WO2002060420A2 - Utilisation d'un ligand de claudine-4 pour la therapie et le diagnostic de tumeurs - Google Patents

Utilisation d'un ligand de claudine-4 pour la therapie et le diagnostic de tumeurs Download PDF

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WO2002060420A2
WO2002060420A2 PCT/EP2002/001017 EP0201017W WO02060420A2 WO 2002060420 A2 WO2002060420 A2 WO 2002060420A2 EP 0201017 W EP0201017 W EP 0201017W WO 02060420 A2 WO02060420 A2 WO 02060420A2
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claudin
carcinomas
ligand
cells
conjugate
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PCT/EP2002/001017
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German (de)
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WO2002060420A3 (fr
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Guido Adler
Thomas Gress
Patrick Michl
Malte Buchholz
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Universität Ulm
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Publication of WO2002060420A3 publication Critical patent/WO2002060420A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the present invention relates to the use of a Claudi ⁇ -4 ligand for the treatment or therapy and / or diagnosis of tumors, a conjugate which comprises a Claudin-4 ligand and at least one chemotherapeutic agent and / or at least one non-radioactive diagnostic agent, and a pharmaceutical composition containing the conjugate according to the invention.
  • a cure is generally not possible with the therapies mentioned above, even when they are combined, and often only some of the patients respond to the therapy (so-called response rates of a maximum of 10 to 20% are achieved, for example, in tumors of the pancreas). There is also a partial Significant reduction in the quality of life of the patient due to the side effects of conventional cytostatics and radiation therapy.
  • various imaging e.g. ultrasound, computed tomography, nuclear magnetic resonance imaging, nuclear medicine procedures
  • endoscopic e.g. gastroscopy, endosonography, colon colonization
  • laboratory chemistry e.g. with tumor markers
  • histological examinations e.g. of biopsy material.
  • the above-mentioned methods are often associated with diagnostic uncertainty, which is why it is often not possible to distinguish a malignant from a benign tumor.
  • fine tissue examinations particularly in the case of tumors rich in electricity, cannot contribute to a definitive clarification.
  • the present invention is therefore based on the object of providing a new system for the therapy and diagnosis of tumor diseases, which makes it possible to control tumors for therapy or diagnosis with corresponding therapeutic agents or diagnostic agents in an efficient manner on the basis of receptor molecules specifically present on the tumor cells (drug - targeting).
  • a claudin-4 ligand for the treatment or therapy and / or diagnosis of tumors.
  • CPE C. perfringens enterotoxin
  • This model corresponds in vivo to acute enterotoxin-mediated gastroenteritis triggered by the C / os.r / d / um bacterium (McCIane, 1996). Mutation analyzes have also shown that the C-terminus of the CPE binds specifically to claudin-4, while the N-terminus triggers the cytotoxic effect.
  • a C-terminal CPE fragment was cloned which, although stably binds to claudin-4, has no cytotoxic properties (Hanna et al., 1989; US Pat. No. 5,695,956). It has also been shown that the non-cytotoxic C-terminal CPE fragment after binding to the receptor causes internalization of claudin-4, which significantly reduces the barrier function of the tight junctions (Sonoda et al., 1999).
  • the present invention is based on the finding that claudin-4 is increasingly expressed in various tumor cells in comparison to healthy tissue (cf. the attached example).
  • Tumors that show increased expression of claudin-4 are e.g. Colon cancer (colon cancer), breast cancer (breast cancer) and pancreatic cancer (pancreatic cancer).
  • WO-A-94/24155 describes the therapeutic use of another Clostridium tox n, namely Clostridium difficile toxin A.
  • Clostridium difficile Toxin A with approximately 2,700 amino acids (molecular weight approximately 308 kDa) has a completely different structure than CPE (319 amino acids), the amino acid sequences of both proteins showing no homology. Therefore, the mechanism of action of Clostridium difficile Toxin A cannot be compared with that of CPE and does not lead to binding to Claudin-4.
  • claudin-4 ligand means a compound that is capable of specifically interacting with claudin-4.
  • the specific binding of the claudin-4 ligand with the receptor can be based on any chemical or physical interactions, for example ionic interactions, hydrogen bonds, Van der Waals bonds and / or hydrophobic interactions.
  • the equilibrium constant of the association reaction between the claudin-4 ligand and the receptor is sufficiently large, which is preferably a value greater than 1x10 5 M "1 , more preferably greater than 1x10 7 M '1 .
  • a preferred Claudin-4 ligand to be used according to the invention is, for example, the above-mentioned C. perfringens enterotoxin (CPE). Due to the cytotoxic effects of C. perfringens enterotoxin, it is suitable, for example, for the targeted destruction of the CPE receptor (ie Claudin 4) expressing tumor cells. However, it is possible to use a fragment or derivative of such a toxin which preferably does not itself have a cytotoxic effect but still specifically interacts with claudin-4. Such fragments include, for example, amino acids 171 to 390, more preferably amino acids 290 to 319, of the above-mentioned CPE (cf. US Pat. No. 5,695,956). Further examples of claudin-4 ligands suitable according to the invention include antibodies directed against claudin-4 and fragments or derivatives thereof as well as the claudin-4 binding peptide compounds disclosed in WO-A-00/26360.
  • the ligand is preferably conjugated to at least one chemotherapeutic agent.
  • the therapeutic agent to be prepared can contain at least one further chemotherapeutic agent in addition to the ligand according to the invention or a conjugate defined above.
  • chemotherapeutic agent means that the substance in question either itself or after its implementation by the metabolism in the respective organism provides a means of inhibiting the growth and / or killing of tumor cells and thus also encompasses the derivatives resulting from these reactions.
  • Particularly suitable chemotherapeutic agents are e.g. Cytostatics.
  • chemotherapeutic agents with which the claudin-4 ligand can be coupled or which can be contained in the therapeutic agent produced using this claudin-4 ligand are antibiotics, anthracyclines, N-nitrosoureas, alkylating agents, antinucleosides, purine or pyrimidine antagonists, folic acid antagonists, taxanes, camptothecins, podophyllotoxin derivatives, vinca alkaloids and platinum compounds.
  • chemotherapeutic agents are selected, for example, from the group consisting of 5-fluorouracil, gemcitabine, c / s-configured platinum (II) complexes, oxaliplatin, irinothecan, mitoxantrone, mitomycin C and taxol.
  • the claudin-4 ligand preferably comprises at least one label or one diagnostic agent.
  • diagnostic agent or “marker” means a substance or a chemical group which, by means of suitable chemical and / or physical measuring methods in the organism in vivo or parts thereof in vitro, for example tissue samples, cells and / or liquids, such as for example Serum is detectable, preferably also quantifiable.
  • Preferred labels or diagnostics contain, for example, one or more radionuclides, ligands comprising one or more radionuclides, preferably complexing such radionuclides, one or more positron emitters, one or more NMR contrast agents, one or more fluorescent compounds or one or more contrast agents near IR range.
  • a tumor can be detected in a biopsy sample by detection of a suitable label, for example a fluorescent label.
  • the Claudin-4 ligand is suitable, for example, for the therapy and / or diagnosis of pancreatic carcinomas, carcinomas of the papilla Vateri, colon carcinomas, rectal carcinomas, gastric carcinomas, ovarian carcinomas and
  • the link between the claudin-4 ligand and the chemotherapeutic or diagnostic agent can either not be cleaved in the body or the link can be pH-dependent and / or enzymatically cleavable.
  • the Claudin-4 ligand in the conjugates defined above is connected to the chemotherapeutic agent or the diagnostic agent via a spacer molecule.
  • the direct link between the chemotherapeutic agent or Diagnostic agent and Claudin-4 ligand executed, the spacer molecule and / or the link between chemotherapeutic agent and the spacer molecule and / or the link between Claudin-4 ligand and the spacer molecule can either not be cleaved in the body or the spacer molecule and / the linkages mentioned Depending on the pH and / or be enzymatically cleavable.
  • spacer molecules eg maleimide, N-hydroxysuccinimide spacers etc.
  • pH-dependent cleavable eg acid-labile bonds
  • enzymatically cleavable linkages eg a peptide which contains the recognition sequence of a protease
  • the present invention further relates to a conjugate comprising a claudin-4 ligand defined above and at least one chemotherapeutic agent and / or at least one non-radioactive diagnostic agent.
  • the conjugate according to the invention preferably contains the chemotherapeutic agents listed above.
  • Preferred non-radioactive diagnostics are selected, for example, from the group consisting of positron emitters, NMR contrast media, fluorescent compounds and contrast media in the near IR range.
  • a pharmaceutical composition which contains the conjugate defined above and optionally at least one pharmaceutically acceptable carrier and / or an auxiliary and / or a diluent.
  • the pharmaceutical composition according to the invention is preferably used for the treatment of tumors, in particular the carcinomas mentioned above.
  • the present invention relates to a method for the treatment of the above-mentioned tumor diseases.
  • the treatment of tumors according to the invention can, for example, be systemic, locoregional or local administration of one using of the therapeutic agent, such as a pharmaceutical composition, made of claudin-4 ligand to a patient.
  • the therapeutic agent such as a pharmaceutical composition, made of claudin-4 ligand to a patient.
  • the "patient” can be, for example, an animal or a human.
  • Systemic administration can comprise an injection, for example a bolus injection, of the pharmaceutical composition containing the conjugate according to the invention, or of a self-cytotoxic toxin listed above.
  • Such pharmaceutical compositions can also be used continuously e.g. administered by infusion.
  • a claudin-4 ligand that is itself cytotoxic, such as the CPE toxin is also used in other claudin-4-expressing tissues.
  • locoregional chemotherapy using the claudin-4 ligand according to the invention for example the CPE toxin or a suitable conjugate thereof, preferably after selective catheterization of the blood vessel supplying the tumor, is applied, as a result of which the selective effect in the tumor is increased.
  • Another possibility is the local intratumoral injection of a toxin or a conjugate or a corresponding pharmaceutical composition mentioned above.
  • FIG. 1 shows the results of studies on the expression of claudin-4 in tissues and cell lines.
  • A is a photograph of a northern blot analysis showing the overexpression of claudin-4 in pancreatic cancer tissues and colon carcinomas.
  • P1 to 5 pancreatic adenocarcinoma, representing 24 of 31 pancreatic adenocarcinoma examined;
  • C1 to 5 colon adenocarcinoma, representative of 12 examined tissues;
  • PV carcinoma of the papilla Vateri;
  • G gastric adenocarcinoma, representative of two examined gastric cancer tissues,
  • B breast adenocarcinoma, representative of four examined breast cancer tissue;
  • S soft tissue sarcoma, representative of two examined sarcoma tissues;
  • CP chronic pancreatitis, representative of nine examined tissues with chronic pancreatitis;
  • NP normal pancreas, representing nine normal pancreatic tissues examined.
  • B is a photograph of a multi-tissue Northern
  • (C) is a photograph of a Northern blot analysis showing expression in various pancreatic cancer cell lines, HFL fibroblasts and Claudin-4 constitutively overexpressing S2-007 cells.
  • (D) is a photograph of Capan-1 cells immunocytochemically stained with an anti-Claudin-4 antibody.
  • FIG. 2 shows the results of investigations of the invasion and proliferation properties of Claudin-4 overexpressing S2-007 cells.
  • A is a graphical representation of the results of an invasion test with parental S2-007 cells and claudin-4 overexpressing S2-007 cells and parental S2-028 cells. The results with parental S2-007 cells also represent mock-transfected S2-007 cells.
  • B is a graphical representation of the results of a soft agar test with the parental S2-007 cells and Claudin-4 overexpressing S2-007 cells as well as parental S2-028 cells.
  • C is a graph the results of a soft agar test with the same cells as in (B), 10 days after plating.
  • FIG. 3 shows the results of studies on the cytotoxic effect of CPE on pancreatic cancer cells in vitro and in vivo.
  • A is a graphical representation of a trypan blue exclusion test demonstrating the effect of CPE on the pancreatic cancer cell lines Capan-1, Panc-1 and MiaPaca-2 as well as on the fibroblast cell line HFL (negative control).
  • B is a graphical representation of results from an 86 Rb release test showing the dose dependence of the cytotoxic effect of CPE on Panc-1 cells.
  • C is a graphical representation of the results of an 86 Rb release test regarding the time dependence of the cytotoxic effect of CPE on Panc-1 cells.
  • FIG. D is another graphical representation of an 86 Rb release test for the cytotoxic effect of CPE on constitutively TGF- ⁇ -overexpressing Panc-1 cells and mock-transfected Panc-1 cells.
  • E is a photographic representation of CPE-treated panc-1 xenografts, demonstrating the effect of CPE in developing necrosis of the xenografts. Necrotic areas appear as weakly stained areas in the center of the H&E stained tumor.
  • F shows the effect of CPE on the growth of Panc-1 cells after 1 day and after 7 days after subcutaneous injection in nude mice.
  • RNA matrix test Examination of the expression pattern of claudin-4 in a number of normal tissues was carried out using a multi-tissue RNA matrix test.
  • the matrix contained RNA from 61 different samples of the human body.
  • a certain Claudin-4 expression could be detected in the gastrointestinal tract, in the respiratory tract and in some exo- and endocrine glands. In most other tissues, for example brain, heart, skeletal muscle, blood, bone marrow and liver, no or only a low expression was found (FIG. 1B).
  • Claudin-4 In order to investigate the importance of Claudin-4 in vitro, 16 pancreatic cancer cell lines were examined for the expression of Claudin-4. Claudin-4 expression was detected in 15 of 16 cell lines examined, with Capan-1 cells being the highest and MiaPaca-2 cells the showed the lowest expression rate (FIG. 1C).
  • Suit-2 cell subclone 028 S2-028
  • S2-007 Subclone 007 (S2-007) (Iwamura et al., 1997) almost no claudin-4 expression was detectable.
  • pancreatic cancer cell lines with TGF-ß were used for 2 to 24 Incubated for hours under serum-free conditions. After prolonged incubation with TGF-ß, claudin-4 expression was significantly reduced in Panc-1 cells (FIG. 1C). In contrast, TGF-ß had no effect on claudin-4 expression in cell lines, such as Imim-PC2 cells, which are known to respond less to TGF-ß (Wenger et al., 1999).
  • Claudin-4 affects the invasion potential of pancreatic cancer cell lines
  • claudin-4 influences the invasive behavior of pancreatic cancer cells.
  • the effects of claudin-4 overexpression on the invasion potential, anchorage-independent growth and proliferation were examined. Invasion tests showed a significantly reduced invasion rate of Claudin-4 overexpressing cells compared to parental S2-007 cells and mock-transfected cells (Fig. 2A). In soft agar tests, claudin-4-transfected cells showed colony formation which was significantly reduced in both number and size in comparison to parental or mock-transfected cells (FIG. 2B, C).
  • Claudin-4 has been identified as a receptor for the Clostridium perfringens enterotoxin (CPE-R) (Katahira et al., 1997). CPE acts on intestinal epithelial cells by increasing membrane permeability, which leads to a loss of osmotic balance and thereby cell death (McCIane et al., 1981).
  • CPE C. perfringens enterotoxin
  • nude mice were inoculated with Claudin-4-expressing Panc-1 cells subcutaneously. Four injections of CPE were then made directly into the tumor within 5 days. The animals were sacrificed two days later and the tumor was examined histologically. Tumor size was compared by measuring tumor length and width before treatment and after killing. All tumors treated with CPE showed a large necrotic area of up to 90% of the histological section. In contrast, no or minimal necrosis was found in all histological samples of the control tumors treated with physiological saline (Fig. 3E). Furthermore, the tumors treated with CPE showed no enlargement, while the size of the control tumors increased significantly (FIG. 3F).
  • CPE is also capable in vivo of selectively destroying Claudin-4-expressing tumor cells.
  • Tissue from human patients with ductal adenocarcinoma of the pancreas, carcinoma tissue from different origins, human pancreatic tissue from organ donors and tissue with chronic pancreatitis were provided by the Department of Surgery at the University of Ulm and the Hungarian Academy of Sciences (Budapest, Hungary). All tissues were obtained after approval from the local ethics committee.
  • Panc-1 and MiaPaca-2 European Collection of Animal Cell Cultures, Salisbury, United Kingdom
  • Capan-1, Capan-2 and AsPC-1 American Type Culture Collection, Manassas, Virginia , USA
  • Patu-Il, Patu8988s and Patu8988t Universality of Marburg, Department of Cell Biology, Marburg
  • HPAF Universality of North Carolina, Durham, North Carolina, USA
  • SKPC-1, lmim-PC1 and lmim-PC2 Institudo Municipial d'lnvestigacio Medica, Barcelona, Spain
  • Suit-2 clones S2- 007 and S2-028 Moyazaki Medical College, Miyazaki, Japan
  • Panc-1 cells stably transfected with TGF-ß and mock-transfected Panc-1 cells were obtained from the Medical Clinic of the University of Rostock.
  • Human fetal lung fibroblasts (HFL) have been obtained from the European Collection of Animal Cell Cultures. All cell lines were kept in DMEM (Gibco-BRL, Düsseldorf) containing 10% fetal calf serum (FCS, Gibco-BRL).
  • Clostridium perfringens Enterotoxin A (CPE) was isolated and purified according to McDonel et al., 1988.
  • the polyclonal antibody against claudin-4 was isolated from rabbits and characterized (Furuse et al., 1999).
  • TGF-ß was purchased from R&D Systems (Minneapolis, Minnesota, USA).
  • the multi-tissue matrix with specific RNA from 61 different human samples was obtained from Clontech (Heidelberg) and hybridized according to the manufacturer's instructions using a [ 33 P] -labeled Claudin-4 probe.
  • the hybridized matrix was scanned with a phosphoimager (MolecularDynamics, Sunnyvale, California, USA) and a quantitative analysis of the results obtained was carried out using the ArrayVision software (Interfocus, Haverhill, United Kingdom).
  • RNA from cell lines was extracted using the RNAeasy kit (Qiagen, Hilden).
  • the RNA from shock-frozen pancreatic tissue was prepared according to Wenger et al., 1999. 30 ⁇ g of total RNA were fractionated in size on a membrane, as described in Gress et al., 1997, separated.
  • the hybridization was carried out with a Claudin-4-specific probe labeled with digoxigenin-11-dUTP using the Dig-labeling kit (Röche Diagnostics, Mannheim). Specific bands were detected using a chemiluminescent substrate (Röche Diagnostics).
  • Claudin-4 specific cDNA was amplified from a pancreatic cDNA pool, the sequence was verified and cloned into the mammalian expression vector pBIG2R.
  • the stable transfection in cells of the pancreatic cancer cell line S2-007 was carried out using the Lipofectin reagent (Gibco-BAL).
  • Clones expressing claudin-4 were extracted using hygromycin (300 ⁇ g / ml) and screening was carried out by Northern blot analysis as described above.
  • samples were homogenized in RIPA protein lysis buffer (0.5 g tissue / ml). The samples were then incubated for 10 minutes at 95 ° C. and 15 ⁇ l of each sample were analyzed by SDS-PAGE according to Giehl et al., 2000. The amount of protein was determined by staining the gel with Coomassie blue. The fractionated proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel) using the Semidry technique.
  • the membranes were washed for one hour at room temperature with a polyclonal rabbit antibody against claudin-4 (dilution 1: 1,000, see Furuse et al., 1999) and then for one hour with a peroxidase-coupled antiserum (Pierce, Rochester, New York, USA). Antibody detection was performed using an enhanced chemiluminescence reaction system (Pierce).
  • the incorporation of [ 3 H] -thymidine was measured in order to determine the cellular proliferation (Giehl et al., 2000).
  • the cells were plated in 24-well plates at a density of 5x10 4 cells per well and grown in DMEM containing 10% FCS (1 ml well) to a 70% confluence. After keeping the cells under nutrient deficiency conditions overnight, 1 ⁇ Ci [ 3 H] thymidine was added.
  • the test was carried out according to Ellenrieder et al., 2000, with the parent S2-007 and S2-028 cell lines as well as with two Claudin-4 overexpressing S2-007 clones and a mock-transfected S2-007 clone. There were 1.5 x 5 cells in DMEM / 1% FCS on Matrigel in 12-well plates with a Base layer of DMEM / 10% FCS plated. The Matrigel invasion was measured after 28 hours by counting the cells fixed and stained with methylene blue.
  • Capan-1 cells were maintained on four-well chamber slides (Nalge Nunc, Naperville, Illinois, USA). After the cell density reached 80% confluence, the cells were fixed in 4% paraformaldehyde. After washing, incubation with 0.2% Triton X-100 and saturation with 1% BSA, the cells were treated with a polyclonal rabbit antibody against Claudin-4 at a dilution of 1: 1,000 (Furuse et al., 1999) for 1 Hour and then incubated with Cy3-conjugated goat anti-rabbit IgG. The samples were examined with a confocal Leica TCS-4 microscope (Leica, Wetzlar).
  • the soft agar tests were carried out according to Giehl et al., 2000. 2x10 4 S2-007 and parental S2-0028 cells as well as two Claudin-4 overexpressing S2-007 clones and a mock-transfected S2-007 clone in DMEM / 0.33% Bacto agar were each on one Base layer of DMEM / 0.5% Bacto agar plated. Anchoring independent growth was determined 2 weeks later by counting the number of colonies.
  • pancreatic cancer cell lines Panc-1, Capan-1 and MiaPaca-2 and the fibroblast cell line HFL were grown to 80% confluency in DMEM containing 10% FCS. After washing and renewing the medium, CPE was added so that the final concentrations ranged from 0.03 to 4 ⁇ g / ml. Unless otherwise stated, 1 ⁇ g / ml was used as the standard concentration. After an incubation for 60 minutes at 37 ° C., floating cells were removed and stored. Adherent cells were trypsinized and collected together with the floating cells. After staining with trypan blue, the survival rate was determined by determining the number of trypan blue positive cells and the total number of cells. 86 Rb release test and pre-incubation with TGF-ß
  • the 86 Rb release test was performed according to Hanna et al., 1991.
  • Pancreatic cells were preincubated with 86 Rb (Amersham Pharmacia, Freiburg) in serum-free DMEM for 2 hours. After removing the medium containing 86 Rb and washing, CPE in various concentrations was added for 15 minutes. The medium was then suctioned off and measured in a ⁇ counter.
  • TGF-ß panc-1 cells were kept in serum-free medium for 24 hours and then preincubated with TGF-ß (10 ng / ml) for a further 20 hours under serum-free conditions before the addition of CPE.
  • Stable TGF-ß-overexpressing Panc-1 cells and mock-transfected control cells were incubated for 24 hours in serum-free medium before adding CPE.
  • NMRI nu / nu mice were grown and kept in a pathogen-free environment. Animals six to eight weeks old were used to produce xenografts. 2x10 6 Panc-1 tumor cells in 0.1 ml DMEM were injected subcutaneously into the right flank. Four weeks after the sc injection, the tumor size was determined by measuring the length and width of the tumor and 2 ⁇ g CPE, dissolved in 50 ml 0.9% NaCl, were injected intratumorally on day 1, 2, 4 and 5. On the seventh day, the mice were sacrificed, the tumor size was determined and the tumors were explanted, stored in 2% formaldehyde and stained with H&E for histological examination. Six mice were treated with CPE, six animals were used as controls, each of which was given 50 ⁇ l of 0.9% NaCl. credentials

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Abstract

La présente invention concerne l'utilisation d'un ligand de Claudine-4 pour le traitement et la thérapie et/ou le diagnostic de tumeurs, un conjugué qui contient un ligand de Claudine-4 et au moins un agent chimiothérapeutique et au moins un agent diagnostique non radioactif, ainsi qu'une composition pharmaceutique qui contient le conjugué selon l'invention.
PCT/EP2002/001017 2001-02-01 2002-01-31 Utilisation d'un ligand de claudine-4 pour la therapie et le diagnostic de tumeurs WO2002060420A2 (fr)

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DE10104551A DE10104551A1 (de) 2001-02-01 2001-02-01 Verwendung eines Claudin-4-Liganden in der Therapie und Diagnose von Tumoren
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US20120071421A1 (en) * 2004-10-14 2012-03-22 Yale University Therapy with clostridium perfringens enterotoxin to treat ovarian and uterine cancer
US8664184B2 (en) * 2004-10-14 2014-03-04 Yale University Therapy with Clostridium perfringens enterotoxin to treat ovarian and uterine cancer
US20150218644A1 (en) * 2004-10-14 2015-08-06 Yale University Therapy with clostridium perfringens enterotoxin to treat ovarian and uterine cancer
US9702010B2 (en) 2004-10-14 2017-07-11 Yale University Therapy with Clostridium perfringens enterotoxin to treat ovarian and uterine cancer
EP1957676A2 (fr) * 2005-12-09 2008-08-20 The Board Of Trustees Of The University Of Arkansas Activites antineoplasiques de l'ellipticine et ses derives
EP1957676A4 (fr) * 2005-12-09 2009-08-05 Univ Arkansas Activites antineoplasiques de l'ellipticine et ses derives

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