WO2008046529A1 - Traitement de tumeurs résistantes à la chimiothérapie ou à la radiothérapie au moyen d'une molécule d'interférence l1 - Google Patents

Traitement de tumeurs résistantes à la chimiothérapie ou à la radiothérapie au moyen d'une molécule d'interférence l1 Download PDF

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WO2008046529A1
WO2008046529A1 PCT/EP2007/008672 EP2007008672W WO2008046529A1 WO 2008046529 A1 WO2008046529 A1 WO 2008046529A1 EP 2007008672 W EP2007008672 W EP 2007008672W WO 2008046529 A1 WO2008046529 A1 WO 2008046529A1
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cells
cell
treatment
tumor
radiotherapy
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PCT/EP2007/008672
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English (en)
Inventor
Peter Altevogt
Alexander Stoeck
Daniela Gast
Susanne SEBENS MÜERKÖSTER
Heiner Schäfer
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Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts
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Priority claimed from PCT/EP2007/003105 external-priority patent/WO2008046459A1/fr
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Publication of WO2008046529A1 publication Critical patent/WO2008046529A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to the treatment of tumors and especially the treatment of tumors at least partially resistant to the treatment with chemotherapeutic drug or to radiotherapy.
  • apoptosis resistance is a hallmark of cancer progression and is frequently observed e.g. in ovarian carcinoma.
  • the standard treatment of advanced cancer is often chemotherapy or radiotherapy.
  • different carcinomas acquire resistance to chemotherapeutic drugs or radiotherapy leading to tumor recurrence and frequent death of the patients.
  • no improvement of the clinical situation is observed.
  • Ll is a type 1 membrane glycoprotein of 200 to 230 kDa structurally belonging to the Ig superfamily (3, the numbering of the references corresponds to the list of example 1). Ll plays a crucial role in axon guidance and cell migration in developing nervous system (4, 5). Recent studies have also implicated Ll expression in the progression of human carcinomas. Ll expression was found on different tumors including lung cancer (6), gliomas (7), melanomas (8, 9), renal carcinoma (10, 1 1), colon carcinoma (12) and carcinomas of the uterine corps, cervix and urinary tract (Huszar M, Moldenhauer G, Gschwend V, Ben-Arie A, Altevogt P, Fogel M.
  • Ll (CD171 ) as a molecular marker for differential diagnosis and targeted therapy.
  • Hum Pathol. 2006 Aug;37(8): 1000-8 Furthermore, it is known in the art that Ll is overexpressed in ovarian and endometrial carcinomas in a stage-dependent manner (13).
  • anti-Ll antibodies for the treatment of ovarian and endometrial tumors (cf. WO 02/04952 and WO 06/013051 and reference (35)).
  • anti-Ll antibodies for the treatment of breast cancer, colon cancer, cervical cancer, melanoma, neuroblastoma, small cell lung cancer, lymphoma has been suggested by Primiano et al. (WO2004037198).
  • the present invention relates to the use of an Ll interfering molecule for the preparation of a medicament for sensitizing tumor cells in a patient for the treatment with a chemotherapeutic drug or with radiotherapy.
  • the invention relates to an Ll interfering molecule for use in a method for sensitizing tumor cells in a patient for the treatment with a chemotherapeutic drug or with radiotherapy.
  • the present invention provides means for overcoming the resistance of tumor cells against these drugs.
  • the cells to be sensitized are at least partially resistant to the treatment with said chemotherapeutic drug or to radiotherapy.
  • the cells to be sensitized are not or not yet resistant to the treatment with said chemotherapeutic drug or to radiotherapy.
  • One consequence of said sensitization could be that the cells are rendered more susceptible to said treatment or that said resistance or partial resistance is prevented.
  • the rationale behing said sensitization of the cells might be that, it has been shown in Example 1 that cells which do not express Ll , or which express Ll only in a low amount before the treatment with a chemotherapeutic drug, strongly express Ll after a treatment period of only 3 weeks with a chemotherapeutic agent. Usually the administration of a chemotherapeutic for the treatment of cancer is repeated over a period of several weeks or months.
  • cancer cells may be treated with an Ll interfering molecule in combination with a chemotherapeutic drug or with radiotherapy even if a resistance against said chemotherapeutic drug or with radiotherapy has not been determined before.
  • the term "sensitizing" is to be understood that after the treatment with the L l interfering molecule, the tumor cells are more susceptible to the treatment with a chemotherapeutic drug or with radiotherapy than before the treatment with an Ll interfering molecule.
  • the term "sensitizing" can be understood that due to the treatment with the Ll interfering molecule, preferably during the treatment with the Ll interfering molecule, the tumor cells are or become more susceptible to the treatment with a chemotherapeutic drug or with radiotherapy than before the treatment with an Ll interfering molecule.
  • the cells before the administration of the L l -interfering molecule, were not susceptible to the treatment or only susceptible to an extend that the treatment with a chemotherapeutic drug or with radiotherapy would not result in the desired therapeutic effect.
  • the tumor cells are capable of expressing Ll and are known to acquire a resistance against the respective chemotherapeutic drug or radiotherapy when treated, preferably repeatedly treated with said chemotherapeutic drug or radiotherapy.
  • the susceptibility is increased by at least 20 %, more preferably by at least 40 % and even more preferably by at least 100 %, preferably as compared to cells not treated with the Ll interfering molecule.
  • chemotherapeutic drugs can be used in the methods and uses of the invention. These compounds fall into several different categories, including, for example, alkylating agents, antineoplastic antibiotics, antimetabolites, and natural source derivatives.
  • alkylating agents examples include busulfan, carboplatin, carmustine, chlorambucil, cisplatin, oxaliplatin, cyclophosphamide (i.e., Cytoxan), dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa.
  • antineoplastic antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, and plicamycin.
  • antimetabolites include fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fludarabine, flurouracil (e.g., 5-fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, thioguanine, folic acid derivatives (e.g. 5-formyl tetrahydrofolic acid), and capecitabine.
  • natural source derivatives include docetaxel, etoposide, irinotecan, taxanes (e.g. paclitaxel or docetaxel), teniposide, topotecan, vinblastine, vincristine, vinorelbine, prednisone, and tamoxifen.
  • chemotherapeutic agents that can be used in the invention include asparaginase and mitotane.
  • C2 ceramide can be used.
  • the chemotherapeutic drug is selected from the group consisting of actinomycin-D, mitomycin C, cisplatin, doxorubicin, etoposide, gemcitabine, verapamil, podophyllotoxin, 5-FU, taxans such as paclitaxel, carboplatin, cyclophosphamide, vinorelbine, oxaliplatin, capecitabine, doxorubicin, and ifosfamide.
  • the term "chemotherapeutic drug” also includes an antibody or fragment thereof being capable of inducing apoptosis in the cell.
  • antibodies include antibodies binding to tyrosin kinases, e.g. the EGF receptor.
  • the term "radiotherapy” refers to each radiation therapy which is commonly used to treat tumors cells.
  • this therapy include ⁇ - rays, X-rays, microwaves, UV radiation as well as the direct delivery of radio-isotopes to or next to tumor cells (brachytherapy).
  • the object of this aspect of the invention is to sensitize tumor cells for the treatment with a chemotherapeutic drug or with radiotherapy. Consequently, in a preferred embodiment, after or during the sensitization with the Ll interfering molecule, the patient is further treated with said chemotherapeutic drug or with said radiotherapy.
  • the regimen for treatment with a chemotherapeutic drug or with radiotherapy is known in the art.
  • the term "Ll interfering molecule” may relate to a molecule which binds to Ll (i.e. an Ll binding molecule).
  • the Ll interfering molecule binding to Ll can bind to Ll extracellularly (e.g. an antibody or an anticalin) or intracellularly (e.g. a low molecular weight molecule).
  • Methods for determining whether a given molecule binds to Ll include e.g. ELISA, Western-Blotting, Immunohistochemistry and FACS staining.
  • Ll interfering molecule may relate to a nucleic acid in the tumor cell encoding or being complementary to Ll coding sequences, e.g. Ll encoding DNA or mRNA or parts thereof and when entering a tumor cell modulates, preferably inhibits Ll expression in the tumor cell.
  • Ll coding sequences e.g. Ll encoding DNA or mRNA or parts thereof
  • Such molecules are discussed below with reference to siRNA, antisense molecules and ribozymes.
  • such inhibition may be completely or partially, e.g. the expression may be reduced by at least 50 % or by at least 80 %.
  • this term also relates to molecules which act downstream in the activity cascade of Ll .
  • the Ll interfering molecule is used to sensitize tumor cells to the treatment with a chemotherapeutic drug or with radiotherapy.
  • Examples 1 and 3 provide an experimental test system for testing whether a given Ll interfering molecule is capable of sensitizing tumor cells.
  • Example 1 demonstrates that siRNA directed against Ll is able to abolish chemoresi stance in cell culture, while Example 3 demonstrates the same fact for anti-Ll antibodies.
  • an L l interfering molecule is a molecule as defined above which is capable of sensitizing tumor cells for the treatment with a chemotherapeutic drug or with radiotherapy. Furthermore, it would be possible to evaluate whether a given molecule is capable of sensitizing tumor cells by performing appropriate clinical studies and determining whether the given compound has a statistical significant effect.
  • said Ll interfering molecule is selected from the group consisting of anti-Ll antibodies, antibody fragments thereof, siRNA, antisense RNA or DNA, ribozymes, low molecular weight molecules, soluble Ll , Ll -binding scaffolds such as anticalins, and Ll ligands or parts thereof.
  • the Ll interfering molecule is an anti-Ll antibody or an antibody fragment thereof.
  • anti-Ll antibodies and not only siRNA can be used for sensitizing tumor cells.
  • the experiments provided in Example 3 demonstrate that anti Ll antibodies are able to abolish chemoresistance in cell culture. Furthermore, the experiments provided in Example 4 demonstrate that pretreatment of cultured cells with anti-Ll antibodies leads to a sensibilization towards apoptosis induced by chemotherapeutics.
  • siRNA acts by blocking expression of the Ll molecule, while for the activity of anti-Ll antibodies, it is important that the Ll molecule itself is present.
  • anti-Ll antibodies apparently mediate a signal through the Ll molecule, because binding of anti-Ll antibodies results in a change in the expression of genes related to apoptosis. Therefore, in the context of the present invention, it has been surprisingly found that although anti-Ll antibodies and siRNA have different modes of action, both agents are capable of sensitizing tumor cells to the treatment with a chemotherapeutic drug. Therefore, these findings allow a generalization to all Ll interfering molecules.
  • antibody or antibody fragment is understood as meaning antibodies (e.g. polyclonal or monoclonal antibodies as well as recombinantly produced antibodies) or antigen-binding parts thereof, which may have been prepared by immortalizing B-cells and/or recombinantly and, where appropriate, modified, such as chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab) or F(ab) 2 fragments (see, for example, EP-Bl-O 368 684, US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884), preferably produced with the help of a FAB expression library.
  • antibodies e.g. polyclonal or monoclonal antibodies as well as recombinantly produced antibodies
  • antigen-binding parts thereof which may have been prepared by immortalizing B-cells and/or recombinantly and, where appropriate, modified
  • the antibody or antibody fragment binds to the extracellular portion of Ll .
  • monoclonal antibodies are used, although it is equally envisaged to use polyclonal antibodies.
  • polyclonal antibodies can be produced according to standard methods as described above and are also commercially available from e.g. Santa Cruz Biotechnology, R&D Systems, Abeam or Signet.
  • protein scaffolds against Ll e.g. anticalins which are based on lipocalin (Beste et al. (1999) Proc.
  • the natural ligand-binding sites of the lipocalins for example the retinol-binding protein or the bilin-binding protein, can be altered, for example by means of a "combinatorial protein design" approach, in such a way that they bind to selected haptens, here to Ll (Skerra, 2000, Biochim. Biophys. Acta, 1482, 337-50).
  • Monoclonal antibodies can, for example, be prepared in accordance with the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299).
  • An alternative to preparing monoclonal antibody-secreting hybridomas a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarily determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • Antibody fragments that contain the idiotypes of the protein can be generated by techniques known in the art.
  • such fragments include, but are not limited to, the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragment that can be generated by reducing the disulfide bridges of the F(ab')2 fragment; the Fab fragment that can be generated by treating the antibody molecular with papain and a reducing agent; and Fv fragments.
  • the Ll interfering molecule binds both soluble and membrane-bound Ll .
  • the Ll interfering molecule is capable of preventing soluble Ll from binding to cell surface receptors including integrins or Ll .
  • Assays for determining whether a given molecule has this capacity are known in the art and include functional assays measuring a reduction of motility or of invasive capacity.
  • siRNAs as tools for RNA interference in the process to down regulate or to switch off gene expression, here Ll gene expression, is e.g. described in Elbashir, S. M. et al. (2001) Genes Dev., 15, 188 or Elbashir, S. M. et al. (2001) Nature, 41 1 , 494.
  • siRNAs exhibit a length of less than 30 nucleotides, wherein the identity stretch of the sense Strang of the siRNA is preferably at least 19 nucleotides.
  • an “antisense” nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a sequence-specific portion of a component protein RNA (preferably mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acid may be complementary to a coding and/or noncoding region of a component protein mRNA.
  • the antisense nucleic acids are of at least six nucleotides and are preferably oligonucleotides, ranging from 6 to about 200 nucleotides. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides.
  • Ribozymes are also suitable tools to inhibit the translation of nucleic acids, here the Eph receptor gene, because they are able to specifically bind and cut the mRNAs. They are e.g. described in Amarzguioui et al. (1998) Cell. MoI. Life ScL 54, 1 175-202; Vaish et al. (1998) Nucleic Acids Res., 26, 5237-42; Persidis (1997) Nat. Biotechnol., 15, 921-2 or Couture and Stinchcomb (1996) Trends Genet., 12, 510-5.
  • LMW molecules are molecules which are not proteins, peptides, antibodies or nucleic acids, and which exhibit a molecular weight of less than 5000 Da, preferably less than 2000 Da, more preferably less than 1000 Da, most preferably less than 500 Da. Such LMWs may be identified in High-Through-Put procedures starting from libraries.
  • the tumor cells are of a type selected from the group consisting of astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,
  • Schwannoma neurofibrosarcoma, medulloblastoma, melanoma cells (e.g. malignant melanoma), pancreatic cancer cells, prostate carcinoma cells, head and neck cancer cells, breast cancer cells, lung cancer cells (e.g. small cancer, non-small cancer), colon cancer cells (e.g.
  • adenocarcinoma of the colon adenocarcinoma of the colon
  • colorectal cancer cells gastrointestinal stromal tumor cells, ovarian cancer cells, endometrial cancer cells, renal cancer cells, neuroblastomas, squamous cell carcinomas, medulloblastomas, hepatoma cells and mesothelioma, epidermoid carcinoma, clear cell adenocarcinoma cells and serous adenocarcinoma of the uterine corps cells, cervix carcinoma cells, urinary tract adenocarcinoma cells, Pheochromocytoma cells, neuroma cells, neurilemoma cells, and paranganglioma cells.
  • the tumor cells are epithelial tumor cells, preferably ovarian cancer cells, endometrial cancer cells, adenocarcinoma of the colon, pancreatic carcinoma cells or small cell lung cancer cells.
  • the tumor cells are melanoma cells.
  • tumor cells this means either the plural (“tumor cells”) or singular (“tumor cell”).
  • tumor cells the plural (“tumor cells”) or singular (“tumor cell”).
  • tumor cells the skilled person will appreciate that in most case mor than one tumor cell (i.e. tumor cells) is treated or sensitized.
  • the Ll interfering molecules are used for the preparation of a pharmaceutical composition or medicament.
  • pharmaceutical composition and “medicament” are used interchangeable.
  • the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a preferred carrier when the pharmaceutical composition is administered orally.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated, in accordance with routine procedures, as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
  • the therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free carboxyl groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxides, etc. .
  • the amount of the therapeutic of the invention, which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • suppositories may contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • a therapeutic of the invention e.g., encapsulation in liposomes, microparticles, and microcapsules: use of recombinant cells capable of expressing the therapeutic, use of receptor-mediated endocytosis (e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc..
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment.
  • This may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the therapeutic can be delivered in a vesicle, in particular a liposome (Langer, 1990, Science 249: 1527-1533), more particular a cationic liposome (WO 98/40052).
  • a liposome Langer, 1990, Science 249: 1527-1533
  • a cationic liposome WO 98/40052
  • the therapeutic can be delivered via a controlled release system.
  • a pump may be used (Langer, supra).
  • a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.
  • the invention also includes a method for sensitizing tumor cells in a patient for the treatment with a chemotherapeutic drug or with radiotherapy, comprising administering to the patient an efficient amount of an LI interfering molecule. All embodiments described above also apply to this method of the invention.
  • the term "effective amount" means that a given molecule or compound is administered in an amount sufficient to obtain a desired therapeutic effect.
  • two compounds are administered in a therapeutic effective amount, this includes that one or each of the compounds may be administered in a subtherapeutic amount, i.e. that the amount of each compound on its own is not sufficient to provide a therapeutic effect, but that the combination of the compounds results in the desired therapeutic effect.
  • each of the compounds on its own is administered in a therapeutically effective amount.
  • the invention relates to the use of an Ll interfering molecule for the preparation of a medicament for the treatment of tumor cells in a patient previously treated with a chemotherapeutic drug or with radiotherapy.
  • the present invention relates to an Ll interfering molecule for use in a method for the treatment of tumor cells in a patient previously treated with a chemotherapeutic drug or with radiotherapy.
  • the term "previously treated” may include patients which have already been treated with a chemotherapeutic drug or with radiotherapy in the course of a separated regimen which has taken place e.g. within the last six or eight months. It also includes patients that already have been treated with the respective chemotherapeutic drug or with radiotherapy in a way that the tumor cells have been become at least partially resistant to said treatment.
  • chemotherapeutic drugs or radiotherapy it is in most cases observed that after an initial response of the tumor to such therapy (tumor mass reduction or stabilization of the disease) the tumors start to progress again. Such progression usually starts upon weeks or months after such therapy. Typically these tumors are then resistant to further treatment with the previously applied chemotherapeutic drug and other treatment modalities are wanted. As described above it has been found that such resistant tumors express Ll and therefore become a target for Ll interfering molecules.
  • the term "previously treated” preferably means that the patient previously received such treatment, such treatment showed an initial effect and at the time of therapy with the Ll interfering molecule the tumor is progressing again.
  • the term "previously treated” may also be seen in a context where the Ll interfering molecule and the chemotherapeutic drug or radiotherapy are used within the same regimen, meaning that the treatments are given within one treatment schedule.
  • in one treatment schedule means that the treatment are applied at the same time, one after another or intermittently, but - in contrast to above - time distances between the individual treatments are short (within one week or within 2-4 days) and, if a treatment success is seen, one does not wait for tumor progression before the next treatment is applied.
  • the invention includes the case where a patient is treated with a chemotherapeutic drug or with radiotherapy and subsequently, preferably within one week or less and more preferably within 2-4 days, a treatment with an Ll interfering molecule is started.
  • a treatment with an Ll interfering molecule is started.
  • several cycles of chemotherapy or radiotherapy on one side and treatment with an Ll interfering molecule are made, with intervals of preferably one week or less and more preferably within 2-4 days.
  • the patient is at least partially resistant to the treatment with said chemotherapeutic drug or with radiotherapy, an effect often observed in the course of said treatment types (see above).
  • the invention relates to the use of an Ll interfering molecule for the preparation of a medicament for the treatment of tumor cells in a patient at least partially resistant to treatment with a given chemotherapeutic drug or with radiotherapy.
  • the present invention relates to an Ll interfering molecule for use in a method for the treatment of tumor cells in a patient at least partially resistant to the treatment with a given chemotherapeutic drug or with a given chemotherapeutic drug or with radiotherapy.
  • the term "resistant to treatment” means that the respective tumor cell does not react to the treatment with a chemotherapeutic drug or with radiotherapy in a complete manner. This means preferably that rather, with respect to this tumor cell, treatment with said chemotherapeutic drug or radiotherapy is rather ineffective or even shows no effects. According to the invention, the term “partially” means that the respective effect is not complete.
  • the invention relates to the use of an Ll interfering molecule for the preparation of a medicament for the treatment of tumor cells in a patient, wherein the Ll interfering molecule is administered in combination with a chemotherapeutic drug or with radiotherapy, preferably wherein the chemotherapeutic drug or the radiotherapy is administered prior to the L 1 interfering molecule.
  • the present invention relates to an Ll interfering molecule for use in a method for the treatment of tumor cells in a patient, wherein the Ll interfering molecule is administered in combination with a chemotherapeutic drug or with radiotherapy, preferably wherein the chemotherapeutic drug or the radiotherapy is administered prior to the Ll interfering molecule.
  • the term "treatment of tumor cells” includes both the killing of tumor cells, the reduction of the proliferation of tumor cells (e.g. by at least 30 %, at least 50 % or at least 90 %) as well as the complete inhibition of the proliferation of tumor cells.
  • this term also relates to the treatment of the respective tumorigenic disease, especially to the treatment of a solid tumor formed by said tumor cells or to the treatment of tumorigenic diseases Furthermore, this term includes the prevention of a tumorigenic disease, e.g. by killing of cells that may or are prone to become a tumor cell in the future
  • the term "in combination with” includes any combined administration of the Ll interfering molecule and the chemotherapeutic drug or radiotherapy. This may include the simultaneous application of the drugs or radiotherapy or, preferably, a separate administration. In case that a separate administration is envisaged, one would preferably ensure that a significant period of time would not expire between the time of delivery, such that the Ll interfering molecule and the chemotherapeutic drug or radiotherapy would still be able to exert an advantageously combined effect on the cell. In such instances, it is preferred that one would contact the cell with both agents within about one week, preferably within about 4 days, more preferably within about 12-36 hours of each other.
  • the rational behind this aspect of the invention is that the administration of chemotherapeutic drugs or the treatment with radiotherapy may lead to an increase of L l expression on the surface of the tumor cells which in turn makes the tumor cells a better target for the Ll interfering molecule. Furthermore, it is shown in example 3 and 4 of the application that the treatment with an Ll interfering molecule (eg. siRNA or an anti-Ll antibody) increases apoptosis in cancer cells treated with a chemotherapeutic agent.
  • an Ll interfering molecule eg. siRNA or an anti-Ll antibody
  • this aspect of the invention also encompasses treatment regimens where an L l interfering molecule is administered in combination with the chemotherapeutic drug or radiotherapy in various treatment cycles wherein each cycle may be separated by a period of time without treatment which may last e.g. for two weeks and wherein each cycle may involve the repeated administration of the Ll interfering molecule and/or the chemotherapeutic drug or radiotherapy.
  • treatment cycle may encompass the treatment with a chemotherapeutic drug or with radiotherapy, followed by e.g. the twice application of the Ll interfering molecule within 2 days.
  • the skilled person will understand that the individual therapy to be applied will depend on the e.g. physical conditions of the patient or on the severity of the disease and will therefore have to be adjusted on a case to case basis.
  • the Ll interfering molecule is administered prior to the chemotherapeutic drug or the radiotherapy.
  • the Ll interfering molecule is used to treat tumor cells.
  • the publication ArIt et al. (number (35) in the reference list to Example 1) as well as Example 2 demonstrate an assay for the killing of tumor cells with an Ll interfering molecule, here an anti-Ll antibody. Consequently, in a preferred embodiment, according to this aspect of the invention, an Ll interfering molecule is a molecule as defined above which is capable of treating tumor cells.
  • the definition of the Ll interfering molecule is as explained above.
  • the Ll interfering molecule is selected from the group consisting of anti-Ll antibodies, siRNA, antisense RNA or DNA, ribozymes, low molecular weight molecules, soluble Ll , anticalins, and Ll ligands.
  • the Ll interfering molecule is further linked to a toxin, with the consequence that upon binding of the anti-Ll antibody to Ll , the toxin exerts its effects on the tumor cell with the result that the tumor cell is treated.
  • the L l interfering molecule is a Ll binding molecule, more preferably an anti-Ll antibody.
  • the term “treatment” refers to all sorts of treatment of tumor cells including killing the tumor cells or stopping the growth of tumor cells. Furthermore, the term also includes the prevention of tumor formation, especially of formation of metastases.
  • the tumor cells might be of the same type as explained above, namely of a type selected from the group consisting of astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, ependymoma,
  • Schwannoma neurofibrosarcoma, medulloblastoma, melanoma cells (e.g. malignant melanoma), pancreatic cancer cells, prostate carcinoma cells, head and neck cancer cells, breast cancer cells, lung cancer cells (e.g. small cancer, non-small cancer), colon cancer cells (e.g.
  • adenocarcinoma of the colon adenocarcinoma of the colon
  • colorectal cancer cells gastrointestinal stromal tumor cells, ovarian cancer cells, endometrial cancer cells, renal cancer cells, neuroblastomas, squamous cell carcinomas, medulloblastomas, hepatoma cells and mesothelioma, epidermoid carcinoma, clear cell adenocarcinoma cells and serous adenocarcinoma of the uterine corps cells, cervix carcinoma cells, urinary tract adenocarcinoma cells, Pheochromocytoma cells, neuroma cells, neurilemoma cells, and paranganglioma cells.
  • the tumor cells are epithelial tumor cells, preferably ovarian cancer cells, endometrial cancer cells, adenocarcinoma of the colon, pancreatic carcinoma cells or small cell lung cancer cells.
  • the tumor cells are melanoma cells.
  • the invention also relates to a method for treating tumor cells in a patient previously treated with a chemotherapeutic drug or with radiotherapy, comprising administering to the patient a therapeutically effective amount of an Ll interfering molecule. Furthermore, the invention relates to a method for treating tumor cells in a patient at least partially resistant to treatment with a given chemotherapeutic drug or with radioterapy, comprising administering to the patient a therapeutically effective amount of an L l interfering molecule. Furthermore, the invention relates to a method for treating tumor cells in a patient, comprising administering to the patient a therapeutically effective amount of an Ll interfering molecule in combination with a chemotherapeutic drug or with radiotherapy.
  • Examples 3 and 4 demonstrate that the treatment with an Ll interfering molecule promotes apoptosis in cancer cells induced by chemotherapeutic agents.
  • promote apoptosis means to increase cellular events that are related to apoptotic cell death (e.g. increase caspase-3/-7 activity in the cell).
  • Example 1 demonstrates that the induction of apoptosis by chemotherapeutic agents is inhibited in cells expressing Ll in comparison to non-Ll expressing cells.
  • the Ll interfering molecule promotes apoptosis in said tumor cell or cells, preferably in tumor cells which have been treated, are treated or are to be treated with a chemotherapeutic drug.
  • the present invention also relates to a method of promoting chemotherapeutic drug or radiotherapy induced apoptosis in a eukaryotic cancer cell by treating said cell with an Ll interfering molecule. Furthermore, the present invention also relates to a method of promoting chemotherapeutic drug induced apoptosis in the tumor cells of a patient by administering an Ll interfering molecule to said patient. In a preferred embodiment, apoptosis is induced by a chemotherapeutic drug. With respect to these methods of the invention, all embodiments described above for the other uses or methods of the invention also apply.
  • A FACS analysis of HEK293 and HEK293-hLl cells. Cells were analysed by cytofluorographic analysis using mAb Ll -I l A to Ll followed by PE-conjugated anti- mouse IgG antibody (B and C). Induction of apoptosis by the indicated compounds and Nicoletti staining. The percentage in region M l of the histogram indicates the percentage of living cells that is graphically depicted in (C).
  • D FACS analysis of CHO and CHO- hLl cells. Cells were analysed as described in (A).
  • E and F Induction of apoptosis by the indicated compounds and the indicated length of time. The rate of apoptosis was determined by Nicoletti staining and the percentage of living cells after treatment is depicted.
  • HEK293 and HEK293-hLl cells were treated with staurosporine for the indicated length of time in the presence or absence of purified soluble L l (lO ⁇ g/ml). Cell survival was determined by Nicoletti staining.
  • B Analysis of FAK phosphorylation in HEK293 cells after the addition of soluble Ll .
  • FIG. 4 Effect of Ll -depletion on apoptosis resistance in OVMz ovarian carcinoma cells
  • OVMz cells were transfected with L l -specific siRNA or control siRNA. After 48 hrs, cells cells were stained with mAb L l- I I A to L l followed by PE-conjugated anti-mouse IgG antibody and subjected to FACS analysis.
  • B Cell lysates were analyzed by Western blot analysis unsing antibodies to the L l ectodomain (mAb Ll -I l A) or the cytoplasmic portion (pcytL l ).
  • the L 1 -32 fragment is the ADAM I O-mediated ectodomain cleavage fragment [ 14].
  • Cisplatm ' treatment augments Ll expression in ml 30 cells
  • A Light microscopy of cisplatin treated and no-treated m l 30 cells. Mote the more elongated morphology after treatment for 3 weeks. The bar represents 10 ⁇ m.
  • B FACS analysis of cisplatin treated m l 30 cells with mAb Ll -I l A against human Ll -CAM. The analysis was carried out as described in Fig. l .
  • C Western blot analysis of cell lysate from cisplatin treated on non-treated m l 30 cells.
  • D FACS analysis of cisplatin treated SW707 colon carcinoma cells cells with mAb L l -I lA against human L l -CAM.
  • Ll expression in carcinomas leads to the production of soluble Ll due to metal loprotease- mediated cleavage by ADAM l O and ADAM 17 [14,20,21].
  • Soluble L l can bind to integrins such as ⁇ 5 ⁇ l and ⁇ v ⁇ 5and trigger ERK activation [23] leading to upregulation of Bcl-2.
  • Ll expression itself can activate ERK via Src and is involved in transcriptional regulation including apoptosis-related genes [16,18].
  • Ll -mediated gene regulation is dependent on ERK-activation [16, 18]and Ll proteolytic processing by ADAMs and ⁇ - secretase with subsequent nuclear translocation of the C-terminal fragment [18].
  • FIG. 7 Functional characterization ofHEK293 cells expressing ItLl wt and mutant Ll
  • A A schematic view of the structure of Ll . Mutant Ll forms containing changes in T1247A and S 1248 A site in the cytoplasmic portion is shown.
  • B FACS analysis of stably transfected HEK293 cells.
  • C Analysis of haptotactic cell migration. Fibronectin or BSA for control were coated onto the backside of Transwell chambers. The indicated stably transfected HEK293 cells were seeded into the top chamber and allowed to transmigrate. The migration of empty vector transfected cells (HEK293-mock) was set to 100%.
  • D Analysis of matrigel cell invasion.
  • mice Stably transfected HEK293 cells were seeded into a 6- well plate and allowed to invade into matrigel.
  • FIG. 8 Biochemical analysis ofhLlwi or hLlmutTS expressing cells
  • A Ll processing and cleavage in transfected HEK293 cells. The cell lysates were analyzed by Western blot with pcyt-Ll recognizing the cytoplasmic portion of Ll . The nomenclature of Ll -cleavage fragments is according to a previous publication (Mechtersheimer et al, 2001).
  • B ELISA analysis. Soluble Ll levels in the medium of HEK293-hLlwt or HEK293-hLl mutTS cells treated with or without PMA stimulation for Ih at 37°C was analyzed. Lysates from both cell lines were used as positive controls.
  • FIG. 9 liLlmutTS-mediated suppression of cell migration and invasion (A) HEK293 cells or HEK293-hL lwt cells were transfected transiently with plasmids (10 ⁇ g DNA) encoding hLl mut, dominant-negative ADAM 10 (ADAM l O-DN) or empty pcDNA3 vector. Control transfection with EGFP-plasmid showed >50% transfection efficiency. 48 h after transfection, cells were analyzed for haptotactic cell migration on fibronectin. Each determination was done in quadruplicates. The MEK specific inhibitor PD59098 was used at a final concentration of 20 ⁇ M.
  • FIG. 1 Adenoviral transduction of KS carcinoma cells with hLlwt and hLlmutTS. Cells were infected with a predetermined amounts of adenovirus (opu/cell). 48 h after transduction with hLlwt or hLl mutTS adenovirus or YFP-TM adenovirus for control, KS cells or the Ll positive ovarian carcinoma cells OVMz, SKOV3ip and MO68 were analyzed for haptotactic cell migration on fibronectin as described in the legend to Fig. l .
  • C The ovarian carcinoma cell lines OVMz and SKOV3ip were transduced with adenovirus as described above and analyzed for matrigel invasion.
  • D ERK 1/2 phosphorylation in OVMz cells was analyzed 48 h after transduction with the indicated adenoviral vectors. Relative band intensities as revealed by densitometric scanning are shown.
  • A Differential gene expression in HEK293, HEK293-hLlwt or HEK293-hLlmutTS cells. mRNAs from cells grown in serum were isolated, transcribed to cDNA and used as template for qPCR (SYBRgreen analysis). The indicated target genes were selected after initial gene chip analysis. Identification of differentially expressed proteins (B) by Western blot analysis using antibodies to cathepsin B and CRABPII with actin as loading control and (C) by FACS analysis with antibodies to the ⁇ 3 integrin subunit, the ⁇ v ⁇ 3 integrin and cathepsin B. Note that ⁇ v ⁇ 5 expression is unaltered and that only small amounts of cathepsin B are detectable at the cell surface. (D) RA inhibits in vitro growth of HEK293 and HEK293-hLl mutTS but not HEK293-hLlwt cells.
  • FIG. 11 Requirement of metalloprotease and presenilin cleavage in Ll -mediated gene regulation (A) Analysis of L 1 -32 cleavage by ⁇ -secretase.
  • Cells were treated for 48 h at 37°C with presenilin inhibitor IX (DAPT) or for control with DMSO.
  • Isolated membranes were incubated for 2 h at 37°C and then separated into pellet or supernatant (SM) fractions by ultracentrifugation.
  • Lanes 1 to 2 show cells treated with DMSO (vehicle).
  • Lanes 3 and 4 show cells preincubated with DAPT.
  • SKOV3ip cells were treated with DAPT either in the presence or absence of the metal loprotease inhibitor TAPI-O for 24 hr.
  • Cells were lysed in BOG lysis buffer and analyzed by Western blot analysis. The cell supernatant was analyzed for soluble Ll using mAb Ll -I lA and the cell lysate was examined for L 1 -32 using pcytLI .
  • C HEK293 or HEK293-hLlwt cells were treated with DMSO, DAPT, TAPI-O or both inhibitors for 96 h. mRNA was transcribed to cDNA and analyzed by qPCR for the genes CRABPII and cathepsin B.
  • D Analysis of ERK phosphorylation in SKOV3ip cells after treatment with the indicated compounds.
  • FIG. 12 Nuclear translocation of Ll-CTF
  • A Analysis of Ll -nuclear translocation by ChIP assay. Soluble chromatin was prepared from the indicated cell lines and immunoprecipitated with pcytLI . The final DNA extractions were amplified by PCR using pairs of primers that cover the promoter region of the indicated genes. An aliquot of extracted DNA was used as input control.
  • B Nuclear localization of L l-CTF in CHO-hLlwt cells (middle row) and and CHO-hL lmutTS cells (bottom row) Ll negative CHO cells were used as control (top row).
  • C Purity of isolated nuclei as revealed by marker protein analysis.
  • D Presence of Ll-CTF in the nucleus.
  • HEK293, HEK-hLlwt or HEK- hLl mutTS cells were cultivated in the presence of 10% FCS or in serum free medium for 24 hr and nuclei were prepared and nuclear fragments were analyzed with pcyt-LI and Western blot.
  • the cells were incubated for 24 hr at 37°C with the indicated purified antibodies to Ll ( 10 ⁇ g/ml) or isotype control IgG.
  • the mAb L 1 -38.12 recognizes only the neural form of human Ll but not the tumor form.
  • Cells were also treated with DMSO (vehicle), or the ERK-specif ⁇ c inhibitor PD59098. Cell lysates were examined for phosphorylation of ERK.
  • Soluble Ll in the supernant and Ll -32 in the cell lysate were analyzed by Western blot.
  • C mRNA was isolated from antibody treated SKOV3ip cells, transcribed to cDNA and analyzed by qPCR for the indicated genes.
  • SKOV3ip cells were injected i.p. into nude mice and after tumor implantation animals were treated with the indicated Ll mAbs or control mAb to EpCAM (HEA- 125). After 30 days the tumor volume was determined and is given as the ratio between X-GaI stained tumor mass and the total sinus. 6 animals were analyzed per group.
  • FIG. 15 LlCAM expression in PT45-Plres and PT45-P1 cells
  • LlCAM expression in chemoresistant PT45-Plres cells is ILl ⁇ dependent (a) PT45-Pl res cells were either left untreated (w/o) or treated with 250 ng/mL ILI-RA for 6 hours. In parallel, PT45-P1 cells were either left untreated (w/o) or treated with 20 ng/mL IL l ⁇ for 6 hours. Ll CAM mRNA levels were analysed by real-time PCR and compared with ⁇ -actin used as control. Data from duplicate measurements are expressed as amount of mRNA in arbitrary units.
  • LlCAM is involved in the mediation of chemoresistance in PT45-Plres cells
  • PT45-P1 res cells were transfected with control siRNA or with two L lCAM specific siRNAs.
  • Western blotting for the detection of full-length LlCAM or of HSP90 as a control for equal protein load was performed (upper panel).
  • siRNA transfected PT45- P Ires cells were treated with 20 ⁇ g/mL etoposide or not for 24 hours and caspase-3/-7 activity was determined, (b) siRNA transfected PT45-Pl res cells were subjected to LlCAM immunostaining (Ll -I l A antibody) or staining with an isotype matched control antibody followed by flow cytometry.
  • siRNA transfected PT45-Pl res cells were analysed by western blotting for the detection of full- length LlCAM, ⁇ v-integrin or HSP90
  • d After overnight siRNA transfection, cells were either left untreated or were either treated with 20 ⁇ g/mL etoposide or with 5 ⁇ g/mL gemcitabine for 24 hours, followed by either AnnexinV/PI staining and flow cytometry (AnnexinV positive cells over basal) or by caspase-3/-7 assay (n-fold induced caspase-3/-7 activity of basal),
  • PT45-Pl res cells were either left untreated (w/o) or were treated with 20 ⁇ g/mL etoposide in the absence (w/o) or presence of either 5 ⁇ g/mL anti LlCAM antibody (Clone L l -I IA) or 5 ⁇ g/mL isotype matched control antibody. After 24 hours,
  • FIG. 18 Knock down of LlCAM abolished chemoresistance in Colo357 and Panel cells
  • Colo357 and Panel cells were either left untransfected (w/o) or were transfected with control siRNA or with Ll CAM specific siRNA.
  • b) Untransfected (w/o) or siRNA-transfected Colo357 and Panel cells were either left untreated or treated with 20 ⁇ g/mL etoposide for 24 hours followed by the analysis of caspase-3/-7 activity (expressed as n-fold induced caspase-3/-7 activity of basal). Means ⁇ SD from three independent experiments are shown.
  • FIG. 19 LlCAM expression induced a chemoresistant phenotype in PT45-P1 cells
  • PT45-P1 cells were either transfected with an empty vector (mock) or with LlCAM.
  • b) Transfected PT45-P1 cells were subjected to LlCAM immunostaining (Ll - 1 1 A antibody) or staining with an isotype matched control antibody followed by flow cytometry.
  • PT45-Pl res cells (a,b) or PT45-P1 cells transfected with L lCAM or an empty control vector (mock) (c,d) were left untreated (w/o) or were either treated with Tapi-0, Tapi-1 , GM6001 or L685,458 (each 10 ⁇ mol/L) for 24 hours.
  • (a,c) Cellular lysates were subjected to western blotting using either the antibody clone UJ 127 from Acris detecting only full- length LlCAM or the pcytLl antibody detecting also the cytoplasmic part of Ll CAM. HSP90 was detected as a control for equal protein load.
  • FIG. 21 LlCAM mediates iNOS induction and NO release in PT45-Plres cells
  • PT45-Plres cells were transfected with a control siRNA or with a Ll CAM specific siRNA.
  • siRNA transfected cells 16h were either left untreated or were treated with 250 ng/mL IL ⁇ RA for 24 hours. Then, supernatants were cleared and subjected to a commercial NO assay. The amount of NO was normalized to equal cell number which was determined in parallel (expressed as ⁇ mol NO/10 3 cells), (c) siRNA transfected cells (16h) were either left untreated or were treated with 250 ng/mL ILl -RA and 20 ⁇ g/mL etoposide, either alone or in combination for 24 hours. Then, cells were analysed for caspase-3/-7 activity expressed as n-fold induced caspase-3/-7 activity of basal. Means ⁇ SD from three independent experiments are shown. * indicates p ⁇ 0.05.
  • PT45-Pl res cells were transfected with a control siRNA or with a Ll CAM spe-cific siRNA. After overnight transfection, cells were either left untreated or were treated with 200 ⁇ mol/L SNAP, 20 ⁇ g/mL etoposide or with a combination of both. After 24 hours, cells were analysed for caspase-3/-7 activity expressed as n-fold induced caspase-3/-7 activity of basal. Means ⁇ SD from three independent experiments are shown. * indicates p ⁇ 0.05.
  • Figure 24 Effect of Ll-IlA on drug induced apoptosis (determined by caspase -3/-7 activity) in alpha98g cells and in CaCo2 cells a98g and CaCO2 cells, respectively, were either left untreated or were treated with 20 ⁇ g/mL etoposide or with 5 ⁇ g/ml gemcitabine in the presence of either 5 ⁇ g/mL isotype matched control antibody (mouse IgG) or 5 ⁇ g/mL anti L l CAM antibody (Clone L l -I IA). After 24 hours, cells were analysed by caspase-3/-7 assay. Data are expressed as n-fold caspase-3/-7 activity of basal. Means ⁇ SD from three independent experiments are shown. * indicates p ⁇ 0.05 when comparing mouse IgG treated versus L l -I l A treated cells.
  • Figure 25 Effect of Ll-IlA on drug induced apoptosis (determined by AnnexinV binding) in alpha98g cells a98g cells, respectively, were either left untreated or were treated with 20 ⁇ g/mL etoposide in the presence of either 5 ⁇ g/mL isotype matched control antibody (mouse IgG) or 5 ⁇ g/mL anti L l CAM antibody (Clone Ll-I l A). After 24 hours, cells were analysed by AnnexinV/PI staining and flow cytometry (expressed as % AnnexinV positive cells over basal).
  • Apoptosis resistance is a hallmark of cancer progression, a phenomenon frequently observed in ovarian carcinoma.
  • Ll adhesion. molecule CD 171
  • Ll expression is a predictor of poor outcome.
  • apoptosis resistance is a hallmark of cancer progression. In ovarian carcinoma, this is frequently observed. Chemotherapy is important in controlling residual disease following cyto-reductive surgery and as neo-adjuvant therapy in patients with advanced disease [I ].
  • the standard chemotherapy for advanced ovarian cancer is currently paclitaxel-carboplatin or paclitaxel-cisplatin which is routinely given together with dexamethasone, a synthetic corticoid [2].
  • dexamethasone a synthetic corticoid
  • ovarian carcinomas often aquire resistance to chemotherapeutic drugs leading to tumor recurrance and frequent death of the patients [1 ,2].
  • a better understanding of molecular mechanisms underlying chemoresi stance is urgently needed.
  • Ll is a type I membrane glycoprotein of 200- 220 kDa structurally belonging to the Ig-superfamily [3]. Ll plays a crucial role in axon guidance and cell migration in the developing nervous system [4,5]. Recent studies have also implicated Ll expression in the progresssion of human carcinomas. Ll expression was found on different tumors including lung cancer [6], gliomas [7], melanomas [8,9], renal carcinoma [10,1 1], and colon carcinoma [12]. We reported before that LI is overexpressed in ovarian and endometrial carcinomas in a stage-dependent manner and that Ll expression was a predictor of poor outcome [13]. A clear mechanism by which Ll expression could contribute to the progression of human tumors is still missing.
  • Ll expression was also found to enhance tumor growth in NOD/SCID mice [12,19] and was found to induce Ll- dependent gene expression [16, 18].
  • ADAM lO metalloproteases
  • ADAM 17 ADAM 17 [21 ,22].
  • the soluble Ll ectodomain, as a product of L I cleavage, is detectable in serum and ascites from ovarian carcinoma patients [13]. Soluble Ll from ascites is a potent inducer of cell migration [23].
  • the ovarian carcinoma cell lines OVMz and m l 30 have been described before [19,20].
  • the human epithelial kidney cell line HEK293 and the Chinese hamster ovary (CHO) cell line stably expressing human Ll (hLl) were established by transfection with superfect (Stratagene, Heidelberg, Germany) and selection for Ll expression with mAb Ll -I lA and magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) or sorting by FACS as described before [19,20]. All cells were cultivated in DMEM supplemented with 10% FBS at 37°C, 5% CO2 and 100% humidity. Experiments with human material were approved by the Ethical commitee of the University of Heidelberg.
  • Antibodies to the ectodomain (mAb Ll -I lA, subclone of mAb UJ 127.1 1) or cytoplasmic domain (pcytLl) of human Ll have been described (10).
  • Antibodies to ERKl , phospho- ERK 1/2, FAK and phosphor-FAK (pi 25) were purchased from BD-Transduction (Heidelberg, Germany).
  • the antibody to phospho-PAKl was purchased from Cell Signaling (New England Biolabs, Frankfurt, Germany).
  • the antibody against Bcl-2 was from Santa Cruz (Heidelberg, Germany). Secondary antibodies were obtained from Dianova (Hamburg, Germany).
  • C2-ceramide, staurosporine and cis-Diammineplatinum(!I)dichloride (cisplatin) were purchased from Sigma (Taufkirchen, Germany).
  • Triton X-100 was from Gerbu (Gaiberg, Germany).
  • Cell pellets were lysed in lysis buffer (20 mM Tris/HCl pH 8.0 containing 1% Triton X-100, 150 mM NaCl, 1 mM PMSF), cleared by centrifugation and mixed with two fold-concentrated reducing SDS- sample buffer.
  • lysis buffer (20 mM Tris/HCl pH 8.0 containing 1% Triton X-100, 150 mM NaCl, 1 mM PMSF
  • siRNA transection Transfection of siRNAs was described before [22].
  • Ll (5'-AGGGAUGG- UGUCCACUUCAAAUU-3') siRNA (SEQ ID NO: l) was synthesized by MWG-Biotech (Ebersberg, Germany). Cells were transfected with annealed siRNAs using Oligofectamine (Life Technologies) and analyzed after the indicated time points.
  • Ll enhances apoptosis resistance in HEK293 cells
  • HEK293 and HEK293-hL l cells were treated with C2-ceramide or staurosporine under serum -free conditions and apoptosis was analyzed by Nicoletti staining.
  • Ll -expressing cells were more resistant against apoptosis induced through both stimuli (Fig. I B and C). 93% of the cells were still viable after 24h treatment with C2-ceramide, compared to only 63% of wild-type HEK.293 cells. Similar differences were observed at later time points (Fig. I B) or after treatment with staurosporine.
  • Ll -expressing cells Under these conditions, 77% of Ll -expressing cells were viable in contrast to only 59% of the parental cells. No differences in the viability could be observed under serum free conditions after 24 hours (Fig. 1 B and C). Cancer cells are often resistant to chemotherapeutic agents due to protection from apoptotis.
  • chemotherapeutic drug cisplatin To study a possible influence of L l expression on apoptosis induced through the chemotherapeutic drug cisplatin, we treated HEK293 and HEK293-hLl cells for various length of time. Under these conditions, Ll expressing cells again showed a more resistant phenotype than Ll negative cells. Approximately 70% of Ll positive cells survived the treatment with cisplatin as opposed to only 40% of Ll -negative cells (Fig ID).
  • Soluble Ll has little protective effect in HEK293 cells
  • soluble Ll can stimulate cell migration and trigger ERK-phosphorylation by binding to integrins [23].
  • the release of soluble Ll is increased by apoptotic stimuli [23]. Therefore, we investigated the role of soluble Ll on apoptosis protection.
  • soluble Ll enhanced survival of both cell lines to a similar degree.
  • soluble Ll could only partially rescue HEK293 cells from apoptosis and the rate was not increased when higher amounts of soluble Ll were added (data not shown).
  • Ll expression M 130 cells were treated cells with increasing amounts of cisplatin over a time period of 3 weeks (l O ⁇ M first week, 15 ⁇ M second week, 20 ⁇ M third week).
  • the long-term treatment altered the morphological phenotype of the cells (Fig. 5A) and clearly decreased cell proliferation (data not shown).
  • the expression of Ll was strongly enhanced as revealed by FACS (Fig. 5B, lower panel) and Western Blot analysis (Fig. 5C). Similar results were obtained in the Ll negative colon carcinoma cells SW707 (Fig. 5D).
  • High-grade ovarian carcinoma is a life-threatening disease with a low five-year survival rate.
  • chemotherapy comprising usually a platinum based drug, such as cisplatin or carboplatin, coupled with paclitaxel. While this treatment course shows promising effects in a high percentage of cases, the development of chemoresistance is a hurdle that significantly reduces successful treatment outcomes.
  • Ll-CAM is associated with poor outcome in ovarian and endometrial carcinomas [13].
  • Ll-mediated gene regulation is also operative in human ovarian carcinomas in situ.
  • Ll long-term tretment with cisplatin upregulated Ll expression might be of some clinical relevance. If such a selection would happen also in situ during chemotherapy, it would enrich for tumor cells with enhanced motility, invasiveness and better growth characteristics. This would be of great disadvantage for the patient.
  • Ll antibodies to Ll have therapeutical potential and can reduce cell proliferation in vitro [12,34], and in vivo growth in a xenograft mouse model for human ovarian carcinoma [35].
  • Ll might be a novel target for antibody-based therapy as second line therapy against aggressive human ovarian tumors. It is feasible that upregulation of Ll by chemotherapeutic drugs like cisplatin might improve the targeting and efficacy of Ll- antibodies.
  • ADAM A Disintegrin And Metalloprotease.
  • BOG ⁇ -octylglycopyranoside.
  • CRABPII cellular retinoic acid-binding protein II.
  • CTF C-terminal fragment.
  • ERK extracellular- signal regulated kinase.
  • hLlwt human Ll wild type.
  • hLlmutS human Ll with a mutation of S1248A
  • hLlmutTS human Ll with mutations of T1247A and S1248A.
  • PAK 1 p21 activated kinase 1.
  • RA retinoic acid.
  • RAR retinoic acid receptor.
  • RIP regulated intramembrane proteolysis.
  • SH3 Src homology 3.
  • TF AP2 ⁇ transcription factor activator protein-2.
  • Ll cell adhesion molecule plays an important role in cell migration, axon growth and guidance in the nervous system. Recent work has also implicated Ll in human carcinoma progression and revealed that Ll -expression augmented cell motility, invasion and tumor growth in nude mice, and upregulated proinvasive genes.
  • Ll-CTF C-terminal fragment of Ll
  • Ll cell adhesion molecule (Ll -CAM) is a 200-220 kDa transmembrane glycoprotein of the immunoglobulin (Ig) superfamily. It is composed of six Ig-like domains and five fibronectin type III repeats followed by a transmembrane region and a highly conserved cytoplasmic tail (1). Ll is involved in the regulation of cell migration, axon outgrowth and guidance during the development of the nervous system (2-5). Recent studies have shown that the Ll molecule also plays an important role in the ontogeny of human tumors (6-13). In melanoma and ovarian/endome trial carcinoma, Ll expression is associated with poor prognosis (8-10). The mechanism by which Ll contributes to tumor progression has not been clearly established.
  • Ig immunoglobulin
  • Ll antibodies to Ll were shown to have therapeutical potential and can reduce cell proliferation in vitro (1 1 ,13), and in vivo growth in a xenograft mouse model for human ovarian carcinoma (14).
  • Ll might be a novel target for antibody-based therapy against aggressive human tumors.
  • a better understanding of Ll signaling in carcinoma cells and the mode of action of Ll antibodies is therefore urgently needed.
  • L 1 can augment tumor growth in NOD/SCID mice (13,15), can enhance cell motility on extracellular matrix proteins (16-18) and invasiveness in matrigel invasion assays (13,19). Interference with Ll expression by genetic manipulation was found to be growth inhibitory in vitro (1 1). Importantly, a recent study has demonstrated that Ll can induce ERK-dependent gene regulation (18). As revealed by gene chip analysis, the presence of Ll upregulated expression of the motility and invasion related proteins Rac and Rho but also the proteases cathepsin B and L and the ⁇ 3 integrin subunit (18). Although ERK activation appears to be a crucial element, it remains unclear whether activated ERK alone or only in cooperation with Ll could lead to the expression of these genes.
  • Ll is cleaved and released from the cell membrane by the metal loprotease ADAMlO (16,20).
  • the soluble Ll ectodomain is also detectable in serum and ascites from ovarian carcinoma patients (9).
  • the involvement of ADAMlO in Ll shedding was recently confirmed in a study using a battery of ADAM-deficient fibroblastic cell lines established from knock-out mice (21). This study showed for the first time that proteolytic cleavage of the extracellular domain of Ll by ADAMlO is followed by intramembrane presenilin-dependent ⁇ -secretase cleavage leading to the generation of a Ll cytoplasmic domain missing the transmembrane region (21).
  • RIP regulated intramembrane proteolysis
  • the cytoplasmic part contains a putative SH3 binding domain with the consensus sequence PINP (Position 1249-1252).
  • the proceeding amino acid S 1248 was previously identified as a phosphorylation site for ERK2 (24).
  • S 1248A site-directed alanine mutagenesis
  • hLlmutS site-directed alanine mutagenesis
  • a second mutant was constructed including the adjacent threonine (T1247A, S 1248A) and was termed hLlmutTS (see Fig.7A). Both mutants were stably expressed in HEK293 cells. FACS analysis revealed expression of all Ll constructs at the cell surface (Fig.7B).
  • HEK293 -hLlmutTS cells were injected into the left flanks of mice. As expected, we observed significantly augmented growth of HEK293-hLlwt tumors in comparison to untransfected HEK293 tumors (Fig.7E). Strikingly, HEK293-hLlmutTS cells did not enhance tumor growth in NOD/SCID mice (Fig.7E).
  • SW707-hLlwt cells augmented tumor growth in vivo in agreement with previous results (13).
  • Cells expressing hLlmutTS showed similar in vivo growth as mock- transfected SW707 cells.
  • T1247/S1248 motif in the CTF of hLl has a significant impact on tumor growth in vivo.
  • Soluble Ll is able to stimulate cell migration (16,21). Indeed, a recombinant Ll-Fc protein enhanced cell migration of untransfected HEK293 cells (four-fold increase) and weakly augmented cell migration of hLlwt expressing cells (Fig.8C). In contrast, cells expressing hLlmutTS showed no Ll-Fc stimulated migration (Fig.8C).
  • soluble Ll released by cells, could drive migration by an autocrine/paracrine loop (9,16). Since hLlwt and hLlmutTS were cleaved from the membrane and released into the supernatant to a similar extent (Fig.8B), we investigated whether soluble hLlmutTS was also functional.
  • Ll functions have been shown to involve ERK1/2 activation (17,24). Indeed, a recent study has demonstrated that Ll expression causes sustained ERX activation, leading to enhanced motility of cells and augmented the activity of ERKl/2-dependent genes (18).
  • HEK293 and HEK293-hLlwt cells showed constitutive phosphorylation of ERK (Fig.8E).
  • Fig.8F hLlmutTS expressing cells
  • the amino acid S 1248 that is mutated in hLlmutTS, comprises an ERK2 phosphorylation site (24).
  • ERK2 could indeed not phoshorylate Ll in this position, we made use of GST-fusion proteins encoding the cytoplasmic part of hLlwt and hLlmutTS. Recombinant Src-kinase could readily phosphorylate both GST-fusion proteins whereas ERK2 could only phosphorylate the GST-hLlwt construct (Fig.8F).
  • Mutant Ll acts in a dominant negative fashion
  • hLlmutTS-adeno also strongly suppressed the matrigel invasion of OVMz and SKOV3ip cells (Fig.9C). Thus, we concluded that hLlmutTS possesses a dominant- negative activity which is specific towards Ll expressing cells.
  • ItLlmutTS alters gene expression in HEK293 cells
  • HEK293-hLlwt cells we observed upregulation of genes such as cathepsin B, ⁇ 3 integrin and the transcription factors HOX A9, AP2 ⁇ .
  • the apoptosis related gene Mdm 2 was also upregulated whereas downregulation was noticed for the retinoic acid (RA) binding protein CRABPlI. and the apoptosis-inducing genes STK 39 and IER 3 (Fig.10A).
  • the same set of genes remained unchanged in hLlmutTS expressing cells and was similar to non-transfected cells (Fig.10A). Similar observations were made in the colon carcinoma cell line SW707.
  • CRABPII that is essential for the nuclear transport of RA and tumor growth suppression (27), was dramatically reduced.
  • CRABPII downregulation in hLlwt cells compared to parental or hLlmutTS cells (Fig.10A).
  • the qPCR results for cathepsin B and CRABPII were confirmed by Western blot (Fig.1 OB) and for cathepsin B and ⁇ 3-integrin by FACS analysis (Fig.10C).
  • CRABPII channels RA to the nucleus.
  • RA binds to its specific receptor RAR and regulates gene expression of RAR elements leading to a decrease in cell proliferation. Therefore, we treated cells with RA and then determined the level of cell proliferation.
  • hLlwt expressing cells were more resistant to RA- mediated growth inhibition than hLlmutTS expressing cells (Figs.10D). Similar results were obtained in SW707 cells.
  • Ll expression causes changes in gene expression leading to altered properties of carcinoma cells.
  • the T1247/S1248 site in Ll is essential for this gene regulation.
  • Metalloprotease and presenilin cleavage are essential for Ll -mediated gene regulation
  • Ll is processed by ⁇ -secretase following initial ectodomain cleavage by ADAMlO (21). Consecutive cleavage by both enzymes is a hallmark of Notch, APP and CD44 signaling that is followed by translocation of the intracellular portion to the nucleus (22).
  • DAPT presenilin inhibitor IX
  • Antibodies to Ll can reverse Ll-dependent gene regulation by interfering with Ll signaling
  • Antibodies to Ll were shown to prevent tumor cell proliferation in vitro (1 1) and tumor growth in vivo in a xenograft mouse model for human ovarian carcinoma (14).
  • mAb Ll-I IA To analyze whether the observed effects were unique for the epitope recognized by mAb Ll-I IA, we produced additional antibodies to Ll .
  • the novel antibody was specific for Ll as confirmed by FACS analysis on SKOV3ip cells and Western blot analysis on tumor cell lysates (Figs.8A and B).
  • Fig.7A mAb Ll-14.10 could efficiently inhibit ERK activation in the presence of serum factors.
  • both mAbs to Ll blocked the invasion of SKOV3ip cells in matrigel (Fig.14C).
  • novel antibody Ll-14.10 was tested in comparison to mAb Ll-I IA and control antibodies to EpCAM (HEAl 25) for the inhibition of tumor growth in nude mice.
  • the novel mAb Ll-14.10 was equal in suppressing tumor growth in vivo compared to Ll-I lA, whereas mAb HEA 125 had no effect on tumor growth (Fig.14C). Discussion
  • Ll is a type 1 transmembrane protein that is expressed by human carcinomas and melanomas and has been linked to poor prognosis in several studies (8-10,12). Ll undergoes regulated proteolysis that takes place at the cell surface and in released exosomes and involves the metal loprotease ADAMlO (16,20,30). Recent studies have shown that Ll is also cleaved by the ⁇ -secretase complex (21). Here we provide evidence that the process of regulated proteolysis is important for Ll -dependent signaling in human tumors.
  • RA can suppress cell proliferation and transcription (32).
  • Ll -mediated gene regulation was dependent on ADAM and presenilin processing as it was blocked in the presence of the respective inhibitors.
  • Chromatin-IP demonstrated that the Ll-CTF was associated with promoter regions of the cathepsin B, ⁇ 3 integrin and CRABPII genes but not with ⁇ -actin promoter. This clearly established a link between Ll-CTF nuclear translocation and Ll - mediated gene regulation.
  • hLlmutTS expressing cells that the ERK1/2 phosphorylation was strongly diminished (Fig.8E).
  • the hLlmutTS-adenovirus could act in a dominant-negative fashion by suppressing ERK activation in Ll expressing carcinoma cells (Fig.9). It remains to be investigated how hLlmutTS mediates this effect.
  • ERK 1/2 are serine-threonine kinases which can phosphorylate many proteins including transcription factors, cytoskeletal proteins, membrane proteins and other kinases (33). ERK 1/2 activation can be distinguished into either transient or sustained modes. The latter mode is required for the translocation of activated ERKl /2 from the cytoplasm to the nucleus where it can regulate gene transcription (25,26,33). Recent reports have demonstrated a close association between Ll and sustained ERK 1/2 activation in carcinoma cells (13,18). Recombinant ERK2 could phosphorylate S1248 and S1204 in the cytoplasmic domain of Ll (24) and both sites were phosphorylated in postnatal rat brain (34).
  • ERK 1/2 is a downstream target of Src.
  • Our data suggest that the loss of the T1247/S1248 motif prevented Src-dependent ERK 1/2 activation.
  • Another possibility is that the interaction with RanBPM is effected in the hLlmutTS expressing cells.
  • RanBPM is a novel Ll -interacting protein that acts as an adaptor protein linking Ll to the ERK pathway (37). It remains to be investigated whether hLlmutTS has lost the ability to bind efficiently to RanBPM.
  • Ll undergoes sequential cleavage by ADAMlO and presenilin and both proteolytic products can be detected in the nucleus.
  • Ll promotes sustained ERK activation leading to nuclear translocation of ERK1/2.
  • Ll-CTF is phosphorylated by activated ERK2 and can join a transcriptional complex that in our example was found to associate with several promoter sites.
  • the hLl mutTS and Ll antibodies reduce sustained activation of ERK and prevent Ll - dependent gene regulation. This offers the possibility to target Ll in positive human carcinomas. The inactivation of Ll might be beneficial for blocking the growth and dissemination of tumors.
  • the ovarian tumor cell lines OVMz, SKOV3ip, the breast cancer cell line KS and SW707 colon carcinoma cells were described before (13,20).
  • the primary ovarian carcinoma cell line MO68 was obtained from Dr. Ingrid Herr (DKFZ, Heidelberg).
  • the human epithelial kidney cell line HEK293, Chinese hamster ovary (CHO) cells and SW707 cells stably expressing human Ll (hLlwt) and mutant Ll (hLlmutS, hLlmutTS) were established by transfection with Superfect (Stratagene, Heidelberg, Germany). All cells were cultivated in DMEM supplemented with 10% FCS at 37°C, 5% CO 2 and 100% humidity. Ll mutagenesis was performed with the QuikChangeTM Site-Directed Mutagenesis KU essentially as described by the manufacturer (Stratagene, Heidelberg, Germany). All constructs were verified by sequencing.
  • YFP-TM adenovirus was a kind gift of Dr.P.Keller (MPI for Cell Biology, Dresden).
  • Antibodies to the ectodomain (Ll-I lA, subclone of mAb UJ 127.1 1) or cytoplasmic domain (pcyt-Ll) of human Ll were described (16).
  • the mAb HEA- 125 to EpCAM was previously described (40).
  • Novel mAb to Ll (mAb Ll -14.10) was obtained after immunization of mice with human Ll-Fc protein comprising the ectodomain of Ll as described (41).
  • Antibodies to ERKl, phospho-ERKl/2, FAK and phospho-FAK (pi 25) were purchased from BD-Transduction (Heidelberg, Germany).
  • the Antibody to phospho- PAK 1 was purchased from Cell Signaling (New England Biolabs, Frankfurt, Germany) and antibodies to Src and Phospho-Src were purchased from Abeam (Biozol Diagnostica, Eching, Germany).
  • the antibody against cathepsin B was from Zymed (Invitrogen, Düsseldorf, Germany) and the antibody to CRABPII was from Santa Cruz (Santa Cruz, Heidelberg, Germany). Secondary antibodies were obtained from Dianova (Dianova, Hamburg, Germany).
  • Antibodies to nucleoporin and BiP/GRP78 were from the organelle kit (BD-Transduction, Heidelberg, Germany). Retinoic acid was obtained from Sigma.
  • the MEK inhibitor PD59098 was obtained from Calbiochem (Bad Soden, Germany).
  • the human Ll-Fc protein has been described (16). Analysis of Ll shedding
  • Assays were carried out as described previously (42). Briefly, cell monolayers in serum- free medium were stimulated at 37°C with or without PMA (50 ng/ml). Supernatants were collected and the cells were removed from the tissue culture plastic surface by treatment with PBS/5 mM EDTA. Cell pellets were lysed in lysis buffer (20 mM Tris/HCl pH 8.0 containing 1 % ⁇ -octylglycopyranoside (BOG). 150 mM NaCl, 1 mM PMSF). cleared by centrifugation and mixed with two-fold concentrated reducing SDS-sample buffer. The detection of soluble Ll in the supernatant by Ll -specific capture ELISA has been described before (Mechterheimer et al, 2001).
  • DNA chip analysis and quantitative PCR mRNA was isolated using the Quiagen RNAeasy mini kit (Quiagen Hilden, Germany).
  • the cDNA array contained 1540 DNA fragments of oncological relevance and 60 control genes (http://www.rzpd.de/products/microarrays/oncochip.shtml).
  • the Phosphorlmager screens were scanned (Fuji FLA-3000, 100 ⁇ m resolution, Fuji BAS-reader software).
  • the primary image analysis (estimation of nVol grey level values for each individual spot) was performed using the ArrayVision software package (Interfocus), which had been adjusted to the 5x5 array before.
  • the background was corrected locally in each 5x5 field by subtracting the empty spot signal (average signal of 3 spots, see above). Normalization was performed via the average signal intensity (without empty spots) on the whole membrane. Two independent hybridizations were performed for each experiment.
  • the cDNA was purified on Microspin G-50 columns (Amersham Biosciences, Freiburg, Germany) and quantitated by NanoDrop spectrophotometer (ND- 1000, Kisker-Biotechnology, Steinfurt, Germany). Primers for qPCR were designed with the DNA Star Program and were produced by MWG (Ebersberg, Germany), ⁇ -actin was used as an internal standard. The PCR reaction was performed with the SYBRgreen mastermix (Applied Biosystems, Darmstadt, Germany). The sequence of primers used is available on request.
  • Fusion proteins comprising the cytoplasmic portion of hLlwt and hLl mutTS (beginning with Fl 142) were constructed using conventional techniques.
  • 2 ⁇ g of purified fusion protein was labelled using 3 P-labelled ⁇ -ATP and recombinant SRC (Biomol, Hamburg. Germany) or recombinant ERK2 (Calbiochem). The reactions were carried out as suggested by the manufacturers.
  • ECM substrates fibronectin, laminin or vitronectin
  • BSA BSA for control.
  • 1 x 10 5 cells were filled into the chambers and allowed to bind. Unbound cells were removed with 80% Percoll and adherent cells were fixed with glutardialdehyde in 90% Percoll. Fixed cells were stained with crystal violet and then extensively washed with ddH 2 O. The dye was eluted in 10% acetic acid and OD was measured at 595 nm using an ELISA plate reader. Each experiment was performed in triplicate and the mean values ⁇ SD are presented. Cell proliferation under low serum was measured by Coulter Counter after 24, 48 and 72 hr. ⁇ -secretase cleavage assay
  • the assay was carried out as described (28).
  • Nuclei purification was done as described (29). Briefly, adherent cells (10 7 ) were trypsinized and washed twice with PBS and buffer A (10 mM Tris-HCl, pH 7.4, 8.3 mM KCl, 1.5 mM MgSO 4 , 1.3 mM NaCl). The cells were resuspended in buffer A and swollen for 30 min on ice. After centrifugation, cells were resuspended in buffer B (Buffer A supplemented with 0.5% NP-40 and 1 mM PMSF). Nuclei and cytosol were prepared by passing the suspension through a 23-gauge needle followed by 20 dounces in a homogenizer.
  • Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature, 334: 701 -703.
  • Ll expressed by glioma cells promotes adhesion but not migration.
  • Overexpression of the cell adhesion molecule Ll is associated with metastasis in cutaneous malignant melanoma. Eur. J. Cancer 38: 1708-1716.
  • Extracellular signal-regulated kinase (ERK)-dependent gene expression contributes to Ll cell adhesion molecule-dependent motility and invasion. J. Biol.
  • Ll-mediated branching is regulated by two ezrin-radixin-moezin (ERM)- binding sites, the RSLE region and a novel juxtamembrane ERM-binding region. J Neurosci. 25: 395-403.
  • ERP ezrin-radixin-moezin
  • RanBPM is an Ll -interacting protein that regulates Ll -mediated mitogen-activated protein kinase activation.
  • ERKl /2 is an endogenous negative regulator of the gamma-secretase activity. FASEB J. 20:157-159.
  • ILl ⁇ - Interleukin 1 beta ILl-RA - Interleukin 1 receptor antagonist
  • iNOS inducible nitric oxide synthase
  • NO - nitric oxide PDAC - pancreatic ductal adenocarcinoma
  • PI propidium iodide
  • RT Reverse transcriptase
  • SNAP S-Nitroso-N-acetyl-D,L- penicillamine
  • Pancreatic ductal adenocarcinoma is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance.
  • induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long term chemotherapy involves an increased ILl ⁇ -dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition.
  • NO nitric oxide
  • Ll CAM an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance.
  • Chemoresistant PT45-Plres cells but not chemosensitive parental PT45-P1 cells, express high levels of LlCAM in an IL ⁇ - dependent fashion.
  • PT45-Plres cells subjected to siRNA mediated LlCAM knock-down exhibited reduced iNOS expression and NO secretion as well as a significant increase of anti-cancer drug induced caspase activation, an effect reversed by the NO donor SNAP.
  • overexpression of LlCAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment.
  • LlCAM ectodomain shedding i.e. by ADAMlO, as reported for other LlCAM related activities, seemed to be dispensable for anti-apoptotic protection by LlCAM.
  • Pancreatic ductal adenocarcinoma is 4-5 th in the rank order of fatal tumor diseases in Western countries with a 5 year survival rate ⁇ 2 % and a still increasing prevalence (Lockhart et al., 2005; Schneider et al., 2005). Due to its largely symptomeless progression, PDAC is diagnosed in an already advanced stage with widespread metastasis, and for 80-90% of the patients no option for a curative surgical resection exist anymore at the time of diagnosis. For these patients, current therapeutical options rely on chemotherapy treatment with 5-fluoruracil or gemcitabine, but solely with palliative intention.
  • melanoma melanoma, glioma, ovarial and colon cancer, gastrointestinal stromal tumors or neuroendocrine pancreatic carcinoma
  • Gast et al., 2005; Gavert et al., 2005; Izumoto et al., 1996; Kaifi et al., 2006a; Kaifi et al., 2006b; Meier et al., 2006 high LlCAM expression could be associated with poor prognosis and short survival times (Fogel et al., 2003; Kaifi et al., 2006a; Kaifi et al., 2006b).
  • LlCAM was initially detected in neuronal cells where it is involved in several biological processes like neuron-neuron adhesion, neurite fasciculation, synaptogenesis, neurite outgrowth on Schwann cells and neuronal cell migration (Brummendorf et al., 1998; Hortsch, 2000; Schachner, 1997).
  • LlCAM is a 200-220 kD glycoprotein and a member of the immunoglobulin superfamily. It consists of six immunoglobulin like domains at the amino terminal end of the molecule followed by five fibronectin type III homologous repeats, a single transmembrane region and a short intracellular domain (Moos et al., 1988). Beside its cell surface localization, LlCAM can also be cleaved by several proteases, i.e. the matrix metalloproteinases ADAMlO and ADAM17 or by ⁇ -secretases (Maretzky et al., 2005).
  • proteases i.e. the matrix metalloproteinases ADAMlO and ADAM17 or by ⁇ -secretases (Maretzky et al., 2005).
  • Soluble LlCAM has been reported to be important for migration of neuronal as well as of tumor cells (Maretzky et al., 2005; Mechtersheimer et al., 2001), and several studies support a role for LlCAM in tumor growth (ArIt et al., 2006), tumor cell invasion and metastasis of melanoma, ovarial and colon cancer (Fogel et al., 2003; Gavert et al., 2005; Mechtersheimer et al., 2001).
  • LlCAM mediated neuroprotection is associated with caspase inhibition (Loers et al., 2005), the aim of the present study was to investigate whether LlCAM is expressed in PDAC and whether it is involved in reduced caspase activation and, thereby, in chemoresi stance of PDAC cells.
  • LlCAM is involved in the mediation of chemoresistance in PT45-Plres cells.
  • LlCAM is directly involved in the mediation of chemoresistance
  • its expression in PT45-Plres cells was blocked by siRNA treatment.
  • Two different LlCAM specific siRNAs were positively tested for reducing LlCAM expression along with an increase of etoposide induced caspase-3/-7 activity (figure 17a).
  • the Ll CAM specific siRNA-2 was used for further experiments.
  • LlCAM immunostaining and flow cytometry (figure 17b) treatment with this siRNA also reduced LlCAM surface expression.
  • the specificity of LlCAM siRNA was verified by the detection of ⁇ v integrin expression in PT45-Plres cells exhibiting unaltered levels after transfection with control or LlCAM siRNA (figure 17c).
  • LlCAM knock down led to a significant apoptosis induction in these cells after treatment with anti-cancer drugs as determined by annexinV staining (figure 17d, left panel) or by a luminescent caspase-3/-7 activity assay (figure 17d, right panel).
  • annexinV staining FIG. 17d, left panel
  • a luminescent caspase-3/-7 activity assay FIG. 17d, right panel
  • LlCAM cleavage is dispensable for induction of chemoresistance in PT45-Plres cells. Since several biological functions of LlCAM depend on its ectodomain cleavage by certain proteinases, yielding soluble LlCAM, we investigated whether Ll CAM cleavage is essential for chemoresistance induction. For this purpose, PT45-Plres cells were either left untreated or treated with the matrix metalloproteinase inhibitors Tapi-0, Tapi-1 or GM6001 or with the ⁇ -secretase inhibitor L685.458.
  • cellular lysates were analysed for Ll CAM cleavage by using either the monoclonal antibody UJ 127 from Acris, detecting the extracellular part of the protein or the pcytLl antibody recognizing the cytoplasmic part of the full length form of LlCAM and of the C-terminal fragment emerging from proteinase cleavage.
  • Incubation of PT45-Plres cells with neither of the inhibitors changed LlCAM expression as indicated by the constant amounts of the full-length form (220 kDa) of LlCAM as well as of its cytoplasmic 32 kD fragment (figure 20a ).
  • LlCAM mediated chemoresistance is linked to enhanced NO release and subsequent caspase inhibition in PT45-Plres cells, as we have recently demonstrated (Sebens Muerkoster et al., 2006)
  • iNOS mRNA expression and NO release were analyzed in PT45-Plres cells subjected to LlCAM knock down.
  • transfection of PT45-Plres cells with LlCAM siRNA clearly reduced the amount of iNOS mRNA.
  • NO levels were significantly diminished in cell culture supernatants of PT45-Plres cells after LlCAM knock down compared to control transfected PT45-Plres cells (from 4.9 to 0.9 ⁇ mol/10 5 cells; figure 21b).
  • NO levels could be decreased in control siRNA transfected PT45-Plres cells by IL ⁇ RA treatment, whereas in these cells with already diminished NO formation during LlCAM knock down no further reducing effect of the ILl-RA on NO levels was observed (figure 21b).
  • etoposide induced caspase activation was increased in control transfected PT45-Plres cells by ILl-RA treatment but not in LlCAM siRNA transfected cells (figure 21c).
  • Chemoresistance ofPT45-Plres cells depends on LlCAM mediated NO secretion.
  • LlCAM expression was suppressed by siRNA transfection in PT45-Plres cells subjected to treatment with etoposide in the absence or presence of the NO donor S-Nitroso-N-acetyl-D,L- penicillamine (SNAP).
  • SNAP treatment restored the chemoresistant phenotype in PT45-Plres cells after LlCAM knock down (figure 22).
  • LlCAM siRNA transfected cells showed a 2.3-fold induction in caspase-3/-7 activity after etoposide treatment compared to 1.6-fold induction in control-siRNA transfectants
  • additional SNAP treatment completely reversed the increased caspase activity during LlCAM knock-down, thus restoring the chemoresistant phenotype.
  • LlCAM is expressed ductal pancreatic adenocarcinoma.
  • tissue sections of human pancreatic adenocarcinomas from 20 patients were analysed for LlCAM expression.
  • LlCAM expression was detectable, showing moderate or strong expression in 5 sections (Table 1 , figure 23).
  • nerves and germinal centers of lymph nodes were intensely stained, whereas normal epithelial cells exhibited no LlCAM expression, at all.
  • the strongest LlCAM expression could be detected in grade 3 tumors (Table 1).
  • LlCAM expression has been similarly seen in chemoresistant Colo357 and Panel cells as well as in PT45-P1 and T3M4 cells derived from continuous coculture with pancreatic stromal fibroblasts, thereby gaining a chemoresistant phenotype (unpublished observations).
  • Drug-induced LlCAM expression seems to be dependent on ILl ⁇ since treatment with the ILl-RA diminished LlCAM levels in PT45-Plres cells and knock down experiments with specific LlCAM siRNA underlined the importance of LlCAM in the induction of chemoresistance in these cells.
  • LlCAM triggered neuroprotection has been shown to be associated with increased phosphorylation of ERK1/2, Akt und Bad as well as inhibition of caspase-9 (Loers et al., 2005).
  • PT45-Plres cells that exhibit increased LlCAM expression and an impaired activity of the initiator caspases-8 and -9 as well as the effector caspases -3 and - 7, accounting for anti-apoptotic protection against cytostatic drugs, do not show significant changes in Akt and ERK 1/2 phosphorylation (data not shown).
  • LlCAM Besides its role in the gain of chemoresistance, LlCAM might also be of importance for invasion and metastasis of PDAC cells, a role which has to be defined yet. Taking all these findings into account, LlCAM represents an interesting therapeutic target to overcome chemoresistance and to concomitantly interfere with the process of metastasis.
  • the human PDAC cell line PT45-P1 as well as its handling were described previously (Kalthoff et al., 1993).
  • PT45-P1 and PT45-Plres cells were kept in culture (37°C, 5 % CO 2 , 85 % humidity) using RPMI 1640 medium (PAA Laboratories, Colbe, Germany) supplemented with 1 % glutamine (Life Technologies, Eggenstein, Germany) and 10 % FCS (Biochrom KG, Berlin, Germany).
  • the generation of PT45-Plres cells was done as described elsewhere (Sebens Muerkoster et al., 2006).
  • the human PDAC cell lines Colo357 and Panel were kindly provided by H. Kalthoff (UKSH-Campus Kiel) and kept in culture using RPMI 1640 medium supplemented with 1 % glutamine, 10 % FCS and 1 % sodium pyrovate (Biochrom).
  • IL-I ⁇ and the IL-I receptor antagonist were obtained from R&D Systems (Wiesbaden, Germany).
  • the matrix metalloproteinase inhibitors GM6001, Tapi-0 and Tapi-1 were obtained from Calbiochem (via Merck Biosciences, Schwalbach/Ts, Germany) and the ⁇ -secretase inhibitor L685,458 was purchased from Sigma-Aldrich Chemie (Taufmün, Germany).
  • S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) was purchased from Alexis (Gr ⁇ nberg, Germany).
  • Etoposide was purchased from Bristol Myers Squibb (M ⁇ nchen, Germany) and gemcitabine from Lilly (Bad Homburg, Germany).
  • the following antibodies were used for the detection of Ll CAM by western blotting: Mouse monoclonal anti LlCAM detecting the full-length 220 kD molecule, soluble 85 kD and 200 kD fragments (clone UJ 127 from Acris Antibodies, Hiddenhausen, Germany) and a rabbit polyclonal anti pcytLl antibody detecting the cytoplasmic part of LlCAM (220 kD, 85 kD, 32 kD fragments) as described previously (Mechtersheimer et al., 2001).
  • apoptosis was determined by staining with annexinV/propidium iodide (Biocarta, Hamburg, Germany) and subsequent fluorescence flow cytometry (GalaxyArgon Plus; DAKO Cytomation, Hamburg, Germany) using the FLOMAX software, and by the detection of caspase-3/-7 activity using a homogeneous luminescent assay (Promega, Mannheim, Germany). All samples were measured in duplicates.
  • PT45-P1 cells were seeded into 6 well plates (2 x 10 5 cells/well), were grown overnight, followed by transfection with 5 ⁇ L/well DIMRIE reagent (Invitrogen) and 0.6 ⁇ g/well of the following plasmids: pcDNA3.1 (mock) or pcDNA3.1- LlCAM (LlCAM).
  • pcDNA3.1 mock
  • LlCAM pcDNA3.1- LlCAM
  • PT45-Plres cells were seeded into 12 well plates (1 x 10 5 cells/well), were grown overnight followed by transfection with 12 ⁇ L/well RNAiFect reagent (Invitrogen) and 2 ⁇ g/well of either Stealth negative control siRNA (Invitrogen) or Stealth LlCAM siRNA (Invitrogen). After overnight transfection, cells were either left untreated or treated as indicated for further 24 hours.
  • NO-assay Cells were seeded for transfection and cultured as described above. 48 hours after transfection, supernations were taken and precleared by centrifugation (5000 rpm, 10 min.) prior to analysis. NO secreted into cell culture supernatants was quantified using the Total nitric oxide (NO) colorimetric assay (R&D Systems). The assay was performed following the manufacturer's instructions. Concentrations of measured NO were normalized to the cell numbers determined in parallel.
  • NO Total nitric oxide
  • Cells were seeded into 6 well and 12 well plates, respectively, and transfected or treated as indicated. Then, cells were washed once with PBS and lysed with 1 volume of 2xSDS sample buffer (128 mmol/L Tris-Base, 4.6 % SDS, 10 % glycerol). Samples were heated for 5 minutes at 95 °C and put on ice for 2 minutes. Protein concentrations were determined using the D c Protein assay (BioRad).
  • a monoclonal antibody (clone UJ 127 from Acris Antibodies) was diluted at a concentration of 0.4 ⁇ g/mL in 5 % nonfat milk powder and 0.05 % Tween in TBS (blotto) and incubated overnight at 4°C.
  • the pcytLl antibody (Mechtersheimer et al., 2001) was used at a concentration of 1 ⁇ g/mL in blotto and incubated overnight at 4°C.
  • a polyclonal rabbit antibody for HSP90 (Santa Cruz, Heidelberg, Germany) was diluted 1 :2000 in blotto.
  • the mouse anti CD51 antibody from Beckman Coulter GmbH (Krefeld, Germany) was used at a concentration of 1 :500 in blotto for detection of human ⁇ v integrin. Incubation with the primary antibodies was performed overnight at 4°C.
  • anti-mouse and anti-rabbit HRP-linked antibodies were used at a dilution of 1 :2000 in blotto-TBST at room temperature for 1 hour. After washing in TBST, blots were developed using the LumiGlo peroxidase detection kit (Cell Signaling). Real-time PCR.
  • RNAse-free water 2 ⁇ g were reverse-transcribed into single-stranded cDNA, as described previously (Schafer et al., 1999).
  • Two ⁇ L of cDNA and 0.2 ⁇ mol/L gene-specific primers were adjusted with RNAse-free water to a volume of 15 ⁇ L.
  • 15 ⁇ L of iQ SYBR Green Supermix 15 ⁇ L were added.
  • Primers for the detection of LlCAM (Gavert et al., 2005) were used under the following conditions: 95°C/1 min; 95°C/1 min, 52°C/30 sec, 72°C/30 sec for 40 cycles: 72°C/10 min.
  • Primers for the detection of iNOS were from Biosource (Ratingen, Germany) and used under the following PCR conditions: 95°C/5 min: 95°C/45 sec, 60°C/45 sec, 72°C/45 sec for 40 cycles; 72°C/10 min.
  • ⁇ -actin was amplified in parallel using primers from BD Biosciences Clontech.
  • the Real-time PCR was performed with a MyiQ Single Color Real-time PCR Detection System (BioRad). Data were collected during annealing steps and were further analysed by using the i-Cycler iQ Optical system software (BioRad). All samples were analysed in duplicates and data are expressed as amount of mRNA in arbitrary units.
  • Ll augments cell migration and tumor growth but not beta3 integrin expression in ovarian carcinomas, lnt J Cancer, 115, 658-65.
  • Gavert N Conacci-Sorrell M, Gast D, Schneider A, Altevogt P, Brabletz T, et al. (2005).
  • Ll a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers. J Cell Biol, 168, 633-42.
  • Ll is a potential marker for poorly-differentiated pancreatic neuroendocrine carcinoma. World J Gastroenterol, 12, 94-8.
  • Tumor-stroma interaction of human pancreatic cancer acquired resistance to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins.
  • Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature, 334, 701-3. Muerkoster S, We obtainedkel K, ArIt A, Witt M, Sipos B, Kruse ML, et al. (2004). Tumor stroma interactions induce chemoresi stance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin- 1 beta. Cancer Res, 64, 1331 -7.
  • the putative apoptosis inhibitor IEX-I L is a mutant nonspliced variant of p22(PRGl /IEX-I) and is not expressed in vivo. Biochem Biophys Res Commun,
  • - no LlCAM staining in tumor cells, ⁇ according to L. H. Sobin, Ch. Wittekind (eds.): TNM Classification of Malignant Tumors Sixth Edition 2002, Wiley-Liss Incorp.
  • the human colon adenocarcinoma cell line CaCo2 were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and the human glioblastoma cell line ⁇ 98g was kindly provided by Peter Altevogt (Heidelberg, Germany). Both cell lineswere kept under the following cell culture conditions: 37°C, 5 % CO 2 , 85 % humidity.
  • MEM medium PAA Laboratories, C ⁇ lbe, Germany
  • glutamine Gibco Life Technologies, Eggenstein, Germany
  • FCS Biochrom KG, Berlin, Germany
  • nonessential amino acids Gibco Life Technologies
  • DMEM medium PAA Laboratories
  • FCS Biochrom KG, Berlin, Germany
  • apoptosis was determined by staining with annexinV/propidium iodide (Biocarta, Hamburg, Germany) and subsequent fluorescence flow cytometry (GalaxyArgon Plus; DAKO Cytomation, Hamburg, Germany) using the FLOMAX software, and by the detection of caspase-3/-7 activity using a homogeneous luminescent assay (Promega, mannheim, Germany). All samples were measured in duplicates.

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Abstract

La présente invention concerne l'utilisation de molécules d'interférence L1, en particulier des anticorps L1 dans le traitement des tumeurs. L'invention concerne en particulier l'utilisation desdites molécules d'interférence L1 sur des cellules tumorales lors du traitement chimiothérapique ou radiothérapique ainsi que l'administration combinée de molécules d'interférence L1 avec des produits chimiothérapiques ou avec la radiothérapie.
PCT/EP2007/008672 2006-10-16 2007-10-05 Traitement de tumeurs résistantes à la chimiothérapie ou à la radiothérapie au moyen d'une molécule d'interférence l1 WO2008046529A1 (fr)

Applications Claiming Priority (6)

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US85174906P 2006-10-16 2006-10-16
US60/851,749 2006-10-16
US85467906P 2006-10-27 2006-10-27
US60/854,679 2006-10-27
PCT/EP2007/003105 WO2008046459A1 (fr) 2006-10-16 2007-04-05 Traitement de tumeurs résistant à la chimiothérapie ou à la radiothérapie grace à des molécules interférant avec l1
EPPCT/EP07/003105 2007-04-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008151819A2 (fr) 2007-06-15 2008-12-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Traitement de tumeurs à l'aide d'un anticorps anti-l1 spécifique
WO2009127414A2 (fr) * 2008-04-16 2009-10-22 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Inhibition de l'angiogenèse et métastase de tumeur
EP2357003A2 (fr) * 2008-11-27 2011-08-17 Korea Research Institute of Bioscience and Biotechnology Composition anticancéreuse comprenant une substance et un agent antitumoraux et présentant des effets inhibiteurs sur l'activité et l'expression de l1cam
EP3047039A1 (fr) * 2013-09-18 2016-07-27 Memorial Sloan-Kettering Cancer Center Inhibition de métastases cancéreuses
EP3493846A4 (fr) * 2016-08-02 2020-07-22 Memorial Sloan-Kettering Cancer Center Traitement d'un cancer métastatique et systèmes modèles pour maladie métastatique

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EP1623995A1 (fr) * 2004-08-06 2006-02-08 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Inhibiteurs de L1 et ADAM10 pour la traitement de carcinomes

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KAIFI JUSSUF T ET AL: "Absence of L1 in pancreatic masses distinguishes adenocarcinomas from poorly differentiated neuroendocrine carcinomas", ANTICANCER RESEARCH, vol. 26, no. 2A, March 2006 (2006-03-01), pages 1167 - 1170, XP009084784, ISSN: 0250-7005 *
KIM RYUNGSA ET AL: "Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy.", CANCER CHEMOTHERAPY AND PHARMACOLOGY, vol. 50, no. 5, November 2002 (2002-11-01), pages 343 - 352, XP002436283, ISSN: 0344-5704 *
LOERS GABRIELE ET AL: "Signal transduction pathways implicated in neural recognition molecule L1 triggered neuroprotection and neuritogenesis", JOURNAL OF NEUROCHEMISTRY, vol. 92, no. 6, March 2005 (2005-03-01), pages 1463 - 1476, XP002436284, ISSN: 0022-3042 *
SEBENS MÜERKÖSTER S ET AL: "Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells.Published on line on 6.11.06", ONCOGENE 26 APR 2007, vol. 26, no. 19, 26 April 2007 (2007-04-26), pages 2759 - 2768, XP002436286, ISSN: 0950-9232 *
STOECK ALEXANDER ET AL: "L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells. AVAILABLE ONLINE ON 9.10.06", GYNECOLOGIC ONCOLOGY FEB 2007, vol. 104, no. 2, 9 October 2006 (2006-10-09), pages 461 - 469, XP005853891, ISSN: 0090-8258 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008151819A2 (fr) 2007-06-15 2008-12-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Traitement de tumeurs à l'aide d'un anticorps anti-l1 spécifique
US8138313B2 (en) 2007-06-15 2012-03-20 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Treatment of tumors using specific anti-L1 antibody
US9260521B2 (en) 2007-06-15 2016-02-16 Medigene Ag Treatment of tumors using specific anti-L1 antibody
WO2009127414A2 (fr) * 2008-04-16 2009-10-22 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Inhibition de l'angiogenèse et métastase de tumeur
WO2009127414A3 (fr) * 2008-04-16 2010-04-22 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Inhibition de l'angiogenèse et métastase de tumeur
EP2357003A2 (fr) * 2008-11-27 2011-08-17 Korea Research Institute of Bioscience and Biotechnology Composition anticancéreuse comprenant une substance et un agent antitumoraux et présentant des effets inhibiteurs sur l'activité et l'expression de l1cam
EP2357003A4 (fr) * 2008-11-27 2013-01-23 Korea Res Inst Of Bioscience Composition anticancéreuse comprenant une substance et un agent antitumoraux et présentant des effets inhibiteurs sur l'activité et l'expression de l1cam
EP3047039A1 (fr) * 2013-09-18 2016-07-27 Memorial Sloan-Kettering Cancer Center Inhibition de métastases cancéreuses
EP3047039A4 (fr) * 2013-09-18 2017-04-05 Memorial Sloan-Kettering Cancer Center Inhibition de métastases cancéreuses
EP3493846A4 (fr) * 2016-08-02 2020-07-22 Memorial Sloan-Kettering Cancer Center Traitement d'un cancer métastatique et systèmes modèles pour maladie métastatique
US11464874B2 (en) 2016-08-02 2022-10-11 Memorial Sloan-Kettering Cancer Center Treating metastatic cancer and model systems for metastatic disease

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