1. A method of determining cancer stem cells comprising detecting cells expressing CLDN 6.

2. The method of claim 1 wherein the presence of cells expressing CLDN6 indicates the presence of cancer stem cells and/or the amount of cells expressing CLDN6 correlates with the amount of cancer stem cells.

3. The method of claim 1 or 2 wherein cells expressing CLDN6 are detected in a sample obtained from a cancer patient.

4. The method of any one of claims 1 to 3 wherein the method comprises quantitative and/or qualitative determination of cells expressing CLDN 6.

5. The method of any one of claims 1 to 4 comprising comparing the amount of cells expressing CLDN6 to the amount of cells expressing CLDN6 in a reference sample or to a predetermined reference range.

6. The method of claim 5, wherein the reference sample is a sample from a patient diagnosed as not having cancer.

7. The method of claim 5, wherein the predetermined reference range is based on a population of patients diagnosed as not having cancer.

8. The method of any one of claims 1 to 7, comprising monitoring the amount of cancer stem cells in a cancer patient.

9. The method of claim 8, wherein monitoring the amount of cancer stem cells in a cancer patient comprises comparing the amount of cancer stem cells in a sample obtained from the cancer patient to the amount of cancer stem cells in a sample obtained earlier from the cancer patient.

10. The method of claim 9, wherein the sample obtained from the cancer patient is a sample taken from the cancer patient during or after administration of a cancer treatment.

11. A method of monitoring the efficacy of a cancer treatment in a cancer patient, comprising:

(i) determining the amount of cancer stem cells in a sample obtained from the cancer patient during or after administration of the cancer treatment; and

(ii) comparing the amount of cancer stem cells in the sample obtained from the cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient,

wherein determining the amount of cancer stem cells in the sample obtained from the cancer patient and/or determining the amount of cancer stem cells in the sample obtained earlier from the cancer patient comprises determining the amount of cells expressing CLDN 6.

12. The method of any one of claims 9 to 11, wherein the sample obtained earlier from the cancer patient is a sample taken from the cancer patient before, during or after administration of a cancer treatment.

13. The method of any one of claims 10 to 12, wherein a stable or decreased amount of cancer stem cells indicates that the cancer treatment is effective.

14. The method of any one of claims 10 to 12, wherein an increase in the amount of cancer stem cells indicates that the cancer treatment is ineffective.

15. The method of any one of claims 10 to 14, wherein the cancer therapy is a cancer therapy directed against cancer stem cells.

16. The method of any one of claims 3 to 15, wherein the sample obtained from the cancer patient is a biological fluid or a tumor tissue biopsy.

17. The method of any one of claims 3 to 11, wherein the sample has been subjected to one or more pre-treatment steps.

18. The method of any one of claims 1-17, wherein the cells expressing CLDN6 are detected or their amount is determined by using an immunoassay.

19. The method of claim 18, wherein the immunoassay is selected from the group consisting of: western blotting, immunohistochemistry, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, immunofluorescence, protein a immunoassays, flow cytometry and FACS analyses.

20. The method of any one of claims 1 to 19, wherein the cells expressing CLDN6 are detected or their amount is determined by using an antibody having the ability of binding to CLDN 6.

21. The method of any one of claims 1 to 20, wherein the cells expressing CLDN6 are cancer cells expressing CLDN6 and/or cells present at a tumor site.

22. A method of treating or preventing cancer comprising inhibiting and/or eliminating cancer stem cells by administering to a cancer patient an antibody having the ability of binding to CLDN 6.

23. The method of claim 22, wherein the cancer stem cells express CLDN 6.

24. The method of claim 22 or 23, further comprising administering chemotherapy and/or radiation therapy.

25. The method of any one of claims 22 to 24, wherein inhibiting and/or eliminating cancer stem cells enhances the anti-cancer effect of chemotherapy and/or radiotherapy.

26. The method of claim 25, wherein the enhancement of the anti-cancer effect of chemotherapy and/or radiation therapy comprises an extension of the lifespan of a cancer patient undergoing chemotherapy and/or radiation therapy.

27. A method of treating or preventing cancer comprising administering to a cancer patient (i) an antibody having the ability of binding to CLDN6 and (ii) chemotherapy.

28. The method of claim 27 wherein the cancer involves cancer stem cells expressing CLDN 6.

29. The method of claim 27 or 28, wherein administration of an antibody having the ability of binding to CLDN6 results in the inhibition or elimination of cancer stem cells expressing CLDN 6.

30. The method of any one of claims 27 to 29, wherein administration of an antibody having the ability of binding to CLDN6 enhances the anti-cancer effect of chemotherapy.

31. The method of claim 30, wherein the enhancement of the anti-cancer effect of chemotherapy comprises an extension of the lifespan of a cancer patient undergoing chemotherapy.

32. The method of any one of claims 22-26 and 29-31, wherein elimination of cancer stem cells results in a cure for cancer.

33. The method of any one of claims 24 to 32, wherein the antibody having the ability of binding to CLDN6 and the chemotherapy are administered in synergistically effective amounts.

34. The method of any one of claims 24 to 33, wherein the chemotherapy is administered at a dose that is lower than the maximum tolerated dose.

35. The method of any one of claims 24 to 34, wherein the chemotherapy comprises administering an agent selected from the group consisting of: taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, prodrugs thereof, salts thereof, and combinations thereof.

36. The method of any one of claims 24 to 35, wherein the chemotherapy comprises administering an agent selected from the group consisting of: paclitaxel, cisplatin, carboplatin, prodrugs thereof, salts thereof, and combinations thereof.

37. The method of any one of claims 22-26 and 28-36, wherein the cancer stem cell is located at a tumor site of the cancer patient.

38. The method according to any one of claims 22 to 37, wherein the cancer is resistant to chemotherapy, in particular if the chemotherapy is administered as monotherapy.

39. The method of any one of claims 22 to 38, wherein the antibody having the ability of binding to CLDN6 has an inhibitory and/or cytotoxic effect on cancer stem cells.

40. The method of claim 39, wherein the antibody having the ability of binding to CLDN6 exerts its inhibitory and/or cytotoxic effect on cancer stem cells by mediating one or more of: complement Dependent Cytotoxicity (CDC) -mediated lysis, Antibody Dependent Cellular Cytotoxicity (ADCC) -mediated lysis, induction of apoptosis, and inhibition of proliferation.

41. The method of any one of claims 22 to 40, wherein the antibody having the ability of binding to CLDN6 is coupled to a therapeutic moiety.

42. The method of claim 41, wherein said therapeutic moiety is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide.

43. The method of claim 41 or 42, wherein the therapeutic moiety acts on slow growing cells.

44. The method of any one of claims 22 to 43, wherein the antibody having the ability of binding to CLDN6 binds to the first extracellular loop of CLDN 6.

45. The method of any one of claims 22 to 44, wherein the antibody having the ability of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

46. A method of treating or preventing cancer comprising administering to a cancer patient an antibody drug conjugate comprising an antibody having the ability of binding to CLDN6 covalently linked to at least one toxin drug moiety through a linker.

47. The method of claim 46, wherein the toxin drug moiety is cell membrane permeable.

48. The method of claim 46 or 47, wherein the toxin drug moiety is a maytansinoid or an auristatin.

49. The method of claim 48, wherein the maytansinoid is selected from the group consisting of DM1 and DM 4.

50. The method of claim 48, wherein the auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

51. The method of any one of claims 46 to 50, wherein the linker is a cleavable linker.

52. The method of any one of claims 46 to 51, wherein the linker is a cathepsin-cleavable linker.

53. The method of any one of claims 46-52, wherein the antibody is linked to the linker through a cysteine thiol group of the antibody.

54. The method of any one of claims 46 to 53, wherein the cancer involves cancer stem cells expressing CLDN 6.

55. The method of any one of claims 46 to 54, wherein administration of the antibody drug conjugate results in the inhibition or elimination of CLDN 6-expressing cancer stem cells.

56. The method of any one of claims 46 to 55, further comprising administering chemotherapy and/or radiation therapy.

57. The method of any one of claims 46-56, wherein administration of the antibody drug conjugate enhances the anti-cancer effects of chemotherapy and/or radiation therapy.

58. The method of any one of claims 46 to 57, wherein the antibody having the ability of binding to CLDN6 binds to the first extracellular loop of CLDN6 in the antibody drug conjugate.

59. The method of any one of claims 46 to 58, wherein the antibody having the ability of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

60. The method of any one of claims 1 to 59, wherein CLDN6 has an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

61. The method of any one of claims 1 to 60, wherein the cancer comprises a primary cancer, an advanced cancer, a metastatic cancer, a recurrent cancer, or a combination thereof.

62. A method of treating or preventing cancer, comprising:

(i) determining cancer stem cells in a cancer patient by a method according to any one of claims 1 to 21, and

(ii) administering a cancer treatment directed to cancer stem cells to the cancer patient.

63. The method of claim 62, wherein the cancer treatment against cancer stem cells comprises performing the method of treating or preventing cancer of any one of claims 22-61.

64. A method of preventing cancer chemoresistance, cancer recurrence or cancer metastasis, in particular during or after cancer treatment, the method comprising treating cancer by a method according to any one of claims 22 to 63.

65. A medical article for use in the treatment or prevention of cancer comprising (i) an antibody having the ability of binding to CLDN6 and (ii) a chemotherapeutic agent.

66. The medical article of claim 65 in the form of a kit comprising a first container comprising the antibody having the ability of binding to CLDN6 and a second container comprising the chemotherapeutic agent.

67. The pharmaceutical product of claim 65 or 66, further comprising printed instructions for using the product to treat or prevent cancer.

68. An antibody drug conjugate comprising an antibody having the ability of binding to CLDN6 covalently linked to at least one toxin drug moiety through a linker.

69. The antibody drug conjugate of claim 68, wherein the toxin drug moiety is cell membrane permeable.

70. The antibody drug conjugate of claim 68 or 69, wherein said toxin drug moiety is a maytansinoid or an auristatin.

71. The antibody drug conjugate of claim 70, wherein the maytansinoid is selected from the group consisting of DM1 and DM 4.

72. The antibody drug conjugate of claim 70, wherein said auristatin is selected from the group consisting of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

73. The antibody drug conjugate of any one of claims 68 to 72, wherein the linker is a cleavable linker.

74. The antibody drug conjugate of any one of claims 68-73, wherein the linker is a cathepsin-cleavable linker.

75. The antibody drug conjugate of any one of claims 68-74, wherein said antibody is linked to said linker through a cysteine thiol group of said antibody.

76. The antibody drug conjugate of any one of claims 68 to 75, wherein said antibody having the ability of binding to CLDN6 in said antibody drug conjugate binds to the first extracellular loop of CLDN 6.

77. The antibody drug conjugate of any one of claims 68 to 76, wherein the antibody having the ability of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

78. A pharmaceutical formulation comprising the antibody drug conjugate of any one of claims 68 to 77, and a pharmaceutically acceptable diluent, carrier or excipient.

79. A pharmaceutical product comprising the antibody drug conjugate of any one of claims 68-77, and a chemotherapeutic agent.

80. The pharmaceutical product of claim 79, in the form of a kit comprising a first container comprising the antibody drug conjugate and a second container comprising the chemotherapeutic agent.

81. The pharmaceutical product of claim 79 or 80, further comprising printed instructions for using the product to treat or prevent cancer.

Background

Conventional cancer treatments are primarily intended to selectively detect and eradicate the majority of rapidly growing cancer cells (i.e., cells that form tumor masses) and to exert their toxic effects on cancer cells primarily by interfering with cellular mechanisms involved in cell growth and DNA replication. Furthermore, standard oncology protocols are primarily designed to administer the highest dose of irradiation or chemotherapeutic agent without undue toxicity, i.e., commonly referred to as the "maximum tolerated dose" (MTD).

Chemotherapy regimens also often involve the administration of a combination of chemotherapeutic agents in an attempt to improve the efficacy of the treatment. Although a wide variety of chemotherapeutic agents may be used, these treatments have a number of disadvantages. For example, chemotherapeutic agents cause significant and often dangerous side effects due to non-specific side effects on rapidly growing cells (whether normal or malignant).

Other types of cancer therapy include surgery, hormonal therapy, immunotherapy, epigenetic therapy, anti-angiogenic therapy, targeted therapy, and radiation therapy to eradicate neoplastic cells (neoplastic cells) in a patient.

However, all conventional approaches for cancer treatment have significant drawbacks for the patient, including lack of efficacy (particularly in terms of long-term outcome) and toxicity. Therefore, new therapies for treating cancer patients are needed.

There is increasing evidence that there is a subpopulation of cancer cells within a tumor that retain stem cell-like properties. This subset is called Cancer Stem Cells (CSCs). Cancer stem cells have similar properties compared to normal stem cells, they have the ability to self-renew and form all heterogeneous cell(s) types of tumors. An effective assay to analyze CSC-like properties of tumor cells is a colony formation assay. Using this assay, the self-renewal capacity and tumor-formation potential of a single tumor cell can be easily examined.

Cancer stem cells are thought to be capable of initiating tumor formation, maintaining tumor growth, and possibly causing dissemination of the tumor to distant organ sites in the body. Cancer stem cells comprise a unique subpopulation of tumors that are more tumorigenic, grow relatively more slowly or quiescently, and tend to be relatively more chemoresistant than the tumor mass, relative to the rest of the cells of the tumor (i.e., the tumor mass). Because conventional cancer therapies target rapidly proliferating cells (i.e., cells that form tumor masses), these therapies are considered to be relatively ineffective at targeting and damaging cancer stem cells. Cancer stem cells may exhibit other characteristics that make them relatively more chemoresistant, such as multidrug resistance and anti-apoptotic pathways. The inability to adequately target and eradicate cancer stem cells would constitute a key reason for the inability to perform standard oncology treatment protocols to ensure long-term benefit in many cancer patients. Thus, cancer stem cells may be the leading cause of cancer metastasis as well as cancer recurrence and drug inefficiency after treatment. Thus, one opportunity to cure cancer is to eliminate cancer stem cells.

Claudin (claudin) is an integral membrane protein located within the tight junction of the epithelium and endothelium. Claudin is predicted to have four transmembrane segments with two extracellular loops, and the N-and C-termini are located in the cytoplasm. The Claudin (CLDN) family of transmembrane proteins plays a key role in the maintenance of epithelial and endothelial tight junctions, and may also play a role in the maintenance of the cytoskeleton and in cell signaling. CLDN6 is expressed in a range of different human cancer cells, whereas expression in normal tissues is restricted to the placenta.

Here we present data demonstrating that expression of CLDN6 is upregulated during the generation of pluripotent cells. Furthermore, CLDN6 was closely related to a known marker of cancer stem cells and CLDN6 positive tumor cells showed increased colony formation. This also demonstrates that treatment with CLDN 6-specific antibodies can overcome chemotherapy resistance of tumors (e.g., ovarian cancer) and that the combination of chemotherapy with CLDN6 antibody treatment has a significant synergistic effect.

The results presented herein indicate that CLDN6 is a novel marker for cancer stem cells and that cancer stem cells can be targeted for diagnostic and therapeutic purposes by targeting CLDN 6.

Disclosure of Invention

In one aspect, the present invention relates to a method of determining cancer stem cells comprising detecting cells expressing CLDN 6.

In one embodiment, the presence of cells expressing CLDN6 indicates the presence of cancer stem cells and/or the amount of cells expressing CLDN6 correlates with the amount of cancer stem cells. In one embodiment, cells expressing CLDN6 are detected in a sample obtained from a cancer patient, e.g., before, during and/or after cancer treatment. In one embodiment, the method comprises quantitative and/or qualitative determination of cells expressing CLDN 6. In one embodiment, the method comprises comparing the amount of cells expressing CLDN6 to the amount of cells expressing CLDN6 in a reference sample or to a predetermined reference range. The reference sample may be a sample from a patient diagnosed not to have cancer. The predetermined reference range can be based on a population of patients diagnosed as not having cancer. In one embodiment, the method comprises monitoring the amount of cancer stem cells in a cancer patient, wherein monitoring the amount of cancer stem cells in a cancer patient preferably comprises comparing the amount of cancer stem cells in a sample obtained from a cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient. In one embodiment, the sample obtained from the cancer patient is a sample taken from the cancer patient during or after administration of a cancer treatment.

In another aspect, the present invention relates to a method of monitoring the efficacy of a cancer treatment in a cancer patient, comprising: i) determining the amount of cancer stem cells in a sample obtained from the cancer patient during or after administration of a cancer treatment; and (ii) comparing the amount of cancer stem cells in the sample obtained from the cancer patient with the amount of cancer stem cells in a sample obtained earlier from the cancer patient, wherein determining the amount of cancer stem cells in the sample obtained from the cancer patient and/or determining the amount of cancer stem cells in the sample obtained earlier from the cancer patient comprises determining the amount of cells expressing CLDN 6.

In one embodiment, the sample obtained earlier from a patient with cancer is a sample taken from the cancer patient before, during or after administration of a cancer treatment.

In one embodiment of the methods of all aspects of the invention, a stable or decreased amount of cancer stem cells indicates that the cancer treatment is effective. In one embodiment of the methods of all aspects of the invention, an increase in the amount of cancer stem cells indicates that the cancer treatment is ineffective. In one embodiment of the methods of all aspects of the invention, the cancer treatment is a cancer treatment directed against cancer stem cells. In one embodiment of the method of all aspects of the invention, the sample obtained from the cancer patient is a biological fluid or a tumor tissue biopsy (biopsy). In one embodiment of the method of all aspects of the invention, the sample has been subjected to one or more pre-treatment steps. In one embodiment of the methods of all aspects of the invention, cells expressing CLDN6 are detected or their amount determined by detecting or determining the amount of CLDN6 protein and/or CLDN6 mRNA. In one embodiment of the methods of all aspects of the invention, cells expressing CLDN6 are detected or their amount is determined by using an immunoassay, wherein the immunoassay is preferably selected from the group consisting of: western blot, immunohistochemistry, radioimmunoassay (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay ("sandwich" immunoassay), immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay (agglutination assay), complement fixation assay (complementary-hybridization assay), immunoradiometric assay (immunoradiometric assay), fluorescent immunoassay, immunofluorescence, protein a immunoassay, flow cytometry and FACS analysis. In one embodiment of the methods of all aspects of the invention, cells expressing CLDN6 are detected or their amount determined by using an antibody having the ability of binding to CLDN 6. In one embodiment of the methods of all aspects of the invention, the cells expressing CLDN6 are cancer cells expressing CLDN6 and/or cells present at the tumor site.

In another aspect, the present invention relates to a method of treating or preventing cancer comprising inhibiting and/or eliminating cancer stem cells by administering to a cancer patient an antibody having the ability of binding to CLDN 6.

In one embodiment, the cancer stem cell expresses CLDN 6. In one embodiment, the method further comprises administering chemotherapy and/or radiation therapy. In one embodiment, inhibiting and/or eliminating cancer stem cells enhances the anti-cancer effect of chemotherapy and/or radiation therapy, wherein the enhancement of the anti-cancer effect of chemotherapy and/or radiation therapy comprises an extension of the life span of a cancer patient undergoing chemotherapy and/or radiation therapy.

In another aspect, the invention relates to a method of treating or preventing cancer comprising administering to a cancer patient (i) an antibody having the ability of binding to CLDN6 and (ii) chemotherapy.

In one embodiment, the cancer involves cancer stem cells expressing CLDN 6. In one embodiment, administration of an antibody having the ability of binding to CLDN6 results in the inhibition or elimination of cancer stem cells expressing CLDN 6. In one embodiment, the administration of an antibody having the ability of binding to CLDN6 enhances the anti-cancer effect of chemotherapy, wherein said enhancement of the anti-cancer effect of chemotherapy preferably comprises an extension of the life span of a cancer patient undergoing chemotherapy.

In one embodiment of the method of all aspects of the invention, the elimination of cancer stem cells results in a cure of the cancer. In one embodiment of the methods of all aspects of the invention, the antibody having the ability of binding to CLDN6 and the chemotherapy are administered in synergistically effective amounts. In one embodiment of the method of all aspects of the invention, the chemotherapy is administered at a dose below the maximum tolerated dose. In one embodiment of the method of all aspects of the invention, the chemotherapy comprises administering an agent selected from the group consisting of: taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, prodrugs thereof, salts thereof, and combinations thereof. In one embodiment of the method of all aspects of the invention, the chemotherapy comprises administering an agent selected from the group consisting of: paclitaxel, cisplatin, carboplatin, prodrugs thereof, salts thereof, and combinations thereof. In one embodiment of the method of all aspects of the invention, the cancer stem cells are located at the tumor site of a cancer patient. In one embodiment of the method of all aspects of the invention, the cancer is resistant to chemotherapy, particularly if said chemotherapy is administered as monotherapy. In one embodiment of the methods of all aspects of the invention, the antibody having the ability of binding to CLDN6 has an inhibitory and/or cytotoxic effect on cancer stem cells (cytoxic effect), wherein the antibody having the ability of binding to CLDN6 exerts its inhibitory and/or cytotoxic effect on cancer stem cells by mediating one or more of the following: complement Dependent Cytotoxicity (CDC) -mediated lysis, Antibody Dependent Cellular Cytotoxicity (ADCC) -mediated lysis, induction of apoptosis, and inhibition of proliferation. In one embodiment of the methods of all aspects of the invention, an antibody having the ability of binding to CLDN6 is coupled to a therapeutic moiety and may be an antibody drug conjugate as described herein. In one embodiment, the therapeutic moiety is a cytotoxic agent, a chemotherapeutic agent, or a radionuclide. In one embodiment, the therapeutic moiety acts on slow-growing cells. In one embodiment of the methods of all aspects of the invention, an antibody having the ability of binding to CLDN6 binds to the first extracellular loop of CLDN 6. In one embodiment of the methods of all aspects of the invention, the antibody having the ability of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

In another aspect, the present invention relates to a method of treating or preventing cancer comprising administering to a cancer patient an antibody drug conjugate comprising an antibody having the ability of binding to CLDN6 covalently linked to at least one toxin drug moiety through a linker.

In one embodiment, the toxin drug moiety is cell membrane permeable. In one embodiment, at least one of the toxin drug moieties acts on slow growing cells. In one embodiment, the toxin drug moiety is a maytansinoid (maytansinoid) or an auristatin (auristatin). In one embodiment, the maytansinoid is selected from the group consisting of DM1 and DM 4. In one embodiment, the auristatin is selected from monomethyl auristatin e (mmae) and monomethyl auristatin f (mmaf). In one embodiment, the linker is a cleavable linker, preferably a cathepsin cleavable linker. In one embodiment, the antibody is linked to the linker through a cysteine thiol group of the antibody.

In one embodiment, the cancer involves cancer stem cells expressing CLDN 6. In one embodiment, administration of the antibody drug conjugate results in the inhibition or elimination of cancer stem cells expressing CLDN 6. In one embodiment, the elimination of the cancer stem cells results in a cure for the cancer. In one embodiment, the cancer stem cell is located at a tumor site of the cancer patient. In one embodiment, the antibody drug conjugate has an inhibitory and/or cytotoxic effect on cancer stem cells, wherein the antibody drug conjugate exerts its inhibitory and/or cytotoxic effect on cancer stem cells, preferably by inducing apoptosis and/or inhibiting proliferation.

In one embodiment, the method further comprises administering chemotherapy and/or radiation therapy.

In one embodiment, administration of the antibody drug conjugate enhances the anti-cancer effect of chemotherapy and/or radiotherapy, wherein the enhancement of the anti-cancer effect of chemotherapy and/or radiotherapy preferably comprises an extension of the life span of a cancer patient undergoing chemotherapy and/or radiotherapy.

In one embodiment, the antibody drug conjugate and the chemotherapy are administered in synergistically effective amounts. In one embodiment, the chemotherapy is administered at a dose below the maximum tolerated dose. In one embodiment, the chemotherapy comprises administering an agent selected from the group consisting of: taxanes, platinum compounds, nucleoside analogs, camptothecin analogs, anthracyclines, prodrugs thereof, salts thereof, and combinations thereof. In one embodiment, the chemotherapy comprises administering an agent selected from the group consisting of: paclitaxel, cisplatin, carboplatin, prodrugs thereof, salts thereof, and combinations thereof. In one embodiment, the cancer is resistant to chemotherapy, particularly if the chemotherapy is administered as monotherapy.

In one embodiment, the antibody having the ability of binding to CLDN6 (particularly when present in the antibody drug conjugate) has an affinity and/or specificity for CLDN6 to suitably allow endocytosis (endocytosis) of the antibody and/or the antibody drug conjugate. In one embodiment, an antibody having the ability of binding to CLDN6 in said antibody drug conjugate binds to the first extracellular loop of CLDN 6. In one embodiment, the antibody of the antibody drug conjugate capable of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

In one embodiment of the methods of all aspects of the invention, CLDN6 has an amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment of the methods of all aspects of the invention, the cancer comprises a primary cancer, an advanced cancer, a metastatic cancer, a recurrent cancer, or a combination thereof.

In another aspect, the invention relates to a method of treating or preventing cancer, comprising: (i) determining cancer stem cells in a cancer patient by the method of the invention and (ii) administering a cancer treatment for the cancer stem cells to the cancer patient. In one embodiment, the cancer treatment directed to cancer stem cells comprises performing the methods of treating or preventing cancer of the present invention.

In another aspect, the invention relates to a method of preventing cancer chemoresistance, cancer recurrence or cancer metastasis (particularly during or after cancer treatment), comprising treating cancer by the method of the invention.

In another aspect, the present invention provides a pharmaceutical preparation (medical prophylaxis) for treating or preventing cancer comprising (i) an antibody having the ability of binding to CLDN6 and (ii) a chemotherapeutic agent. The antibody having the ability of binding to CLDN6 and the chemotherapeutic agent may be present in the pharmaceutical preparation in a mixture or separately from each other. The pharmaceutical product may be in the form of a kit comprising a first container comprising the antibody having the ability of binding to CLDN6 and a second container comprising the chemotherapeutic agent. The pharmaceutical product may also include printed instructions for using the product (particularly using the product in the methods of the invention) to treat or prevent cancer. Various embodiments of pharmaceutical preparations, in particular antibodies and chemotherapeutic agents capable of binding to CLDN6, are described herein.

In a particular aspect, the invention provides a pharmaceutical preparation comprising (i) an antibody having the ability of binding to CLDN6 and (ii) paclitaxel. The antibody having the ability of binding to CLDN6 and paclitaxel may be present in the pharmaceutical preparation in a mixture or separately from each other. The pharmaceutical product may be used for the treatment or prevention of cancer, for example ovarian cancer. The pharmaceutical product may be in the form of a kit comprising a first container comprising the antibody having the ability of binding to CLDN6 and a second container comprising paclitaxel. The pharmaceutical product may also include printed instructions for use of the product, particularly for use in the methods of the invention, in the treatment or prevention of cancer, such as ovarian cancer. Various embodiments of a medical preparation, in particular an antibody having the ability of binding to CLDN6, are described herein.

In another aspect, the present invention provides an antibody drug conjugate comprising an antibody having the ability of binding to CLDN6 covalently linked to at least one toxin drug moiety through a linker.

In one embodiment, the toxin drug moiety is cell membrane permeable. In one embodiment, at least one of the toxin drug moieties acts on slow growing cells. In one embodiment, the toxin drug moiety is a maytansinoid or an auristatin. In one embodiment, the maytansinoid is selected from the group consisting of DM1 and DM 4. In one embodiment, the auristatin is selected from monomethyl auristatin e (mmae) and monomethyl auristatin f (mmaf). In one embodiment, the linker is a cleavable linker, preferably a cathepsin cleavable linker. In one embodiment, the antibody is linked to the linker through a cysteine thiol group of the antibody.

In one embodiment, the antibody having the ability of binding to CLDN6 (particularly when present in the antibody drug conjugate) has an affinity and/or specificity for CLDN6 to suitably allow endocytosis of the antibody and/or the antibody drug conjugate. In one embodiment, an antibody having the ability of binding to CLDN6 in said antibody drug conjugate binds to the first extracellular loop of CLDN 6. In one embodiment, the antibody of the antibody drug conjugate capable of binding to CLDN6 comprises: comprises a polypeptide consisting of SEQ ID NO: 5 or a fragment thereof and a light chain variable region (VH) comprising an amino acid sequence represented by SEQ ID NO: 4 or a fragment thereof, or a light chain variable region (VL).

In another aspect, the invention provides a pharmaceutical formulation (pharmaceutical formulation) comprising an antibody drug conjugate of the invention, and a pharmaceutically acceptable diluent, carrier or excipient.

In another aspect, the invention provides a pharmaceutical product comprising an antibody drug conjugate of the invention, and a chemotherapeutic agent. Preferably, the pharmaceutical product is for use in the treatment or prevention of cancer. The antibody drug conjugate and the chemotherapeutic agent may be present in the pharmaceutical product in a mixture or separately from each other. The pharmaceutical product may be in the form of a kit comprising a first container comprising the antibody drug conjugate and a second container comprising the chemotherapeutic agent. The pharmaceutical product may also include printed instructions for using the product (particularly using the product for use in the methods of the invention) to treat or prevent cancer. Various embodiments of pharmaceutical products (particularly antibody drug conjugates and chemotherapeutic agents) are described herein.

In a particular aspect, the invention provides a pharmaceutical product comprising an antibody drug conjugate of the invention and paclitaxel. The antibody drug conjugate and paclitaxel may be present in the pharmaceutical product in a mixture or separately from each other. The pharmaceutical product may be used for the treatment or prevention of cancer, for example ovarian cancer. The pharmaceutical product may be in the form of a kit comprising a first container comprising the antibody drug conjugate and a second container comprising paclitaxel. The pharmaceutical product may also include printed instructions for using the product, particularly using the product for use in the methods of the invention, to treat or prevent cancer, such as ovarian cancer. Various embodiments of pharmaceutical products (particularly antibody drug conjugates) are described herein.

The invention also provides agents and compositions described herein, such as antibody drug conjugates, antibodies capable of binding to CLDN6, and/or chemotherapeutic agents for use in the methods described herein. For example, the invention also provides antibody drug conjugates or antibodies having the ability of binding to CLDN6 for administration with chemotherapeutic agents (e.g., paclitaxel).

In one embodiment, the antibody having the ability of binding to CLDN6 is a monoclonal antibody, a chimeric antibody or a humanized antibody or a fragment of an antibody. In one embodiment, the antibody mediates cell killing when bound to CLDN6 of a cell, particularly CLDN6 expressed by the cell on its cell surface, wherein the cell is preferably a cancer stem cell, e.g., a cancer stem cell of a cancer described herein.

According to the invention, the cancer is preferably selected from: ovarian cancer (particularly ovarian adenocarcinoma and ovarian teratocarcinoma), lung cancer including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC) (particularly squamous cell lung cancer and adenocarcinoma), Large Cell Carcinoma (LCC), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer (particularly basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (particularly malignant polymorphic adenoma), sarcoma (particularly synovial sarcoma and carcinosarcoma), cholangiocarcinoma, bladder cancer (particularly transitional cell carcinoma and papillary carcinoma), renal cancer (particularly renal cell carcinoma) including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small intestine cancer including ileal carcinoma (particularly small intestine adenocarcinoma and ileal adenocarcinoma), placental cancer, cervical cancer, testicular cancer (particularly testicular seminoma, teratocarcinoma and testicular embryonal carcinoma), uterine cancer, choriocarcinoma, and carcinoma, Germ cell tumors such as teratocarcinoma or embryonal carcinoma (particularly germ cell tumors of the testis and ovary), and metastatic forms thereof.

According to the invention, cancer cells and/or cancer stem cells expressing CLDN6 are preferably cells of a cancer as described herein.

In one embodiment, the cancer described herein is CLDN6 positive. In one embodiment, the cancer cells of the cancers described herein are CLDN6 positive. In one embodiment, the cancer cells of the cancers described herein express CLDN6 on their cell surface.

In one embodiment, the cancer described herein comprises a primary cancer, an advanced cancer, a metastatic cancer, a recurrent cancer, or a combination thereof, e.g., a combination of a primary cancer and a metastatic cancer. In one embodiment, the cancer is partially or completely refractory to chemotherapy (e.g., paclitaxel monotherapy). In one embodiment, the cancer is ovarian cancer, particularly ovarian cancer that is partially or completely refractory to chemotherapy (e.g., paclitaxel monotherapy).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Drawings

FIG. 1: CLDN6 mRNA was expressed in human iPS cells.

Human Foreskin Fibroblasts (HFFs) were transfected with Lipofectamine RNAiMAX (Life Technologies) without RNA (no RNA control) or with reprogramming cocktail (unmodified OSKMNL + EBK + miR-mix) and cells were collected on days 5, 12 and 19 post-treatment. RNA was extracted, transcribed into cDNA, and then analyzed by quantitative real-time RT-PCR using the ABI PRISM 7300 sequence detection system and software (Applied Biosystems (Qiagen) with QuantiTect SYBR green Kit). Fold induction of CLDN6 expression is shown for cells treated with the reprogramming mixture (black bars) relative to HFF cells from day 1 of treatment (grey bars). CLDN6 mRNA expression was normalized to mRNA expression of the housekeeping gene HPRT 1. OSKMNL-transcription factors OCT4, SOX2, KLF4, cMYC, NANOG and (und) LIN28, EBK-IFN-escape proteins E3, K3 and B18R, miR-mix-miRNA-302 a/B/c/d and 367.

FIG. 2: CLDN6 was expressed on the surface of human iPS cells.

HFF cells were transfected with either no RNA (no RNA control) or a reprogramming mixture (unmodified OSKMNL + EBK + miR-mix) and harvested on days 5(a), 12(B) and 19(C) post-treatment. Cells were stained with 1 μ g/ml of CLDN 6-specific IMAB027-AF647 and SSEA-4-V450 antibodies (2.5 μ l each tested, purchased from BD) for 30 min at 4 ℃ and analyzed for surface expression by flow cytometry. The experiment was repeated twice and representative dot plots are shown. OSKMNL ═ transcription factors OCT4, SOX2, KLF4, cMYC, NANOG and LIN28, EBK ═ IFN-escape proteins E3, K3 and B18R, miR-mix ═ miRNA-302a/B/c/d and 367.

FIG. 3: surface expression of CLDN6 in ovarian cancer cell lines.

To analyze CLDN6 expression, 1E6 cells were stained with 1 μ g/ml of IMAB027-AF647 for 30 minutes at 4 ℃ and surface expression was analyzed by flow cytometry. COV318 cells are shown in (A). The experiment was performed in triplicate and a representative dot plot is shown. In (B), PA-1 cells stably transfected with a control vector (PA-176) or with a vector expressing shRNA against CLDN6 (clones PA-150 and PA-154) are shown. The experiment was performed in triplicate and a representative dot plot was given. shRNA is small hairpin RNA.

FIG. 4: CLDN6 is important for colony formation of ovarian cancer cells.

To analyze clone formation behavior (clonogenic behavior), COV318, PA-150 and PA-154 cells were stained with 1. mu.g/ml of IMAB027-AF647 for 30 minutes at 4 ℃ and then 700 (COV318) or 500 (PA-150/54) CLDN6 positive or CLDN 6-negative cells were sorted into 6-well plates. Cells were allowed to colony for 14 days and then stained with 0.5% crystal violet for 20 minutes. (A) Representative pictures of each cell line are shown. (B) Quantification of colonies was performed by manual counting. The mean and standard deviation of three independent experiments are shown.

FIG. 5: CLDN6 was co-expressed with CSC markers CD24, CD90 and CD44 in ovarian cancer cell line COV 318.

1E6 COV318 cells were stained with antibodies against different surface markers in the FACS group (panel) according to Table 1 for 30 min at 4 ℃ and analyzed for CSC marker expression by flow cytometry. The experiment was performed in triplicate. A representative dot plot of CLDN6 co-localization with different established CSC markers is shown in (a). Percentage of co-localization of CD44, CD24, CD90 and CLDN6 positive cells calculated in (B) using different gating (gate) strategies shown on the x-axis of the graph. The mean and standard deviation of triplicates are shown.

FIG. 6: enrichment of cells expressing CLDN6 resulted in the accumulation of established CSC markers.

COV318 cells were stained with 0.5 μ g/ml IMAB027 and APC conjugated goat anti-human IgG secondary antibody (1: 300) and then CLDN6 positive and CLDN6 negative fractions were separated by FACS sorting. The cells of both fractions were allowed to expand for 10 days. 1E6 cells from each fraction were stained with antibodies against different surface markers in the FACS groups according to Table 1 for 30 min at 4 ℃. The experiment was performed in triplicate. Representative dot plots of the expression levels of different CSC markers in CLDN6 positive and CLDN6 negative fractions and their co-localization with CLDN6 are shown in (a). In (B), the percentage of CSC marker expression levels are shown as a graph, and enrichment factors (fold expression) for the relevant markers CD44, CD90 and CD24 were calculated by comparing the percentage of positive cells in CLDN6 positive and CLDN6 negative fractions.

FIG. 7: the CLDN6 high expressing cell line showed enrichment for CSC markers compared to CLDN6 low expressing cells.

1E6 cells of ovarian cancer cell lines OV90(A) and PA-1(B) or testicular cancer cell lines NEC-8(C) and NEC-14(D) highly expressed by CLDN6 were stained with antibodies against different surface markers in the FACS groups shown in Table 1 for 30 minutes at 4 ℃ and CSC marker expression was analyzed by flow cytometry. The experiment was performed in triplicate and representative dot plots are shown.

FIG. 8: the antitumor effect of IMAB027 in combination with paclitaxel in an early xenograft tumor model.

Subcutaneous human ES-2 xenograft tumors ectopically expressing human CLDN6 were treated by intraperitoneal (i.p.) injection of paclitaxel at 15mg/kg on days 3, 10 and 17 post-transplantation. Antibody maintenance therapy was started on day 4 with 35mg/kg of IMAB027 injected three times a week (alternating intravenous (i.v.)/i.p./i.p.). (A) Mean tumor growth kinetics (± SEM) after treatment with IMAB027 (white squares), paclitaxel (grey circles), a combination of IMAB027 and paclitaxel (black squares), or vehicle control (white circles). Arrows mark the time point at which treatment begins. (B) Survival curves of treated mice. Group size: n is 12.

FIG. 9: antitumor effect of IMAB027 in combination with cisplatin in an advanced xenograft tumor model.

Subcutaneous human NEC14 xenograft tumors were grown to-100 mm before treatment began³The median size of (d). Mice were treated by intraperitoneal injection of 1mg/kg cisplatin daily from day 6 to 10 post-transplantation, and 35mg/kg IMAB027 (to administer IMAB027 three times a week starting on day 6)i.v./i.p./i.p. alternating) as maintenance therapy. (A) Mean tumor growth kinetics (± SEM) after treatment with IMAB027 (closed circles), cisplatin (open squares), a combination of IMAB027 and cisplatin (closed squares), or vehicle control (open circles). Arrows mark the time point at which treatment begins. (B) Size of individual tumors in mice at day 24 post-transplantation (mean ± standard deviation). (C) Survival curves of treated mice. Group size: n is 19. P value: p < 0.05; p is less than 0.01 and P is less than 0.001.

FIG. 10: antitumor effect of IMAB027 in combination with carboplatin in an advanced xenograft tumor model.

As depicted in figure 9, IMAB027 alone or in combination with cytostatic drugs was used to treat advanced human NEC14 xenograft tumors. Mice were treated by intraperitoneal bolus injection (bolus injection) of 30mg/kg of carboplatin (instead of cisplatin) on days 6, 13, and 20. (A) Mean tumor growth kinetics (± SEM) after treatment with IMAB027 (closed circles), carboplatin (open squares), combinations of IMAB027 with carboplatin (closed squares) or vehicle control (open circles). Arrows mark the time point at which treatment begins. (B) Size of individual tumors in mice at day 24 post-transplantation (mean ± standard deviation). (C) Survival curves of treated mice. Group size: n is 19. P value: p < 0.05; p is less than 0.01 and P is less than 0.001.

FIG. 11: CLDN6 is important for the sphere forming behavior of ovarian cancer cells.

To analyze the effect of CLDN6 on spheroid formation, CLDN6 positive and CLDN6 negative COV318 cells were separated by fluorescence activated cell sorting after staining with 0.5 μ g/mi IMAB 027. COV318 cells positive for CLDN6 and negative for CLDN6 were grown in ultra-low attachment plates (ultra low attachment plates) under conditions of sphere formation (serum-free DMEM/F12 medium with 0.4% bovine serum albumin, 20ng/ml basic fibroblast growth factor, 10ng/ml epidermal growth factor and 5 μ g/mi insulin). (A) Representative pictures of first generation spheres of CLDN6 positive (CLDN6+) and CLDN6 negative (CLDN6-) COV318 cells at days 3, 8 and 19 post-sort. (B) Representative pictures of second generation spheres obtained from single cells of CLDN6+ first generation spheres of (a) at day 22 post sorting.

FIG. 12: enrichment of CLDN6 positive cells after treatment with platinum derivatives.

COV318 cells were treated with 500ng/ml cisplatin or 2000ng/ml carboplatin for 4 days. After treatment, cells were allowed to grow for 3 days (white bars) and 6 days (black bars), respectively, in the absence of cell growth inhibitory drugs. Expression of CLDN6 was analyzed by flow cytometry using the CLDN 6-specific antibody IMAB027 and an isotype control antibody. Expression of treated COV318 cells relative to untreated cells is shown. For evaluation, values of isotype controls were subtracted from CLDN6 staining.

FIG. 13: enrichment of CLDN6 positive cells after intraperitoneal transplantation.

COV318 cells were injected intraperitoneally in athymic nude mice. Mice that produced ascites were euthanized and both ascites and solid tumors were collected for further characterization. Isolated cells were analyzed for expression of CLDN6 immediately after preparation and after maintaining them in culture for several passages. (A) Flow cytometry analysis of CLDN6 expression of parental COV318 cells was performed using the CLDN6 specific antibody IMAB027 and isotype control. (B) At different time points after isolation, CLDN6 was expressed on cells from ascites and from solid tumors of the ovary, liver, stomach, pancreas and diaphragm (ascites on days 5 and 35; solid tumors on days 12 and 29). The fluorescence intensity is shown on the X-axis. The number of events displayed on the Y-axis is scaled (scale) to a percentage of the maximum number of events.

FIG. 14: CLDN6 is associated with an ovarian cancer stem cell marker in a primary tumor sample.

mRNA expression levels of CLDN6 and various described ovarian cancer stem cell markers were analyzed by qRT-PCR for 42 ovarian cancer samples using the Fluidigm detection system and software. Spearman correlation analysis was performed to analyze the correlation of CLDN6 with cancer stem cell specific markers. A scatter plot of significant correlation (P value ≦ 0.05) is shown in (A). A summary of all correlations is shown in (B).

FIG. 15: IMAB027 mediated ADCC following treatment with carboplatin and paclitaxel.

The ADCC activity of IMAB027 in combination with chemotherapy was analyzed using COV362(Luc) target cells. Thus, cells were treated with the indicated concentrations of carboplatin, gemcitabine, paclitaxel, doxorubicin, or topotecan for 4 days. After treatment, cells were allowed to grow for 3 days (A-D) and 10 days (E-J), respectively, in the absence of cytostatic drugs. Control cells were not cultured with cytostatic agents. PBMCs from healthy donors were used with IMAB027 (black line) or isotype control antibody (grey line) at-40: 1 ratio of effector (PBMC) to target cells ADCC experiments were performed (A, C, E, G, I). Data points (n ═ 4 replicates) are plotted as mean ± SD. Expression of CLDN6 was analyzed by flow cytometry using IMAB027 (B, D, F, H, J). Black dashed lines demonstrate expression of CLDN6 in untreated cells, and gray solid bars represent expression of CLDN6 after treatment.

FIG. 16: the antitumor effect of IMAB027 in combination with PEB treatment in a very advanced xenograft tumor model.

Subcutaneous human NEC14 xenograft tumors were grown to a very advanced stage in nude mice. Tumor treatment with PEB (cisplatin, etoposide and bleomycin) and IMAB027 began on day 13. Mice receiving the PEB regimen were treated by intraperitoneal injection with 1mg/kg cisplatin and 5mg/kg etoposide on days 13, 14, 15, 16, and 17, and 10mg/kg bleomycin on days 13, 17, and 21. The antibody IMAB027 was administered by i.v./i.p./i.p. alternating injections of 35mg/kg three times a week on days 13 to 101 post-transplantation. The vehicle control group received 0.9% NaCl solution and drug substance buffer instead. Mice were monitored for a total of 220 days. (A) (B) mean tumor growth kinetics (± SEM) of untreated mice and mice treated with IMAB027, PEB or a combination of PEB and IMAB 027. The arrow marks the time point at which treatment begins (Dunn multiple comparison test: Tmax, P < 0.001). (C) Survival curves for untreated mice and mice treated with IMAB027, PEB or a combination of PEB and IMAB027 (Mantel-Cox test: P < 0.05; P < 0.01). Group size: n is 14.

FIG. 17: relative binding affinities and cytotoxicity of IMAB027, IMAB027-DM1 and IMAB 027-vcMAE.

(A) Binding of IMAB027, IMAB027-DM1 and IMAB027-vcMMAE to OV90 cells endogenously expressing CLDN6 was measured by flow cytometry analysis. (B) IMAB027-DM1 and IMAB027-vcMMAE mediated dose-response curves for reduction of OV90 cell viability. Tumor cells were incubated with IMAB027-DM1 or IMAB 027-vcMAE for 72 hours. The decrease in cell viability was measured using XTT-based viability assays. Data points (n ═ 3 replicates) are plotted as mean ± SD. MFI: mean fluorescence intensity.

FIG. 18: antitumor effect of IMAB027-DM1 conjugate on advanced xenograft tumors.

Nude mice bearing established subcutaneous human OV90 xenograft tumors were treated 10 days post-transplantation with a single intravenous dose injection of IMAB027-DM1 or vehicle control at 1.78mg/kg, 5.33mg/kg, or 16 mg/kg. Subcutaneous tumor size was measured twice weekly (mean + SEM). Group size: n is 5, v: p < 0.05, v: p is less than 0.01.

FIG. 19: range of doses of IMAB027-DM1 and IMAB027-vcMMAE conjugate against advanced OV90 xenograft tumors were determined.

Nude mice that had established subcutaneous human OV90 xenograft tumors were treated 10 days post-transplantation with either IMAB027-DM1, IMAB027-vcMMAE, a single dose intravenous injection of vehicle, or a multiple dose injection of IMAB 027. (A) Tumor growth in mice treated with 1.33, 2.67 and 5.33mg/kg of IMAB027-DM1 i.v. (top) or with 4, 8 or 16mg/kg of IMAB027-vcMMAE i.v. (bottom), compared to vehicle control and IMAB027(35mg/kg, i.v./i.p. weekly). Subcutaneous tumor size was measured twice weekly (mean + SEM). (B) Kaplan-Meier survival curves for mice treated with vehicle or IMAB 027-vcMAE at 4, 8, or 16 mg/kg. When the tumor volume reaches 1400mm ³At that time, or if the tumor becomes ulcerative, the mice are sacrificed. Group size: n is 10, v: p < 0.05, v: p < 0.01, Tzhang: p is less than 0.001.

FIG. 20: the range of doses of IMAB027-vcMMAE conjugate against advanced PA-1 xenograft tumors was determined.

15 days after transplantation, a single dose intravenous injection of IMAB027-vcMMAE, vehicle control, or a multiple dose injection of IMAB027Nude mice with established subcutaneous human PA-1 xenograft tumors were treated. (A) Mean tumor growth (± SEM) and (B) Kaplan-Meier survival curves in mice treated with vehicle control, IMAB027(35mg/kg, i.v./i.p. weekly), or IMAB027-vcMMAE at 4, 8, or 16 mg/kg. When the tumor volume reaches 1400mm³At that time, or if the tumor becomes ulcerative, the mice are sacrificed. Group size: n is 8, v: p < 0.05, v: p is less than 0.01. (C) Representative immunohistochemical staining of CLDN6 in PA-1 xenograft tumor sections at different time points post-transplantation.

FIG. 21: antitumor effect of IMAB027-vcMMAE on advanced MKN74 xenograft tumors.

Nude mice with established subcutaneous human MKN74 xenograft tumors were treated 7 days post-transplantation with 16mg/kg of IMAB027-vcMMAE or vehicle control intravenous injection. (A) Mean tumor growth (± SEM) and (B) Kaplan-Meier survival curves for mice treated with vehicle control or IMAB 027-vcMMAE. When the tumor volume reaches 1400mm ³At that time, or if the tumor becomes ulcerative, the mice are sacrificed. Group size: n is 10. (C) Flow cytometry analysis of CLDN6 expression on MKN74 tumor cells prior to transplantation and representative immunohistochemical staining of untreated MKN74 xenograft tumors on day 31 post-transplantation. It is a new method for preparing the compound: p < 0.01, Tzhang: p is less than 0.001.

FIG. 22: antitumor effects of IMAB027-DM1 and IMAB027-vcMMAE on advanced intraperitoneal metastatic human ovarian tumors.

Nude mice were implanted intraperitoneally with the luciferase-ectopically expressing human ovarian cancer cell line PA-1 (Luc). Animals were treated by intraperitoneal injection with 16mg/kg IMAB027-DM1, IMAB027-vcMMAE or vehicle control on day 14 post-transplantation after the formation of intraperitoneal metastatic xenograft tumors. After administration of fluorescein by luminescence activity using the IVIS luminea imaging system, the growth of metastases was determined. (A) Quantification of metastatic load (metastasis load) in mice treated with IMAB027-DM1, IMAB027-vcMMAE or vehicle. (B) In vivo whole body luminescence images of nude mice at day 28 post-transplantation. Group size: n-8 (carrier) or n-9 (IMAB027-DM1, IMAB027-vcMMAE), vijra: p < 0.01, Tzhang: p is less than 0.0001.

FIG. 23: endocytosis of human cancer cells to CLDN 6-bound antibodies.

Endocytosis of CLDN 6-binding IMAB027, chimAB5F2D2, or isotype control antibody was determined using a cytotoxicity-based assay that relies on the co-internalization (co-internalization) of the binding target antibody with saporin (saporin) -conjugated anti-human IgG Fab fragment (FabZap). PA-1, OV90 or NEC14 human cancer cells were incubated with IMAB027, chimAB5F2D2 or an isotype control antibody and anti-human FabZap for 72 hours. (A) IMAB027/FabZap and chimAB5F2D2/FabZap mediated dose-response curves for the reduction of PA-1, OV90 and NEC14 cell viability, respectively. Data points (n ═ 3 replicates) are plotted as mean ± SD. (B) Comparison of IMAB027 normalized EC50 (relative EC50) and maximum (relative maximum) for flow cytobinding and endocytosis.

Detailed Description

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

Hereinafter, elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to form additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the invention to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and/or preferred elements. Moreover, any arrangement or combination of elements described herein is to be considered disclosed in the specification unless the context clearly dictates otherwise.

Preferably, terms used herein such as "A multilingual collaboration of biotechnology terms: (IUPAC Recommendations), "H.G.W.Leuenberger, B.Nagel, and H.K6lbl eds., Helvetica Chimica Acta, CH-4010Basel, Switzerland (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the literature of the art (see, e.g., Molecular Cloning: Arabidopsis Manual, second edition, eds., J.Sambrook et al, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

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

Throughout this specification, several documents are cited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, guidelines, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Claudins (claudins) is a family of proteins that are the most important components of tight junctions, where claudins form a paracellular barrier that controls molecular flow in the intercellular spaces between epithelial cells. Claudins are four-transmembrane proteins, with both the N-and C-termini located in the cytoplasm. The first extracellular loop, designated EC1 or ECL1, consists on average of 53 amino acids, while the second extracellular loop, designated EC2 or ECL2, consists of about 24 amino acids. Cell surface proteins of the claudin family (e.g., CLDN6) are expressed in tumors of various origins and are particularly suitable as target structures in connection with antibody-mediated cancer immunotherapy due to their selective expression (not expressed in normal tissues associated with toxicity) and localization to the plasma membrane.

CLDN6 has been identified to be differentially expressed in tumor tissues, the only normal tissue expressing CLDN6 being the placenta, where small amounts of CLDN6 were detected at the RNA level. CLDN6 has been found to be expressed in, for example, ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, renal cell carcinoma, and bladder cancer.

In various embodiments of the invention, cancer diseases associated with expression of CLDN6 include ovarian cancer (particularly ovarian adenocarcinoma and ovarian teratoma), lung cancer including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC) (particularly squamous cell lung cancer and adenocarcinoma), gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer (particularly basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (particularly malignant polymorphic adenoma), sarcoma (particularly synovial sarcoma and carcinosarcoma), cholangiocarcinoma, bladder cancer (particularly transitional cell carcinoma and papillary carcinoma), kidney cancer (particularly renal cell carcinoma) including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small cell carcinoma including ileocecal carcinoma (particularly small intestine adenocarcinoma and ileal adenocarcinoma), testicular embryonic carcinoma, placental choriocarcinoma, cervical carcinoma, testicular cancer (particularly testicular seminoma, seminoma, Teratoma of the testis and embryonal testicular cancer), uterine cancer, germ cell tumors such as teratoma or embryonal cancer (particularly germ cell tumors of the testis), and metastatic forms thereof. In one embodiment, the cancer disease associated with expression of CLDN6 is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, the ovarian cancer is carcinoma or adenocarcinoma. Preferably, the lung cancer is a carcinoma or adenocarcinoma, and preferably a bronchiolar carcinoma, such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

The term "CLDN" as used herein means claudin and includes CLDN 6. Preferably, the claudin is human claudin.

The term "CLDN 6" preferably refers to human CLDN6, and in particular to a CLDN comprising SEQ ID NO: 1 or SEQ ID NO: 2 or a variant of said amino acid sequence, preferably consisting of SEQ ID NO: 1 or SEQ ID NO: 2 or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably comprises SEQ ID NO: 1 or SEQ ID NO: 2, and more preferably amino acids at positions 28 to 80, and even more preferably amino acids at positions 28 to 76. The second extracellular loop of CLDN6 preferably comprises SEQ ID NO: 1 or SEQ ID NO: 2, preferably from amino acid 141 to 159, more preferably from amino acid 145 to 157. The first and second extracellular loops preferably form extracellular portions of CLDN 6.

According to the invention, the term "variant" particularly refers to mutants, splice variants, conformations, isoforms, allelic variants, species variants (species variants) and species homologues (species homolog), particularly those occurring in nature. Allelic variants refer to changes in the normal sequence of a gene, the significance of which is usually not apparent. For a given gene, whole gene sequencing typically identifies multiple allelic variants. Species homologs are nucleic acid or amino acid sequences of a different species origin than the given nucleic acid or amino acid sequence. The term "variant" shall encompass any post-translationally modified variants and conformational variants.

According to the present invention, the term "claudin-positive cancer" or similar terms means a cancer that involves cancer cells expressing claudin, preferably expressing claudin on the surface of said cancer cells. CLDN6 is expressed on the surface of a cell and is readily bound by CLDN 6-specific antibodies added to the cell if CLDN6 is located on the surface of the cell.

"cell surface" is used according to its usual meaning in the art and thus includes the exterior of a cell which is susceptible to binding by proteins and other molecules. For example, a transmembrane protein having one or more extracellular portions is considered to be expressed on the cell surface.

In the context of the present invention, the term "extracellular portion" refers to a portion of a molecule (e.g. a protein) that faces the extracellular space of a cell, and preferably is accessible to the outside of the cell, e.g. by an antigen binding molecule (e.g. an antibody) located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

The terms "portion" or "segment" are used interchangeably herein to refer to a continuous element. For example, a portion of a structure (e.g., an amino acid sequence or a protein) refers to a contiguous element of the structure. The parts, portions or fragments of a structure preferably comprise one or more of the functional properties of said structure. For example, the portion, portion or fragment of an epitope or peptide is preferably immunologically equivalent to the epitope, peptide from which it is derived. A part or fragment of a protein sequence preferably comprises at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids of said protein sequence.

According to the invention, CLDN6 is not substantially expressed in cells if the expression level is lower compared to the expression in placental cells or placental tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%, or even lower, of the expression in the placental cells or placental tissue. Preferably, CLDN6 is not substantially expressed in a cell if the level of expression is no more than 2-fold, preferably 1.5-fold, of the level of expression in a non-cancerous tissue (other than placenta), and preferably is no more than the level of expression in said non-cancerous tissue. Preferably, CLDN6 is not substantially expressed in a cell if the expression level is below the detection limit and/or if the expression level is too low to be bound by CLDN 6-specific antibodies added to the cell.

According to the present invention, CLDN6 is expressed in cells if the expression level exceeds the expression level in non-cancerous tissues (except placenta) preferably by more than 2-fold, preferably by more than 10-fold, 100-fold, 1000-fold or 10000-fold. Preferably, CLDN6 is expressed in a cell if the expression level is above the detection limit and/or if the expression level is high enough to enable binding by CLDN 6-specific antibodies added to the cell. Preferably, CLDN6 expressed in a cell is expressed or exposed on the surface of the cell.

It has been found that CLDN6 expression is detectable only as mRNA in the placenta, while no protein is detectable at all. Thus, statements made herein regarding expression of CLDN6 in the placenta preferably refer to expression of mRNA.

According to the present invention, the term "disease" refers to any pathological condition, including cancer, in particular those forms of cancer described herein. Any reference herein to cancer or a particular form of cancer also includes cancer metastasis thereof. In a preferred embodiment, the disease to be treated according to the present application relates to cells expressing CLDN6, in particular to cancer stem cells expressing CLDN 6.

According to the invention, "a disease associated with cells expressing CLDN 6" or similar expressions means that CLDN6 is expressed in cells of a diseased tissue or organ. In one embodiment, expression of CLDN6 is increased in cells of a diseased tissue or organ as compared to the state in a healthy tissue or organ. An increase means an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is seen only in diseased tissue, while expression is inhibited in corresponding healthy tissue. According to the present invention, diseases associated with cells expressing CLDN6 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those wherein cancer cells express CLDN 6.

As used herein, "cancer disease" or "cancer" includes diseases having the following characteristics: dysregulated cell growth, proliferation, differentiation, adhesion and/or migration. By "cancer cell" is meant an abnormal cell that grows by rapid, uncontrolled cellular proliferation and continues to grow after the stimulus that initiated the new growth ceases. Preferably, a "cancer disease" is characterized by cells expressing CLDN6, in particular cancer stem cells expressing CLDN 6.

According to the present invention, the term "cancer" includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, otorhinolaryngological (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, and metastases thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or metastases of the above-mentioned cancer types or tumors. According to the present invention, the term cancer also includes cancer metastasis.

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

An "adenocarcinoma" is a cancer derived from glandular tissue. This tissue is also part of a large class of tissue known as epithelial tissue. Epithelial tissues include the skin, glands, and various other tissues lining the body's cavities and organs. Embryologically, the epithelium is derived from ectoderm, endoderm and mesoderm. The cells classified as adenocarcinomas do not necessarily have to be part of the gland, as long as they have secretory properties. This form of cancer may occur in some higher mammals, including humans. Well differentiated adenocarcinomas tend to resemble the glandular tissue from which they are derived, while poorly differentiated adenocarcinomas may not. By staining the cells from the tissue biopsy, the pathologist will determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinoma can arise in many tissues of the body due to the ubiquitous nature of glands in the body. Although each gland may not secrete the same substance, as long as the cell has exocrine function, it can be considered glandular and thus its malignant form is named adenocarcinoma. Given sufficient time, malignant adenocarcinomas invade other tissues and often metastasize. Ovarian adenocarcinoma is the most common type of ovarian cancer. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinomas and endometrioid adenocarcinomas.

By "metastasis" is meant the spread of cancer cells from their original site to another site in the body. The formation of metastases is a very complex process and relies on malignant cells detaching from the primary tumor, invading the extracellular matrix, penetrating the endothelial basement membrane into body cavities and blood vessels, and subsequently infiltrating the target organs after transport through the blood. Finally, the growth of new tumors at the target site is dependent on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components can remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastasis", which refers to metastasis distant from the primary tumor and regional lymph node system. In one embodiment, the term "metastasis" according to the invention refers to lymph node metastasis.

Refractory cancer is a malignant tumor to which a particular treatment is ineffective, either initially unresponsive to the treatment, or becomes unresponsive over time. The terms "refractory", "non-responsive" or "resistant" are used interchangeably herein.

The term "cancer stem cell" as used herein refers to a cell that may be an ancestor of a hyperproliferative cancer cell. Cancer stem cells have the ability to regrow tumors as evidenced by their ability to form tumors in immunodeficient mice. Cancer stem cells are also generally slow growing relative to the tumor mass, i.e., cancer stem cells are generally quiescent. In certain (but not all) embodiments, the cancer stem cells may represent only a portion of the tumor, e.g., about 0.1% to 10%. Cancer stem cells may have one or more of all of the following characteristics or properties: (i) may have the ability to initiate and/or maintain tumor growth, (ii) may generally have fewer mutations than tumor mass (e.g., due to slow growth and thus less DNA replication-related errors, improved DNA repair, and/or epigenetic/non-mutagenic changes that contribute to its malignancy), (iii) may have many of the characteristics of (a) normal stem cells (e.g., characteristics of normal stem cells such as similar cell surface antigens and/or intracellular expression profiles, self-renewal programs, multidrug resistance, immature phenotypes, etc.) and may be derived from (a) normal stem cells; (iv) may be a source of metastases, (v) may be slow growing or quiescent, (vi) may be tumorigenic (e.g., as determined by NOD/SCID implantation experiments), (vii) may be relatively resistant (i.e., chemoresistant) compared to conventional treatments, and (viii) may include subpopulations of tumors (e.g., relative to tumor mass).

By "treating" is meant administering a treatment (e.g., a compound or composition or combination of compounds or compositions) to a subject to prevent or eliminate a disease, including reducing the size or number of tumors in the subject, arresting or slowing the disease in the subject, inhibiting or slowing the development of new disease in the subject, reducing the frequency or severity of symptoms and/or relapses in a subject currently suffering from or previously suffering from the disease, and/or extending, i.e., increasing or extending, the lifespan of the subject. In particular, the term "treatment of a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or inhibiting the progression or worsening, or preventing or delaying the onset of a disease or its symptoms.

In the context of the present invention, terms such as "protection" or "prevention" refer to the prevention or treatment of both the onset and/or spread of a disease in a subject, and in particular to minimizing the chance of causing a disease in a subject or delaying the development of a disease. For example, a subject at risk for cancer would be a candidate for treatment to prevent cancer.

By "at risk" is meant a subject identified as having a higher likelihood of developing a disease (particularly cancer) than normal, as compared to the general population. In addition, subjects who have or are currently suffering from a disease (particularly cancer) have an increased risk of developing the disease because such subjects may continue to develop the disease. Subjects that currently have or have had cancer also have an increased risk of cancer metastasis.

According to the present invention, the term "patient" means a subject, particularly a subject suffering from a disease, including humans, non-human primates or other animals, particularly mammals, e.g., cows, horses, pigs, sheep, goats, dogs, cats or rodents (e.g., mice and rats). In a particularly preferred embodiment, the patient is a human.

In the context of administering a treatment, the term "combination" as used herein refers to the use of more than one treatment or therapeutic agent. The use of the term "combination" does not limit the order of treatment or therapeutic agents administered to a subject. The treatment or therapeutic agent may be administered prior to, concurrently with, or after the administration of the second treatment or therapeutic agent to the patient. Preferably, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or within a time interval such that the therapeutic or therapeutic agents may act together. In a particular embodiment, the therapeutic or therapeutic agents are administered to the subject in a sequence, amount, and/or within a time interval such that they provide an increased benefit over if administered otherwise (particularly independently of each other). Preferably, the added benefit is a synergistic effect.

By "target cell" is meant any unwanted cell, such as a cancer cell, in particular a cancer stem cell. In some preferred embodiments, the target cell expresses CLDN 6.

According to the present invention, the term "chemotherapy" refers to treatment with one or more chemotherapeutic agents or a combination of chemotherapeutic agents (e.g., cytostatic or cytotoxic agents). Chemotherapeutic agents according to the invention include cytostatic compounds and cytotoxic compounds.

According to the present invention, the term "chemotherapeutic agent" includes taxanes (e.g., paclitaxel and docetaxel) and platinum compounds (e.g., cisplatin and carboplatin) and combinations thereof. Preferred combinations, particularly for the treatment of ovarian cancer, may include a combination of a taxane and a platinum compound, for example a combination of paclitaxel and carboplatin. More preferred combinations, particularly for treating ovarian cancer, particularly ovarian germ cell tumors, and/or for treating germ cell tumors, particularly ovarian cancer and testicular germ cell tumors, can include combinations of platinum compounds (e.g., cisplatin) with etoposide and/or bleomycin. According to the present invention, reference to a chemotherapeutic agent includes any prodrug, such as an ester, salt or derivative (e.g. a conjugate of said agent). Examples are conjugates of the agent with a carrier substance, e.g., protein-bound paclitaxel (e.g., albumin-bound paclitaxel). Preferably, the salt of the agent is a pharmaceutically acceptable salt.

Taxanes are a class of diterpene compounds that were first derived from natural sources (e.g., plants of the genus Taxus), but some have been artificially synthesized. The main mechanism of action of taxanes is the disruption of microtubule function, thereby inhibiting the process of cell division. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

According to the present invention, the term "docetaxel" refers to a compound having the following formula:

specifically, the term "docetaxel" refers to the compound 1, 7 β, 10 β -trihydroxy-9-oxo-5 β, 20-epoxyytax-11-ene-2 α, 4, 13 α -triyl-4-ethyl ester-2-benzoate-13- { (2R, 3S) -3- [ (tert-butoxycarbonyl) -amino ] -2-hydroxy-3-phenylpropyl ester }.

According to the present invention, the term "paclitaxel" refers to a compound having the formula:

specifically, the term "paclitaxel" refers to the compound (2 α, 4 α, 5 β, 7 β, 10 β, 13 α) -4, 10-bis- (acetoxy) -13- { [ (2R, 3S) -3- (benzoylamino) -2-hydroxy-3-phenylpropionyl ] oxy } -1, 7-dihydroxy-9-oxo-5, 20-epoxyytax-11-ene-2-benzoic acid ester.

According to the present invention, the term "platinum compound" refers to a compound having a structure comprising platinum, such as a platinum complex, and includes compounds such as cisplatin, carboplatin, and oxaliplatin.

The term "cisplatin" refers to the compound cis-diamminedichloroplatinum (II) (CDDP) of the formula:

the term "carboplatin" refers to the compound cis-diammine (1, 1-cyclobutanedicarboxylic acid) platinate (II) of the formula:

the term "oxaliplatin" refers to a compound which is a platinum compound complexed with a diaminocyclohexane carrier ligand of the formula:

in particular, the term "oxaliplatin" refers to the compound [ (1R, 2R) -cyclohexane-1, 2-diamine ] (ethanediato) -O, O') platinum (II). Oxaliplatin for injection is also commercially available under the trade name Eloxatine.

Additional chemotherapeutic agents contemplated for use in the present invention (whether used alone or in combination with other chemotherapeutic agents (e.g., taxanes or platinum compounds)) include, but are not limited to: nucleoside analogs, camptothecin analogs, and anthracyclines.

The term "nucleoside analog" refers to a structural analog of a nucleoside, including the classes of both purine analogs and pyrimidine analogs.

The term "gemcitabine" is a compound that is a nucleoside analog of the formula:

in particular, the term refers to the compounds 4-amino-1- (2-deoxy-2, 2-difluoro-. beta. -D-erythro-pentofuranosyl) pyrimidin-2 (1H) -one or 4-amino-1- [ (2R, 4R, 5R) -3, 3-difluoro-4-hydroxy-5- (hydroxymethyl) oxolan (oxolan) -2-yl ] -1, 2-dihydropyrimidin-2-one.

The term "nucleoside analog" includes fluoropyrimidine derivatives such as fluorouracil and prodrugs thereof. The term "fluorouracil" or "5-fluorouracil" (5-FU or f5U) (sold under the tradenames Adrucil, Carac, Efudix, Efludex and Fluoroplex) are compounds that are pyrimidine analogs of the formula:

in particular, the term refers to the compound 5-fluoro-1H-pyrimidine-2, 4-dione.

The term "capecitabine" (Xeloda, Roche) refers to a chemotherapeutic agent, which is a prodrug that is converted to 5-FU in tissue. Orally administrable capecitabine has the formula:

in particular, the term refers to the compound [1- (3, 4-dihydroxy-5-methyltetrahydrofuran-2-yl) -5-fluoro-2-oxo-1H-pyrimidin-4-yl ] carbamic acid pentyl ester.

The term "folinic acid" or "leucovorin" refers to a compound used in synergistic combination with the chemotherapeutic agent 5-fluorouracil. Thus, if reference is made herein to administration of 5-fluorouracil or a prodrug thereof, such administration in one embodiment may include administration with folinic acid. Folinic acid has the following formula:

in particular, the term refers to the compound (2S) -2- { [4- [ (2-amino-5-formyl-4-oxo-5, 6, 7, 8-tetrahydro-1H-pteridin-6-yl) methylamino ] benzoyl ] amino } glutaric acid.

According to the present invention, the term "camptothecin analogue" refers to a derivative of the compound camptothecin (CPT; (S) -4-ethyl-4-hydroxy-1H-pyrano [3 ', 4': 6, 7] indolino [1, 2-b ] quinoline-3, 14- (4H, 12H) -dione). Preferably, the term "camptothecin analog" refers to a compound comprising the structure:

preferred camptothecin analogues according to the present invention are inhibitors of the dnase topoisomerase i (topo i). Preferred camptothecin analogs according to the present invention are irinotecan and topotecan.

Irinotecan is a drug that prevents DNA unwinding (unwinding) by inhibiting topoisomerase I. From a chemical point of view, it is a semi-synthetic analogue of the natural alkaloid camptothecin having the following formula:

specifically, the term "irinotecan" refers to the compound (S) -4, 11-diethyl-3, 4, 12, 14-tetrahydro-4-hydroxy-3, 14-dioxo-1H-pyrano [3 ', 4': 6, 7] -indolizino [1, 2-b ] quinolin-9-yl- [1, 4 '-dipiperidine ] -1' -carboxylic acid ester.

Topotecan is a topoisomerase inhibitor of the formula:

specifically, the term "topotecan" refers to the compound (S) -10- [ (dimethylamino) methyl ] -4-ethyl-4, 9-dihydroxy-1H-pyrano [3 ', 4': 6, 7 indolizino [1, 2-b ] quinoline-3, 14(4H, 12H) -dione monohydrochloride.

Anthracyclines are a class of drugs commonly used in cancer chemotherapy, which are also antibiotics. Structurally, all anthracyclines share a common tetracyclic 7, 8, 9, 10-tetrahydrotetracene-5, 12-quinone (four-ring 7, 8, 9, 10-tetrahydrotetracene-5, 12-quinone) structure and generally require glycosylation at specific sites.

Anthracyclines preferably perform one or more of the following mechanisms of action: 1. inhibition of DNA and RNA synthesis by insertion between base pairs of the DNA/RNA strand, thereby preventing replication of rapidly growing cancer cells; 2. inhibition of topoisomerase II, preventing supercoiled DNA relaxation and thereby blocking DNA transcription and replication; 3. free oxygen radicals are generated which damage DNA and cell membranes mediated by iron.

According to the present invention, the term "anthracyclines" preferably refers to agents which preferably induce apoptosis by inhibiting the recombination of DNA with topoisomerase II, preferably anticancer agents.

Examples of anthracyclines and anthracycline analogs include, but are not limited to, daunorubicin (daunorubicin), doxorubicin (adriamycin), epirubicin, idarubicin, daunorubicin, pyrarubicin (pyrarubicin), valrubicin, N-trifluoro-acetyldoxorubicin-14-valerate, aclacinomycin, morpholinodoxorubicin (morpholino-DOX), cyanomorpholino-doxorubicin (cyano-morpholino-DOX), 2-pyrrolo-doxorubicin (2-PDOX), 5-iminodaunorubicin, mitoxantrone, and aclacinomycin A (aclacinomycin). Mitoxantrone is a member of the anthracendione class of compounds, an anthracycline analog that lacks the sugar moiety of the anthracycline but retains a planar polycyclic aromatic ring structure that allows insertion into DNA.

In the context of the present invention, a particularly contemplated anthracycline is epirubicin. Epirubicin is an anthracycline drug having the formula:

and are commercially available under the trade names elence (usa), Pharmorubicin or Epirubicin Ebewe (elsewhere). Specifically, the term "epirubicin" refers to the compound (8R, 10S) -10- [ (2S, 4S, 5R, 6S) -4-amino-5-hydroxy-6-methyl-Alk-2-yl]Oxy-6, 11-dihydroxy-8- (2-hydroxyacetyl) -1-methoxy-8-methyl-9, 10-dihydro-7H-tetracene-5, 12-dione. Epirubicin is favored over the most prevalent anthracycline doxorubicin in some chemotherapeutic regimens because epirubicin appears to cause fewer side effects.

The term "etoposide" refers to a semisynthetic derivative of podophyllotoxin which exhibits anti-tumor activity. Etoposide inhibits DNA synthesis by forming a complex with topoisomerase II and DNA. This complex induces double-stranded DNA breaks and prevents repair by topoisomerase II binding. Cumulative breaks in DNA prevent entry into the mitotic phase of cell division and lead to cell death. Etoposide has the formula:

in particular, the term refers to the compound 4 '-demethyl-epipodophyllotoxin 9- [4, 6-O- (R) -ethylidene- β -D-glucopyranoside ], 4' - (dihydrogenphosphate).

The term "bleomycin" refers to a glycopeptide antibiotic produced by Streptomyces verticillata (Streptomyces verticillatus) bacteria. When used as an anticancer agent, it acts on the principle of generating a break in DNA. Bleomycin preferably includes compounds having the formula:

if the chemotherapy according to the invention is administered in combination with an antibody having the ability of binding to CLDN6, which may be present in the conjugate with at least one toxin drug moiety, i.e. as an antibody drug conjugate, it is preferred to administer the chemotherapy (as a mixture or as a separate component) prior to and/or simultaneously with the administration of the antibody. Preferably, administration of the chemotherapy is initiated prior to administration of the antibody. Preferably, the chemotherapy increases expression of CLDN6 in cancer cells (e.g., cancer stem cells) and is initiated or administered prior to administration of the antibody such that the anti-tumor activity of the antibody is enhanced. Preferably, chemotherapy is administered beginning at least 2 days, at least 4 days, at least 6 days, at least 8 days, at least 10 days, at least 12 days, or at least 14 days prior to the first administration of the antibody. Administration of the chemotherapy may continue during administration of the antibody or stop before or during administration of the antibody, e.g., 1 to 3 days, 1 to 7 days, 1 to 10 days, or 1 to 14 days before antibody administration. Preferably, the chemotherapeutic agent comprises a taxane (e.g., paclitaxel or docetaxel) and/or a platinum compound (e.g., cisplatin or carboplatin).

The term "antigen" refers to a substance, e.g., a protein or peptide, that comprises an epitope against which an immune response is and/or will be elicited. In a preferred embodiment, the antigen is a tumor-associated antigen (e.g., CLDN6), i.e., a cancer cell component that can be derived from the cytoplasm, cell surface and nucleus, particularly those antigens that are preferably produced in large amounts within the cell or as surface antigens on cancer cells.

In the context of the present invention, the term "tumor-associated antigen" or "tumor antigen" preferably refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and/or organs or in a specific developmental stage, as well as to a protein that is expressed or aberrantly expressed in one or more tumor or cancer tissues. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of cancer cells and is preferably not or only rarely expressed in normal tissues.

The term "epitope" refers to an antigenic determinant in a molecule, i.e., refers to the portion of a molecule that is recognized by the immune system (e.g., by an antibody). For example, an epitope is a discrete three-dimensional site on an antigen that is recognized by the immune system. Epitopes are usually composed of chemically active surface groups of molecules (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not lost. Epitopes of a protein (e.g., CLDN6) preferably comprise a continuous or discontinuous portion of the protein and are preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, e.g., epitopes may preferably be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

The term "antibody" includes glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as any molecule comprising an antigen-binding portion of the glycoprotein. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, fragments or derivatives of antibodies, including but not limited to single chain antibodies (e.g., scFv's) and antigen binding antibody fragments (e.g., Fab and Fab' fragments), and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen binding antibody fragments and derivatives as described herein. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "monoclonal antibody" as used herein refers to a preparation (preparation) of antibody molecules of a single molecular component. Monoclonal antibodies exhibit a single binding specificity and affinity. In one embodiment, the monoclonal antibody is produced by a hybridoma, which includes a B cell obtained from a non-human animal (e.g., a mouse) fused to an immortalized cell.

The term "recombinant antibody" as used herein includes all antibodies produced, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) or hybridomas produced therefrom in which the immunoglobulin genes are transgenic or transchromosomes, (b) antibodies isolated from host cells transformed to express the antibodies (e.g., from transfectomas), (c) antibodies isolated from recombinant, combinatorial antibody libraries, and (d) antibodies produced, expressed, produced or isolated by any other means involving splicing of immunoglobulin gene sequences with other DNA sequences.

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

The term "humanized antibody" refers to a molecule having an antigen binding site substantially from an immunoglobulin of a non-human species, wherein the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only Complementarity Determining Regions (CDRs) grafted (graft) onto the appropriate framework regions in the variable domain. The antigen binding site may be wild-type or modified by one or more amino acid substitutions, for example to make it more similar to a human immunoglobulin. Some forms of humanized antibodies retain all of the CDR sequences (e.g., humanized mouse antibodies that contain all six CDRs from the mouse antibody). Other forms have one or more CDRs that have been altered relative to the original antibody.

The term "chimeric antibody" refers to an antibody in which a portion of each of the heavy and light chain amino acid sequences is homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular class, while the remaining segments of the chain are homologous to the corresponding sequences in another species or belonging to another class. Typically, the variable regions of both the light and heavy chains mimic the variable regions of an antibody from one mammalian species, while the constant portions are homologous to antibody sequences from another species. One clear advantage of such chimeric forms is that the variable regions can be conveniently generated from currently known sources using readily available B cells or hybridomas from non-human host organisms, in combination with which the constant regions are derived from, for example, human cell preparations. The variable region has the advantage of being easy to prepare and the specificity is not affected by the source, whereas since the constant region is human, the antibody will have a lower probability of eliciting an immune response in a human subject upon injection than if the constant region is from a non-human source. However, the definition is not limited to this specific example.

Antibodies can be from different species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

Antibodies described herein include IgA, e.g., IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, kappa, or IgG1, lambda isotype (i.e., IgG1, kappa, lambda), IgG2a antibody (e.g., IgG2a, kappa, lambda), IgG2b antibody (e.g., IgG2b, kappa, lambda), IgG3 antibody (e.g., IgG3, kappa, lambda), or IgG4 antibody (e.g., IgG4, kappa, lambda).

As used herein, "heterologous antibody" is defined as a transgenic organism that produces such an antibody. The term refers to antibodies having an amino acid sequence or coding nucleic acid sequence corresponding to an amino acid sequence or coding nucleic acid sequence found in an organism not consisting of the transgenic organism and typically from a different species than the transgenic organism.

The term "heterologous hybrid antibody" as used herein refers to an antibody having light and heavy chains of different biological origin. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

The antibodies described herein are preferably isolated. As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds CLDN6 is substantially free of antibodies that specifically bind antigens other than CLDN 6). An isolated antibody specifically binds to an epitope, isoform or variant of human CLDN6, however, it may have cross-reactivity to other related antigens, e.g., from other species (e.g., CLDN6 species homologs). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of "isolated" monoclonal antibodies refers to antibodies having different specificities and combined with a well-defined composition or mixture.

The term "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") of an antibody or similar terms refer to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, HL, CL and CH domains; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH domains; (iv) (ii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments consisting of VH domains (Ward et al, (1989) Nature 341: 544-546); (vi) (vii) an isolated Complementarity Determining Region (CDR), and (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by synthetic linkers using recombinant methods into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed by the term antibody " Antigen binding fragments ". Another example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for use in the same manner as intact antibodies.

The term "binding domain" characterizes a structure associated with the present invention (e.g., an antibody) that binds to/interacts with a given target structure/antigen/epitope. Thus, according to the present invention, a binding domain represents an "antigen-interaction-site".

For the purposes of the present invention, the term "antibody" encompasses all antibodies and antibody derivatives (e.g., antibody fragments) as described herein. The term "antibody derivative" refers to any modified form of an antibody (e.g., a conjugate of an antibody with another agent or antibody) or an antibody fragment.

Naturally occurring antibodies are typically monospecific, i.e., they bind to a single antigen. The invention includes antibodies that bind to a target cell (by binding to CLDN6) and to a second entity, such as a cytotoxic cell (e.g., by binding to the CD3 receptor). The antibodies of the invention may be bispecific or multispecific, e.g., trispecific, tetraspecific, etc.

The term "bispecific molecule" is intended to include substances with two different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, and (b) a receptor (e.g., an Fc receptor on the surface of an effector cell). The term "multispecific molecule" is intended to include substances with two or more different binding specificities. For example, the molecule can bind to or interact with (a) a cell surface antigen, (b) a receptor (e.g., an Fc receptor on the surface of an effector cell), and (c) at least one other component. Thus, the term "antibody capable of binding to CLDN 6" includes, but is not limited to, bispecific, trispecific, tetraspecific and other multispecific molecules directed to CLDN6 and other targets (e.g., Fc receptors on effector cells). The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to pair the two domains on the same chain, thereby facilitating the pairing of the domains with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger, P., et al (1993) Proc. Natl. Acad. Sci. USA 90: 6444-.

In the context of the present invention, an "antibody having the ability of binding to CLDN 6" is preferably capable of eliciting immune effector functions as described herein. Preferably, the immune effector function is directed against a cell (e.g. a cancer stem cell) bearing on its surface the tumor associated antigen CLDN 6.

In the context of the present invention, the term "immune effector function" includes any function mediated by a component of the immune system which results, for example, in inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably, the immune effector function results in killing of cancer cells, in particular cancer stem cells. Such functions include Complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), antibody dependent cell mediated phagocytosis (ADCP), induction of apoptosis of cells bearing the tumor-associated antigen, cytolysis of cells bearing the tumor-associated antigen, and/or inhibition of proliferation of cells bearing the tumor-associated antigen. The bound substance also acts only by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies block the function of tumor-associated antigens or induce apoptosis only by binding to tumor-associated antigens on the surface of cancer cells.

According to the invention, the antibody may be conjugated to a therapeutic moiety or agent, such as a toxin drug moiety, particularly a cytotoxin, a drug (e.g. an immunosuppressant) or a radioisotope. Cytotoxins or cytotoxic agents include any agent that is harmful to and specifically kills cells. Examples include: paclitaxel (taxol), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (tenoposide), vincristine, vinblastine, colchicin (colchicin), doxorubicin, daunorubicin, dihydroxy anthrax (anthracacin) dione, mitoxantrone, mithramycin, actinomycin D, amanitin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents suitable for forming antibody conjugates include, but are not limited to: antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil dacarbazine (decarbazine)), alkylating agents (e.g., mechlorethamine (mechlororethamine), thiopentalbutyric chlorambucil (thioepa chlorembucil), melphalan (melphalan), carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and Anthranomycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic or radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressive agent. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide, or ricin a. Particularly preferred toxin drug moieties according to the invention are compounds which inhibit microtubule assembly and have an antiproliferative and/or cytotoxic effect.

According to the present invention, antibodies conjugated to therapeutic moieties or agents (e.g., cytotoxins) and acting on slow growing or quiescent cells (e.g., cancer stem cells) are particularly preferred. Such therapeutic moieties include those that act on mRNA and/or protein synthesis. Several inhibitors of transcription are known. For example, actinomycin D, which is both a transcription inhibitor and a DNA damaging agent, is inserted into DNA, thereby inhibiting the initiation of transcription. Flavopiridol targets the elongation phase of transcription. Alpha amanitin binds directly to RNA polymerase II, resulting in inhibition of both the initiation and extension stages.

The antibodies may also be conjugated with a radioisotope (e.g., iodine-131, yttrium-90, or indium-111) to produce cytotoxic radiopharmaceuticals.

The antibody conjugates of the invention are useful for modifying a given biological response, and the drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety may be a peptide, protein or polypeptide having a desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, e.g., abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; proteins, for example, tumor necrosis factor or interferon-gamma; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. Further preferred drug moieties according to the invention are curcumin, salinomycin and sulforaphane.

Techniques For conjugating these therapeutic moieties to Antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (ed.), pp 243-56 (Alan R.Liss, Inc.1985); hellstrom et al, "Antibodies For Drug Delivery," Controlled Drug Delivery (2 nd edition), Robinson et al (ed.), pages 623-53 (Marcel Dekker, Inc. 1987); thorpe, "Antibody Carriers Of Cytotoxin Agents In Cancer Therapy: a Review ", Monoclonal Antibodies' 84: biological And Clinical Applications, Pinchereit et al, pp.475-506 (1985); "Analysis, Results, And d Future Therapeutic Of The Therapeutic Use Of radioactive Antibody In Cancer Therapy", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al (eds.), pages 303-16 (Academic Press 1985), And Thurpe et al, "The prediction And Cytoxic Properties Of Antibody-Toxin Conjugates", immunological Rev., 62: 119-58(1982).

In a preferred embodiment, the antibody according to the invention is conjugated to one or more maytansinoid molecules.

Maytansinoids are potent microtubule-targeting compounds that inhibit the proliferation of cells at mitosis. Maytansinoids are derivatives of maytansinoids, which are 19-membered ansa-macrolides (ansa macrolides) linked to a chlorinated benzene ring. Maytansine has the formula:

it has been found that certain microorganisms also produce maytansinoids, such as maytansinol (maytansinol) and C-3 maytansinol esters (U.S. Pat. No.4,151,042). For example, in U.S. Pat. nos. 4,137,230; 4,248,870, respectively; 4,256,746, respectively; 4,260,608, respectively; 4,265,814, respectively; 4,294,757, respectively; 4,307,016, respectively; 4,308,268, respectively; 4,308,269, respectively; 4,309,428, respectively; 4,313,946, respectively; 4,315,929, respectively; 4,317,821, respectively; 4,322,348, respectively; 4,331,598, respectively; 4,361,650, respectively; 4,364,866, respectively; 4,424,219, respectively; 4,362,663, respectively; and 4,371,533 and Kawai et al (1984) chem.pharm.Bull.3441-3451 (which is incorporated herein by reference) have reported synthetic maytansinol and maytansinol analogues.

Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Particularly preferred maytansinoids according to the invention are the thiol-containing derivatives of maytansine, for example DM1 and DM 4. Such thiol-containing derivatives of maytansine include compounds in which the methyl group bound to the carbonyl group is substituted with a free thiol-containing group (e.g., the group-R-SH, where R represents an alkylene or other carbon-containing group atom).

DM1, also known as mertansine, is a maytansinoid of the formula:

in particular, the term "mertansine" or "DM 1" refers to Compound N^2′-deacetyl N^2′- (3-mercapto-1-oxopropyl) -maytansine.

"DM 4" refers to Compound N^2′-deacetyl-N^2′- (4-methyl-4-mercapto-1-oxopentyl) -maytansine.

anti-CLDN 6 antibody-maytansinoid conjugates are prepared by chemically linking an anti-CLDN 6 antibody to a maytansinoid molecule without significantly impairing the biological activity of the antibody or the maytansinoid molecule. An average of 3 to 4 maytansinoid-conjugated molecules per antibody molecule, although it is expected that even one toxin/antibody molecule would enhance cytotoxicity relative to the use of naked antibody.

In this regard, the term "antibody covalently linked to at least one toxin drug moiety" includes situations where one or more molecules of the same drug are covalently linked to an antibody molecule and where different drugs are covalently linked to an antibody molecule. In the latter case, one or more molecules of each different drug may be linked to an antibody molecule or a combination thereof (e.g., one molecule of one drug is linked while several molecules of another drug are linked).

In some embodiments of the invention, antibodies are conjugated to dolastatin (dolastatin) or dolastatin peptide analogs and derivatives, auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588, incorporated herein by reference). Auristatins are synthetic analogs of dolastatin 10, a natural product from the marine mollusk hare (Dolabela auricularia). Like maytansinoids, auristatins are microtubule disruptors. The dolastatin or auristatin drug moiety can be linked to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety.

Exemplary auristatin embodiments include monomethyl auristatin drug moieties, such as MMAE and MMAF, which are preferably N-terminally linked.

MMAE, also known as monomethyl auristatin E, has the following formula:

in particular, the term "MMAE" refers to the compound (S) -N- ((3R, 4S, 5S) -1- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methylamino) butanamide. MMAE is in fact desmethyl auristatin E, i.e. the N-terminal amino group has only one methyl substituent, rather than two substituents as auristatin E itself.

Particularly preferred according to the invention are antibody-vc auristatin conjugates, such as antibody-vcMMAE conjugates. According to the present invention, the term "antibody-vc auristatin" or "vcMMAE" refers to an antibody-drug conjugate (ADC) comprising an auristatin (e.g., MMAE) linked to an antibody by a lysosomally cleavable dipeptide valine-citrulline (vc).

MMAF, also known as monomethylauristatin M, refers to the compound (S) -2- ((2R, 3R) -3- ((S) -1- ((3R, 4S, 5S) -4- ((S) -N, 3-dimethyl-2- ((S) -3-methyl-2- (methylamino) butyrylamino) butyramido) -3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl) -3-methoxy-2-methylpropanamide) -3-phenylpropionic acid.

There are many linking groups known in the art for the preparation of antibody-drug conjugates.

In one embodiment of the invention, the antibody is linked to the drug by a bifunctional crosslinking reagent. As used herein, "bifunctional crosslinking reagent" refers to a reagent having two reactive groups, one of which is capable of reacting with an antibody and the other of which is capable of reacting with a drug to link the antibody to the drug, thereby forming a conjugate. Any suitable bifunctional crosslinking reagent may be used with the present invention, so long as the linking reagent provides targeting properties for maintaining the drug, e.g., cytotoxicity and antibody. Preferably, the linker molecule links the drug and the antibody via a chemical bond such that the drug and the antibody are chemically coupled (e.g., covalently bound) to each other.

In one embodiment, the bifunctional crosslinking reagent comprises a non-cleavable linker. A non-cleavable linker is any chemical moiety capable of linking a drug (e.g., a maytansinoid) to an antibody in a stable, covalent manner. Preferably, the non-cleavable linker is non-cleavable under physiological conditions (in particular within a cell). Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, under which conditions the drug or antibody remains active. Suitable crosslinking reagents that form a non-cleavable linker between the drug and the antibody are well known in the art. In one embodiment, the drug is linked to the antibody via a thioether bond. Examples of non-cleavable linkers include linkers having a maleimide-based or haloacetyl moiety for reaction with a drug (e.g., with a sulfhydryl group of a maytansinoid). Such bifunctional crosslinking agents are well known in the art and include, but are not limited to: n-succinimidyl-4- (maleimidomethyl) cyclohexanecarboxylate (SMCC) and N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxy- (6-amidohexanoate) are "long chain" analogs of SMCC (LC-SMCC). Preferably, the bifunctional crosslinking reagent is SMCC. Using such linkers, a drug (e.g., mertansine) can be attached to an amino group of an antibody (e.g., the free NH2 group of a lysine residue) via 4- (3-mercapto-2, 5-dioxo-1-pyrrolidinylmethyl) -cyclohexanecarboxylic acid. Each antibody drug conjugate molecule may comprise a single antibody molecule bound to several molecules of mertansine.

In a particularly preferred embodiment, the linking reagent is a cleavable linker. Preferably, the cleavable linker is cleavable under physiological conditions (in particular within a cell). Examples of suitable cleavable linkers include disulfide linkers, acid labile linkers, photolabile linkers, peptidase labile linkers, and esterase labile linkers. Disulfide-containing linkers are cleavable linkers produced by disulfide exchange, which can occur under physiological conditions. An acid labile linker is a linker cleavable at acidic pH. For example, certain intracellular compartments (e.g., endosomes and lysosomes) have an acidic pH (pH 4-5) and provide conditions suitable for cleavage of acid-labile linkers. Light labile linkers are useful on body surfaces as well as in many body cavities where light is available. In addition, infrared light can penetrate tissue. Peptidase-labile linkers can be used to cleave certain peptides inside or outside of cells. In one embodiment, the cleavable linker is cleaved under mild conditions, i.e. under intracellular conditions wherein the activity of the cytotoxic agent is not affected.

In a particularly preferred embodiment, the linker is a linker comprising or consisting of the dipeptide valine (Val) -citrulline (Cit) (vc), which is cleaved by cathepsins in tumor cells.

According to the present invention, the term "cancer therapy directed against cancer stem cells" refers to any therapy that can be used to target and preferably kill and/or impair the proliferation or viability of cancer stem cells. Such treatments include: i) antibodies, antibody fragments and proteins (e.g., antibodies or antibody conjugates capable of binding to CLDN6 as described above) targeted to naked or conjugated to a therapeutic moiety targeted at the surface of certain cells of cancer stem cells (e.g., CLDN6) or ii) small molecules that impair proliferation or viability of cancer stem cells. In a specific embodiment, the agent that binds to the antigen is expressed at a higher level on cancer stem cells than on normal stem cells. In a specific embodiment, the agent specifically binds to a cancer stem cell antigen.

According to the present invention, the term "binding" preferably refers to specific binding.

According to the present invention, an antibody is capable of binding to a predetermined target if the antibody has significant affinity for the predetermined target and binds to the predetermined target in a standard assay. "affinity" or "binding affinity" is generally determined by an equilibrium dissociation constant (K)_D) To measure. Preferably, the term "significant affinity" means at 10^-5M or less, 10^-6M or less, 10^-7M or less, 10^-8M or less, 10^-9M or less, 10^-10M or less, 10^-11M or less or 10^-12Dissociation constant (K) of M or less_D) Binding to a predetermined target.

In a standard assay, an antibody is (substantially) unable to bind to a target if it does not have significant affinity for the target and does not bind, in particular does not bind detectably, to the target. Preferably, the antibody is unable to detectably bind to the target if present at a concentration of up to 2 μ g/ml, preferably 10 μ g/ml, more preferably 20 μ g/ml, especially 50 or 100 μ g/ml or more. Preferably, the K if the antibody binds to the target so as to bind to the predetermined target to which the antibody is capable of binding_DCompared with at least 10 times, 100 times and 10 times³10 times of⁴10 times of⁵Multiple or 10 times⁶Multiple K_DBound, the antibody does not have significant affinity for the target. For example, K if an antibody binds to a target to which the antibody is capable of binding_DIs 10^-7M, K to which the antibody binds to a target without significant affinity therefor_DIs at least 10^-6M、10^-5M、10^-4M、10^{- 3}M、10^-2M or 10^-1M。

In a standard assay, an antibody is specific for a predetermined target if it is capable of binding to the target while being incapable of binding to other targets, i.e., has no significant affinity for, nor significantly binds to, the other targets. According to the invention, an antibody has specificity for CLDN6 if it is capable of binding to CLDN6 while being (substantially) incapable of binding to other targets. Preferably, if the affinity and binding of the antibody to such other target does not significantly exceed the affinity or binding of a protein not associated with CLDN6 (e.g., Bovine Serum Albumin (BSA), casein, Human Serum Albumin (HSA), or a non-dense protein transmembrane protein such as an MHC molecule or transferrin receptor or any other designated polypeptide), The antibody is specific for CLDN 6. Preferably, K if the antibody binds to the target so as to bind to a non-specific target_DAt least 10 times, 100 times and 10 times lower than the other³10 times of⁴10 times of⁵Multiple or 10 times⁶Multiple K_DAnd binding, the antibody is specific for the predetermined target. For example, K if an antibody binds to its specific target_DIs 10^-7M, K to which the antibody binds to its unspecific target_DIs at least 10^-6M、10^-5M、10^-4M、10^-3M、10^-2M or 10^-1M。

Binding of the antibody to the target can be determined experimentally using any suitable method; see, e.g., Berzofsky et al, "Antibody-Antibody Interactions" Fundamental Immunology, Paul, W.E., eds., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W.H.Freeman and Company New York, N Y (1992) and the methods described herein. Affinity can be readily determined using conventional techniques, e.g., by equilibrium dialysis; by using the BIAcore 2000 instrument, using the general method described by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by other methods known to the skilled person. For example, the compounds can be prepared by Scatchard et al, Ann n.y.acad.scl, 51: 660(1949) analyzing the affinity data. The affinity of a particular antibody-antigen interaction measured may be different if measured under different conditions (e.g., salt concentration, pH). Thus, affinity and other antigen-binding parameters (e.g., K) _D、IC₅₀) The measurement of (a) is preferably performed with a standardized solution of antibody and antigen and a standardized buffer.

As used herein, "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.

As used herein, "isotype switching" refers to the phenomenon whereby the class or isotype of an antibody changes from one Ig class to another.

The term "naturally occurring" as used herein when applied to an object refers to the fact that the object is visible in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

The term "rearrangement" as used herein refers to a configuration of a heavy or light chain immunoglobulin locus in which the V segments are located immediately adjacent to the D-J or J segments, respectively, in a conformation encoding a substantially intact VH or VL domain. Rearranged immunoglobulin (antibody) loci can be identified by comparison to germline DNA; the rearranged locus will have at least one recombinant heptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to a configuration in which the V segment is not recombined and thus is not immediately adjacent to a D or J segment.

Preferably, binding of an antibody having the ability of binding to CLDN6 to cells expressing CLDN6 induces or mediates killing of cells expressing CLDN 6. Cells expressing CLDN6 are preferably cancer stem cells, and in particular cells of the cancer diseases described herein, such as cancer stem cells of ovarian cancer. Preferably, the antibody induces or mediates cell killing by inducing one or more of Complement Dependent Cytotoxicity (CDC) mediated lysis, Antibody Dependent Cellular Cytotoxicity (ADCC) mediated lysis, apoptosis, and inhibition of proliferation of cells expressing CLDN 6. Preferably, ADCC-mediated cell lysis occurs in the presence of effector cells, which in particular embodiments are selected from the group consisting of monocytes, mononuclear cells, NK cells and PMNs. Inhibition of cell proliferation can be measured in vitro by determining the proliferation of cells in an assay using bromodeoxyuridine (5-bromo-2-deoxyguanosine, BrdU). BrdU is a synthetic nucleoside that is an analog of thymidine and, during DNA replication, can be incorporated into newly synthesized DNA of replicating cells (in the S phase of the cell cycle) to replace thymidine. The incorporated chemical is detected using, for example, an antibody specific for BrdU, indicating that the cell is actively replicating its DNA.

In some preferred embodiments, the antibodies described herein may be characterized by one or more of the following properties:

a) specific for CLDN 6;

b) a binding affinity for CLDN6 of about 100nM or less, preferably, about 5 to 10nM or less, more preferably, about 1 to 10nM or less;

c) the ability to induce or mediate CDC on CLDN6 positive cells;

d) the ability to induce or mediate ADCC on CLDN6 positive cells;

e) the ability to inhibit growth of CLDN6 positive cells;

f) the ability to induce apoptosis of CLDN 6-positive cells.

In one embodiment, the antibody having the ability of binding to CLDN6 has the ability to bind to an epitope present in CLDN6, preferably an epitope located within the extracellular domain of CLDN6, in particular within the first extracellular loop, preferably amino acids 28 to 76 of CLDN6 or the second extracellular loop, preferably amino acids 141 to 159 of CLDN 6. In some specific embodiments, an antibody having the ability of binding to CLDN6 binds to an epitope not present on CLDN9 but present on CLDN 6. Preferably, an antibody having the ability of binding to CLDN6 binds to an epitope not present on CLDN4 and/or CLDN3 but present on CLDN 6. Most preferably, an antibody having the ability of binding to CLDN6 binds to an epitope not present on CLDN proteins other than CLDN6 but present on CLDN 6.

An antibody having the ability of binding to CLDN6 preferably binds to CLDN6 but not to CLDN9 and preferably does not bind to CLDN4 and/or CLDN 3. Preferably, an antibody having the ability of binding to CLDN6 is specific for CLDN 6. Preferably, an antibody having the ability of binding to CLDN6 binds to CLDN6 expressed on the surface of a cell. In some particularly preferred embodiments, an antibody having the ability of binding to CLDN6 binds to a native epitope of CLDN6 present on the surface of a living cell.

In a preferred embodiment, the antibody having the ability of binding to CLDN6 comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 3. 5, 7, 9 and fragments thereof, and a heavy chain variable region (VH).

In a preferred embodiment, the antibody having the ability of binding to CLDN6 comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 4. 6, 8, 10, 11, 12 and fragments thereof, and a light chain variable region (VL).

In certain preferred embodiments, an antibody having the ability of binding to CLDN6 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following possibilities (i) to (vii):

(i) the VH comprises the amino acid sequence represented by SEQ ID NO: 3 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 4 or a fragment thereof,

(ii) the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 6 or a fragment thereof,

(iii) The VH comprises the amino acid sequence represented by SEQ ID NO: 7 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 8 or a fragment thereof, or a pharmaceutically acceptable salt thereof,

(iv) the VH comprises the amino acid sequence represented by SEQ ID NO: 9 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 10 or a fragment thereof, or a pharmaceutically acceptable salt thereof,

(v) the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 4 or a fragment thereof,

(vi) the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 11 or a fragment thereof,

(vii) the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 12 or a fragment thereof.

In a particularly preferred embodiment, the antibody having the ability of binding to CLDN6 comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) of:

the VH comprises the amino acid sequence represented by SEQ ID NO: 5 or a fragment thereof and VL comprises the amino acid sequence set forth by SEQ ID NO: 4 or a fragment thereof.

The term "fragment" particularly refers to one or more Complementarity Determining Regions (CDRs), preferably at least the CDR3 variable region of the heavy chain variable region (VH) and/or the light chain variable region (VL). In one embodiment, the one or more Complementarity Determining Regions (CDRs) are selected from the group of complementarity determining regions CDR1, CDR2, and CDR 3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region (VH) and/or the light chain variable region (VL).

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises the CDRs and intervening framework regions (intervening framework regions) thereof. Preferably, the portion will also comprise at least about 50% of one or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Antibody construction by recombinant DNA techniques can result in the introduction of linker-encoded residues at the N-or C-terminus of the variable region for ease of cloning or other manipulation steps, including the introduction of linkers to link the variable regions of the invention with other protein sequences, including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein tags.

In one embodiment, an antibody comprising one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein comprises the CDRs in a human antibody framework.

Reference herein to an antibody comprising a particular chain or a particular region or sequence in its heavy chain preferably refers to the situation wherein all heavy chains of said antibody comprise said particular chain, region or sequence. This applies correspondingly also to the light chain of the antibody.

It is understood that the antibodies described herein can be delivered to a patient by administering a nucleic acid (e.g., RNA) encoding the antibody and/or by administering a host cell comprising a nucleic acid (e.g., RNA) encoding the antibody. Thus, when administered to a patient, the nucleic acid encoding the antibody may be present in naked form or in a suitable delivery vehicle (e.g., in the form of liposomes or viral particles), or within the host cell. Over a longer period of time, the provided nucleic acids can generate antibodies in a sustained manner to mitigate the instability at least partially observed for therapeutic antibodies. Nucleic acids for delivery to a patient can be produced recombinantly. If a nucleic acid not present in the host cell is administered to a patient, it is preferably taken up by the cells of the patient expressing the antibody encoded by the nucleic acid. If the nucleic acid is present in the host cell, the cell administers the nucleic acid to the patient, preferably by expression from the host cell in the patient to produce an antibody encoded by the nucleic acid.

The term "nucleic acid" as used herein is intended to include DNA and RNA, e.g., genomic DNA, eDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA, (IVT RNA) or synthetic RNA.

The nucleic acid may be comprised in a vector. The term "vector" as used herein includes any vector known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors such as phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as Bacterial Artificial Chromosomes (BACs), Yeast Artificial Chromosomes (YACs) or P1 Artificial Chromosomes (PACs). The vector includes an expression vector and a cloning vector. Expression vectors include plasmids and viral vectors and typically contain the desired coding sequences and appropriate DNA sequences required for expression of the operably linked coding sequences in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in an in vitro expression system. Cloning vectors are generally used to design and amplify a desired DNA fragment and may lack the functional sequences required to express the desired DNA fragment.

In the context of the present invention, the term "RNA" refers to a molecule comprising, preferably consisting entirely or essentially of, ribonucleotide residues. "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations may include, for example, the addition of non-nucleotide material to the end or interior of the RNA (e.g., at one or more nucleotides of the RNA). The nucleotides in the RNA molecule may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

According to the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA" and to "transcripts" which can be produced using DNA as a template and encode peptides or proteins. An mRNA typically comprises a 5 'untranslated region (5' -UTR), a protein or peptide coding region, and a 3 'untranslated region (3' -UTR). mRNA has a limited half-life in cells and in vitro. Preferably, the mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to the skilled worker. For example, there are a number of commercially available in vitro transcription kits.

In one embodiment of the invention, the RNA is a self-replicating RNA, e.g., a single-stranded self-replicating RNA. In one embodiment, the self-replicating RNA is a positive-sense single-stranded RNA. In one embodiment, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, the self-replicating RNA is alphaviral (alphaviral) genomic RNA or RNA from an alphaviral genome. In one embodiment, the self-replicating RNA is a viral gene expression vector. In one embodiment, the virus is a Semliki forest (Semliki forest) virus. In one embodiment, the self-replicating RNA comprises one or more transgenes, at least one of which encodes an antibody described herein. In one embodiment, if the RNA is or is derived from a viral RNA, the transgene may partially or completely replace a viral sequence, such as a viral sequence encoding a structural protein. In one embodiment, the self-replicating RNA is an in vitro transcribed RNA.

The genome of alphavirus is a positive-sense single-stranded RNA (ssRNA (+)) that encodes two Open Reading Frames (ORFs) for a large polyprotein. The ORF at the 5' end of the genome encodes the nonstructural proteins nSP1 to nSP4(nsP1-4), which are translated and processed into RNA-dependent RNA polymerase (replicase); the ORF at the 3' end encodes the structural proteins-capsid and glycoprotein. Both ORFs are separated by a so-called subgenomic promoter (SGP) which controls transcription of the structural ORF. When used as a gene vector, the structural proteins behind SGP are usually replaced by transgenes. To package these vectors into viral particles, the structural proteins are typically expressed in trans from helper constructs. Alphaviruses replicate exclusively at the RNA level in the cytoplasm of infected cells. Following infection, the ssRNA (+) genome serves as mRNA for translation of the nsP1234 polyprotein precursor, which is autoproteolytically processed into fragments nsP123 and nsP4 at an early stage in the virus life cycle. Fragments nsP123 and nsP4 form a negative-strand replicase complex that transcribes negative-strand RNA from the genomic RNA template. At a later stage, the nsP1234 polyprotein is cleaved extensively into single proteins that assemble into the (+) strand replicase complex that synthesizes the new (+) strand genome, as well as subgenomic transcripts that encode structural proteins or transgenes. Subgenomic RNAs as well as new genomic RNAs are capped and polyadenylated and thus recognized as mrnas upon infection of target cells. Only the new genomic RNA contains a packaging signal that ensures that the genomic RNA is specifically packaged into and out of the geminivirus. The attractiveness of alphavirus replicons for vectoriology is based on the positive orientation of the capped and polyadenylated RNA genome. Transferable replicon RNA is readily synthesized in vitro, whereby addition of a cap analog (cap-analog) to an in vitro transcription reaction can effect capping and the poly-a tail can be encoded as a poly-T track (poly-Ttrack) on the plasmid template. In Vitro Transcribed (IVT) replicons are transfected by conventional transfection techniques and even small amounts of the starting IVT RNA are rapidly propagated. Within a few hours after transfer, the transgene located downstream of the SGP is transcribed into about 40.000 to 200.000 copies of very high copy number subgenomic RNA per cell, and thus, it is not surprising that the recombinant protein is strongly expressed. Depending on the particular purpose, the IVT replicon can be transfected directly into the target cell, or packaged into an alphavirus particle with a helper vector that provides the structural gene in trans. Transfer into the skin or muscle results in high and sustained local expression, while strongly inducing humoral and cellular immune responses.

In order to increase the expression and/or stability of the RNA used according to the invention, it may be modified, preferably without altering the sequence of the expressed peptide or protein.

In the case of RNA, the term "modification" as used according to the present invention includes any modification of the RNA that does not occur naturally in said RNA.

In one embodiment of the invention, the RNA used according to the invention does not contain uncapped 5' -triphosphates. Removal of such uncapped 5' triphosphates can be achieved by treating the RNA with a phosphatase.

The RNA according to the invention may have modified naturally occurring or synthetic ribonucleotides to increase its stability and/or reduce cytotoxicity. For example, in one embodiment 5-methylcytidine is partially or fully (preferably fully) substituted with cytidine in the RNA used according to the invention. Alternatively or additionally, in one embodiment, pseudouridine is partially or fully (preferably fully) substituted for uridine in the RNA used according to the invention.

In one embodiment, the term "modifying" refers to providing an RNA having a 5 '-cap or 5' -cap analog. The term "5 '-cap" refers to the cap structure found at the 5' -end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by an unusual 5 '-to 5' -triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. The term "conventional 5 '-cap" refers to the naturally occurring RNA 5' -cap, preferably to the 7-methylguanosine cap (m 7G). In the context of the present invention, the term "5 '-cap" includes 5' -cap analogs that resemble the structure of an RNA cap and are modified to have the ability to stabilize RNA if attached thereto (preferably in vivo and/or in a cell).

RNA having a 5 '-cap or 5' -cap analogue can be provided by in vitro transcription of a DNA template in the presence of the 5 '-cap or 5' -cap analogue, wherein the 5 '-cap is co-transcriptionally incorporated into the resulting RNA strand, or for example, RNA can be produced by in vitro transcription and the 5' -cap post-transcriptionally attached to the RNA using a capping enzyme (e.g., that of vaccinia virus).

The RNA may include further modifications. For example, a further modification of the RNA used in the present invention may be an extension or truncation of the naturally occurring poly (a) tail or a change in the 5 ' -or 3 ' -untranslated region (UTR), e.g. the introduction of a UTR unrelated to the coding region of said RNA, e.g. the insertion of one or more 3 ' -UTRs, preferably two copies from a globin gene (e.g. α 2-globin, α 1-globin, β -globin, preferably β -globin, more preferably human β -globin).

Thus, in order to increase the stability and/or expression of the RNA used according to the invention, it may be modified to occur in association with a polyA sequence, preferably 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, in particular 100 to 150 adenosine residues in length. In a particularly preferred embodiment, the poly-A sequence is about 120 adenosine residues in length. Furthermore, the incorporation of two or more 3 '-untranslated regions (UTRs) into the 3' -untranslated regions of RNA molecules can result in enhanced translation efficiency. In a specific embodiment, the 3' -UTR is from the human β -globin gene.

Preferably, if the RNA is delivered to (i.e. transfected into) a cell, particularly a cell present in vivo, the RNA expresses the protein or peptide it encodes.

The term "transfection" refers to the introduction of nucleic acids (in particular RNA) into cells. For the purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, wherein the cells may be present in a subject (e.g., a patient). Thus, according to the present invention, the cells used to transfect the nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue and/or organism of a patient. According to the invention, transfection may be transient or stable. For some applications of transfection, even only transient expression of the transfected genetic material may be sufficient. Since the nucleic acid introduced during transfection is not normally integrated into the nuclear genome, the foreign nucleic acid will be diluted or degraded by mitosis. Cells that allow for the amplification of nucleic acid episomal (episomal) dramatically reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its progeny, stable transfection must occur. The RNA can be transfected into cells to transiently express the protein it encodes.

The term "stability" of an RNA refers to the "half-life" of the RNA. "half-life" refers to the period of time required to eliminate half the activity, amount, or number of a molecule. In the context of the present invention, the half-life of an RNA is indicative of the stability of said RNA. The half-life of an RNA may affect the "expression duration" of the RNA. It can be expected that RNA with a long half-life will be expressed long-term.

In the context of the present invention, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription", wherein the term "in vitro transcription" refers to RNA (in particular mRNA) which is synthesized in vitro in a cell-free system, preferably using suitable cell extracts. Preferably, the cloning vector is used to produce transcripts. These cloning vectors are generally designated as transcription vectors and are encompassed by the term "vector" according to the invention.

According to the present invention, the term "translation" refers to the process in the ribosomes of a cell by which the strand of messenger RNA directs the assembly of amino acid sequences to form a peptide or protein.

According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and/or peptides or proteins, for example by transcription and/or translation. With respect to RNA, the term "expression" or "translation" refers in particular to the production of peptides or proteins. It also includes partial expression of nucleic acids. Furthermore, expression may be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "abnormal expression".

According to the present invention, "aberrant expression" or "aberrant expression" means that expression is altered, preferably increased, compared to a reference (e.g., a condition in a subject that does not have a disease associated with aberrant or aberrant expression of a certain protein, such as a tumor antigen). An increase in expression means an increase of at least 10%, in particular at least 20%, at least 50% or at least 100%, or more. In one embodiment, expression is seen only in diseased tissue, while expression is inhibited in healthy tissue.

The term "specifically expressed" means that the protein is expressed substantially only in a specific tissue or organ. For example, tumor antigens are specifically expressed in the placenta means that the protein is predominantly expressed in the placenta and is not expressed in other tissues or is not expressed to a significant degree in other tissues or organ types. Thus, proteins that are exclusively expressed in cells of the placenta and to a lesser extent in any other tissue are specifically expressed in cells of the placenta. In some embodiments, the tumor antigen may also be specifically expressed under normal conditions in more than one tissue type or organ (e.g., in 2 or 3 tissue types or organs, but preferably in no more than 3 different tissue or organ types). In this case, the tumor antigen is then specifically expressed in these organs.

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

Some aspects of the invention rely on the adoptive transfer of host cells transfected in vitro with nucleic acids (e.g., antibody-encoding RNA as described herein) and transferred to a recipient (e.g., a patient), preferably after ex vivo expansion from low precursor frequency to clinically relevant cell numbers. According to the invention, the host cells used for the treatment may be autologous (autologous), allogeneic (allogenic) or syngeneic (syngeneic) with the recipient to be treated.

The term "autologous" is used to describe anything from the same subject. For example, "autograft" refers to a graft of tissue or organ from the same subject. Such processes are advantageous because they overcome immunological barriers that would otherwise lead to rejection.

The term "allogeneic" is used to describe anything from different individuals of the same species. When the genes at one or more loci are not identical, two or more individuals are said to be allogeneic with respect to each other.

The term "syngeneic" is used to describe anything from individuals or tissues of the same genotype, i.e., the same inbred strain of the same egg twin or animal, or tissue thereof.

The term "heterologous" is used to describe something that is made up of a number of different elements. As an example, the transfer of bone marrow from one individual to a different individual constitutes a xenograft. A heterologous gene is a gene from a different subject.

According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to polypeptides comprising two or more amino acids, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more and at most preferably 8, 10, 20, 30, 40 or 50, in particular 100, covalently linked by peptide bonds. The term "protein" refers to a large peptide, preferably to a peptide having more than 100 amino acid residues, but in general the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

The teachings herein given with respect to a particular amino acid sequence (e.g., those set forth in the sequence listing) are to be construed as also referring to variants of the particular sequence that produce a sequence functionally equivalent to the particular sequence, e.g., an amino acid sequence that exhibits the same or similar properties as the properties of the particular amino acid sequence. One important property is to retain the binding of the antibody to its target or to maintain the effector functions of the antibody. Preferably, the sequence that is a variant with respect to the specific sequence retains binding of said antibody to CLDN6 when it replaces the specific sequence in the antibody and preferably retains the function of the antibody as described herein, e.g., CDC mediated lysis or ADCC mediated lysis.

For example, the sequences shown in the sequence listing may be modified to remove one or more, preferably all, free cysteine residues, in particular by replacing cysteine residues by amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine or methionine, most preferably alanine or serine.

It will be appreciated by those skilled in the art that modifications may be made to the sequences of the CDRs, hypervariable regions and variable regions, in particular, without losing the ability to bind to CLDN 6. For example, the CDR regions are identical to or highly homologous to regions of the antibodies specified herein. "highly homologous" can be expected in the CDR 1 to 5, preferably 1 to 4, for example, 1 to 3 or 1 or 2 replacement. In addition, the hypervariable and variable regions may be modified such that they exhibit substantial homology with regions of the antibodies specifically disclosed herein.

For the purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. Amino acid deletion variants comprising a deletion at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncated variants.

Amino acid insertion variants include the insertion of a single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site of the amino acid sequence, but random insertions and appropriate screening of the resulting product are also possible.

Amino acid addition variants include amino-terminal and/or carboxy-terminal fusions of one or more amino acids (e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids).

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be in any position of the protein.

Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue at that position. It is preferred to modify the amino acid sequence at positions that are not conserved between homologous proteins or peptides and/or to replace the amino acid with another amino acid having similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the replacement of one of the families of amino acids with which its side chain is related. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

Preferably, the degree of similarity between a given amino acid sequence and a variant amino acid sequence of said given amino acid sequence is at least about 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Preferably, the degree of similarity or identity is given over a region of amino acids that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if a reference amino acid sequence consists of 200 amino acids, it is preferred to give a degree of similarity or identity, preferably consecutive amino acids, to at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids. In some preferred embodiments, the degree of similarity or identity is given over the entire length of the reference amino acid sequence. Alignments for determining sequence similarity (preferably sequence identity) can be performed using tools known in the art, preferably using optimal sequence alignments, e.g., using Align, using standard settings, preferably EMBOSS: : needle, Matrix: blosum62, Gap Open (Gap Open)10.0, Gap extended (Gap extended) 0.5.

"sequence similarity" refers to the percentage of amino acids that are identical or that show conservative amino acid substitutions. "sequence identity" between two amino acid sequences means the percentage of amino acids that are identical between the sequences.

The term "percent identity" is intended to mean the percentage of amino acid residues that are identical between the two sequences to be compared, obtained after optimal alignment, which percentage is purely statistical and the differences between the two sequences are randomly distributed over their entire length. Sequence comparisons between two amino acid sequences are routinely performed by comparing their sequences after optimal alignment, either by segment or by "comparison window" to identify and compare local regions of sequence similarity. In addition to manual generation, optimal alignment of sequences for comparison can be generated by: the local homology algorithm of Smith and Waterman, 1981, Ads app. math.2, 482, the similarity search method of Neddleman and Wunsch, 1970, j.mol.biol.48, 443, Pearson and Lipman, 1988, proc.natl acad.sci.usa 85, 2444, or a Computer program using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575Science Drive, Madison, Wis.).

Percent identity was calculated by the following method: the number of positions that are identical between two sequences being compared is determined, divided by the number of positions being compared, and the result multiplied by 100 to obtain the percent identity between the two sequences.

The term "cell" or "host cell" preferably refers to an intact cell, i.e., a cell that has an intact membrane and has not released its normal intracellular components (e.g., enzymes, organelles, or genetic material). The intact cells are preferably living cells, i.e. living cells capable of performing their normal metabolic function. Preferably, according to the present invention, the term refers to any cell that can be transfected with an exogenous nucleic acid. Preferably, the nucleic acid is expressed in a recipient when the cell is transfected with the exogenous nucleic acid and transferred to the recipient. The term "cell" includes bacterial cells; other useful cells are yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from gram-negative bacterial strains, such as Escherichia coli (Escherichia coli), Proteus (Proteus) and Pseudomonas (Pseudomonas), and gram-positive bacterial strains, such as strains of Bacillus (Bacillus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus), and Lactococcus (Lactococcus). Suitable fungal cells include cells from Trichoderma (Trichoderma), Neurospora (Neurospora) and Aspergillus (Aspergillus) species. Suitable yeast cells include cells from Saccharomyces (Saccharomyces) such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces pombe) such as Schizosaccharomyces pombe, Pichia (Pichia) such as Pichia pastoris and Pichia methanolica (Pichia methanolica) and Hansenula species. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293HEK, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins may also be used. Mammalian cells, such as those from humans, mice, hamsters, pigs, goats, and primates, are particularly preferred for adoptive transfer. Cells can be derived from a wide variety of tissue types and include primary cells and cell lines, such as cells of the immune system, particularly antigen presenting cells (e.g., dendritic cells and T cells), stem cells (e.g., hematopoietic stem cells and mesenchymal stem cells), and other cell types. Antigen presenting cells are cells that display antigen on their surface in the case of the major histocompatibility complex. T cells can recognize this complex using their T Cell Receptor (TCR).

The term "transgenic animal" refers to an animal having a genome comprising one or more transgenes (preferably heavy and/or light chain transgenes) or transchromosomes (integrated or not integrated into the animal's native genomic DNA), and which is preferably capable of expressing the transgene. For example, a transgenic mouse may have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome such that the mouse produces human anti-CLDN 6 antibody when immunized with CLDN6 antigen and/or cells expressing CLDN 6. The human heavy chain transgene may be integrated into the chromosomal DNA of a mouse, as is the case with transgenic mice (e.g., HuMAb mice (e.g., HCo7 or HCol2 mice)), or the human heavy chain transgene may be maintained extrachromosomally, as is the case with transchromosomal (e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal mice can be capable of producing multiple isotypes (e.g., IgG, IgA, and/or IgE) of human monoclonal antibodies directed against multiple isotypes of CLDN6 by undergoing V-D-J recombination and isotype switching.

As used herein, "reduce" or "inhibit" means the overall reduction in a level (e.g., expression level or cell proliferation level) or the ability to cause an overall reduction, preferably 5% or greater, 10% or greater, 20% or greater, more preferably 50% or greater, most preferably 75% or greater.

The term, e.g. "increase", "increase" or "enhancement" preferably means an increase or enhancement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.

Although some considerations regarding the mechanism behind antibody potency are provided below, the present invention should not be considered to be limited in any way.

The antibodies described herein preferably interact with a component of the immune system, preferably via ADCC or CDC. The antibodies described herein can also be used to target a payload (e.g., a radioisotope, drug, or toxin) to kill tumor cells directly or can be used in conjunction with traditional chemotherapeutic agents to attack tumors through complementary mechanisms of action that can include an impaired anti-tumor immune response due to cytotoxic side effects of the chemotherapeutic agent on T lymphocytes. However, the antibodies described herein may also act simply by binding CLDN6 on the cell surface, for example to block cell proliferation.

Antibody-dependent cell-mediated cytotoxicity

ADCC describes the cell killing ability of effector cells (particularly lymphocytes) as described herein, which preferably require target cells labeled with an antibody.

ADCC preferably occurs when the antibody binds to an antigen on tumor cells and the antibody Fc domain engages with Fc receptors (fcrs) on the surface of immune effector cells (engage). Several families of Fc receptors have been identified, and particular cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism that directly induces varying degrees of direct tumor destruction that leads to antigen presentation and induces tumor-directed T cell responses. Preferably, the in vivo induction of ADCC will elicit tumor-directed T cell responses and host-derived antibody responses.

Complement dependent cytotoxicity

CDC is another method of cell killing that can be directed by antibodies. IgM is the most potent isotype used for complement activation. Both IgG1 and IgG3 were also effective in directing CDC through the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex results in the exposure of multiple C1q binding sites immediately adjacent to the CH2 domain involved in an antibody molecule (e.g., an IgG molecule) (C1q is one of the three subcomponents of complement C1). Preferably, the exposed C1q binding sites convert the previous low affinity C1q-IgG interaction into a high affinity interaction, which triggers a cascade of events involving a series of other complement proteins and results in the proteolytic release of effector cell chemotactic/activating agents C3a and C5 a. Preferably, the complement cascade ultimately forms a membrane attack complex that creates pores in the cell membrane that facilitate free passage of water and solutes inside and outside the cell.

The antibodies described herein can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., Kohler and Milstein, Nature 256: 495 (1975). Although in principle, a somatic hybridization protocol is preferred, other techniques can be used to generate monoclonal antibodies, for example, viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries.

A preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. The generation of hybridomas in mice is a well established protocol. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known.

Other preferred animal systems for preparing hybridomas secreting monoclonal antibodies are rat and rabbit systems (e.g., as 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, the human monoclonal antibodies can be produced using transgenic or transchromosomal mice carrying portions of the human immune system rather than the mouse system. These transgenic and transfectant colored mice include mice referred to as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice". The production of human antibodies in such transgenic mice can be performed as described in detail in WO2004035607 for CD 20.

Yet another strategy for generating monoclonal antibodies is to isolate the gene encoding the antibody directly from lymphocytes producing antibodies of defined specificity, see, e.g., Babcock et al, 1996; the inorganic strand for generating monoclonal antibodies from single, isolated rare and growing antibodies of defined specific properties. Details of Recombinant Antibody Engineering can also be found in Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K.C.Lo Antibody Engineering ISBN 1-58829-092-1.

To produce antibodies, mice can be immunized as described using vectors from the antigen sequence (i.e., the sequence to which the antibody is directed) conjugated to the peptide, an enriched preparation of recombinantly expressed antigen or fragment thereof, and/or cells expressing the antigen. Alternatively, mice can be immunized with DNA encoding an antigen or fragment thereof. If immunization with a purified preparation or enriched preparation of an antigen does not produce antibodies, the immune response can also be promoted by immunizing the mouse with cells (e.g., cell lines) that express the antigen.

Immune responses can be monitored throughout the course of the immunization protocol with plasma and serum samples obtained by tail vein or retro-orbital bleeds. Mice with sufficient titers of immunoglobulin can be used for fusion. Mice can be boosted intraperitoneally or intravenously with antigen expressing cells 3 days prior to sacrifice and removal from the spleen to increase the rate of specific antibody secreting hybridomas.

To generate monoclonal antibody-producing hybridomas, spleen and lymph node cells can be isolated from immunized mice and fused with a suitable immortalized cell line (e.g., a mouse myeloma cell line). The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened for antibody secreting hybridomas by ELISA. Antibodies specific for the antigen can be identified by immunofluorescence and FACS analysis using antigen expressing cells. Antibody secreting hybridomas can be replated (replated), screened again, and subcloned by limiting dilution if monoclonal antibodies are still positive. The stable subclones can then be cultured in vitro in tissue culture medium to produce antibodies for characterization.

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

For example, in one embodiment, a gene of interest (e.g., an antibody gene) can be ligated into an expression vector (e.g., a eukaryotic expression plasmid), for example, by using the GS gene expression systems disclosed in WO 87/04462, WO 89/01036, and EP 338841, or other expression systems known in the art. The purified plasmid with the cloned antibody gene can be introduced into a eukaryotic host cell, e.g., a CHO cell, NS/0 cell, HEK293T cell, or HEK293 cell, or other eukaryotic cell (e.g., a plant-derived cell, a fungal, or a yeast cell). The method for introducing these genes may be a method described in the art, for example, electroporation, lipofectine, lipofectamine, or others. After these antibody genes are introduced into the host cells, the cells expressing the antibodies can be identified and selected. These cells represent transfectomas whose expression levels can be expanded and scaled up to produce antibodies thereafter. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Alternatively, the cloned antibody gene may be expressed in other expression systems, including prokaryotic cells, e.g., microorganisms such as e. Furthermore, antibodies can be produced in non-human transgenic animals, e.g., in the milk of sheep and rabbits or in eggs, or in transgenic plants; see, e.g., Verma, r., et al (1998) j.immunol.meth.216: 165-181; pollock, et al (1999) j.immunol.meth.231: 147-; and Fischer, R, et al (1999) biol. chem.380: 825-839.

Fitting together

Murine antibodies are highly immunogenic in humans, resulting in reduced therapeutic efficacy when repeatedly used. The major immunogenicity is mediated by the heavy chain constant region. If individual antibodies are chimeric or humanized, the immunogenicity of murine antibodies in humans can be reduced or avoided altogether. Chimeric antibodies are antibodies in which different portions are from different animal species, for example, those having a variable region from a murine antibody and a human immunoglobulin constant region. The variable regions of the heavy and light chains of murine antibodies are joined to the constant regions of the human heavy and light chains to obtain chimerism of the antibodies (e.g., as described by Kraus et al, in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, the chimeric antibody is produced by linking a human kappa light chain constant region to a murine light chain variable region. In another preferred embodiment, a chimeric antibody can be produced by linking a human λ light chain constant region to a murine light chain variable region. Preferred heavy chain constant regions for the production of chimeric antibodies are IgG1, IgG3 and IgG 4. Other preferred heavy chain constant regions for the production of chimeric antibodies are IgG2, IgA, IgD and IgM.

Humanization

Antibodies interact with a target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequence within a CDR is more diverse than the sequence outside the CDR among antibodies. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that comprise CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al (1998) Nature 332: 323-327; Jones, P. et al (1986) Nature 321: 522-525; and Queen, C. et al (1989) Proc. Natl. Acad. Sci. U.S. A.86: 10029-10033). Such framework sequences are available from public DNA databases that include germline antibody gene sequences. These germline sequences differ from the mature antibody gene sequences in that they do not contain the fully assembled variable genes that are formed by v (d) J junctions during B cell maturation. Germline gene sequences will also differ from the sequences of the high affinity secondary antibody repertoire (secondary recombinant antibody) at individual places evenly across the variable region.

Standard binding assays (e.g., ELISA, Western blot, immunofluorescence, and flow cytometry analysis) can be used to determine the ability of an antibody to bind antigen.

To purify the antibody, the selected hybridomas were cultured in two-liter spinner flasks to purify the monoclonal antibody. Alternatively, antibodies can be produced in a dialysis-based bioreactor. The supernatant may be filtered and concentrated (if necessary) before affinity chromatography on protein G-sepharose or protein a-sepharose. The eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer can be changed to PBS and the concentration can be determined by OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be divided into aliquots and stored at-80 ℃.

Site-directed mutagenesis or multi-site-directed mutagenesis can be used to determine whether a selected monoclonal antibody binds to a unique epitope.

Isotype ELISAs can be performed using various commercially available kits (e.g., Zymed, Roche Diagnostics) to determine the isotype of the antibody. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates were allowed to react with either monoclonal antibody or purified isotype control for two hours at ambient temperature. The wells can then be reacted with mouse IgG1, IgG2a, IgG2b, or IgG3, IgA, or mouse IgM specific peroxidase conjugated probes. After washing, the plates were developed with ABTS substrate (1mg/ml) and analyzed at OD 405 to 650. Alternatively, the isosttip mouse monoclonal antibody isotype kit (Roche, cat. No.1493027) can be used as described by the manufacturer.

Flow cytometry can be used to demonstrate the presence of antibodies in the serum of immunized mice or the presence of monoclonal antibodies binding to live cell expressed antigens. Cell lines expressing antigen either naturally or after transfection and negative controls lacking antigen expression (cultured under standard growth conditions) can be mixed with varying concentrations of monoclonal antibody in hybridoma supernatant or in PBS containing 1% FBS and can be incubated at 4 ℃ for 30 minutes. After washing, the APC-or Alexa 647-labeled anti-IgG antibody can be allowed to bind to the antigen-binding monoclonal antibody under the same conditions as the primary antibody staining. Samples were analyzed by flow cytometry using FACS instruments using light scattering and side scattering properties to gate individual living cells. A co-transfection method may be employed to distinguish antigen-specific monoclonal antibodies from non-specific binders in a single measurement. Cells transiently transfected with plasmids encoding antigen and fluorescent markers can be stained as described above. Transfected cells can be detected in a different fluorescent channel than antibody-stained cells. Since most transfected cells express both transgenes simultaneously, antigen-specific monoclonal antibodies preferentially bind to cells expressing fluorescent markers, while non-specific antibodies bind to untransfected cells in comparable ratios. Alternative assays using fluorescence microscopy may be used in addition to or in place of flow cytometry assays. Cells can be stained exactly as described above and examined by fluorescence microscopy.

Immunofluorescence microscopy analysis can be used to demonstrate the presence of antibodies in the serum of immunized mice or the presence of monoclonal antibodies binding to live cells expressing the antigen. For example, cell lines expressing antigen either spontaneously or after transfection and negative controls lacking antigen expression were cultured under standard growth conditions in DMEM/F12 medium supplemented with 10% Fetal Calf Serum (FCS), 2mM L-glutamine, 100IU/ml penicillin, and 100. mu.g/ml streptomycin in chamber slides (chamber slides). The cells were then fixed with methanol or paraformaldehyde or left untreated. The cells can then be reacted with monoclonal antibodies against the antigen for 30 minutes at 25 ℃. After washing, the cells were reacted with Alexa 555-labeled anti-mouse IgG secondary antibodies (Molecular Probes) under the same conditions. Then, the cells were examined by fluorescence microscopy.

Cell extracts from appropriate negative controls and antigen-expressing cells can be prepared and subjected to Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked, and probed with the monoclonal antibody to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and visualized with ECL substrate.

Antibody reactivity with antigen can also be tested by immunohistochemistry in a manner well known to the skilled artisan, for example, using paraformaldehyde or acetone fixed frozen sections or paraformaldehyde fixed paraffin embedded tissue sections from non-cancerous or cancerous tissue samples obtained from patients during routine surgical procedures or from mice bearing xenograft tumors inoculated with cell lines that spontaneously express antigen or express antigen after transfection. For immunostaining, antibodies reactive with antigen can be incubated, followed by addition of horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the supplier's instructions.

Antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CLDN 6. In vitro testing of monoclonal antibody activity will provide a preliminary screen prior to in vivo model testing.

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells or other effector cells from healthy donors can be purified by Ficoll Hypaque density centrifugation followed by lysis of the contaminating red blood cells. The washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum, or 5% heat-inactivated human serum, and treated with different effector cells: ratio of target cells to ⁵¹Cr-tagged target cells expressing CLDN6 are pooled. Alternatively, the target cells may be labeled with fluorescence enhancing ligand (BATDA). The highly toxic fluorescent chelate formed by the enhancing ligand released from the dead cells and europium can be measured with a fluorometer. Another alternative technique may utilizeTransfection of target cells with luciferase. The added lucifer yellow can then be oxidised by live cells only. Purified anti-CLDN 6 IgG was then added at different concentrations. Irrelevant human IgG can be used as a negative control. Depending on the type of effector cell used, the assay can be carried out for 4 to 20 hours at 37 ℃. Can be measured in culture supernatant⁵¹Release of Cr or presence of EuTDA chelate to determine cell lysis of the sample. Alternatively, the luminescence resulting from the oxidation of lucifer yellow may be used as a measure of viable cells.

anti-CLDN 6 monoclonal antibodies can also be tested in various combinations to determine whether the use of multiple monoclonal antibodies enhances cell lysis.

Complement Dependent Cytotoxicity (CDC)

Monoclonal anti-CLDN 6 antibodies can be tested for their ability to mediate CDC using a variety of known techniques. For example, complement serum can be obtained from blood in a manner known to the skilled artisan. Different methods can be used to determine CDC activity of a mAb. For example, it can be measured ⁵¹Cr release or increased membrane permeability may be evaluated using Propidium Iodide (PI) exclusion assay. Briefly, target cells can be washed and 5X 10⁵The cells were incubated with different concentrations of mAb for 10 to 30 minutes at room temperature or 37 ℃. Then, serum or plasma was added to a final concentration of 20% (v/v), and the cells were incubated at 37 ℃ for 20 to 30 minutes. All cells from each sample can be added to the PI solution in the FACS tube. The mixture can then be immediately analyzed by flow cytometry analysis using FACSArray.

In one alternative assay, induction of CDC may be determined from adherent cells. In one embodiment of the assay, the assay is performed 24 hours prior to the assay, at 3X 10⁴Density of wells cells were seeded in tissue culture flat-bottomed microtiter plates. The following day, growth medium was removed and cells were incubated with antibody in triplicate. Control cells were incubated with growth medium or growth medium containing 0.2% saponin (saponin) to determine background lysis and maximum lysis, respectively. After incubation for 20 min at room temperature, the supernatant was removed and the cells were added with human plasma or serum in 20% (v/v) DMEM (pre-warmed to 37 ℃) and re-incubated at 37 ℃Incubate for 20 min. All cells from each sample were added to a solution of propidium iodide (10 μ g/ml). The supernatant was then replaced with PBS containing 2.5. mu.g/ml ethidium bromide and the fluorescence emission after excitation at 520nm was measured using Tecan Safire at 600 nm. Percent specific lysis was calculated as follows: % specific cleavage-x 100 (sample fluorescence-background fluorescence)/(maximum cleavage fluorescence-background fluorescence).

Induction of apoptosis and inhibition of cell proliferation by monoclonal antibodies

Monoclonal anti-CLDN 6 antibodies can be incubated with, for example, CLDN6 positive tumor cells or CLDN6 transfected tumor cells at 37 ℃ for about 20 hours to test for the ability to trigger apoptosis. Cells can be harvested, washed with annexin-V binding buffer (BD biosciences), and incubated with annexin-V (BD biosciences) conjugated with FITC or APC for 15 minutes in the dark. All cells from each sample can be added to a PI solution (10 μ ug/ml in PBS) in FACS tubes and immediately evaluated by flow cytometry (as above). Alternatively, general inhibition of cell proliferation by monoclonal antibodies can be detected using commercially available kits. DELFIA cell proliferation kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of incorporation of 5-bromo-2-deoxyuridine (BrdU) during DNA synthesis of proliferating cells in a microplate. Europium-labeled monoclonal antibodies were used to detect incorporated BrdU. Cells were fixed using a fixing solution and the DNA was denatured to enable detection of the antibody. Unbound antibodies are washed away and DELFIA inducer is added to dissociate the europium ions from the labeled antibodies into solution, where they form highly fluorescent chelates with the components of the DELFIA inducer. In the assay, the fluorescence measured using time-resolved fluorescence is proportional to the synthesis of DNA in the cells of each well.

Preclinical study

The binding substances described herein can also be detected in an in vivo model (e.g., in immunodeficient mice bearing xenograft tumors seeded with a cell line expressing CLDN 6) to determine their efficacy in controlling the growth of CLDN expressing tumor cells.

In vivo studies using the antibodies described herein can be performed after xenografting CLDN 6-expressing tumor cells into immunocompromised mice or other animals. The antibody can be administered to tumor-free mice, followed by injection of tumor cells to measure the effect of the antibody in preventing the formation of tumors or tumor-related symptoms. The antibodies can be administered to tumor-bearing mice to determine the therapeutic effect of each antibody in reducing tumor growth, metastasis, or tumor-related symptoms. Antibody administration can be combined with administration of other substances (e.g., cytostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors, or other antibodies) to determine the synergistic efficacy and potential toxicity of the combination. Animals may be vaccinated with antibodies or control reagents and thoroughly studied for symptoms that may be associated with CLDN6 antibody treatment to analyze antibody-mediated toxic side effects. Possible side effects of in vivo administration of CLDN6 antibodies include, inter alia, toxicity in tissues expressing CLDN6, including placenta. Antibodies recognizing CLDN6 in humans and other species (e.g., mice) are particularly useful for predicting potential side effects mediated by the administration of monoclonal CLDN6 antibodies in humans.

The method can be performed according to the Protocols of Epitope Mapping of Glenn E.Morris (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, maps the epitope recognized by the antibody.

The compounds and agents described herein may be administered in any suitable pharmaceutical composition.

The pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the antibody described herein and optionally other agents discussed herein to produce a desired response or desired effect.

The pharmaceutical compositions are generally provided in homogeneous dosage forms and may be prepared in a manner known per se. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

The pharmaceutical composition may comprise salts, buffer substances, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the active ingredients of the pharmaceutical composition.

Pharmaceutically acceptable salts are useful in the preparation of pharmaceutically acceptable salts and are also included in the present invention. Such pharmaceutically acceptable salts include, by way of non-limiting example, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, for example, sodium, potassium or calcium salts.

Suitable buffering substances for use in the pharmaceutical composition include acetic acid in salt, citric acid in salt, boric acid in salt and phosphoric acid in salt.

Preservatives suitable for use in the pharmaceutical composition include benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

Injectable formulations may contain pharmaceutically acceptable excipients, for example, Ringer lactate.

The term "carrier" refers to an organic or inorganic component, natural or synthetic in nature, in which the active components are combined to facilitate, enhance or effect the application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a patient.

Carrier materials which can be used for parenteral administration are, for example, sterile water, Ringer lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "excipient" as used herein is intended to mean all substances that may be present in a pharmaceutical composition but are not active ingredients, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents or coloring agents.

The agents and compositions described herein may be administered by any conventional route, for example, by parenteral administration, including by injection or infusion. Administration is preferably parenteral, e.g., intravenous, intraarterial, subcutaneous, intradermal, or intramuscular.

Compositions suitable for parenteral administration generally comprise a sterile aqueous or nonaqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible carriers and solvents are Rjnger's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solution or suspension medium.

The agents and compositions described herein are administered in an effective amount. An "effective amount" refers to an amount that alone or in combination with other dosages achieves a desired response or desired effect. In the case of treatment of a particular disease or a particular condition, the desired response preferably involves inhibiting the progression of the disease. This includes slowing the progression of the disease and, in particular, interrupting or reversing the progression of the disease. The desired response in treating a disease or condition may also be to delay or prevent the onset of the disease or condition. In particular, the term "effective amount" refers to a therapeutic amount sufficient to result in the prevention of the development, recurrence or onset of cancer and one or more symptoms thereof, the reduction of the severity, duration of cancer, the amelioration of one or more symptoms of cancer, the prevention of the development of cancer, the induction of regression of cancer and/or the prevention of metastasis of cancer. In one embodiment of the invention, the amount treated is effective to achieve stabilization, reduction or elimination of the cancer stem cell population and/or eradication, elimination or control of primary cancer, metastatic cancer and/or recurrent cancer.

An effective amount of an agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient (including age, physiological condition, size and weight), the duration of the treatment, the type of concomitant therapy (if any), the particular route of administration, and like factors. Thus, the dosage of an agent described herein administered may depend on a number of such parameters. In cases where the patient does not respond adequately to the initial dose, a higher dose (or an effective higher dose obtained by a different, more localized route of administration) may be used.

The agents and compositions described herein may be administered to a patient to treat or prevent a cancer disease, for example, a cancer disease characterized by the presence of CLDN 6-expressing cancer stem cells as described herein.

The agents and compositions provided herein can be used alone or in combination with conventional treatment regimens, such as surgery, irradiation, chemotherapy, and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

The treatment of cancer represents a field where combination strategies are particularly desirable, since the combined action of two, three, four or even more cancer drugs/treatments often produces a synergistic effect that is significantly more influential than a monotherapy approach. Thus, in another embodiment of the invention, Cancer therapy may be effectively combined with a variety of other drugs, among which are combinations such as with conventional tumor therapy, multi-epitope strategies, additional Immunotherapy, and therapeutic approaches targeting angiogenesis or apoptosis (for review see, e.g., Andersen et al, 2008: the combination of the vaccination with other therapies. Cancer Immunology, 57 (11): 1735) 1743). Sequential administration of different agents may inhibit the growth of cancer cells at different checkpoints (check points), while other agents may, for example, inhibit neovascularization, survival or metastasis of malignant cells, which may potentially convert cancer into a chronic disease. The following list provides non-limiting examples of some anti-cancer drugs and treatments that may be used in combination with the present invention:

1. Chemotherapy

Chemotherapy is a standard of care for many types of cancer. The most common chemotherapeutic agents act by killing rapidly dividing cells, one of the main properties of cancer cells. Thus, the combination with conventional chemotherapeutic agents (e.g., alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antineoplastic agents that affect cell division or DNA synthesis) can significantly improve the therapeutic effect of the present invention by suppressing cells by scavenging, restarting the immune system, by making tumor cells more susceptible to immune-mediated killing, or by additionally activating cells of the immune system. Synergistic anti-cancer effects of chemotherapy and vaccination-based immunotherapeutic drugs have been demonstrated in several studies (see, e.g., Quoix et al 2011: Therapeutic administration with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B tertiary. Lancet Oncol.12: 1125-33; see also Liseth et al 2010: Combination of interactive chemotherapy and anticancer drugs in the laboratory of human therapeutics: the chemotherapeutic experiment. J biomedical technology 2010: 6920979; see also Hirooka et al 2009: A administration of clinical chemotherapy and immunotherapy for biological cancer: 69). There are hundreds of chemotherapeutic drugs available that are basically suitable for combination therapy. Some (non-limiting) examples of chemotherapeutic agents that may be combined with the present invention are: carboplatin (Parapelatin), cisplatin (Platinol, Platinol-AQ), crizotinib (Xalkori), cyclophosphamide (Cytoxan, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), erlotinib (Tarceva), etoposide (VePesid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), liposome-encapsulated Doxorubicin (DOXIL), methotrexate (Folex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), nexanib (Nexavar), sunitinib (Suten), topotecan (Hycamtin), trabectedin (Yondeldeldridin) (Yondelia), vincristine (Oncovin, Vincrasar S) and vinblastine (Velbumin).

2. Surgery

Cancer surgery-the process of removing tumors-remains the basis for cancer treatment. Surgery may be combined with other cancer treatments to remove any remaining tumor cells. Combining surgical procedures with subsequent immunotherapy has proven a promising approach several times.

3. Radiation of radiation

Radiation therapy remains an important component of cancer therapy, and about 50% of all cancer patients receive radiation therapy during the course of their disease. The main goal of radiation therapy is to cause cancer cells to lose their proliferative (cell division) potential. The types of radiation used to treat cancer are photon radiation (X-rays and gamma rays) and particle radiation (electrons, protons, and neutron beams). There are two ways to deliver radiation to the site of cancer. External beam radiation is delivered from outside the body to the tumor site by targeting high energy rays (photon, proton or particle radiation). Internal radiation or brachytherapy is delivered directly into the tumor site from inside the body by radioactive sources sealed in a catheter or seed. Radiation therapy techniques suitable for combination with the present invention are, for example, fractionation (fractionation) (radiation therapy delivered in a fractionated protocol, e.g., fractions of 1.5 to 3Gy administered daily over several weeks), 3D conformal radiation therapy (3 DCRT; delivery of radiation to total tumor volume), intensity modulated radiation therapy (IMRT; intensity modulation of multiple radiation beams controlled by a computer), image guided radiation therapy (IGRT; including techniques that allow for corrected pre-radiotherapy imaging), and stereotactic volume radiation therapy (SRBT, delivering extremely high individual doses of radiation over a few treatment fractions). For a review of radiotherapy, see Baskar et al 2012: cancer and radiation therapy: current advances and future directionalities. int.j Med sci.9 (3): 193-199.

4. Antibodies

Antibodies (preferably monoclonal antibodies) achieve their therapeutic effect on cancer cells through a variety of mechanisms. They may have a direct role in producing apoptosis or programmed cell death. They can block components of signal transduction pathways (e.g., growth factor receptors), effectively preventing proliferation of tumor cells. In cells expressing monoclonal antibodies, they can cause the formation of anti-idiotype antibodies. Indirect effects include the recruitment of cytotoxic cells, such as monocytes and macrophages. This type of antibody-mediated cell killing is referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind complement, resulting in direct cytotoxicity, known as Complement Dependent Cytotoxicity (CDC). As described, for example, in Gadri et al 2009: synthetic effect of dendritic cell vaccination and anti-CD20 anti treatment in the therapy of hormone lymphoma. J Immunother.32 (4): 333-40, combining surgical methods with immunotherapeutic drugs or methods is a successful approach. The following list provides some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be combined with the present invention: abafuzumab (CA-125), abciximab (CD41), adalimumab (EpCAM), aftuzumab (Aftuzumab) (CD20), pertuzumab (Alacizumab pegol) (VEGFR2), pentoxymumab (CEA), Amatuximab (MORAB-009), Maanamumab (TAG-72), aprezumab (HLA-DR), abciximab (CEA), bazedoxifen (phosphatidyl serine), betuzumab (CD22), Belimumab (BAFF), bevacizumab (VEGF-A), Bivatuzumab mertansine (CD44 v6), Blinatumumab (CD19), betuximab (CD30 TNFRSF8), Mokatuzumab (mucin Canada), Rituzumab (MUC1), Pentuzumab (CTX), prostate cancer (IGF), Eptuzumab (EGFR), Eptuzumab-3), Eptuzumab (EGFR-3), and EGFR (EGFR-CTX) receptor, Claudiximab (claudin), Clivatuzumab tetraxetan (MUC1), cetuximab (TRAIL-R2), daclizumab (CD40), Dalotuzumab (insulin-like growth factor I receptor), dinolizumab (RANKL), dimuzumab (B-lymphoma cells), Drozitumab (DR5), exemestane (GD3 ganglioside), edrecolomab (EpCAM), erlotinumab (SLAMF7), edratuzumab (PDL192), Entuximab (NPC-1C), epratuzumab (CD22), rituximab (HER 2/neuu, CD3), Iretuzumab (integrin alpha v beta 3), Farletuzumab (receptor 1), FBTA 24 (CD20), Ficlatuzumab (900105), IGF 1 (IGF 1-beta receptor), TGF-1 (TGF-beta 6851), TGF-glycoprotein alpha v beta-1), Farituzumab (TGF-2), Farituzumab (TGF-beta-6851), Farituzumab (TGF-beta-2), TGF-beta-6851), TGF-beta-1, TGF-beta-5, TGF-beta-5, and pharmaceutically acceptable salts (TGF-beta-D), pharmaceutically acceptable salts, and pharmaceutically acceptable salts thereof, pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts thereof, and pharmaceutically acceptable salts thereof, pharmaceutically, Girentuzimab (carbonic anhydrase 9(CA-IX)), Glembumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), Ibrumumab (VEGFR-1), agovacizumab (CA-125), Intuximab ravtansine (SDC1), Intetumumab (CD51), Onintuzumab (CD22), ipilimumab (CD152), Iratumumab (CD30), Rabeuzumab (CEA), lexamu monoclonal antibody (TRAIL-R7), ribavirin (hepatitis B surface antigen), trastuzumab (CD33), Lorvotuzumab mertansine (CD56), lucakamumab (CD40), luciximab (CD23), Maruzumab (TRAIL-R1), matuzumab (EGFR), Meripuzumab (IL-5), Rituzumab (CD74), Nemaduzumab (CD 8984), Nemaduzumab (CD 3), Nemaduzumab (EGFR (CD 6336), Nemaduzumab (Gx), Nemaduzumab (EGFR-R6342), Nemaduzumab (EGFR), Nemaduzumab (R635), Nemaduzumab (R3), Nemaduzumab (R) (R635), Nemaduzumab (R) or (R) (E (R) (E) (R) (E) (R) (E) (R) (E) (R) (E) (R) (E) (R) (E (R) (E (R) (E (R) (Tab) of Tab (R) (E (R) (Tab) of Tab (G) (Tab (R) (Tab) of Tab (G) of Tab (G (K) of Tab (G (K) of Tab (G (K) of Tab (G (K) of Tab (III (G, Nivolumab (IgG4), ofatumumab (CD20), Olaratumab (PDGF-R α), Onaruzumab (human scatter factor receptor kinase), Oportuzumab monatox (EpCAM), Oagovacizumab (CA-125), Oxelumab (OX-40), panitumumab (EGFR), Patrituzumab (HER3), Pemtumumab (MUC1), pertuzumab (HER2/neu), pertuzumab (adenocarcinoma antigen), Potuzumab (vimentin), Racomotuzumab (N-glycolneuraminic acid), Radreutuzumab (fibronectin extra domain-B), Revimentin (rabies virus glycoprotein), Ramouzumab (VEGFR2), Rilotumumab (Rilotuzumab) (HER2/neu), Rituximab (HGF 20), Rotuzumab (IGF-1 receptor), mAb (CD200), Saxizuzumab (FAP), Ab (FAP 84), Tatuzumab (IL 19), and Tatuzumab (IL 19), Tenatumomab (tenascin C), Teprotumumab (CD221), Cetuzumab (CTLA-4), gazezumab (TRAIL-R2), TNX-650(IL-13), tositumomab (CD20), trastuzumab (HER2/neu), TRBS07(GD2), Teximumab (CTLA-4), Cemouleukin-mab (EpCAM), Ublituximab (MS4A1), Urelumab (4-1BB), Voluximab (integrin alpha 5 beta 1), Votuzumab (tumor antigen CTAA16.88), Zatuzumab (EGFR), and Zatuzumab (CD 4).

5. Cytokines, chemokines, co-stimulatory molecules, fusion proteins

Another embodiment of the invention is the use of a pharmaceutical composition encoded by an antigen of the invention in combination with a cytokine, chemokine, co-stimulatory molecule and/or fusion protein thereof to elicit a beneficial immunomodulatory or tumor-suppressive effect. To increase the infiltration of immune cells into the tumor and to promote movement of antigen presenting cells to the tumor draining lymph nodes, a variety of chemokines having the structure C, CC, CXC and CX3C can be used. Some of the most promising chemokines are, for example, CCR7 and its ligands CCL19 and CCL21, in addition CCL2, CCL3, CCL5 and CCL 16. Other examples are CXCR4, CXCR7, and CXCL 12. In addition, co-stimulatory or regulatory molecules, such as B7 ligands (B7.1 and B7.2), are useful. Other cytokines are also useful, such as interleukins in particular (e.g., IL-1 to IL17), interferons (e.g., IFN α 1 to IFN α 8, IFN α 10, IFN α 13, IFN α 14, IFN α 16, IFN α 17, IFN α 21, IFN β 1, IFNW, IFNE1, and IFNK), hematopoietic factors, TGF (e.g., TGF- α, TGF- β, and other members of the TGF family), and finally, members of the tumor necrosis factor family of receptors and their ligands and other stimulatory molecules, including but not limited to: 4-1BB, 4-1BB-L, CD137, CD137L, CTLA-4GITR, GITRL, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2, p75NGF-R, DR6, IT.. beta. R, RANK, EDAR1, XEDAR, Fn114, Troy/Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT and CD95(Fas/APO-1), glucocorticoid-induced TNFR-related protein, TNF receptor-related apoptosis-mediating protein (TRAMP) and death receptor 6(DR 6). CD40/CD40L and OX40/OX40L are important targets for combination immunotherapy because they act directly on T cell survival and proliferation. For a review see Lechner et al 2011: chemokines, costimulatory molecules and fusion proteins for the immunological therapy of microorganisms 3(11), 1317-1340.

6. Bacterial treatment

Researchers have used anaerobic bacteria, such as Clostridium novyi (Clostridium novyi), to deplete the interior of oxygen-poor tumors. When anaerobic bacteria come into contact with the oxidised side of the tumour, they die, which means that they are harmless to the rest of the body. Another strategy is to use anaerobic bacteria that have been transformed with enzymes that can convert nontoxic prodrugs into toxic drugs. As the bacteria proliferate in the necrotic and hypoxic regions of the tumor, the enzyme is expressed only in the tumor. Therefore, systemically applied prodrugs are only metabolized to toxic drugs in tumors. This has been demonstrated to be effective with the nonpathogenic anaerobic bacterium Clostridium sporogenes (Clostridium sporogenes).

7. Kinase inhibitors

Another large group of potential targets for complementary cancer treatments include kinase inhibitors, since the growth and survival of cancer cells is closely associated with dysregulation of kinase activity. To restore normal kinase activity and thereby reduce tumor growth, a wide range of inhibitors are being used. The set of targeted kinases includes receptor tyrosine kinases such as BCR-ABL, B-Raf, EGFR, HER-2/ErbB2, IGF-IR, PDGFR-alpha, PDGFR-beta, c-Kit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK; cytoplasmic tyrosine kinases such as c-SRC, c-YES, Abl, JAK-2; serine/threonine kinases such as ATM, Aurora a & B, CDK, mTOR, PKCi, PLK, B-Raf, S6K, STK11/LKB1 and lipid kinases such as P13K, SK 1. Small molecule kinase inhibitors such as PHA-739358, nilotinib, dasatinib and PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxanib (semaxinib) (SU5416), vatalanib (PTK787/ZK222584), Sutent (SU11248), sorafenib (BAY 43-9006), and leflunomide (SU 101). For more information see, e.g., Zhang et al 2009: nature Reviews Cancer 9, 28-39.

Toll-like receptors

Members of the Toll-like receptor (TLR) family are important links to innate and adaptive immunity and the action of many adjuvants is dependent on the activation of TLRs. A number of vaccines that have been established against cancer incorporate ligands for TLRs to boost vaccine response. In addition to TLR2, TLR3, TLR4, particularly TLR7 and TLR8, have been examined for use in passive immunotherapy approaches for cancer treatment. Closely related TLRs 7 and 8 contribute to anti-tumor responses by affecting immune cells, tumor cells and tumor microenvironment, and can be activated by nucleoside analog structures. All TLRs have been used as independent immunotherapeutic or cancer vaccine adjuvants and can be synergistically combined with the formulations and methods of the present invention. For more information see van Duin et al 2005: triggering TLR signaling in vaccation. trends in Immunology, 27 (1): 49-55.

9. Angiogenesis inhibitors

In addition to therapies targeting immunomodulatory receptors affected by tumor-mediated escape mechanisms (escape mechanisms) and immunosuppression, there are therapies that target the tumor environment. Angiogenesis inhibitors prevent the widespread growth of blood vessels (angiogenesis) required for tumor survival. For example, angiogenesis promoted to meet the increased nutrient and oxygen requirements of tumor cells can be blocked by targeting different molecules. Non-limiting examples of angiogenesis-mediated molecules or angiogenesis inhibitors that may be combined with the present invention are soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptor VEGFR1, VEGFR2 and co-receptor neuropilin-1 and neuropilin 2)1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin (angiostatin) and related molecules, endostatin, angiostatin, calreticulin, platelet factor 4, TIMP and CDAI, Meth-1 and Meth-2, IFN- α, IFN- β and IFN- γ, CXCL10, IL-4, IL-12 and IL-18, prothrombin (tricyclic domain-2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, mammostatin (statin), a proliferator-related protein, restin (restin bead) and drugs such as bevacizin bead, Itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN- α, platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonist, angiostatic steroid + heparin, cartilage derived angiostatic factor, matrix metalloproteinase inhibitor, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, prolactin α V β 3 inhibitor, linamide, taquinomod (tasquinimod), for reviews see Schoenfeld and Dranoff 2011: hum vaccine, (9): 976-81.

10. Small molecule targeted therapeutic drug

Small molecule targeted therapeutic drugs are often inhibitors of enzymatic domains on mutated, overexpressed, or other key proteins within cancer cells. Prominent and non-limiting examples are the tyrosine kinase inhibitors imatinib (Gleevec/Glivec) and gefitinib (Iressa). The use of small molecules targeting certain kinases (e.g. sunitinib malate and/or sorafenib tosylate) in combination with vaccines for cancer treatment is also described in the previous patent application US 2009004213.

11. Virus-based vaccines

There are many virus-based cancer vaccines available or under development that can be used in combination therapy methods with the formulations of the present invention. One advantage of using such viral vectors is their inherent ability to elicit an immune response, an inflammatory response that occurs as a result of viral infection that generates the danger signals required for immune activation. The ideal viral vector should be safe and should not introduce an anti-vector immune response so as to enhance the anti-tumor specific response. Recombinant viruses such as vaccinia virus, herpes simplex virus, adenovirus, adeno-associated virus, retrovirus, and fowlpox virus have been used in animal tumor models and have begun human clinical trials based on their encouraging results. A particularly important virus-based vaccine is the virus-like particle (VLP), which is a small particle that contains certain proteins from the outer shell of the virus. Virus-like particles do not contain any genetic material from the virus and do not cause infection, but they can be constructed to present tumor antigens on their outer shell. VLPs may be derived from a variety of viruses, such as hepatitis b virus or other virus families, including the Parvoviridae (partoviridae) (e.g., adeno-associated viruses), the Retroviridae (Retroviridae) (e.g., HIV), and the Flaviviridae (e.g., hepatitis c virus). For a general review see Sorensen and Thompsen 2007: virus-based immunology of cancer: what do we knock now and where are we gouging apmis 115 (11): 1177-93; virus-like particles against cancer are described in buonakuro et al 2011: development in virus-like particle-based Vaccines for inducing diseases and cancer. expert Rev Vaccines10 (11): 1569-83; and Guillen et al 2010: virus-like particles as vaccine antigens and adjuvants: application to viral diseases, cancer immunological and infectious disease predictive protocols, procedia in genetics 2(2), 128, 133.

12. Multi-epitope strategy

The use of multiple epitopes shows promising results for vaccination. Rapid sequencing technologies combined with intelligent algorithmic systems enable the use of tumor mutant groups (mutanomes) and may provide multiple epitopes for personalized vaccines that may be combined with the present invention. For more information see 2007: the correlation of statistical color candidates with matched dense cells loaded with multiple major or custom complex class I peptides.J. Immunother 30: 762- > 772; furthermore, Castle et al 2012: (iii) expanding the tissue for tumor vaccation. cancer Res 72 (5): 1081-91.

13. Adoptive T cell transfer

For example, in Rapoport et al 2011: combination immunological use of adaptive T-cell transfer and molecular inhibition of therapy of the basic of hTERT and survivin after ASCT for myeloma. blood 117 (3): 788-97 combinations of tumor antigen vaccination and T cell metastasis are described.

14. Peptide-based targeted therapy

The peptides may bind to cell surface receptors or the extracellular matrix affected around the tumor. Radionuclides attached to these peptides (e.g., RGD) eventually kill cancer cells if the nuclide decays near the cell. Oligomers or polymers of these binding motifs are of particular great interest, as they may lead to enhanced tumor specificity and avidity. For a non-limiting example, see Yamada 2011: peptide-based cancer therapy for pro-state cancer, shadow cancer, and major gliomas. Nihon Rinsho 69 (9): 1657-61.

15. Other treatments

There are many other cancer treatments that can be combined with the present invention to produce a synergistic effect. Non-limiting examples are apoptosis-targeted therapy, hyperthermia (hyperthermia), hormone therapy, telomerase therapy, insulin-enhancing therapy, gene therapy and photodynamic therapy.

Various methods known in the art can be used to detect and/or determine the amount of cells expressing CLDN 6.

For example, immunoassays can be used to detect expression of CLDN6 protein in cells or on the surface of cells. According to the present invention, immunoassays include, but are not limited to: western blot, immunohistochemistry, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, immunofluorescence, protein a immunoassay, flow cytometry or FACS analysis.

In one embodiment, the cells are bound to one or more labeled antibodies capable of binding to CLDN6 prior to detecting and/or determining the amount.

Alternatively, expression of CLDN6 mRNA may be detected or the amount of CLDN6 mRNA may be determined to detect and/or determine the amount of cells expressing CLDN 6.

In certain embodiments of the invention, the sample obtained from the patient for detecting and/or determining the amount of cells expressing CLDN6 is a biological fluid including, but not limited to: blood, bone marrow, serum, urine, or interstitial fluid (interstitial fluid). In other embodiments, the sample from the patient is a tissue sample (e.g., a tissue biopsy from a subject having or suspected of having cancerous tissue). Most preferably, the sample is a tissue biopsy of a tumor.

According to the methods of the invention, the sample may be a biological sample that has been subjected to one or more pretreatment steps prior to detecting and/or determining the amount of cells expressing CLDN 6. In certain embodiments, the biological fluid is pretreated by centrifugation, filtration, precipitation, dialysis, or chromatography, or by a combination of these pretreatment steps. In other embodiments, the tissue sample is pre-treated by freezing, chemical fixation, paraffin embedding, dehydration, permeabilization (permerabilization), or homogenization, followed by centrifugation, filtration, precipitation, dialysis, or chromatography, or by a combination of these pre-treatment steps.

The amount of cancer stem cells in a sample can be expressed, for example, as a percentage or quantification of total cells or total cancer cells in the sample with respect to area (e.g., cells in each field of view), volume (e.g., cells per ml), or weight (e.g., cells per ml).

The amount of cancer stem cells in the test sample can be compared to (a) the amount of cancer stem cells in the reference sample. In one embodiment, the reference sample is a sample obtained from a subject undergoing treatment at an earlier time point (e.g., prior to receiving treatment as a baseline reference sample, or at an earlier time point when receiving treatment). In this embodiment, the desired result of the treatment is a reduction in the amount of cancer stem cells in the test sample as compared to the reference sample. In another embodiment, the reference sample is obtained from a healthy subject who has no detectable cancer, or from a patient who is in remission for the same type of cancer. In this embodiment, the desired result of the treatment is that the test sample has the same amount of cancer stem cells or less amount of cancer stem cells detected in the reference sample. In a particular embodiment, a steady or decreased amount of cancer stem cells relative to the amount of (previously detected) cancer stem cells determined earlier for the subject indicates an improvement in the subject's prognosis or a positive response to treatment, whereas an increased amount of cancer stem cells relative to earlier indicates the same or worse prognosis, and/or failure to respond to treatment.

In some embodiments, a combination of a cell surface marker (e.g., CLDN6) with other markers typical of cancer stem cells is used to determine the amount of cancer stem cells in a sample.

The invention also provides kits comprising one or more containers filled with reagents for detecting, measuring or monitoring cells expressing CLDN 6. In one embodiment, the kit optionally comprises instructions for using the agent (particularly using the agent in the methods of the invention) to determine cancer stem cells or to monitor the efficacy of a cancer treatment by detecting and/or determining the amount of cells expressing CLDN 6. In one embodiment, the kit comprises an agent that specifically binds to CLDN6 protein or CLDN6 mRNA. In some embodiments, the agent is an antibody or antibody fragment. In other embodiments, the agent is a nucleic acid. For nucleic acid detection, the kit typically comprises, but is not limited to, a probe specific for CLDN6 mRNA. For quantitative PCR, the kit typically comprises preselected primers specific for a CLDN6 nucleic acid sequence. The quantitative PCR kit may also contain enzymes suitable for amplifying nucleic acids (e.g., polymerases such as Taq) as well as deoxynucleotides and buffers required for amplification of the reaction mixture. The quantitative PCR kit may further comprise a probe specific for a CLDN6 nucleic acid sequence. In some embodiments, the quantitative PCR kit further comprises components suitable for reverse transcription of RNA, said components comprising an enzyme for reverse transcription reaction (e.g., a reverse transcriptase) and primers, and deoxynucleotides and buffers required for the reverse transcription reaction.

In certain embodiments, the agent is detectably labeled. In addition, the kit may contain instructions for performing the assay and for interpreting and analyzing the data generated by the performance of the assay.

Based on the results obtained (i.e., whether cancer stem cells are present or whether the amount of cancer stem cells is stable or decreased), the medical professional may choose a particular cancer treatment, e.g., one directed to cancer stem cells, or may choose to continue the treatment. Alternatively, based on the results, there are no cancer stem cells or no increase in the amount of cancer stem cells, the medical professional may choose to administer a cancer treatment that does not target cancer stem cells or to continue, alter, or stop the treatment.

In certain embodiments of the invention, if it is determined that the reduction in the cancer stem cell population is insufficient to allow comparison of the cancer stem cell population in a sample obtained from a patient undergoing cancer treatment with a sample previously obtained from the patient, the medical professional has a number of options to adjust the treatment. For example, the medical professional may increase the dosage of the cancer treatment, the frequency of administration, the duration of administration, or any combination thereof. In a particular embodiment, after the determination, the patient may be administered an additional cancer treatment in place of or in combination with the first treatment.

In other embodiments, the medical professional may choose not to adjust the cancer treatment if it is determined that the reduction in the cancer stem cell population can be used to compare the cancer stem cell population in a sample obtained from a patient undergoing the cancer treatment with a sample previously obtained from the patient. For example, the medical practitioner can choose a dose that does not increase the cancer treatment, the frequency of administration, the duration of administration, or any combination thereof. In addition, the medical personnel may choose to add additional treatments or combination treatments.

The invention is further illustrated by the following examples, which are not to be construed as limiting the scope of the invention.

Examples

Example 1: expression of CLDN6 on the surface of induced human pluripotent stem cells

To analyze whether CLDN6 is expressed in human induced pluripotent stem cells (ipscs), human neonatal fibroblast cells (human bioscs 6) were expressed in real-time quantitative RT-PCR) at various time points using ABI pr7300 sequence detection System and software (Applied Biosystems with QuantiTect SYBR Kit (ism qin)) using either a reprogramming mixture (unmodified OSKMNL + EBK + miR-mix; consisting of the transcription factors OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 for In Vitro Transcription (IVT) of OSKMNL, and miRNA mix consisting of the IFN-escape proteins E3, K3 and B18R for EBK, or mock-transfected HFF (RNA-free control) according to the protocol described in PCT/EP 2012/04673. Cells were cultured in Nutristem serum-free medium (Stemgent, Cambridge (MA)) supplemented with 10ng/ml bFGF and 0.5. mu.M Thiazovin. The reprogramming mixture was transfected with Lipofectamine RNAiMAX (Life Technologies) on days 1, 2, 3, 4, 8, 9, 10 and 11 of the experiment. As a control, cells were treated with Lipofectamine RNAiMAX only (no RNA control). On day 19 of treatment, we detected almost 6000-fold significant upregulation of CLDN6 compared to untreated HFF, whereas on day 12 of treatment, we observed approximately 2000-fold upregulation of CLDN6 (fig. 1). Thus, CLDN6 is expressed in human induced pluripotent stem cells (ipscs).

Flow cytometry was used to check whether CLDN6 was also expressed on the surface of ipscs. As iPSCs were grown on HFF feeder cells, we combined staining analysis of SSEA-4, a widely accepted stem cell marker, to ensure that iPSCs could be specifically detected. For this purpose, HFF cells treated with reprogramming mix or mock control (no RNA) were collected on days 5, 12 and 19 of treatment and stained with 1 μ g/ml CLDN 6-specific IMAB027-AF647 and 2 μ l ssea-4 antibody for 30 min at 4 ℃ and analyzed for surface expression by flow cytometry. A Viability Dye (Viabilitydye) 7-AAD was also included in our staining protocol to allow the exclusion of dead cells from our analysis. The experiment was repeated twice and 50.000 events were recorded from each sample using a BD Canto II flow cytometer. The recorded cells were analyzed using FlowJo software and a representative dot plot is shown (figure 2).

No CLDN6 was detected on the surface of HFFs neither treated with nor with the reprogramming mixture on day 5. Unexpectedly, we found that 15% of the HFF expressed SSEA-4, regardless of treatment with the reprogramming mixture. This can be explained by the fact that the HFFs used are neonatal fibroblasts and it is likely that these cells retain some positivity to SSEA-4. On day 12 of treatment, about 63% of the treated HFFs were positive for SSEA-4 and we observed about 15% of CLDN6-SSEA-4 double positive fraction. On day 19 of treatment, 15% of the treated HFFs were positive for CLDN6 and SSEA-4, representing a different subpopulation. It is speculated that the CLDN6-SSEA-4 positive subset only labeled ipscs, whereas the CLDN6-SSEA-4 negative subset was considered HFF feeder cells or not reprogrammed cells, whereas SSEA-4 single positive cells represented cells at the start of reprogramming.

Since we found 15% of HFF cells to be positive for SSEA-4 but not for CLDN6, CLDN6 is presumed to represent a more specific marker for human ipscs than SSEA-4. SSEA-4 is also expressed in nascent HFF, whereas CLDN6 appears to be specifically expressed only in all reprogrammed HFF cells representing iPSC fractions.

Thus, CLDN6 is specifically expressed on the surface of human ipscs.

Example 2: CLDN6 important for colony formation of ovarian cancer cells

An effective assay to analyze CSC-like properties of tumor cells is a colony formation assay. Using this assay, the self-renewal capacity and tumor-forming efficacy of single tumor cells can be easily examined. To analyze whether CLDN6 plays a role in tumor formation, we selected, on the one hand, COV318, an ovarian tumor cell line showing only a subpopulation of CLDN6 positive cells, and on the other hand, PA-1, a uniformly CLDN6 expressing cell line carrying stable lentiviral small hairpin RNAs (shrna) that mediate knockdown of CLDN6 (clone PA-150, PA-154); refer to fig. 3.

Cells were stained with 1 μ g/ml IMAB027-AF647 for CLDN6 at 4 ℃ for 30 minutes before sorting for their expression of CLDN6 by FACS (fluorescence activated cell sorting) using a BD FACSAria cell sorter. 500(PA-150, PA-154) or 700(COV318) cells of a positive or negative subset of CLDN6 were sorted directly into wells of a 6-well plate and grown for up to 14 days until sufficient colonies were formed. Media was changed twice weekly. Colonies were stained with 10% ethanol containing 0.5% crystal violet and fixed for 20 minutes, washed three times with distilled water and allowed to air dry. Photographs were taken and colonies were counted manually. At least 50 cells were considered to be one colony. In fig. 4, representative colony formation assays for COV318 and CLDN 6-knockdown cell lines PA-150 and 54 cells are shown.

Interestingly, CLDN6 negative cells showed significantly lower colony formation in both cell lines compared to CLDN6 positive cells. From these results, we conclude that CLDN6 plays an important role in colony forming ability, an important feature of cancer stem cells.

Example 3: CLDN6 is co-expressed with CSC markers CD24, CD90 and CD44 in ovarian cancer cell lines

The use of specific surface marker expression profiles is a common strategy for the identification and isolation of CSCs from solid tumors and cell lines. Surface markers used in the literature for the isolation of CSCs from ovarian cancer include CD44, CD24, CD90, CD34, CD117, and CD 133. To analyze whether we could identify CSC subpopulations in ovarian cancer cell lines containing small subpopulations of CLDN 6-positive cells, we established FACS panels containing antibodies against these surface markers (table 1). Furthermore, we also contemplate the detection of CLDN6 antibodies in the panel to study the percentage of CLDN6 co-localization with established CSC markers to judge the potential of CLDN6 to act as a CSC marker. For this purpose, 1E6 cells of the cell line COV318 were stained with the indicated amount of antibody (see Table 1) for 30 minutes at 4 ℃ before their expression profile of surface markers was analyzed by flow cytometry. In our country We also contemplate viability dyes in the staining protocolSo that dead cells were excluded from our analysis. The experiment was performed in triplicate and 50.000 events were recorded from each sample using a BD Canto II flow cytometer. The recorded cells were analyzed using FlowJo software.

Table 1: CSC FACS panel. FACS panels for analysis of CSC markers and expression of CLDN6 in ovarian cancer cell lines are shown. The amount of antibody and conjugated fluorochrome used for the corresponding marker are listed.

FACS analysis showed that COV318 cells expressed CSC markers CD44, CD90 and CD24 subsets and that CLDN6 was at least partially co-localized with all three markers (fig. 5A). We then used different gating strategies to calculate the percentage of co-localization of all four markers. First, we calculated the percentage of CD44, CD24, CD90 and CLDN6 positive cells in the whole live cell population. We found that 0.18% of the viable cells were positive for all four markers. We then calculated the percentage of CD44, CD24 and CD90 positive cells in the live cell population, which may represent CSC fractions. We found that 0.23% of the cells were positive for all three markers when compared to the whole live cell population, whereas when set to CLDN6 positive sub-population, we found that 20.1% of the cell fraction was triple positive, indicating an 87-fold concentration of 3 markers in the CLDN6 positive sub-fraction. In the last step, we calculated the percentage of CLDN6 positive cells in the whole live cell population on the one hand and in the CD44/CD24/CD90 positive subpopulation on the other hand. We found that the concentration of cells expressing CLDN6 changed from 0.91% in the whole cell population to 66.87% in the CSC fraction, indicating a 74-fold increase (fig. 5B).

Taken together, these data indicate that CLDN6 accumulated in CSC fractions, whereas CSC markers were enriched in the CLDN 6-positive subpopulation. These findings indicate that CLDN6 is a marker for CSCs.

Example μ example 4: enrichment of cells expressing CLDN6 resulted in the accumulation of established CSC markers CD44, CD24 and CD90 Product of large quantitiesCSC fractions isolated in cell lines and tumors have been shown to be often enriched for CSC markers, such as CD44 and CD 24. To analyze the potential of CLDN6 to serve as a novel CSC marker, we investigated whether cell isolates of CLDN6 positive fractions from a large population of cells resulted in the accumulation of established ovarian CSC markers.

For this purpose, COV318 cells were stained with 0.5 μ g/ml IMAB027 at 4 ℃ for 30 minutes, then incubated with a goat anti-human IgG secondary antibody (1: 300) at 4 ℃ for 10 minutes, after which CLDN6 positive and CLDN6 negative cell fractions were isolated from COV318 cells by FACS sorting using a BD FACSAria cell sorter. The selected cells were then expanded under standard growth conditions for 10 days. These two subpopulations of 1E6 cells were stained for CSC markers CD44, CD24, CD90, CD34, CD117 and CD133 at 4 ℃ for 30 min (see table 1 for details) and their expression profiles of surface markers were analyzed by flow cytometry. 50.000 events were recorded from each sample using a BD Canto II flow cytometer, and the recorded cells were analyzed using FlowJo software.

FACS analysis showed that after 10 days of culture under standard conditions, about 50% of cells in CLDN 6-positive sorted fraction remained positive for CLDN6, while CLDN 6-negative sorted cells were completely negative for CLDN 6. Importantly, we found that CLDN6 positive fractions showed accumulation of CSC markers CD44, CD24 and CD90 compared to CLDN6 negative cell fractions of COV318 cells. Representative dot plots for the different samples are shown in fig. 6A. Further quantification of the expression levels of these markers revealed 99-fold enrichment of CD44, 8-fold enrichment of CD90 and 33-fold enrichment of CD24 when comparing CLDN 6-positive and CLDN 6-negative subpopulations (fig. 6B).

These results demonstrate that CLDN6 can be used as a selection marker for the isolation of CSC fractions from a large number of cell lines, indicating that CLDN6 is a novel CSC marker.

Example 5: CLDN6 high expressing cell lines showed enrichment of CSC markers compared to CLDN6 low expressing cellsCollection

CLDN6 has been shown to be highly expressed in germ cell tumors, ovarian adenocarcinoma, and some cancers with the primary phenotype (primary phenotype). Where CLDN6 is a CSC marker, we would expect to accumulate cells with CSC-like characteristics in such cell lines or tumors, thus accumulating CSC marker positive cells.

We studied 4 high expression cell lines of CLDN 6: expression levels of established CSC markers for ovarian cancer cell lines OV90 and PA-1 and for testicular cancer cell lines NEC-8 and NEC-14. For this purpose, 1E6 cells of each cell line were stained for the surface markers CD44, CD24, CD90, CD34, CD117 and CD133 and CLDN6 at 4 ℃ for 30 minutes (see in particular table 1) and then the expression profile of the cells was analyzed by flow cytometry. The experiment was performed in triplicate, 50.000 events were recorded from each sample using a BD Canto II flow cytometer and the recorded cells were analyzed using FlowJo software. By using viable dyesCounterstaining was performed to exclude dead cells from the analysis. A representative dot plot for each sample is shown in fig. 7.

FACS analysis showed that about 95% of all cell lines studied were positive for CLDN 6. As expected, in addition to CLDN6, these CLDN6 high expressing cell lines also showed accumulation of established CSC markers, with OV90 cells showing high expression of CD44, CD133, CD24 and CD117, PA-1 cells showing high expression of CD44, CD133, CD90 and CD117, and NEC-8 and NEC-14 cells showing increased expression levels of the markers CD133, CD90, CD24 and CD 117.

These results indicate that CLDN6 high expressing cell line is enriched for CSC-like cells and further support that CLDN6 is a CSC marker.

Example 6: treatment of advanced human xenograft tumors with CLDN6 antibody in combination with chemotherapeutic drugs to synergize In the same way, the growth of tumor cells is inhibited and the survival is prolonged

Human cancer cell lines were transplanted to Hsd: athymic nude-Foxn1^nuA mouse. After tumor establishment, tumor-bearing mice were grouped and received CLDN 6-specific monoclonal antibody (IMAB027), chemotherapeutic drugs, or a combination of both. The control group received antibody buffer (vehicle control).

In particular, for the treatment of human ES-2(CLDN6) xenograft tumors, at 37 ℃ with 5% CO₂In a wet incubator of (1), human ovarian cancer cell line ES-2 stably transfected with human CLDN6 was cultured in minimal essential medium (Life Technologies) containing 1 × non-essential amino acid solution (Life Technologies), 700 μ G/ml G418(Life Technologies), and 10% FCS (Life Technologies). For transplantation, to 6-week-old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the hypochondrium with 5X 10 cells in 200. mu.l PBS⁶ES-2(CLDN6) cells. On day 3 after subcutaneous tumor inoculation, mice were treated with saline control group, antibody group or drug monotherapy group and antibody/cytostatic drug combination treatment group (n-12 per group). Paclitaxel or saline control, 15mg/kg, was administered on days 3, 10 and 17 post-transplant. Antibody maintenance treatment was started on day 4 with a bolus injection of 35mg/kg IMAB027 or vehicle control (IMAB027 buffer) three times a week (alternating i.v./i.p./i.p.). Tumor burden and animal health were monitored twice weekly. When the tumor volume reaches the maximum 1400mm ³Or when the tumor becomes ulcerative, the mice are sacrificed. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test.

For treatment of advanced human NEC14 xenograft tumors, according to the supplier's instructions, 5% CO at 37 ℃₂In a humidified incubator containing 10% FCS (Life technologies) in RPMI 1640 medium GlutaMAX^TMHuman testicular germ cell tumor cell line NEC14 was cultured (Life Technologies). For transplantation, to 6-8 week old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the hypochondrium 2X 10 in 200. mu.l PBS⁷NEC14 cells. In the late treatment study, tumors grew to a volume of 50mm³To 150mm³And dividing the mice into a control group, an antibody group or a cytostatic drug monotherapy group and an antibody/cell before treatmentGrowth inhibitory drug combination treatment groups (n-19 per group). 6 days after transplantation, drug alone or combination or vehicle control (saline) was administered as follows: i.p bolus injection of 1mg/kg cisplatin injection at 6, 7, 8, 9 and 10 days; i.p. bolus 30mg/kg carboplatin and antibody maintenance therapy, three antibody bolus 35mg/kg IMAB027 or vehicle control (IMAB027 buffer) three times a week (alternating i.v./i.p./i.p.) on days 6, 13 and 20. Tumor burden was monitored twice weekly. When the tumor volume reaches the maximum 1400mm ³Mice were sacrificed when tumors became ulcerative. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test.

Treatment of human ES-2(CLDN6) xenograft tumors ectopically expressing human CLDN6 with paclitaxel had no effect and showed no antitumor activity compared to the control group. In contrast, IMAB027 inhibits tumor growth and prolongs survival in mice. Combination treatment with IMAB027 with paclitaxel synergistically inhibited tumor growth (fig. 8).

Furthermore, both cisplatin and IMAB027 as single agents were able to significantly reduce tumor growth in animals bearing NEC14 tumors. However, after initial tumor growth inhibition, we observed recurrent tumor growth in most animals. In the method of combination therapy, cisplatin and IMAB027 act synergistically, not only to inhibit tumor growth, but also to cause complete NEC14 tumor regression (remission). Most impressively, survival data show the therapeutic efficacy of IMAB027 in combination with cisplatin. In contrast to the single agent approach, almost all mice treated with IMAB027 together with cisplatin survived for 90 days after tumor transplantation (figure 9).

Other platinum derivatives (e.g. carboplatin) showed only very limited antitumor efficacy in the advanced treatment of mice with NEC14 xenograft tumors. However, the combination of carboplatin with IMAB027 resulted in a synergistic tumor suppressive effect, as well as a highly potent tumor growth inhibition and prolongation of survival (fig. 10).

Thus, the combination of CLDN 6-specific antibodies with chemotherapeutic drugs increases the inhibition of tumor growth and prolongs the survival of mice transplanted with human tumor cells. The combination of the antibody with the chemotherapeutic drug produces a synergistic effect on the inhibition of tumor cell growth and prolonged survival.

Example 7: CLDN6 is a CSC marker

Example 7.1: CLDN6 is important for the sphere forming behavior (spheres forming behavior) of ovarian cancer cells

Another useful assay to analyze CSC-like properties of tumor cells is a sphere formation assay. Using this assay, the ability of cells to anchor independent growth (a characteristic feature of CSCs) can be readily examined. To analyze whether CLDN6 plays a role in anchorage-independent growth of tumor cells, we selected COV318 cells (an ovarian tumor cell line containing only a subpopulation of CLDN 6-positive cells). COV318 cells were sorted for their CLDN6 expression and populations of CLDN6 positive and negative cells were allowed to form spheres under stem cell specific conditions for 21 days. The sphere formation assay showed that CLDN 6-positive COV318 cells showed the ability to form spheres while CLDN 6-negative cells almost completely died when cultured under stem cell specific conditions (fig. 11A). These results indicate that CLDN6 positive fractions represent an enriched population of stem cells that exhibit the ability to anchor independent growth.

To determine the ability of CLDN 6-positive COV318 cells to undergo many cycles of cell division while retaining their undifferentiated state, we analyzed the ability of the spheres to produce second generation spheres. For this purpose, first generation spheres of CLDN6 positive COV318 cells (day 22 post-seeding) were dissociated into single cells and then replated. 23 days after re-plating, we can clearly observe that second generation spheres have formed and that these newly formed spheres are morphologically more regular than the original first generation spheres (FIG. 11B). These observations further confirmed that the CLDN6 positive fraction of COV318 cells represents a stem cell fraction of this cell line.

Taken together, these results clearly demonstrate that CLDN6 plays a significant role in anchorage-independent growth of ovarian cancer cells, an important feature of cancer stem cells.

Example 7.2: enrichment of CLDN 6-positive COV318 cells after in vitro treatment with chemotherapeutic drugs

Ovarian cancer cell line COV318 showed heterologous expression of CLDN6 and a very small subpopulation of cells (-0.3-0.5%) expressed CLDN 6. In each case, in vitro treatment of COV318 cells with platinum derivatives yielded a residual cell population with a higher percentage (> 2%) of CLDN6 positive cells (fig. 12). The specific accumulation of CLDN6 positive cells after treatment indicates that these cells may have a selective survival or growth advantage during chemotherapy. Resistance to conventional chemotherapy is a characteristic of CSCs.

Example 7.3: enrichment of CLDN6 positive COV318 cells in vivo

In previous studies, we found that COV318 cells injected subcutaneously into the flank of athymic nude mice were weakly tumorigenic. In contrast, mice receiving COV318 cells by intraperitoneal injection developed malignant ascites and peritoneal tumors within more than 100 days. Most cells isolated from ascites and tumors were positive for CLDN6 (fig. 13B, top panel) compared to the parental COV318 cells (fig. 13A). After culture under standard conditions, COV318 cells again lost CLDN6 expression in vitro (fig. 13B, bottom panel). This means that CLDN 6-positive COV318 cells exhibit a higher capacity to form tumors, indicating that a small CLDN 6-positive subpopulation comprises CSCs.

Example 7.4: CLDN6 is associated with ovarian cancer stem cell markers in primary tumor samples

Since our previous results show that CLDN6 co-localizes with some CSC markers of ovarian cancer cell lines, we next addressed such a problem: whether CLDN6 expression also correlates with CSC markers in primary tumor samples from ovarian cancer patients.

To this end, mRNA expression of CLDN6 and selected markers described in the literature that are specific for cancer stem cells in ovarian cancer (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1AI, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch1, Pou5F1, CD133, Snail, Sox2) were analyzed by qRT-PCR in 42 human ovarian cancer samples. Subsequent correlation analysis of CLDN6 with these markers was performed using Spearman's r.scatter plots (Spearman's r.scatter plots). A scatter plot from significant correlations and a summary of all correlations are shown in fig. 14.

In the ovarian cancer samples analyzed, CLDN6 was found to be positively correlated with CTCFL, LIN28B, CD24, GNL3 and EPCAM, while CD44 was found to be negatively correlated with CLDN6 (fig. 14A). No significant correlation was observed for all other studied markers (fig. 14B).

These results further support that CLDN6 is a CSC marker.

Example 8: enhancement of anti-tumor activity of anti-CLDN 6 antibody by combination with chemotherapeutic drugs

Example 8.1: effect of chemotherapeutic Agents on IMAB027 mediated ADCC

The effect of chemotherapeutic agents on IMAB027 mediated ADCC was analyzed with COV362(Luc) cells, which were pretreated with carboplatin, gemcitabine, paclitaxel, doxorubicin, and topotecan, respectively. As shown by flow cytometry, pretreatment of target cells resulted in an increase in CLDN6 protein levels on the cell surface (fig. 15B, D, F, H and J). The maximal cell lysis was increased 3-fold for cells treated with chemotherapeutic agents compared to untreated target cells (fig. 15A, C, F, G and I). In summary, combination with chemotherapeutic drugs may enhance the antitumor activity of IMAB 027.

Example 8.2: the combination of multiple chemotherapeutic PEBs (cisplatin, etoposide and bleomycin) with IMAB027 efficiently increased the inhibition of tumor growth and prolonged survival of mice transplanted with the human tumor cell line NEC14

Hsd: athymic nude-Foxn 1^nuThe mouse is transplanted with human testicular germ cell tumor cell line NEC14 subcutaneously. Mice with very advanced tumors were randomized and treated with antibody IMAB027, multi-chemotherapy PEB (cisplatin, etoposide and bleomycin) or a combination of IMAB027 and PEB.

Multiple chemotherapies reduced tumor growth very significantly compared to untreated and IMAB027 treated mice (figure 16A). However, after the initial response of the tumor to PEB treatment, tumor growth began in most animals on day 30. Only in combination processes in which PEB acts synergistically with IMAB027Sustained inhibition of tumor growth was achieved (fig. 16B). Untreated mice showed median survival of 30 days, whereas median survival of 34 and 97 days was observed in mice treated with IMAB027 and PEB, respectively. In the group treated with PEB, 3/14 (21%) mice showed complete tumor regression and 1/14 (7%) mice exhibited-30 mm at the end of the study³The residual tumor mass of (a). Surprisingly, complete tumor regression was observed in 12/14 (86%) mice treated with PEB in combination with IMAB 027. 11 mice were cured without any relapse within 6 months and one tumor-free mouse was euthanized on day 93 due to poor overall health (fig. 16C). This study showed that animals with very advanced human NEC14 testicular tumors that received PEB in combination with IMAB027 had significantly longer survival and significantly higher response rates than animals treated with multiple chemotherapies alone.

Example 9: anti-CLDN 6 antibody-drug conjugates are very effective in treating CLDN 6-expressing tumors

Example 9.1: in vitro binding and antitumor Activity of toxin conjugated IMAB027

Relative binding affinities of IMAB 027-drug conjugate IMAB027-DM1 and IMAB027-vcMMAE were tested using flow cytometry on ovarian cancer cell lines OV 90. In saturation binding experiments, the concentration of antibody was plotted against Median Fluorescence Intensity (MFI) and EC50 (the concentration of antibody binding to half of the binding sites at equilibrium), and the maximum binding was calculated by non-linear regression. Compared to unconjugated IMAB027, IMAB 027-drug conjugates showed similar low EC50 values and saturation of binding was achieved at low concentrations (fig. 17A). The cytotoxic activity of IMAB 027-drug conjugates was determined using the XTT proliferation assay with OV90 cells. The dose-response curves showed similar inhibition of tumor cell growth in vitro by IMAB027-DM1 and IMAB027-vcMMAE (fig. 17B). In summary, IMAB027 conjugated to DM1 or vcMMAE bound to CLDN6 positive target cells with similar relative affinity efficiently induced killing of tumor cells.

Example 9.2: treatment of advanced human xenograft tumors with toxin-conjugated IMAB027 anti-CLDN 6 antibody inhibited tumor cell growth, prolonged survival and mediated complete tumor regression

IMAB027, an antibody specific for CLDN6 conjugated to a cytotoxic drug, was tested for anti-tumor activity in mice transplanted with CLDN6 positive human cancer cell lines. In this targeting approach, IMAB027 was linked to maytansinoids DM1 or auristatin E (MMAE) and used in athymic nude-Foxn 1 with advanced xenograft tumors^nuTumor growth was monitored in mice.

Treatment of advanced human OV90 ovarian subcutaneous xenograft tumor model：

The anti-tumor effect of toxin-conjugated IMAB027 was tested in mice bearing an advanced human xenograft tumor of homologous CLDN6 expression. Treatment was started on day 10 after tumor cell transplantation. IMAB027-DM1 and IMAB027-vcMMAE very significantly inhibited tumor growth of OV90 ovarian cancer cell xenografts homologously expressing CLDN6 after a single bolus i.v. (fig. 18 and 19A). More importantly, a single application of 16mg/kg IMAB027-vcMMAE resulted in complete regression of the tumor in 60% of treated mice (fig. 19B).

NightFoxnlTreatment of human PA-1 ovarian subcutaneous xenograft tumor model：

Toxin-conjugated IMAB027 was also tested for anti-tumor effect in an advanced xenograft tumor model with heterologous CLDN6 expression. Following subcutaneous transplantation, PA-1 xenograft tumors lost CLDN6 expression after a certain period of time (fig. 20C). Treatment was started on day 15. At that time, PA-1 xenograft tumors began to lose CLDN6 expression. Animals received 4, 8 or 16mg/kg IMAB027-vcMMAE by i.v. single bolus injection. Control animals received either unconjugated IMAB027 or vehicle control buffer instead.

Treatment with IMAB027-vcMMAE inhibited tumor growth of PA-1 xenografts and prolonged survival of tumor-bearing mice very significantly, while IMAB027 or IMAB027-DM1 (data not shown) did not affect PA-1 tumor growth (fig. 20A and B).

The reduced level of CLDN6 positive tumor cells in tumor masses may be responsible for the weak antitumor activity of IMAB027 and IMAB027-DM1 in this in vivo tumor model. IMAB027-DM1 was conjugated to membrane impermeable toxin DM1 via a non-cleavable linker. In contrast, IMAB027-vcMMAE is conjugated to the cell membrane permeable toxin MMAE via a cathepsin-cleavable linker. After cell treatment, the release of MMAE in membrane-permeable form is beneficial for killing tumor cells lacking specific epitopes (bystatder effect). Thus, treatment with IMAB027-vcMMAE was very effective in eradicating PA-1 tumors containing both CLDN6 positive and CLDN6 negative cells.

In summary, IMAB027-vcMMAE kills human xenograft tumors with heterologous CLDN6 expression very efficiently by target cell activation killing of bystander cells.

Treatment of advanced human MKN74 stomach subcutaneous xenograft tumor model：

In contrast to PA-1 xenograft tumors that lost CLDN6 expression in advanced tumors, MKN74 xenograft tumors acquired CLDN6 expression. As shown by flow cytometry using the CLDN 6-specific antibody IMAB027, < 0.3% of cells of the gastric cancer cell line MKN74 were CLDN6 positive in vitro. Interestingly, in athymic nude mice, a large number of tumor cells showed CLDN6 expression after subcutaneous transplantation (fig. 21C). Treatment of established MKN74 xenograft tumors with 16mg/kg IMAB027-vcMMAE resulted in very significant inhibition of tumor growth and prolonged survival (fig. 21A and B).

The inhibition of tumor growth observed with IMAB027-vcMMAE may be caused by killing of target cells by activation of bystander cells.

Treatment of advanced human PA-1 ovarian intraperitoneal xenograft tumor model：

In addition to treatment of xenograft tumors by subcutaneous injection (s.c.), the anti-tumor activity of toxin-conjugated IMAB027 antibodies was tested in an in vivo monitoring using ectopically luciferase expressing PA-1 cells in an i.p. xenograft tumor model (fig. 22). On day 14 after tumor cell transplantation, mice received 16mg/kg IMAB027-DM1 or 16mg/kg IMAB027-vcMMAE via a single bolus i.v.

Measurements of bioluminescent intensity in vivo showed that tumor growth of peritoneal PA-1 metastases was inhibited after treatment with IMAB027-DM 1. Furthermore, IMAB027-vcMMAE showed significantly higher antitumor effect than IMAB027-DM1 or vehicle, with complete regression of peritoneal tumors in 100% of the animals (fig. 22).

In summary, IMAB027-vcMMAE and IMAB027-DM1 were very effective and showed no toxic side effects in the concentration range tested in vivo. IMAB027-DM1 significantly inhibited tumor growth in subcutaneous xenograft tumors and reduced tumor growth in peritoneal xenograft tumors. IMAB027-vcMMAE very significantly inhibited tumor growth and prolonged survival in animals bearing subcutaneous or peritoneal human xenograft tumors with homologous or even heterologous CLDN6 expression. Most impressively, a significant fraction of tumor-bearing animals were cured after treatment with MMAE-conjugated antibodies. The excellent antitumor activity of IMAB027-vcMMAE (particularly observed in animals whose tumors exhibit expression of heterologous CLDN 6) demonstrates that IMAB027-vcMMAE conjugates are suitable for the treatment of tumors with a low percentage of CLDN6 positivity.

Example 9.3: endocytosis of CLDN 6-specific antibodies is dependent on affinity and CLDN6 binding epitope

The cytotoxic potency of toxin-conjugated antibodies is strictly dependent on their targeting-mediating potential for internalization. Therefore, the production of antibodies with high endocytosis rate is an important key factor in the development of toxin conjugated antibodies.

The efficiency of endocytosis was tested in vitro by incubating human cancer cells endogenously expressing CLDN6 with CLDN6 reactive monoclonal chimeric antibodies and anti-human Fab fragments conjugated with the toxin saporin. Internalization of CLDN 6-bound antibody/Fab-saporin complexes results in specific killing of cells and can be measured using a cell viability assay. Screening for different CLDN 6-reactive antibodies demonstrated that endocytosis is not only dependent on the binding affinity of the antibody, but also on the epitope. We observed that binding of CLDN 6-specific antibodies to an epitope within the first extracellular loop of CLDN6 supports endocytosis in OV-90 and PA-1 human cancer cells. Notably, CLDN6 reactive antibody 5F2D2 with similar or higher affinity but binding to another epitope showed lower cytotoxic potential in this assay (fig. 23).

Example 10: materials and methods used in examples 7 to 9 above cell culture：

COV362(Luc) and PA-1(Luc) cells stably expressing a fluorescent reporter gene were generated by stably transfecting the cell lines COV362(ECACC, 07071910) and PA-1(ATCC, CRL-1572) with firefly luciferase, respectively.

NEC14(JCRB, 0162) and MKN74(JCRB, 0255) cells were cultured in RPMI 1640 medium (Gibco, 61870-010) supplemented with 10% heat-inactivated FCS (Gibco, 10270-106). COV318(ECACC, 07071903) and COV362(Luc) cells were cultured in DMEM (Gibco, 41965-039) containing 2mM GlutaMAX (Gibco, 35050-038) and 10% heat-inactivated FCS. PA-1 and PA-1(Luc) cells were cultured in MEM (Gibco, 31095-. OV90(ATCC, CRL-11732) cells were cultured in a 1: 1 mixture of MCB105(Sigma, M6395) and 199 medium (Sigma, M4530) supplemented with 1.5g/l sodium bicarbonate and 15% heat-inactivated FCS. At 37 ℃ and 5% CO₂Cells were cultured under the conditions described above.

Expression of CLDN6 was determined by flow cytometry:

the cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300- ⁶The concentration of individual cells/ml was resuspended in FACS buffer. Mu.l of the cell suspension was incubated with anti-CLDN 6 antibody IMAB027 or isotype control human IgG1 antibody (Sigma, I5154) at a concentration of 2.5. mu.g/ml for 30 minutes at 4 ℃. Cells were washed three times with FACS buffer and F (ab') conjugated to APC diluted 1: 200 in FACS buffer₂Fragment goat anti-human IgG (Jackson ImmunoResearch, 109-. Cells were washed twice and resuspended in FACS buffer. Binding was analyzed by flow cytometry using the BD FACS Array (BD Biosciences) and FlowJo software (Tree Star Inc.). Live/dead cells (live/dead) dye propidium iodide (Sigma, P4864) was used to exclude dead cells from the assay.

Treatment of COV318 cells with platinum derivatives：

COV318 cells (1.2X 10 per 100mm cell culture dish)⁶Individual cells) were grown under standard conditions. After 24 hours, cells were treated with 0.5. mu.g/ml cisplatin or 2. mu.g/ml carboplatin and incubated for 96 hours. The medium was changed and the treated cells were grown under standard growth conditions. After 3 to 6 days, cells were analyzed by flow cytometry for CLDN6 expression.

Antibody Dependent Cellular Cytotoxicity (ADCC) after treatment with cytostatic agents:

The effect of carboplatin and paclitaxel on IMAB 027-mediated ADCC was determined using the human ovarian carcinoma cell line COV362 stably transfected with luciferase as a reporter. COV362(Luc) cells (3X 10 per 150mm cell culture dish)⁶Individual cells) were grown under standard conditions. After 24 hours, cells were treated with 5ng/mL paclitaxel, 20. mu.g/mL carboplatin, 25ng/mL gemcitabine, 20ng/mL doxorubicin, or 7.5ng/mL topotecan and incubated for 4 days. The medium was changed and the treated cells were grown under standard conditions for an additional 3 days (for carboplatin and gemcitabine) or 10 days (for paclitaxel, doxorubicin or topotecan).

Cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300-054) and adjusted to a concentration of 2X 10 in DMEM containing 2mM glutamine (Gibco, 25030-081) and 20mM HEPES (Gibco, 15630-056)⁵Individual cells/ml. Each hole is 1 multiplied by 10⁴Individual cells were seeded into white 96-well PP plates and incubated at 37 ℃ and 5% CO₂Incubate for-5 hours.

PBMCs (peripheral blood mononuclear cells) were isolated from human donor blood samples by density gradient centrifugation using Ficoll Hypaque (GE Healthcare, 17144003). Interphase (interphase) -containing PBMCs were isolated and cells were washed 3 times with PBS containing 2mM EDTA. PBMC were prepared at 1.6X 10 ⁷The cells/mL concentration were resuspended in X-Vivo 15 medium (Lonza, BE04-418Q) and stored at 37 ℃ and 5% CO₂Next, until the measurement is performed.

To the cells 25 μ Ι imab027 and isotype control antibody were added at the indicated concentrations. Thereafter, 25. mu.l of PBMC suspension was added and incubated at 37 ℃ and 5% CO₂The cells were incubated for 24 hours.

After adding 10. mu.l of 8% Triton X-100(Sigma, T8787) in PBS to the total lysis control and 10. mu.l PBS to the maximum viable cell control, 50. mu.l of fluorescein cocktail (in ddH) was added to the samples₂3.84mg/ml D-fluorescein (Sigma Aldrich, 50227) and 160mM HEPES) in O, and the cells were incubated for 90 minutes at room temperature in the dark. Bioluminescence was measured with a luminometer (Infinite M200, TECAN). The result is expressed as an integrated digital relative light unit.

Specific lysis was calculated as follows:

maximum viable cells: 10 μ l PBS, no antibody

Total cracking: 10 μ L of 8% Triton X-100 in PBS, no antibody.

Intraperitoneal transplantation of COV318 cells in athymic nude mice:

the human ovarian cancer cell line COV318 was tested for in vivo tumorigenicity and accumulation of CLDN6 positive cells in mice. Thus, 2X 10 resuspended in PBS ⁷One COV318 cell was injected intraperitoneally into a 6-8 week old female Hsd: athymic nude-Foxn 1^nuIn mice. Mice were monitored daily. Animals were euthanized once life-threatening symptoms were evident. Tumor and ascites cells were isolated and cultured for further analysis. Tumors were dissociated mechanically, sieved and washed with DMEM medium (Gibco, 41965-. To obtain a single cell suspension, tumor cells were treated with accutase (Life Technologies, A11105-01) at 37 ℃ for 30 minutes, passed through a 40 μm cell percolator, and washed with DMEM medium. Ascites fluid was collected and contaminating red blood cells were removed by using ACK (ammonium-chloride-potassium) lysis buffer (Invitrogen, a 10492-01). Under the standard conditions of the reaction, the reaction solution is,tumor and ascites cells were grown using standard COV318 medium supplemented with penicillin/streptomycin (Gibco, 15140). Cells were screened for CLDN6 expression by flow cytometry.

Treatment of very advanced human NEC14 xenograft tumors:

for transplantation, to 6-8 week old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the hypochondrium 2X 10 in 200. mu.l PBS⁷NEC14 cells. In a very advanced treatment study, tumors were grown for 13 days to a maximum volume of 170mm ³And mice were divided into control, IMAB027, PEB (cisplatin, etoposide, bleomycin) and IMAB027/PEB groups (n ═ 14 per group) prior to treatment.

13 days after transplantation, drugs were administered as follows: a bolus i.p. 1mg/kg cisplatin at days 13, 14, 15, 16 and 17; bolus i.p 5mg/kg etoposide on days 13, 14, 15, 16 and 17; i.p.bolus injection of 10mg/kg bleomycin at days 13, 17 and 21. IMAB027 was administered by an i.v./i.p./i.p. alternating rapid bolus injection of 35mg/kg IMAB027 three times a week from day 13 to day 101. As vehicle controls, mice received drug buffer instead of antibody or 0.9% NaCl solution instead of PEB, respectively.

Tumor burden and animal health were monitored twice weekly. When the tumor reaches a volume of 1400mm³Mice were sacrificed when tumors became ulcerative. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test.

Sphere formation assay：

For the sphere formation assay, COV318 cells were stained with 0.5 μ g/ml IMAB027 for CLDN6 for 30 minutes at 4 ℃, followed by incubation with a goat anti-human IgG secondary antibody (1: 300) for 10 minutes at 4 ℃, followed by sorting of the cells for CLDN6 expression using a BD FACSAria cell sorter. Then 1X 10 ⁶Individual CLDN6 positive or negative sorted cells were seeded into 6-well ultra-low attachment plates (Co. sub.) (Co. sub.sub.sub.32 medium in serum-free DMEM/F12 medium containing 0.4% bovine serum albumin, 20ng/ml basic fibroblast growth factor, 10ng/ml epidermal growth factor and 5. mu.g/ml insulinrning) and allowing the cells to form spheres for 21 days under conditions specific for these stem cells. The medium was changed every other day without destroying the spheres and representative pictures were taken periodically.

To produce second generation spheres, first generation spheres of CLDN6 positive COV318 cells (day 22 post-seeding) were dissociated into single cells and then replated into wells of 6-well ultra-low attachment plates. In addition, the medium was changed every other day without destroying the formed spheres, and representative pictures were taken periodically.

^TMQuantitative real-time RT-PCR analysis was performed using the Bio Mark HD system (Fluidigm):

using Bio Mark^TMHD System (Fluidigm) with Stem cell specific factors for selected ovarian cancers (CTCFL, LIN28B, CD24, GNL3, EpCAM, CD44, ABCG2, ALDH1A1, AMACR, ATXN1, BMI1, BMP4, CD34, CD117, Myd88, Nanog, Notch1, Pou5F1, CD133, Snail, Sox2) by qRT-PCRGene expression assays (Life technologies) were performed on 42 human ovarian cancer samples. RNA was isolated from ovarian cancer samples using RNeasy Mini kit (Qiagen) according to the instructions of the respective manufacturers and cDNA was synthesized using PrimeScript RT Reagent kit (Takara Bio Inc.). According to Advanced Development Protocol 28-Rapid Gene expression analysis UsingAssays rev A2-samples were prepared and analyzed. Loading onto the 96.96Gene Expression Dynamic Array IFC was accomplished by IFC Controller HX. By Fluidigm BioMark^TMThe HD system analyzes the chip array. TaqMan PreAmp MasterMix was purchased from Applied Biosystems. The data set was evaluated according to the Δ Δ Ct method. The correlation analysis of CLDN6 with selected ovarian cancer stem cell markers was performed using Spearman' sr. By aligning the correlation coefficientsTo assess the significance of the correlation value. P values for multiple tests were adjusted using Benjamini and Hochberg methods, and adjusted P values ≦ 0.05 were considered significant.

Toxin conjugated CLDN6 antibodies

Toxin conjugation of monoclonal antibodies was performed in Piramal Healthcare (Grangemouth, UK).

For DM1 conjugation, naked antibody was modified with SMCC (6 × molar concentration) reacting with the free NH2 residue of lysine group by incubation in PBS (pH 7.2) for 1 hour at room temperature. The modified antibody was then dialyzed into 35mM citrate buffer (ph5.0) and the linker to antibody ratio was determined using a reverse phase elman's assay. DM1(6 x molar concentration) was conjugated to the maleimide moiety of the SMCC linker via its thiol group by incubation at room temperature for 17 hours. The conjugated antibody was dialyzed into formulation buffer (20mM His, 85mg/ml sucrose, pH5.8) and stored at-80 ℃. The drug-antibody ratio was analyzed by UV spectrometry, the monomer content by SEC-HPLC and the free drug content by RP-HPLC.

For MMAE conjugates, the naked antibody was dialyzed into PBS (ph7.2) and modified by thiolation of the free NH2 group of the lysine residue at room temperature for 2 hours using the trout's reagent (2-Iminothiolane) (20 × molar concentration). Subsequently, thiolated antibody was dialyzed into 35mM citrate buffer (pH 5.5) and the linker-to-antibody ratio was determined using a reverse phase elman assay. By incubation at room temperature for 15 hours, vcMMAE (6 x molar concentration) was conjugated to the thiol group of the thiolated antibody through valine of the cathepsin-cleavable linker. The conjugated antibody was dialyzed into formulation buffer (20mM His, 85mg/ml sucrose, pH 5.8) and stored at-80 ℃. The drug-antibody ratio was analyzed by UV spectrometry, the monomer content by SEC-HPLC and the free drug content by RP-HPLC.

Determination of relative binding affinity by flow cytometry：

With 0.05% trypsinEDTA (Gibco, 25300-054), washing the cells with FACS buffer (PBS containing 2% FCS (Gibco, 10270-106) and 0.1% sodium azide (Applichem, A1430)), and washing at 2X 10⁶The concentration of individual cells/ml was resuspended in FACS buffer. IMAB027, IMAB027-DM1 or IMAB 027-vcMAE (titration series of 0.1ng/ml to 20. mu.g/ml) was incubated with 100. mu.l of the cell suspension at 4 ℃ for 30 minutes. The cells were then washed three times with FACS buffer and incubated with anti-human IgG (Jackson ImmunoResearch, 109-. Subsequently, the cells were washed twice and resuspended in 100. mu.l of FACS buffer. Binding was analyzed by flow cytometry using BD FACSArray (BD Biosciences) and FlowJo software (Tree Star Inc.).

Viability assay with toxin conjugated IMAB027：

The effect of viability of IMAB027-DM1 and IMAB027-vcMMAE on human tumor Cell lines was determined in vitro using a colorimetric assay (Cell promotion Kit XTT from Applichem) which measures metabolic activity of cells.

OV90 cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300-. After 24 hours, a concentration series of DM 1-conjugated IMAB027 and MMAE-conjugated IMAB027 or isotype control antibody diluted in 50 μ l of medium was added. Cells were cultured for 3 to 7 days until untreated cells reached approximately 80% confluency. Cell viability analysis was performed using the Applichem CellProlification Kit II (Applichem, A8088-1000) according to the manufacturer's instructions. After 3 to 5 hours incubation with XTT reagent, 100 μ Ι of cell supernatant was transferred to a new 96-well assay plate and absorbance was measured at 480nm (reference 630nm) using a spectrophotometer (Tecan). Viability was calculated using the following equation:

blank: cell-free medium and XTT

Maximum viable cells: cells, media and XTT

ECS0 values were determined by non-linear regression using GraphPad Prism 6 software.

Treatment of advanced subcutaneous OV90 xenograft tumors：

The human ovarian cancer cell line OV90 was cultured under standard conditions. For transplantation, to 6-8 week old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the hypochondrium with 1X 10 cells in 200. mu.l PBS⁷OV90 cells. In a late stage treatment study, tumors were allowed to grow for 10 days and would have an established 50mm before treatment³To 150mm³Mice with volumetric tumors were randomly divided into vehicle and antibody groups (n ═ 10). Tumor volume was monitored every two weeks (TV ═ length x width²)/2). TV is expressed in mm³So that a growth curve of the tumor is constructed over time.

In an initial dose range determination study, animals were injected i.v. with a single injection of vehicle, IMAB027-DM1(1.78mg/kg, 5.33mg/kg or 16mg/kg) and dissected on day 35 post-implantation. In a second dose range determination study, animals were given i.v. a single injection of vehicle, IMAB 027-vcMAE (4mg/kg, 8mg/kg or 16mg/kg) or IMAB027-DM1(1.33mg/kg, 2.67mg/kg or 5.33 mg/kg). IMAB027 was administered three times per week by i.v./i.p./i.p. alternating bolus injections of IMAB027 at 35 mg/kg. When the tumor reaches more than 1400mm³Or when an ulcer forms, the mice are sacrificed. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test. Survival was analyzed using the Mantel Cox test.

Treatment of advanced subcutaneous PA-1 xenograft tumors and immunohistochemistry of tumor sections：

The human ovarian cancer cell line PA-1 was cultured under standard conditions. For transplantation, to 6-8 week old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the hypochondrium with 1X 10 cells in 200. mu.l PBS⁷PA-1 cells. In the late treatment study, tumors were allowed to grow for 15 days before treatment and would have established 40mm³To 120mm³Mice with volumetric tumors were randomly divided into vehicle and antibody groups (n ═ 10). Tumor volume was monitored every two weeks (TV ═ length x width²)/2)。TV is expressed in mm³So that a growth curve of the tumor is constructed over time.

I.v. single injection of vehicle, IMAB 027-vcMAE (4mg/kg, 8mg/kg or 16mg/kg) or IMAB027-DM1(4mg/kg, 8mg/kg or 16mg/kg) into animals. IMAB027 was administered three times per week by i.v./i.p./i.p. alternating bolus injections of IMAB027 at 35 mg/kg. When the tumor reaches more than 1400mm³Or when an ulcer forms, the mice are sacrificed. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test. Survival was analyzed using the Mantel Cox test.

To analyze expression of CLDN6 during tumor establishment and progression, PA-1 tumors of untreated mice were dissected at days 7, 14 and 56, respectively, fixed with formalin and embedded in paraffin. From each sample FFPE (formalin fixed paraffin embedded) block, 4 μm tissue sections were prepared, mounted on sticky slides (SuperFrost Ultra Plus, Thermo Fisher Scientific) and baked at 60 ℃ for 60 minutes. FFPE tissue sections were deparaffinized prior to staining. The sections were boiled in 10M citric acid (pH 6.0) supplemented with 0.05% Tween-20 for 10 minutes at 120 ℃. By 0.3% H in PBS ₂O₂Incubated for 15 minutes to quench endogenous peroxidase. After washing with PBS, non-specific antibody binding sites were blocked with blocking buffer (10% goat serum in PBS) for 30 minutes at room temperature, followed by overnight incubation with 0.2 μ g/ml of the first rabbit anti-claudin-6 antibody (IBL-America, 18865) diluted in blocking buffer. The samples were then washed 3 times with PBS and incubated with the respective second ready-to-use antibodies (Power Vision HRP goat anti-rabbit; Immunologic) for 30 minutes at room temperature. Visualization was performed using chromogenic substrate solution (Vector Red; Vector Laboratories) for 4: 30 minutes. After counterstaining with hematoxylin, dehydration and mounting, sections were analyzed using a Leica DM2000 microscope.

Treatment of advanced subcutaneous MKN74 xenograft tumors and immunohistochemistry of tumor sections：

The human gastric cancer cell line MKN74 was cultured under standard conditions. For transplantation, to 6-8 week old females Hsd: athymic nude-Foxn 1^nuMice were inoculated subcutaneously in the flank at 200. mu.m1X 10 in lPBS⁷And (3) MKN74 cells. In late treatment studies, tumors were allowed to grow for 7 days and would have established 200 ± 30mm before treatment³Mice with volumetric tumors were randomly divided into vehicle and antibody groups (n ═ 10). Tumor volume was monitored every two weeks (TV ═ length x width ²)/2). TV is expressed in mm³So that a growth curve of the tumor is constructed over time.

Animals received vehicle control buffer or 16mg/kg IMAB027-vcMMAE on day 8 by a single i.v. bolus injection. When the tumor reaches more than 1400mm³Or when an ulcer forms, the mice are sacrificed. Inhibition of tumor growth was analyzed using the Kruskal-Wallis test and the ex vivo Dunn multiple comparison test. The Mann-Whithney test and the post hoc Dunn multiple comparison test were used to analyze the inhibition of tumor growth. Survival was analyzed using the Mantel Cox test.

CLDN6 targeted expression on MKN74 cells was analyzed by flow cytometry prior to transplantation and by histochemistry on dissected untreated MKN74 xenografts on day 31.

For immunohistochemistry, tissue sections of 3 μm thickness were prepared, mounted on slides, and air dried at room temperature for 90 minutes. All tissue sections were fixed in acetone for 10 min at-20 ℃ and washed in PBS for 5 min. Endogenous peroxidase was quenched by incubation with 0.03% hydrogen peroxide (Dakocytomation EnVision System, K4011) for 15 minutes. After washing with PBS, non-specific antibody binding sites were blocked with blocking buffer (10% goat serum in PBS) for 30 min at room temperature followed by incubation with 5. mu.g/ml IMAB027-FITC for 60 min at room temperature. The samples were then washed 3 times with PBS and incubated with respective second ready-to-use antibodies (Bright Vision poly HRP anti-rabbit IgG, Immunologic, DPVR-110HRP) for 30 minutes at room temperature. Visualization was performed using chromogenic substrate solution (Vector Red; Vector Laboratories) for 2: 30 minutes. After counterstaining with hematoxylin, dehydration and mounting, sections were analyzed using a Leica DM2000 microscope.

Treatment of advanced intraperitoneal PA-1(Luc) xenograft tumors：

The anti-tumor activity of toxin conjugated IMAB027 antibody was studied in vivo using human ovarian teratocarcinoma cell line PA-1(Luc) stably expressing firefly luciferase as a light emitting reporter as an intraperitoneal xenograft tumor model. Previous transplantation experiments showed that intraperitoneal inoculation of PA-1(Luc) cells produced intraperitoneal tumor nodules.

Resuspend 1X 10 in PBS⁷Individual PA-1(Luc) cells were injected intraperitoneally into female Hsd: athymic nude-Foxn 1^nuIn (1). Bioluminescence imaging began on day 14 post tumor cell inoculation, and was performed weekly thereafter until the end of the study. D-fluorescein (Perkinelmer, 122796) was dissolved in sterile water and injected intraperitoneally (150mg/kg, injection volume 200. mu.l) 5 minutes prior to imaging with the IVIS Lumina imaging System (Advanced Molecular Vision). Mice were anesthetized with isoflurane and placed in the dark room of IVIS luminea and photons emitted for an integration time of 1 minute were quantified. The intensity of transmitted light from PA-1 cells expressing luciferase in animals was expressed as a pseudo-color image (pseudocolor image), where blue was the lowest intensity bioluminescent signal and red was the highest intensity bioluminescent signal. Grayscale photographic images of mice were obtained under LED low illumination. The images were overlaid using Living Image software (Xenogen). For all images, comparable illumination settings were used. To quantify the bioluminescence, regions of interest (ROIs) were determined and the total flux (total flux) of the respective ROIs was measured using photon/second (p/s) units. Background bioluminescence values obtained from non-signaling areas on the animals were subtracted from the respective bioluminescence values of each animal.

On day 14, mice were randomized and treated by intraperitoneal administration of 16mg/kg of IMAB027-DM1 or IMAB027-vcMMAE, respectively. Control animals received vehicle buffer. Tumor growth was monitored weekly by bioluminescence imaging from a ventral view and subsequent analysis of total flux (photons/sec) in the region of interest covering the abdomen of the mice.

Endocytosis：

Endocytosis of bound CLDN6 antibody was determined using a cytotoxicity-based endocytosis assay that relies on the co-internalization of a targeted binding antibody and saporin-conjugated anti-human IgG Fab fragments (Fab-ZAP humans, Advanced Targeting Systems, IT-51). Saporin is a ribosome inactivating protein that, upon internalization, inhibits the biosynthesis of proteins, thus leading to cell death.

PA-1 cells were harvested with 0.05% trypsin/EDTA (Gibco, 25300-³Each cell/well was seeded in 50. mu.l growth medium in 96-well culture plates. After 24 hours, a volume of 25 μ l each of Fab-ZAP was added, followed by IMAB027 or isotype control antibody. CLDN6 antibody was administered in a 6 or 8-step dilution series, with constant concentrations of Fab-ZAP (Fab-ZAP: antibody ratio from 3: 1 to 6561: 1). CO humidified at 37 ℃ ₂The cells were cultured in the incubator for another 72 hours. Cell viability was then analyzed using cell proliferation kit II from AppliChem (AppliChem, a8088-1000) according to the manufacturer's instructions. The absorbance was measured at 480nm (reference 630nm) using a spectrophotometer (Tecan).

Sequence listing

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<120> diagnosis and treatment of cancer involving cancer stem cells

<130> 342-79 PCT

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<151> 2013-07-31

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