and conformational variants.

[0078] The term "CLDN9" preferably refers to human CLDN9, and more particularly to proteins comprising the amino acid sequence described in Sequence ID No. 2 of the sequence listing or variants of said amino acid sequence.

[0079] The term "CLDN4" preferably refers to human CLDN4, in particular to proteins comprising the amino acid sequence described in Sequence ID No. 4 of the sequence listing or variants of said amino acid sequence.

[0080] The term "CLDN3" preferably refers to human CLDN3, in particular to proteins comprising the amino acid sequence described in Sequence ID No. 3 of the sequence listing or variants of said amino acid sequence.

[0081] CLDN6 has been shown to be expressed in, for example, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cell carcinoma, and bladder cancer. CLDN6 is expressed in ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma; gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, particularly basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, particularly malignant pleomorphic adenoma; sarcoma, particularly synovial sarcoma and carcinosarcoma; cholangiocarcinoma; bladder cancer, particularly transitional cell carcinoma and papillary carcinoma; kidney cancer, particularly It can detect and target germ cell tumors, particularly germ cell tumors of the testis, including renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestinal cancer including ileal cancer, especially small intestinal adenocarcinoma and ileal adenocarcinoma, embryonal testicular cancer, placental choriocarcinoma, cervical cancer, testicular cancer, especially testicular seminoma, testicular teratoma and testicular embryonic carcinoma, uterine cancer, teratomas or embryonic cancers, and their metastatic forms. In one embodiment, cancerous diseases associated with CLDN6 expression are selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, ovarian cancer is carcinoma or adenocarcinoma. Preferably, lung cancer is carcinoma or adenocarcinoma, and preferably bronchiolar cancer such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

[0082] According to the present invention, cells expressing CLDN6 are preferably characterized by cell surface membrane-bound CLDN6, that is, CLDN6 is bound to the cell surface. Furthermore, according to the present invention, cellular CLDN6 is preferably cell surface membrane-bound CLDN6. Cells expressing CLDN6 or cells characterized by CLDN6 and its binding to the cell surface are preferably cancer cells, preferably cancer cells derived from cancer as described herein.

[0083] The term "bound to the cell surface" means that a tumor-associated antigen, such as CLDN6, is positioned bound to the cell's plasma membrane, with at least a portion of the tumor-associated antigen facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located outside the cell. In this context, "part" preferably consists of at least four, preferably at least eight, preferably at least twelve, and more preferably at least twenty amino acids. Binding may be direct or indirect. For example, binding may be due to interaction with one or more transmembrane domains, one or more lipid anchors, or other proteins, lipids, saccharides, or other structures found on the outer leaflet of the cell's plasma membrane. For example, a tumor-associated antigen bound to the cell surface may be a transmembrane protein having an extracellular portion, or a protein bound to the cell surface by interacting with another protein that is a transmembrane protein.

[0084] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and therefore includes the outside of the cell that is accessible for binding by proteins and other molecules.

[0085] According to the present invention, CLDN6 is substantially not expressed in cells if its expression level is lower than that in placental cells or placental tissue. Preferably, the expression level is less than 10% of the expression in placental cells or placental tissue, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05%, or even lower. Preferably, CLDN6 is substantially not expressed in cells if its expression level is less than twice, preferably less than 1.5 times, the expression level in non-cancerous tissue other than the placenta, and preferably not exceeds the expression level in said non-cancerous tissue. Preferably, CLDN6 is substantially not expressed in cells if its expression level is below the detection limit and / or is too low to allow binding by a CLDN6-specific antibody applied to the cell.

[0086] According to the present invention, CLDN6 is expressed in cells if its expression level is preferably more than 2 times, preferably more than 10 times, more than 100 times, more than 1000 times, more than 10000 times, or more than 10000 times higher than its expression level in non-cancerous tissues other than the placenta. Preferably, CLDN6 is expressed in cells if its expression level is above the detection limit and / or if its expression level is high enough to allow binding by a CLDN6-specific antibody applied to the cell. Preferably, the CLDN6 expressed in cells is expressed or exposed on the surface of the cell.

[0087] The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and encompasses any molecule containing its antigen-binding moiety. The term “antibody” includes, but is not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, monoclonal antibodies and antibody fragments or derivatives, including antigen-binding antibody fragments such as scFv and Fab and Fab' fragments, as well as all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives 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 hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0088] The antibodies described herein may be human antibodies. As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germ cell immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germ cell immunoglobulin sequences (mutations introduced, for example, by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).

[0089] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from an immunoglobulin from a non-human species, while the rest of the molecule's immunoglobulin structure is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may consist of a complete variable domain fused to a constant domain, or only a complementation-determining region (CDR) transplanted into an appropriate framework region within the variable domain. The antigen-binding site may be wild-type or modified by one or more amino acid substitutions, for example, to more closely resemble a human immunoglobulin. Some forms of humanized antibodies preserve all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). Other forms have one or more altered CDRs compared to the original antibody.

[0090] The term "chimeric antibody" refers to an antibody in which portions of the amino acid sequences of the heavy and light chains are homologous to the corresponding sequences of an antibody from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequences of an antibody from another species or class. Typically, the variable regions of both the light and heavy chains mimic the variable region of an antibody from one species of mammal, while the constant region is homologous to the sequence of an antibody from another species. One clear advantage of such a chimeric morphology is that the variable region can be conveniently induced from currently known sources using readily available non-human host organism-derived B cells or hybridomas, for example, in combination with a constant region derived from human cell preparations. The variable region has the advantage of being easy to prepare and its specificity is not affected by the source, although the human constant region is less likely to induce an immune response from human subjects than a constant region from a non-human source when the antibody is injected. However, the definition is not limited to this particular example.

[0091] The terms "antigen-binding portion" (or simply "binding portion") or "antigen-binding fragment" (or simply "binding fragment") of an antibody refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide crosslinks at a hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of VL and VH domains of one arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs), and (vii) combinations of two or more isolated CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers that allow them to be produced as single-chain proteins (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) by recombination, where the VL and VH regions pair up to form a monovalent molecule. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of the antibody. Further examples include binding-domain immunoglobulin fusion proteins, which include (i) a binding-domain polypeptide fused to an immunoglobulin hinge-domain polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region.The binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application Publication No. 2003 / 0118592 and U.S. Patent Application Publication No. 2003 / 0133939. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0092] The antibodies described herein may be monoclonal antibodies. As used herein, the term “monoclonal antibody” refers to a preparation of an antibody molecule with a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, monoclonal antibodies are produced by a hybridoma containing B cells obtained from a non-human animal, such as a mouse, fused to immortalized cells.

[0093] The antibodies described herein may be recombinant antibodies. As used herein, the term “recombinant antibody” includes all antibodies produced, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to an immunoglobulin gene, or hybridomas produced therefrom; (b) antibodies isolated from host cells transformed to express an antibody, e.g., transfectomas; (c) antibodies isolated from a recombinant combinatorial antibody library; and (d) antibodies produced, expressed, created or isolated by any other means including splicing an immunoglobulin gene sequence with another DNA sequence.

[0094] As used herein, the term “transfectoma” includes recombinant eukaryotic host cells that express antibodies, such as CHO cells, NS / O cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast cells.

[0095] As used herein, "heterologous antibody" is defined with respect to a transgenic organism that produces such an antibody. This term refers to an antibody having an amino acid sequence or a coding nucleic acid sequence corresponding to that recognized in an organism not composed of a transgenic organism, and generally an antibody derived from a species other than a transgenic organism.

[0096] As used herein, "heterohybrid antibody" refers to an antibody having light and heavy chains of different biological origins. For example, an antibody having a human heavy chain associated with a mouse light chain is a heterohybrid antibody.

[0097] The present invention includes all antibodies and antibody derivatives described herein that are subsumed within the term "antibody" for the purposes of the present invention. The term "antibody derivative" refers to any modified form of an antibody, such as a conjugate of an antibody with another agent or another antibody, or an antibody fragment.

[0098] The antibodies described herein are preferably isolated. As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities. Furthermore, an isolated antibody may be substantially free of other cellular materials and / or chemical substances.

[0099] According to the present invention, an antibody has a significant affinity for a given target in a standard assay and is capable of binding to the given target when binding to the given target. "Affinity" or "binding affinity" is often measured by the equilibrium dissociation constant (K D ). Preferably, the term "significant affinity" refers to binding to a given target with a dissociation constant (K -5 of 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less. D )

[0100] If an antibody does not have significant affinity for the target in a standard assay and does not significantly bind to the target, particularly if it does not bind detectably, it cannot (substantially) bind to the target. Preferably, the antibody does not bind detectably to the target when present at concentrations up to 2 μg / ml, preferably up to 10 μg / ml, more preferably up to 20 μg / ml, particularly 50 μg / ml or 100 μg / ml or higher. Preferably, the antibody has a K for binding to a predetermined target to which it can bind. D At least 10 times, 100 times, 10 3 double, 10 4 double, 10 5 double, or 10 6 K is twice as high D When binding to the target, it does not have significant affinity for the target. For example, K regarding the binding of an antibody to a target to which the antibody can bind. D 10 -7 If M, then K is related to binding to targets for which the antibody does not have significant affinity. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 It is M.

[0101] An antibody is specific to a given target if it can bind to a given target but cannot bind to other targets, i.e., it does not have significant affinity for other targets in a standard assay and does not significantly bind to other targets. According to the present invention, an antibody is specific to CLDN6 if it can bind to CLDN6 but cannot (substantially) bind to other targets, particularly other CLDN proteins such as CLDN9, CLDN4 and / or CLDN3, and / or proteins other than claudin proteins, preferably proteins other than CLDN6. Preferably, an antibody is specific to CLDN6 if its affinity and binding to such other targets do not significantly exceed its affinity or binding to claudin-independent proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins such as MHC molecules or transferrin receptors, or any other specific polypeptide. Preferably, an antibody is specific to CLDN6 if its affinity and binding to targets to which it is not specific is not significantly greater than its affinity or binding to claudin-independent proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins such as MHC molecules or transferrin receptors, or any other specific polypeptide. D At least 10 times, 100 times, 10 3 double, 10 4 double, 10 5 double, or 10 6 K is twice as low D When binding to a predetermined target, the antibody is specific to that predetermined target. For example, the K of the antibody regarding binding to a target that is specific to the antibody. D 10 -7 If M is the case, then K is related to the binding of the antibody to a non-specific target. D at least 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 It is M.

[0102] The binding of an antibody to its target can be experimentally determined using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described herein. Affinity can be readily determined using conventional techniques, e.g., by equilibrium dialysis; by using a BIAcore 2000 instrument with general procedures outlined by the manufacturer; by radioimmunoassay using a radiolabeled target antigen; or by other methods known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NYAcad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may differ when measured under different conditions, e.g., salt concentration, pH. Therefore, affinity and other antigen-binding parameters, e.g., K D ,I C 50 The measurement is preferably performed using standardized solutions of the antibody and antigen, as well as a standardized buffer.

[0103] The term "competition" refers to competition between two antibodies for binding to a target antigen. If two antibodies do not block each other for binding to the target antigen, such antibodies do not compete, which is an indicator that the antibodies do not bind to the same portion, i.e., epitope, of the target antigen. Methods for testing antibody competition for binding to a target antigen are well known to those skilled in the art. One example of such a method is the so-called cross-competition assay, which can be performed, for example, as ELISA or by flow cytometry. For example, an ELISA-based assay can be performed by coating a well of an ELISA plate with one of the antibodies, adding the competing antibody and the His-tagged antigen / target, and detecting whether the added antibody inhibited the binding of the His-tagged antigen to the coated antibody by, for example, adding a biotinylated anti-His antibody, followed by streptavidin-poly-HRP, further developing the reaction with ABTS, and measuring the absorbance at 405 nm. For example, a flow cytometry assay can be performed by incubating cells expressing an antigen / target with an excess of unlabeled antibody, then incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin, and finally analyzing the cells by flow cytometry.

[0104] Two antibodies possess "same specificity" when they bind to the same antigen and the same epitope. Whether an antibody under test recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibodies bind to the same epitope, can be tested by various methods known to those skilled in the art, for example, based on competition among antibodies for the same epitope. Competition between antibodies can be detected by cross-blocking assays. For example, a competitive ELISA assay can be used as a cross-blocking assay. For instance, a target antigen can be coated into wells of a microtiter plate, and the antigen-binding antibody and a candidate competitive test antibody can be added. The amount of antigen-binding antibody bound to the antigen in the wells indirectly correlates with the binding ability of the candidate competitive test antibody competing for binding to the same epitope. Specifically, the greater the affinity of the candidate competitive test antibody for the same epitope, the less antigen-binding antibody will bind to the antigen-coated well. The amount of antigen-binding antibody bound to a well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0105] An antibody that competes with another antibody, for example, an antibody containing heavy and light chain variable regions as described herein, for binding to an antigen, or an antibody that has specificity to an antigen of another antibody, for example, an antibody containing heavy and light chain variable regions as described herein, may be a variant of the heavy and / or light chain variable regions as described herein, for example, a modified CDR and / or an antibody containing a degree of identity as described herein.

[0106] As used herein, “isotype” refers to an antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. Antibodies according to 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), and IgM antibodies. However, other antibody isotypes, including IgG1, IgA1, IgA2, secreted IgA, IgD, and IgE antibodies, are also included in the present invention.

[0107] As used herein, "isotype switching" refers to the phenomenon in which the class or isotype of an antibody changes from one Ig class to one of other Ig classes.

[0108] As used herein, the term “reconstructed” refers to the configuration of a heavy-chain or light-chain immunoglobulin locus in which the V segment is located directly adjacent to the DJ or J segment in a conformation that essentially encodes a complete VH or VL domain, respectively. Reconstructed immunoglobulin (antibody) loci can be identified by comparison with germ cell DNA; a reconstructed locus has at least one recombinant heptamer / nonamer homologous element.

[0109] As used herein with respect to the V segment, the terms “unrecombined” or “germline configuration” refer to a configuration in which the V segment has not been recombined so as to be directly adjacent to a D or J segment.

[0110] According to the present invention, antibodies may be derived from a variety of species, including but not limited to mice, rats, rabbits, guinea pigs, and humans. Antibodies also include chimeric molecules in which an antibody constant region derived from one species, preferably humans, is combined with an antigen-binding site derived from another species. Furthermore, antibodies include humanized molecules in which an antigen-binding site of an antibody derived from a non-human species is combined with a constant region and framework region of human origin.

[0111] Antibodies can be produced by various techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies can, in principle, be used, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using antibody gene libraries.

[0112] The preferred animal strain for preparing hybridomas that secrete monoclonal antibodies is the mouse strain. Hybridoma production in mice is a well-established procedure. Immune protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0113] Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are rat and rabbit systems (see, for example, Spieker-Polet et al., Proc. Natl. Acad. Sci. USA. 92:9348 (1995), and also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)).

[0114] In yet another preferred embodiment, human monoclonal antibodies against CLDN6 can be produced using transgenic or transchromosomal mice that carry a portion of the human immune system rather than a mouse lineage. These transgenic and transchromosomal mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.” Production of human antibodies in such transgenic mice can be carried out as detailed with respect to CD20 in International Publication No. 2004 035607.

[0115] Another strategy for producing monoclonal antibodies is to directly isolate the gene encoding the antibody from lymphocytes that produce antibodies of defined specificities; see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. For more information on recombinant antibody engineering, see also Welschof and Kraus, Recombinant antibodydes for cancer therapy ISBN-0-89603-918-8 and Benny KCLo Antibody Engineering ISBN 1-58829-092-1.

[0116] To generate antibodies against CLDN6, mice can be immunized with carrier-bound peptides derived from the CLDN6 sequence, recombinantly expressed CLDN6 antigen or enriched preparations thereof, and / or cells expressing CLDN6 or its fragments, as described above. Alternatively, mice can be immunized with DNA encoding full-length human CLDN6 or its fragments. If antibodies are not obtained by immunization using purified or enriched preparations of the CLDN6 antigen, mice can also be immunized with cells expressing CLDN6, such as cell lines, to promote an immune response.

[0117] The immune response can be monitored throughout the course of the immunization protocol using plasma and serum samples obtained from the tail vein or postorbital hemorrhage. Mice with sufficient titers of anti-CLDN6 immunoglobulin can be used for fusion. To increase the proportion of hybridomas secreting specific antibodies, mice can be boosted with CLDN6-expressing cells via intraperitoneal or intravenous routes 3–5 days before sacrificial death and splenectomy.

[0118] To generate hybridomas that produce monoclonal antibodies against CLDN6, cells can be isolated from lymph nodes, spleen, or bone marrow obtained from immunized mice and fused to suitable immortalized cell lines, such as mouse myeloma cell lines. The resulting hybridomas can then be screened for antigen-specific antibody production. Individual wells can be screened by ELISA for antibody-secreting hybridomas. Antibodies specific to CLDN6 can be identified by immunofluorescence and FACS analysis using CLDN6-expressing cells. If antibody-secreting hybridomas are reseeded and screened again, and still positive for anti-CLDN6 monoclonal antibodies, they can be subcloned by limiting dilution. Stable subclones can then be cultured in vitro to generate antibodies in tissue culture medium for characterization.

[0119] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods well known in the art (Morrison, S. (1985) Science 229:1202).

[0120] For example, in one embodiment, one or more genes of interest, such as an antibody gene, can be ligated to an expression vector, such as a eukaryotic expression plasmid, as used by the GS gene expression system disclosed in International Publication No. 87 / 04462, International Publication No. 89 / 01036, and European Patent No. 338841, or other expression systems well known in the art. The purified plasmid containing the cloned antibody gene can be introduced into eukaryotic host cells such as CHO cells, NS / O cells, HEK293T cells, or HEK293 cells, or other eukaryotic cells such as plant-derived cells, fungal or yeast cells. The method used to introduce these genes may be one of the methods described in the art, such as electroporation, lipofectin, or lipofectamine. After introducing these antibody genes into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomas, and their expression levels can then be amplified to scale up for antibody production. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.

[0121] Alternatively, the cloned antibody gene can be expressed in other expression systems, including prokaryotic cells such as Escherichia coli (E. coli). Furthermore, antibodies can be produced in transgenic non-human animals such as sheep and rabbit milk or chicken eggs, 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.

[0122] Chimeric antibodies are antibodies derived from different animal species, such as those having a variable region derived from a mouse antibody and a human immunoglobulin constant region. Antibody chimerization is achieved by ligating the variable regions of the heavy and light chains of a mouse antibody with the constant regions of the heavy and light chains of a human antibody (as described, for example, in Kraus et al., Methods in Molecular Biology series, Recombinant antibody for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by ligating the human κ light chain constant region with the mouse light chain variable region. In another preferred embodiment, a chimeric antibody can be produced by ligating the human λ light chain constant region with the mouse 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 the production of chimeric antibodies are IgG2, IgA, IgD, and IgM.

[0123] Antibodies interact with target antigens primarily through amino acid residues located in six heavy and light chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse among individual antibodies than sequences outside of CDRs. Since CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific native antibodies by constructing expression vectors containing CDR sequences from specific native antibodies transplanted into framework sequences from different antibodies 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. USA. 86:10029-10033). Such framework sequences are available from public DNA databases containing germline antibody gene sequences. These germline sequences differ from mature antibody gene sequences because they do not contain fully constructed variable genes formed by V(D)J junctions during B cell maturation. Germline gene sequences also differ from high-affinity secondary repertoire antibody sequences, evenly distributed across individual positions throughout the variable region. For example, somatic mutations are relatively rare in the amino-terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the antibody's binding properties. Therefore, it is not necessary to obtain the entire DNA sequence of a particular antibody to reconstruct an intact recombinant antibody with binding properties similar to the original antibody (see International Publication 99 / 45962). Typically, partial heavy and light chain sequences across the CDR region are sufficient for this purpose. The partial sequences are used to determine which germline variable and ligation gene segments contributed to the recombinant antibody variable gene. The germline sequences are then used to fill in the missing portions of the variable region. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the final antibody properties. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification.Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and then combined by PCR amplification to create a fully synthetic variable region clone. This process offers several advantages, such as the removal or inclusion of specific restriction sites or the optimization of specific codons.

[0124] To create synthetic V sequences with the same amino acid coding ability as the natural sequence, a duplicate set of synthetic oligonucleotides can be designed using the nucleotide sequences of heavy and light chain transcripts from hybridomas. The synthetic heavy and κ chain sequences can be differentiated from the natural sequence in three ways: by interrupting a sequence of repeating nucleotide bases to facilitate oligonucleotide synthesis and PCR amplification; by incorporating an optimal translation initiation site according to the Kozak rule (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and by creating a HindIII site upstream of the translation initiation site.

[0125] For both the heavy and light chain variable regions, the optimized coding chain and corresponding non-coding chain sequences are decomposed into 30-50 nucleotides approximately midway through the corresponding non-coding oligonucleotides. Thus, for each chain, the oligonucleotides can be constructed into overlapping double-stranded sets spanning 150-400 nucleotide segments. These pools are then used as templates to generate 150-400 nucleotide PCR amplification products. Typically, a single variable region oligonucleotide set is split into two pools and amplified separately to produce two overlapping PCR products. These overlapping products are then joined by PCR amplification to form the complete variable region. It may also be desirable to include overlapping fragments of the heavy or light chain constant region in the PCR amplification to generate fragments that can be easily cloned into expression vector constructs.

[0126] Next, the reconstructed chimeric or humanized heavy and light chain variable regions are combined with cloned promoter, leader, translation start, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form an expression vector construct. The heavy and light chain expression constructs are combined into a single vector, which is then transfected into host cells simultaneously, sequentially, or separately, and subsequently fused to form host cells expressing both chains. Plasmids for use in constructing human IgGκ expression vectors are described. Plasmids can be constructed to allow reconstruction of complete heavy and light chain minigenes using PCR-amplified V heavy chain and Vκ light chain cDNA sequences. These plasmids can be used to express fully human or chimeric IgG1,κ or IgG4,κ antibodies. Similar plasmids can be constructed for the expression of other heavy chain isotypes or antibodies containing the λ light chain.

[0127] Therefore, in another aspect of the present invention, structurally related humanized anti-CLDN6 antibodies are produced using the structural features of the anti-CLDN6 antibodies described herein, while retaining at least one functional property of the antibodies of the present invention, such as binding to CLDN6. More specifically, one or more CDR regions of a mouse monoclonal antibody can be recombinantly combined with known human framework regions and CDRs to produce further recombinant humanized anti-CLDN6 antibodies.

[0128] The ability of an antibody to bind to CLDN6 can be determined using standard binding assays, such as ELISA, Western blotting, immunofluorescence, and flow cytometry.

[0129] 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 peptide. The peptide or protein used for immunization can be used to determine the specificity of the hybridoma supernatant or to analyze serum titers.

[0130] Flow cytometry can be used to demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells. Cell lines that express the antigen naturally or after transfection, and negative controls lacking antigen expression (grown under standard growth conditions), can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatant or PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, APC or Alexa647-labeled anti-IgG antibodies can be bound to antigen-binding monoclonal antibodies under the same conditions as primary antibody staining. Samples can be analyzed by flow cytometry using a FACS instrument that gates single living cells using light and lateral scattering properties. To distinguish antigen-specific monoclonal antibodies from non-specific binding antibodies in a single measurement, a simultaneous transfection method can be used. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained as described above. Transfected cells can be detected on different fluorescence channels than cells stained with the antibody. Since the majority of transfected cells express both transgenes, antigen-specific monoclonal antibodies selectively bind to cells expressing the fluorescent marker, while non-specific antibodies bind to non-transfected cells at a similar rate. Alternative assays using fluorescence microscopy can be used in addition to, or instead of, flow cytometry assays. Cells can be accurately stained and examined by fluorescence microscopy as described above.

[0131] Immunofluorescence microscopy can be used to demonstrate the presence of anti-CLDN6 antibodies in the serum of immunized mice, or the binding of monoclonal antibodies to living cells expressing CLDN6. For example, cell lines expressing CLDN6 spontaneously or after transfection, and negative controls lacking CLDN6 expression, are grown in chamber slides under standard growth conditions in DMEM / F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin, and 100 μg / ml streptomycin. The cells can then be fixed with methanol or paraformaldehyde, or left untreated. The cells can then be reacted with a monoclonal antibody against CLDN6 at 25°C for 30 minutes. After washing, the cells can be reacted with Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. The cells can then be examined by fluorescence microscopy.

[0132] When cells are fixed with methanol or paraformaldehyde and permeabilized with Triton X-100, the total CLDN6 levels of the cells can be observed. Surface localization of CLDN6 can be investigated in live cells and impermeable paraformaldehyde-fixed cells. Furthermore, the targeting of CLDN6 to tight junctions can be analyzed by co-staining with tight-junction markers such as ZO-1. Additionally, the effects of antibody binding within the cell membrane and CLDN6 localization can be investigated.

[0133] Anti-CLDN6 IgG can be further tested for reactivity with the CLDN6 antigen by Western blotting. Simply put, cell extracts can be prepared from cells expressing CLDN6 and a suitable negative control, and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and spread on an ECL substrate.

[0134] Anti-CLDN6 mouse IgG can be further tested for reactivity with the CLDN6 antigen by immunohistochemistry using methods well known to those skilled in the art, for example, using paraformaldehyde or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from mice carrying xenograft tumors inoculated with cell lines expressing CLDN6 spontaneously or after transfection. For immunostaining, an antibody reactive to CLDN6 can be incubated, followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody according to the distributor's instructions.

[0135] One particularly preferred method for assaying CLDN6 in the method of the present invention is immunohistochemistry or IHC. Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of tissue sections, for example, in the tissues referred to 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 achieved in many ways. In the most common example, antibodies are conjugated to enzymes such as peroxidase that can catalyze a color reaction. Alternatively, antibodies can be tagged to fluorophores such as fluorescein or rhodamine.

[0136] Sample preparation is crucial for preserving cell morphology, tissue structure, and the antigenicity of the target epitope. This requires proper tissue collection, fixation, and sectioning. Paraformaldehyde is typically used for fixation. Depending on the purpose and thickness of the experimental sample, thin sections (approximately 4–40 μm) are cut from the tissue of interest, or the entire tissue is used if it is not thick enough and translucent. Sectioning is usually performed using a microtome, and the sections are placed on glass slides.

[0137] The sample may require additional steps, including deparaffinization and antigen retrieval, to make the epitope available for antibody binding. Surfactants such as Triton X-100 are commonly used in immunohistochemistry to lower surface tension, allowing for better and more uniform coverage of the sample with less reagent.

[0138] The direct method of immunohistochemical staining uses one labeled antibody that directly binds to the antigen to be stained. The more common indirect method of immunohistochemical staining uses one antibody against the antigen to be probed and a second labeled antibody against the first antibody.

[0139] To reduce background staining in IHC, the sample is incubated with a buffer that blocks the reaction site to which the primary or secondary antibody could otherwise bind. The primary antibody is induced against the antigen of interest and is typically unbound (unlabeled), while the secondary antibody is induced against the immunoglobulin of the primary antibody species. The secondary antibody usually binds to a linker molecule such as biotin that recruits the reporter molecule, or it binds directly to the reporter molecule itself. Common blocking buffers include normal serum, skim milk powder, BSA or gelatin, and commercially available blocking buffers.

[0140] Reporter molecules vary depending on the nature of the detection method, with the most common being chromogenic and fluorescent detection via enzymes and fluorophores, respectively. Using chromogenic reporters, the enzyme label reacts with the substrate to produce a darkly colored product that can be analyzed with a standard light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are two of the most widely used enzymes as labels for protein detection. Arrays of chromogenic, fluorescent, and chemiluminescent substrates can be used with either DAB or BCIP / NBT enzymes. Fluorescent reporters are small organic molecules used for IHC detection. For chromogenic and fluorescent detection methods, densitometry analysis of the signal can provide semi-quantitative and fully quantitative data, relating the level of the reporter signal to the level of protein expression or localization, respectively.

[0141] After immunohistochemical staining of a target antigen, secondary staining is often applied to provide contrast that helps highlight the primary stain. Many of these stains are specific to separate cell compartments or antigens, while others stain entire cells. Both chromogenic and fluorescent dyes can be used in IHC to provide a vast number of reagents to suit any experimental design. Hematoxylin, Hoechst stain, and DAPI are commonly used.

[0142] The mapping of antibody-recognized epitopes can be performed as detailed in "Epitope Mapping Protocols (Methods in Molecular Biology)" ISBN-089603-375-9 by Glenn E. Morris and "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 by Olwyn MrWestwood and Frank C. Hay.

[0143] In relation to the present invention, the term “immune effector function” includes any function mediated by components of the immune system that results in inhibition of tumor growth and / or inhibition of tumor development, including suppression of tumor dissemination and metastasis. Preferably, the immune effector function results in the death of cancer cells. Preferably, in relation to the present invention, the immune effector function is an antibody-mediated effector function. Such functions include complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis of tumor-associated antigen-carrying cells, for example by binding of an antibody to a surface antigen, inhibition of CD40L-mediated signaling, for example by binding of an antibody to a CD40 receptor or CD40 ligand (CD40L), and / or inhibition of proliferation of tumor-associated antigen-carrying cells, preferably ADCC and / or CDC. Therefore, antibodies capable of mediating one or more immunoeffector functions can mediate cell death, preferably by inducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotyped adhesion, and / or phagocytosis, and more preferably by inducing CDC-mediated lysis and / or ADCC-mediated lysis. Antibodies can also exert their effects simply by binding to tumor-associated antigens on the surface of cancer cells. For example, an antibody may block the function of a tumor-associated antigen or induce apoptosis simply by binding to a tumor-associated antigen on the surface of cancer cells.

[0144] ADCC represents the cell-killing ability of effector cells, particularly lymphocytes, and preferably requires antibody-marked target cells. ADCC preferably occurs when an antibody binds to an antigen on cancer cells, and the antibody's Fc domain engages with an Fc receptor (FcR) on the surface of an immune effector cell. Several families of Fc receptors have been identified, and certain cell populations characteristically express defined Fc receptors. ADCC can be considered a mechanism that directly induces varying degrees of immediate tumor destruction and also leads to the induction of antigen presentation and tumor-targeting T cell responses. Preferably, in vivo induction of ADCC results in tumor-targeting T cell responses and further host-derived antibody responses.

[0145] CDC is another method of cell death that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also very effective in directing CDC via the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex involves the C of an antibody molecule, such as an IgG molecule. H This results in the exposure of multiple C1q binding sites in close proximity to the 2 domains (C1q is one of the three subcomponents of complement C1). Preferably, these exposed C1q binding sites convert the previously low-affinity C1q-IgG interaction to a high-affinity one, initiating a cascade of events involving a series of other complement proteins, resulting in the proteolytic release of effector cell chemotactic / activators C3a and C5a. Preferably, the complement cascade terminates by forming a membrane invasion complex that can induce apoptosis by creating pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell.

[0146] In relation to the present invention, the term “immune effector cells” refers to cells that exert effector functions during an immune response. For example, such cells secrete cytokines and / or chemokines to kill microorganisms, secrete antibodies, recognize cancer cells, and optionally eliminate 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.

[0147] According to the present invention, "nucleic acid" is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcription RNA (IVT RNA) or synthetic RNA. According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. According to the present invention, nucleic acids may be single-stranded or double-stranded and may be in the form of molecules that are linear or covalently closed to form a circle. Nucleic acids can be used for introduction into cells, i.e., cell transfection, in the form of RNA which can be prepared, for example, by in vitro transcription from a DNA template. RNA can be further modified before application by sequence stabilization, capping, and polyadenylation.

[0148] The nucleic acids described herein may be included in vectors. As used herein, the term “vector” includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phages, viral vectors such as adenoviruses or baculoviruses, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors, including plasmids and viral vectors, generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of an operablely linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0149] For expressing antibodies, a vector type can be used in which the antibody chain resides in a different vector, or in which the antibody chain resides in the same vector.

[0150] As used herein, the term “RNA” means a molecule containing ribonucleotide residues. “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. This term includes isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA; essentially pure RNA; synthetic RNA; recombinantly produced RNA; and modified RNA, which differs from native RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, for example, at one or more nucleotides of RNA, to the ends (one or both) or internally. Nucleotides in an RNA molecule may also include non-standard nucleotides, such as nucleotides not found in nature, or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0151] According to the present invention, the term "RNA" includes, and preferably relates to, "mRNA," meaning "messenger RNA," and relates to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA typically includes a 5' untranslated region, a protein or peptide coding region, and a 3' untranslated region.

[0152] In relation to the present invention, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA.

[0153] The nucleic acids described in accordance with the present invention are preferably isolated. According to the present invention, the term "isolated nucleic acid" means that the nucleic acid is (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel electrophoresis, or (iv) synthesized, for example by chemical synthesis. Isolated nucleic acids are nucleic acids that can be used for manipulation by recombinant DNA technology.

[0154] According to the present invention, nucleic acids may exist alone or in combination with other nucleic acids, which may be homogeneous or heterogeneous. In a preferred embodiment, the nucleic acid is functionally linked to an expression regulatory sequence, which may be homogeneous or heterogeneous. The term "homogeneous" means that the nucleic acid is functionally linked naturally in nature, while the term "heterogeneous" means that the nucleic acid is not functionally linked in nature.

[0155] Nucleic acids and regulatory sequences are "functionally" linked to each other if they are covalently linked such that the expression or transcription of the nucleic acid is under the control or influence of the regulatory sequence. When a nucleic acid is translated into a functional protein using a regulatory sequence functionally linked to a coding sequence, the induction of the regulatory sequence results in the transcription of the nucleic acid without causing a frameshift in the coding sequence or making it impossible for the coding sequence to be translated into a desired protein or peptide.

[0156] The term “expression regulatory sequence” or “expression regulatory element” according to the present invention includes promoters, ribosome binding sites, enhancers, and other regulatory elements that regulate gene transcription or mRNA translation. In certain embodiments of the present invention, expression regulatory sequences can be regulated. The exact structure of an expression regulatory sequence may vary depending on the species or cell type, but generally includes a 5' untranscribed sequence and 5' and 3' untranslated sequences involved in the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, or a CAAT sequence. More specifically, a 5' untranscribed expression regulatory sequence includes a promoter region containing a promoter sequence for transcriptional regulation of functionally linked nucleic acids. An expression regulatory sequence may also include an enhancer sequence or an upstream activating sequence.

[0157] According to the present invention, the terms “promoter” or “promoter region” relate to a nucleic acid sequence located upstream (5' end) of the expressed nucleic acid sequence and controlling the expression of the sequence by providing a recognition and binding site for RNA polymerase. The “promoter region” may include further recognition and binding sites for further factors involved in the regulation of gene transcription. Promoters can control the transcription of prokaryotic or eukaryotic genes. Furthermore, promoters can be “inducible,” meaning they can initiate transcription in response to an inducer, or they can be “constitutive,” meaning transcription is not controlled by an inducer. Genes under the control of an inducible promoter are not expressed or are expressed only to a small extent in the absence of an inducer. In the presence of an inducer, the gene is switched on or the level of transcription increases. This is generally mediated by the binding of a specific transcription factor.

[0158] Preferred promoters according to the present invention include SP6, T3 and T7 polymerase promoters, human U6 RNA promoter, CMV promoter, and artificial hybrid promoters (e.g., CMV) in which one or more portions are fused to a portion or more of the promoter of a gene for another cellular protein, such as human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and which may or may not include additional introns (one or more).

[0159] The term “expression” is used herein in its broadest sense and includes the production of RNA, or the production of RNA with proteins or peptides. With respect to RNA, the terms “expression” or “translation” particularly refer to the production of peptides or proteins. Expression can be transient or stable. According to the present invention, the term “expression” also includes “ectopic expression” or “abnormal expression.”

[0160] "Ectopic expression" or "abnormal expression," according to the present invention, means that the expression is altered, preferably increased, compared to the state of a subject without a disease associated with ectopic or abnormal expression of a reference, such as a specific protein, such as a tumor-associated antigen. An increase in expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100%, or more. In one embodiment, expression is observed only in diseased tissue, while expression is suppressed in healthy tissue.

[0161] The term "specifically expressed" means that a protein is expressed 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 not expressed in other tissues or to a significant degree in other tissue or organ types. Thus, a protein that is exclusively expressed in gastric mucosal cells and expressed to a significantly lower degree in other tissues is specifically expressed in gastric mucosal cells. In some embodiments, tumor-associated antigens may also be specifically expressed in two or more tissue types or organs under normal conditions, for example, two or three tissue types or organs, but preferably three or fewer different tissue or organ types. In this case, the tumor-associated antigen is specifically expressed in these organs.

[0162] The term "translation" according to the present invention relates to a process in the ribosome of a cell in which a chain of messenger RNA instructs the construction of an amino acid sequence to produce a protein or peptide.

[0163] According to the present invention, the term "~coding nucleic acid" means that, when present in a suitable environment, preferably within a cell, the nucleic acid can be expressed to produce the protein or peptide it encodes.

[0164] The term "peptide" includes oligopeptides and polypeptides and refers to substances containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably nine or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty-one or more, and preferably eight, ten, twenty-five, and especially up to 100 amino acids, which are covalently linked by peptide bonds. The term "protein" refers to a large peptide, preferably a peptide having more than 100 amino acid residues, but generally the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

[0165] Preferably, the proteins and peptides described in accordance with the present invention are 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 an essentially purified state. The term “essentially purified” means that the protein or peptide essentially does not contain other substances to which it is bound in nature or in vivo.

[0166] The teachings given herein with respect to a specific amino acid sequence, for example, one shown in a sequence listing, should be interpreted as also relating to a functionally equivalent sequence, such as a modification of the specific sequence, i.e., a variant, resulting in an amino acid sequence exhibiting the same or similar properties as the specific amino acid sequence. One important property is retention of antibody binding to the target. Preferably, a modified sequence with respect to a specific sequence retains antibody binding to the target when substituting the specific sequence in the antibody.

[0167] Those skilled in the art will understand that the CDR sequence, the hypervariable region, and the variable region sequence can be modified without losing their ability to bind to the target. For example, the CDR sequence is identical or highly homologous to the CDR sequence specified herein.

[0168] The "high degree of homology" is intended to allow for 1 to 5 substitutions, preferably 1 to 4, for example, 1 to 3 or 1 or 2 substitutions.

[0169] The term "mutant" as used in this invention includes mutants, splice mutants, conformational mutants, isoforms, allelic mutants, species mutants, and species homologs, particularly those occurring in nature. Allelic mutants are associated with normal changes in the sequence of a gene, but their significance is often unclear. Complete gene sequencing often identifies numerous allelic mutants of a given gene. A species homolog is a nucleic acid sequence or amino acid sequence that originates from a different species than that of a given nucleic acid sequence or amino acid sequence.

[0170] For the purposes of the present invention, "mutants" of amino acid sequences include amino acid insertion mutants, amino acid addition mutants, amino acid deletion mutants, and / or amino acid substitution mutants. Amino acid deletion mutants, which include deletions at the N-terminus and / or C-terminus of a protein, are also called N-terminal and / or C-terminal cleavage mutants.

[0171] Amino acid insertion mutants involve the insertion of one or more amino acids into a specific amino acid sequence. In amino acid sequence mutants with insertions, one or more amino acid residues are inserted at specific locations in the amino acid sequence, but random insertions are also possible, provided that the resulting products are properly screened.

[0172] Amino acid addition mutants include amino-terminal and / or carboxyl-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0173] Amino acid deletion mutants are characterized by the removal of one or more amino acids from a sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be located at any position in the protein.

[0174] Amino acid substitution mutants are characterized by the removal of at least one residue in a sequence and the insertion of another residue in its place. It is preferable that the modification is located at a position in the amino acid sequence that is not conserved among homologous proteins or peptides, and / or that the amino acid is replaced with another amino acid having similar properties. Preferably, the amino acid change in a protein mutant is a conserved amino acid change, i.e., a substitution of a similarly charged or uncharged amino acid. Conserved amino acid changes include substitutions of one of the families of amino acids whose side chains are related. Naturally occurring amino acids are generally classified into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

[0175] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is 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 total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably with optimal sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extension 0.5.

[0176] "Sequence similarity" indicates the proportion of amino acids that are identical or represent a conserved amino acid substitution. "Sequence identity" between two amino acid sequences indicates the proportion of amino acids that are identical between those sequences.

[0177] The term "identity percentage" is intended to indicate the percentage of amino acid residues that are identical between two sequences being compared, obtained after optimal alignment. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across their entire length. Sequence comparisons between two amino acid sequences are typically performed by comparing them after optimal alignment, and the comparison is done segment by segment or "comparison window" by window to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be achieved manually, by local homology algorithms (Smith and Waterman, 1981, Ads App.Math.2, 482; Neddleman and Wunsch, 1970, J.Mol.Biol.48, 443; Pearson and Lipman, 1988, Proc.Natl Acad.Sci.USA 85, 2444; or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0178] The identity percentage is calculated by determining the number of identical positions in the two sequences being compared, dividing this number by the total number of positions being compared, and multiplying the result by 100 to obtain the identity percentage between these two sequences.

[0179] According to the present invention, homologous amino acid sequences exhibit identity of at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% of the amino acid residues.

[0180] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences for preparing proteins and peptides with substitutions, additions, insertions, or deletions is described in detail, for example, Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, such as solid-phase synthesis and similar methods.

[0181] The present invention includes derivatives of peptides or proteins as described herein, which are included in the terms “peptide” and “protein.” According to the present invention, “derivatives” of proteins and peptides are modified forms of proteins and peptides. Such modifications include any chemical modifications, as well as single or multiple substitutions, deletions, and / or additions of any molecules related to proteins or peptides, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, “derivatives” of proteins or peptides include modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term “derivative” also extends to all functional chemical equivalents of the proteins and peptides. Preferably, modified peptides have increased stability and / or increased or decreased immunogenicity.

[0182] According to the present invention, amino acid sequences, peptides or proteins, variants, derivatives, modified forms, fragments, parts or portions thereof preferably each have the functional properties of the amino acid sequence, peptide or protein from which they are derived, i.e., they are functionally equivalent. In one embodiment, amino acid sequences, peptides or proteins, variants, derivatives, modified forms, fragments, parts or portions thereof are immunologically equivalent to the amino acid sequence, peptide or protein from which they are derived. In one embodiment, the functional properties are immunological properties.

[0183] The term “derived from” means, according to the present invention, that a particular entity, in particular a particular sequence, is present in the object, in particular a living organism or molecule, from which it originates. In the case of an amino acid sequence, in particular a particular sequence region, “derived from” means, in particular, that the relevant amino acid sequence originates from the amino acid sequence in which it exists.

[0184] The terms “cell” or “host cell” preferably refer to an intact cell, i.e., a cell with an intact membrane that does not release its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, i.e., a living cell capable of performing its normal metabolic functions. The term “cell” according to the present invention includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred. Cells may originate from a number of tissue types and may include primary cells and cell lines. The term “cell” includes noncancerous cells and cancer cells, such as cancer cells of the oncogenic types disclosed herein.

[0185] Cells containing nucleic acid molecules preferably express peptides or proteins encoded by the nucleic acid.

[0186] "Target cells" refer to cells that are targeted by immune responses, such as antibodies. Target cells include undesirable cells such as cancer cells as described herein. In preferred embodiments, target cells are cells that express CLDN6. Cells that express CLDN6 typically include cancer cells.

[0187] The term “transgenic animal” refers to an animal having a genome that includes one or more transgenes, preferably antibody heavy chain and / or light chain transgenes, or a transchromosome (which may or may not be 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 a human anti-CLDN6 antibody when immunized with cells expressing the CLDN6 antigen and / or CLDN6. The human heavy chain transgene may be integrated into the mouse’s chromosomal DNA, as in transgenic mice, e.g., HuMAb mice such as HCo7 or HCol2 mice, or the human heavy chain transgene may be maintained extrachromosomally, as in transchromosomal (e.g., KM) mice described in International Publication No. 02 / 43478. Such transgenic and transchromosomal mice may be able to produce multiple isotypes (e.g., IgG, IgA, and / or IgE) of human monoclonal antibodies against CLDN6 by undergoing VDJ recombination and isotype switching.

[0188] The term "immunologically equivalent" means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit and / or exert the same or essentially the same immunological properties with respect to the type of immunological action, 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 connection with the present invention, the term "immunologically equivalent" is preferably used with respect to the immunological action or properties of a peptide or peptide variant or antibody used for immunization. A particular immunological property is the ability to generate an immune response by binding to an antibody and, where appropriate, preferably by stimulating antibody production. For example, if an amino acid sequence induces an immune response, preferably an antibody, that has specificity to react with a reference amino acid sequence, such as a reference amino acid sequence that forms part of CLDN6, when exposed to the immune system of a subject, the amino acid sequence is immunologically equivalent to the reference amino acid sequence.

[0189] The present invention provides a method for detecting the presence of CLDN6 antigen in a sample or for 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 for the formation of a complex between the antibody and CLDN6. The formation of the complex is then detected, and the difference in complex formation between the sample and the control sample indicates the presence of CLDN6 antigen in the sample.

[0190] The above method is particularly useful for diagnosing CLDN6-related diseases, such as cancerous diseases, including the cancerous diseases described herein. Preferably, an amount of CLDN6 in a sample greater than the amount of CLDN6 in a reference or control sample indicates the presence of a CLDN6-related disease in the subject from which the sample originates, particularly in humans.

[0191] When used in the manner described above, the antibodies described herein may be provided with labels that function to (i) provide a detectable signal; (ii) interact with a second label to alter a detectable signal provided by the first or second label, e.g., FRET (fluorescence resonance energy transition); (iii) affect mobility, e.g., electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters; or (iv) provide a capture moiety, e.g., affinity, antibody / antigen, or ionic complex formation. Suitable labels are fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobar labels, enzyme labels, particle labels, especially metal particle labels, magnetic particle labels, polymer particle labels, organic small molecules such as biotin, ligands or binding molecules for receptors such as cell adhesion proteins or lectins, and labeled sequences containing nucleic acids and / or amino acid residues that can be detected by the use of binders. The labels include, but are not limited to, radioactive diagnostic materials including barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate, as well as positron emitters such as fluorine-18 and carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, and radionuclides for nuclear magnetic resonance such as fluorine and gadolinium.

[0192] According to the present invention, a “reference,” such as a reference sample or reference organism, can be used to relate and compare the results obtained from the test sample or test organism using the method of the present invention. Typically, a reference organism is a healthy organism, particularly one that is not suffering from a disease such as cancer. A “reference value” or “reference level” can be determined empirically from the references by measuring a sufficiently large number of references. Preferably, the reference value is determined by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.

[0193] As used herein, the terms “reduce” or “inhibit” relate to the ability to produce an overall reduction of a level preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term “inhibit” or similar phrases include complete or essentially complete inhibition, i.e., reduction to zero or essentially zero.

[0194] Terms such as “increase” or “boost” preferably relate to an increase or boost of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

[0195] The active substances, compositions, and methods described herein can be used to diagnose subjects with diseases. The diseases that can be diagnosed include all diseases that express CLDN6. Particularly preferred diseases are cancerous diseases, such as those described herein.

[0196] According to the present invention, the term "disease" refers to any pathological condition, including cancerous diseases, and in particular the forms of cancerous diseases described herein.

[0197] The term "normal" as used in the terms "normal tissue" or "normal state" refers to the state in healthy tissue or a healthy subject, i.e., a non-pathological state, and "healthy" preferably means non-cancerous.

[0198] According to the present invention, “a disease involving cells expressing CLDN6” means that CLDN6 is expressed in cells of diseased tissue or organ. In one embodiment, the expression of CLDN6 is increased compared to its state in healthy tissue or organ. An increase means an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or more. In one embodiment, the expression is observed only in diseased tissue, and the expression in healthy tissue is suppressed. According to the present invention, diseases involving or associated with cells expressing CLDN6 include cancerous diseases, particularly the forms of cancer described herein.

[0199] According to the present invention, the terms “tumor” or “tumor disease” refer to a swelling or lesion formed by the abnormal growth of cells (called neoplastic cells or tumor cells). “Tumor cells” means abnormal cells that grow by rapid, uncontrolled proliferation and continue to grow after the stimulus that initiated new growth has ceased. Tumors exhibit a partial or complete absence of structural mechanisms and functional coordination with normal tissue and typically form a distinct tissue mass that can be benign, premalignant, or malignant.

[0200] A benign tumor is a tumor that lacks all three malignant characteristics of cancer. Therefore, by definition, a benign tumor does not grow aggressively indefinitely, does not invade surrounding tissues, and does not spread (metastasize) to non-adjacent tissues. Common examples of benign tumors include nevi and uterine fibroids.

[0201] The term "benign" refers to mild, non-progressive diseases, and indeed, many types of benign tumors are harmless to health. However, some neoplasms defined as "benign tumors" because they lack the invasive characteristics of cancer can still have adverse effects on health. Examples include tumors that cause "mass effects" (compression of vital organs essential for life, such as blood vessels) or "functional" tumors of endocrine tissue that can overproduce certain hormones (examples include thyroid adenomas, adrenocortical adenomas, and pituitary adenomas).

[0202] Benign tumors are typically surrounded by an outer surface that inhibits their ability to behave malignantly. In some cases, certain “benign” tumors can later develop into malignant tumors, which arises from further genetic changes in a subpopulation of neoplastic cells in the tumor. A prominent example of this phenomenon is the tubular adenoma, a common type of colon polyp that is a significant precursor to colon cancer. The cells of a tubular adenoma, like most tumors that often progress to cancer, exhibit specific abnormalities in cellular maturation and appearance known collectively as dysplasia. These cellular abnormalities are not seen in benign tumors that rarely or never become cancerous, but are seen in other precancerous tissue abnormalities that do not form individual masses, such as precancerous lesions of the cervix. Some authorities prefer to refer to dysplastic tumors as “premalignant” and reserve the term “benign” for tumors that rarely or never develop into cancer.

[0203] A neoplasm is an abnormal tissue mass resulting from neoplasia. Neoplasia (from the Greek word for new growth) is the abnormal proliferation of cells. The growth of cells outpaces the growth of the surrounding normal tissue and does not coordinate with the normal tissue. The growth continues in the same excessive manner even after the cessation of stimulation. It usually causes a lump or tumor. Neoplasms can be benign, premalignant, or malignant.

[0204] The term "tumor growth" or "tumor development" according to the present invention relates to the tendency of a tumor to increase in size and / or the tendency of tumor cells to proliferate.

[0205] Cancer (medical term: malignant neoplasm) is a class of diseases characterized by uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body via the lymphatic system or bloodstream). These three malignant characteristics of cancer distinguish it from benign tumors, which are self-limiting and do not invade or metastasize. Most cancers form tumors, but some cancers, such as leukemia, do not. According to this invention, the terms “cancer” and “tumor” or “cancer disease” and “tumor disease” are used herein to refer to diseases characterized by uncontrolled growth of cells and, if applicable, invasion and / or metastasis.

[0206] Preferably, the “cancer disease” according to the present invention is characterized by cells expressing CLDN6. The cells expressing CLDN6 are preferably cancer cells, and preferably cancer cells of tumors and cancers as described herein. Preferably, such cells are cells other than placental cells.

[0207] Cancers are classified by the type of cells that resemble the tumor, and therefore by the tissue from which the tumor is presumed to originate. These are histology and location, respectively.

[0208] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, hematological cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, digestive tract cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose, and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as their metastases. Examples include lung carcinoma, breast carcinoma, prostate carcinoma, colon carcinoma, renal cell carcinoma, cervical carcinoma, or metastases of the above cancer types or tumors. The term "cancer" according to the present invention also includes cancer metastases.

[0209] The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC has three main subtypes: squamous cell lung cancer, adenocarcinoma, and large cell lung cancer. Adenocarcinoma accounts for about 10% of lung cancers. This cancer is usually found in the periphery of the lung, while small cell lung cancer and squamous cell lung cancer both tend to be located more centrally.

[0210] According to the present invention, "carcinoma" is a malignant tumor derived from epithelial cells. This group is the most common type of cancer, including the most common forms of breast cancer, prostate cancer, lung cancer, and colon cancer.

[0211] Adenocarcinoma is a type of cancer that develops in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands, and various other tissues that line the inside of body cavities and organs. Epithelium is embryologically derived from the ectoderm, endoderm, and mesoderm. To be classified as adenocarcinoma, cells do not necessarily have to be part of a gland, as long as they have 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, although poorly differentiated ones may not. Pathologists determine whether a tumor is adenocarcinoma or some other type of cancer by staining cells from a biopsy. Adenocarcinoma can occur in many tissues of the body due to the ubiquity of glands in the body. Each gland may not secrete the same substance, but as long as it has an exocrine function to cells, it is considered a gland, and therefore its malignant form is named adenocarcinoma. Malignant adenocarcinoma invades other tissues and often metastasizes if it has enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian cancer. Ovarian adenocarcinoma includes serous and mucinous adenocarcinoma, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

[0212] "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and subsequent transport by the blood before invading the target organ. Finally, the growth of a new tumor at the target site, i.e., a secondary or metastatic tumor, depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor, as tumor cells or components may remain and exhibit metastatic potential. In one embodiment, the term "metastasis" according to this invention refers to "distant metastasis," which is a metastasis that is far from the primary tumor and the regional lymph node system.

[0213] The cells of a secondary or metastatic tumor are similar to the cells of the original tumor. This means that, for example, if ovarian cancer metastasizes to the liver, the secondary tumor will consist of abnormal ovarian cells, not abnormal liver cells. Therefore, the tumor in the liver is called metastatic ovarian cancer, not liver cancer.

[0214] Recurrence or relapse occurs when a person becomes ill again with a condition they have had in the past. For example, if a patient has had a neoplastic disease, has been successfully treated for the disease, and then develops the disease again, the newly developed disease may be considered a recurrence or relapse. However, according to the present invention, recurrence or relapse of cancer may, but not necessarily, occur at the site of the original cancer. For example, if a patient has an ovarian tumor and has been successfully treated, recurrence or relapse may be the development of an ovarian tumor or a tumor at a site different from the ovary. Tumor recurrence or relapse also includes situations in which the tumor develops at a site different from the original tumor site and situations in which it develops at the original tumor site. Preferably, the original tumor that the patient was treated for is a primary tumor, and the tumor at a site different from the original tumor site is a secondary or metastatic tumor.

[0215] "To treat" means administering a compound or composition to a subject in order to prevent or eliminate a disease, including reducing the size or number of tumors in the subject; to stop or delay a disease in the subject; to suppress or delay the onset of a new disease in the subject; to reduce the frequency or severity of symptoms and / or recurrences in a subject who currently has or has previously had a disease; and / or to extend, i.e., increase, the lifespan of the subject.

[0216] In particular, the term “treatment of disease” includes the cure, reduction of duration, improvement, prevention, slowing or inhibiting of progression or worsening of a disease or its symptoms, or prevention or delaying of its onset.

[0217] "At risk" refers to subjects, or patients, who are identified as having a higher-than-usual likelihood of developing a disease, particularly cancer, compared to the general population. Furthermore, subjects who have had or currently have a disease, particularly cancer, are at high risk of developing the disease because they continue to be at risk of developing it. Subjects who currently have or have had cancer are also at high risk of cancer metastasis.

[0218] The term "immunotherapy" refers to treatments that involve a specific immune response.

[0219] In relation to the present invention, terms such as “protect,” “prevent,” “preventive,” “preventive,” or “protective” relate to the prevention or treatment of the onset and / or transmission of a disease in a subject, or both, particularly minimizing the likelihood of the subject developing a disease or delaying its onset. For example, as described above, a person at risk of tumors would be a candidate for a treatment that prevents tumors. Immunotherapy can be carried out using any of a variety of techniques in which the active ingredient works to remove antigen-expressing cells from the patient.

[0220] In certain embodiments, immunotherapy may be active immunotherapy, where treatment relies on in vivo stimulation of the endogenous host immune system that responds to disease cells by administering immune response modifiers (such as immunoreactive peptides and nucleic acids).

[0221] In other embodiments, the immunotherapy may be passive immunotherapy, and the treatment involves the delivery of an active agent (e.g., effector cells) that has an established tumor immunoreactivity that can directly or indirectly mediate an antitumor effect and does not necessarily depend on an intact host immune system.

[0222] The term "in vivo" refers to the conditions in the subject.

[0223] The terms “subject,” “individual,” “organism,” or “patient” are used interchangeably and relating to vertebrates, preferably mammals. For example, in relation to the present invention, mammals include humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, and horses, laboratory animals such as mice, rats, rabbits, and guinea pigs, and captive animals such as zoo animals. The term “animal” as used herein also includes humans. The term “subject” may also include a patient having a disease, preferably a disease as described herein, i.e., an animal, preferably a human.

[0224] According to the present invention, “sample” can be any sample useful in accordance with the present invention, particularly biological samples such as tissue samples containing bodily fluids and / or cell samples, which can be obtained by conventional methods, for example by tissue biopsy including punch biopsy, and by collecting blood, bronchial aspirate, sputum, urine, feces or other bodily fluids. According to the present invention, the term “sample” also includes processed samples such as fractions or isolates of biological samples, for example nucleic acids and peptide / protein isolates. Preferably, the sample includes cells or tissue of the organ being examined, for example, to be diagnosed for cancer. For example, if the cancer being diagnosed is lung cancer, the sample would include cells or tissue obtained from the lungs.

[0225] According to the present invention, the sample can be any sample such as a biological sample derived from a patient, including or expected to include a tumor or cancer cells. The biological sample can be any tissue sample such as blood, a tissue sample obtained from a primary tumor or tumor metastasis, or any other sample containing a tumor or cancer cells.

[0226] The present invention will be described in detail by the following drawings and examples, which are for illustrative purposes only and are not intended to be limiting. Further embodiments included in the present invention will be accessible to those skilled in the art by the description and examples.

Example

[0227] The techniques and methods used herein are carried out by methods described herein or known per se, for example as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (l989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods, including the use of kits and reagents, are carried out according to the manufacturer's information unless otherwise indicated.

[0228] [Example 1] Materials and Methods Mapping of Antibody Binding Sites (Figure 4A) Mapping of the epitope recognized by an antibody can be carried out as detailed in "Epitope Mapping Protocols (Methods in Molecular Biology)" by Glenn E. Morris ISBN-089603-375-9 and "Epitope Mapping: A Practical Approach" by Olwyn M.R. Westwood, Frank C. Hay, Practical Approach Series 248.

[0229] Epitope mapping with biotinylated peptides (Figure 4B) Peptide ELISA was performed to identify the antibody binding sites of the monoclonal mouse lead antibody and the polyclonal rabbit serum from IBL. Biotinylated overlapping peptides covering the C-terminal sequence of CLDN6 were bound to SA-coated plates. Purified mumAB (1 μg / ml) or IBL rabbit serum (1 μg / ml) was applied to the antigen-coated ELISA plates, and unbound antibodies were washed away. Bound antibodies were detected using the corresponding enzyme-labeled secondary antibodies (alkaline phosphatase goat anti-mouse IgG (1+2a+2b+3) or alkaline phosphatase goat anti-rabbit IgG F(ab)2) and ABTS enzyme substrate, and the signal intensities were analyzed.

[0230] To compare the signal intensities of mumAB 58-4B-2 and rabbit serum (IBL), the maximum binding of the antibody to C-terminal peptide 19 was defined as 100%.

[0231] The binding strength of each antibody to one peptide was calculated independently by comparing it with the maximum binding of each test system (mouse or rabbit).

[0232] The binding of mumAB was analyzed in triplicate in three independent experiments. The binding of IBL serum was analyzed in triplicate in two independent experiments.

[0233] Isotype determination To determine the isotype of the purified antibody, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, catalog number 1493027) was used as described by the manufacturer.

[0234] Western blotting The newly prepared anti-CLDN6 IgG can be further tested for specific binding to the CLDN6 antigen by Western blotting. Simply put, cell extracts from cells expressing CLDN3, 4, 6, or 9, along with a suitable negative control, can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and spread on an ECL substrate.

[0235] histology Immunohistochemistry (IHC) was performed on glass slides of tissue samples fixed in 4% buffered formalin and embedded in paraffin. Paraffin embedding was performed according to a standard protocol.

[0236] After deparaffinization and rehydration, all slides were subjected to antigen recovery by boiling at 95-98°C for 30 minutes in 0.1 M Tris / 0.01 M EDTA buffer (pH 9.0) supplemented with 15 mM sodium azide, followed by quenching of endogenous peroxidase with 3% H2O2 supplemented with 15 mM NaN3. 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, the slides were incubated at room temperature for 1 hour with 1.0 or 5.0 μg / ml of diagnostic monoclonal mouse anti-CLDN6 antibody 58-4B. Antibody binding was visualized using a ready-to-use solution containing polymer-based horseradish peroxidase-labeled secondary antibody (Histofine MAX PO(M) / UIO MAX PO(M), Nichirei, Japan). Subsequently, the sections were counterstained with Meyer's hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and subjected to evaluation by evaluators.

[0237] Histological evaluation All samples were analyzed for the relative proportion of positively stained tumor cells to all visible tumor cells in each section. Stain intensity was classified as negative (-), weakly positive (1+), moderately positive (2+), and strongly positive (3+). Only membrane staining was considered positive. Ovarian cancer tissue served as a positive control for each stain. Furthermore, embryonic kidney tissue from 29-day-old New Zealand white rabbits was found to exhibit strong positive staining intensity. This was used as an internal staining intensity standard for staining positivity (2+~3+).

[0238] [Example 2] Production of monoclonal antibodies The objective of this project was to produce a mouse monoclonal CLDN6-specific antibody capable of detecting CLDN6-expressing tumor cells in ovarian cancer or other cancerous tissues, including primary peritoneal tumors or Fallopian tube tumors (FFPE) tissue.

[0239] To produce highly specific, high-affinity diagnostic CLDN6 antibodies, it was essential to initiate immunization protocols using a variety of different immunogens (Table 1) and adjuvants. During this project, approximately 130 mice (C57BL / 6 and BALB / c) were subjected to various immunization strategies to induce an α-CLDN6 immune response (Table 2).

[0240] To activate the mouse immune system and overcome immune tolerance, recombinant proteins encoding different portions of human CLDN6 (polyhistidine (HIS) tagged and glutathione-S-transferase (GST) tagged) were used, expressed as recombinant fusion proteins with various epitopes that promote peptide complex and affinity purification (Table 1).

[0241] Of the 20 different immune strategies, the best results were achieved by treating mice with GST-tagged CLDN6 C-terminal recombinant protein combined with various adjuvants (Table 2) (Table 2).

[0242] On the day of fusion, mouse splenocytes were isolated and fused to the mouse myeloma cell line P3X63Ag8.653 (ATCC). The fusion of mouse cells to the myeloma cell line followed the standard protocol published by Kohler and Milstein in 1975. After hypoxanthine-aminopterin-thymidine (HAT) selection, the supernatant was tested by ELISA for the secretion of antibodies that recognize the antigen used for immunization.

[0243] Sixty-nine fusions were performed, and over 25,000 hybridoma cell clones were screened. 402 hybridoma cells from the ELISA-positive supernatant were subcloned to produce monoclonal hybridomas. The supernatant of the subcloned hybridoma cells was re-screened by ELISA for binding to the claudin 6 antigen. 88 positive clones (hybridoma cells that bound to the antigen but not to the skeleton or tag) were grown, and their specificity was further analyzed by Western blotting of the supernatant.

[0244] Three lead candidates (58-4B, 58-1B, and 58-3A) were derived from an 11-step immunization strategy (immunization No. 10 for 123 days). Three days prior to splenectomy, mice were boosted with immunization to activate target B cells, followed by fusion No. 58 (Table 3).

[0245] [Example 3] Western blot screening of monoclonal antibodies To test whether the ELISA positive antibodies in the supernatant could specifically bind to recombinant claudin 6, Western blot analysis was performed. Antibodies that bind to CLDN6 but not to other tagged proteins were purified, and cells were grown and cryopreserved. The antibodies were purified by FPLC using MABselect protein A affinity resin. The purified antibodies selected by Western blot screening were re-analyzed by Western blot to evaluate their binding to CLDN6 positive tumor cell lines (PA-1, NEC-8) and HEK293 cells transfected with CLDN3, 4, 6 or 9 (Figure 2). Furthermore, the antibodies were evaluated for their ability to bind to antigens in formalin-fixed paraffin-embedded tissues (FFPE) by immunohistochemistry. The supernatants of 33 hybridomas that specifically bind to claudin 6 were purified and further analyzed by immunohistochemistry.

[0246] [Example 4] Histological Analysis of Western Blot Positive Antibodies The purpose of this experiment was to test the CLDN6 specificity and sensitivity of the antibodies. This was done using FFPE ovarian cancer tissues expressing CLDN6 (Figure 3).

[0247] In the initial experiment, purified antibodies that tested positive in the first Western blot screening were analyzed at concentrations of 1 μg / ml and 5 μg / ml in ovarian cancer FFPE sections. A laboratory-standard, established overnight staining protocol, including citrate recovery buffer and a recovery temperature of 120°C, was used. Antibodies that showed tissue-specific CLDN6 staining without producing high levels of nonspecific background staining were further titrated to 0.2, 1, 2, and 5 μg / ml in various ovarian cancer tissues to further evaluate the sensitivity and specificity of these antibodies. In the next development stage, since the differences in staining intensity of different antibodies could not be clearly evaluated at a concentration of 0.2 μg / ml, newly prepared antibodies were directly tested at higher concentrations of 1 and 5 μg / ml. Antibodies that produced a strong signal on the cell membrane of ovarian tumor tissue in selected tissues and produced little to no background in adjacent healthy tissue were selected for further titration experiments and specificity analysis. The following three antibodies met these criteria and were selected as lead candidates for further investigation: 58-1B, 58-3A, and 58-4B.

[0248] [Example 5] Epitope mapping of mumAb (Figure 4) Peptide ELISA was performed to identify antibody-binding epitopes on CLDN6. Each purified antibody was tested with peptides that overlapped with 11 amino acids covering the C-terminal sequence of CLDN6. Antibodies 46-5A, 58-1B, 58-3A, 58-4B, and 58-9B showed specific binding to the epitope covered by peptide AISRGPSEYPTKNYV (peptide 7; Figure 4A). The binding sites of these antibodies were further characterized using biotinylated peptides in which 14 of the 15 amino acids overlapped.

[0249] Surprisingly, antibodies 58-1B, 58-3A, and 58-4B, as well as the subclone 58-4B-2 that binds to the sequence EYPTKNY (Figure 4B), can bind to CLDN6 in FFPE cancer tissue with high specificity at low background levels. In contrast, antibody 46-5A was able to bind to a peptide that does not contain the sequence EYPTKNY, but contains a portion of it, namely the sequence EYPTK (Figure 4B). A commercially available rabbit anti-CLDN6 polyclonal antiserum (IBL) binds to a peptide containing the sequence RGPS (Figure 4C).

[0250] [Example 6] Analysis of antibody specificity using a normal tissue panel Antibodies that produced a strong signal to the tested ovarian cancer tissue (58-1B, 58-3A, and 58-4B) were analyzed in various relevant normal, i.e., healthy donor tissues (e.g., lung, testis, uterus, and ovaries) to ensure high CLDN6 target specificity, using established laboratory staining protocols. Staining of these selected tissues was performed using established laboratory staining protocols (overnight; recovery temperature 120°C, citrate recovery buffer).

[0251] None of the three selected antibodies produced nonspecific staining signals in the normal tissue panel tested, except for a faint nonspecific signal in a single peripheral region of one ovarian tissue sample. This weak signal was only visible at the higher of the two concentrations tested (1 and 5 μg / ml) and did not indicate ovarian substructure.

[0252] [Example 7] Comparison of antibody sensitivity using ovarian cancer and normal tissue panel samples. Previous experiments did not show significant differences in staining patterns or intensity between antibodies 58-3A, 58-1B, and 58-4B. Therefore, the antibodies were subjected to staining experiments using a more clinically oriented, i.e., time-efficient, staining protocol. To simulate the staining procedures applied in a standard pathology laboratory, a one-day protocol was established, including a one-hour primary antibody incubation step and a water bath (98°C) recovery step. Furthermore, two different recovery buffers were compared to test whether different recovery conditions could affect the sensitivity of the test antibodies for CLDN6 detection.

[0253] For all antibodies, using a 1-day protocol adapted with Tris buffer for recovery, compared to standard citrate recovery buffer, resulted in stronger signals and more positively stained tumor cells. The strongest staining signal was produced with 58-4B, reaching up to 80% of 3+ stained tumor cells, while candidate 58-1B had the weakest staining intensity, reaching only 30% of 3+ stained tumor cells.

[0254] A staining protocol using Tris buffer for recovery, which yielded the most sensitive staining signal, was then applied to staining a normal tissue panel to test the specificity of the selected antibody under standard clinical laboratory conditions.

[0255] The best results regarding CLDN6 specificity and sensitivity in FFPE tissue were achieved by using the 58-4B diagnostic antibody candidate. No signal was detected in normal tissue. All other antibodies produced a signal in Reinke crystals of testicular samples. Furthermore, the 58-3A antibody produced weak staining of blood vessels. The 58-1B antibody showed a stronger signal (1+) in normal tissue of the testes and ovaries.

[0256] In all samples analyzed, mumAb 58-4B performed better in that it showed strong staining with no background staining in the relevant tumor tissue, while mumAb 58-3A and 58-1B candidates showed strong staining in the relevant tumor tissue but were accompanied by higher background signals. In particular, the signal generated using mumAb 58-1B was high intensity but had the disadvantage of high background staining.

[0257] For future use in a clinical setting, the corresponding hybridomas for the production of diagnostic antibody candidates were adapted to serum-free medium. Unfortunately, clone 58-3A could not be adapted to serum-free conditions. Therefore, antibody candidate 58-3A was also excluded from further histological testing, and 58-4B remained as the final lead candidate.

[0258] [Example 8] Histological detailed analysis and antibody characterization In the initial stage, to detect differences in target sensitivity and target specificity between lots, 58-4B antibody lots produced under serum-free conditions were compared with antibody lots produced under serum conditions using a normal tissue panel TMA (MNO961). Staining was performed using a clinic-standard 1-day protocol with royalty-free components (water bath recovery, Tris buffer for recovery).

[0259] Faint staining was detected in some normal tissue samples for both antibody lots (see Table 4). Compared to antibodies produced under serum conditions, the serum-free antibody lot showed fewer artificial spots and was cleaner in the analyte samples. In the testes, staining signals were observed in Reinke crystals with both antibody lots.

[0260] In the second stage, antibodies produced in serum-free samples were tested on a TMA panel for ovarian cancer and compared to commercially available IBL antibodies. These commercially available antibodies had already been used in previous test staining. Polyclonal IBL antibodies were used with the test protocol (overnight, recovery temperature 120°C, citrate recovery buffer). 58-4B antibodies were used with a standard clinic protocol (1-day protocol, water bath recovery, Tris buffer for recovery).

[0261] Of the 72 spotted TMA ovarian cancer cases, only one sample was positive for IBL antibody. In contrast, when stained with 58-4B antibody, 14 out of 72 samples were tested for CLDN6 positivity.

[0262] Unfortunately, the quality of commercially available ovarian cancer TMA samples was poor, and the staining signal in positive tumor samples was weak. Therefore, we repeated the comparison with tumor resection specimens from Dr. Dhaene, which show larger tumor areas and are of high quality, including lung, uterine, and testicular tumor samples.

[0263] Compared to staining using polyclonal IBL antibodies, 58-4B staining resulted in more sensitive and specific detection of CLDN6 in tumor samples, meaning that more tumor cells were positive and more strongly stained tumor cells were detected.

[0264] [Example 9] Sequence analysis of lead antibodies The sequences of antibodies 58-1B, 58-3A, and 58-4B are shown in Figure 5 and below.

[0265] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0266] [Table 7]

[0267] [Table 8]

[0268] Additional sheet on biological materials

[0269] Further identification of deposited items: 1) The name and address of the depositary institution for the deposited items are as follows: Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Inhoffenstr.7 B 38124 Braunschweig DE [Table 9]

[0270] Additional information regarding all of the above deposits: - Mouse (Mus musculus) splenocyte fused with mouse (Mus musculus) myeloma Ag8.653 - Hybridoma secreted antibodies against human CLDN6

[0271] 2) Depositor: All of the above deposits were made by: Ganymed Pharmaceuticals AG At Goldgrube 12 55131 Mainz DE

Claims

1. An antibody or its antigen-binding fragment, (i) The antibody or its antigen-binding fragment is bound to a peptide consisting of the amino acid sequence EYPTKNY (SEQ ID NO: 38), and / or (ii) The antibody or its antigen-binding fragment binds to claudin 6 (CLDN6), and the antibody or its antigen-binding fragment binds to CLDN6 by binding to an epitope consisting of the amino acid sequence EYPTKNY (SEQ ID NO: 38), and / or (iii) The antibody or its antigen-binding fragment is bound to a peptide consisting of the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and / or (iv) The antibody or its antigen-binding fragment binds to claudin 6 (CLDN6), and the antibody or its antigen-binding fragment binds to CLDN6 by binding to an epitope consisting of the amino acid sequence EYPTKNYV (SEQ ID NO: 29), and / or (v) The antibody or its antigen-binding fragment binds to a peptide consisting of the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), and the antibody or its antigen-binding fragment does not bind to a peptide consisting of the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14), and / or (vi) The antibody or its antigen-binding fragment binds to claudin 6 (CLDN6), and the antibody or its antigen-binding fragment binds to CLDN6 by binding to an epitope consisting of the amino acid sequence AISRGPSEYPTKNYV (SEQ ID NO: 15), and does not bind to the peptide consisting of the amino acid sequence TSAPAISRGPSEYPT (SEQ ID NO: 14). (vii) The antibody or its antigen-binding fragment contains the following peptide: PAISRGPSEYPTKNY (SEQ ID NO: 22), AISRGPPSEYPTKNYV (SEQ ID NO: 15), ISRGPPSEYPTKNYV (SEQ ID NO: 23), SRGGPPSEYPTKNYV (SEQ ID NO: 24), RGPPSEYPTKNYV (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) bind to one or more of these, and the antibody or its antigen-binding fragment does not bind to the peptide consisting of the amino acid sequence TSAPAISRGPPSEYPT (SEQ ID NO: 14). The antibody or its antigen-binding fragment is as follows: (a) an antibody heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 40, and an antibody light chain variable region containing the amino acid sequence described in SEQ ID NO: 41; (b) A variable region of the antibody heavy chain containing the amino acid sequence described in SEQ ID NO: 42, and a variable region of the antibody light chain containing the amino acid sequence described in SEQ ID NO: 43; (c) an antibody heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 44, and an antibody light chain variable region containing the amino acid sequence described in SEQ ID NO: 45; or (d) A variable antibody heavy chain region containing the amino acid sequence described in SEQ ID NO: 42, and a variable antibody light chain region containing the amino acid sequence described in SEQ ID NO: 45, including, An antibody or its antigen-binding fragment.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, IgA1, IgA2, IgD, or IgE.

3. An antibody or antigen-binding fragment according to claim 1 or 2, conjugated to a detectable label.

4. The label is an indicator enzyme, a radioactive label, a fluorescent phosphopone, or a paramagnetic particle, or the label comprises alkaline phosphatase, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate, fluorine 18, carbon 11, iodine 123, technetium 99m, iodine 131, indium 111, fluorine, or gadolinium, the antibody or antigen-binding fragment according to claim 3.

5. A method for detecting CLDN6 in a sample or determining the amount of CLDN6, (i) A step of contacting a sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and (ii) A step of detecting the formation of a complex between the antibody or its antigen-binding fragment and CLDN6, or determining the amount of the complex. A method that includes this.

6. A method for detecting cells expressing CLDN6, (i) A step of contacting a cell sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, and (ii) A step of detecting the formation of a complex between the antibody or its antigen-binding fragment and CLDN6 expressed by the cells in the sample. A method that includes this.

7. A method for detecting or monitoring cancer cells expressing CLDN6, (i) A step of contacting a biological sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, (ii) A step of detecting the formation of a complex between the antibody or its antigen-binding fragment and CLDN6, and / or determining the amount of the complex. A method that includes this.

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