RU2816850C2 - Antibodies for treating cancer, in which claudin 6 is expressed - Google Patents

Abstract

FIELD: biotechnology; chemistry.

SUBSTANCE: invention relates to biochemistry, in particular to a method of producing an antibody or an antigen-binding fragment thereof, which binds to CLDN6. Also disclosed is a recombinant nucleic acid encoding the produced antibody; vector containing said nucleic acid; recombinant cell containing said nucleic acid; polypeptide encoded by said nucleic acid. Disclosed is a method of producing said recombinant eukaryotic host cell.

EFFECT: invention enables to efficiently obtain an antibody or its antigen-binding fragment, which binds to CLDN6.

32 cl, 10 ex, 1 tbl, 37 dwg

Description

In the last decade, antibodies have been successfully introduced into clinical practice for use in the treatment of cancer; they appear to be the most promising therapeutic agent in oncology. Compared with conventional cancer drugs, antibody-based therapeutics have potentially higher specificity and fewer side effects because antibodies accurately distinguish between normal and neoplastic cells and rely on less toxic immunological antitumor mechanisms such as complement activation. and mobilization of cytotoxic cells of the immune system.

Claudins are integral membrane proteins located at tight junctions between the epithelium and endothelium. It is believed that the claudin molecule has four transmembrane segments with two extracellular “loops”, and the N- and C-termini are located in the cytoplasm. Transmembrane proteins of the claudin family (CLDN) play a key role in maintaining tight junctions between the epithelium and endothelium, and are also possibly involved in cytoskeletal maintenance and cellular signaling mechanisms. The expression of these proteins differs significantly between normal and tumor cells, which, along with their membrane localization, makes them an attractive target for cancer immunotherapy; The use of antibody-based therapeutics to target claudins in cancer treatment promises high specificity of therapeutic effects.

However, the clinical application of claudin-targeted therapeutics faces several obstacles. Due to the ubiquitous presence of claudins in the body and their important role in maintaining tight junctions, high specificity of claudin-targeted therapeutic interventions is required to maximize treatment specificity and minimize systemic toxicity.

Publication WO 2009/087978 relates to antibodies directed against caludin-6 and their use as anti-cancer agents. In particular, monoclonal antibodies designated AB3-1, AE1-16, AE49-11 and AE3-20 are described. However, as shown by fluorescence-activated cell sorting (FACS) analysis described in Example 5, none of these antibodies were specific for claudin-6. Antibodies AE3-20 interacted with CLDN9, whereas antibodies AE1-16 and AE49-11 showed significant reactivity against CLDN9 and also interacted with CLDN4. The strength of AB3-1 antibody binding to CLDN6 was the same as to CLDN9. Example 7 describes that administration of AE49-11 antibodies to mice with model tumors suppressed tumor growth and prolonged life. But given the nonspecificity of the antibodies used in this example, it remains unclear whether the observed effect was actually due to the binding of these antibodies to CLDN6.

Thus, CLDN6-specific antibodies have not yet been described that selectively bind to the surface of cells in which CLDN6 is expressed. And antibody-based therapeutics targeting CLDN6 require specific antibodies.

The amino acid sequence comparison of CLDN3, CLDN4, CLDN6 and CLDN9 shown in Figure 1 demonstrates the high conservation of CLDN6 relative to other claudins. The high degree of homology of CLDN6 with other claudins, especially CLDN9 and CLDN4, and the fact that WO 2009/087978 failed to obtain antibodies specific for CLDN6 suggest that it is not possible to obtain antibodies that specifically bind to CLDN6.

Disclosure of the Invention

The experimental results described herein confirm that in humans, CLDN6 is expressed in a variety of cancer cells, whereas in normal tissues its expression is limited to the placenta.

This invention describes for the first time the successful production of antibodies that are specific for CLDN6 and are capable of binding to the surface of intact cells in which CLDN6 is expressed. Fluorescence-activated cell sorting (FACS) studies of intact cells expressing CLDN6 demonstrated specific binding of anti-CLDN6 antibodies, whereas in cells expressing other claudin proteins, particularly CLDN3, CLDN4 and CLDN9, or cells in which neither of these proteins are formed, no binding was observed at all. Thus, the present invention unexpectedly showed that it is possible to obtain antibodies that specifically interact with CLDN6 on the surface of cells in which CLDN6 is expressed, but do not exhibit significant reactivity against other highly homologous claudins as antigens.

In general, the present invention provides antibodies useful as therapeutic agents for the treatment and/or prevention of diseases in which cells expressing CLDN6 play a role, in which CLDN6 is associated with their cell surface, including diseases accompanied by the formation of tumors, in particular cancer, such as ovarian cancer, including ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), especially squamous cell lung carcinoma and adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, including malignant polymorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular small intestine adenocarcinoma and ileal adenocarcinoma, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, particularly testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, such as teratocarcinoma or embryonal carcinoma, particularly testicular germ cell tumor, and their metastasizing forms.

One embodiment of the present invention provides antibodies capable of binding to CLDN6 associated with the surface of cells in which CLDN6 is expressed. Preferably, these antibodies are unable to substantially bind to CLDN9 associated with the surface of cells in which CLDN9 is expressed. Preferably, these antibodies are unable to bind substantially to CLDN4 associated with the surface of cells in which CLDN4 is expressed and/or are unable to bind substantially to CLDN3 associated with the surface of cells in which CLDN3 is expressed. Most preferably, these antibodies are not capable of binding to any claudin other than CLDN6 associated with the surface of cells in which the specified claudin is expressed, and are specific for CLDN6. Preferably, said cells in which said claudin is expressed are intact, in particular with an unpermeabilized cell membrane, and said claudin associated with the cell surface has a native conformation, i.e. not denatured. Preferably, these antibodies are capable of binding to one or more CLDN6 epitopes in their native conformation.

In one embodiment of the present invention, the antibodies of the invention are capable of binding to an epitope located within the extracellular portion of CLDN6, wherein said portion of CLDN6 preferably comprises any of the following amino acid sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO : 14 and SEQ ID NO: 15, preferably the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, more preferably the amino acid sequence of SEQ ID NO: 6. Preferably, the antibodies of the invention are capable of binding to an epitope located within one of the following amino acids sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15, preferably the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

In one embodiment of the present invention, the antibodies of the invention are capable of binding to CLDN6 by interacting with at least one, preferably more than one, for example two, three, four or five, preferably all amino acid residues selected from the group consisting of Thr33, Phe35, Gly37, Ser39, Ile40 and Leu151, preferably by interaction with at least one, preferably more than one, preferably all amino acid residues selected from the group consisting of Thr33, Phe35, Gly37, Ser39 and Ile40, more preferably by interaction with at least one, preferably more than one, preferably all amino acid residues selected from the group consisting of Phe35, Gly37, Ser39 and Ile40, or consisting of Thr33, Phe35, Gly37 and Ser39, and in particular by interaction with at least one, preferably more than one, preferably all, amino acid residues selected from the group consisting of Phe35, Gly37, and Ser39. Preferably, the antibodies of the invention do not react with one or more, preferably all, amino acid residues selected from the group consisting of Glu154, Ala155, Arg158 and Gly161, and preferably do not interact with one or more, preferably all amino acid residues selected from the group consisting of Arg158 and Gly161.

The interaction between antibodies and CLDN6, particularly in its native conformation, can be studied by alanine scanning-based mutagenesis. Mutant CLDN6 can be characterized by their ability to bind to specific monoclonal antibodies. The impaired binding of a specific monoclonal antibody to mutant CLDN6 suggests that the amino acid residue at which the mutation has occurred is important for contact with the antibody. You can define binding, for example. by flow cytometry.

In one embodiment of the present invention, the antibodies of the invention are produced by a method comprising the step of immunizing an animal with a peptide having an amino acid sequence being any of the following: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15. preferably the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or an immunologically equivalent peptide or nucleotide sequence or host cell in which said peptide is expressed.

In other embodiments of the present invention, the CLDN6 to which the inventive antibody is capable of binding has the amino acid sequence SEQ ID NO: 2 or the amino acid sequence SEQ ID NO: 8. It is particularly preferred that the inventive antibody is capable of binding to CLDN6 having the amino acid sequence SEQ ID NO: 2, and with CLDN6 having the amino acid sequence of SEQ ID NO: 8.

In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising at least one, preferably two, more preferably all three CDR (complementarity determining region) sequences from the antibody heavy chain sequence selected from SEQ ID NO: 34, 36, 38 and 40 or their variants. In Figure 25, the CDR sequences in the above-mentioned antibody heavy chain sequences are highlighted with a square frame.

In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising the sequence CDR3 Xaa1 Gly Xaa2 Val Xaa3, where Xaa1 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, Xaa2 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, and Xaa3 is any amino acid, preferably Leu or Phe, more preferably Leu. In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising the sequence CDR3 Asp Xaa1 Gly Xaa2 Val Xaa3 or Xaa1 Gly Xaa2 Val Xaa3 Asp, where Xaa1, Xaa2 and Xaa3 represent the amino acids specified above. In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising the sequence CDR3 Asp Xaa1 Gly Xaa2 Val Xaa3 Asp, where Xaa1, Xaa2 and Xaa3 represent the amino acids specified above. In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising the sequence CDR3 Ala Arg Asp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, where Xaa1, Xaa2 and Xaa3 represent the amino acids specified above. In one embodiment of the present invention, the proposed antibody according to the above embodiments contains a CDR1 sequence corresponding to SEQ ID NO: 47 or a variant thereof, and/or a CDR2 sequence corresponding to SEQ ID NO: 48 or a variant thereof.

In one embodiment of the present invention, the antibody of the invention comprises a heavy chain comprising a sequence from an antibody heavy chain selected from SEQ ID NOs: 34, 36, 38 and 40 or variants thereof.

In one embodiment of the present invention, the antibody of the invention comprises a light chain comprising at least one, preferably two, more preferably all three CDR sequences from the antibody light chain sequence selected from SEQ ID NOs: 35, 37, 39 and 41 or variants thereof. In Figure 26, the CDR sequences in the above-mentioned antibody light chain sequences are highlighted with a square box.

In one embodiment of the present invention, the antibody of the invention comprises a light chain comprising the sequence CDR3 Arg Xaa1 Xaa2 Xaa3 Pro, where Xaa1 is any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2 is any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferably Tyr, Ser or Asn, most preferably Asn, and Xaa3 is any amino acid, preferably Ser or Tyr, more preferably Tyr. In one embodiment of the present invention, the antibody of the present invention contains a light chain comprising the sequence CDR3 Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro, where Xaa1, Xaa2 and Xaa3 represent the amino acids specified above. In one embodiment of the present invention, the antibody of the present invention contains a light chain comprising the sequence CDR3 Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, where Xaa1, Xaa2 and Xaa3 represent the amino acids specified above. In one embodiment of the present invention, the antibody according to the above embodiments comprises a light chain comprising a CDR1 sequence corresponding to SEQ ID NO: 52 or a variant thereof, and/or a CDR2 sequence corresponding to SEQ ID NO: 53 or a variant thereof.

In one embodiment of the present invention, the antibody of the invention comprises a light chain comprising an antibody light chain sequence selected from SEQ ID NOs: 35, 37, 39, 41, 54 and 55 or variants thereof.

In various embodiments of the present invention, the antibody of the invention comprises a heavy chain as defined above and a light chain as defined above.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising at least one, preferably two, more preferably all three CDR sequences from the antibody heavy chain sequence SEQ ID NO: x or a variant thereof, and

(ii) a light chain comprising at least one, preferably two, more preferably all three light chain CDR sequences of the antibody SEQ ID NO: x+1 or a variant thereof;

where x is chosen from 34, 36, 38 and 40.

In Figures 25 and 26, respectively, the indicated CDR sequences in the above-mentioned antibody heavy and light chain sequences are highlighted in square frames.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising a CDR3 sequence selected from the group consisting of Xaa1 Gly Xaa2 Val Xaa3, Asp Xaa1 Gly Xaa2 Val Xaa3, Xaa1 Gly Xaa2 Val Xaa3 Asp, Asp Xaa1 Gly Xaa2 Val Xaa3 Asp and Ala Arg Asp Xaa1 Gly Xaa2 Val Xaa3 Asp Tyr, where Xaa1 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, Xaa2 is any amino acid, preferably an aromatic amino acid, more preferably Phe or Tyr, most preferably Tyr, and Xaa3 is any amino acid, preferably Leu or Phe, more preferably Leu.

(ii) an antibody light chain comprising a CDR3 sequence selected from the group. consisting of Arg Xaa1 Xaa2 Xaa3 Pro, Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro, Gln Gln Arg Xaa1 Xaa2 Xaa3 Pro Pro Trp Thr, where Xaa1 represents any amino acid, preferably Ser or Asn, most preferably Ser, Xaa2 represents any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more preferably Tyr, Ser or Asn, most preferably Asn, and Xaa3 is any amino acid, preferably Ser or Tyr, more preferably Tyr.

In one embodiment of the present invention, the proposed antibody according to one of the above embodiments contains: (i) an antibody heavy chain comprising a CDR1 sequence corresponding to SEQ ID NO: 47 or a variant thereof and/or a CDR2 sequence corresponding to SEQ ID NO: 48 or its variant and/or (ii) an antibody light chain comprising a CDR1 sequence corresponding to SEQ ID NO: 52, or a variant thereof and/or a CDR2 sequence corresponding to SEQ ID NO: 53, or a variant thereof.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising a heavy chain sequence selected from SEQ ID NOs 34, 36, 38 and 40 or variants thereof, preferably SEQ ID NO: 36 or a variant thereof, and

(ii) a light chain comprising an antibody light chain sequence selected from SEQ ID NOs 35, 37, 39, 41, 54 and 55 or variants thereof, preferably SEQ ID NO: 35 or a variant thereof.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising the heavy chain sequence of SEQ ID NO: 36 or a variant thereof, and

(ii) a light chain comprising an antibody light chain sequence selected from SEQ ID NOs 35, 54 and 55 or variants thereof.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising the heavy chain sequence of SEQ ID NO: 36 or a variant thereof, and

(ii) a light chain comprising an antibody light chain sequence selected from SEQ ID NOs 54 and 55 or variants thereof.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising the heavy chain sequence of SEQ ID NO: 36 or a variant thereof, and

(ii) a light chain comprising the antibody light chain sequence of SEQ ID NO 35 or a variant thereof.

In one embodiment of the present invention, the proposed antibody contains:

(i) a heavy chain comprising the heavy chain sequence of SEQ ID NO: x or a variant thereof, and

(ii) a light chain comprising the antibody light chain sequence SEQ ID NO x+1 or a variant thereof,

where x is chosen from 34, 36, 38 and 40.

In preferred embodiments of the present invention, the antibody of the invention comprises a heavy chain comprising a gamma-1 heavy chain constant region, preferably a gamma-1 heavy chain constant region of a human antibody, for example a sequence such as SEQ ID NO: 25, and/or contains a light chain comprising a kappa light chain constant region of an antibody, preferably a human kappa light chain constant region, such as a sequence such as SEQ ID NO: 27.

In preferred embodiments of the present invention, the provided antibodies have one or more of the following activities: (i) kill cells in which CLDN6 is expressed, (ii) inhibit proliferation of cells in which CLDN6 is expressed, (iii) inhibit colony formation of cells in which CLDN6 is expressed , (iv) mediate remission, i.e. reduction in size, preferably complete remission, i.e. complete disappearance of established tumors, (v) prevent the formation or restoration of pre-existing tumors, and (vi) inhibit metastasis of cells in which CLDN6 is expressed. Accordingly, the antibodies of this invention can be used to achieve one or more of the above phenomena, particularly when administered to a patient. Killing cells and/or inhibiting one or more of their activities can be used for therapeutic purposes, as described herein. In particular, killing cells, inhibiting cell proliferation, and/or inhibiting the formation of cell colonies can be used to treat or prevent cancer, including the formation of metastases. Inhibition of cell proliferation, colony formation and/or metastasis can be used, in particular, to treat or prevent the formation of cancer metastases and the metastatic spread of cancer cells. Preferably, the antibodies of the present invention mediate cell killing by causing complement-dependent cytotoxicity (CDC)-mediated lysis, antibody-dependent cellular cytotoxicity (ADCC)-mediated lysis, apoptosis, homotypic adhesion and/or phagocytosis, preferably causing complement-dependent cytotoxicity-mediated lysis ( CDC), and/or lysis mediated by antibody-dependent cellular cytotoxicity (ADCC). The present invention also includes embodiments in which the antibodies of the invention exhibit their activities described herein, such as killing cells and/or inhibiting one or more cellular activities, such as cell proliferation and/or colony formation, without causing complement-dependent cytotoxicity (CDC)-mediated lysis. , lysis, antibody-dependent cellular cytotoxicity (ADCC), apoptosis, homotypic adhesion and/or phagocytosis. Thus, the antibodies of this invention can exert their effect. simply by binding to CLDN6 on the cell surface and thus blocking cell proliferation, for example. In one embodiment of the present invention, the antibodies of the invention do not cause cell lysis mediated by complement-dependent toxicity.

Preferably, lysis mediated by antibody-dependent cell cytotoxicity occurs in the presence of effector cells, which in particular embodiments of the present invention are selected from the group consisting of monocytes, mononuclear cells, NK cells (natural killer cells) and polymorphonuclear leukocytes (PMN), and phagocytosis is carried out by macrophages.

The activity of inhibiting or reducing the proliferation of cells in which CLDN6 is expressed, preferably cancer cells, can be measured in vitro by determining the proliferation of cancer cells in which CLDN6 is expressed using a bromodeoxyuridine (5-bromo-2-deoxyuridine) assay. BrdU). Bromodeoxyuridine is a synthetic nucleotide that is an analogue of thymidine, which can be incorporated into newly synthesized DNA of a replicating cell (during the S phase of the cell cycle), replacing thymidine during DNA replication. By determining the amount of incorporated bromodecosiuridine, for example, using antibodies specific to it, it is possible to identify cells in which active DNA replication occurs.

The activity of inhibiting or reducing colony formation by cells expressing CLDN6, preferably cancer cells, can be measured in vitro using a clonogenic assay. The clonogenic test is a microbiological method for studying the effectiveness of various agents on cell viability and proliferation. It is often used in cancer research to determine the effects of drugs and radiation on proliferating tumor cells. Such an experiment involves three main steps: (i) exposing a sample of cells, particularly cancer cells, to be studied, (ii) seeding the cells in a tissue culture vessel, and (iii) growing the cells. The resulting colonies are fixed, stained and counted. Colony formation is important because it is associated with the ability of individual tumor cells to colonize organs, forming metastases. The colony formation inhibitory activity of antibodies reflects their potential effectiveness in inhibiting metastasis. Antibodies having such activity, ie, the ability to inhibit or reduce colony formation in a clonogenic test, are particularly useful for treating or preventing metastasis and metastatic spread of cancer cells, particularly those types discussed herein.

In preferred embodiments of the present invention, the antibodies exhibit one or more immunoeffector functions against cells bearing CLDN6 in its native conformation, wherein the one or more effector functions are preferably selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) ), induction of apoptosis and inhibition of proliferation; the preferred effector functions are antibody-dependent cell-mediated cytotoxicity and/or complement-dependent cytotoxicity.

Preferably, said one or more activities or one or more immunoeffector functions exhibited by said antibodies are induced by the binding of said antibodies to CLDN6, preferably to an epitope located within an extracellular portion of CLDN6, wherein said extracellular portion of CLDN6 preferably comprises any amino acid sequence of the following: SEQ ID NO : 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15, the amino acid sequence of SEQ ID NO: 6 is preferred or SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 6 is more preferred.

According to the present invention, cells in which CLDN6 is expressed preferably have CLDN6 associated with their surface. Cells in which CLDN6 is expressed or cells bearing CLDN6 in its native conformation on their surface are preferably tumor cells, for example cancer cells, preferably cancer cells in cancers selected from the group consisting of ovarian cancer, including ovarian adenocarcinoma and teratocarcinoma of the ovary, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), especially squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, including malignant polymorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, such as teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumor, and metastatic forms thereof.

One or more therapeutic effectors, such as radiolabels, cytotoxins, therapeutic enzymes, apoptotic agents, and the like, can be attached to the antibodies of the present invention to provide targeted cytotoxicity, i.e. destruction of tumor cells.

In one embodiment of the present invention, the antibodies of the invention (i) bind to cells that express CLDN6 and that have CLDN6 associated with the cell surface and (ii) do not bind to cells that do not express CLDN6 and that do not have CLDN6, associated with the cell surface. Antibodies of the present invention preferably (i) mediate the killing and/or inhibit the proliferation of cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with the cell surface, and (ii) do not mediate the killing and/or inhibit the proliferation of cells in which express CLDN6 and which do not have cell surface associated CLDN6.

In a particularly preferred embodiment of the present invention, the antibodies of the invention bind to native CLDN6 epitopes found on the surface of living cells, such as SEQ ID NO: 6 or 7. In other preferred embodiments of the invention, the antibodies of the invention are specific for cancer cells that express CLDN6 and do not bind to cancer cells that do not express CLDN6.

Antibodies of this invention can be obtained from various types of living organisms, including but not limited to mouse, rat, rabbit, guinea pig and human. Antibodies of this invention also include hybrid molecules in which a constant region derived from one species of living organism, preferably human, is combined with an antigen binding site derived from another species. Also, the antibodies of this invention include humanized molecules in which non-human antigen-binding sites are combined with constant and framework regions of human origin.

Antibodies of this invention include polyclonal and monoclonal antibodies and IgG2a (eg, IgG2a, κ, λ), IgG2b (eg, IgG2b, κ, λ), IgG3 (eg, IgG3, κ, λ) and IgM antibodies. But other antibody isotypes are also included in this invention, including IgG1, IgA1, IgA2, secretory IgA, IgD, IgE. Antibodies of the invention may be full length antibodies or antigen binding fragments thereof, including, for example, Fab, F(ab') ₂ , Fv, single chain Fv fragments, or bispecific antibodies. In addition, antigen binding fragments include a hybrid immunoglobulin binding domain comprising (i) a polypeptide that forms a binding domain (eg, a heavy chain variable region or a light chain variable region) that is linked to a polypeptide that forms the hinge region of the immunoglobulin molecule, (ii) a constant an immunoglobulin heavy chain CH2 region connected to the hinge region; and (iii) an immunoglobulin heavy chain constant region CH3 connected to a CH2 constant region. Such fusion proteins are also described in US patent applications No. 2003/0118592 and 2003/0133939.

The antibodies of this invention are preferably monoclonal, hybrid, human or humanized antibodies or antibody fragments. Antibodies of this invention include fully human antibodies. Such antibodies can be produced by non-human transgenic animals, for example transgenic mice capable of producing multiple isotypes of human monoclonal antibodies against CLDN6, by V-D-J recombination and isotype switching. Such a transgenic animal for the production of polyclonal antibodies, such as those described in US Patent Application No. 2003/0017534, may also be a rabbit.

Antibodies of this invention are preferably released from the complex with CLDN6 with a dissociation constant (KD) of approximately 1-100 nM or less. Preferably, the antibodies of this invention do not cross-react with related cell surface antigens and thereby do not inhibit their function.

In preferred embodiments of the present invention, the provided antibodies have one or more of the following properties:

a) specificity for CLDN6;

b) binding affinity for CLDN6 is about 100 nM or less. preferably about 5-10 nM or less, and more preferably about 1-3 nM or less;

c) the ability to cause complement-dependent cytotoxicity to cells. in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface;

d) the ability to inhibit the proliferation of cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface;

e) the ability to induce apoptosis of cells in which CLDN6 is expressed and which differ in that CLDN6 is associated with their surface;

f) the ability to induce homotypic adhesion of cells in which CLDN6 is expressed and which differ in that CLDN6 is associated with their surface;

g) the ability to cause antibody-dependent cell-mediated cytotoxicity against cells in which CLDN6 is expressed and which have CLDN6 associated with their surface, in the presence of effector cells;

h) the ability to prolong the life of a patient having tumor cells that express CLDN6 and which are characterized in that CLDN6 is associated with their surface;

i) the ability to reduce the number of cells that express CLDN6 and that have CLDN6 associated with their surface;

j) the ability to aggregate CLDN6 on the surface of living cells.

Preferred antibodies described herein are those produced by (or derived from) hybridoma cells deposited in the Collection of Microorganisms and Cell Cultures of Germany at the German Institute. Leibniz (DSMZ) (Inhoffenstrasse 7B, 38124 Braunschweig, Germany, Germany) and having the following designations and registration numbers:

1. GT512muMAB 59A, registration number DSM ACC3067, deposited 06/21/2010;

2. GT512muMAB 60A, registration number DSM ACC3068, deposited 06/21/2010;

3. GT512muMAB 61D, registration number DSM ACC3069, deposited 06/21/2010;

4. GT512muMAB 64A, registration number DSM ACC3070, deposited 06/21/2010;

5. GT512muMAB 65A, registration number DSM ACC3071, deposited 06/21/2010;

6. GT512muMAB 66B, registration number DSM ACC3072, deposited 06/21/2010;

7. GT512muMAB 67A, DSM registration number ACC3073. deposited 06/21/2010;

8. GT512muMAB 55A, registration number DSM ACC3089, deposited 07/31/2010; or

9. GT512muMAB 89A, registration number DSM ACC3090, deposited 07/31/2010.

The designations of antibodies of this invention herein reflect the designation of the antibody itself and/or the clone that produces it, for example muMAB 59A.

Also preferred are antibodies having the specificity of antibodies produced by or derived from the hybridomas described above, in particular those containing an antigen binding region or antigen binding site, in particular a variable region identical to or having a high degree of homology with them. It is meant that the preferred antibodies of this invention have a CDR region identical to the corresponding region of the antibodies produced by or derived from the hybridomas described above. By high homology is meant that each CDR region may have from 1 to 5, preferably from 1 to 4, for example from 1 to 3 or from 1 to 2 substitutions. Particularly preferred are antibodies that are hybrid and humanized forms of antibodies produced by or derived from the hybridomas described above.

Thus, the antibodies of the present invention may be selected from the group consisting of i) antibodies produced by clones deposited under registration number DSM ACC3067 (GT512muMAB 59A), DSM ACC3068 (GT512muMAB 60A), DSM ACC3069 (GT512muMAB 61D), DSM ACC3070 ( GT512MUMAB 64A), DSM ACC3071 (GT512MUMAB 65A), DSM ACC3072 (GT512MUMAB 66B), DSM ACC3073 (GT512MUMAB 67A), DSM ACC3089 (GT512MUMAB 55A) or DSM ACC3090 (GT512 mumab 89a), or received from them; (ii) antibodies that are hybrid or humanized forms of antibodies according to paragraph (i); (iii) antibodies having the antibody specificity of (i); (iv) antibodies containing an antigen-binding site or an antigen-binding site as in the antibodies of (i). The antigen binding site or antigen binding site, as in the antibodies of (i), may include a variable region of the antibodies of (i).

The present invention also relates to cells, such as hybridoma cells, that produce the antibodies described herein.

Preferred hybridoma cells are available in the DSMZ collection (Inhoffeenstr. 7B, 38124 Braunschweig, Germany) with any of the following designations and registration numbers:

1. GT512muMAB 59A, registration number DSM ACC3067, deposited 06/21/2010;

2. GT512muMAB 60A, registration number DSM ACC3068, deposited 06/21/2010;

3. GT512muMAB 61D, registration number DSM ACC3069, deposited 06/21/2010;

4. GT512muMAB 64A, registration number DSM ACC3070, deposited 06/21/2010;

5. GT512muMAB 65A, registration number DSM ACC3071, deposited 06/21/2010;

6. GT512muMAB 66B, registration number DSM ACC3072, deposited 06/21/2010;

7. GT512muMAB 67A, DSM registration number ACC3073. deposited 06/21/2010;

8. GT512muMAB 55A, registration number DSM ACC3089, deposited 08/31/2010; or

9. GT512muMAB 89A, registration number DSM ACC3090, deposited 08/31/2010.

Antibodies against CLDN6 can be modified, combined or co-expressed with carriers of other specificities. In one specific embodiment of the present invention, bispecific or polyspecific antibodies are provided having at least one first specificity for CLDN6 (e.g., an anti-CLDN6 antibody or mimetic thereof) and a second specificity for an effector cell, such as an Fc receptor binding specificity (e.g. Fc-γ, including Fc-γRI, or any other Fc receptor) or with a receptor on T cells, such as CD3.

Accordingly, the present invention includes bispecific and polyspecific molecules that bind to both CLDN6 and the Fc receptor or a receptor on T cells, such as CD3. Examples of Fc receptors are IgG receptor, Fcγ receptor, such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Other Fc receptors, such as IgA receptors (eg, FcαRI) may also serve as targets. The Fc receptor is preferably located on the surface of effector cells, such as monocytes, macrophages or activated mononuclear cells. In one preferred embodiment of the present invention, the bispecific and polyspecific molecules bind to the Fc receptor at a site different from the receptor's binding site for immunoglobulins (eg, IgG or IgA), such that binding of the bispecific and polyspecific molecules is not blocked at physiological levels of immunoglobulins.

In yet another embodiment of the present invention, the anti-CLDN6 antibodies of the invention are modified, coupled to, or co-expressed with other functional molecules, such as other peptides or proteins (eg, Fab' fragments). Thus, the antibodies of this invention can be functionally linked (for example, by chemical addition, fusion of genetic material, non-covalent association, etc.) with one or more other molecular entities, for example with other antibodies (for example, to obtain bispecific or polyspecific antibodies), cytotoxic agents, cellular ligands or antigens (eg, to obtain an immunoconjugate, such as an immunotoxin). Antibodies of this invention can be combined with other therapeutic agents, for example radioactive isotopes, small molecule anticancer drugs, recombinant cytokines or chemokines. Thus, the present invention covers a variety of antibody conjugates, bispecific and polyspecific molecules and fusion proteins, all of which are capable of binding to cells in which CLDN6 is expressed and/or to cells characterized in that CLDN6 is associated with their surface; all of these agents can be used to target these cells.

In general, in the context of this invention, all derivatives of antibodies, such as their conjugates, bispecific and polyspecific molecules and fusion proteins, are herein included in the term "antibodies".

In another embodiment, the present invention provides CLDN6 binding proteins that are not derived from immunoglobulin domains, including single chain proteins. Such binding proteins and methods for their preparation are described, for example, in Binz et al. (2005) Nature Biotechnology 23 (10): 1257-1268, incorporated herein by reference. It should be noted that everything said herein regarding immunoglobulins or binding molecules derived therefrom applies correspondingly to binding molecules derived from non-immunoglobulin domains. In particular, using such binding molecules derived from non-immunoglobulin domains, it is possible to block CLDN6 in cells in which the specified target is expressed and which are characterized in that the specified target is associated with their surface; in this way, the therapeutic effect described herein with respect to the antibodies of this invention can be achieved, in particular the inhibition of one or more of the many tumor cell activities described herein, such as proliferation, can be achieved. It is also possible (though not necessary) to impart antibody effector functions to such non-immunoglobulin binding molecules by, for example, coupling them to the Fc region of antibodies.

In general, this invention covers the treatment and/or diagnosis of diseases, in particular cancer, by targeting CLDN6, which is expressed in cells and associated with their surface. This approach allows for the selective targeting and/or elimination of such cells and thereby minimizing the negative impact on normal cells that do not express CLDN6 and that do not have CLDN6 associated with their surface. Preferred diseases for treatment or diagnosis are those involving cells that express CLDN6 and that are characterized by CLDN binding, such as cancers, in particular cancers of the type described herein.

In one embodiment, the present invention provides compositions, for example pharmaceutical and diagnostic compositions/kits, comprising antibodies or combinations of antibodies according to this invention. The pharmaceutical composition of this invention may contain a pharmaceutically acceptable carrier and, if necessary, one or more auxiliary components, stabilizing agents, etc. In one specific embodiment of this invention, such a composition includes a combination of antibodies that bind to distinct epitopes or that have different functional properties, for example, capable of causing complement-dependent and/or antibody-dependent cell-mediated cytotoxicity and the ability to induce apoptosis. In this embodiment of the present invention, the antibodies can be used in combination, for example in the form of a pharmaceutical composition containing two or more anti-CLDN6 monoclonal antibodies. For example, anti-CLDN6 monoclonal antibodies that have different but complementary activities can be combined in a single treatment to achieve the desired therapeutic effect. In one preferred embodiment of the present invention, the composition includes anti-CLDN6 antibodies that mediate complement-dependent cytotoxicity together with other anti-CLDN6 antibodies. which mediate apoptosis. In another embodiment of the present invention, the composition includes anti-CLDN6 antibodies that mediate highly effective killing of target cells in the presence of effector cells, together with other anti-CLDN6 antibodies. which inhibit the proliferation of cells expressing CLDN6, characterized in that CLDN6 is associated with their surface.

The present invention also includes the simultaneous or sequential administration of two or more anti-CLDN6 antibodies, at least one of which is a hybrid anti-CLDN6 antibody and at least one other antibody is a human anti-CLDN6 antibody, which antibodies bind to the same or different epitopes of CLDN6. Preferably, the patient is first administered the anti-CLDN6 hybrid antibodies and then the human anti-CLDN6 antibodies, with the human anti-CLDN6 antibodies preferably administered over a period of time, eg as maintenance therapy.

Antibodies, conjugates, bispecific/multispecific molecules and compositions of this invention can be used in various methods to inhibit the proliferation of cells in which CLDN6 is expressed and which differ in that CLDN6 is associated with their surface, and/or to selectively kill cells in which it is expressed CLDN6 and which are characterized in that CLDN6 is associated with their surface by exposing these cells to an effective amount of said antibodies, conjugates, bispecific/multispecific molecules and compositions such that the proliferation of said cells is inhibited and/or they die. In one embodiment of the present invention, such a method includes killing cells that express CLDN6 and that have CLDN6 bound to their surface, optionally in the presence of effector cells, for example, by complement-dependent cytotoxicity, apoptosis, antibody-dependent cytotoxicity , phagocytosis, or through the combined action of these mechanisms. Cells that express CLDN6, which are distinguished by the fact that CLDN6 is associated with their surface and which can be inhibited or killed by the antibodies of this invention, include cancer cells.

The antibodies, conjugates, bispecific/multispecific molecules and compositions of this invention can be used to treat and/or prevent various diseases that involve cells that express CLDN6 and that have CLDN6 bound to their surface by administering said antibodies to patients suffering from these diseases. Examples of diseases. that can be treated (eg, alleviate a disease) or prevented in this way include, but are not limited to those listed herein, diseases in which tumors form. Examples of diseases in which tumors form and which can be treated and/or prevented in this manner include cancers, such as ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell carcinoma of the lung and adenocarcinoma, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, including malignant multiform adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, for example teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumor, and metastatic forms thereof.

In another aspect, the present invention provides a method of treating or preventing diseases or disorders involving cells that express CLDN6 and which are characterized in that CLDN6 is associated with their surface, comprising administering to a patient antibodies, conjugates, bispecific/polyspecific molecules and compositions according to this invention. Preferably, these diseases and disorders are those in which tumors form, and in specific embodiments of the present invention they are selected from the group consisting of ovarian cancer, including ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), especially squamous cell lung carcinoma and adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, especially basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in including malignant polymorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal - cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, such as teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumor, and metastatic forms thereof. Preferably, CLDN6 is expressed on the surface of said cells.

This invention includes the use of the agents and compositions described herein for the prevention and treatment of cancer, ie, for the treatment of patients who suffer from any cancer disease or who are at increased risk of developing such a disease. In one aspect of the present invention, methods of inhibiting tumor growth are provided, comprising administering one or more of the agents and compositions described herein.

Preferably, the agents and compositions described herein are administered to patients in such a way that the therapeutically active substance does not or substantially does not enter tissues or organs that do not contain tumors that express CLDN6 and that have CLDN6 associated with their surface , such as placental tissue or placenta. Therefore, the agents and compositions described herein can be administered locally.

In one aspect of the present invention, the antibodies described herein are provided for use in the methods of treatment described herein. In one embodiment of the present invention, a pharmaceutical composition described herein is provided for use in the methods of treatment described herein.

In one specific embodiment of this invention, the patient to whom the proposed antibodies are administered additionally receives chemotherapy or radiation therapy or agents that modulate, i.e. enhance or inhibit the expression or activity of Fc receptors, for example Fcγ receptors, for example cytokines. Cytokines to be administered during this treatment include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-γ) and tumor necrosis factor (TNF). Chemotherapeutic agents for administration during this treatment include, but are not limited to, antineoplastic agents, for example doxorubicin, cisplatin, Taxotere, 5-fluorouracil. methotrexate, gemcitabine and cyclophosphamide.

In another aspect, the present invention relates to the immunization of animals, for example mice, with human CLDN6 or a peptide fragment thereof to produce antibodies. Preferred peptides for immunization are selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 15 and immunologically equivalent peptides.

Wild-type animals, as well as transgenic animals (which are not human), can be immunized with purified or enriched preparations of CLDN6 antigens or peptide fragments thereof, and/or nucleic acids and/or cells in which CLDN6 or a fragment thereof is expressed. Preferably, for this purpose, transgenic animals (which are non-human) should be capable of producing multiple isotypes of human monoclonal antibodies against CLDN6 (eg, IgG, IgA or IgM) by V-D-J recombination and isotype switching. Isotype switching can occur in a classical or non-classical way.

In another aspect of the present invention, animal (non-human) B cells are provided, as described above. The isolated B cells can then be immortalized by fusion with already immortalized cells to create a source (eg, hybridoma) of the antibodies of the present invention. Such hybridomas producing antibodies of this invention are also included within its scope.

In another aspect, this invention relates to methods for diagnosing, detecting and monitoring the course of cancer, including detecting and/or quantifying CLDN6 or cells in which CLDN6 is expressed and which have CLDN6 associated with their surface, in samples of biological material taken from a patient using the antibodies of this invention. Samples of biological material can be taken from a patient with the onset or development of cancer, or suspected of having such a disease, or at an increased risk of such a disease.

In one embodiment of the method for diagnosing, detecting and monitoring the course of an oncological disease according to this invention, a sample of biological material and/or a control/comparison sample is taken from a tissue or organ corresponding to the tissue or organ to be diagnosed, detected or monitored for a cancer lesion. disease; for example, the cancer to be diagnosed, detected or monitored is ovarian cancer and a biological sample and/or control/comparison sample is taken from ovarian tissue. Such organs and tissues are described herein, for example, in connection with various cancers and diseases.

In one embodiment of the method for diagnosing, detecting and monitoring the course of an oncological disease according to this invention, a sample of biological material and/or a control/comparison sample is taken from a tissue or organ in the cells of which, in the absence of a tumor, CLDN6 is not expressed and they do not have a pronounced presence CLDN6 on its surface. Preferably, said tissue is not placental.

As a rule, the level of target molecules in a sample of biological material is compared with some level taken as a control or baseline, and then a deviation from this latter is considered an indication of the presence and/or stage of cancer in a given patient. The level taken for comparison may be that which occurs in a control sample (for example, in a sample of healthy tissue from the same patient) or the average value established from the results of determination in a number of healthy individuals. By deviation from the specified level taken for comparison is meant any significant change, for example a decrease or increase of not less than 10%, 20% or 30%, preferably not less than 40% or 50% or even more. Preferably, the biological material sample contains CLDN6 or cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface, or the number of CLDN6 or cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface is increased compared to the level taken for comparison, this indicates the presence of cancer.

Typically, detection and/or quantification in the methods of this invention involves the use of labeled antibodies that specifically bind to target molecules.

In one particular aspect, the present invention relates to a method for detecting, i.e. determining the location or distribution of an oncological disease in the body, for example which tissues or organs are affected, which involves administering to the patient antibodies of the present invention coupled with a detectable label. Labeling of an organ or tissue in a specified patient may indicate the presence or increased risk of developing a tumor in that tissue or organ.

As noted above, the antibodies of this invention can be obtained directly from the hybridoma cells in which the antibodies are expressed, or by cloning and recombinant expression in a host cell (eg, CHO cells or lymphocytes). Examples of host cells also include microorganisms such as Escherichia coli and fungi such as yeast. Alternatively, said antibodies can be produced recombinantly in transgenic animals (which are not human) or plants. The present invention also includes embodiments in which said antibodies are produced by immunization or vaccination in situ (in the body of a patient) using the immunization methods described herein.

This invention also relates to nucleic acids. comprising genes or nucleotide sequences encoding antibodies or fragments thereof, such as any chain of an antibody molecule, as described herein. These nucleic acids can be incorporated into a vector, such as a plasmid, cosmid, virus, bacteriophage, or other vector, using, for example, genetic engineering techniques commonly used for such purposes. The vector may also include other genes, such as marker genes, which will allow selection in suitable host cells and under suitable conditions. In addition, the vector may include regulatory elements to ensure proper expression of the coding sequences in a suitable host cell. Such regulatory elements are known to those skilled in the art, including a promoter, alternatively spliced exon cassettes, and translation initiation codons.

Preferably, the nucleic acids of the present invention are operably linked to the above-mentioned sequences that provide regulation of expression in eukaryotic or prokaryotic cells. Regulatory elements that enable expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.

Methods for generating the nucleic acid molecules of the present invention and vectors comprising these molecules, introducing said vectors into suitably selected host cells, and causing expression of the desired sequences are well known in the art.

In another aspect, the present invention relates to host cells carrying the nucleic acids or vectors described herein.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of drawings

Figure 1. Sequence alignment of CLDN3, CLDN4, CLDN6 and CLDN9

Figure 2. Immunofluorescence analysis of blood serum collected from mice immunized to obtain antibodies specific against CLDN6

(A) Unfixed CHO-K1 cells co-transfected with nucleic acid encoding CLDN6 along with GFP fluorescent protein were tested with mouse monoclonal anti-CLDN6 antibodies (R&D Systems, MAB3656). CLDN6 is localized on the plasma membrane of transfected cells and can serve as a target for specific antibodies on living cells. (B) Serum from mice used to generate F3-6C3-H8 hybridomas contained antibodies that bind to CLDN6 on the surface of unfixed CHO-K1 cells co-transfected with nucleic acid encoding CLDN6 along with GFP fluorescent protein.

Figure 3. Determination of endogenous expression of claudins in HEK293T cells by Western blotting

Protein lysates of HEK293T cells transfected with nucleic acids encoding CLDN3, CLDN4, CLDN6 and CLDN9, respectively, or control cells (which were subjected to the same manipulations, but without the nucleic acid) were analyzed by Western blotting using ready-made commercially available antibodies against CLDN3(A) (Invitrogen, Cat No. 34-1700), anti-CLDN4(A) (Zymed, 32-9400), anti-CLDN6(A) (ARP, 01-8865) and anti-CLDN9(A) (Santa Cruz, sc-17672). These antibodies detected expression of the corresponding targets only in appropriately transfected HEK293T cells. In untransfected HEK293T cells, no endogenous expression of any of the listed claudins was observed.

Figure 4. Determination of the specificity of prepared commercially available anti-CLDN antibodies by flow cytometry.

The binding of commercially available anti-CLDN antibodies to HEK293T cells transfected with nucleic acids encoding CLDN3, CLDN4, CLDN6 and CLDN9, respectively, and to untransfected HEK293T cells was determined by flow cytometry. Only ready-made antibodies against CLDN3 turned out to be specific to their target.

Figure 5. Determination of the specificity of anti-CLDN antibodies obtained according to this invention by flow cytometry

Flow cytometry was used to determine the binding of antibodies in the supernatants of monoclonal subclones of hybridomas with HEK293T cells simultaneously transfected with two vectors, one of which encoded CLDN6, CLDN3, CLDN4 or CLDN9, and the other a fluorescent marker.

(A) Antibodies in the supernatant of the monoclonal hybridoma subclone F3-6C3-H8 bound specifically to cells transfected with CLDN6 but not to cells transfected with CLDN3, CLDN4, and CLDN9, respectively. In contrast, antibodies in the supernatant of the monoclonal hybridoma subclone F4-4F7-F2 bound to cells transfected with CLDN6 or CLDN9. Antibodies in the supernatant of the monoclonal hybridoma subclone F3-6C3-H8 also bound to cells transfected with the CLDN6 (I143V)-SNP variant.

(B) Antibodies from the supernatant of the monoclonal hybridoma subclone F3-7B3-B4 bound to cells transfected with CLDN6, CLDN3, or CLDN9. Monoclonal antibodies in the supernatant of the hybridoma subclone F3-3F7-A5 bound to cells transfected with CLDN6, CLDN4 or CLDN9

Figure 6. Binding specificity of murine anti-CLDN6 mAbs muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A

The binding of anti-CLDN6 antibodies to human CLDN6, 3, 4, 9 and CLDN6 SNP (single nucleotide polymorphism) variant I143V proteins was determined by flow cytometry using HEK293T cells transiently expressing the corresponding human claudin. These cells were simultaneously transfected with a fluorescent marker to distinguish untransfected cells (populations Q1 and Q3) from transfected ones (populations Q2 and Q4). The antibody concentration was taken to saturate the binding site with CLDN6 (25 μg/ml). Expression of human CLDN6, 3, 4, 9 and CLDN6-SNP(I143V) proteins was confirmed using commercially available monoclonal antibodies against human claudin-6 (R&D Systems, MAB3656), human claudin-3 (R&D Systems, MAB4620) and claudin -4 people (R&D Systems, MAB 4219).

Figure 7. Relative affinity of murine anti-CLDN6 monoconal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A

To determine relative affinity, the binding of anti-CLDN6 antibodies to human CLDN6 protein stably expressed on the surface of HEK293 cells was analyzed by flow cytometry. Based on data on the saturation of binding sites, a graph of the FACS signal (average fluorescence intensity) versus antibody concentration was constructed. EC ₅₀ (antibody concentration at which half of their binding sites are occupied at equilibrium) was calculated using the nonlinear regression method. For the CLDN6-specific antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A, very low EC ₅₀ values were obtained (200-500ng/ml) and saturation of binding sites was achieved at low concentrations.

Figure 8. Complement dependent cytotoxicity (CDC) activity of murine anti-CLDN6 monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A

The complement-dependent cytotoxicity activity of anti-CLDN6 antibodies was determined in a luciferase system that detects endogenous adenosine triphosphate in unlysed cells. CHO-K1 cells stably expressing human CLDN6 were incubated with various concentrations of muMAB 59A, 60A, 61D, 64A, 65A, 66B, and 67A antibodies. For MuMAB antibodies 59A, 60A, 61D, 64A, 65A, 66B, and 67A, dose-dependent complement-dependent cytotoxicity was observed and occurred at low antibody concentrations.

Figure 9. Complement-dependent cytotoxicity (CDC) activity of mouse anti-CLDN6 monoclonal antibodies muMAB 65A and 66B against NEC8 cells with endogenous CLDN6 expression and NEC8 LVTS2 54 cells (CLDN6 knockdown)

Mouse anti-CLDN6 monoclonal antibodies muMAB 65A and 66B caused cytotoxicity to NEC8 cells in a dose-dependent manner. The target specificity of muMAB 65A and 66B was confirmed in NEC8 LVTS2 54 cells (CLDN6 knockdown).

Figure 10. Therapeutic effect of murine anti-CLDN6 monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A in early tumor xenograft development in mice where mice were inoculated with the NEC8 tumor cell line.

Athymic Nude-Foxn1 ^nu mice were injected with NEC8 cells with endogenous expression of CLDN6. Mouse monoclonal antibodies against CLDN6 muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A suppressed tumor growth compared to the control group, which received saline instead of antibodies.

Figure 11. Binding specificity of anti-CLDN6 hybrid monoclonal antibodies chimAB 61D, 64A, 67A and 89A

The binding of anti-CLDN6 antibodies to human CLDN6, 3, 4 and 9 proteins was determined by flow cytometry using HEK293 cells stably expressing the corresponding human claudin. The antibody concentration saturates the binding site (25 μg/ml). Expression of human CLDN3, 4, 6 and 9 proteins was confirmed using commercially available monoclonal antibodies against human claudin-3 (R&D Systems, MAB4620) and human claudin-4 (R&D Systems, MAB 4219), and CLDN6/9-reactive mouse monoclonal antibodies muMAB 5F2D2, respectively. For the negative control, the same manipulations were performed under the same conditions, but without the first antibodies.

Figure 12. Relative affinity of anti-CLDN6 monoclonal fusion antibodies chimAB 61D, 64A, 67A and 89A to HEK293-CLDN6 cells

The binding of anti-CLDN6 antibodies to human CLDN6 stably expressed on the surface of HEK293 cells was determined by flow cytometry. The results of the experiment with saturation of binding sites were presented in the form of a graph, plotting the FACS signal (average fluorescence intensity) on the ordinate axis, and the antibody concentration on the abscissa axis. EC ₅₀ (antibody concentration at which half of their binding sites are occupied at equilibrium) was calculated using the nonlinear regression method. For the CLDN6 specific antibodies chimAB 64A and 89A, very low EC ₅₀ values were obtained (450-600 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 67A and 61D were characterized by low (1000 ng/ml) and medium (2300 ng/ml) EC ₅₀ values, respectively.

Figure 13. Relative affinity of anti-CLDN6 monoclonal fusion antibodies chimAB 61D, 64A, 67A and 89A to NEC8 cells

To determine the affinity of anti-CLDN6 antibodies to tumor cells that endogenously express human CLDN6, the binding of these antibodies to NEC8 (testicular cancer) cells was determined by flow cytometry. For the CLDN6 specific antibodies chimAB 64A and 89A, very low EC ₅₀ values were obtained (600-650 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 61D and 67A were characterized by medium (1700 ng/ml) and high (6100 ng/ml) EC ₅₀ values, respectively.

Figure 14. Relative affinity of monoclonal fusion antibodies against CLDN6 chimAB 61D, 64A, 67A and 89A to OV90 cells

To determine the affinity of anti-CLDN6 antibodies to tumor cells that endogenously express human CLDN6, the binding of these antibodies to OV90 (ovarian cancer) cells was determined by flow cytometry. For the CLDN6 specific antibodies chimAB 64A and 89A, very low EC ₅₀ values were obtained (550-600 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 61D and 67A were characterized by average EC ₅₀ values (1500 ng/ml and 2300 ng/ml, respectively).

Figure 15. Complement dependent cytotoxicity (CDC) activity of anti-CLDN6 monoclonal fusion antibodies chimAB 61D, 64A, 67A and 89A against wild-type and knockdown NEC8 cells

The complement-dependent cytotoxicity activity of anti-CLDN6 antibodies was determined in a luciferase system that detects endogenous adenosine triphosphate in unlysed cells. Wild-type NEC8 cells (NEC8 LVTS2 77) with ectopic luciferase expression were incubated with various concentrations of chimAB 61D, 64A, 67A, and 89A antibodies. ChimAB antibodies 61D, 64A, 67A and 89A affected NEC8 cells in a dose-dependent manner, whereas in the case of CLDN6 knockdown NEC8 cells (NEC8 LVTS2 54), none of these antibodies caused nonspecific lysis. These results demonstrate the target-specific effector functions of chimAB 61D, 64A, 67A, and 89A.

Figure 16. Antibody-dependent cell-mediated cytotoxicity (ADDC) activity of anti-CLDN6 monoclonal fusion antibodies chimAB 61D, 64A, 67A and 89A against wild-type and knockdown NEC8 cells

Antibody-dependent cell-mediated cytotoxicity (ADDC) activity of anti-CLDN6 antibodies was determined in a luciferase system that detects endogenous adenosine triphosphate in unlysed cells. Wild-type NEC8 cells (NEC8 LVTS2 77) were incubated with various concentrations of chimAB 61D, 64A, 67A, and 89A antibodies. ADDC activity of chimAB 61D, 64A, 67A, and 89A antibodies was dose dependent and occurred even at low antibody concentrations. To demonstrate the target specificity of the antibodies, NEC8 cells stably knocked down for CLDN6 (NEC8 LVTS2 54) were used.

Figure 17. Long-term therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 61D, 64A and 67A in mice with xenografts of NEC8 tumor cells when exposed to early stages of tumor development

Athymic Nude-Foxn1 ^nu mice with xenografts of NEC8 cells, in which CLDN6 is endogenously expressed, received antibodies specific to CLDN6 for 46 days. After this exposure, tumor growth was monitored for 60 days. Even after discontinuation of this immunotherapy, no tumor growth was observed in mice treated with muMAB 61D, 64A, and 67A monoclonal antibodies.

Figure 18. Therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 89A in mice with xenografts of NEC8 tumor cells when exposed to early stages of tumor development

We used athymic Nude-Foxn1 ^nu mice with xenografts of NEC8 cells in which CLDN6 is endogenously expressed. Scattered circles represent tumor volumes at various time points during antibody treatment during early development of NEC8 xenografts in Nude-Foxn1 ^nu nude mice. Compared to controls, mice treated with muMAB 89A showed inhibition of tumor growth (A). The administration of antibodies specific to CLDN6 or PBS (control) lasted 47 days, during which tumor growth was recorded over the next 51 days. At the end of the experiment, mice treated with muMAB89A showed no tumors, unlike controls treated with PBS (B).

Figure 19. Therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 64A in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development

Scattered circles represent tumor volumes at various time points during antibody treatment late in the development of NEC8 xenografts in Nude-Foxn1 ^nu nude mice. Immunotherapy with the murine anti-CLDN6 monoclonal antibody muMAB 64A inhibited the growth of solid tumors arising from NEC8 xenografts compared with both control groups, one receiving PBS without antibodies and the other receiving nonspecific antibodies.

Figure 20. Long-term therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 64A in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development

15 days after the injection of tumor cells 15, mice received CLDN6-specific antibodies muMAB 64A for 45 days. Tumor growth was recorded for another 49 days (A). A graph representing mouse survival shows that animals treated with the CLDN6-specific antibody muMAB 64A lived longer (B).

Figure 21. Therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 61D, 67A and 89A in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development

Scattered circles, triangles, and squares represent tumor volumes at various time points during antibody treatment late in the development of NEC8 xenografts. Mouse monoclonal antibodies against CLDN6 muMAB 61D, 67A and 89A suppressed tumor growth - in contrast to control treatments (PBS without antibodies and nonspecific antibodies.

Figure 22. Long-term therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 61D, 67A and 89A in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development

17 days after the injection of tumor cells, mice received CLDN6-specific antibodies muMAB 61D, 67A and 89A for 42 days. During the immunotherapy period and for another 49 days, tumor growth was recorded (A). A graph representing mouse survival shows that animals treated with the CLDN6-specific antibodies muMAB 61D and 67A lived longer (B).

Figure 23. Therapeutic effect of mouse anti-CLDN6 monoclonal antibodies muMAB 64A and 89A in mice bearing xenografts of wild-type NEC8 tumor cells and NEC8 stably knocked down CLDN6 cells.

muMAB antibodies 64A and 89A were therapeutic only in mice with wild-type NEC8 xenografts, but no such effect was observed in animals injected with CLDN6 knockdown NEC8 cells, demonstrating the target specificity of these antibodies in vivo.

Figure 24. High-resolution mapping of epitopes interacted with by chimAB antibodies 61D, 64A, 67A and 89A

Alanine mutant variants are designated “wild-type residue number alanine” or “wild-type residue number glycine” when alanine is present at this position in the wild-type protein; amino acids are named with single letter designations. For interaction with CLDN6-specific hybrid antibodies chimAB 61D, 64A, 67A, and 89A, amino acid residues F35, G37, S39, and, probably, T33 of the first extracellular domain of CLDN6 are important. Amino acid residue 140 is essential for the binding of chimAB 89A and is involved in the binding of chimAB 61D and 67A. In addition, amino acid residue L151 of the second extracellular domain is involved in the interaction with chimAB 67A antibodies. Although immunofluorescence analysis confirmed the expression of CLDN6 mutant variants P28A, W30A, G49A, L50A, W51A, C54A, and C64A, they did not produce membrane staining. Therefore, interactions of the antibodies of this invention with these amino acids cannot be excluded. In general, the identified epitopes are consistent with those DNA and peptides of the EC1 domain of CLDN6 that were used for immunization.

Figure 25. Alignment of the amino acid sequences of the heavy chain variable region of the CLDN6-specific antibodies of the present invention.

The complementarity determining regions (HCDR1, HCDR2 and HCDR3) are shown with a frame.

Figure 26. Amino acid sequence alignment of the light chain variable region of the CLDN6-specific antibodies of the present invention.

The complementarity determining regions (LCDR1, LCDR2 and LCDR3) are shown with a frame.

Figure 27. Binding specificity of monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS

The binding specificity of anti-CLDN6 antibodies was determined by flow cytometry using HEK293T cells transiently transfected with human CLDN6, 3, 4 and 9, respectively. To distinguish untransfected from transfected cells, cells were simultaneously transfected with a fluorescent marker. The antibody concentration was saturating the binding site (100 μg/ml). Expression of human CLDN3, 4, 6 and 9 proteins was confirmed using commercially available monoclonal antibodies against human claudin-3 (R&D Systems, MAB4620) and human claudin-4 (R&D Systems, MAB 4219), and CLDN6/9-reactive mouse monoclonal antibodies muMAB 5F2D2, respectively. Monoclonal hybrid antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS bound to CLDN6, but did not interact with CLDN3, 4 and 9, respectively.

Figure 28. Relative binding affinities of anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to HEK293-CLDN6 cells

To assess relative affinity, the binding of anti-CLDN6 antibodies to human CLDN6 stably expressed on the surface of HEK293 cells was determined by flow cytometry. Based on the results of the experiment with saturation of binding sites, a graph was constructed of the dependence of the FACS signal (average fluorescence intensity) on the antibody concentration. EC ₅₀ (antibody concentration at which half of their binding sites are occupied at equilibrium) was calculated using the nonlinear regression method. CLDN6-specific antibodies had similar low EC ₅₀ values and saturation of binding sites was achieved at low concentrations.

Figure 29. Relative binding affinities of anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to NEC8 cells

To assess relative affinity, the binding of anti-CLDN6 antibodies to tumor cells that endogenously express human CLDN6, namely NEC8 (testicular cancer) cells, was determined by flow cytometry. Compared with the CLDN6-specific antibodies chimAB 64A, mAb206-SUBG, and mAb206-SUBS, the variant with the mAb206-LCC light chain combination showed three times greater affinity for NEC8 cells. In all cases, saturation of binding sites was achieved at low concentrations.

Figure 30. Relative binding affinities of anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to OV90 cells.

The binding affinity of antibodies against CLDN6 to human OV90 cells (ovarian cancer) was determined by flow cytometry. Antibodies specific for CLDN6 had similar low EC ₅₀ values. The variant with the mAb206-LCC light chain combination exhibited the highest binding strength to OV90 cells.

Figure 31a. Complement-dependent cytotoxicity (CDC) activity of anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC and mAb206-SUBG against wild-type NEC8 cells and NEC8 knockdown cells.

The complement-dependent cytotoxicity activity of anti-CLDN6 antibodies was determined in a luciferase system that detects endogenous adenosine triphosphate in unlysed cells. Wild-type NEC8 cells with ectopic luciferase expression were incubated with various concentrations of chimAB 64A, mAb206-LCC, and mAb206-SUBG antibodies. These antibodies affected NEC8 cells in a dose-dependent manner, whereas in the case of CLDN6 knockdown NEC8 cells (NEC8 LVTS2 54), none caused nonspecific lysis. These results demonstrate the target-specific effector functions of chimAB 64A, mAb206-LCC, and mAb206-SUBG.

Figure 31b. Activity of monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS, manifested by complement-dependent cytotoxicity (CDC) against NEC8 cells.

These antibodies induced CDC against NEC8 cells in a dose-dependent manner. Compared to chimAB 64A antibodies, variants with amino acid substitutions mAb206-SUBG and mAb206-SUBS had similar activity.

Figure 32a. Anti-CLDN6 monoclonal hybrid antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS exhibit antibody-dependent cell-mediated cytotoxicity (ADDC) activity against NEC8 and OV90 cells.

The activity of antibodies against CLDN6, manifested by antibody-dependent cell-mediated cytotoxicity, was determined in a luciferase system that detects endogenous adenosine triphosphate in unlysed cells. NEC8 and OV90 cells were incubated with various concentrations of anti-CLDN6 fusion antibodies. In both tumor cell lines, all of these antibodies induced ADCC even at low concentrations, and the effect of ADDC was dose dependent.

Figure 32b. The activity of monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC and mAb206-SUBG, manifested by antibody-dependent cell-mediated cytotoxicity (ADDC), against wild-type NEC8 cells and NEC8 knockdown cells.

To demonstrate the target specificity of the antibodies, NEC8 cells stably knocked down for CLDN6 were used.

Figure 33. Therapeutic effect of monoclonal hybrid antibodies against CLDN6 mAb206-LCC, mAb206-SUBG and mAb206-SUBS in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development.

Nude-Foxn1 ^nu athymic mice with NEC8 cell xenografts were used. 17 days after the injection of tumor cells, mice were injected with CLDN6-specific antibodies mAb206-LCC, mAb206-SUBG and mAb206-SUBS, and then tumor growth was recorded. Scattered circles, triangles, and squares represent tumor volumes at various time points during antibody treatment late in the development of NEC8 xenografts. Compared with the control (mice received nonspecific antibodies), monoclonal hybrid antibodies against CLDN6 mAb206-LCC, mAb206-SUBG and mAb206-SUBS suppressed tumor growth.

Figure 34. Therapeutic effect of monoclonal hybrid antibodies against CLDN6 mAb206-LCC, mAb206-SUBG and mAb206-SUBS in mice with xenografts of NEC8 tumor cells when exposed to late stages of tumor development

For comparison with Fig. 33 data on tumor growth over time are presented in more detail. It can be seen that monoclonal hybrid antibodies against CLDN6 are able to suppress tumor growth (A). The survival curve shows that mice treated with CLDN6-specific antibodies lived longer (B).

Figure 35. High-resolution mapping of epitopes interacted with by chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS antibodies

Alanone mutant variants are named “wild-type residue number alanine”; amino acids are named with single letter designations. For interaction with CLDN6-specific hybrid antibodies, amino acid residues F35, G37, S39, and, probably, T33 of the first extracellular domain of CLDN6 are important. Antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS were characterized by the same binding characteristics.

Figure 36. Therapeutic effect of murine anti-CLDN6 monoclonal antibody muMAB 64A on metastasis in mice bearing NEC8 tumor cell xenografts.

Nude-Foxn1 ^nu athymic mice were injected into the tail vein of NEC8 cells, and three days later these animals received muMAB 64A antibodies specific to CLDN6. After 8 weeks, the tumor burden in the lung tissue was determined using polymerase chain reaction. Mouse monoclonal antibodies against CLDN6 muMAB 64A, compared with control (PBS without antibodies), caused a clear inhibition of tumor growth.

Figure 37. Immunohistochemical staining of human cancerous and normal tissues using monoclonal antibodies muMAB 64A, mAb206-LCC and mAb206-SUBG.

In contrast to normal tissues, sections of ovarian and testicular tumors showed pronounced uniform staining. Very intense membrane staining occurred in malignant epithelial cells, whereas adjacent stromal cells and nonmalignant epithelial cells were not stained. These results clearly indicate that the CLDN6-specific antibodies of the present invention specifically bind to malignant cells obtained from tumor patients. (Explanation: number of tissues stained with antibodies / number of tissues examined).

Definitions of terms

To facilitate understanding of the present invention, we will first define some terms. Additional definitions are given as the invention progresses.

In the following description and in the accompanying claims, the words “includes” and “comprises” and their derivatives (and synonyms, such as “includes”) are to be understood to mean (unless otherwise stated) that the terms, integers or steps or groups of members, integers or stages are included in the group under consideration, but it is possible that it also includes other members, integers or stages or groups of members, integers or stages. The definite and indefinite articles and their analogues in the description of this invention and especially in the claims imply both the singular and the plural unless otherwise stated or unless clearly inconsistent with the particular context. Value ranges are provided here to provide an abbreviation for all values within a given range, rather than specifying each value individually. And, unless otherwise stated, each individual value is included herein as if it were individually stated. All methods described herein can be performed in any desired order unless otherwise noted or unless clearly inconsistent with the particular context. Any example given herein and taken together, as well as the use here of expressions such as "such as" or "for example" or "including" serve only to illustrate the present invention and do not impose limitations on the scope of the invention unless specified other. Nothing in this specification should be construed as indicating any unclaimed element essential to the practice of the invention.

Claudins are a family of proteins that are very important components of tight junctions, where they form a pericellular barrier that regulates the flow of substances in the intercellular space of the epithelium. Claudins are transmembrane proteins; The claudin molecule crosses the membrane four times, with both the N-terminus and the C-terminus located in the cytoplasm. The first extracellular loop consists of an average of 53 amino acid residues, the second - of about 24 amino acid residues. In the claudin family, proteins CLDN6 and CLDN9 are most similar to each other. As used herein, the abbreviation “CLDN” stands for “claudin” and includes the proteins CLDN6, CLDN9, CLDN4 and CLDN3. Preferably, the CLDN is a human claudin.

The designation "CLDN6" preferably refers to human CLDN6, in particular to (i) a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or encoding the amino acid sequence of SEQ ID NO: 8, for example, a nucleic acid containing the nucleotide sequence SEQ ID NO: 1, or (ii) to a protein containing the amino acid sequence of SEQ ID NO: 2 or containing the amino acid sequence of SEQ ID NO: 8. The first extracellular loop of CLDN6 preferably contains amino acid residues 28 to 80, more preferably amino acid residues 28 at the 76 amino acid sequence shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 8, for example the amino acid sequence shown in SEQ ID NO: 7. The second extracellular loop of CLDN6 preferably contains amino acid residues 138 to 160, preferably amino acid residues 141 to 159, more preferably amino acid residues 145 to 157 of the amino acid sequence shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 8, for example the amino acid sequence shown in SEQ ID NO: 6 Said first and second extracellular loops preferably form the extracellular portion of CLDN6.

The designation "CLDN9" preferably refers to human CLDN9, in particular to (i) a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 or (ii) a protein containing the amino acid sequence of SEQ ID NO: 9. First extracellular loop CLDN9 preferably contains amino acid residues 28 to 76 of the amino acid sequence shown in SEQ ID NO: 9. The second extracellular loop of CLDN9 preferably contains amino acid residues 141 to 159 of the amino acid sequence shown in SEQ ID NO: 9. Said first and second extracellular loops preferably form the extracellular part of CLDN9.

The designation "CLDN4" preferably refers to human CLDN4, in particular to (i) a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 or (ii) a protein containing the amino acid sequence of SEQ ID NO: 10. First extracellular loop CLDN4 preferably contains amino acid residues 28 to 76 of the amino acid sequence shown in SEQ ID NO: 10. The second extracellular loop of CLDN4 preferably contains amino acid residues 141 to 159 of the amino acid sequence shown in SEQ ID NO: 10. Said first and second extracellular loops preferably form the extracellular part of CLDN4.

The designation "CLDN3" preferably refers to human CLDN3, in particular to (i) a nucleic acid containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 or (ii) a protein containing the amino acid sequence of SEQ ID NO: 11. First extracellular loop CLDN3 preferably contains amino acid residues 27 to 75 of the amino acid sequence shown in SEQ ID NO: 11. The second extracellular loop of CLDN9 preferably contains amino acid residues 140 to 158 of the amino acid sequence shown in SEQ ID NO: 11. Said first and second extracellular loops preferably form the extracellular part of CLDN3.

The CLDN sequences described above include any variants of the said sequences, in particular mutant forms, splice variants, conformations, isoforms, allelic variants, variants and homologous forms of various species origin, including those that occur in nature. Allelic variants refer to changes in the normal sequence of a gene, the meaning of which is often unclear. When determining the complete nucleotide sequence of a gene, numerous variants of this gene are often identified. Homologous forms of different species origin are the corresponding nucleotide or amino acid sequences found in different species. The term "claudin" (denoted "CLDN") covers (i) splicing variants of CLDN, (ii) post-translational modification variants of CLDN, in particular various glycosylation variants, such as glycosylation of the N-terminal region of the polypeptide chain, (iii) conformational variants of CLDN, (iv ) cancer-associated and non-cancer-associated CLDN variants. Preferably, CLDN is present in its native conformation.

Expression of CLDN6 has been detected, e.g. in tumor tissue in ovarian cancer, lung cancer, stomach, breast, liver, pancreas, skin, melanomas, head and neck cancer, sarcomas, bile duct cancer, renal cell cancer and bladder cancer. CLDN6 is a particularly preferred target for the treatment and/or prevention of ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, as well as lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), including squamous cell carcinoma and lung adenocarcinoma , cancer of the stomach, breast, liver, pancreas, skin, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, including malignant polymorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer, including ileal cancer, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, for example teratocarcinoma or embryonal carcinoma, in particular germ cell carcinoma testicular tumors and their metastatic forms. In one embodiment of the present invention, the cancer associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. preferably the ovarian cancer is a carcinoma or adenocarcinoma. Preferably, the lung cancer is a carcinoma or adenocarcinoma, preferably a bronchiolar cancer, such as bronchiolar carcinoma or bronchiolar adenocacinoma. In one embodiment of the present invention, the tumor cells associated with the expression of CLDN6 are cancer cells of the specified diseases.

The term "section" refers to a part. When applied to specific structures such as an amino acid sequence or a protein, the term "region" can mean a continuous or integral part of the specified structure. Preferably, the amino acid sequence region comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the amino acid residues of said amino acid sequence. Preferably, if the section is a component, said component consists of 2, 3, 4, 5, 6, 7, 8 or more parts of a structure, each part being a continuous element of that structure. For example, a constituent amino acid sequence may be formed from 2, 3, 4, 5, 6, 7, 8, or more, preferably no more than 4 parts of said amino acid sequence, each part preferably containing at least 5 contiguous amino acid residues, according to at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, preferably at least 30 contiguous amino acid residues of a given amino acid sequence.

The terms “part” and “fragment” are used interchangeably herein and refer to continuous elements. For example, part of a structure such as an amino acid sequence or a protein refers to a contiguous element of the structure. A section, part or fragment of a structure preferably contains one or more functional properties of said structure. For example, a region, part or fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or peptide from which it is derived.

The term "extracellular region of CLDN" in the context of this invention refers to the portion of claudin that faces the extracellular space and is preferably accessible from the outside of said cell, for example to antibodies external to the cell. Preferably, the term refers to one or more extracellular loops or portions thereof, or any another extracellular portion of the CLDN, which is preferably specific to said CLDN, preferably said portion comprising at least 5, at least 8, at least 10, at least 15, at least 20, at least 30 or at least 50 amino acid residues or more.

The term “cell surface bound/associated CLDN” should be understood to refer to native CLDN, i.e. CLDN in its non-denatured form, preferably in its natural conformation. Preferably, the term "cell surface bound/associated CLDN" means that there is an association between the protein and the plasma membrane and that it is located on the cell membrane where at least a portion of the CLDN, preferably an extracellular region, faces the extracellular space of said cell and is accessible outside of it, for example for antibodies located outside the cell. The connection can be direct or indirect. For example, the association may be through one or more transmembrane domains, one or more lipid anchoring structures, and/or through interaction with some other protein, lipid, saccharide, or other structure located on the outer layer of the cell's plasma membrane. For example, CLDN associated with the cell surface may be a transmembrane protein, i.e. a protein embedded in a membrane (integral membrane) having an extracellular region, or CLDN may be a protein associated with the cell surface through interaction with another protein that is transmembrane.

CLDN6 is associated with the surface of a cell if it is located on the surface of a cell and is available for binding to CLDN6-specific antibodies added to such cells. In preferred embodiments of the present invention, cells characterized in that CLDN6 is associated with their surface are cells in which which CLDN6 is expressed. It should be emphasized that if CLDN6 is expressed in a cell, then the CLDN6 associated with the cell surface can only be from among these protein molecules.

The expression “CLDN-bearing cell” preferably means that said cell has claudin molecules on its surface, i.e. it is connected to the surface of the specified cell.

The phrases “cell surface” or “cell surface” are used in their usual sense in the art, i.e. include the outer surface of the cell, accessible for binding to proteins and other substances from the outside.

The expression “CLDN expressed on the cell surface” means that claudin produced in a given cell becomes associated with its surface.

For purposes of the present invention, cells are considered to have no significant expression of CLDN6 and no significant amount of this protein associated with the cell surface if the level of expression and amount of CLDN6 on the cell surface is less than that of placental cells or placental tissue. Preferably, the expression level and amount of CLDN6 on the cell surface should be less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression level and amount of this protein on the cell surface in placental cells or in placental tissue (or even below). Preferably, the cells do not have significant expression of CLDN6 and do not have a significant amount of this protein associated with the cell surface if the level of expression and amount of CLDN6 on the cell surface exceeds those in non-tumor, non-cancerous tissues other than placental tissue by no more than 2 times, preferably no more than 1.5-fold and preferably not greater than the expression level and amount of cell surface CLDN6 in said non-tumor non-cancerous tissues. Preferably, cells do not have significant expression of CLDN6 and do not have significant amounts of this protein associated with the cell surface if the expression level and amount of CLDN6 on the cell surface is below the detection limit of the detection method and/or if the expression level and amount of CLDN6 on the cell surface is too low in order for CLDN6-specific antibodies added to these cells to bind.

For the purposes of this invention, cells are considered to express CLDN6 and have this protein on the cell surface if the level of expression and amount of CLDN6 on the cell surface exceeds those in non-tumor non-cancerous tissues other than placental tissue, preferably by more than 2-fold, preferably by 10-fold. 100, 1000 or 10,000 times. Preferably, CLDN6 is expressed in cells and the protein is present on the cell surface if the expression level and amount of CLDN6 on the cell surface exceeds the detection limit of the detection method and/or if the expression level and amount of CLDN6 on the cell surface is sufficient to allow CLDN6-specific antibodies to bind added to these cells. Preferably, CLDN6 expressed in cells is displayed on their cell surface.

The term “raft” means a microdomain of the lipid bilayer of the cell membrane, enriched in cholesterol and sphingolipids, which is located in the outer layer of the plasma membrane of the cell. Some proteins are capable of binding into such domains, forming aggregates or focal adhesion sites, which affects the functioning of proteins. For example, the transfer of CLDN6 molecules into such structures after the protein has bound to the antibodies of this invention results in the formation of high density antigen (CLDN6)-antibody complexes in the plasma membrane. The high density of antigen (CLDN6)-antibody complexes is important for efficient activation of the complement system during complement-dependent cytotoxicity.

As used herein, the term “disease/illness” refers to any pathological condition, including cancer, in particular those cancers referred to herein.

The expression "diseases involving cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface" according to this invention means that the expression and amount of this protein on the cell surface in the affected tissue or organ is preferably increased compared to the state in healthy organ or tissue. By increase is meant an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least at least 1000%, at least 10,000% or even more. In one embodiment of the present invention, CLDN6 is expressed and present on the cell surface only in diseased tissue, and its expression is suppressed in healthy tissues. According to the present invention, diseases involving cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface include oncological diseases, such as cancer. Also according to the present invention, oncological diseases, such as cancers, are preferably those in which CLDN6 is expressed in tumor cells or cancer cells and this protein is associated with their cell surface.

As used herein, the terms “cancer,” “tumor-associated disease,” or “oncogenic disease” include diseases characterized by dysregulation of cellular growth, proliferation, differentiation, adhesion and/or migration, which can lead to the formation of tumors and/or metastases. or to the emergence of a tendency towards their formation. “Tumor cells” are understood as abnormal cells that are characterized by rapid, uncontrolled proliferation and continued cell growth and that continue to multiply even after the factor that initiated cell growth has ceased to act.

A “tumor” is defined as an abnormal group of cells or tissue that grows rapidly through uncontrolled proliferation and continues to grow after the cessation of the agent that caused the cell growth. Tumors are characterized by a partial or complete lack of structural organization and functional coordination with normal tissue; Typically, a tumor is a discrete mass of tissue that may be benign, precancerous, or malignant.

Preferably, the “cancer disease”, “tumor-associated disease” or “oncogenic disease” of the present invention is a cancer disease, i.e. malignant diseases, and a tumor cell is a cancer cell. Preferably, the “oncological disease”, “tumor-associated disease” or “oncogenic disease” is characterized in that there are cells that express CLDN6 and are associated with their surface, and tumor cells express CLDN6 and are associated with their surface.

Cells in which CLDN6 is expressed and which have the protein associated with their surface are preferably tumor cells or cancer cells, preferably those tumors and cancers described herein. Preferably, such cells are not placental cells.

Preferred cancers and malignancies of the present invention are selected from the group consisting of ovarian cancer, including ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), especially squamous cell lung carcinoma and adenocarcinoma, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, including malignant polymorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, bladder cancer, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma, including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer , including ileal cancer, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum, embryonal testicular carcinoma, placental site choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonal testicular cancer, uterine cancer, germ cell tumors, e.g. teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumor, and their metastatic forms.

The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Non-small cell lung carcinoma is divided into three main subtypes: squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Adenocarcinoma accounts for approximately 10% of all lung cancer cases. Cancerous lesions of this type are usually visible on the periphery of the lungs - in contrast to small cell and squamous cell carcinomas, which are characterized by a more central localization.

Skin cancer is a malignant growth of the skin. The most common skin cancers are basal cell carcinoma, squamous cell carcinoma, and melanoma. Malignant melanoma is a very aggressive disease. It is caused by the uncontrolled growth of pigment cells called melanocytes.

According to this invention, carcinoma (cancer) is a malignant tumor originating from the epithelial cells of a particular organ.

Bronchiolar carcinoma is a lung cancer believed to originate from the epithelium of the small airways (bronchioles); In this disease, neoplastic tissue extends along the walls of the alveoli and grows in small masses within the alveoli. Some cells contain mucin, which is also present in the alveoli, where there are also denudated cells.

Adenocarcinoma is a tumor originating from glandular tissue. Glandular tissue refers to epithelial tissue, which includes the skin, glands, and various linings of cavities and organs. During embryonic development, the epithelium originates from the ectoderm, endoderm, or mesoderm. In order for cells to be called adenocarcinoma, they do not have to be part of the gland - it is enough that they are capable of secretion. This type of carcinoma occurs in higher animals and humans. Well-differentiated adenocarcinoma resembles the glandular tissue from which it originates, whereas poorly differentiated adenocarcinoma may not resemble it at all. By staining the cells in the biopsy material, the pathologist can determine whether the tumor in question is an adenocarcinoma or another malignancy. Adenocarcinomas can occur in various tissues of the body, since certain glands are present throughout the body. Each gland has its own specific secretion composition, but any cell that has an exocrine function is considered glandular, and its malignant form is called adenocarcinoma. Malignant adenocarcinomas grow into other tissues and often metastasize if they develop long enough. The most common type of ovarian carcinoma is adenocarcinoma. Ovarian adenocarcinoma includes serous, mucinous, clear cell and endometrioid adenocarcinoma.

Cystadenocarcinoma is a malignant form of superficial epithelial-stromal tumor, a type of ovarian cancer.

Superficial epithelial-stromal tumors are a group of neoplastic formations of the ovary that are thought to originate from the superficial epithelium of the ovary (modified peritoneum) or from ectopic endometrioid or fallopian tube tissue. Such tumors make up the majority of all ovarian cancers.

Teratocarcinoma is a germ cell tumor that is a mixture of teratoma and embryonal carcinoma, or choriocarcinoma, or both. Choriocarcinoma is a malignant trophoblastic tumor, highly aggressive, usually arising from the placenta. It is characterized by early spread through the blood to the lungs.

Sarcoma is a cancer of connective tissue (bone, cartilage, fat), in which proliferation of cells originating from the mesoderm occurs, in contrast to carcinomas, whose origin is epithelial. Synovial sarcoma is a fairly rare cancer of soft tissue in the joints of the extremities.

Renal cell carcinoma, also known as renal cell carcinoma or renal cell adenocarcinoma, is a cancer of the kidney that develops in the lining of the proximal convoluted tubule, where blood is filtered and waste is removed. Today, renal cell carcinoma is the most common kidney cancer in adults; Of all tumors of the genitourinary system, this cancer has the highest mortality rate. Renal cell carcinoma is divided into clear cell and papillary. Clear cell renal cell carcinoma is more common than other forms of renal cell carcinoma. Clear cell renal cell carcinoma cells appear very pale or clear under a microscope. Papillary renal cell carcinoma is also common. In most cases, the tumor forms characteristic small finger-like projections (papillae).

A germ cell tumor is a neoplasm that originates from germline cells. Germ tumors can be malignant (cancerous) or benign. Germline cells are normally found in the gonads (ovaries or testes). Germ tumors that occur outside the gonads (eg, in the head and neck, mouth, pelvis; in fetuses, newborns, and young children - most often along the midline of the body, particularly at the tip of the coccyx) may be birth defects arising due to errors in the course of individual development.

There are two main classes of germ cell tumors: seminomas and nonseminoma tumors. The latter include teratocarcinomas, embryonal carcinomas, yolk sac tumors, choriocarcinomas, and differentiated teratomas. Most cell lines derived from nonseminoma tumors are equivalent to embryonal carcinomas, i.e. they are composed entirely of stem cells that do not differentiate under normal conditions, although some such tumors may respond to differentiation inducers such as retinoic acid.

Metastasis is the result of cancer cells spreading from the site of the original malignant lesion to other parts of the body. Metastasis, i.e. the formation of metastases is a very complex process that depends on the separation of malignant cells from the primary tumor, their penetration into the intercellular matrix, penetration through the basement membrane and endothelium into the body cavities and into the lumen of blood vessels, transport by blood and inclusion in target organs. When a new tumor has formed, its growth depends on the germination of its blood vessels. Metastases often form even after the primary tumor is removed, because tumor cells or components capable of metastasizing may remain in the body. In one embodiment of the present invention, the term "metastasis" refers to distant metastases, i.e. located outside the area of spread from the primary tumor through the lymphatic vessels and nodes in the region of the affected organ.

The cells of the secondary tumor (metastasis) are the same as those in the primary tumor. This means, for example, that if ovarian cancer metastasizes to the liver, the secondary tumor consists of abnormal ovarian cells rather than abnormal liver cells. Accordingly, this tumor in the liver is called metastatic ovarian cancer and not liver cancer.

The term “treatment” means administering to a patient a substance or composition described herein for the purpose of preventing or eliminating a disease, including reducing the tumor size or number of tumors in that patient; stopping or slowing the progression of the disease; inhibiting or slowing the development of a new disease in a given patient; reducing the incidence or severity of symptoms and/or recurrence in a patient who currently has or has previously had cancer; and/or extension, i.e. increasing the patient's life expectancy.

The term “treating a disease” includes curing, shortening the duration of a disease state, reducing its severity, preventing the development or inhibiting the progression of a disease process or deterioration of a patient's condition, or delaying the onset of a disease or its symptoms.

The expressions “to be at risk”, “there is a risk of developing a disease”, “increased risk of developing a disease” mean that for a given individual (patient) the probability of developing a particular disease, in particular cancer, is higher than “normal”, i.e. e. average in this population. In addition, the risk of developing a disease, particularly cancer, is increased for individuals who have had the disease in the past or currently have it, since such an individual may continue to develop the disease. Individuals who have had or currently have cancer also have an increased likelihood of cancer metastasis.

The term "immunotherapy" refers to treatments that use certain immunological processes. As used herein, the terms “protect,” “prevent,” “prophylactic,” “prophylactic,” or “protective” refer to preventing or treating, or both, the occurrence and/or spread of a tumor in an individual. In the context of the present invention, the term "immunotherapy" preferably refers to active immunization or vaccination of a tumor. The prophylactic use of immunotherapy, for example the administration of a composition according to this invention, preferably protects the patient from the development of tumor growth. The therapeutic use of immunotherapy, for example the therapeutic administration of a composition according to this invention, can lead to inhibition of tumor development/growth, including inhibition of tumor development/growth, in particular to interruption of tumor progression, which preferably leads to its elimination. The use of immunotherapy for therapeutic purposes can protect an individual from, for example, the spread or metastasis of an existing tumor.

The term "immunization" or "vaccination" refers to the process of introducing an antigen to an individual in order to elicit an immune response for the purpose of treatment or prevention.

The terms “individual”, “individual”, “diseased”, “patient”, “treatment/prevention target” or “organism” are used interchangeably herein and refer to vertebrates, preferably mammals. For example, mammals in the context of this invention are humans and other primates, domestic animals (dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (mice, rats, rabbits, guinea pigs, etc. .), as well as animals kept in captivity (for example, in a zoo). The term "animal" as used herein also includes a human. The terms “individual”, “individual”, “object of treatment/prevention” may also include the meaning “patient/sick”, i.e. an animal, preferably a human, suffering from a disease, preferably a disease associated with the expression of CLDN6, preferably an oncogenic disease, such as cancer.

The term "adjuvant" refers to substances that prolong or enhance or accelerate the immune response. The compositions of this invention preferably exert their effect without the addition of adjuvants. However, the compositions of this patent application may contain any known adjuvant. Adjuvants include various substances, for example, oil emulsions (for example, Freund's adjuvant and its modifications), mineral substances (for example, alum), products of bacterial origin (for example, Bordetella pertussis toxin), liposomes, immunostimulating complexes. Examples of adjuvants: monophosphoryl lipid-A (MPL SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18, and QS-L1 (So et al. , 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants, complete Freund's adjuvants, vitamin E, montanide, alum, CpG oligonucleotides (Krieg et al., 1995, Nature 374: 546-549) and various emulsions such as “water in oil”, obtained from oils subject to biodegradation, such as squalene and/or tocopherol.

According to the present invention, the sample can be any one that can be used according to the present description, in particular a biological sample, for example a tissue sample, including body fluids and/or cellular samples, and which can be obtained by conventional methods, for example by biopsy, including needle biopsy, by collection of blood, bronchial aspirate, sputum, urine, feces or other body fluids. For purposes of this invention, the term "biological sample" also includes fractions of biological samples.

The term "antibody" refers to glycoproteins containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any substance/molecule containing an antigen-binding site. The term "antibodies" includes monoclonal antibodies and fragments or derivatives thereof, including (the examples listed herein are not limiting): human monoclonal antibodies, humanized monoclonal antibodies, hybrid monoclonal antibodies, single chain antibodies, such as scFv, and antigen binding antibody fragments, for example Fab and Fab' fragments; the term also includes all recombinant forms of antibodies (eg, antibodies expressed in prokaryotes), deglycosylated antibodies, and any antigen-binding fragments and derivatives described herein. Each antibody heavy chain consists of a variable region (abbreviated VH) and a constant region. Each light chain consists of a variable region (abbreviated VL) and a constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), between which there are more structurally conserved regions called framework (FR). Each variable region of both the light and heavy chains is formed by three CDRs and four FRs, arranged in the following order (counting from the N-terminus to the C-terminus of the polypeptide chain): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the light and heavy chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of an immunoglobulin molecule to tissues or factors of the body's own, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the complement system.

In the present invention, the expression “at least one of the CDR sequences” preferably means at least one CDR3 sequence. The expression “antibody CDR sequences” preferably refers to the CDR1, CDR2 and CDR3 regions of the heavy or light chain of an antibody.

According to the present invention, if a particular antibody chain is stated to contain a particular CDR sequence, such as a CDR3 sequence, it means that the particular CDR sequence either forms a CDR region, such as the CDR3 region of the specified antibody chain (i.e., the CDR region consists of the specified specific sequence CDR), or forms part of a CDR region, such as the CDR3 region of the specified antibody chain (that is, the CDR region contains the specified specific CDR sequence).

If the present invention specifies that an antibody contains a particular heavy chain and/or a particular light chain, for example a chain containing particular CDR sequences, then it is preferred that both heavy chains and/or both light chains of the antibody each consist of a particular heavy chain and/or or a specific light chain.

The term “humanized antibodies” refers to molecules that have an antigen-binding region derived primarily from a non-human immunoglobulin and the remainder of the molecule is based on the sequence and/or structure of a human immunoglobulin. In such an antibody, the antigen-binding region may contain either the entire variable domains attached to the constant domains, or only the complementarity determining regions (CDRs) attached to the framework regions of the variable domains. The antigen binding sites may be native or modified by one or more amino acid substitutions, for example modified to be more similar to human immunoglobulins. Some forms of humanized antibodies retain all CDR sequences (for example, a humanized mouse antibody that contains all six CDR sequences from a mouse antibody). Other forms of humanized antibodies have one or more CDR sequences that are changed from the original antibody.

The term "hybrid antibodies" refers to those antibodies in which one part of each amino acid sequence of both the light and heavy chains is homologous to the corresponding sequences of antibodies derived from a particular animal species or belonging to a particular class, while the rest of the same is homologous to the corresponding sequences of other antibodies. Typically, the variable regions of both the light and heavy chains are similar to the variable regions of antibodies originating from one species of mammal, and the constant portions are homologous to sequences of antibodies originating from another species. One of the distinct advantages of such hybrid forms is that the variable region can be derived from known sources, which is convenient since readily available B cells or hybridomas from non-human organisms can be used in combination with constant regions derived from , for example, preparations of human cells. If variable regions are relatively easy to obtain and their specificity is independent of source, then constant regions, being human, are less likely to elicit an immune response in humans when administered with such antibodies than would constant regions originating from another living organism. However, this definition is not limited to this specific example.

The term “antigen-binding site” (antigen-binding region) of an antibody (or simply “binding site”) as used herein refers to one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function can be performed by antibody fragments, i.e. its entire sequence is not required for this. Examples of binding fragments encompassed by the term "antigen-binding region" include: (i) Fab fragments - monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F(ab') ₂ fragments - divalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments, consisting of the VL and VH domains of one arm of the antibody molecule, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), consisting of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which can optionally be connected by a synthetic linker structure. Also, although the two domains of the Fv fragment - VL and VH - are encoded by separate genes, they can be connected using recombinant DNA technology using a linker structure, which allows them to be obtained as a single polypeptide chain in which the VL and VH regions form a pair, forming a monovalent molecule (called single chain Fv, abbreviated scFv); see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also encompassed by the term "antigen-binding region" of an antibody. Another example is binding domain-immunoglobulin fusion proteins containing (i) a polypeptide that is a binding domain attached to a polypeptide that is the hinge region of an immunoglobulin molecule, (ii) a heavy chain constant region of an immunoglobulin molecule CH2 attached to the hinge region, and (iii) a CH3 immunoglobulin heavy chain constant region attached to a CH2 constant region. The polypeptide forming the binding domain may be a heavy chain variable region or a light chain variable region. Immunoglobulin binding domain fusion proteins are also described in US patent applications 2003/0118592 and 2003/0133939. These antibody fragments are prepared using conventional methods known to those skilled in the art; the possibility of practical use of such fragments is revealed in the same way as intact antibodies.

The term "epitope" refers to the antigenic determinant of a molecule, that is, that part of the molecule that is recognized by the immune system, for example, which is recognized by antibodies. For example, epitopes are discrete spatial structures on an antigenic molecule that are recognized by the immune system. In the context of the present invention, the epitope preferably comes from the CLDN protein. Epitopes typically consist of chemically active surface moieties, such as amino acid residues or sugar side chains, and have specific properties in terms of spatial structure and electrical charge. There are conformational and non-conformational epitopes: the former lose their ability to bind in the presence of denaturing solvents, while the latter do not. Epitopes of proteins such as claudins preferably contain continuous (sequential) or composite (discontinuous, combined) regions of these proteins and contain preferably from 5 to 100, preferably from 5 to 50, more preferably from 8 to 30, most preferably from 10 to 25 amino acid residues; for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues in length.

The term “discontinuous epitope” as used herein means a conformational epitope on a protein antigenic molecule formed by at least two distinct regions of the primary structure (amino acid sequence) of that antigenic protein.

The term "dual-specific molecule" ("bispecific molecule") refers to any agent, such as a protein, peptide, or protein or peptide complex, that has two different binding specificities. For example, such a molecule may bind (interact) with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" refers to any agent, such as a protein, peptide, or protein or peptide complex, that has more than two different binding specificities. For example, such a molecule may bind (interact) with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell (b) and (c) at least one other component. Accordingly, the present invention includes, but is not limited to, molecules with two, three, four, and multiple binding specificities directed against CLDN6 and other targets, such as Fc receptors on effector cells. The term "bispecific antibodies" also includes diantibodies. Diantibodies are bivalent bispecific antibodies in which the VH and VL domains are synthesized as a single polypeptide chain, but using a linker structure too short to allow two domains of the same chain to join, thereby forcing complementary domains of different chains to join, so that two antigen binding sites are created (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).

As used herein, the term “heteroantibodies” refers to two or more antibodies whose derivatives or antigen-binding regions are linked together, at least two of which have different specificities. These various specificities include specificity for binding to an Fc receptor on effector cells and specificity for binding to an antigen or epitope on a target cell, such as a tumor cell.

The antibodies described herein may be human. The term “human antibody” as used herein refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations arising by random or site-directed mutagenesis in vitro or by somatic mutations in vivo.

The term “monoclonal antibodies” as used herein refers to antibody preparations comprising molecules of the same composition and structure. A monoclonal antibody has a single binding specificity and affinity for a specific epitope. In one embodiment of the present invention, monoclonal antibodies are produced by hybridomas comprising B cells derived from non-human animals, such as mice, fused with immortalized cells.

The term “recombinant antibodies” as used herein includes all antibodies produced, expressed, synthesized or isolated by methods utilizing recombination, for example (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for immunoglobulin genes, or from a hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example from a transfectome, (c) antibodies isolated from a recombinant antibody combinatorial library, and (d) antibodies , obtained, expressed, synthesized or isolated by any other means using splicing of immunoglobulin genes with other DNA sequences.

The term “transfectome” as used herein includes recombinant eukaryotic host cells in which antibodies are expressed, for example CHO cells, NS/0, HEK293, HEK293T, plant cells or fungi, including yeast.

The term “heterologous antibodies” as used herein is defined with respect to the transgenic organism producing such antibodies. This term refers to antibodies having an amino acid sequence or coding nucleotide sequence corresponding to that of an organism that is not transgenic, and usually originating from an organism of a species other than that of the transgenic organism.

The term “heterohybrid antibodies” as used herein refers to antibodies in which the light chains and heavy chains are derived from different organisms. For example, antibodies in which the heavy chain is human and the light chain is mouse are heterohybrid.

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

The antibodies described herein are preferably isolated. By isolated antibodies, as used herein, we mean antibodies substantially free of other antibodies with different antigen specificities (eg, isolated antibodies that specifically bind CLDN6 are substantially free of antibodies that specifically bind antigens other than CLDN6). Isolated antibodies that specifically bind to an epitope, isoform or variant of human CLDN6 may, however, exhibit cross-reactivity against other, related antigens, for example from other species (eg, homologous to CLDN6). In addition, the isolated antibody is substantially free of other cellular material and/or chemicals. In one embodiment of the present invention, “combination of isolated monoclonal antibodies” means antibodies with different specificities combined in a specific composition.

According to this invention, antibodies are capable of binding to a predetermined target if they have significant affinity for the specified predetermined target and bind to the specified predetermined target in standard analytical systems such as those described herein. Preferably, the antibodies are capable of binding to a target if the binding of said antibodies to said target on the surface of intact cells can be detected and measured by flow cytometry (FACS). Preferably, the binding of antibodies to the specified target can be detected and measured at an antibody concentration of 10 μg/ml or less, 5 μg/ml or less, or 2 μg/ml or less. Preferably, antibody binding to said target can be detected and measured at antibody concentrations of 50 nM or less, 30 nM or less, or 15 nM or less. Affinity (binding affinity) is often quantified by the equilibrium constant of the dissociation reaction ( _KD ). Preferably, the term "significant affinity" refers to binding to a predetermined target for which (K _D ) = 10 ^-5 M or less, 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. For the antibodies of the present invention, the EC ₅₀ value for binding to CLDN6 is preferably 6500 ng/ml or less, 3000 ng/ml or less, 2500 ng/ml or less, 2000 ng/ml or less, 1500 ng/ml or less, 1000 ng/ml or less, 500 ng/ml or less, 400 ng/ml or less, 300 ng/ml or less, 200 ng/ml or less, or 100 ng/ml or less.

Antibodies are (virtually) unable to bind to a given target unless there is significant affinity for the target and the antibodies are not found to bind to the target using standard assay systems. Preferably, antibodies are incapable of essentially binding to a given target if analysis by flow cytometry (FACS) does not detect binding to the specified target on the surface of intact cells. Preferably, the binding of antibodies to the specified target is not detected at antibody concentrations up to 2 μg/ml, preferably up to 5 μg/ml, preferably up to 10 μg/ml, preferably up to 20 μg/ml, more preferably up to 50 μg/ml, especially up to 100 μg/ml or up to 150 μg/ml, up to 200 μg/ml or more. Preferably, antibody binding to said target is not detected at antibody concentrations up to 15 nM, preferably up to 30 nM, preferably up to 50 nM, preferably up to 100 nM, preferably up to 150 nm or up to 170 nm, up to 300 nm, up to 600 nm, up to 1000 nm , up to 1300 nm or more. Preferably, antibody binding to the target is not detected at the saturating concentration of antibodies for binding to the target, i.e., CLDN6. Preferably, there is no significant antibody affinity for the target if K_D at least 10, 100, 10³, 10⁴, 10⁵ or 10⁶ times greater than K_D to bind to a predetermined target to which the antibodies are capable of binding. For example, if K_D for the binding of these antibodies to a predetermined target with which they are able to bind is 10^-7 M, then K_D for these antibodies to bind to a target for which there is no significant affinity, there will be at least 10^-6 M,10^-5 M,10^-4 M,10^-3 M,10^-2 M or 10^-1 M.

Antibodies are specific for a predetermined target if they are able to bind to said predetermined target but are unable to bind to other targets, i.e. do not have significant affinity for other targets and do not show significant binding to other targets in standard analytical systems. According to the present invention, antibodies are specific for CLDN6 if they are able to bind to CLDN6, but are not able to bind to other targets, in particular to other claudin proteins other than CLDN6, for example CLDN9, CLDN4, CLDN3 and CLDN1. Preferably, the antibodies are specific for CLDN6 if the affinity for and binding to other claudin proteins other than CLDN6, such as CLDN9, CLDN4, CLDN3 and CLDN1, does not significantly exceed the affinity and binding to non-claudin proteins, such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin transmembrane proteins, such as MHC proteins or transferrin receptor or any other known polypeptide. Preferably, antibodies are specific for a predetermined target if they bind to said target with a K _D that is at least 10, 100, 10 ³ , 10 ⁴ , 10 ⁵ or 10 ⁶ times less than the K _D for binding to the target, for which these antibodies are not specific. For example, if the K _D for binding of these antibodies to a predetermined target for which they are specific is 10 ^-7 M, then the K _D for binding of these antibodies to a target for which they are not specific will be at least 10 ^-6 M. 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

The binding of antibodies to the target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, NY (1992), and the methods described herein. Affinity can be easily determined using methods commonly used for this purpose, for example by equilibrium dialysis; using BIAcore 2000 biosensors using a standard procedure according to the manufacturer's instructions; by radioimmunoassay using a radiolabeled target antigen or by any other route known to those skilled in the art. Affinity data analysis can be performed, for example, according to the method described in Scatchard et al., Ann NY Acad. ScL 51:660 (1949). The numerical values of the affinity parameters for a given antibody-antigen interaction can vary depending on the conditions for their determination, for example, on the salt concentration and pH value. Thus, the experimentally determined values of affinity or other parameters characterizing the interaction of an antibody with an antigen can vary depending on the determination conditions, for example, on the salt concentration and pH value. Thus, when measuring affinity and other parameters characterizing the interaction of an antibody with an antigen, such as K _D and IC ₅₀ , it is preferable to use standardized solutions of antibodies and antigens and standard buffer solutions.

A unique property of the antibodies of this invention is their ability to bind cell surface claudin 6. This is demonstrated by flow cytometry analysis of cells expressing claudin 6.

In order to determine the binding of monoclonal antibodies to living cells in which claudins are expressed, flow cytometry can be used. Briefly, proceed as follows. Cells of the desired lines expressing membrane-bound claudins (grown under standard culture conditions) are mixed with a solution of antibodies (at various concentrations) in PBS containing 2% heat-inactivated FCS and 0.1% NaN ₃ at 4°C for 30 min. The cells are then washed and reacted with fluorescently labeled second antibodies under the same conditions as the first antibodies. Samples are analyzed by FACS, recording forward and side scatter of light from each individual cell and determining the binding of labeled antibodies.

The term "binding" according to this invention refers preferably to specific binding, as defined above.

As used herein, the term “isotype” refers to a class of antibodies (eg, IgM or IgG1) that are encoded by heavy chain constant region genes.

As used herein, the term “isotype switching” refers to the phenomenon that the synthesis of immunoglobulins switches from one isotype (class) to another.

The term “natural”/“naturally occurring” is used in this document to refer to objects that can be found naturally (in nature). For example, it is a polypeptide or nucleotide sequence found in an organism (including viruses), that can be isolated from a natural source, that has not been intentionally modified by humans in a laboratory setting, and that occurs in nature.

The term “rearranged” as used herein refers to immunoglobulins with a heavy or light chain configuration such that the V segment is located immediately adjacent to the D-J or J segment, corresponding to substantially the entire VH or VL domain, respectively. The rearranged immunoglobulin (antibody) gene locus can be identified by comparison with the DNA of germline cells: the rearranged locus must have at least one recombined homologous heptamer/nonamer element.

The term “unrearranged” or “germline configuration” as used herein in relation to a V segment refers to a configuration in which the V segment has not recombined to be in close proximity to a D or J segment.

The term "nucleic acid molecule" as used herein refers to DNA and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA. Nucleic acid molecules can be used for introduction into cells (transfection), for example in the form of RNA, which can be obtained in vitro by transcription from a DNA template. Before use, RNA can be modified by sequence stabilization, capping, and polyadenylation.

The nucleic acids of this invention are preferably isolated. The term "isolated nucleic acid" as used herein means that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example by digestion and fractionation by gel electrophoresis or (iv) synthesized, for example chemically. The isolated nucleic acid is used for use in recombinant DNA techniques.

The nucleic acids of this invention can be used alone or in combination with other nucleic acids, which may be homologous or heterologous. In preferred embodiments of the present invention, the nucleic acids are operably linked to the sequences. expression regulators that may be homologous or heterologous to a given nucleic acid, the term “homologous” meaning that the nucleic acid is also naturally (naturally) operably linked to expression regulatory sequences, and the term “heterologous” meaning that the nucleic acid is not functionally linked naturally to expression control sequences.

A nucleic acid, such as an RNA and/or protein or peptide expression nucleic acid, and expression control sequences are operably linked to each other if they are covalently linked such that the expression or transcription of the nucleic acid is controlled or influenced by the data regulatory sequences. If a given nucleic acid is to be translated to form a functional protein, then in the presence of an expression regulatory sequence that is operably linked to a coding sequence, induction of that regulatory sequence results in transcription of that nucleic acid without a frameshift in that coding sequence or the coding sequence cannot translated to form the desired protein or peptide.

The term "expression control sequence" (regulatory sequence) as used herein includes promoters, ribosome binding sites, enhancers, and other regulatory elements that influence gene transcription or messenger RNA translation. In some specific embodiments of the present invention, expression control sequences may themselves be regulated. The structure of sequences regulating expression can be different: it depends on the type of living organism or cell type, but as a rule it contains 5'-non-transcribed and 5'- and 3'-non-translated sequences that are involved in the initiation of transcription and translation , respectively, for example TATA box, cap sequence, CAAT sequence, etc. In particular, the 5' non-transcribed regulatory sequence contains a promoter region that includes a promoter sequence for regulating the transcription of an operably linked nucleic acid. Expression control sequences may also contain enhancer sequences or upstream activating sequences (UAS).

In this invention, the term "promoter" or "promoter region" refers to the nucleotide sequence located "upstream", i.e. in the 5` direction from the nucleotide sequence that is expressed. and regulating the expression of this sequence, containing a site for recognition and binding to RNA polymerase. The promoter region may include other recognition and binding sites for other factors involved in the regulation of transcription of a given gene. A promoter can regulate the transcription of a prokaryotic or eukaryotic gene. Also, the promoter can be inducible and initiate transcription in response to an inducing agent, or the promoter can be constitutive (not inducible) and then transcription is not regulated by the inducing agent. A gene regulated by an inducible promoter is not expressed or only expressed to a small extent in the absence of the inducing agent. In the presence of an inducing agent, the gene is “turned on” or the level of transcription increases. This is usually mediated by the binding of a specific transcription factor.

Promoters preferred in this invention include the promoters for the SP6, T3 and T7 polymerases, the human U6 promoter, the CMV promoter, and artificial hybrid promoters (e.g., CMV) in which a portion(s) are coupled to a portion(s) promoters of genes encoding other cellular proteins, for example human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), including or not including additional intron(s).

As used herein, the term “expression” is used in its most general meaning and includes the production of RNA or RNA and protein/peptide. It also involves partial expression of nucleic acids. Expression can occur temporarily (transient expression) or permanently (stable expression). As used herein, the term “expression” also includes the terms “aberrant expression” and “abnormal expression”.

"Aberrant expression" or "abnormal expression" as used herein means altered expression, preferably increased, compared to a level considered normal, preferably compared to the level of expression in normal cells (non-tumorigenic) or in a healthy individual. An increase in expression is considered to be an increase in its level by at least 10%, in particular by at least 20%, at least 50% or at least 100%. In one embodiment of the present invention, expression occurs only in diseased tissue, whereas in normal tissue it is suppressed.

In a preferred embodiment of the present invention, the nucleic acid molecule is present in a vector where it is provided with a promoter that regulates the expression of the nucleic acid. The term “vector” as used herein is used in its most general sense and includes any adjuvant that allows said nucleic acid, for example, to enter prokaryotic and/or eukaryotic cells and, if desired, integrate into the genome. Such vectors are preferably replicated and/or expressed in cells. Vectors include plasmids, phagemids, bacteriophages, or viral genomes. The term "plasmid" as used herein generally refers to constructs of extrachromosomal genetic material, typically circular double-stranded DNA, that can replicate independently of chromosomal DNA.

As a vector for the expression of antibodies, one can use vectors with which the heavy chain and the light chain of the antibody are represented by different vectors, or vectors with which the heavy chain and the light chain of the antibody are presented in one vector.

Statements made herein regarding specific nucleic acids and amino acid sequences, such as those set forth in the Sequence Listing, also include modifications, i.e., variations, of the specific sequences that result in sequences that are functionally equivalent to the specific sequences, such as amino acid sequences having properties identical to (or similar to) those particular sequences and nucleotide sequences encoding those amino acid sequences having properties identical to (or similar to) those of the amino acid sequences encoded by those particular nucleotide sequences. One important property is the retention of the ability to bind to its target or support antibody effector functions, such as complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC). Preferably, when a sequence modified from a particular sequence replaces that particular sequence in an antibody molecule, the ability of said antibody to bind to the corresponding target and preferably the functions of said antibody as described herein are retained.

Likewise, statements made herein regarding specific antibodies or hybridomas producing specific antibodies should also apply to antibodies that differ in the amino acid sequence and/or nucleotide sequence encoding it, modified from the amino acid sequence and/or nucleotide sequence encoding it of those particular antibodies. , but functionally equivalent to them. An important property is the preservation of the binding of antibodies to their target or the preservation of their effector function. Preferably, the sequence modified from any particular sequence, replacing the latter in the antibody molecule, retains the ability of the antibody to bind to its target and preferably the function of the antibody, eg, CDC- or ADCC-mediated lysis.

Those skilled in the art will understand that, in particular, CDR sequences, hypervariable regions and variable regions can be modified without losing the ability to bind the target. For example, the CDR regions will be identical or highly homologous to the regions of the antibodies described herein. The term “highly homologous” implies that 1 to 5, preferably 1 to 4, eg 1 to 3 or 1 or 2, substitutions can be made in the CDR. In addition, hypervariable and variable regions can be modified so that they are substantially homologous to certain regions of specific antibodies described herein.

It should be appreciated that the specific nucleic acids described herein also include nucleic acids modified to optimize codon frequency in a particular host cell or organism. Differences in codon frequency between organisms can lead to a variety of problems regarding heterologous gene expression. As a result of codon optimization by changing one or more nucleotides in the original sequence, it is possible to optimize the expression of a given nucleic acid, in particular the optimization of translation efficiency, in a homologous or heterologous host organism in which the given nucleic acid is to be expressed.

According to the present invention, a variant, derivative, modified form or fragment of a nucleotide sequence, amino acid sequence or peptide preferably has a functional property of the nucleotide sequence, amino acid sequence or peptide from which it is derived. Such functional properties include interaction or binding with other molecules. In one embodiment of the present invention, a variant, derivative, modified form or fragment of a nucleotide sequence, amino acid sequence or peptide is immunologically equivalent to the nucleotide sequence, amino acid sequence or peptide from which it is derived.

Preferably, the degree of identity of any nucleotide sequence and a nucleotide sequence modified from or a variant thereof should be at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98% or 99%.

With regard to nucleotide sequence variants corresponding to CLDN6, the degree of identity is preferably taken over a region of at least about 300, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600 or at least about 630 nucleotides. In preferred embodiments of the present invention, the degree of identity is taken over the entire length of the nucleotide sequence to which comparison is made, for example those nucleotide sequences that are shown in the Sequence List. Preferably, the two nucleotide sequences are capable of hybridizing to each other and forming a stable double helix, preferably under conditions in which specific hybridization of the polynucleotides is possible (harsh conditions). Stringent hybridization conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, FM Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65°C in buffer solution (3.5 x SSC, 0.02% Ficol, 0.02% PVP , 0.02% bovine serum albumin, 2.5 mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2 mM EDTA). The abbreviation SSC denotes a solution of 0.15 M sodium chloride//0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA was transferred is washed, for example, twice with SSC at room temperature and then with 0.1-0. 5 × SSC/0.1 × SDS at temperatures up to 68°C.

The term “variant” as used herein also includes mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologues, particularly those existing in nature. An allelic variant is a change in the normal nucleotide sequence of a gene, and the meaning of this change often remains unclear. When determining the complete sequence of a gene, numerous allelic variants of a given gene are often identified. A species homologue is a nucleotide or amino acid sequence of a different species origin than that of a given nucleotide or amino acid sequence.

For the purposes of this invention, amino acid sequence variants include variants due to insertions or deletions, additions and/or substitutions of amino acid residues. Variants resulting from deletion, including a deletion at the N-terminus and/or the C-terminus of the protein, are also called N-terminal or C-terminal truncated variants.

Variants resulting from insertions of amino acid residues include insertions of one or two or more amino acid residues in a particular amino acid sequence. In the case of amino acid sequence variants containing an insertion, one or more amino acid residues are inserted at a specific location in the amino acid sequence, although insertions at random locations are also possible with due screening of the resulting product.

Variations resulting from the addition of amino acid residues include the addition of one or more amino acid residues (eg, 1, 2, 3, 5, 10, 20, 30, 50 or more) to the amino and/or carboxyl terminus of the polypeptide chain.

Variants resulting from deletion of amino acid residues are characterized in that one or more amino acid residues (eg, 1, 2, 3, 5, 10, 20, 30, 50 or more) are removed from the polypeptide chain. Deletions are possible at any position of the polypeptide chain.

Variants resulting from the replacement of amino acid residues are characterized in that at least one amino acid residue is removed from the polypeptide chain, and another amino acid residue is introduced in its place. Preference is given to changes at positions in the amino acid sequence that are not conserved among homologous proteins or peptides and/or it is preferred that the amino acid residues being replaced and those being replaced have similar properties. It is preferred that amino acid substitutions in protein variants be conservative, that is, that the amino acid residue being replaced and the amino acid residue being replaced have a similar electrical charge. A conservative amino acid substitution implies that the replacing amino acid belongs to the same group in terms of side chain properties as the one being replaced. Naturally occurring amino acids are generally divided into four groups: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and polar. charge-bearing (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids.

With respect to the amino acid sequence corresponding to SEQ ID NO: 37, the term “variant” refers in particular to a sequence in which the cysteine residue at position 436 is replaced by another amino acid residue from those listed above, preferably glycine, alanine, serine, threonine, valine or leucine.

The degree of similarity, preferably identity, between a given amino acid sequence and a sequence modified from or a variant thereof, for example between significantly homologous amino acid sequences, is preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably taken to be a portion of the polypeptide chain that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the amino acid sequence being compared. For example, if the amino acid sequence being compared consists of 200 amino acid residues, then the degree of similarity or identity is preferably taken to be at least about 20, at least about 40, at least about 60, at least about 80, to at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, preferably contiguous. With respect to CLDN6 polypeptide chain variants, the degree of similarity or identity is preferably taken over a region consisting of at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200 or at least about 210 amino acid residues. In preferred embodiments of the present invention, the degree of similarity or identity is taken over the entire length of the amino acid sequence to which comparison is made, for example the amino acid sequences shown in the Sequence List. To align sequences to determine their similarity, preferably identity, tools known in the art can be used, preferably using the best alignment, for example using Align programs, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acid residues that are identical or represent conserved amino acid substitutions. "Sequence identity" when comparing two polypeptides or nucleotide sequences indicates the proportion of amino acid residues or nucleotides that are the same in the sequences being compared.

“Identity (percentage)” is obtained after the best alignment, and this value is purely statistical and the differences between the two compared sequences are distributed evenly over the entire length of the sequences. Comparison of two nucleotide or amino acid sequences is usually made after an optimal alignment, with the comparison being made using a comparison segment or window to identify and compare local areas of sequence similarity. Optimal alignment of sequences for comparison can be done, in addition to “manually”, using the local homology algorithm of Smith and Waterman (see Smith, Waterman, 1981, Ads App. Math. 2, 482), CC using the local homology algorithm of Needleman and Unch (Neddleman , Wunsch, 1970, J. Mol. Biol. 48, 443), using the Pearson and Lipman similarity search method (Pearson, Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444) or using computer programs, in which these algorithms are used (GAP, BESTFIT, FASTA, BLAST P, BLAST N, TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA).

Identity (percentage) is calculated by determining the number of identical positions in the two sequences being compared and dividing it by the number of positions being compared and multiplying the result by 100, so that the resulting percentage identity of the two sequences is obtained.

Conservative substitutions can be made, for example, based on the similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the amino acid residues in question. For example: (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine); (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine; (c) positively charged (basic) amino acids include arginine, lysine and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. As a rule, substitutions are made within groups (a)-(d). In addition, glycine and proline can replace each other, since both amino acids are capable of disrupting the correct alpha-helical structure of the polypeptide chain. Some preferred substitutions are made within the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Knowing the genetic code and having recombinant DNA technology and the ability to synthesize it, it is quite possible for one skilled in the art to create DNA encoding conserved variants of amino acid sequences.

The present invention includes antibodies in which changes are made affecting the Fc region in order to change the functional or pharmacokinetic properties of these antibodies. Such changes may result in decreased or increased C1q binding and complement-dependent cytotoxicity, or increased or decreased FcγR binding and antibody-dependent cellular cytotoxicity. It is possible, for example, to substitute one or more amino acid residues in the heavy chain constant region, which would cause a change in effector function while maintaining antigen-binding ability compared to modified antibodies (for comparison, see US patents 5,624,821 and 5,648,260).

The half-life of antibodies in vivo can be increased by modifying the salvage receptor binding epitope in the immunoglobulin constant domain or similar domain so that the molecule does not contain an intact CH2 domain or an intact Fc region (for comparison, see US Patents 6,121,022 and 6,194,551. Time The half-life of antibodies can also be increased by mutations in the Fc region, for example by replacing the threonine at position 252 with leucine, the threonine at position 254 with serine, or the threonine at position 256 with phenylalanine (for comparison, see US Pat. No. 6,277,375).

To change the effector function of antibodies, the glycosylation pattern of antibody molecules can also be modified. For example, expression of antibodies can be carried out in transfectomes where there is no attachment of fucose, which is normally attached to the asparagine residue at position 297 of the region; as a result, the affinity of the Fc region for the Fc receptor will increase, which in turn will lead to increased antibody-dependent cellular cytotoxicity in the presence of natural killer (NK) cells; for comparison, see Shield et al. (2002) JBC, 277: 26733. Galactosylation can be modified to modify complement-dependent cytotoxicity.

In another embodiment of the present invention, mutations are generated randomly throughout the entire anti-CLDN6 antibody coding sequence or some portion thereof, for example, by saturation mutagenesis, and the resulting modified anti-CLDN6 antibodies can be tested for target binding activity.

As used herein, the terms “cell” and “host cell” preferably refer to an intact cell, that is, a cell with an intact and unaltered membrane that has not lost its normal intracellular components, such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, that is, a living cell capable of normally performing its inherent metabolic functions. Preferably, the terms of this invention refer to any cell that can be transformed or transfected using exogenous nucleic acid. The term “cell” as used herein includes prokaryotic cells (eg, Ecsherichia coli) or eukaryotic cells (eg, dendritic cells, B cells, CHO, COS, K562, HEK293, HELA, yeast and insect cells). The exogenous nucleic acid within such a cell may be (i) on its own in a freely distributed state, (ii) incorporated into a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. Particularly preferred are mammalian cells, such as mouse, hamster, pig, goat and primates, including humans. The cells of this invention can be derived from a variety of tissue types and include embryonic cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments of the present invention, the cells are antigen presenting cells, such as dendritic cells, monocytes or macrophages. The term “host cell” as used herein preferably refers to cells into which the recombinant expression vector has been introduced.

A cell containing a nucleic acid molecule preferentially expresses the peptide or protein encoded by the nucleic acid.

The term "transgenic animal" refers to animals whose genome contains one or more foreign genes (transgenes), preferably antibody heavy and/or light chain genes, or foreign chromosomes (transchromosomes), whether or not integrated into the natural genomic DNA of that animal, and which are preferentially capable of expressing these transgenes. For example, a transgenic mouse may have a transgene or transchromosome—a human antibody light chain gene and a human heavy chain gene or human chromosome with a nucleotide sequence encoding the heavy chain, such that the mouse, after immunization with CLDN6 antigen and/or cells in which CLDN6 is expressed, , human antibodies against CLDN6 are produced. The human heavy chain transgene may be integrated into the mouse chromosomal DNA, as in the case of transgenic mice, such as HuMAb, HCo7, or HCol2 mice, or the human heavy chain transgene may exist outside the chromium, as in the case of transchromosomal mice (eg, KM) described in the publication WO 02/43478. Such transgenic and transchromosomal mice may be capable of producing multiple isotypes of human anti-CLDN6 monoclonal antibodies (eg, IgG, IgA, and/or IgE) through V-D-J recombination and isotype switching.

The words “reduce” or “inhibit” as used herein mean the ability to cause an overall reduction in the level of, for example, cell proliferation, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, most preferably by 75% or more. The term "inhibit" (or similar expressions) includes complete or substantially complete inhibition, that is, reduction to zero or substantially zero.

The words "increase" or "amplification" preferably refer to an increase or amplification by at least 10%, preferably by at least 20%, preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%, even more preferably at least 80% and most preferably at least 100%. These words may also refer to circumstances where at time zero there is no detectable signal for a particular substance or state and at some time later than time zero there is a detectable signal for that substance or state.

The term “immunologically equivalent” means that an immunologically equivalent agent (molecule), such as an immunologically equivalent amino acid sequence, exhibits the same or substantially the same immunological properties and/or has the same or substantially the same immunological effects, for example with respect to the type of immunological effect ( the induction of a humoral and/or cellular immune response, the intensity and/or duration of the induced immunological reaction) or in relation to the specificity of the induced immunological reaction. In the context of the present invention, the term "immunologically equivalent" is preferably used in relation to the immunological properties or effects of the peptide or peptide variant taken for immunization. In particular, the immunological property is the ability to bind to antibodies and induce an immune response, preferably by stimulating the formation of antibodies. For example, an amino acid sequence is immunologically equivalent to another comparison sequence if said amino acid sequence, when presented to the immune system of an individual, produces an immunological response, preferably the formation of antibodies that specifically react with the comparison sequence, for example, an amino acid sequence forming part of CLDN6.

The term “immune effector functions” as used herein includes any functions mediated by components of the immune system that result in inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, the immune effector functions result in the killing of tumor cells. Preferably, immune effector functions in the context of this invention are mediated by antibodies. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), induction of apoptosis in cells bearing tumor-associated antigens, for example by binding of antibodies to a surface antigen, and/or inhibition of proliferation of cells bearing tumor-associated antigens. tumors, preferably ADCC and/or CDC. Thus, antibodies capable of mediating one or more immune effector functions are preferably capable of mediating cell killing by causing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion and/or phagocytosis, preferably causing CDC-mediated lysis and/or ADCC-mediated lysis. Antibodies may also act simply by binding to tumor-associated antigens on the surface of tumor cells. For example, antibodies can block the function of tumor-associated antigens by binding to tumor-associated antigens on the surface of tumor cells.

Detailed Description of the Invention

Mechanism of action of mAbs

Although the following offers considerations regarding the mechanism underlying the therapeutic action of antibodies, they should not be construed as limiting the present invention in any way.

Antibodies described herein can interact with components of the immune system, preferably through cytotoxicity - ADCC or CDC. The antibodies of this invention can also be used to target a desired agent (eg, a radioactive isotope, drug or toxin) to directly kill tumor cells or used in synergistic combination with conventional chemotherapeutic agents, targeting the tumor in complementary ways that may include an antitumor immune response, possibly weakened due to the cytotoxic side effects of chemotherapy drugs affecting T lymphocytes. However, the effect of the antibodies of this invention may also occur simply by binding to CLDN6 on the surface of cells, which blocks cell proliferation.

Antibody-dependent cell-mediated cytotoxicity

ADCC characterizes the ability of the effector cells described herein, in particular lymphocytes, to kill other cells, which preferably requires that the target cells be labeled with antibodies.

Preferably, ADCC occurs when antibodies bind to an antigen on tumor cells and the Fc domain of the antibody molecule binds to the Fc receptor (FcR) on the surface of the effector cell. Several families of Fc receptors have been identified; Certain cell populations are characterized by the expression of certain Fc receptors. ADCC can be viewed as a mechanism designed to directly cause - to varying degrees - immediate tumor destruction, and leading to antigen presentation and induction of an antitumor T-cell response. Preferably, induction of ADCC in vivo should result in an antitumor T cell response and an antibody response involving the body's own antibodies.

Complement-dependent cytotoxicity

CDC is another antibody-mediated cell killing pathway. The most effective antibody isotype for activating complement is IgM. IgG1 and IgG3 are also very effective at directing CDC through the classical pathway of complement activation. In this cascade of reactions, the preferential formation of antigen-antibody complexes results in the availability of multiple C1q binding sites in the vicinity of the C _H 2 domain of the involved antibody molecules, such as IgG (C1q is one of the three proteins whose complex is the C1 component of the complement system). Preferably, when these C1q binding sites become accessible, the previously low affinity of IgG for C1q increases substantially, which initiates a cascade of reactions involving a number of other complement proteins and leading to the proteolytic release from the effector cell of the chemotactic/activating agents C3a and C5a. Preferably, the complement cascade ends with the formation of a complex that attacks the membrane of the target cell and creates pores in it, facilitating the free entry of water and solutes into and out of the cell.

Antibody formation

Antibodies of this invention can be produced by various methods, including conventional monoclonal antibody technology, for example the standard Kohler-Milstein somatic cell hybridization method (see G. Kohler, C. Milstein, Nature 256: 495, 1975). Although somatic cell hybridization is the preferred method for producing the antibodies of this invention, other methods for producing monoclonal antibodies, such as viral or oncogenic transformation of B cells or phage display using antibody gene libraries, can in principle be used.

The preferred animal system for producing monoclonal antibody-producing hybridomas is the murine system. The production of hybridomas in mice is a well-established laboratory procedure. Protocols for immunization of mice and methods for isolating activated splenocytes for hybridization are known in the art. Hybridization pairs (eg, murine myeloma cells) and cell fusion techniques are also known.

Preferred animal systems for the production of monoclonal antibody-secreting hybridomas in this invention also include rat and rabbit systems (for example, those described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995) ; see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)).

In yet another preferred embodiment of the present invention, human monoclonal antibodies directed against CLDN6 can be produced using transgenic or transchromosomal mice carrying elements of the human immune system instead of the corresponding murine ones. These transgenic and transchromosomal mice include animals designated HuMAb and KM, respectively; in this document, both are collectively referred to as transgenic. The production of human antibodies using such transgenic mice can be done as described in detail for CD20 in WO2004 035607.

Another approach to obtaining monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes that produce specific antibodies (see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy ). Details of the production of recombinant antibodies can also be found in the works of M. Welschof, J. Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

Immunization

To obtain antibodies against CLDN6, mice can be immunized with carrier-conjugated peptides derived from the amino acid sequence of CLDN6, enriched with a preparation of CLDN6 antigen expressed as a recombinant protein, or fragments thereof, and/or cells in which CLDN or fragments thereof are expressed, as described herein document. Alternatively, mice can be immunized with DNA encoding the complete human polypeptide chain or fragments thereof. If immunization with a purified or enriched CLDN6 antigen preparation does not produce the desired antibodies, mice can be immunized with cells in which CLDN6 is expressed, for example taken from a cell culture of an appropriate line, to stimulate an immune response.

The immune response can be monitored during immunization using plasma and serum samples from the tail vein or retroorbital venous sinus. For cell fusion, animals with a sufficiently high titer of immunoglobulins against CLDN6 are selected. To enhance the production of specific antibodies by hybridomas, mice are injected with cells expressing CLDN6 by intraperitoneal or intravenous injection 3-5 days before sacrifice and spleen collection.

Creation of hybridomas producing monoclonal antibodies

To obtain hybridomas that produce monoclonal antibodies against CLDN6, cells are isolated from lymph nodes or spleens taken from immunized mice. and fuse them with immortalized cells of a suitable line, such as mouse myeloma cells. The resulting hybridomas are tested for the production of antigen-specific antibodies. The contents of individual cells are examined by enzyme-linked immunosorbent assay (ELISA) for the presence of antibody-secreting hybridomas. Antibodies specific for CLDN6 can be identified using immunofluorescence assays and FACS using cells in which CLDN6 is expressed. Hybridomas that produce the desired antibodies are subcultured, retested, and those that produce monoclonal antibodies against CLDN6 can be subcloned by serial dilution. Stable subclones are then cultured in vitro to obtain antibodies in the culture medium and characterize them.

Generation of transfectomes producing monoclonal antibodies

Antibodies of this invention can also be produced in transfectomes of host cells, using, for example, a combination of recombinant DNA technology and gene transfection techniques well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment of the present invention, the gene(s) of interest, such as antibody genes, are inserted into an expression vector, such as a eukaryotic cell plasmid, as described for the GS gene expression system in WO 87/04462 and WO 89/01036, and in European patent 338 841, or use another expression system that is well known in the art. Purified plasmids containing cloned antibody genes are introduced into eukaryotic host cells, such as CHO, NS/0, HEK293T or HEK293 cells, or other eukaryotic cells, such as plant cells or fungal cells, including yeast. To introduce the genes of this invention, methods described in the literature can be used, such as electrocution, the use of lipofectin, lipofectamine, etc. The host cells in which the introduced antibody genes are expressed can be identified and selected. The expression level of antibody genes in transfected cells (transfectomes) can be increased, then these cells can be multiplied and the antibodies they produce can be obtained in the required quantities. Recombinant antibodies are isolated and purified from the supernatant of the culture medium and/or cells.

Alternatively, cloned antibody genes of the present invention may be expressed in other expression systems, including prokaryotic cells, such as microorganisms, including Escherichia coli. Also, the antibodies of this invention can be produced by transgenic animals, for example in the milk of sheep and rabbits or in the eggs of chickens, or in transgenic plants; see, for example, Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380:825-839.

Expression of intact antibodies using partial sequences (humanization, chimerization)

a) Chimerization

Mouse monoclonal antibodies labeled with toxic agents or radioactive isotopes can be used to treat humans. Unlabeled murine antibodies are highly immunogenic in humans when used repeatedly, which reduces the therapeutic effect. Immunogenicity is mainly due to the constant regions of the heavy chains of antibody molecules. The immunogenicity of murine antibodies in humans can be reduced or even eliminated if these antibodies are chimerized or humanized. Hybrid (or chimeric) antibodies are antibodies in the molecules of which different regions originate from animals of different species, for example, the variable region is from a mouse antibody, and the constant region is from a human immunoglobulin. Chimerization of antibodies is achieved by combining the heavy and light chains of the variable region of a mouse antibody with the heavy and light chains of the constant region of a human antibody (for example, as described in Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0- 89603-918-8). In one preferred embodiment of the present invention, the hybrid antibodies combine a human kappa light chain constant region and a murine light chain variable region. In another preferred embodiment of the present invention, the hybrid antibodies are formed by joining the human lambda light chain constant region to the murine light chain variable region. For the production of hybrid antibodies, the constant regions of the heavy chains IgG1, IgG3 and IgG4 are preferred. For the production of hybrid antibodies, the heavy chain constant regions of IgG2, IgA, IgD and IgM are also preferred.

b) Humanization

Antibodies interact with target antigens primarily through amino acid residues located in six complementarity determining regions (CDRs). Therefore, amino acid sequences within the CDR vary more widely between antibodies than sequences outside the CDR. Because CDR sequences mediate the majority of interactions between an antibody and an antigen, it is possible to express recombinant antibodies that mimic the properties of certain natural antibodies by creating expression vectors that include CDR sequences from certain natural antibodies combined with framework sequences from other 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. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from publicly available DNA databases containing nucleotide sequences of germline antibody genes. These gametic sequences differ from mature antibody gene sequences because they do not have fully assembled variable sequences, which are formed by joining of V(D)J segments during B cell maturation. Gametic nucleotide sequences also differ from the sequences corresponding to the high-affinity antibodies of the secondary repertoire throughout the variable region. For example, somatic mutations are relatively rare near the N-terminus of framework region 1 and near the C-terminus of framework region 4. In addition, many somatic mutations do not significantly affect antibody binding. For this reason, in order to reconstitute an intact recombinant antibody with binding properties like the original antibody, it is not necessary to obtain the complete DNA sequence of the antibody (see WO 99/45962). For this purpose, coverage of the CDR regions of the partial sequences of the heavy and light chains is usually sufficient. The partial sequence is used to determine which germline variable and connecting gene segments (gametic sequence) are involved in the formation of the recombined antibody variable genes. The gametic sequence is then used to fill in the missing portions of the variable regions. The heavy and light chain leader sequences are cleaved off during protein maturation and do not contribute to the properties of the final antibody. To fill in missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or polymerase chain reaction (PCR) amplification. Alternatively, the entire variable region can be synthesized as a set of short overlapping oligonucleotides and combined by PCR amplification to create a fully synthetic variable region clone. This process has a number of advantages: certain restriction sites are eliminated or included, or certain codons are optimized.

Transcripts of heavy and light chain nucleotide sequences from hybridomas are used to assemble a set of overlapping synthetic oligonucleotides to create synthetic V sequences capable of encoding the same amino acid sequences as natural nucleotide sequences. Synthetic heavy chain and kappa sequences may differ from natural sequences in three respects: strings of repeating nucleotides are interrupted to facilitate oligonucleotide synthesis and PCR amplification; include optimal Kozak translation initiation sites (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); HindIII endonuclease restriction sites are located towards the 5' end of the nucleotide sequence from the translation initiation sites.

For both light and heavy chain variable regions, the optimized coding and corresponding non-coding sequences are divided into segments of 30-50 nucleotides approximately in the middle of the corresponding non-coding oligonucleotide. Thus, for each strand, oligonucleotides can be assembled into overlapping double-stranded sets spanning segments of 150–400 nucleotides. These pools are then used as templates to obtain PCR amplification products of 150-400 nucleotides. Typically, one set of variable region oligonucleotides produces two pools that are amplified separately to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may be desirable to also include an overlapping fragment of the heavy or light chain constant region in the PCR amplification to obtain fragments that are easily cloned into expression vector constructs.

The engineered hybrid or humanized heavy and light chain variable regions are then combined with the cloned promoter and leader sequences, translation initiation site, constant region, 3' untranslated sequence, polyadenylation site, and transcription termination site to form an expression vector construct. Expression constructs for the heavy and light chains can be combined in a single vector and transfected jointly, sequentially or separately into host cells, which are then fused to produce a host cell in which both chains are expressed. Plasmids are known for use in the construction of expression vectors for human IgGκ. Plasmids can be constructed such that PCR amplified cDNA sequences of the heavy chain and kappa light chain variable regions can be used to reconstitute full heavy and light chain minigenes. These plasmids can be used to express fully human or hybrid IgG1, kappa or IgG4, kappa antibodies. Such plasmids can be constructed to express other heavy chain isotypes or to express antibodies containing lambda light chains.

Thus, in another aspect of the present invention, the structural properties of anti-CLDN6 antibodies are used to create structurally related humanized anti-CLDN6 antibodies 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 murine monoclonal antibodies can be combined recombinantly with known human framework regions and CDR regions to produce additional recombinant humanized anti-CLDN6 antibodies of the present invention.

Binding to cells in which the antigen is expressed

The ability of antibodies of this invention to bind to CLDN6 can be determined using standard analytical methods such as those presented in the examples - for example, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, flow cytometry.

Isolation and determination of the properties of antibodies

To purify anti-CLDN6 antibodies, selected hybridomas are grown in two-liter monoclonal antibody purification roller flasks. Alternatively, antibodies against CLDN6 are produced in bioreactors with dialysis. The supernatant is filtered and, if necessary, concentrated, after which affinity chromatography is carried out with protein G or protein A immobilized on Sepharose. The purity of the eluted IgG is checked by gel electrophoresis and high-performance liquid chromatography. The buffer solution is exchanged with PBS, the concentration is determined by optical density at a wavelength of 280 nm (extinction coefficient 1.43). Aliquots of monoclonal antibodies are stored at -80°C.

To determine whether selected anti-CLDN6 monoclonal antibodies bind to a given epitope, site-directed mutagenesis and multiple site-directed mutagenesis can be used.

Isotype determination

To determine the isotype of purified antibodies, an enzyme-linked immunosorbent assay (ELISA) can be used using various commercially available kits (eg, Zymed, Roche Diagnostics). The wells of a microtiter plate are coated with anti-mouse Ig. After blocking, react with monoclonal antibodies or a purified control (isotype) sample at room temperature for 2 hours. Mouse IgG1, IgG2a, IgG2b or IgG3, IgA, or mouse IgM-specific peroxidase conjugates are then added to the wells. After washing, the plates were treated with 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) as substrate (1 mg/ml) and the absorbance was determined at 405-650 nm. Or use a ready-made IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) according to the manufacturer's instructions.

Flow cytometry

Flow cytometry 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 in which CLDN6 is expressed. Samples of cell lines that express CLDN6 naturally or after transfection and negative controls (cells without CLDH6 expression grown under standard culture conditions) are mixed with various concentrations of monoclonal antibodies in the hybridoma supernatant or in PBS containing 1% FBS and incubated at 4°C for 30 minutes. After washing, the CLDH6-binding monoclonal antibodies are coupled to allophycocyanin (APC)- or Alexa647-labeled anti-IgG antibodies under the same conditions as the primary antibody staining. Samples are analyzed using a FACS flow cytometer, determining the parameters of forward (front) and side light scattering of single living cells. To distinguish the binding of monoclonal antibodies specific for CLDN6 from non-specific binding in each individual dimension, a co-transfection method can be used. Cells were transiently transfected with plasmids encoding CLDN6 and a fluorescent marker and stained as described above. Transfected cells are detected in a fluorescence detection channel that is different from that of cells stained with antibodies. Because most transfected cells express both transgenes, monoclonal antibodies specific for CLDN6 bind preferentially to cells expressing the fluorescent marker, whereas nonspecific antibodies bind in comparable amounts to untransfected cells. Fluorescence microscopy can be used in addition to or instead of flow cytometry. To use a fluorescence microscope, cells are stained as described above.

Immunofluorescence microscopy

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 in which CLDN6 is expressed. For example, samples of cell lines in which CLDN6 is expressed spontaneously or after transfection and a negative control (cells without CLDH6 expression) are grown in slide chambers under standard culture conditions in DMEM/F12 medium containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and l00 μg/ml streptomycin. Then the cells are fixed with methyl alcohol or paraformaldehyde or left untreated. Cells were then reacted with anti-CLDN6 monoclonal antibodies for 30 min at 25°C. After washing, cells are bound to secondary anti-mouse IgG antibodies labeled with Alexa555 (Molecular Probes) under the same conditions. The cells are then examined using fluorescence microscopy.

The total level of CLDN6 in cells can be determined. when cells are fixed with methyl alcohol or paraformaldehyde and treated with Triton X-100 detergent to increase permeability. In living cells and in cells treated with paraformaldehyde but with normal permeability, the localization of CLDN6 can be studied. Additionally, CLDN6 binding at tight junction sites can be examined by staining with tight junction markers such as ZO-1. The effects of antibody binding and cell membrane localization of CLDN6 can also be studied.

Western blotting

The interaction of anti-CLDN6 immunoglobulin G with CLDN6 antigen can also be tested using Western blotting. Briefly do the following. Extracts of cells expressing CLDN6 and corresponding negative controls are prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The electrophoresis-separated antigens are transferred to nitrocellulose membranes, blocked, and detected with monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG and peroxidase staining with enhanced chemiluminescence (ECL) substrate.

Immunocytochemical analysis

The interaction of anti-CLDN6 immunoglobulin G with the CLDN6 antigen can also be tested by immunocytochemistry, methods of which are well known to those skilled in the art. For example, frozen sections of paraformaldehyde- or acetone-fixed tissue, or paraffin-embedded sections of paraformaldehyde-fixed tissue, sampled from tumor or healthy tissue from a patient during surgery or from mice bearing tumor xenografts inoculated with spontaneously or posttransfectionally expressed cell lines are used. CLDN6. For immunostaining to detect antibodies that can interact with CLDN6, horseradish peroxidase conjugates with goat anti-mouse or anti-rabbit antibodies (manufactured by DAKO) can be used according to the manufacturer's instructions

Antibody activity in vitro: phagocytosis and death of target cells

In addition to specifically binding to CLDN6, antibodies against this protein are tested for their ability to mediate phagocytosis and death of cells that express CLDN6 and that differ in that CLDN6 is bound to their surface. The results of testing the activity of monoclonal antibodies in vitro serve as an initial screening prior to testing in in vivo models.

Antibody-dependent cell-mediated cytotoxicity (ADCC):

In short, they do the following. polymorphonuclear cells (PMN), natural killer (NK) cells, monocytes, mononuclear cells or other effector cells are purified by density gradient centrifugation in Ficoll Hypaque medium, followed by lysis of red blood cells. The washed effector cells are suspended in RPMI containing 10% heat-inactivated fetal bovine serum or 5% heat-inactivated human serum and mixed with ⁵¹ Cr-labeled target cells that express CLDN6 and that have CLDN6 bound to their surface. at different quantitative ratios of effector cells and target cells. Alternatively, target cells are labeled with a fluorescence enhancement ligand (BATDA). A fluorimeter is used to measure the fluorescence of the europium chelate complex with an enhancing ligand. Alternatively, transfection of target cells with luciferase can be used. The added Lucifer yellow dye will only be oxidized by viable cells. Purified anti-CLDN6 immunoglobulins G are then added at various concentrations. Other human IgGs can be used as negative controls. Incubation (at 37°C) takes from 4 to 20 hours depending on the type of effector cells taken. Cytolysis in the samples was determined by measuring the release of ⁵¹ Cr in the presence of EuTDA chelate in the culture supernatant. Or a measure of the number of viable cells can be luminescence resulting from the oxidation of Lucifer yellow.

It is also tested whether anti-CLDN6 monoclonal antibodies enhance cytolysis in various combinations with other antibodies.

Complement-Dependent Cytotoxicity Anti-CLDN6 monoclonal antibodies are tested for their ability to mediate complement-dependent cytotoxicity using a variety of methods known in the art. For example, blood serum containing complement is prepared by methods known to those skilled in the art. Various methods can be used to determine the activity of monoclonal antibodies against complement-dependent cytotoxicity. For example, the release of ⁵¹ Cr can be measured or the change in membrane permeability can be assessed using propidium iodide (PI). Briefly do the following. Target cells are washed and 5 x 10 ⁵ /ml incubated with various concentrations of monoclonal antibodies for 10-30 minutes at room temperature or at 37°C. Serum or plasma is added to a final concentration of 20% (v/v) and the cells are incubated at 37°C for 20-30 min. From each sample, all cells are placed in propidium iodide solution in FACS tubes and immediately analyzed by flow cytometry using a FACS plate.

Alternatively, the induction of complement-dependent cytotoxicity can be determined on adherent cells. In one embodiment of this approach, 24 hours before the experiment, cells are seeded onto flat-bottomed microtiter plates at a density of 3 x 10 ⁴ /well. The next day, the culture medium is removed and the cells are incubated with antibodies (in three parallel experiments). Control cells are incubated with culture medium or with culture medium containing 0.2% saponin to determine background lysis and maximum lysis, respectively. After 20 min of incubation at room temperature, the supernatant was removed and 20% (v/v) human serum or plasma in DMEM (warmed to 37°C) was added to the cells and incubated for an additional 20 min at 37°C. All cells from each sample are placed in a solution of propidium iodide (10 μg/ml). The supernatants were then replaced with PBS containing 2.5 μg/ml ethidium bromide and fluorescence emission was measured at 600 nm (excitation wavelength 520 nm) using a Tecan Safire microplate reader. The percentage of specific lysis is calculated as % specific lysis = (sample fluorescence - background fluorescence) / (fluorescence at maximum lysis - background fluorescence) x 100.

Inhibition of cell proliferation by monoclonal antibodies

To test the ability of anti-CLDN6 monoclonal antibodies to induce apoptosis, they can, for example, be incubated with tumor cells harboring CLDN6 or with tumor cells transfected with CLDN6. Incubation is carried out at 37°C for 20 hours. Cells were then harvested, washed with Annexin V binding buffer (BD biosciences), and incubated with fluorescein isothiocyanate (FITC) or allophycocyanin (APC)-conjugated annexin V (BD biosciences) for 15 min in the dark. From each sample, all cells were placed in propidium iodide (PI) solution (10 μg/ml in PBS) in FACS tubes and immediately performed with flow cytometry (as described above). It is also possible to determine the general inhibition of cell proliferation by monoclonal antibodies using ready-made commercial kits. Thus, the DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) allows for immunological analysis without the use of radioactive isotopes based on the quantitative determination of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the synthesized DNA of proliferating cells on microplates. BrdU incorporation is detected using europium-labeled monoclonal antibodies. To detect antibodies, cells are fixed and DNA denatured using a fixative solution. Unbound antibodies are washed and the DELFIA inducer is added to dissociate europium ions, which pass from the labeled antibodies into the solution, where they form intensely fluorescent chelate complexes with the components of the DELFIA inducer. The resulting measurement (with time resolution) characterizing fluorescence in each cell is proportional to the intensity of DNA synthesis in the cells.

Preclinical studies

Monoclonal antibodies that bind to CLDN6 can also be tested in in vivo models (eg, immunodeficient mice bearing xenograft tumors inoculated with CLDN6-expressing cells, possibly after transfection), to determine their effectiveness in regulating tumor proliferation. cells in which CLDN6 is expressed.

In in vivo studies using immunocompromised mice and tumor cell xenografts expressing CLDN6, antibodies of the present invention can be used. These antibodies are administered to tumor-free mice, which are then injected with tumor cells and the effect of the antibodies in preventing the formation of tumors or tumor-related symptoms is assessed. The antibodies under study are also administered to mice with tumors to evaluate their therapeutic effect, i.e. reducing tumor growth, metastasis or tumor-related symptoms. The use of antibodies of this invention can be combined with the use of other substances used as cytotoxic drugs, growth factor inhibitors, cell cycle or angiogenesis blocking agents, or other antibodies to evaluate the synergistic effect and potential toxicity of such combinations. To study the toxic side effects mediated by the antibodies of this invention, animals are administered the appropriate antibodies or regulatory agents and the symptoms that may be associated with the therapeutic effects of anti-CLDN6 antibodies are assessed in detail. Possible side effects of in vivo use of anti-antibodies include, but are not limited to, toxicity to tissues in which CLDN6 is expressed, including the placenta. Antibodies that recognize CLDN6 in humans and other species, such as mice, are particularly useful in predicting possible side effects mediated by the use of monoclonal antibodies against CLDN6 in humans.

Epitope mapping

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

I. Bispecific/polyspecific substances that bind to CLDN6

In another embodiment of the present invention, anti-CLDN6 antibodies are modified or coupled to other functional molecules, such as peptides or proteins (eg, Fab' fragments) to create bispecific or polyspecific molecules that bind to multiple target binding sites or epitopes. For example, an antibody of the present invention may be operably linked (eg, by chemical conjugation, association of genetic material, non-covalent linkage, or otherwise) to one or more other binding molecules, such as another antibody, peptide, or binding mimetic.

Accordingly, the present invention includes bispecific and polyspecific substances comprising at least one first binding specificity for CLDN6 and a second binding specificity for a second target epitope. In one specific embodiment of the present invention, the second target epitope is an Fc receptor, such as the human Fc-γRI (CD64) or Fc-α (CD89) receptors, or a T cell receptor, such as CD3. Thus. This invention includes bispecific and polyspecific substances capable of binding both to effector cells (for example, monocytes, macrophages or polymorphonuclear cells) in which Fc-γR, Fc-αR or Fc-αR is expressed, and to target cells in which CLDN6 and which differ in that CLDN6 is bound to their surface. These bispecific and multispecific substances make cells that express CLDN6 and that differ in that CLDN6 is associated with their surface target effector cells and initiate Fc receptor-mediated activity of effector cells, such as phagocytosis of cells that express CLDN6 and that differ in that that CLDN6 is associated with their surface, antibody-dependent cell-mediated cytotoxicity, cytokine release, or superoxide anion formation.

Bispecific and polyspecific agents of this invention may also have a third binding specificity, in addition to the Fc binding specificity and the CLDN6 binding specificity. In one embodiment of the present invention, the third binding specificity relates to a portion directed to an enhancing factor (EF), for example a substance that binds to a cell surface protein involved in cytotoxic activity and thereby enhances the immune response directed to target cells. The portion of the molecule of the multispecific agent that binds to the enhancing factor may be an antibody, a functional antibody fragment, or a ligand. binding to a given substance, such as an antigen or a receptor, and thereby causing an enhanced effect of binding determinants for the Fc receptor or antigen of the target cell. The enhancing factor binding portion of the multispecific agent molecule may bind to the Fc receptor or antigen of the target cell. Or, the part of the molecule of a multispecific agent that binds to the enhancing factor may bind to a structure different from the two that comprise the first and second specificities of the agent. For example, the enhancing factor-binding portion of a multispecific agent molecule may bind to a cytotoxic T cell (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or another cell of the immune system, leading to increased immune response). response directed against the target cell).

In one embodiment of the present invention, bispecific and polyspecific agents contain at least one antibody providing binding specificity, including, for example, Fab, Fab', F(ab') ₂ , Fv or single chain Fv. The antibody may also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single chain construct as described in Ladner et al., US Pat. No. 4,946,778. This antibody may also be a fusion immunoglobulin binding domain, as described in US patent applications 2003/0118592 and 2003/0133939.

In one embodiment of the present invention, bispecific and polyspecific agents have a specificity for binding to an Fc-γR or Fc-αR present on the surface of effector cells and a second specificity for binding to a target cell antigen, such as CLDN6.

In one embodiment of the present invention, Fc receptor binding specificity is provided by a monoclonal antibody whose binding is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight gamma chain genes found on chromosome 1. These genes collectively encode twelve transmembrane or soluble receptor isoforms, which are grouped into three classes of Fc-γ receptors: Fc-γRI (CD64 ), Fc-γRII (CD32) and Fc-γRIII (CD16). In one preferred embodiment of the present invention, the Fc-γ receptor is a human high-affinity Fc-γRI.

In another preferred embodiment of the present invention, Fc receptor binding specificity is provided by an antibody that binds to a human IgA receptor, for example Fc-α receptor (Fc-αRI (CD89)), which binding is preferably not blocked by human immunoglobulin A (IgA). The term “IgA receptor” refers to the gene product of a single α gene (Fc-αRI), located on chromosome 19. This gene is known to encode several alternatively spliced isoforms with molecular weights ranging from 55 to 110 kDa. Fc-αRI (CD89) is constitutively expressed on monocytes/macrophages, granulocytes (eosinophils and neutrophils), but not on effector cells. Fc-αRI has a moderate affinity for IgA1 and IgA2, which is increased by cytokines such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16: 423-440). Four Fc-αRI-specific monoclonal antibodies have been identified (designated A3, A59, A62 and A77) that bind to Fc-αRI outside the IgA binding domain (Monteiro, R. C. et al. (1992) J. Immunol. 148: 1764).

In another embodiment of the present invention, the bispecific agents comprise two monoclonal antibodies of the invention having complementary activities, eg, one antibody acts primarily by inducing complement-dependent cytotoxicity and the other primarily induces apoptosis.

The term “effector cell-specific antibodies” as used herein refers to antibodies or functional antibody fragments that bind to the Fc receptor of effector cells. Preferably, the antibodies used in this invention bind to the Fc receptor of effector cells at a site that does not bind endogenous immunoglobulins.

As used herein, the term "effector cell" is used to refer to cells of the immune system that participate in the effector phase of an immune response, as opposed to the antigen recognition phase and the activation phase of effector elements. Examples of immune system cells involved in the effector phase of the immune response include cells of myeloid and lymphoid origin, such as lymphocytes (eg, B cells and T cells, including cytolytic T cells (CTL)), killer cells, natural killer (NK) cells -cells), macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells and basophils. Some effector cells express specific Fc receptors and these cells perform specific functions. In preferred embodiments of the present invention, effector cells are capable of causing antibody-dependent cell-mediated cytotoxicity (ADCC), for example, neutrophils can cause ADCC. For example, monocytes and macrophages in which FcR is expressed are involved in the specific killing of target cells and in presenting antigen to other components of the immune system or in binding antigen-presenting cells. In other embodiments of the present invention, effector cells can engulf a target antigen, target cell, or microorganism by phagocytosis. The expression of specific FcRs on effector cells can be regulated by humoral factors such as cytokines. Thus, it was found that the expression of Fc-γRI is regulated, namely enhanced, by γ-interferon. As a result of this increased expression, the cytotoxic activity of Fc-γRI-bearing cells directed against target cells increases. Effector cells can phagocytose or lyse the target antigen or target cells.

The term “target cell” as used herein means any undesired cell in an individual (eg, human or animal) that may be a target for the antibodies of the present invention. In preferred embodiments of the present invention, the target cells are cells in which CLDN6 is expressed or overexpressed and which are characterized in that CLDN6 is associated with their surface. Cells that express CLDN6 and that are characterized by having CLDN6 associated with their surface typically include tumor cells.

II. Immunoconjugates

In another aspect, the invention provides anti-CLDN6 antibodies conjugated to a therapeutic structure or agent, eg, a cytotoxin, a drug (eg, an immunosuppressant), or a radioactive isotope. Such conjugates are referred to herein as immunoconjugates. Immunoconjugates comprising one or more cytotoxins are referred to herein as immunotoxins. Cytotoxins or cytotoxic agents include any agent that is harmful to living cells, in particular killing them. Examples of such substances include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and their analogues or substances homologous to them.

Therapeutic agents suitable for forming the immunoconjugates of this invention include, but are not limited to the examples provided herein, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine), alkylating agents (e.g., meclorethamine , thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin, formerly known as daunomycin, and doxorubicin), antibiotics (eg, dactinomycin, formerly known as actinomycin, bleomycin, mithramycin and anthramycin) and agents that inhibit mitosis (eg, vincristine and vinblastine). In one preferred embodiment of the present invention, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment of the present invention, the therapeutic agent is an immunosuppressant. In yet another embodiment of the present invention, the therapeutic agent is granulocyte-macrophage colony-stimulating factor (GM-CSF). In one preferred embodiment of the present invention, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin A.

Antibodies of this invention can also be conjugated to radioactive isotopes, such as iodine-131, yttrium-90 or indium-111, to create cytotoxic radioactive drugs for the treatment of disorders associated with CLDN6, such as cancer. The antibody conjugates of this invention can be used to modify a particular biological response, and the therapeutic construct should not be considered limited to classical chemotherapeutic agents. For example, this structure may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1, interleukin-2, interleukin-6, granulocyte-macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or other growth factors.

Techniques for conjugating such structures having a therapeutic effect with antibodies are well known in the art; see, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pincheraet al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

In yet another embodiment of the present invention, the antibodies are attached to a chelating linker structure, such as tiuxetan, which allows a radioactive isotope to be attached to the antibody.

III. Pharmaceutical compositions

In another aspect, the present invention provides compositions, for example pharmaceutical compositions, containing one variant of antibodies or a combination of antibodies according to this invention. These pharmaceutical compositions may contain pharmaceutically acceptable carriers and diluents, as well as any other known additional components and excipients in accordance with generally accepted procedures described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment of the present invention, the present compositions include a combination of multiple (e.g., two or more) isolated antibodies of the present invention acting through different pathways, for example, a single antibody that predominantly causes complement-dependent cytotoxicity is combined with another antibody that preferentially induces apoptosis.

The pharmaceutical compositions of this invention can be used as part of a combination therapy, that is, combined with other therapeutic agents. For example, such combination therapy may include a composition of this invention together with at least one anti-inflammatory agent or with at least one immunosuppressive agent. In one embodiment of the present invention, such therapeutic agents include one or more anti-inflammatory agents, such as steroid drugs or non-steroidal anti-inflammatory drugs. Preferred agents include, for example, aspirin and other salicylates, cyclooxygenase-2 (Cox-2) inhibitors, for example, rofecoxib (Vioxx), celecoxib (Celebrex), non-steroidal anti-inflammatory drugs, for example, ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen ( Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin).

In another embodiment of the present invention, such therapeutic agents include agents that cause depletion or functional inactivation of regulatory T cells; these are low-dose cyclophosphamide, anti-CTLA4, anti-interleukin-2 or anti-interleukin-2 receptor antibodies.

In yet another embodiment of the present invention, such therapeutic agents include chemotherapeutic drugs, for example, taxol derivatives, Taxotere, gemcitabine, 5-fluorouracil, doxorubicin (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan, Procytox, Neosar). In another embodiment of the present invention, the antibodies of the invention may be administered to a subject in combination with chemotherapeutic agents that preferably exhibit therapeutic efficacy in patients with cancer, such as the cancers described herein.

In yet another embodiment of the present invention, the present antibodies can be administered to an individual in parallel with radiation therapy and/or autologous peripheral blood or bone marrow stem cell transplantation.

As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial or antifungal agents, osmotic pressure agents and absorption delaying agents, and other agents that are physiologically compatible. Preferably, the carrier of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active substance, such as antibodies, bispecific and polyspecific compounds, may be coated with a material to protect the active substance from acids and other natural factors that can inactivate the active substance.

The term “pharmaceutically acceptable salt” as used herein refers to salts that retain the biological activity of the parent compound and do not introduce any undesirable toxic effects (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19).

Examples of such salts include acid salt adducts and alkaline salt adducts. Acid salt adducts include derivatives of non-toxic inorganic acids, for example hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acids, etc., as well as derivatives of non-toxic organic acids, for example aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Alkaline salt adducts include derivatives of alkali and alkaline earth metals, for example sodium, potassium, magnesium, calcium, etc., as well as derivatives of non-toxic organic amines, for example N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and so on.

The compositions of the present invention can be administered to an individual by various methods known in the art, in which those skilled in the art will understand that the route and/or method of administration will vary depending on the desired results. The active substance may be combined with a carrier. which will prevent its rapid release, for example the active substance may be formulated in a controlled release dosage form, including implants, transdermal patches and microcapsule systems enclosing the active substance. Biodegradable and biocompatible polymers can be used here, for example ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Methods for preparing such preparations are well known in the art; see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Some methods of introducing an active substance into the body may require that the substance be coated with or administered together with a material that prevents inactivation. For example, the active substance may be administered to the individual in a suitable carrier, for example liposomes, or in a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water emulsions (CGF Inc.) as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for preparation of an injectable solution or dispersion immediately prior to administration. The use of such media and agents for pharmaceutically active substances is known in the art. Except in cases of incompatibility of conventional vehicles or agents with the active substance of this invention, any vehicles and agents can be used in the pharmaceutical compositions of this invention. These compositions may also contain additional active substances.

In general, therapeutic compositions must be sterile and stable under the conditions of manufacture and storage. The compositions of this invention may be presented as a solution, microemulsion, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethyl alcohol, polyhydric alcohol (For example, glycerin, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. The desired fluidity is maintained, for example, by using a coating material such as lecithin, by achieving the required particle size (in the case of dispersions) and by using surfactants. In many cases, it is preferable to include agents that provide the desired osmotic pressure, such as sugars, polyhydric alcohols (eg, mannitol, sorbitol) or sodium chloride. Prolongation of absorption of a composition administered by injection is achieved by including in its composition an agent that delays absorption, for example monosubstituted salts of stearic acid and gelatin.

Sterile injection solutions can be prepared by combining the required amount of the active substance in a suitable solvent with one or a combination of the above ingredients and subsequent sterilization by microfiltration.

Typically, dispersions are prepared by incorporating the active substance into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those mentioned above. In the case of sterile powders for the preparation of sterile injection solutions, the preferred methods for their preparation are vacuum drying and freeze drying (lyophilization), which allow the active substance to be obtained in powder form along with any additional desired ingredient from their solution, previously sterilized by filtration.

The dosage and mode of administration are selected to ensure the optimal desired response (for example, therapeutic effect). For example, a dose may be administered as a single dose or in divided doses over a period of time, or the dose may be proportionally decreased or increased depending on the therapeutic indications in a particular situation. It is particularly preferable to formulate the composition for parenteral administration as a single dosage form to facilitate administration and ensure uniformity of dosage. As used herein, the term unit dosage form means physically separate units convenient for single administration to an individual in need; each unit contains a predetermined amount of active substance, calculated to achieve the desired therapeutic effect, together with the desired pharmaceutical carrier.

Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble substances such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) fat-soluble substances, such as ascorbyl palmitate, hydroxyanisole butylate (BHA), hydroxytoluene butylate (HNT), lecithin, propyl gallate, α-tocopherol, etc.; (3) metal complexing agents (chelating agents), such as citric acid, ethylenediaminetetraacetate (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

The therapeutic compositions of this invention include those intended for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. These preparations may be conveniently presented as a unit dosage form and may be manufactured by any method known in the pharmaceutical art. The amount of active ingredient combined with the carrier material to obtain a unit dosage form varies depending on the individual needs of the patient and the specific route of administration of the drug. Generally, the amount of active ingredient combined with the carrier material to produce a unit dosage form should be such as to produce a therapeutic effect.

Formulations of this invention suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or aerosol formulations containing suitable carriers known in the art. Dosage forms for topical and transdermal administration of the compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalable preparations. The active substance can be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if necessary, with any preservatives, buffering agents or propellants.

The term “parenteral administration” and the expression “administered parenterally” as used herein mean methods of administering a drug to the body other than the enteral and local routes; they typically involve injections and include (but are not limited to those listed here) intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subaracnoidal, intraspinal, epidural, and intrasternal injections and infusions. Zia .

Examples of aqueous and non-aqueous carriers suitable for use in the pharmaceutical compositions of this invention include water, ethyl alcohol, polyhydric alcohols (for example, glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (for example, olive oil ), injectable organic esters (eg ethyl oleate). Proper fluidity is maintained, for example, by using coating materials such as lecithin, by achieving the required particle size (in the case of dispersions) and by using surfactants.

The compositions of this invention may also contain additional components, for example preservatives, wetting agents, emulsifying and dispersing agents. The presence of microorganisms can be prevented either by sterilization in one way or another, or by including antibacterial and antifungal agents in the composition, for example parabens, chlorobutanol, phenolsorbic acid, etc. It may also be desirable to include in the composition agents that provide the desired osmotic pressure, for example sugars, sodium chloride, etc. In addition, prolongation of absorption of the pharmaceutical form administered by injection is achieved by the inclusion of absorption delaying agents, for example monosubstituted aluminum stearate and gelatin.

Regardless of the route of administration chosen, the compounds of this invention, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions of this invention are formulated into pharmaceutically acceptable dosage forms using methods known to those skilled in the art.

The specific content of active ingredients in the pharmaceutical compositions of this invention and the dosage of the drug can be varied so that the active ingredient is present in an effective amount to produce the desired therapeutic effect in a particular patient for a particular formulation of the drug and for the chosen route of administration, without at the same time causing toxic effect on a given individual. Selecting the concentration of the active ingredient in the drug and dosage

of the drug are determined by a number of pharmacokinetic factors, including the activity of the specific composition used according to this invention, the method and mode of administration of the drug, the rate of elimination of this compound from the body, the duration of the course of treatment, other drugs, substances and/or materials used in combination with these compositions, age , gender, body weight, general and specific health of the patient, medical history, as well as other factors well known in the medical field.

It will not be difficult for doctors and veterinarians familiar with this field of technology to determine and prescribe the amount of the desired pharmaceutical composition that is effective in a particular case. For example, the use of the compounds of this invention in pharmaceutical compositions in humans or animals can be started at doses below those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally speaking, a suitable daily dose of the composition of this invention will be one at which the amount of active substance is minimal to effectively provide a therapeutic effect. Such an effective dose will generally depend on the factors discussed above. Preferred routes of administration are intravenous, intramuscular, intraperitoneal or subcutaneous, with the site of administration preferably proximal to the target location. If desired, an effective daily dose of the therapeutic composition may be administered in two, three, four, five, six or more divided doses spaced at appropriate intervals throughout the day, using unit dosage forms if necessary. Although the compound of this invention can be administered by itself, it is preferable to administer it as part of a pharmaceutical preparation (composition).

In one embodiment of the present invention, the antibodies of the invention are administered to a patient by infusion, preferably slowly over an extended period of time, for example over 24 hours, to reduce toxic side effects. Administration by continuous infusion can also be carried out over a period of 2 to 24 hours, for example from 2 to 12 hours, repeating, if necessary, one or more times after, for example, 6 or 12 months. The dosage can be determined or adjusted as appropriate by measuring the amount of anti-CLDN6 monoclonal antibodies in the bloodstream after administration of a biological sample using anti-idiotypic antibodies that target anti-CLDN6 antibodies.

In another embodiment of the present invention, the antibodies of the invention are administered in a maintenance regimen, for example once a week for 6 months or longer.

In yet another embodiment of the present invention, the antibodies of the invention are administered in a regimen comprising a single infusion of anti-CLDN6 antibodies followed by an infusion of anti-CLDN6 antibodies conjugated to a radioactive isotope. This scheme can be repeated, for example 7-9 days later.

In one embodiment of the present invention, therapeutic substances are included in liposomes. In a more preferred embodiment of the present invention, the liposomes carry a targeting structure. In the most preferred embodiment, the therapeutic substances in liposomes are administered in a loading dose, with the site of administration proximal to the desired site of the body, for example the site of a tumor. The composition should be sufficiently liquid to be easily administered by syringe. And it must be stable under the conditions of manufacture and storage, protected from contamination by microorganisms such as bacteria and fungi.

In yet another embodiment of the present invention, the antibodies of the invention are processed in this manner. to prevent or reduce their transfer across the placenta. This is achieved by methods known in the art, for example by PEGylation of antibodies or using F(ab)2' fragments. Reference may also be made to Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation. J. Immunol. Methods, 152: 177-190; "Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann. Allergy Asthma Immunol. 74: 279-283.

The therapeutically effective dose for oncology therapy can be determined by the objective response of the tumor, which can be complete or partial. Complete response (CR) is the absence of clinical, radiological or any other signs of disease. Partial response (PR) is a reduction in tumor size by more than 50%. The duration of objective tumor response is characterized by the average time to progression.

The therapeutically effective dose of a drug for oncology therapy can also be determined by its ability to stabilize the progression of the disease. The ability of a substance to suppress cancer can be assessed using animal models, which can predict the drug's effectiveness against human tumors. Alternatively, this property of the composition can be assessed by studying the ability of the substance to inhibit cell proliferation or apoptosis by in vitro methods known to those skilled in the art. A therapeutically effective amount of a therapeutic agent may reduce the size of a tumor or otherwise reduce symptoms of a disease in a patient. One of ordinary skill in the art would be able to determine this amount based on factors such as the patient's body weight, severity of symptoms, the specific composition of the formulation, and the chosen route of administration.

The composition of this invention must be sterile and liquid enough to be administered by syringe. In addition to water, the carrier may be isotonic buffered saline, ethyl alcohol, polyhydric alcohol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity is maintained, for example, by using coating materials such as lecithin, by achieving the required particle size (in the case of dispersions) and by using surfactants. In many cases, it is preferable to include agents that provide the desired osmotic pressure, such as sugars, polyhydric alcohols (eg, mannitol or sorbitol) and sodium chloride. Prolonged absorption of the injectable composition is achieved by the inclusion of absorption delaying agents, such as aluminum stearate or gelatin.

When the active substance is properly protected as described above, it can be administered orally, for example with an inert diluent or digestible edible carrier.

IV. Applications and methods of this invention

The antibodies of this invention (including immunoconjugates, bispecific/multispecific agents, compositions and other derivatives described herein) have a variety of therapeutic uses relevant to the treatment of disorders affecting cells in which CLDN6 is expressed and which are characterized in that CLDN6 connected to their surface. For example, the antibodies of this invention can be administered to cultured cells, eg in vitro or ex vivo, or to human patients, eg in vivo, to treat or prevent various disorders described herein. Preferred targets include human patients with disorders that can be corrected or that can be alleviated by killing affected cells, particularly cells that have altered expression of CLDN6 compared to normal cells and/or altered association of CLDN6 with their surface.

For example, in one embodiment of the present invention, the antibodies of the invention are used to treat a patient with an oncogenic disorder, for example a disorder characterized by the presence of cells that express CLDN6 and that have CLDN6 associated with their surface. Examples of oncogenic diseases that can be treated and/or prevented are all cancers and tumors that express CLDN6, including those described herein.

The pharmaceutical compositions and methods of treatment of this invention can also be used for immunization or vaccination to prevent the diseases described herein.

In another embodiment of the present invention, the antibodies of the invention are used to determine the level of CLDN6 or specific forms of CLDN6, or the number of cells containing CLDN6 on the surface of their membrane, and the values thus obtained are then associated with certain diseases or symptoms such as those described above. Alternatively, the antibodies of this invention are used to deplete or disrupt the function of cells that express CLDN6 and that have CLDN6 associated with their surface; thereby revealing the important participation of these cells in the development of the disease. This is achieved by incubating the test and control samples with antibodies against CLDN6 under conditions that allow the formation of a complex between the antibodies taken and CLDN6. Any complexes formed as a result of the interaction of the taken antibodies with CLDN6 are identified, and the experimental and control (reference) samples are compared using them.

First, the antibodies of this invention are tested for their binding activity relevant to therapeutic and diagnostic applications in vitro. For example, this can be done using flow cytometry as described herein.

The antibodies of this invention can induce in vivo or in vitro one or more of the following biological activities: inhibition of proliferation and/or differentiation of cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface; killing cells that express CLDN6 and that have CLDN6 associated with their surface; mediating phagocytosis of cells in which CLDN6 is expressed and which differ in that CLDN6 is associated with their surface, or directed at them by antibody-dependent cellular cytotoxicity in the presence of effector cells; mediating complement-dependent cytotoxicity directed at cells that express CLDN6 and that have CLDN6 bound to their surface in the presence of complement; mediating apoptosis of cells in which CLDN6 is expressed and which differ in that CLDN6 is associated with their surface; induction of homotypic adhesion; and/or inducing trafficking into lipid rafts following CLDN6 binding.

In one specific embodiment of the present invention, the antibodies are used in vivo or in vitro for the treatment, prevention or diagnosis of various diseases associated with CLDN6. Examples of diseases associated with CLDN6 include, but are not limited to, cancers, such as those described herein.

As discussed above, the anti-CLDN6 antibodies of the present invention can be administered to a patient along with one or more other therapeutic agents, such as cytotoxic agents, radioactive agents, anti-angiogenesis agents, and/or immunosuppressants, in order to attenuate the induction of an immune response against the anti-CLDN6 antibodies. this invention. These antibodies can be combined with one or another agent, forming an immunocomplex, or they can be administered separately from the agent taken. In the latter case (separate administration), the antibodies can be administered before, after or simultaneously with an additional agent or can be combined with other known therapeutic approaches, for example, with anti-cancer therapy, such as radiation therapy. Such therapeutic agents include, but are not limited to, anti-neoplasia agents such as those mentioned above. Administration of the anti-CLDN6 antibodies of the present invention along with chemotherapeutic agents produces dual anticancer effects through different mechanisms, resulting in a cytotoxic effect on tumor cells. Such co-administration may solve the problem of developing drug resistance or changing the antigenic properties of the tumor, which makes it unable to interact with these antibodies.

Compositions (eg, antibodies, polyspecific and bispecific agents, immunoconjugates) of this invention containing complement binding sites, such as complement binding sites in IgG1, -2 or -3 or IgM molecules, can also be used in the presence of complement. In one embodiment of the present invention, ex vivo treatment of a population of cells comprising target cells with the binding agent of the present invention and corresponding effector cells is supplemented by the addition of complement or serum containing complement. Binding of complement proteins promotes phagocytosis of target cells carrying on their surface the binding agent of this invention. In another embodiment of the present invention, cells bearing on their surface the compositions of this invention are subject to lysis with the participation of complement. In yet another embodiment of the present invention, the present compositions do not activate complement.

The compositions of this invention can be administered to the body along with complement. Accordingly, compositions containing antibodies, polyspecific and bispecific agents, and serum or complement are included within the scope of this invention. These compositions have the advantage that complement is in close proximity to antibodies, bispecific and polyspecific molecules.

Alternatively, the antibodies, polyspecific and bispecific agents of the invention, and serum or complement are administered separately. Binding of the compositions of the invention to target cells may cause the CLDN6 antigen-antibody complex to be transported into a lipid raft of the cell membrane. This transfer creates a high density of antigen-antibody complexes that can effectively activate and/or enhance complement-dependent cytotoxicity.

Also included within the scope of this invention are kits comprising antibody compositions (eg, antibodies and immunoconjugates) and instructions for their use. Such a kit may also contain one or more additional reagents, such as an immunosuppressive agent, a cytotoxic agent, or a radiotoxic agent, or one or more additional antibodies of the invention (eg, antibodies with complementary activity).

Accordingly, patients receiving antibody compositions of the present invention may be additionally administered (before, concurrently with, or after administration of the antibodies) another therapeutic agent, such as a cytotoxic or radiotoxic agent, that enhances or prolongs the therapeutic effect of the antibodies of the present invention.

In other embodiments of the present invention, the patient may additionally receive an agent that modulates, for example enhancing or inhibiting, the expression or activity of Fc-γ or Fc-α receptors by; such an agent may be, for example, a cytokine. Preferred cytokines in this regard include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factor (TNF). Important agents for enhancing the therapeutic efficacy of antibodies and pharmaceutical compositions described herein include β-glucans, which are branched homopolysaccharides consisting of glucose units; they are produced by a variety of plants (eg cereal seeds) and microorganisms such as bacteria, algae and fungi (including yeast). Fragments of β-glucan molecules produced by living organisms can also be used. Preferred β-glucans are polymers of β(1,3) glucose in which at least some of the main chain glucose residues, for example 3-6% of the main chain glucose residues, have β(1,6) branches.

In one specific embodiment, the present invention provides methods for detecting the presence of CLDN6 antigen in a sample or measuring the amount of CLDN6 antigen, comprising treating the test sample and a control sample with antibodies that specifically bind to CLDN6 under conditions that allow the antibody or portion thereof to form a complex with CLDN6 . The formation of such a complex is detected, and the difference in the corresponding indicator between the analyzed and control samples serves as a measure of the presence of the CLDN6 antigen in the sample.

In yet another embodiment of the present invention, a method is provided for detecting the presence or measuring the number of cells that express CLDN6 and that have CLDN6 associated with their surface, in vivo or in vitro. This method includes (i) introducing into the test subject (patient) a composition according to this invention conjugated with a detectable marker; (ii) exposing the subject of analysis (the patient) to an agent for detecting said marker in order to identify regions containing cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface.

The above methods can be used, in particular, for diagnosing diseases in which CLDN6 is important and/or determining the location of lesions in CLDN6-related diseases, such as cancer. Preferably, an indication that an individual, particularly a human, has a CLDN6-related disease is the presence of CLDN6 in a test sample taken from said individual in an amount greater than that in a control sample.

When used in the methods mentioned above, the antibodies described herein can carry a label that can perform the following functions: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, such as fluorescence resonance energy transfer (FRET); (iii) influence mobility, eg electrophoretic mobility, by changing charge, hydrophobicity, shape or other physical properties, or (iv) provide/create binding structure, eg provide affinity, antigen/antibody interaction or ionic complex formation. The following structures are suitable as such labels: fluorescent labels, luminescent labels, chromophore labels, radioactive labels, isotopic labels (preferably stable), isobaric labels, enzyme labels, corpuscular labels (in particular metal particles), magnetic corpuscular labels, polymer corpuscular tags, low molecular weight organic compounds (eg, biotin), receptor ligands or binding compounds (eg, adhesion proteins or lectins), sequences including nucleic acids and/or amino acid residues that can be detected using binding agents, and the like. Labels include (but are not limited to those listed here) barium sulfate, yocetamic acid, iopanoic acid, calcium iopodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate, and imaging agents including positron sources (e.g., fluorine-18 and carbon-11), γ-ray sources (eg iodine-123, technetium-99m, iodine-131 and indium-111), nuclear magnetic resonance nuclides (eg fluorine and gadolinium).

In another embodiment of the present invention, immunoconjugates are used to target compounds (eg, therapeutic agents, tags, cytokines, radioactive substances, immunosuppressants, etc.) to cells bearing CLDN6 on their surface by attaching such compounds to antibodies. Thus, the present invention also provides methods for detecting ex vivo or in vitro the location of cells in which CLDN6 is expressed and which are characterized in that CLDN6 is associated with their surface, for example, tumor cells circulating in the bloodstream.

The present invention is further illustrated by examples, which should not be considered limiting the scope of the present invention.

Examples

The techniques and methods used in this invention were carried out or described herein in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods, including the use of kits and reagents, were performed according to the information provided by the manufacturer unless otherwise stated.

Example 1: Quantification of CLDN6 Expression in Normal Tissues, Cancer Tissues and Cell Lines by Real-Time Polymerase Chain Reaction (RT-PCR)

Total cellular RNA was isolated from frozen tissue samples and cancer cell lines using the RNeasy Mini Kit (Qiagen); For priming, oligo(dT ₁₈ ) reverse transcription Superscript II (GIBCO/Lifetech) was used according to the manufacturer's instructions. The integrity of the resulting cDNA was verified by amplifying the p53 transcript in 30 cycles of PCR. The results were normalized to the HPRT gene and the expression value of CLDN6 was calculated using ΔΔCT.

For each normal tissue, samples taken from three individuals were examined. In normal tissues, only trace amounts of CLDN6 transcripts were detected after 40 cycles of RT-PCR. The only normal tissue with CLDN6 expression slightly above the detection threshold was the placenta.

We found that, in contrast to normal tissues, in samples from tumor tissue, the expression level of CLDN6 is high in ovarian cancer (adenocarcinoma), lung cancer (non-small cell, with the highest frequency and expression level in adenocarcinomas), gastric cancer, breast cancer, liver cancer , pancreatic cancer, skin cancer (basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (malignant pleomorphic adenoma), sarcoma (synovial sarcoma and carcinosarcoma), bile duct cancer, renal cell carcinoma (clear cell carcinoma and papillary carcinoma ) and uterine cancer, as well as in cell lines A2780 (ovarian cancer), NIH-OVCAR3 (ovarian cancer), HCT-116 (colon cancer), EFO-27 (ovarian cancer), CPC-N (SCLC), NCI- H552 (NSCLC), SNU-1 (stomach cancer), KATOIII (stomach cancer), YAPC (pancreatic cancer), AGS (stomach cancer), FU97 (stomach cancer), MKN7 (stomach cancer).

Example 2: Quantification of CLDN6 Expression in Normal Tissues, Cancer Tissues and Cell Lines by Western Blot

For Western blot analysis, 20 μg of total protein isolated from cells lysed with Lemley lysis buffer (Lemley P.V.) was taken. Extracts were diluted in reducing sample buffer (Roth), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and then electroblotted onto a polyvinylidene fluoride (PVDF, Pall Corporation) membrane. Immunostaining was performed with polyclonal antibodies against CLDN6 (ARP) and β-actin (Abcam), then the primary antibodies were detected using horseradish peroxidase conjugated to goat anti-mouse and goat anti-rabbit secondary antibodies (Dako).

For each normal tissue, tissue lysates from five individuals were examined. CLDN6 protein expression was not detected in any of the normal tissues examined. In contrast to normal tissues, high levels of CLDN6 protein expression were observed in tumor tissue samples from ovarian and lung cancer. Expression of CLDN6 was detected in the cell lines NIH-OVCAR3 (ovarian cancer), MKN7 (stomach cancer), AGS (stomach cancer), CPC-N (small cell lung cancer), HCT-116 (colon cancer), FU97 (stomach cancer) , NEC8 (embryonic testicular carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (teratocarcinoma of the ovary).

Example 3 Immunohistochemical (IHC) Analysis of CLDN6 Expression in Normal Tissues and Cancer Tissues

Paraffin-embedded tissue sections (4 μm) were incubated for 1 hour at 58°C on a heating stage (HI 1220, Leica). Paraffin was removed from the sections by incubating the slides with the sections in Roticlear solvent (Roth) twice for 10 min at room temperature. The sections were then rehydrated in decreasing concentrations of alcohol (99%, 2 x 96%, 80% and 70%, 5 min each). To unmask antigenic determinants, preparations were boiled at 120°C, 15 psi (lb/ ⁱⁿ² ) for 15 minutes in citrate buffer solution (10 mM, pH 6.0) with 0.05% Tween-20. Immediately after boiling, the slides were incubated in phosphate buffered saline (PBS) for 5 min. Endogenous peroxidase activity was blocked using hydrogen peroxide (0.3%) in methyl alcohol for 15 min at room temperature. To avoid nonspecific binding, the preparations were blocked with goat serum (10%) in PBS for 30 min at room temperature. The slides were then incubated with CLDN6-specific polyclonal antibodies (1 μg/ml) (ARP) overnight at 4°C. The next day, slides were washed with PBS at room temperature 3 times for 5 min each, followed by incubation with 100 μl of secondary antibodies (PowerVision ready-to-use anti-rabbit poly-HRP IgG (ImmunoLogic)) for 1 hour at room temperature. Then the preparations were washed with PBS at room temperature 3 times for 5 min. Final staining was performed using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame, CA, USA). Sections were counterstained with hematoxylin for 90 seconds at room temperature. The preparations were dehydrated in increasing concentrations of ethyl alcohol (70%, 80%, 2 x 96% and 99%, 5 min each) and incubated in xylene for 10 min, after which they were filled with X-tra Kit (Medite Histotechnic).

No CLDN6 protein expression was detected in normal tissues of the lung, ovary, stomach, colon, pancreas, liver, duodenum, and kidney. In contrast to normal tissue, tumor tissue sections showed intense or at least noticeable staining in cancers of the ovary, lung, skin, pancreas, stomach, breast, bladder (transitional cell carcinoma), cervix, testis (seminoma), and uterus. Staining was clearly focused on the plasma membrane of malignant epithelial cells, whereas adjacent stromal cells and nonmalignant epithelial cells were not stained. These results indicate that the CLDN6 protein is localized to the plasma membrane of malignant cells.

Example 4: Production of mouse anti-CLDN6 antibodies

a. Preparation of expression vectors encoding the complete CLDN6 protein and CLDN6 fragments

Non-natural codon optimized DNA sequences (SEQ ID NO: 3) encoding the complete polypeptide chain of CLDN6 (US National Center for Biotechnology Information (NCBI) registration number NP_067018.2, SEQ ID NO: 2) were obtained by chemical synthesis (GENEART AG, Germany) and cloned into the pcDNA3.1/myc-His vector (Invitrogen, USA), resulting in the p3953 vector. The insertion of a stop codon allowed the CLDN6 protein to be expressed without fusion with the myc-His tag encoded by this vector. CLDN6 expression was checked by Western blotting, flow cytometry, and immunofluorescence analysis using a commercially available anti-CLDN6 antibody (ARP, 01-8865; R&D Systems, MAB3656).

In addition, a codon optimized DNA sequence (SEQ ID NO: 4) encoding a putative extracellular domain 2 (EC2) fragment of CLDN6 (SEQ ID NO: 6) was obtained as an association with a signal peptide derived from the N-terminal leader sequence of the immunoglobulin κ chain , followed by 4 additional amino acid residues to provide the correct signal peptidase cleavage site (SEQ ID NO: 5) and cloned it into the pcDNA3.1/myc-His vector, resulting in the p3974 vector. Prior to immunization, EC2 fragment expression was confirmed by immunofluorescence microscopy on transiently transfected and paraformaldehyde (PFA)-fixed CHO-K1 cells using a commercially available anti-myc antibody (Cell Signaling, MAB 2276).

b. Generation of cell lines with stable expression of CLDN6

Cell lines HEK293 and P3X63Ag8U.1, in which CLDN6 was stably expressed, were obtained according to standard methods using the p3953 vector.

c. Immunization

Balb/c mice were immunized with 25 μg of p3974 plasmid DNA together with 4 μl of polyethylenimine mannose (PEI-Man; in vivo-jetPEI™-Man, manufactured by PolyPlus Transfection) (150 mM aqueous solution of PEI-Man with 5% glucose) by i.p. injections on days 0, 16, and 36. On days 48 and 62, animals were immunized (by intraperitoneal injection) with P3X63Ag8U.1 myeloma cells transfected with the p3953 vector to stably express CLDN6. Cells that were injected into mice on day 62 were exposed to radioactive radiation (3000 rad) before injection. The presence of antibodies against CLDN6 in the serum of experimental animals was determined by immunofluorescence microscopy between days 20 and 70, using CHO-K1 cells transfected with nucleic acid encoding CLDN6 together with GFP fluorescent protein. To do this, 24 hours after transfection, paraformaldehyde-fixed and unfixed cells were incubated with serum (1:100 dilution) from immunized dilution mice for 45 min at room temperature. Cells were washed, incubated with Alexa555 fluorophore-labeled anti-mouse immunoglobulins (Molecular Probes) and analyzed using a fluorescence microscope.

Antibodies specific for CLDN6 were determined in serum samples collected from mice used to produce the F3-6C3-H8 hybridoma; see figure 2.

To generate monoclonal antibodies, mice with a significant immune response against CLDN6 were immunized 4 days before spleen collection (by intraperitoneal injection) with HEK293 cells (2 x 10 ⁷ ) stably transfected with the p3953 vector.

d. Preparation of hybridomas producing mouse monoclonal antibodies against CLDN6

Splenocytes isolated from immunized mice in an amount of 6 x 10 ⁷ cells were fused with mouse myeloma cells line P3X63Ag8.653 (ATCC, CRL 1580) in an amount of 3 x 10 ⁷ using PEG 1500 (Roche, CRL 10783641001). Cells were plated on flat-bottomed microtiter plates (approximately 5 x 10 ⁴ per well) and cultured for about two weeks in RPMI selection medium containing 10% heat-inactivated fetal calf serum, 1% fusion and cloning supplement (HFCS, Roche, CRL 11363735), 10 mM HEPES, 1 mM sodium pyruvate, 4.5% glucose, 0.1 mM 2-mercaptoethanol, 1 x penicillin/streptomycin and 1 x HAT (hypoxanthine, aminopterin, thymidine) (Invitrogen, CRL 21060). After 10-14 days, each well was checked by flow cytometry for the presence of monoclonal antibodies against CLDN6. Hybridomas secreting the desired antibodies were subcloned by serial dilution and retested for the presence of monoclonal antibodies against CLDN6. Stable subclones were cultured to obtain small amounts of antibodies in the culture medium and characterize them. At least one clone from each hybridoma was selected that retained the activity of the original cells (checked by flow cytometry). For each clone, a stock of nine ampoules was made and stored in liquid nitrogen.

Example 5. Binding properties of hybridoma supernatants and monoclonal antibodies

a. Qualitative control of transiently transfected HEK293T cells by (i) Western blotting and (ii) flow cytometry

(i) HEK293T cells were transfected with nucleic acid encoding CLDN3, CLDN4, CLDN6 and CLDN9, respectively, or all manipulations were performed without nucleic acid as a control. The expression of CLDN3, CLDN4, CLDN6, or CLDN9 in HEK293T cells was determined by Western blotting. For this purpose, 24 hours after transfection, cells were collected and lysed. The lysate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred (by blot) to a nitrocellulose membrane and stained with antibodies against CLDN3(A) (Invitrogen, 34-1700), against CLDN4(A) (Zymed, 32-9400), against CDLN6(A) (ARP, 01-8865) or anti-CLDN9(A) (Santa Cruz, sc-17672), which specifically bind to the C-terminal region of the corresponding claudin under denaturing conditions. They were incubated with peroxidase-labeled secondary antibodies, developed with electrochemiluminophore (ECL reagent), and a LAS-3000 spectrometer (Fuji) was used for visualization. Bands corresponding to the expected molecular masses of CLDN3, CLDN4, CLDN6, and CLDN9 were observed only in the transfected cells and not in the control (Fig. 3); this demonstrates that HEK293T cells do not endogenously express any of the claudins and are therefore a useful tool for determining the cross-reactivity of anti-CLDN6 antibodies.

(ii) HEK293T cells from (i) were also analyzed by flow cytometry using anti-CLDN antibodies recognizing native epitopes (mouse anti-CLDN3 IgG2a (R&D, MAB4620), mouse anti-CLDN4 IgG2a (R&D, MAB4219), mouse anti-CLDN6 IgG2b (R&D , MAB3656)). These antibodies, manufactured by Sigma (product numbers M9144 and M8894), served as isotype controls. The specificity of these anticlaudin antibodies was determined using HEK293T cells transiently transfected with nucleic acids encoding CLDN3, CLDN4, CLDN6, and CLDN9, respectively. Antibodies against CLDN6 showed cross-reactivity with CLDN3, CLDN4 and CLDN9. Antibodies against CLDN4 showed cross-reactivity with CLDN3, CLDN6 and CLDN9. Anti-CLDN3 antibodies bound specifically to CLDN3 (Fig. 4).

b. Determination of the specificity of monoclonal antibodies obtained according to this invention by flow cytometry

HEK293T cells were transfected simultaneously with two vectors, one encoding various claudins and the other a fluorescent marker. 24 hours after transfection, cells were harvested using trypsin (0.05%)/EDTA and washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide). Cells were transferred to U-bottom microtiter plates at 2 x ¹⁰⁵ cells per well and incubated with hybridoma supernatants for 60 min at 4°C. Cells were then washed with FACS buffer and incubated with specific secondary antibodies (anti-mouse IgG 1+2a+2b+3, Dianova, 115-135-164) conjugated to allophycocyanin (APC). The cells were then washed twice and binding was determined using a BD FACS Array flow cytometer/bioanalyzer (Figure 5). Fluorescent marker expression is plotted on the horizontal axis, and antibody binding is plotted on the vertical axis. A commercially available mouse IgG2b anti-CLDN6 antibody (R&D, MAB3656) served as a positive control; Antibodies from Sigma (product catalog number M8894) served as isotype controls.

Monoclonal antibodies in supernatants from hybridoma subclones F3-6C3-H2, F3-6C3-H8, F3-6C3-H9, F3-6C3-D8 and F3-6C3-G4 (all derived from hybridoma F3-6C3) were specific for CLDN6 and did not bind to CLDN9, CLDN3, and CLDN4. In fig. 5A shows example results for the hybridoma subclone F3-6C3-H8. Monoclonal antibodies in the supernatant from the hybridoma subclone F3-6C3-H8 also bound to cells transfected with the CLDN6 variant (I143V)-SNP. Monoclonal antibodies in the supernatant from the hybridoma subclone F4-4F7-F2 bound to both CLDN6 and CLDN9 (Fig. 5A). Monoclonal antibodies in the supernatant from the hybridoma subclone F3-7B3-B4 bound to CLDN6, CLDN3, and CLDN9 (Fig. 5B). Monoclonal antibodies in the supernatant from the hybridoma subclone F3-3F7-A5 bound to CLDN6, CLDN4, and CLDN9 (Fig. 5B).

Example 6: Production and testing of monoclonal antibodies against CLDN6

a. Preparation of expression vectors encoding extracellular domain 1 of CLDN6

A codon optimized DNA sequence (SEQ ID NO: 12) encoding a fragment of CLDN6, presumably corresponding to the extracellular domain 1 (EC1) of this protein (SEQ ID NO: 7), was obtained as a combination with a signal peptide derived from the N-terminal leader sequence of the immunoglobulin κ chain followed by 4 additional amino acid residues to provide the correct signal peptidase cleavage site (SEQ ID NO: 13) and cloned into the pcDNA3.1/myc-His vector, resulting in the p3974 vector. Prior to immunization, EC1 fragment expression was confirmed by immunofluorescence microscopy on transiently transfected and paraformaldehyde (PFA)-fixed CHO-K1 cells using a commercially available anti-myc antibody (Cell Signaling, MAB 2276).

b. Immunization

Balb/c mice were immunized with 25 μg of p3973 plasmid DNA along with 4 μl of polyethylenimine mannose (PEI-Man; in vivo-jetPEI™-Man, manufactured by PolyPlus Transfection) (150 mM aqueous solution of PEI-Man with 5% glucose) by intraperitoneal injection on days 0 and 14. On days 28 and 44, animals were immunized (by subcutaneous injection) with KLH protein-conjugated peptides SEQ ID NO: 14 and SEQ ID NO: 15 (100 μg each in PBS, manufactured by JPT Peptide Technologies GmbH, Germany ) together with an HPLC purified oligonucleotide (PTO-CpG-ODN, 25 μg in PBS; 5'-TCCATGACGTTCCTGACGTT; produced by Eurofins MWG Operon, Germany). On days 64, 77 and 97, animals were immunized (by intraperitoneal injection) with P3X63Ag8U.1 myeloma cells (2x10 ⁷ ) transfected with the p3953 vector for stable expression of CLDN6. Before immunization, cells were treated with mitomycin-C (2.5 μg/ml, Sigma-Aldrich, M4287). On days 64 and 97, cells were injected with HPLC-purified oligonucleotide PTO-CpG-ODN (50 μg in PBS), and on day 77 with Freund's incomplete adjuvant.

To generate monoclonal antibodies, mice with a significant immune response against CLDN6 were immunized four days before spleen collection (by intraperitoneal injection) with HEK293 cells (2x10 ⁷ ) stably transfected with the p3953 vector.

c. Screening of monoclonal antibodies against CLDN6

Flow cytometry

To test the binding of monoclonal antibodies to CLDN6 and its homologues, HEK293T cells were transiently transfected with the corresponding plasmid encoding a particular claudin, and its expression was determined by flow cytometry. To distinguish transfected from nontransfected cells, HEK293T cells were simultaneously transfected with a fluorescent marker. 24 hours after transfection, cells were harvested with trypsin (0.05%)/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2 x 10 ⁶ cells/ml. This cell suspension (100 μl) was incubated with the appropriate antibodies at the indicated concentration for 30 min at 4°C. Cross-reactive antibodies were used to determine the expression of CLDN6 and CLDN9. Commercially available mouse anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) antibodies served as positive controls; Mouse IgG2a (Sigma, M9144) and IgG2b (Sigma, M8894) served as isotype controls, respectively. Cells were washed three times with FACS buffer and incubated with specific secondary antibodies (anti-mouse IgG 1+2a+2b+3, Dianova, 115-135-164) conjugated to allophycocyanin (APC) for 30 min at 4°C. The cells were then washed twice and resuspended in FACS buffer. Binding was determined using a BD FACSArray flow cytometer/bioanalyzer. Fluorescent marker expression is plotted on the horizontal axis, and antibody binding is plotted on the vertical axis.

Complement-dependent cytotoxicity (CDC)

Complement-dependent cytotoxicity (CDC) was determined by measuring intracellular adenosine triphosphate (ATP) content in unlysed cells after addition of human complement to target cells incubated with anti-CLDN6 antibodies. A highly sensitive method using a luciferase-catalyzed luminescent reaction was used to quantify ATP.

CHO-K1 cells stably transfected with CLDN6 (CHO-K1-CLDN6) were harvested with trypsin (0.05%)/EDTA solution, washed twice with X-Vivo 15 medium (Lonza, BE04-418Q) and suspended at a concentration of 1 x 10 ⁷ cells/ml in X-Vivo 15 medium. This cell suspension (250 μl) was transferred to an electroporation cuvette (0.4 cm) and mixed with 7 μg of in vitro transcribed RNA encoding luciferase. Cells were electroporated at 200 V and 300 μF capacitance using a Gene Pulser Xcell electroporation system (Bio Rad). After electroporation, cells were resuspended in 2.4 ml of prewarmed D-MEM/F12 medium (1:1) with GlutaMax-I medium (Invitrogen, 31331-093) containing 10% (v/v) FCS, 1% (v/v) volume) penicillin/streptomycin and 1.5 mg/ml selective antibiotic G418. The cell suspension was placed into wells (50 μl per well) of a 96-well white polypropylene plate and incubated at 37°C in a 7.5% CO ₂ atmosphere. 24 hours after electroporation, 50 μl of mouse monoclonal antibodies against CLDN6 (indicated concentrations) in a mixture of 60% RPMI medium (containing 20 mM HEPES) and 40% human serum (from six healthy donors) was added to the cells. For the “total lysis” control, 10 μl per well of a mixture of 8% (v/v) Triton X-100 in PBS was added; to control the “maximum number of viable cells”, 10 μl per well of PBS was added; 10 μl per well of PBS was also added to the experimental samples. Incubated for 80 min at 37°C in an atmosphere of 7.5% CO ₂ , then 50 μl per well of “luciferin mixture” (3.84 mg/ml D-luciferin, 0.64 U/ml adenosine triphosphatase, 160 mM HEPES in double distilled water). The plate was incubated in the dark for 45 min at room temperature. Luminescence was measured using an Infinite M200 luminometer (TECAN). Data are presented as digital signal integration results in relative light units (RLU).

NEC8 cells were electroporated at 200 V and 400 μF capacitance and cultured in a mixture of RPMI 1640 and GlutaMAX-I media (Invitrogen, 61870) containing 10% (v/v) FCS.

Specific lysis was calculated as follows:

maximum viable cell count: 10 µl PBS, no antibody; total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibody.

Exposure to antibodies in early stages

For early antibody exposure, Nude-Foxn1 ^nu nude mice were injected subcutaneously into the flank with 2 × ¹⁰⁷ NEC8 cells in 200 μl PBS. There were ten females in each group, 6-8 weeks old. Three days after this inoculation, animals were administered (alternating intravenous and intraperitoneal injections) 200 μg of purified mouse monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B, and 67A twice a week for 46 days. Specimens injected with PBS served as negative controls. Tumor sizes were determined twice a week: Tumor volume (TV) = (length × width ² )/2. TV was expressed in cubic millimeters (mm ³ ) and a tumor growth curve was plotted over time. When the tumor exceeded 1500 mm ³ , the animal was sacrificed.

results

Mouse monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B, and 67A bound strongly to human CLDN6 proteins and CLDN6 SNP (single nucleotide polymorphism) variant I143V, but no binding to CLDN3, 4, and 9 was observed (Fig. 6).

For MuMAB antibodies 59A, 60A, 61D, 64A, 65A, 66B and 67A, very low EC ₅₀ values were obtained (200-500 ng/ml) and saturation of binding sites was achieved at low concentrations (Fig. 7).

For MuB antibodies 59A, 60A, 61D, 64A, 65A, 66B, and 67A, dose-dependent CDC was observed and occurred at low antibody concentrations (Fig. 8). Anti-CLDN6 antibodies muMAB 65A and 66B induced CDC against NEC8 cells in a dose-dependent manner (Fig. 9). The target specificity of muMAB 65A and 66B was confirmed by experiments with NEC8 LVTS2 54 cells (CLDN6 knockdown).

Also, muMAB antibodies 59A, 60A, 61D, 64A, 65A, 66B and 67A suppressed tumor growth in mice injected with NEC8 cells (Fig. 10).

Example 7. Production and testing of hybrid monoclonal antibodies against CLDN6

a. Preparation of hybrid mouse-human monoclonal antibodies

To generate the fusion antibodies, nucleotide sequences encoding the murine antibody heavy and light chain variable regions, including leader sequences, were amplified by polymerase PCR using the primers listed in the table below.

Nucleotide sequences. corresponding to the murine heavy chains were linked at an ApaI restriction site (5'-GGGCCC-3') to a nucleotide sequence corresponding to the N-terminal part of the human Fcγ1 chain in the expression vector. Nucleotide sequences corresponding to the murine κ chain variable regions, including the leader sequence, were cloned upstream of the constant region using the BsiWI restriction site. The correctness of the resulting construct and the orientation for the upstream promoter in the vector sequence corresponding to the constant region was verified by sequencing. Due to the position of the ApaI restriction site, the amplification of the variable region including the leader sequence in any case includes the first 11 nucleotides of the human γ1 constant region coding sequence together with the ApaI site sequence. The nucleotide sequence corresponding to the human γ1 heavy chain constant region is designated SEQ ID NO: 24; the amino acid sequence of the human γ1 constant region thus expressed is designated SEQ ID NO: 25. The nucleotide sequence encoding the κ light chain constant portion is designated SEQ ID NO: 26; the corresponding amino acid sequence is designated SEQ ID NO: 27.

Table 1. Mouse hybridoma lines used for antibody cloning muMAB Isotype Primer SEQ ID NO: Heavy chain 64A IgG2a 17, 18 89A IgG2a 17, 19 61D IgG2a 17, 20 67A IgG2a 17, 20 Light chain 64A IgG 21, 22 89A IgG 21, 23 61D IgG 21, 22 67A IgG 21, 22

The corresponding mouse regions of hybrid monoclonal antibodies are designated with the prefix “chim“ (from English chimeric - hybrid), for example chimAB 64A.

Amplification of nucleotide sequences corresponding to the mouse variable regions of the light and heavy chains, including leader sequences, was carried out by PCR with rapid amplification of cDNA ends (“step-out”), described in the work of Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6). To do this, total RNA was obtained from hybridoma cell lines (see Table 1) using standard methods known to those skilled in the art, for example using the RNeasy Mini Kit (Qiagen). Single-stranded cDNA was prepared using the template-switching method also described by Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6, 1558). In addition to the (dT)30 oligomer (SEQ ID NO: 28), a hybrid DNA/RNA oligomer (SEQ ID NO: 29) was used to serve as a 5' adapter for changing templates during cDNA strand polymerization. In this adapter oligomer, the last three nucleotides were not deoxyribo-nucleotides, but ribonucleotides. The subsequent step-out PCR used an antisense oligomer targeting the mouse κ chain constant region or the γ chain subclass 2a constant region (SEQ ID NO: 30 and 31, respectively). The IgG subclass of murine monoclonal antibodies produced by the hybridoma cell lines was previously determined using an IsoStrip kit (Roche), and the appropriate antisense oligomer was selected accordingly (see Table 1). The sense oligomer in the step-out PCR, which included two oligomers (SEQ ID NO: 32 and 33), was a mixture of primers.

Then, the identified nucleotide sequences corresponding to the mouse variable regions, including leader sequences, were amplified by PCR excluding the 5'-terminal untranslated region (5'-UTR) and the mouse 3'-constant region, adding restriction sites at the ends, which made it possible to subcloning into the prepared expression vectors carrying nucleotide sequences corresponding to human constant regions. In addition, the sense oligomers provided the Kozak consensus sequence (5'-GCCGCCACC-3'), and the antisense oligomers for the heavy chain variable regions included the first 11 nucleotides of the human γ1 heavy chain constant region in addition to the ApaI restriction site (see Table 1, SEQ ID NO: 17-23). Nucleotide sequences corresponding to the variable regions of κ light chains, including leader sequences, were cloned using HindIII and BsiWI restriction endonucleases; the heavy γ chain variable regions required the enzymes HindIII and ApaI.

Nucleotide sequences were also amplified. corresponding to other variable regions of the light and heavy chains of murine antibodies and other hybrid monoclonal antibodies against CLDN6 were obtained as described above.

b. Production of hybrid monoclonal antibodies against CLDN6

Hybrid monoclonal antibodies were transiently expressed in HEK293T cells (ATCC CRL-11268) transfected with plasmid DNA encoding the light and heavy chains of the corresponding antibodies. 24 hours before cell transfection in the amount

8 × 10 ⁷ were sown on culture dishes (145 mm) and grown in 25 ml of HEK293T medium (DMEM/F12 + GlutaMAX-I, 10% FCS, 1% penicillin/streptomycin). Plasmid DNA was dissolved in HEK293T media without additives (20 μg in 5 ml per plate). 75 μl of polyethylenimine (PEI) (1 mg/ml) (Polyscience, 23966) was added and the DNA:PEI mixture was incubated for 15 min at room temperature. This transfection mixture was then added dropwise to the cells. 24 hours after transfection, HEK293T medium was replaced with Pro293a medium (Lonza, BE12-764Q) containing penicillin/streptomycin (1%). For optimal expression, transfected cells were cultured for 96-120 hours at 37°C in an atmosphere containing 7.5% CO ₂ ). The supernatant was collected and the hybrid antibodies were purified by fast protein liquid chromatography (FPLC) using Protein A columns. Antibody concentrations were determined and purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

c. Study of the properties of hybrid monoclonal antibodies against CLDN6

Flow cytometry

To determine the specificity and affinity of CLDN6-specific hybrid monoclonal antibodies, their binding to HEK293 cells stably transfected with CLDN3, 4, 6, or 9, respectively, and to tumor cell lines with endogenous CLDN6 expression was studied by flow cytometry. Cells were collected using 0.05% trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2 × ¹⁰⁶ cells/ml. This cell suspension in an amount of 100 μl was incubated with the appropriate antibodies at the indicated concentrations for 60 min at 4°C. Hybrid cross-reactive antibodies (chimAB 5F2D2) were used to determine the expression of CLDN6 and CLDN9 A. Commercially available mouse anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) antibodies served as positive controls; human IgG1-κ (Sigma, I5154) served as a negative control. Cells were washed three times with FACS buffer and incubated for 30 min at 4°C with specific secondary antibodies, respectively goat anti-human IgG Fc-γ conjugated to allophycocyanin (APC) (Dianova, 109-136-170), or with anti-mouse IgG 1+2a+2b+3a conjugated to APC (Dianova, 115-135-164). Cells were washed twice and resuspended in FACS buffer. Binding was determined using a BD FACSArray flow cytometer/bioanalyzer.

Complement-dependent cytotoxicity (CDC)

Complement-dependent cytotoxicity (CDC) was determined by measuring intracellular adenosine triphosphate (ATP) content in unlysed cells after addition of human complement to target cells incubated with anti-CLDN6 antibodies. A highly sensitive method using a luciferase-catalyzed luminescent reaction was used to quantify ATP.

For this study, we used wild-type NEC8 cells (carrying CLDN6) and NEC8 cells knocked down for this protein (lacking CLDN6); both were stably transduced with the luciferase expression construct. Cells were collected using 0.05% trypsin/EDTA, diluted to a concentration of 2 × ¹⁰⁵ cells/ml with a mixture of RPMI and GlutaMax-I media (Invitrogen, 61870-010) containing 10% (v/v) FCS. Cells at 1 x 10 ⁴ were seeded onto a white polypropylene 96-well plate and incubated for 24 hours at 37°C in an atmosphere of 5% CO ₂ . Subsequently, 50 μl of anti-CLDN6 monoclonal fusion antibodies (at the indicated concentrations) in a mixture of RPMI medium containing 20 mM HEPES (60%) and human serum obtained from six healthy donors (40%) was added to the cells. To control for total lysis, 10 μl per well of an 8% (v/v) solution of Triton X-100 in PBS was added; to control the maximum number of viable cells, 10 μl per well of PBS was added; 10 μl per well of PBS was also added to the test samples. Incubated for 80 min at 37°C in an atmosphere of 7.5% CO ₂ , then 50 μl per well of “luciferin mixture” (3.84 mg/ml D-luciferin, 0.64 U/ml adenosine triphosphatase, 160 mM HEPES in double distilled water). The plate was incubated in the dark for 45 min at room temperature. Luminescence was measured using an Infinite M200 luminometer (TECAN). Data are presented as digital signal integration results in relative light units (RLU).

Specific lysis was calculated as follows:

maximum viable cell count: 10 µl PBS, no antibody; total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibody.

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Antibody-dependent cell-mediated cytotoxicity (ADDC) was determined by measuring intracellular adenosine triphosphate (ATP) content in unlysed cells following the addition of human peripheral blood mononuclear cells (PBMCs) to target cells incubated with anti-CLDN6 antibodies. A highly sensitive method using a luciferase-catalyzed luminescent reaction was used to quantify ATP.

For this study, we used wild-type NEC8 cells (carrying CLDN6) and NEC8 cells knocked down for this protein (lacking CLDN6); both were stably transduced with the luciferase expression construct. Cells were collected using 0.05% trypsin/EDTA, diluted to a concentration of 2 × ¹⁰⁵ cells/ml with a mixture of RPMI and GlutaMax-I media (Invitrogen, 61870-010) containing 10% (v/v) FCS and 20 mM Hepes. Cells in an amount of 1 × 10 ⁴ were seeded onto a white polypropylene 96-well plate and incubated for 4 hours at 37°C in an atmosphere of 5% CO ₂ .

Peripheral blood mononuclear cells were isolated from human donor blood by density gradient centrifugation using Ficoll/Hypaque (GE Healthcare, 17144003). The layer containing PBMC was collected, and the cells were washed twice with PBS/EDTA (2 mM). PBMC in the amount of 1 × 10 ⁸ cells were added to 50 ml of X-Vivo 15 medium (Lonza, BE04-418Q) containing 5% heat-inactivated human serum (Lonza, US14-402E) and incubated for 2 hours at 37°C C in an atmosphere of 5% CO ₂ .

4 hours after this manipulation of the target cells (NEC-8), monoclonal fusion antibodies against CLDN6 in PBS solution were added to the cells at the indicated concentrations. Non-adherent PBMCs, which separated from adherent monocytes after 2 hours of incubation, were collected and diluted with X-vivo 15 medium to a concentration of 8 × 10 ⁶ cells/ml. This cell suspension (25 μl) was added to target cells and anti-CLDN6 monoclonal fusion antibodies. The plates were incubated for 24 hours at 37°C in an atmosphere of 5% CO ₂ .

After 24 hours of incubation, to control “total lysis,” a solution of Triton X-100 (8% v/v) in PBS was added in an amount of 10 μl per well; to control the “maximum number of viable cells,” 10 μl of PBS per well was added; PBS 10 μl per well was also added to the test samples. Then 50 μl per well of “luciferin mixture” (3.84 mg/ml D-luciferin, 0.64 U/ml adenosine triphosphatase, 160 mM HEPES in double distilled water) was added. The plate was incubated in the dark for 30 min at room temperature. Luminescence was measured using an Infinite M200 luminometer (TECAN). Data are presented as digital signal integration results in relative light units (RLU).

Specific lysis was calculated as follows:

maximum viable cell count: 10 µl PBS, no antibody; total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibody.

d. results

Anti-CLDN6 fusion monoclonal antibodies chimAB 61D, 64A, 67A, and 89A bound strongly to human CLDN6, but no binding was observed to CLDN3, 4, and 9 (Fig. 11).

When bound to human CLDN6 stably expressed on the surface of HEK293 cells, anti-CLDN6 hybrid monoclonal antibodies chimAB 64A and 89A had very low EC ₅₀ values (450-600 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 67A and 61D were characterized by low (1000 ng/ml) and medium (2300 ng/ml) EC ₅₀ values, respectively (Fig. 12).

When bound to CLDN6 endogenously expressed in NEC8 cells, the anti-CLDN6 hybrid monoclonal antibodies chimAB 64A and 89A had very low EC ₅₀ values (600-650 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 61D and 67A were characterized by medium (1700 ng/ml) and high (6100 ng/ml) EC50 values, respectively (Fig. 13).

When bound to CLDN6 endogenously expressed in OV90 cells, the anti-CLDN6 hybrid monoclonal antibodies chimAB 64A and 89A had very low EC ₅₀ values (550-600 ng/ml) and saturation of binding sites was achieved at low concentrations. Antibodies chimAB 61D and 67A had average EC ₅₀ values (1500 ng/ml and 2300 ng/ml, respectively) (Fig. 14).

Anti-CLDN6 fusion monoclonal antibodies chimAB 61D, 64A, 67A and 89A exhibited complement-dependent cytotoxicity activity against NEC-8 cells in a dose-dependent manner (FIG. 15).

Anti-CLDN6 hybrid monoclonal antibodies chimAB 61D, 64A, 67A, and 89A exhibited antibody-dependent cell-mediated cytotoxicity activity against NEC-8 cells, which was dose dependent and cytotoxicity occurred even at low antibody concentrations (Fig. .16).

These results clearly demonstrate the specificity of these hybrid monoclonal antibodies for CLDN6.

Example 8 Treatment Using Anti-CLDN6 Monoclonal Antibodies

Treatment in the early stages

For early antibody exposure, Nude-Foxn1 ^nu nude mice were injected subcutaneously into the flank with 2 × ¹⁰⁷ NEC8 cells in 200 μl of RPMI medium (Gibco). There were ten females in each group, 6-8 weeks old. Three days after inoculation of tumor cells, the animals were administered twice a week by intravenous or intraperitoneal injection (alternating) 200 μg of purified mouse monoclonal antibodies muMAB 89A for seven weeks. A group of individuals that were injected with the same volume of PBS served as a negative control. Tumor sizes were determined twice a week: tumor volume (TV) = (length × width ² )/2). TV was expressed in mm ³ and graphs of tumor growth over time were plotted. When the tumor size exceeded 1500 mm ³ , the animal was sacrificed.

Treatment in later stages

For exposure to antibodies at late stages of development of xenograft tumors, athymic female mice aged 6-8 weeks of the Nude-Foxn1 ^nu line were injected by subcutaneous injection into the flank of NEC8 cells in an amount of 2 × 10 ⁷ in 200 μl of RPMI medium (Gibco). Tumor sizes were determined twice a week: tumor volume (TV) = (length × width ² )/2). TV was expressed in mm ³ and graphs of tumor growth over time were plotted. 15-17 days after inoculation of tumor cells, animals were divided into groups for exposure; in each group there were 8 individuals with tumors of the same size - more than 80 mm ³ . For five weeks, mice were treated twice weekly by intravenous or intraperitoneal injection (alternating) with 200 μg of purified mouse monoclonal fusion antibodies. A group of individuals that were injected with nonspecific antibodies served as a negative control. When the tumor size exceeded 1500 mm ³ , the animal was sacrificed.

When mice bearing xenografts of NEC8 tumor cells were exposed to muMAB 61D, 64A, and 67A murine monoclonal antibodies early in tumor development, no tumor growth was observed even after immunotherapy was stopped (Fig. 17).

When mice bearing xenografts of NEC8 tumor cells were exposed to the mouse monoclonal antibody muMAB 89A early in tumor development, tumor growth was suppressed and there were no noticeable tumors in mice treated with muMAB89A at the end of the study (Fig. 18).

When mice with xenografts of NEC8 tumor cells were exposed to muMAB 64A murine monoclonal antibodies at late stages of tumor development, tumor growth was suppressed (Fig. 19).

When mice with xenografts of tumor cells of the NEC8 line were exposed to mouse monoclonal antibodies muMAB 64A at late stages of development, the life expectancy of experimental individuals increased (Fig. 20).

When mice with xenografts of NEC8 tumor cells were exposed to mouse monoclonal antibodies muMAB 61D, 67A and 89A at late stages of tumor development, inhibition of tumor growth was achieved (Fig. 21).

When exposed to mice with xenografts of NEC8 tumor cells, individuals treated with the CLDN6-specific antibodies muMAB 61D or 67A at later stages of tumor development lived longer (Fig. 22).

When exposed late to mice with wild-type NEC8 cell xenografts and stable CLDN6 knockdown, muMAB 64A and 89A antibodies had a therapeutic effect only in mice with wild-type NEC8 cell xenografts, and in mice with CLDN6 knockdown NEC8 cell xenografts these antibodies were not present, demonstrating the specificity of anti-CLDN6 antibodies in vivo (Fig. 23).

Example 9: High Resolution Epitope Mapping of Anti-CLDN6 Monoclonal Antibodies

When mapping epitopes using enzyme-linked immunosorbent assay (ELISA), specific monoclonal antibodies against CLDN6 bound only very weakly to linear epitopes, indicating that they have conformational epitopes. To study the interaction of the antibodies described herein with CLDN6 in its native conformation, site-directed mutagenesis in mammalian cell cultures was used for epitope mapping. Alanine scanning mutagenesis was performed at amino acid residues 27-81 and 137-161 in the first and second extracellular domains, respectively. After transient expression in HEK293T cells, the CLDN6 mutants were assessed for their ability to bind to specific monoclonal antibodies. The impaired binding of the specific monoclonal antibody to the CLDN6 mutant indicated that the affected amino acid residue was important for contact and/or spatial structure. Binding was determined by flow cytometry. To distinguish transfected cells from non-transfected ones, transfection was carried out simultaneously with a fluorescent marker.

In the CLDN6 protein, amino acid residues important for interaction with specific anti-CLDN6 hybrid antibodies were identified by alanine scanning. Substitutions for alanine and glycine were obtained by site-specific mutagenesis (GENEART AG, Germany). To test the binding of monoclonal fusion antibodies to wild-type CLDN6 and its mutant variants, HEK293T cells were transiently transfected with the appropriate plasmids encoding each claudin, and expression was determined by flow cytometry. To distinguish transfected from nontransfected cells, HEK293T cells were simultaneously transfected with a fluorescent marker. 24 hours after transfection, cells were harvested using trypsin (0.05%)/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2 x 10 ⁶ cells/ml. This cell suspension (100 μl) was incubated with antibodies at a concentration of 10 μg/ml for 45 min at 4°C. A commercially available mouse anti-CLDN6 antibody (R&D, MAB3656) was used as a control to determine cell surface expression of CLDN6 mutants. The cells were washed three times with FACS buffer and incubated with specific second antibodies - goat anti-human IgG Fc-γ (Dianova, 109-136-170) or anti-mouse IgG 1+2a+2b+3a (Dianova, 115-135-164), - conjugated with allophycocyanin (APC) for 30 minutes at 4°C. The cells were then washed twice and resuspended in FACS buffer. Binding to transfected cells was determined using a BD FACS Array flow cytometer/bioanalyzer. Fluorescent marker expression was plotted along the horizontal axis, and antibody binding was plotted along the vertical axis. The average signal intensity for binding of CLDN6-specific monoclonal fusion antibodies to CLDN6 mutant variants was expressed as a percentage relative to binding to wild-type CLDN6. If an amino acid residue was key for binding, then after its mutation, binding did not occur; if the amino acid residue had a supporting value, then after mutation the binding was only reduced compared to the binding of wild-type CLDN6.

High-resolution epitope mapping showed that amino acid residues F35, G37, S39 and, probably, T33 of the first extracellular domain of CLDN6 are important for interaction with specific hybrid antibodies against CLDN6 chimAB 61D, 64A, 67A and 89A. Amino acid residue I40 is essential for binding to chimAB 89A antibodies and is involved in binding to chimAB 61D and 67A. In addition, amino acid residue L151 of the second extracellular domain of CLDN6 is important for interaction with chimAB 67A (Fig. 24).

Example 10: Production and Validation of Enhanced Anti-CLDN6 Monoclonal Antibodies

It turned out that antibodies 64A, having excellent properties in terms of binding to CLDN6 and effective treatment of tumors, contain a free cysteine residue at position 46 of the light chain, which significantly impairs such properties of the protein as solubility, stability and the ability to form aggregates. A free cysteine residue is also undesirable for other reasons, such as regulation. Therefore, we attempted to create 64A-based antibodies that do not contain a free cysteine residue at position 46 without losing their desirable properties.

Hybrid antibodies mAb206-LCC were obtained, which combine the chimAB 64A heavy chain and the chimAB 61D light chain. As a result of amino acid substitutions, MAb206-SUBG and mAb206-SUBS were obtained, in which the cysteine at position 46 of the chimAB 64A light chain was replaced by glycine and serine, respectively.

Production of monoclonal hybrid antibodies against CLDN6 in HEK293T cells

Monoclonal fusion antibodies were transiently expressed in HEK293T cells (ATCC CRL-11268) transfected with plasmid DNA encoding the light and heavy chains of the corresponding antibodies, as described in section b of Example 7.

Preparation of monoclonal hybrid antibodies against CLDN6 of CHO cells adapted to growth in suspension

CHO cells adapted to growth in suspension were subcultured in a serum-free medium in a CO ₂ incubator with a shaker and a humidified working chamber. One day before transfection, cells were seeded in serum-free medium in shake flasks.

On the day of transfection, cells were centrifuged (5 min at 200 g) and resuspended in fresh DMEM (Invitrogen, 41965-039) in shake flasks. DNA and transfection reagent were added to the cells and mixed gently by shaking. After cumulative cultivation in a CO ₂ incubator, the cells were diluted with serum-free nutrient medium and further cultivated in an incubator shaker. On days 0, 2, 4, and 6, cells were fed with ready-made CHO CD EfficientFeed™ A and B, respectively (Invitrogen, A1023401 and A1024001). When cell viability dropped below 90%, fusion antibodies were harvested. The resulting antibodies were purified by flash protein liquid chromatography (FPLC) using protein A columns. Antibody concentration was determined by absorbance at 280 nm, and purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Flow cytometry

The binding of specific hybrid monoclonal antibodies against CLDN6 to cells in which their target is expressed was determined by flow cytometry. Cells were collected using 0.05% trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2 × ¹⁰⁶ cells/ml. This cell suspension in an amount of 100 μl was incubated with the appropriate antibodies at the indicated concentrations for 30 min at 4°C. ChimAB 5F2D2 antibodies were used to determine the expression of CLDN6 and CLDN9. Commercially available mouse anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) antibodies served as positive controls; human IgG1-κ (Sigma, I5154) served as a negative control. Cells were washed three times with FACS buffer and incubated for 30 min at 4°C with specific secondary antibodies, respectively goat anti-human IgG Fc-γ conjugated to allophycocyanin (APC) (Dianova, 109-136-170), or with anti-mouse IgG 1+2a+2b+3a conjugated to APC (Dianova, 115-135-164). Cells were washed twice and resuspended in FACS buffer. Binding was determined using a BD FACSArray flow cytometer/bioanalyzer.

Complement-dependent cytotoxicity (CDC)

Complement-dependent cytotoxicity (CDC) was determined by measuring intracellular adenosine triphosphate (ATP) content in unlysed cells after adding human complement to target cells incubated with anti-CLDN6 antibodies as described in Example 7.

Antibody-dependent cell-mediated cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADDC) was determined by measuring intracellular adenosine triphosphate (ATP) content in unlysed cells following the addition of human peripheral blood mononuclear cells (PBMCs) to target cells incubated with anti-CLDN6 antibodies. A highly sensitive method using a luciferase-catalyzed luminescent reaction was used to quantify ATP.

For this study, we used wild-type NEC8 cells (carrying CLDN6) and NEC8 cells knocked down for this protein (lacking CLDN6); both were stably transduced with luciferase RNA, and OV90 cells were transiently transfected with in vitro transcribed RNA encoding luciferase. Cells were collected using 0.05% trypsin/EDTA solution, diluted to a concentration of 2 × 10 ⁵ cells/ml (NEC-8 wild type and CLD6 knockdown) or 1 × 10 ⁶ cells/ml (OV90) with the appropriate growth medium containing additional 20 mmM Hepes. Cells in quantities of 1 × 10 ⁴ or 5 × 10 ⁴ were seeded onto a white polypropylene 96-well plate and incubated at 37°C in an atmosphere of 5% CO ₂ .

Peripheral blood mononuclear cells were isolated from human donor blood by density gradient centrifugation using Ficoll/Hypaque (GE Healthcare, 17144003). The layer containing PBMC was collected, and the cells were washed twice with PBS/EDTA (2 mM). PBMC in the amount of 1 × 10 ⁸ cells were added to 50 ml of X-Vivo 15 medium (Lonza, BE04-418Q) containing 5% human serum (from six healthy donors) and incubated for 2 hours at 37°C in an atmosphere with 5% CO ₂ . 4 hours after this manipulation, monoclonal hybrid antibodies against CLDN6 (25 μl) in PBS solution were added to the cells at the indicated concentrations. Non-adherent PBMC, which separated from adherent monocytes after 2 hours of incubation, were collected and diluted with X-vivo 15 medium to a concentration of 1.6 × 10 ⁷ cells/ml (NEC-8 wild-type or CLDN6 knockdown cells) or 4 × 10 ⁷ cells/ml (OV90 cells). This cell suspension (25 μl) was added to target cells and anti-CLDN6 monoclonal fusion antibodies. The plates were incubated for 24 hours at 37°C in an atmosphere of 5% CO ₂ .

After 24 hours of incubation, to control “total lysis,” a solution of Triton X-100 (8% v/v) in PBS was added in an amount of 10 μl per well; to control the “maximum number of viable cells,” 10 μl of PBS per well was added; PBS 10 μl per well was also added to the test samples. Then 50 μl per well of “luciferin mixture” (3.84 mg/ml D-luciferin, 0.64 U/ml adenosine triphosphatase, 160 mM HEPES in double distilled water) was added. The plate was incubated in the dark for 30 min at room temperature. Luminescence was measured using an Infinite M200 luminometer (TECAN). Data are presented as digital signal integration results in relative light units (RLU).

Specific lysis was calculated as follows:

maximum viable cell count: 10 µl PBS, no antibody; total lysis: 10 µl 8% (v/v) Triton X-100 in PBS, no antibody.

Treatment in later stages

To target the late stages of development of tumors arising from xenografts of tumor cells with antibodies, athymic female mice of the Nude-Foxn1 ^nu line, 6-8 weeks old, were injected by subcutaneous injection into the flank with 2 × 10 ⁷ NEC8 cells in 200 μl of RPMI medium (Gibco) . Tumor sizes were determined twice a week: tumor volume (TV) = (length × width ² )/2). TV was expressed in mm ³ and graphs of tumor growth over time were plotted. 17 days after tumor cell inoculation, animals were divided into treatment groups; in each group there were 8 individuals with tumors of the same size - more than 80 mm ³ . For five weeks, mice were treated twice weekly by intravenous or intraperitoneal injection (alternating) with 200 μg of purified mouse monoclonal fusion antibodies. A group of individuals that were injected with nonspecific antibodies served as a negative control. When the tumor size exceeded 1500 mm ³ , the animal was sacrificed.

Metastasis research

To study metastasis, NEC8 cells were harvested and filtered through a mesh (70 μm) to remove large cellular aggregates. Nude-Foxn1 ^nu female nude mice, 6 weeks old, were treated with 4 × 106 ^NEC8 cells by tail vein injection. Three days after the injection of tumor cells, twice a week the animals were administered by intravenous or intraperitoneal injection (alternating) purified mouse monoclonal antibodies muMAB 64A in an amount of 200 μg in PBS or PBS without antibodies. After 8 weeks, mice were sacrificed, lungs were removed, and samples were frozen in liquid nitrogen.

Genomic DNA was isolated from frozen tissue according to the instructions of the Blood & Cell Culture DNA Midi Kit (Qiagen, 13343). Lung tissue was homogenized using Ultra Torax T8.10 dispersant (IKA-Werke). To avoid contamination of human genomic DNA, lung genomic DNA was obtained under sterile conditions using quantitative polymerase chain reaction (qPCR). To determine the tumor burden of human NEC8 cells in mouse lung tissue samples, a standard curve was generated with defined amounts of human NEC8 cell genomic DNA and mouse lung genomic DNA. The following dilution series were used (NEC8 cell DNA/mouse lung DNA): 200/0, 40/160, 8/192, 1.6/198.4, 0.32/199.68, 0.064/199.94, 0.013/ 200 and 0/200 ng. For qPCR, 200 ng of genomic DNA, 2xSYBR Green dye (Qiagen, 204145), 16 nmol sense primer (GGGATAATTTCAGCTGACTAAACAG, Eurofins) and 16 nmol antisense primer (TTCCGTTTAGTTAGGTGCAGTTATC, Eurofins) were taken in a total volume of 50 μl; The 7300 Real Time PCR-System (Applied Biosystems, USA) was used for analysis. For a negative control, water was taken instead of DNA. Quantitative PCR was carried out under the following conditions: 15 min at 95°C (1 cycle), 30 s at 95°C / 30 s at 62°C / 30 s at 72°C (40 cycles), 15 s at 95°C / 30 s at 60°C / 15 s at 95°C (1 cycle). All qPCR reactions were performed in triplicate. Tumor burden was determined by the standard curve method and calculated using the software supplied with the 7300 System instrument (Applied Biosystems).

High-resolution epitope mapping

To study the interaction of the antibodies of this invention with CLDN6 in its native conformation, the method of site-directed mutagenesis in mammalian cell cultures was used to map epitopes. Alanine scanning was performed on amino acid residues 27-81 and 137-161 of the first and second extracellular domains of CLDN6, respectively. Substitutions to alanine were carried out by site-specific mutagenesis (GENEART AG, Germany). After transient expression in HEK293T cells, the CLDN6 mutants were assessed for their ability to bind to specific monoclonal antibodies. The impaired binding of the specific monoclonal antibody to the CLDN6 mutant indicated that the affected amino acid residue was important for contact and/or spatial structure. Binding was determined by flow cytometry. To distinguish transfected from nontransfected cells, transfection was performed simultaneously with a fluorescent marker. 24 hours after transfection, cells were harvested with trypsin (0.05%)/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2 × 10 ⁶ cells/ml. This cell suspension (100 μl) was incubated with antibodies at a concentration of 10 μg/ml for 30 min at 4°C. A commercially available mouse anti-CLDN6 antibody (R&D, MAB3656) was used as a control to determine cell surface expression of CLDN6 mutants. The cells were washed three times with FACS buffer and incubated with specific second antibodies - goat anti-human IgG Fc-γ (Dianova, 109-136-170) or anti-mouse IgG 1+2a+2b+3a (Dianova, 115-135-164), - conjugated with allophycocyanin (APC) for 30 minutes at 4°C. The cells were then washed twice and resuspended in FACS buffer. Binding to transfected cells was determined using a BD FACS Array flow cytometer/bioanalyzer. Fluorescent marker expression was plotted along the horizontal axis, and antibody binding was plotted along the vertical axis. The average signal intensity for binding of CLDN6-specific monoclonal fusion antibodies to CLDN6 mutant variants was expressed as a percentage relative to binding to wild-type CLDN6. If an amino acid residue was key for binding, then after its mutation, binding did not occur; if the amino acid residue had a supporting value, then after mutation the binding was only reduced compared to the binding of wild-type CLDN6.

Immunohistochemical analysis

Cryosections of tissue (4 μm) immediately after preparation were fixed with acetone for 10 min at a temperature of -20°C. The sections were then dried for 10 min at room temperature and stored at −80°C. Before use, sections were warmed (10 min at room temperature) and rehydrated in PBS for 5 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS for 15 min at room temperature. To avoid nonspecific binding, the preparations were incubated with goat serum (10%) in PBS for 30 min at room temperature. The slides were then incubated with the CLDN6-specific mouse monoclonal antibody muMAB 64A (0.2 μg/ml in PBS containing 10% goat serum) overnight at 4°C. The next day, the slides were washed with PBS (3 times for 5 min) at room temperature and incubated with secondary antibodies (100 μl of commercially available anti-mouse IgG (ImmunoLogic) conjugated to a polymer-horseradish peroxidase complex (PowerVision)) for an hour at room temperature. temperature. The slides were then washed with PBS (3 times for 5 min) at room temperature. Final staining was completed within one and a half minutes using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame, CA, USA). Sections were counterstained with hematoxylin for 90 seconds at room temperature. The preparations were dehydrated in increasing concentrations of ethyl alcohol (70%, 80%, 2 × 96% and 99%, 5 min each) and incubated in xylene for 10 min, after which they were filled with X-tra Kit (Medite Histotechnic).

To use the monoclonal hybrid antibodies mAb206-LCC and mAb206-SUBG in human tissues, they were labeled with fluorescein isothiocyanate (FITC, Squarix GmbH, Germany). Cryosections were prepared and processed as described above (fixation, blocking of endogenous peroxidase, blocking of nonspecific binding sites). The slides were then incubated with mAb206-LCC-FITC and mAb206-SUBG-FITC antibodies (1 μg/ml in PBS containing 10% goat serum) for an hour at room temperature in the dark. After this, the preparations were washed with PBS (3 times for 5 min) at room temperature and incubated with 200 μl of rabbit anti-FITC-HRP antibodies (AbD Serotec, diluted 1:300 in PBS containing 10% goat serum) for 30 min at room temperature. temperature. Wash with PBS (3 times for 5 min) at room temperature, after which the substrate is reacted for 2 min 30 using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame, CA, USA). Counterstaining, dehydration, and embedding in media were performed as described above.

A flow cytometry study of the binding specificity of anti-CLDN6 monoclonal fusion antibodies using HEK293T cells transiently transfected with human CLDN6, 3, 4 and 9 showed that chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS monoclonal fusion antibodies bind to CLDN6 , but do not interact with CLDN3, 4 and 9, respectively (Fig. 27).

Regarding binding to human CLDN6 stably expressed on the surface of HEK293 cells, the anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS had similarly low EC ₅₀ values and saturation of binding sites was achieved at low concentrations (Fig. 28).

The binding affinity (affinity) of monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to tumor cells in which human CLDN6 is endogenously expressed was assessed by binding to tumor cells of the NEC8 line (testicular cancer), which was determined by flow cytometry. Compared with the CLDN6-specific antibodies chimAB 64A, mAb206-SUBG, and mAb206-SUBS, the variant with the mAb206-LCC light chain combination showed three times greater affinity for NEC8 cells. In all cases, saturation of binding sites was achieved at low concentrations (Fig. 29).

A flow cytometry study of the binding specificity of monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to human OV90 cells (ovarian cancer) showed that antibodies specific to CLDN6 were characterized by similar low EC ₅₀ values. The light chain combination mAb206-LCC exhibited the highest binding strength to OV90 cells (Fig. 30).

In NEC-8 cells, anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC, and mAb206-SUBG exhibited complement-dependent cytotoxicity (CDC) activity in a dose-dependent manner. And with NEC-8 cells knocked down for CLDN6, none of these antibodies caused nonspecific lysis. This result demonstrates the target-specific effector function of chimAB 64A, mAb206-LCC and mAb206-SUBG antibodies (Fig. 31a).

CDC activities of chimAB 64A, mAb206-LCC, mAb206-SUBG, and mAb206-SUBS antibodies against NEC8 cells were dose-dependent. Compared to chimAB 64A, the amino acid substitution variants mAb206-SUBG and mAb206-SUBS had similar CDC activity against NEC8 cells (Figure 31b).

Monoclonal hybrid antibodies against CLDN6 chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS had dose-dependent activity manifested by antibody-dependent cell-mediated cytotoxicity (ADCC), and caused ADCC against tumor cells of the NEC8 and OV90 lines even at low concentrations (Fig. 32a).

Fig. 32b demonstrates the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of anti-CLDN6 monoclonal fusion antibodies chimAB 64A, mAb206-LCC and mAb206-SUBG against wild-type and NEC8 knockdown NEC8 cells. To demonstrate the target specificity of the antibodies, NEC8 cells stably knocked down for CLDN6 were used.

In experiments with xenografts of NEC8 tumor cells in mice, exposure to CLDN6-specific antibodies mAb206-LCC, mAb206-SUBG and mAb206-SUBS at late stages of tumor development suppressed tumor growth compared to control exposures (Fig. 33).

The tumor growth curves presented in Fig. 34a show that monoclonal hybrid antibodies against CLDN6 mAb206-LCC, mAb206-SUBG and mAb206-SUBS are able to suppress tumor growth. The survival graph (Fig. 34b) demonstrates the increase in lifespan of mice treated with CLDN6-specific antibodies.

High-resolution mapping of epitopes showed that for the interaction of CLDN6-specific hybrid antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS with CLDN6, amino acid residues F35, G37 and S39 are important, as well as, probably, T33 of the first extracellular domain of CLDN6 . The binding features of chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS were the same (Fig. 35).

To study the therapeutic effect of mouse monoclonal antibodies against CLDN6 muMAB 64A on metastasis, a model with tumor cell xenografts was used: Nude-Foxn1 ^nu athymic mice were injected into the tail vein of NEC8 cells, and three days later these animals received CLDN6-specific antibodies muMAB 64A. After 8 weeks, the tumor burden in the lung tissue was determined using polymerase chain reaction. The mouse monoclonal antibody against CLDN6 muMAB 64A, compared with the control (PBS without antibodies), caused a clear inhibition of tumor growth (Fig. 36).

Immunohistochemical staining of human cancerous and normal tissues using monoclonal antibodies muMAB 64A, mAb206-LCC and mAb206-SUBG showed that, in contrast to normal tissues, strong uniform staining was observed in sections of ovarian and testicular tumors. Very intense membrane staining occurred in malignant epithelial cells, whereas adjacent stromal cells and non-malignant epithelial cells were not stained (Fig. 37). These results clearly indicate that the CLDN6-specific antibodies of the present invention specifically bind to malignant cells obtained from tumor patients. (Explanation: number of tissues stained with antibodies/number of tissues examined).

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LIST OF SEQUENCES

<110> Ganymed Pharmaceuticals AG et al.

<120> Antibodies for treatment of cancer

<130> 342-64 PCT

<150>EP 11 004 004.5

<151> 2011-05-13

<150>US 61/486,071

<151> 2011-05-13

<160> 57

<170> Patent In version 3.5

<210> 1

<211> 1369

<212> DNA

<213> Homo sapiens

<400> 1

cgacactcgg cctaggaatt tcccttatct ccttcgcagt gcagctcctt caacctcgcc 60

atggcctctg ccggaatgca gatcctggga gtcgtcctga cactgctggg ctgggtgaat 120

ggcctggtct cctgtgccct gcccatgtgg aaggtgaccg ctttcatcgg caacagcatc 180

gtggtggccc aggtggtgtg ggagggcctg tggatgtcct gcgtggtgca gagcaccggc 240

cagatgcagt gcaaggtgta cgactcactg ctggcgctgc cacaggacct gcaggctgca 300

cgtgccctct gtgtcatcgc cctccttgtg gccctgttcg gcttgctggt ctaccttgct 360

ggggccaagt gtaccacctg tgtggaggag aaggattcca aggcccgcct ggtgctcacc 420

tctgggattg tctttgtcat ctcaggggtc ctgacgctaa tccccgtgtg ctggacggcg 480

catgccatca tccgggactt ctataacccc ctggtggctg aggcccaaaa gcgggagctg 540

ggggcctccc tctacttggg ctgggcggcc tcaggccttt tgttgctggg tggggggttg 600

ctgtgctgca cttgcccctc gggggggtcc cagggcccca gccattacat ggcccgctac 660

tcaacatctg cccctgccat ctctcggggg ccctctgagt accctaccaa gaattacgtc 720

tgacgtggag gggaatgggg gctccgctgg cgctagagcc atccagaagt ggcagtgccc 780

aacagctttg ggatgggttc gtaccttttg tttctgcctc ctgctatttt tcttttgact 840

gaggatattt aaaattcatt tgaaaactga gccaaggtgt tgactcagac tctcacttag 900

gctctgctgt ttctcaccct tggatgatgg agccaaagag gggatgcttt gagattctgg 960

atcttgacat gcccatctta gaagccagtc aagctatgga actaatgcgg aggctgcttg 1020

ctgtgctggc tttgcaacaa gacagactgt ccccaagagt tcctgctgct gctgggggct 1080

gggcttccct agatgtcact ggacagctgc cccccatcct actcaggtct ctggagctcc 1140

tctcttcacc cctggaaaaa caaatgatct gttaacaaag gactgcccac ctccggaact 1200

tctgacctct gtttcctccg tcctgataag acgtccaccc cccagggcca ggtcccagct 1260

atgtagaccc ccgcccccac ctccaacact gcacccttct gccctgcccc cctcgtctca 1320

ccccctttac actcacattt ttatcaaata aagcatgttt tgttagtgc 1369

<210> 2

<211> 220

<212>PRT

<213> Homo sapiens

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Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu

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Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210> 3

<211> 805

<212> DNA

<213> Artificial

<220>

<223> codon-optimized nucleic acid sequence encoding human claudin 6

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caagcgcgtc aattaaccct cactaaaggg aacaaaagct gttaattaac taaggtacca 60

agcttgccac catggccagc gccggcatgc agatcctggg agtggtgctg accctgctgg 120

gctgggtgaa cggcctggtg tcctgcgccc tgcccatgtg gaaagtgacc gccttcatcg 180

gcaacagcat cgtggtggcc caggtcgtgt gggagggcct gtggatgagc tgtgtggtgc 240

agagcaccgg ccagatgcag tgcaaggtgt acgacagcct gctggccctg cctcaggatc 300

tgcaggccgc cagagccctg tgtgtgatcg ccctgctggt cgccctgttc ggcctgctgg 360

tgtacctcgc tggcgccaag tgcaccacct gtgtggagga aaaggacagc aaggcccggc 420

tggtcctgac aagcggcatc gtgttcgtga tcagcggcgt gctgacactg atccccgtgt 480

gctggaccgc ccacgccatc atccgggact tctacaaccc tctggtggcc gaggcccaga 540

agagagagct gggcgccagc ctgtatctgg gatgggccgc ctcaggactg ctgctgctgg 600

gcggaggcct gctgtgctgt acatgtccta gcggcggctc ccagggccct agccactaca 660

tggcccggta cagcaccagc gcccctgcca tcagcagagg ccccagcgag taccccacca 720

agaactacgt gtgataggaa ttcgagctct tatggcgcgc ccaattcgcc ctatagtgag 780

tcgtattacg tcgcgctcac tggcc 805

<210> 4

<211> 165

<212> DNA

<213> Artificial

<220>

<223> codon-optimized nucleic acid sequence encoding the predicted

extracellular loop 2 (EC2) of human claudin 6

<400> 4

ggcgcgccaa ggtaccaagc ttgccaccat ggaaaccgac accctgctgc tgtgggtgct 60

gctcctgtgg gtcccaggct ctacaggcga cgccgcccag cccagagact tctacaaccc 120

cctggtggcc gaggcccaga agctcgagtc tagagggtta attaa 165

<210> 5

<211> 38

<212>PRT

<213> Artificial

<220>

<223> predicted 2nd extracellular loop of human claudin 6 containing an

Ig kappa leader sequence

<220>

<221> SIGNAL

<222> (1)..(21)

<223> Ig kappa leader sequence

<220>

<221> DOMAIN

<222> (26)..(38)

<223> predicted 2nd extracellular loop (EC2) of human claudin 6

<400> 5

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Arg Asp Phe Tyr Asn Pro Leu

20 25 30

Val Ala Glu Ala Gln Lys

35

<210> 6

<211> 13

<212>PRT

<213> Homo sapiens

<400> 6

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

1 5 10

<210> 7

<211> 53

<212>PRT

<213> Homo sapiens

<400> 7

Pro Met Trp Lys Val Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala

1 5 10 15

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

20 25 30

Gly Gln Met Gln Cys Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln

35 40 45

Asp Leu Gln Ala Ala

50

<210> 8

<211> 220

<212>PRT

<213> Homo sapiens

<220>

<221> VARIANT

<222> (143)..(143)

<223> human claudin 6 I143V polymorphism

<400> 8

Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu

1 5 10 15

Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210> 9

<211> 217

<212>PRT

<213> Homo sapiens

<400> 9

Met Ala Ser Thr Gly Leu Glu Leu Leu Gly Met Thr Leu Ala Val Leu

1 5 10 15

Gly Trp Leu Gly Thr Leu Val Ser Cys Ala Leu Pro Leu Trp Lys Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala

195 200 205

Ser Gly Leu Asp Lys Arg Asp Tyr Val

210 215

<210> 10

<211> 209

<212>PRT

<213> Homo sapiens

<400> 10

Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu

1 5 10 15

Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr

195 200 205

Val

<210> 11

<211> 220

<212>PRT

<213> Homo sapiens

<400> 11

Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly

1 5 10 15

Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val

85 90 95

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

100 105 110

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

115 120 125

Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg

130 135 140

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

145 150 155 160

Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly

165 170 175

Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr

180 185 190

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

195 200 205

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

210 215 220

<210> 12

<211> 159

<212> DNA

<213> Artificial

<220>

<223> codon-optimized nucleic acid sequence encoding the predicted

extracellular loop 1 (EC1) of human claudin 6

<400> 12

cccatgtgga aagtgaccgc cttcatcggc aacagcatcg tggtggccca ggtggtctgg 60

gagggcctgt ggatgagctg cgtggtgcag agcaccggcc agatgcagtg caaggtgtac 120

gacagcctgc tggccctgcc tcaggatctg caggctgct 159

<210> 13

<211> 106

<212>PRT

<213> Artificial

<220>

<223> predicted 1st extracellular loop of human claudin 6 containing an

Ig kappa leader sequence

<220>

<221> Signal

<222> (1)..(21)

<223> Ig kappa leader sequence

<220>

<221> Domain

<222> (26)..(78)

<223> predicted 1st extracellular loop (EC1) of human claudin 6

<400> 13

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ala Ala Gln Pro Pro Met Trp Lys Val Thr Ala

20 25 30

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

35 40 45

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

50 55 60

Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Leu Glu

65 70 75 80

Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn

85 90 95

Met His Thr Gly His His His His His

100 105

<210> 14

<211> 13

<212>PRT

<213> Homo sapiens

<400> 14

Pro Met Trp Lys Val Thr Ala Phe Ile Gly Asn Ser Ile

1 5 10

<210> 15

<211> 15

<212>PRT

<213> Homo sapiens

<400> 15

Met Trp Lys Val Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala

1 5 10 15

<210> 16

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Oligonucleotide

<400> 16

tccatgacgt tcctgacgtt 20

<210> 17

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 17

gagaaagctt gccgccacca tgggatggag ctggatcttt ctc 43

<210> 18

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 18

gagagggccc ttggtggagg ctgaagagac tgtgagagtg gtg 43

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 19

gagagggccc ttggtggagg ctgaggagac tgtgagagtg gtg 43

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 20

gagagggccc ttggtggagg ctgaggagac tgtgagagtg gtg 43

<210> 21

<211> 46

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 21

gagaaagctt gccgccacca tgcattttca agtgcagatt ttcagc 46

<210> 22

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 22

cacacgtacg tttgatttcc agcttggtgc ctc 33

<210> 23

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 23

cacacgtacg tttgatttcc agcttggtgc ctc 33

<210> 24

<211> 993

<212> DNA

<213> Homo sapiens

<400> 24

gcctccacca agggcccaag cgtgttcccc ctggccccca gcagcaagag caccagcggc 60

ggcacagccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgagc 120

tggaacagcg gagccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagagcagc 180

ggcctgtaca gcctgagcag cgtggtgacc gtgcccagca gcagcctggg cacccagacc 240

tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagag agtggagccc 300

aagagctgcg acaagaccca cacctgcccc ccctgcccag ccccagagct gctgggcgga 360

cccagcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag caggaccccc 420

gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc cagaggtgaa gttcaactgg 480

tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagagagga gcagtacaac 540

agcacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600

gaatacaagt gcaaggtctc caacaaggcc ctgccagccc ccatcgaaaa gaccatcagc 660

aaggccaagg gccagccacg ggagccccag gtgtacaccc tgccccccag ccgggaggag 720

atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc cagcgacatc 780

gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac ccccccagtg 840

ctggacagcg acggcagctt cttcctgtac agcaagctga ccgtggacaa gtccaggtgg 900

cagcagggca acgtgttcag ctgcagcgtg atgcacgagg ccctgcacaa ccactacacc 960

cagaagtccc tgagcctgag ccccggcaag tag 993

<210> 25

<211> 330

<212>PRT

<213> Homo sapiens

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 26

<211> 324

<212> DNA

<213> Homo sapiens

<400> 26

cgtacggtgg ccgctcccag cgtgttcatc ttccccccca gcgacgagca gctgaagtcc 60

ggcaccgcca gcgtggtgtg cctgctgaac aacttctacc cccgggaggc caaggtgcag 120

tggaaggtgg acaacgccct gcagagcggc aacagccagg agagcgtcac cgagcaggac 180

agcaaggact ccacctacag cctgagcagc accctgaccc tgagcaaggc cgactacgag 240

aagcacaagg tgtacgcctg cgaggtgacc caccagggcc tgtccagccc cgtgaccaag 300

agcttcaaca ggggcgagtg ctag 324

<210> 27

<211> 107

<212>PRT

<213> Homo sapiens

<400> 27

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

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

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 28

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<220>

<221> misc_feature

<222> (31)..(32)

<223> any nucleotide

<400> 28

tttttttttt tttttttttt tttttttttt nn 32

<210> 29

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 29

aagcagtggt atcaacgcag agtacgcggg 30

<210> 30

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 30

ctgctcactg gatggtggga agatgg 26

<210> 31

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 31

acaggggcca gtggatagac cgatg 25

<210> 32

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 32

gtaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

<210> 33

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 33

gtaatacgac tcactatagg gc 22

<210> 34

<211> 117

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH sequence

<400> 34

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

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Tyr Gly Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 35

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL sequence

<400> 35

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Val Tyr

35 40 45

Ser Thr Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Gly Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 117

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH sequence

<400> 36

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

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Thr Val Ser Ser

115

<210> 37

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL sequence

<400> 37

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 38

<211> 117

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH sequence

<400> 38

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

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ile Ile Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Thr Val Ser Ser

115

<210> 39

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL sequence

<400> 39

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 40

<211> 117

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH sequence

<400> 40

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

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Leu Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Tyr Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 41

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL sequence

<400> 41

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met

20 25 30

His Trp Phe Gln Leu Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Asn Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 42

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Any amino acid, preferably Leu or Phe, more preferably Leu

<400> 42

Xaa Gly Xaa Val Xaa

15

<210> 43

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Any amino acid, preferably Leu or Phe, more preferably Leu

<400> 43

Asp Xaa Gly Xaa Val Xaa

15

<210> 44

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Any amino acid, preferably Leu or Phe, more preferably Leu

<400> 44

Xaa Gly Xaa Val Xaa Asp

15

<210> 45

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Any amino acid, preferably Leu or Phe, more preferably Leu

<400> 45

Asp Xaa Gly Xaa Val Xaa Asp

15

<210> 46

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Any amino acid, preferably an aromatic amino acid, more

preferably Phe or Tyr, most preferably Tyr

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Any amino acid, preferably Leu or Phe, more preferably Leu

<400> 46

Ala Arg Asp Xaa Gly Xaa Val Xaa Asp Tyr

1 5 10

<210> 47

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR1 sequence

<400> 47

Gly Tyr Ser Phe Thr Gly Tyr Thr

15

<210> 48

<211> 8

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VH CDR2 sequence

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> Any amino acid, preferably Thr, Ser or Ile, most preferably Thr

<400> 48

Ile Asn Pro Tyr Asn Gly Gly Xaa

15

<210> 49

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> Any amino acid, preferably Ser or Asn, most preferably Ser

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more

preferably Tyr, Ser, or Asn, most preferably Asn

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably Ser or Tyr, more preferably Tyr

<400> 49

Arg Xaa Xaa Xaa Pro

15

<210> 50

<211> 7

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> Any amino acid, preferably Ser or Asn, most preferably Ser

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more

preferably Tyr, Ser, or Asn, most preferably Asn

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Any amino acid, preferably Ser or Tyr, more preferably Tyr

<400> 50

Gln Arg Xaa Xaa Xaa Pro Pro

15

<210> 51

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL CDR3 sequence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably Ser or Asn, most preferably Ser

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> Any amino acid, preferably Tyr, Ser, Ile, Asn or Thr, more

preferably Tyr, Ser, or Asn, most preferably Asn

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> Any amino acid, preferably Ser or Tyr, more preferably Tyr

<400> 51

Gln Gln Arg Xaa Xaa Xaa Pro Pro Trp Thr

1 5 10

<210> 52

<211> 5

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL CDR1 sequence

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> Any amino acid, preferably Ser or Asn, most preferably Ser

<400> 52

Ser Ser Val Xaa Tyr

15

<210> 53

<211> 3

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL CDR2 sequence

<400> 53

Ser Thr Ser

1

<210> 54

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL Sequence

<400> 54

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Gly Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 55

<211> 107

<212>PRT

<213> Artificial Sequence

<220>

<223> Antibody VL Sequence

<400> 55

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 56

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 56

gggataattt cagctgacta aacag 25

<210> 57

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 57

ttccgtttag ttaggtgcag ttatc 25

<---

Claims (41)

1. A method for producing an antibody or an antigen-binding fragment thereof that binds to CLDN6 bound to the surface of a cell expressing CLDN6, the method comprising the steps of: (a) culturing a host cell transformed to express an antibody or an antigen-binding fragment thereof; and (b) isolating an antibody or antigen-binding fragment thereof expressed by the host cell; wherein the transformed host cell contains: (i) a nucleic acid sequence encoding a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody heavy chain having amino acid sequences at positions 26-33, positions 51-58 and positions 97-106 of SEQ ID NO: 36, respectively, or a variant thereof; and a nucleic acid sequence encoding a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody light chain having amino acid sequences at positions 27-31, positions 49-51 and positions 88-97 of SEQ ID NO: 35, respectively, or a variant thereof, wherein the variant comprises heavy chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: 47, 48 and 46, respectively, and light chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: 52, 53 and 51 respectively. 2. The method according to claim 1, characterized in that the said method includes transformation of the host cell with an expression vector containing the specified nucleic acid sequence, and the expression vector contains a promoter sequence, a leader sequence, a translation initiation sequence, a light chain constant region, a constant heavy chain region, 3' untranslated sequence, polyadenylation sequence, or transcription termination sequence. 3. The method according to claim 1 or 2, characterized in that the antigen-binding fragment is a Fab, F(ab') ₂ , Fv or single-chain Fv. 4. Method according to any one of paragraphs. 1-3, wherein the cell is a HEK293 cell, a HEK293T cell, a dendritic cell, a B cell, a K562 cell, a HELA cell, a CHO cell, an NS/0 cell, a lymphocyte cell, or a eukaryotic cell. 5. A recombinant nucleic acid encoding a polypeptide that binds to CLDN6 associated with the surface of a cell that expresses CLDN6, wherein the recombinant nucleic acid comprises a nucleic acid sequence encoding a polypeptide comprising CDR1, CDR2 and CDR3 regions of an antibody heavy chain having amino acid sequences in positions 26-33, positions 51-58 and positions 97-106 of SEQ ID NO: 36, respectively, or a variant of said antibody heavy chain CDR1, CDR2 and CDR3 regions; and a nucleic acid sequence encoding a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody light chain having amino acid sequences at positions 27-31, positions 49-51 and positions 88-97 of SEQ ID NO: 35, respectively, or a variant of these CDR1 regions , CDR2 and CDR3 of the antibody light chain, wherein said variants comprise heavy chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: 47, 48 and 46, respectively, and light chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: : 52, 53 and 51, respectively. 6. The recombinant nucleic acid according to claim 5, characterized in that the nucleic acid sequence encodes a polypeptide containing a heavy chain variable region having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region having the amino acid sequence of SEQ ID NO: 35. 7. The recombinant nucleic acid according to claim 5, characterized in that the nucleic acid sequence encodes a polypeptide containing a heavy chain variable region having the amino acid sequence of SEQ ID NO: 36, and a light chain variable region having the amino acid sequence of SEQ ID NO: 54 or 55. 8. A recombinant cell for expressing a CLDN6 binding polypeptide associated with the surface of a cell expressing CLDN6, wherein the cell is a human cell and it contains a recombinant nucleic acid according to any one of claims. 5-7, operably linked to expression control sequences, and said polypeptide is encoded by said recombinant nucleic acid. 9. The recombinant cell of claim 8, wherein the cell is a HEK293 cell, a HEK293T cell, a dendritic cell, a B cell, a K562 cell, a HELA cell, or a lymphocyte cell. 10. A polypeptide that binds to CLDN6 associated with the surface of a cell expressing CLDN6, wherein the polypeptide is encoded by a recombinant nucleic acid according to any one of claims. 5-7 or can be obtained from a recombinant cell according to claim 8 or 9. 11. The polypeptide according to claim 10, containing the hinge region of an immunoglobulin. 12. An antibody that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the antibody is expressed by the recombinant cell of claim 8 or 9. 13. An antigen binding fragment that binds to CLDN6 associated with the surface of a cell expressing CLDN6, wherein the antigen binding fragment is expressed by the recombinant cell of claim 8 or 9. 14. The antigen binding fragment according to claim 13, which is a Fab, F(ab') ₂ , Fv or single chain Fv. 15. Antigen-binding fragment according to claim 13 or 14, characterized in that the antigen-binding fragment is fused to the hinge region of the immunoglobulin. 16. A recombinant nucleic acid encoding a polypeptide containing the CDR1, CDR2 and CDR3 regions of an antibody heavy chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the recombinant nucleic acid contains a nucleic acid sequence encoding a polypeptide containing the CDR1, CDR2 regions and an antibody heavy chain CDR3 having the amino acid sequences of SEQ ID NOs: 47, 48 and 46, respectively. 17. A recombinant cell for expressing a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody heavy chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the cell is a human cell and it contains the recombinant nucleic acid of claim 16, operably linked to expression control sequences, and said polypeptide is encoded by said recombinant nucleic acid. 18. The recombinant cell of claim 17, wherein the cell is a HEK293 cell, a HEK293T cell, a dendritic cell, a B cell, a K562 cell, a HELA cell, or a lymphocyte cell. 19. A polypeptide that binds CLDN6 bound to the surface of a cell expressing CLDN6, comprising the CDR1, CDR2 and CDR3 regions of the heavy chain of an antibody that binds CLDN6 bound to the surface of a cell expressing CLDN6, wherein the polypeptide is encoded by the recombinant nucleic acid of claim 16 or may be obtained from a recombinant cell according to claim 17 or 18. 20. A recombinant nucleic acid encoding a polypeptide containing the CDR1, CDR2 and CDR3 regions of an antibody light chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the recombinant nucleic acid contains a nucleic acid sequence encoding a polypeptide containing the CDR1, CDR2 regions and an antibody light chain CDR3 having the amino acid sequences of SEQ ID NOs: 52, 53 and 51, respectively. 21. A recombinant nucleic acid encoding a polypeptide containing an antibody light chain variable region that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the recombinant nucleic acid contains a nucleic acid sequence encoding a polypeptide containing an antibody light chain variable region having an amino acid sequence SEQ ID NO: 54 or 55. 22. A recombinant cell for expressing a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody light chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the cell is a human cell and it contains the recombinant nucleic acid of claim 20, operably linked to expression control sequences, and said polypeptide is encoded by said recombinant nucleic acid. 23. A recombinant cell for expressing a polypeptide containing an antibody light chain variable region that binds to CLDN6 bound to the surface of a cell expressing CLDN6, wherein the cell is a human cell and it contains the recombinant nucleic acid of claim 21 operably linked to the sequences expression control, and said polypeptide is encoded by said recombinant nucleic acid. 24. The recombinant cell of claim 23, wherein the cell is a HEK293 cell, a HEK293T cell, a dendritic cell, a B cell, a K562 cell, a HELA cell, or a lymphocyte cell. 25. A polypeptide that binds CLDN6 bound to the surface of a cell expressing CLDN6, comprising the CDR1, CDR2 and CDR3 regions of the light chain of an antibody that binds CLDN6 bound to the surface of a cell expressing CLDN6, wherein the polypeptide is encoded by the recombinant nucleic acid of claim 20 or may be obtained from a recombinant cell according to claim 22 or 24. 26. A polypeptide that binds CLDN6 bound to the surface of a cell expressing CLDN6, comprising a light chain variable region of an antibody that binds CLDN6 bound to the surface of a cell expressing CLDN6, wherein the polypeptide is encoded by the recombinant nucleic acid of claim 21 or can be produced from a recombinant cell according to claim 23 or 24. 27. A method for producing a recombinant eukaryotic host cell, comprising the steps of: A. transforming a eukaryotic cell (i) with an expression vector containing a recombinant nucleic acid according to any one of claims. 5-7, or (ii) an expression vector containing a recombinant nucleic acid containing a nucleic acid sequence encoding a polypeptide containing CDR1, CDR2 and CDR3 regions of an antibody heavy chain having amino acid sequences at positions 26-33, positions 51-58 and positions 97-106 SEQ ID NO: 36, respectively, or a variant thereof, and an expression vector containing a recombinant nucleic acid containing a nucleic acid sequence encoding a polypeptide containing the CDR1, CDR2 and CDR3 regions of an antibody light chain having amino acid sequences at positions 27- 31, provisions 49-51 and provisions 88-97 SEQ ID NO: 35, respectively, or a variant thereof; and b. obtaining a transformed cell, wherein the transformed cell contains a recombinant nucleic acid or recombinant nucleic acids, wherein the variant comprises heavy chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: 47, 48 and 46, respectively, and light chain CDR1, CDR2 and CDR3 regions having amino acid sequences according to SEQ ID NO: 52, 53 and 51, respectively. 28. The method of claim 27, wherein the recombinant eukaryotic host cell is a human cell. 29. The method of claim 27 or 28, wherein the cell is a HEK293 cell, a HEK293T cell, a dendritic cell, a B cell, a K562 cell, a HELA cell, or a lymphocyte cell. 30. A vector for expressing a polypeptide that binds to CLDN6 associated with the surface of a cell expressing CLDN6, wherein the vector contains a nucleic acid sequence according to any one of claims. 5-7, and expression control sequences to which said nucleic acid sequence is operably linked. 31. A vector for expressing a polypeptide comprising the CDR1, CDR2 and CDR3 regions of an antibody heavy chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, the vector comprising the nucleic acid sequence of claim 16 and expression control sequences with which the sequence the specified nucleic acid is operably linked. 32. A vector for expressing a polypeptide containing the CDR1, CDR2 and CDR3 regions of an antibody light chain that binds to CLDN6 bound to the surface of a cell expressing CLDN6, the vector comprising the nucleic acid sequence of claim 20 or 21 and expression control sequences with which the sequence of said nucleic acid is operably linked.

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