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WO2006125645A2 - Inhibiteurs de la mmp-15 dans le traitement du cancer - Google Patents

Inhibiteurs de la mmp-15 dans le traitement du cancer Download PDF

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Publication number
WO2006125645A2
WO2006125645A2 PCT/EP2006/004997 EP2006004997W WO2006125645A2 WO 2006125645 A2 WO2006125645 A2 WO 2006125645A2 EP 2006004997 W EP2006004997 W EP 2006004997W WO 2006125645 A2 WO2006125645 A2 WO 2006125645A2
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mmp
agent
expression
antibody
cancer
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WO2006125645A3 (fr
Inventor
Axel Ullrich
Reimar Abraham
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U3 PHARMA AG
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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U3 PHARMA AG
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96486—Metalloendopeptidases (3.4.24)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to an agent that affects matrix- metalloproteinase 15 (MMP-15) activity and a method of diagnosing, preventing and/or treating a hyperproliferative disease such as precancerous or cancerous cell growth.
  • MMP-15 matrix- metalloproteinase 15
  • apoptosis has been widely recognized as a hallmark of cancer.
  • Tumors have to evolve mechanisms for escaping various apoptotic stimuli such as an attack of the immune system, lack of growth factors or detachment from the basal membrane (Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57-70).
  • Oncogenes like c-MYC, E2F1 and E1A can induce apoptosis early in tumour development and therefore, the induction of apoptosis serves as a defence mechanism of the body against oncogenic transformation.
  • successful chemotherapy of cancer patients kills tumor cells through apoptosis (Evan, G. I., and Vousden, K. H. (2001) Nature 411, 342-348).
  • MMP matrix-metalloproteinase
  • MMP's play diverse roles in cellular processes ranging from degradation of extracellular matrix to cleavage of cell surface receptors and ligands. They are well characterized for their contribution to the metastatic phenotype of cancer cells and can be inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).
  • TIMPs can also play a role in the activation of MMP's (Egeblad, M., and Werb, Z. (2002) Nat Rev Cancer 2, 161-174).
  • MMP inhibitors A lot of effort has been put in the development of MMP inhibitors and their use in cancer therapy, the rationale being that inhibition of MMP's should inhibit their pro-metastatic effect. Those inhibitors were broad band small molecule inhibitors of the catalytic activity of a number of MMP's. All of these inhibitors failed clinical trials due to lack of effect.
  • MMP- inhibitors Today it is known that MMP's actually play diverse and sometimes opposing roles in cancer development. Therefore, a reason for the failure of MMP- inhibitors might be their lack of specificity and lack of biological knowledge of which MMP's have to be targeted in cancer therapy.
  • the problem underlying the present invention was to provide agents and methods which are suitable for diagnosing, preventing and/or treating a hyperproliferative disease associated with MMP expression.
  • a first aspect relates to a method of preventing and/or treating a disorder associated with MMP-15 expression, particularly a hyperproliferative disease, comprising administering to a subject in need thereof an effective amount of an agent that affects matrix-metalloproteinase 15 (MMP-15) activity by reducing MMP-15 expression and/or interacting with the MMP-15 expression product.
  • MMP-15 matrix-metalloproteinase 15
  • the present invention illustrates the use of an agent that affects MMP-15 activity, i.e. an agent that is capable of reducing or inhibiting the activity of the matrix-metalloproteinase MMP-15/MT2-MMP for inhibition of cell proliferation and hence as a treatment for hyperprolifeative disorders.
  • the present invention provides evidence that among other mechanisms an agent that is capable of reducing or inhibiting the activity of the matrix-metalloproteinase MMP-15 modulates the apoptotic threshold in cells expressing MMP-15.
  • MMP-15 is a member of the membrane-type sub-family of matrix metalloproteinases. It has been shown to cleave several extracellular matrix proteins, confer invasive capacity through collagen and take part in the activation of MMP-2.
  • WO02/063037 describes the identification of MMP-15 as a candidate anti-apoptotic protein in cancer due to its upregulation in apoptosis resistant sub-clones of the human cervical carcinoma cell line HeIa S3.
  • MMP-15 by RNA interference mediated downregulation of MMP- 15 mRNA leads to increased cell death upon various apoptosis inducers in HeIa S3, the apoptosis-resistant sub-clones of HeIa S3 and in three more cancer cell lines derived from breast cancer (MCF-7), prostate cancer (PC-3) and lung cancer (Calu-6) as is shown in the present invention.
  • MCF-7 breast cancer
  • PC-3 prostate cancer
  • Calu-6 lung cancer
  • ectopic expression of MMP-15 in HeIa S3 leads to a reduced apoptotic rate.
  • the in vivo importance of these observations is demonstrated by an online search in microarray gene expression datasets which showed a higher expression of MMP-15 in lung adenocarcinoma compared to normal lung.
  • the present invention relates to an agent that affects the activity of a Matrix-Metalloprotease (MMP) which is selected from the group consisting of MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11 , MMP-12, MMP-13, MMP-14, MMP-16, MMP-17, MMP-18, MMP- 19, MMP-20, MMP-24, MMP-25, MMP-26, MMP-27 and MMP-28, wherein said agent reduces the MMP-15 expression and/or interacts with the MMP expression produced.
  • MMP Matrix-Metalloprotease
  • the term "activity" refers to the biological function of a M M P, which influences the phenotype of a cell, in particular but not limited to cancer phenotypes like evasion of apoptosis, self sufficiency in growth signals, cell proliferation, tissue invasion and metastasis, insensitivity to anti-growths signals and sustained angiogenesis. This activity may be for example achieved by the proteolytic function of the MMP or its ability to interact with other proteins or to trigger further downstream signalling pathways by other means.
  • reducing or inhibiting relates to direct binding of an agent to MMP15 or a nucleic acid coding thereof. By binding to MMP-15, the agent reduces or inhibits the biological function of the MMP as described above.
  • apoptotic threshold means the limit of apoptotic signals that has to be crossed in order for a cell to enter apoptosis. It relates to the fact that in particular cancer cells face a number of apoptotic signals which would drive the cells into apoptosis if the cancer cells had not developed anti- apoptotic mechanisms which balance those signals. Overexpression or uncontrolled activity of MMP-15 may be such an anti-apoptotic mechanism that inhibits cell death. Lowering the apoptotic threshold by inhibiting the anti-apoptotic mechanisms exerted by MMP-15 would tip the balance towards the apoptotic signals and ultimately kill the cancer cells.
  • antisense molecule refers to a molecule that is designed to specifically bind to RNA, resulting in the formation of a RNA-DNA or RNA- RNA hybrid and an arrest in DNA replication, reverse transcription or messenger RNA translation. Antisense molecules based on a selected sequence can specifically interfere with expression of the corresponding gene. Antisense molecules are typically generated within the cell by expression from antisense constructs that contain the antisense strand as the transcribed strand.
  • small RNA interference refers to a double-stranded RNA that dominantly silences gene expression in a sequence-specific manner by causing the corresponding endogenous RNA to be degraded.
  • siRNA triggers the degradation of mRNA that matches its sequence, thereby repressing expression of the corresponding gene.
  • siRNAs to a cell can trigger RNA intereference in mammalian cells (Elbashir, S. M., et al. NATURE 411 :494-498 (2001 )), and the invention therefore contemplates the use of siRNAs to target degradation of mRNA encoding a MMP-15 polypeptide.
  • microRNA refers to a sequence specific posttranscriptional regulator of gene expression which is characterised by inhibiting the translation of a target mRNA.
  • the term "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • carriers includes carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically acceptable carrier is an aqueous phi buffered solution or a liposome (a small vesicle composed of various types of lipids, phospholipids and/or surfactants which is useful for delivery of a drug to a mammal).
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • low molecular weight (less than about 10 residues) polypeptides proteins such as serum albumin, ge
  • pharmaceutical composition refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient (The McGraw-Hill Dictionary of Chemical Terms, Parker, S., Ed., McGraw-Hill, San Francisco (1985), incorporated herein by reference).
  • precancerous and cancerrous refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth, comprising, hyperplasia and neoplasisa, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • an anti-plastic agent or “cancer treatment” refers to a treatment that is presently known in the art as therapeutically effective in neoplastic diseases.
  • An agent that affects MMP-15 activity may lower the apoptotic threshold in cells in cells expressing MMP-15.
  • the agent that affects MMP-15 activity acts on the nucleic acid level, either on the transcription or on the gene itself. On the gene level said agent may cause a partial or complete gene inactivation, for example by gene disruption.
  • Reducing or inhibiting transcription may comprise application of antisense molecules, for example DNA or RNA molecules or RNA analogues, ribozymes, small RNA molecules capable of RNA interference (siRNA) or miRNAs.
  • precursor RNA molecules of siRNA or DNA molecules encoding the latter may be suitable. Particularly suitable are siRNA molecules comprising the nucleic acid SEQ ID NO:1 UGA CGC AGC CUA CAC CUA C.
  • Antisense molecules are typically generated within a cell by expression from antisense constructs that contain an antisense strand to the transcribed strand. Antisense production and uses thereof are discussed extensively in the literature and are widely known and available to one skilled in the art.
  • the agent that affects the activity of MMP- 15 acts on the protein level.
  • the agent may be an antibody or a fragment thereof, directed against said MMP-15.
  • the antibody of the present invention may be a monoclonal or polyclonal antibody, as well as a recombinant antibody, e.g. single chain antibody or a fragment thereof, which contains at least one antigen-binding site, an antibody fragment such as a Fab, Fab ' or F(ab ' ) 2 fragment or a recombinant fragment such as a scFv fragment and a humanized antibody or a human antibody.
  • a Fab, Fab ' or F(ab ' ) 2 fragment or a recombinant fragment such as a scFv fragment and a humanized antibody or a human antibody.
  • the application of chimeric antibodies, humanized antibodies or human antibodies is especially preferred.
  • Polyclonal antibodies can be obtained by methods well known in the art. Any animal, which is known to produce antibodies can be immunized with a MMP-15 polypeptide. Antibody containing sera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using methods as for example, ELISA or FACS.
  • Monoclonal antibodies produced by the hybridoma method are first described by Kohler et al., Nature, 256:495 (1975). Monoclonal antibodies can also be produced by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567) or may be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991), for example.
  • Humanized forms of the antibodies may be generated according to the methods known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma or recombinant cell line, which produces the above described monoclonal antibodies or binding fragments thereof.
  • the antibody is coupled either to a detectable label, as radioisotopes or enzymatic labels (biotin, avidin and the like) or to an effector, such as calicheamicin, Auristatin-PE, a
  • radioisotope or a toxic chemotherapeutic agent such as geldanamycin and maytansine.
  • these antibody conjugates are useful in targeting cells, e.g. cancer cells, expressing MMP-15 for elimination.
  • the linking of MMP-15 antibodies of the invention to radioisotopes e.g. provides advantages to tumor treatments. Unlike chemotherapy and other forms of cancer treatment, radioimmunotherapy or the administration of a radioisotope-antibody combination directly targets the cancer cells with minimal damage to surrounding normal, healthy tissue.
  • Preferred radioisotopes include g. 3 H 1 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 1, 131 I).
  • antibodies used in the invention can be conjugated with toxic chemotherapeutic drugs such as geldanamycin (Mandler et al., J. Natl. Cancer Inst, 92(19), 1549-51 (2000)) and maytansine, for example, the maytansinoid drug, DM1 (Liu et al., Proc. Natl. Acad. Sci. U.S.A. 93:8618- 8623 (1996)) and auristatin-PE.
  • DM1 Liu et al., Proc. Natl. Acad. Sci. U.S.A. 93:8618- 8623 (1996)
  • auristatin-PE auristatin-PE.
  • Different linkers that release the drugs under acidic or reducing conditions or upon exposure to specific proteases can be employed.
  • Anti-MMP-15 antibodies can be conjugated as described in the art.
  • Reducing or inhibiting of MMP-15 activity on the protein level may be also achieved by application of small molecule inhibitors.
  • a screening procedure to identify molecules affecting MMP-15 activity can comprise measuring apoptosis in response to a compound that is capable of inducing apoptosis, as for example TRAIL, in a cell line expressing MMP-15 as well as in a cell line not expressing MMP-15 in the presence of a small molecule inhibitor.
  • Molecules that increase the apoptotic rate in the MMP-15 expressing cell line only are small molecule inhibitors in accordance with the present invention.
  • a screening method to determine the apoptotic rate involves growing cells (for example in a cell culture dish) that either naturally or recombinantly express MMP-15 and cells that naturally or recombinantly do not express MMP-15, e. g. MMP-15 knockdown cells.
  • the test compound is added and the mixture is incubated for an appropriate amount of time.
  • An apoptosis inducing agent is added to the cells and the rate of apoptosis of the MMP-15 expressing cells is compared to those cells that do not express MMP-15 using methods known to those skilled in the art.
  • Molecules that increase the apoptotic rate in the MMP-15 expressing cells only are inhibitors in accordance with the present invention.
  • Still a further aspect relates to a method of identifying new agents that lower the apoptotic threshold in a cell and/or inhibit cell proliferation comprising screening for agents capable of reducing or inhibiting MMP-15 activity.
  • the present invention is suitable for diagnosing, preventing and/or treating a hyperproliferative disorder or any condition that would benefit from treatment with an agent capable of reducing or inhibiting MMP-15 activity.
  • the disorder is characterised by uncontrolled MMP-15 activation.
  • Preferred examples are gastrointestinal cancer, pancreas cancer, ovarian cancer, stomach cancer, endometrial cancer, salivary gland cancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer, bladder cancer, glioma, melanoma or other cancer associated with MMP-15 expression.
  • Most preferred examples are lung cancer, breast cancer, prostate cancer, cervical cancer and lymphoma.
  • a disorder which is caused or associated with MMP-15 expression can be identified by measuring MMP-15 expression, e.g. on the mRNA level (RT- PCR, Northern Blot, cDNA Array analysis, SAGE, in situ hybridization etc.) and/or on the protein level (Western Blot, Immunohistochemistry, Immunoflurosecence, Immunoprecipitation, FACS, Protein Array). The presence of such a disorder may also be indicated by uncontrolled MMP-15 activity.
  • the present invention relates to a method of modulating the apoptotic threshold or inhibiting cell proliferation by applying an agent that affects MMP-15 activity.
  • the method of the invention can be carried out in vitro, ex vivo or in vivo.
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one agent according to the present invention and a pharmaceutically acceptable carrier, diluent and/or adjuvants and, optionally, a container enclosing these.
  • Pharmaceutical compositions according to the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose.
  • about 1 ⁇ g/kg to 15 mg/kg of the at least one antigen binding protein according to the invention may be administered to a patient in need thereof, e. g. by one or more separate administration or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • the dose of a pharmaceutical composition according to the present invention to be administered depends on a variety of factors. These are, for example, the nature of the agent, the tumor type or the route of administration. It should be emphasized that the present invention is not limited to a certain dose or mode of administration.
  • a pharmaceutical composition according to the present invention is administered systemically, most preferably by injection into the circulation system.
  • the potency of the pharmaceutical composition of the invention is based on the binding of the at least one agent that reduces or inhibits MMP-15 activity. Preferably, this binding leads to a reduction of the MMP-15 mediated signal transduction and/or to a modulation of the apoptotic threshold.
  • the present invention relates to a method of treating a hyperproliferative disease in a subject in need thereof, comprising the steps: (i) treating said subject with an antineoplastic therapy such as chemotherapy, radiation, treatment with TRAIL, FAS-ligand, TNF- ⁇ or the use of an antineoplastic agent and
  • step ii) administering to said subject an effective amount of an agent that affects the activity of MMP-15, by reducing MMP-15 expression and/or interacting with the MMP-15 expression product.
  • the steps i) and ii) can be performed subsequential (step i) before step ii) or vice versa) as well as simultaneously.
  • a pharmaceutical composition according to the present invention is combined with one or more antineoplastic agents or a further cancer treatment.
  • Said combination for example may enhance the effect of a treatment of a disorder, such as cancer and is capable of inhibiting abnormal cell growth, migration or invasion.
  • the further antineoplastic agent or cancer treatment is selected from the group of therapeutic proteins including but not limited to antibodies or immunomodulatory proteins.
  • the further antineoplastic agent or cancer treatment is selected from the group of small molecule inhibitors or chemotherapeutic agents consisting of mitotic inhibitors, kinase inhibitors, DNA-damaging agents like DNA-alkylating agents, intercalating antibiotics or topoisomerase inhibitors, as well as anti-metabolites, growth factor inhibitors, cell cycle inhibitors, enzymes, histone deacetylase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, e. g. anti-androgens, and antiangiogenesis agents.
  • the antineoplastic agent or cancer treatment is radiation, treatment can be achieved either with an internal (brachytherapy BT) or external (external beam radiation) source.
  • the present invention relates to a method of diagnosing a disease associated with the expression of MMP-15, comprising the steps: (i) determining the expression of MMP-15 in a test sample and (ii) comparing said expression to the expression of a control sample.
  • the expression is determined in a sample comprising precancerous or cancerous cells, e.g. from a human tumor patient.
  • the sample may be derived from tissue sections, biopsy samples or body fluids.
  • the expression of MMP-15 in the sample to be tested can be compared to a normal sample, which is for example a normal tissue.
  • Expression can be determined on the gene level by methods known in the art as for example RT-PCR 1 DNA arrays, Northern Blot or other methods suitable for detecting MMP-15 DNA or RNA nucleic acids.
  • MMP-15 expression can be determined by ELISA, FACS, Immunohistochemistry, Western Blot or other immunological methods.
  • the present invention relates to a kit for diagnosing, preventing and/or treating a disorder associated with MMP-15 expression, comprising at least one agent that affects MMP-15 activity.
  • the inventive kit can comprise at least one further active agent, e.g. an antineoplastic agent as described above.
  • FIG. 1 shows the apoptotic rate of HeIa S3 and three HeIa S3 sub-clones upon cell death induction by the Fas agonistic antibody CH-11 , TRAIL and serum starvation.
  • the sub-clones were selected for resistance against CH- 11 mediated i.e. Fas-induced apoptosis as described in Patent Application WO02/063037.
  • FIG. 2 shows increased cell death on siRNA mediated MMP-15 downregulation in 2 of the 3 resistant clones upon CH-11 treatment (A) and in all clones upon TRAIL treatment (B) and serum starvation (C).
  • FIG. 2D shows the reduced mRNA level of MMP-15 with MMP-15 targeted siRNA. A correction fator was applied in order to correct to the lower MMP-15 downregulation rate in the resistant clones.
  • FIG. 3A shows decreased cell death in HeIa S3 ectopically expressing MMP- 15 upon CH-11 , TRAIL and starvation.
  • FIG. 3B shows the protein expression of MMP-15 in empty vector infected cells and MMP-15 infected cells.
  • FIG. 4A shows increased cell death upon siRNA mediated MMP-15 downregulation and TRAIL treatment in the cancer cell lines MCF-7, PC-3 and Calu-6 but not MeI Juso.
  • FIG. 4B shows the reduced mRNA level in these cell lines upon treatment with MMP-15 siRNA
  • FIG. 5 shows the increased expression of MMP-15 in a total of 254 tumor samples and 52 normal lung samples in 3 independent microarray datasets generated by Battarcharjee et al., (2001) Proc Natl Acad Sci U S A 98, 13790-13795 (A), Beer et al., (2002) Nat Med 8, 816-824 (B) and Garber et al., (2001) Proc Natl Acad Sci U S A 9S, 13784-13789 (C)
  • S3 cells were infected with empty vector pLXSN or pLXSN-MMP15-His/myc. Subsequently, cells were selected for 10 days with 0.5 mg/ml G418 and cloned. The clones were checked for MMP-15 expression by lmmunoblotting using an ⁇ -myc antibody for detection.
  • A For cell growth assays, the clones were seeded in 12 wells at 150000/well, grown for 72 hours in complete growth medium (Ham's F12 with 10% FCS), trypsinized and counted using a Neubauer hematocytometer.
  • B Cells were lysed and the lysates resolved by SDS-PAGE followed by immunoblot analysis using ⁇ -myc antibodies and ⁇ - ⁇ -actin antibodies as a loading control.
  • FIG. 7 Expression level of MMP-15. HeIa S3 and HeIa S3 clone 14 cells were grown in complete growth medium (Ham's F12 with 10% FCS) to 70% confluency. RNA was isolated and quantitative real-time PCR performed in triplicates as described in Materials and Methods. Error bars represent standard error of the mean.
  • AB916 ⁇ -MMP15 antibodies inhibit basal cell growth of HeIa S3 clone 14.
  • Cells were grown in duplicate in complete growth medium (Ham's F12 with 10% FCS) with 20 ⁇ g/ml AB916 ⁇ -MMP15 antibody or with 20 ⁇ g/ml AB9161 ⁇ -MMP15 antibody or no antibody as controls for 72 hours. Subsequently, cells were trypsinized and counted using a hematocytometer. Values represent cell numbers per well (24 well).
  • Example 1 The versatility of the present invention is illustrated by, but not limited to, the following examples.
  • Example 1 The versatility of the present invention is illustrated by, but not limited to, the following examples.
  • Cell death was measured counting the percentage of hypodiploid cells using flow cytometry as described (4). Alternatively, cell death was measured by dye exclusion determining the percentage of dead cells that took up propidium iodide. Flow cytometry was used gating for the cells that were bigger than the smallest cells (by forward scatter) of the healthy cell population, thus excluding cell debris. Propidium iodide fluorescence was measured in the FL3 channel on a FacsCalibur (Becton Dickinson) or EpicsXL (Beckman- Coulter) flow cytometer. For both assays, cell culture supernatant and trypsinized cells were pooled. Background values without cell death stimulus were subtracted for CH-11 and TRAIL induced cell death.
  • FacsCalibur Becton Dickinson
  • EpicsXL Beckman- Coulter
  • the correction factor for the siRNA experiments in HeIa S3 and the resistant clones was calculated by dividing the fold downregulation (i.e. expression level of MMP-15 with GL2 siRNA divided by expression level with MMP- 15 siRNA) of HeIa S3 by the fold downregulation of the respective clone. This yielded factors 1.5, 1.3 and 1.6 for clones 14, 36 and 58, respectively.
  • MMP-15 was amplified from HeIa S3 cDNA by PCR using primers flanking the coding region and sequenced. The protein sequence contained a GIy > Arg substitution at position 609 as published previously (5). MMP-15 was cloned into pcDNA 3.1/ myc-His (Invitrogen) and subcloned into pLXSN (Clontech) to yield myc-His tagged MMP-15.
  • RNA and cDNAarray experiments were used: total RNA was extracted from exponentially growing cells kept under normal growth conditions with FCS using guanidinium thiocyanate as described (6). Afterwards, mRNA was isolated with the help of oligo-dT cellulose and reverse transcribed using oligo-dT primers (7). Single stranded cDNA was purified with the help of the PCR purification kit from Qiagen. For quantitative real-time PCR, total RNA was isolated using the RNeasy Kit (Qiagen) and cDNA prepared using ABI Reverse Transcription Reagents with random hexamer primers according to the manufacturer's protocols. This cDNA was used without further purification in quantitative real-time PCR.
  • Real-time PCR was performed using the PCR Master Mix (ABI) and ' "Assays on Demand” (ABI) for MMP-15 and ⁇ - Actin as a loading control according to the ' supplier's instructions on an ABI Prism 7000. Expression levels were calculated according to the following formula: 1000 * 2 (Ct Actin - Ct MMP- 15). Cell lysis and immunoblotting were performed as described previously (8).
  • Fig 2D demonstrates that the MMP-15-directed siRNA works in reducing the MMP-15 mRNA level compared to control firefly luciferase-directed siRNA (GL2).
  • Fig. ZA shows that this reduction of the MMP-15 level increased the cell death rate in clones 14 and 36.
  • the marginal enhancement of cell death in HeIa S3 is statistically not significant (at P ⁇ 0.05, t-Test).
  • siRNA oligonucleotides can have off-target effects (9). Hence, usage of only one siRNA does not guarantee that the observed phenotype is truly generated by knock down of the siRNA target.
  • Fig. 3A shows that cell death induced through FAS 1 TRAIL and serum starvation could indeed be inhibited by MMP-15 overexpression.
  • MMP-15 overexpression alone is sufficient to protect from apoptosis.
  • siRNA mediated knockdown of MMP-15 enhanced cell death induced by TRAIL in all cell lines but MeI Juso where the increase in cell death rate did not achieve statistical significance (at P ⁇ 0.05, t- Test). Incidentally, this cell line also has the lowest MMP-15 expression of all studied cell lines.
  • MMP-15 can protect cells of various cancer types from cell death in vitro
  • the question whether deregulation of MMP-15 expression has a role in the promotion of actual cancer of patients is still open.
  • Previous studies showed a stronger expression of MMP-15 compared to normal tissue in a subset of primary breast cancer (11 ), pancreatic cancer (12), papillary thyroid cancer (13) and astrocytic tumors (14).
  • the sample sizes of these studies were small, which is why we decided to inspect published DNA microarray data for MMP-15 expression.
  • MMP-15 GeneBank Acc.No NM_002428
  • GST Glutathione-S-Transferase
  • MMP15Ex MMP15 extracellular domain
  • MMP15cat MMP15 catalytic domain
  • the cloning vector pSj26(mod) (Seiffert et al., 1999) is utilized that is designed for the eukaryotic expression and secretion of recombinant fusion proteins and is derived from the pCDNA3 cloning vector (Invitrogen, Groningen, The Netherlands) by inserting the complete DNA sequence coding for Schistosoma japonicum glutathione-S-transferase (GST) (Pharmacia Biotech, Freiburg, Germany) in the Xhol and Apal sites of pCDNA3.
  • GST Schistosoma japonicum glutathione-S-transferase
  • the extracellular or catalytic domain of MMP15 is PCR amplified and cloned into pSj26(mod).
  • Primer design is accomplished with the software VectorNTI (Invitrogen) according to the manufacture manual.
  • the resulting pSj26(mod)- MMP15Ex or -MMP15cat expression plasmid is transfected into 293 cells (ATCC CRL-1573) by the calcium phosphate DNA coprecipitation method.
  • Cells are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. After selection with 1 mg/ml G418 (Sigma, Deisenhofen, Germany) for two weeks, surviving clones are tested for expression and secretion of the fusion protein by Western blot analysis with antibodies against GST. High- expressing cells are used to produce MMP15Ex or MMP15cat.
  • DMEM Dulbecco's modified Eagle's medium
  • Monoclonal antibodies are raised by immunization of four- to eight-week old female Balb/c mice either with purified recombinant MMP15Ex/MMP15cat GST fusion protein as described before or with NIH3T3 cells, stably expressing MMP15 cells (NIH3T3-MMP15) which are generated by methods known in the art (e.g. Molecular Cloning, Sambrook & Russell, 3 rd Edition, Chapter 16).
  • the mice are injected intramuscularly 3 times in 14-day intervals with 50 ⁇ g protein diluted 1 :2 in ABM-2 adjuvants (PanSystems, Aidenbach, Germany).
  • the spleens are removed 4 days after the last injection for fusion with the SP210 myeloma cell line.
  • the resulting hybridomas are grown in RPMI 1640 culture medium containing 10% FCS, antibiotics, and hypoxanthine, aminopterin, and thymidine (HAT) (Sigma).
  • Culture supernatants are screened by flow cytometry on NIH3T3-MP15 cells stably expressing MMP15 which are generated by methods well known in the art (e.g. Molecular Cloning, Sambrook & Russell, 3 rd Edition, Chapter 16).
  • Positive hybridomas secreting antibodies that selectively recognize the transfectant, but not the parental NIH-3T3 cells, are cloned by limiting dilution.
  • MMP15-reactive clones are cultured in serum-free medium supplemented with 1 % Nutridoma (Roche, Germany), and antibodies are purified from supernatants using Protein G-Sepharose columns (Pharmacia Biotech, Freiburg, Germany).
  • MMP15 antibody influences cell proliferation
  • RNA was isolated using the RNeasy Kit 5 (Qiagen), and cDNA was prepared using ABI Reverse Transcription Reagents with random hexamer primers according to the manufacturer's protocols. This cDNA was used without further purification in quantitative real-time PCR.
  • Realtime PCR was performed using the PCR Master Mix (ABI) and "Assays on Demand” (ABI) for MMP-15 and P-Actin as a loading control according to the o supplier's instructions on an ABI Prism 7000. Expression levels were calculated according to the following formula: 1000* 2 (ctAcUn - ctMMp - 15 ⁇ References

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Abstract

L'invention concerne un agent qui affecte l'activité de la matrice-métalloprotéase-15 (MMP-15) et une méthode diagnostique, de prévention et/ou de traitement d'une maladie hyperproliférative de type croissance de cellules cancéreuses ou précancéreuses.
PCT/EP2006/004997 2005-05-24 2006-05-24 Inhibiteurs de la mmp-15 dans le traitement du cancer Ceased WO2006125645A2 (fr)

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US6387901B1 (en) * 1999-07-06 2002-05-14 Pfizer Inc Alkyne containing metalloproteinase inhibitors
EP1364066A2 (fr) * 2001-02-02 2003-11-26 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procede d'identification d'acides nucleiques fonctionnels
WO2003091258A1 (fr) * 2002-04-26 2003-11-06 Pfizer Products Inc. Inhibiteurs de la metalloproteinase n-substitues-heteroaryloxy-aryl-spiro-pyrimidine-2,4,6-trione

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