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WO2006018654A1 - Régulation de la prolifération et de la mort de cellules - Google Patents

Régulation de la prolifération et de la mort de cellules Download PDF

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Publication number
WO2006018654A1
WO2006018654A1 PCT/GB2005/003247 GB2005003247W WO2006018654A1 WO 2006018654 A1 WO2006018654 A1 WO 2006018654A1 GB 2005003247 W GB2005003247 W GB 2005003247W WO 2006018654 A1 WO2006018654 A1 WO 2006018654A1
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hecth9
inhibitor
cells
antibody
polypeptide
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PCT/GB2005/003247
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English (en)
Inventor
Kristian Helin
Federica Marinoni
Emanuela Grassilli
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Cancer Research Technology Limited
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Priority claimed from GB0418630A external-priority patent/GB0418630D0/en
Application filed by Cancer Research Technology Limited filed Critical Cancer Research Technology Limited
Publication of WO2006018654A1 publication Critical patent/WO2006018654A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • the invention relates to proteins involved in cell proliferation and apoptosis, particularly proliferation and apoptosis of tumour cells.
  • Materials and methods for producing such proteins are provided, as are assays for identifying inhibitors of the protein and methods for producing such inhibitors .
  • cyclins and cdks cyclin-dependent kinases
  • the cyclins and cdks act at various points throughout the cell cycle to permit the cell to move through the cell cycle at the appropriate time.
  • Different cyclin/cdk complexes control different stages of the cell cycle, and progression through the cycle is regulated by controlled destruction of cyclins by the ubiquitin-regulated proteolysis pathway.
  • the ubiquitin pathway works by targeting proteins for destruction by conjugating them to ubiquitin, which directs the conjugated proteins to the proteasome, which breaks the protein down into short fragments.
  • Ubiquitin conjugation and deconjugation to substrate proteins is carried out by multiple proteins, which each play a specific role in the cascade. Conjugation is initiated by an activating enzyme (El) that activates the C- terminal residue of ubiquitin.
  • a carrier protein (E2) then transfers the activated ubiquitin to a ligase enzyme (E3) that ligates the activated ubiquitin to a substrate protein.
  • E2 and E3 proteins are specific for different substrates.
  • Degradation of cyclins is not the only role played by ubiquitin and proteins of the ubiquitin protein degradation pathway in the regulation of cell proliferation. They may also play a role by targeting the destruction of other proteins involved in cell cycle regulation, such as p27 or p53 (see Wong et al . , 2003) . The involvement played by such proteins is likely to be variable and wide-ranging due to the large number of potential substrates .
  • tumours which are resistant to apoptosis may fail to respond effectively to traditional chemotherapeutic drugs .
  • the invention provides materials and methods relating to proteins, which regulate cell proliferation and apoptosis.
  • the invention provides HectH9 polypeptides and nucleic acids, agonists and inhibitors thereof, and their use in assays and methods of treatment.
  • the inventors used a yeast two-hybrid screen to search for proteins, which interact with CDC6, a cell cycle regulator. CDC ⁇ is required for DNA replication and is thus essential for S phase entry.
  • the screen identified a part of HectH9, a putative E3 ubiquitin ligase.
  • the full HectH9 sequence was cloned and shown to be upregulated in a range of human cancers. Inhibition of HectH9 protein production strikingly reduced the proliferation of certain cancer cells.
  • HectH9 can enhance the activity of chemotherapeutic drugs by inducing or increasing apoptosis when administered in combination with chemotherapeutic drugs.
  • Schwarz et al report the cloning of a human homologue to rat protein UREBl corresponding to a portion of approximately 800 amino acids of AB002310. They predict the human protein to be an E3 ubiquitin ligase and named it HectH9.
  • the hect (ubiquitin ligase) domain of HectH9 was produced by in vitro translation and shown to interact with the ubiquitin carrier proteins UbcH5 and UbcH7.
  • WO 03/063688 discloses a HectH9 nucleotide and predicted amino acid sequence. No function is demonstrated for the predicted HectH9 protein, as the protein is not made but merely predicted from the HectH9 nucleotide sequence. Accordingly, in a first aspect, the invention provides an isolated polypeptide comprising a HectH9 sequence. Preferably, said HectH9 sequence is the amino acid sequence shown in Fig 8 or a variant thereof. In preferred embodiments, a HectH9 polypeptide of the invention has HectH9 activity as described below.
  • the invention also provides isolated polynucleotides encoding HectH9 polypeptides of the invention.
  • a preferred polynucleotide comprises the nucleotide sequence of Fig 7.
  • expression vectors and host cells comprising HectH9 polynucleotides as described herein, and a method of producing a HectH9 polypeptide comprising culturing said host cells and recovering the polypeptide from the host cell culture.
  • Another aspect of the invention provides antibodies raised against a HectH9 polypeptide of the invention or fragment thereof.
  • Pharmaceutical compositions comprising an inhibitor of HectH9 activity, an anti-HectH9 antibody, a HectH9 polynucleotide or a HectH9 polypeptide in combination with a pharmaceutically acceptable excipient are also provided.
  • the invention provides in vitro methods of identifying an agonist or inhibitor of HectH9 activity.
  • the method comprises determining the efficiency of ubiquitinylation of a substrate by a HectH9 polypeptide in the presence and absence of a candidate agonist or inhibitor.
  • the method comprises determining the efficiency of thioester bond formation between ubiquitin and the Hect domain of a HectH9 polypeptide in the presence and absence of a candidate agonist or inhibitor.
  • the method further comprises the step of administering the inhibitor to cells in culture and determining the effect of the inhibitor on cell proliferation or apoptosis.
  • the Hect H9 inhibitor may be administered in combination with a chemotherapeutic drug or other agent which induces apoptosis.
  • the cells are tumour cells. They may be primary tumour cells or tumour cells lines such as MCFlOA, T47D, MDAMB468, SKBR3, BT20, MCF7, BT549, W138, Tig3, MRC5, IMR90, A549, CALU, SW480, ht29, DLDl, HCT116 or HeLa.
  • Cell proliferation may be assayed by, for example, inspection of treated cultures, FACS analysis or measurement of BrdU incorporation in comparison with control (untreated) cultures.
  • Apoptosis may be assayed by, for example, microscopic inspection of treated cultured, FACS analysis, or measurement of caspase cleavage .
  • HectH9 polypeptide in the identification of an agonist or inhibitor of HectH9.
  • the invention further provides a method, which includes, following identification of an agonist or inhibitor of HectH9 activity as described above, formulation of the inhibitor into a composition including at least one additional component.
  • the invention provides for the use of an inhibitor of HectH9 in the manufacture of a medicament for treating a hyperproliferative disease.
  • the medicament is for administration in conjunction with a chemotherapeutic drug.
  • the invention provides a method of treating a hyperproliferative disease comprising administering to a patient an effective amount of an inhibitor of HectH9.
  • the method of treatment further comprises the administration of one or more chemotherapeutic drugs in addition to the inhibitor of HectH9.
  • the invention also provides a method of inhibiting cell proliferation comprising inhibiting HectH9 production or activity in a population of cells, preferably by treating said population with an inhibitor of HectH9 activity.
  • the method comprises administration of an inhibitor of HectH9 expression or activity to a patient.
  • the method may further comprise the administration of one or more chemotherapeutic drugs.
  • the method may further comprise the administration of other anticancer agents, for example small molecule inhibitors such as bortezomib and erlotinib (VelcadeTM and TarcevaTM) and antibodies, in particular monoclonal antibodies, such as trastuzamab, cetuximab and bevacizumab (HerceptinTM, ErbituxTM and AvastinTM) .
  • Also provided is a method of inducing or accelerating cell death comprising inhibiting HectH9 production or activity in a population of cells, preferably by treating said population with an inhibitor of HectH9 production or activity.
  • the method further comprises treating the cells with one or more chemotherapeutic drugs
  • the chemotherapeutic drug is preferably administered at the same time as the inhibitor of HectH9, or otherwise concurrent with inhibition of HectH9. However, the chemotherapeutic drug may also be administered shortly before or after an inhibitor of HectH9.
  • a chemotherapeutic drug may be, for example, an alkylating agent; an anthrocytic antibacterial; a glucocorticoid; or an inhibitor of protein, DNA or RNA synthesis.
  • Preferred chemotherapeutic drugs include 5- fluorouracil and oxaliplatinum.
  • a preferred hyperproliferative disease is cancer, including sarcoma and carcinoma e.g adenocarcinoma, for example colon cancer, lung cancer, prostate cancer, liver cancer, pancreas cancer, thyroid cancer, lymphoma, glioblastoma or breast cancer.
  • sarcoma and carcinoma e.g adenocarcinoma, for example colon cancer, lung cancer, prostate cancer, liver cancer, pancreas cancer, thyroid cancer, lymphoma, glioblastoma or breast cancer.
  • the disease cells lack functional p53. Lack of functional p53 may be detected by, for example, sequencing of the p53 gene exons. Cells lacking functional p53 are resistant to many chemotherapeutic drugs . Inhibitors of HectH9 may be particularly useful in inducing apoptosis in cells which lack functional p53.
  • Inhibitors of HectH9 include small molecules, an anti- HectH9 antibody, an antisense RNA, shRNA or siRNA.
  • the invention also provides a method of detecting increased HectH9 expression comprising determining the level of HectH9 mRNA or protein in a test cell sample and comparing said level with that of a control cell sample.
  • the method is performed in vitro.
  • the test cell sample may be, for example, a polyp, adenoma or cancer cell sample, and may comprise colon, lung or breast cells.
  • HectH9 protein may be detected using, amongst others, an anti-HectH9 antibody or RT-PCR.
  • the sequence of HectH9 cDNA was predicted by using genomic sequence and EST databases by assembly of 7 fragments that partially overlap. The existence of the predicted cDNA has been confirmed by RT-PCR (right hand panel) .
  • the cDNA sequence is 13653 nt long and encodes a protein of 4374 amino acids.
  • the protein contains 3 domains: a ubiquitin associated domain UBA, a WE domain and the catalytic HECT domain (Homologous of E_ ⁇ -AP c:arboxy-t.erminal) . Specific antibodies were generated, which by Western blotting recognize one band of the expected size (482 kDa) .
  • B. The cloned cDNA encodes a protein of the expected size. Control (NT) and HA-HectH9 transfected cells were probed with an anti-HA and anti-HECT antibodies as indicated.
  • Fig. 2 HectH9 is overexpressed in human tumours.
  • ISH In situ hybridisation analysis
  • Fig. 3 HectH9 is overexpressed in a subset of human cancer cell lines.
  • Fig. 4 HectH9 downregulation leads to growth arrest and morphological changes in HeLa cells.
  • HectH9 may confer a growth advantage to cells we designed siRNA oligos to interfere for HectH9 expression.
  • Three oligos were tested in HeLa cells, which have a high exprssion of HectH9. Oligos 1 and 3 caused a complete abrogation of protein expression 48 hours after transfeetion.
  • Fig. 5 HectH9 depletion impairs the growth of cancer cells but not primary fibroblasts.
  • Fig. 6 Identification of putative interactors
  • A SDS-PAGE showing proteins immunoprecipitated by endogenous HectH9. To identify putative interactors or targets of HectH9 mass spectrometry analysis was performed on the bands indicated on the gel to identify the immunoprecipitated proteins. The proteins identified included human plectin (PLEl), involved in cytoskeleton organization.
  • B Plectin and HectH9 were immunoprecipitated from cells and probed with anti-HectH9 and plectin antibodies as shown. This shows that plectin and HectH9 interact in vivo. Lane b is a negative control, performed with beads only without adding antibody
  • C, D The UBA domain of HectH9 fused to GST was used to pull down putative interactors.
  • the domain used is UBA since it has been shown that other UBA domains are able to interact with ubiquitinated proteins. Pull down and mass spectrometry analysis let us identify other cytoskeleton proteins as putative interactors. Among them: Drebrin, a-actinin4, Myosin IC, vimentin and actin.
  • Fig 10 Caspase cleavage and Bax expression in HCT116 cells
  • the invention provides an isolated polypeptide comprising a HectH9 sequence.
  • said HectH9 sequence comprises the amino acid sequence shown in Fig 8 or a variant thereof.
  • the invention also provides HectH9 polynucleotides.
  • a HectH9 polynucleotide encodes the HectH9 amino acid sequence shown in Fig 8 or a variant thereof.
  • the HectH9 polynucleotide comprises the nucleic acid sequence shown in Fig 7.
  • HectH9 polypeptides and nucleic acids of the invention may be variants of the amino acid and nucleic acid sequences shown in Figs 7 and 8.
  • a variant HectH9 polypeptide will have at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in Fig 8 or a fragment thereof. Identity is determined as described below.
  • a variant HectH9 nucleic acid will have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleic acid sequence shown in Fig 7 or a fragment thereof.
  • a HectH9 polypeptide of the invention will have HectH9 activity.
  • the HectH9 activity may be one or more of ubiquitin ligase activity; ability to promote cell proliferation (in particular in one or more of HeLa, T47D and MDA-MB468 cells); ability to induce cellular morphological changes (in particular in one or more of HeLa, T47D and MDA-MB468 cells) ; and ability to inhibit apoptosis induced by chemotherapeutic drugs.
  • the HectH9 polypeptide will have all these activities, or at least ubiquitin ligase activity and ability to promote cell proliferation and/or ability to promote proliferation in one or more of HeLa, T47D and MDA-MB468 cells.
  • a HectH9 polypeptide may correspond to the full-length amino acid sequence of Fig 8, or a variant thereof as defined above, or may be a fragment of said Fig 8 sequence or variant. Such a fragment may have, for example, about or greater than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000 or 4000 amino acids.
  • a HectH9 polypeptide comprises amino acids 1318-1354 and/or 1612-1692 and/or 4003-4374 of the sequence shown in Fig 8.
  • the HectH9 polypeptide amino acid at the position corresponding to position 249 of the amino acid sequence shown in Fig 8 is not threonine.
  • a HectH9 polynucleotide may correspond to the full-length amino acid sequence of Fig I 1 or a variant thereof as defined above, or may be a fragment of said Fig 7 sequence or variant. Such a fragment may have, for example, about or greater than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 9500, 9600, 9700, 9800, 9900, 10000, 11000, 12000, 13000 or 14000 base pairs.
  • a HectH9 polypeptide as described herein may be expressed as part of a fusion protein, for example with an epitope tag at the C- or N-terminus of the expressed HectH9 polypeptide.
  • Suitable epitope tags include a Glu-Glu epitope tag, a His tag, a Flag tag, a HA tag or a GST tag.
  • the epitope tag may be any peptide sequence, which enables the fusion protein to be purified using a ligand that binds to the tag.
  • the ligand may be, for example, a chelated nickel ion or an antibody directed to the tag sequence.
  • a HectH9 polynucleotide or polypeptide of the invention is isolated.
  • ⁇ Isolated' as used herein, indicated that the polypeptide or polynucleotide is isolated from at least one component of its natural environment.
  • an isolated polypeptide or polynucleotide may include a recombinant HectH9 gene or its product expressed within a host cell.
  • the isolated HectH9 polynucleotide or polypeptide is substantially purified.
  • a purified HectH9 polynucleotide or polypeptide of the invention is over 50%, over 60%, over 70%, over 80%, over 90%, over 95%, over 98% or over 99% pure.
  • An inhibitor of HectH9 may be an inhibitor of HectH9 activity or an inhibitor of HectH9 protein or mRNA production.
  • the term "inhibitor” includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of HectH9, or reduces the expression of HectH9 protein or mRNA.
  • Inhibitors of HectH9 activity include small molecules, a dominant negative form of HectH9 or a neutralising antibody to HectH9.
  • a dominant negative HectH9 could be, for example, a catalytically inactive HectH9 protein (e.g wherein the cysteine in the catalytic domain is mutated to another residue) , or a HectH9 lacking or mutant in the domain required for substrate interaction.
  • the overexpression of a substrate domain will prevent HectH9 from ubiquitylating its substrate (s) .
  • Antibodies may include poly- and monoclonal antibodies, antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.
  • Inhibitors of HectH9 protein or mRNA production include ribozymes, triple helix nucleic acid, a HectH9 antisense RNA or a HectH9 siRNA or shRNA as described below.
  • Inhibitors of HectH9 may be identified by various methods.
  • a test compound is assayed for the ability to disrupt the formation of thioester bonds between the hect domain of HectH9 and ubiquitin.
  • the assay includes a HectH9 polypetide comprising the
  • Hect domain, El and E2 and ubiquitin, along with the test compound The formation of thioester bonds is detected in the presence and the absence of the test compound to determine the effect of the test compound on HectH9 activity.
  • a test compound is assayed for the ability to inhibit the ubiquitination of a substrate by HectH9.
  • the assay includes a HectH9 polypetide comprising the Hect domain and substrate binding domain, El and E2 and ubiquitin, along with the test compound.
  • the degree of ubiquitinylation is detected in the presence and the absence of the test compound to determine the effect of the test compound on HectH9 activity.
  • the HectH9 HECT domain is in-vitro translated using TNT® T7 Coupled Reticulocyte Lysate System (L4610) (Promega) .
  • Ubiquitin thioester formation is assayed using 5 ⁇ l of translation mixture incubated with 1 ⁇ g of GSTubiquitin fusion protein 5' at room temperature and detected by SDS-PAGE. Before loading on SDS/PAGE, reactions were also incubated with or without DTT (final concentration 10OmM) that destroys the thioester bond.
  • Endogenous HectH9 is immunoprecipitated as described below.
  • a putative substrate is in-vitro translated using TNT® T7 Coupled Reticulocyte Lysate System (L4610) (Promega) .
  • the immunoprecipitated HectH9 (bound to sepharose beads) is incubated with in-vitro translation mixture for 30 minutes. Subsequently, the reaction is loaded on a SDS page to assess if the putative substrate has been modified by HectH9 activity.
  • the E2, UbcH7 in case of HectH9 is not supplied.
  • the E2s present in the reaction mixture or in the cells should be sufficient to support the reaction. If they are not, UbcH7 could be supplied, as a GST fusion protein in the in vitro assays or by transfection in the in vivo assay. Accession nos for reagents which may be required are: El: gi:88969; E2 specific for HectH9 (UbcH7) :gi : 1064915
  • IP Protein immunoprecipitation
  • the samples are rotated with the beads for at least 90 minutes at 4 degrees C, them washed 3-4 times in ImI ElA buffer by centrifuging at 5000rpm for 1 minute. Beads are resuspended in 40 microlitres of SDS sample buffer for SDS -PAGE, or resuspended in buffer for the assays above.
  • the HectH9 substrate (s) may be identified by immunoprecipitation and mass spectrometry as described below.
  • Potential substrates include myc, plectin, MAP7, keratin, drebrin, myosin Ic, vimentin, actin and alpha- actinin 4 (Butkevich et al., 2004; Shirao, 1995; Shirao et al., 1994; Shirao et al . , 1988; Honda et al., 1998)) .
  • a functional assay may be performed wherein the ability of a candidate compound to inhibit the ability of HectH9 to induce cell proliferation and/or morphological changes is assayed. Detection of cell proliferation and morphological changes may be achieved using the methods described below. Small molecules and mimetics
  • HectH9 polypeptides or portions thereof can be used to screen for small molecule inhibitors or agonists of HectH9 activity.
  • Small molecules contemplated include synthetic organic or inorganic compounds, peptides and peptide-like molecules and nucleic acids.
  • "Small molecule” is defined herein to have a molecular weight below about 500 Daltons.
  • Candidate compounds identified by the assays described above may be optimised, or further compounds obtained, by designing mimetics as described below.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • ⁇ chemotherapeutic drug' indicates any drug used to treat cancer.
  • Chemotherapeutic drugs are preferably selectively destructive of cancer cells . Most act by targeting only proliferating cells, which preferentially works on cancer cells due to their high proliferation rate. For example, they may act by disrupting the cell cycle, leading to apoptosis of proliferating cells.
  • chemotherapeutic drugs are toxic to cancer cells, i.e. they induce apoptosis or necrosis, but they may also act by inhibiting cell proliferation.
  • chemotherapeutic drugs are:
  • Alkylating agents which alkylate DNA and inhibit DNA synthesis such as mustargen-nitrogen mustard, cyclophosphamide (Cytoxan, melphalan (alkeran, chlorambucil (leukeran, cis-platinum ("non-classical” alkylating agent, carbo-platinum (“non-classical alkylating agent, carmustine (BCNU) , thiotepa and busulfan (myleran) .
  • Vinca alkaloids and related substances such as vincristine, vinblastine and plant alkaloids such as VP- 16 (etoposide) .
  • Anthracycline antibiotics such as doxorubicin
  • Glucocorticoids such as prednisone/prednisolone and triamcinolone (vetalog)
  • Inhibitors of protein/DNA/RNA synthesis such as methotrexate, ⁇ -thioguanine, 5-fluorouracil (5-FU) cytosine arabinoside (ara-C, cytosar) , L-asparaginase (Elspar) , dacarbazine (DTIC), hydroxyurea (hydrea) and procarbazine (matulane) .
  • Miscellaneous agents such as paclitaxel .
  • bortezomib VelcadeTM
  • proteasome inhibitor a proteasome inhibitor
  • erlotinib TarcevaTM
  • Other such agents include antibodies, in particular monoclonal antibodies such as trastuzamab
  • HerceptinTM an anti-Her2 antibody
  • cetuximab an anti-EGFR antibody
  • AvastinTM an anti-VEGF antibody.
  • nucleic acid sequences that are complementary in sequence to a coding sequence of a gene can inhibit production of the protein product from the gene. It is not known exactly how this occurs, but it is thought that the antisense nucleic acid sequences hybridise to cellular mRNA, forming a double stranded molecule. The cell does not translate the mRNA in this double-stranded form, so translation is inhibited. Antisense nucleic acids may have other effects, including inhibition of transcription and splicing inhibition.
  • 'antisense 1 nucleic acid indicates a nucleic acid sequence, which is sufficiently complementary to the RNA molecule for which the antisense nucleic acid is specific to cause molecular hybridisation between the antisense nucleic acid and the mRNA such that translation of the mRNA is inhibited. Such hybridisation must occur under in vivo conditions, that is, inside the cell.
  • Oligomers of about fifteen nucleotides or greater and molecules that hybridise to the AUG initiation codon are particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into cells.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage.
  • RNA target Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469- 471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997) .
  • Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides .
  • the base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex.
  • RNA interference e.g., PCT publication No. WO 97/33551.
  • RNA interference is a process of sequence- specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • dsRNA double-stranded RNA
  • RNAi is mediated by short double-stranded RNA molecules (small interfering RNAs or siRNAs) .
  • siRNAs may be introduced into a cell as short RNA oligonucleotides of 10-30bp, or as longer dsRNAs which are subsequently cleaved to produce siRNAs.
  • the RNA may be introduced into the cell as RNA, or may be transcribed from a DNA or RNA vector.
  • the siRNA is between 10 and 30 bp in length, more preferably between 19 and 24bp, most preperably 20, 21, 22 or 23 bp in length.
  • the siRNA has the sequence AAGTACAGGCCATGCAGAGC or AAGGGCAAAATGCAGAGCAGG.
  • the siRNA has an overhang at one or both ends of one or more deoxythymidine bases, preferably 1, 2 or 3 thymidine bases. The overhang is not to be interpreted as part of the siRNA sequence. Where present, it serves to increase the stability of the siRNA within cells by reducing its susceptibility to degradation by nucleases .
  • shRNAs are more stable than synthetic siRNAs.
  • a shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target.
  • the shRNA is then processed into an siRNA which degrades the target gene mRNA and suppresses expression.
  • shRNAs can be produced within a cell by transfecting the cell with a DNA construct encoding the shRNA sequence under control of a RNA polymerase III promoter, such as the human Hl or 7SK promoter.
  • the shRNA may be synthesised exogenously and introduced directly into the cell.
  • the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length.
  • the stem of the hairpin is preferably between 19 and 30 base pairs in length.
  • the stem may contain G-U pairings to stabilise the hairpin structure.
  • SiRNA and shRNA molecules may be synthesized using standard solid or solution phase synthesis techniques which are known in the art.
  • Linkages between nucleotides may be phosphodiester bonds or alternatives, for example, linking groups of the formula P(O)S, (thioate) ; P(S)S, (dithioate) ; P (O) NR 1 2; P(O)R 1 ; P(O)OR6; CO; or CONR 1 2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O- or-S-.
  • Modified nucleotide bases can be used in addition to the naturally occurring bases, and may confer advantageous properties on siRNA molecules containing them.
  • modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing.
  • the provision of modified bases may also provide siRNA molecules which are more, or less, stable than unmodified siRNA.
  • modified nucleotide base' encompasses nucleotides with a covalently modified base and/or sugar.
  • modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3 'position and other than a phosphate group at the 5 'position.
  • modified nucleotides may also include 2 ' substituted sugars such as 2 ' -0-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2 ' -halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose .
  • 2 ' substituted sugars such as 2 ' -0-methyl- ; 2-0- alkyl ; 2-0-allyl ; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro- ; 2 ' -halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars
  • Modified nucleotides include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles . These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4,N4-ethanocytosine, 8-hydroxy-N6- methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1- methyladenine, 1- methylpseudouracil, 1-methylguanine, 2, 2-dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7-
  • LNA-related and PNA-related molecule may also be used.
  • PNA Peptide Nucleic Acid
  • PNA is an analogue of DNA in which the backbone is a pseudopeptide rather than a sugar. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The neutral backbone of PNA results in stronger binding and greater specificity than normally achieved.
  • LNA locked nucleic acid
  • LNA is a high-affinity nucleic acid analogue.
  • LNA is a bicyclic nucleic acid where a ribonucleoside is linked between the 2 '-oxygen and the 4 ' -carbon atoms with a methylene unit. Oligonucleotides containing LNA nucleotides are very stable when bound to complementary DNA and RNA. They also have improved mismatch discrimination.
  • siRNA molecules, shRNA molecules or longer dsRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector as described below.
  • a siRNA for inhibiting production of HectH9 may be provided, for example, in the form of synthesised short dsRNA molecules, in the form of longer dsRNA molecules which are processed within the cell, or in the form of a vector encoding said RNA.
  • Antibodies may be raised against HectH9 polypeptides described herein. Antibodies may be monoclonal or polyclonal and may be produced by a variety of methods known in the art. Antibodies may be raised against a full-length HectH9 protein or a fragment thereof. A fragment may comprise, for example, a Hect domain, a UBA domain or a WE domain as defined herein. Alternatively, short polypeptide fragments of 10-50 amino acids may be used to raise antibodies.
  • Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the HectH9 polypeptide.
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • an antibody specific for HectH9 may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the nanoparticles, or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • ⁇ monoclonal antibody' refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e. the individual antibodies comprising the population are identical apart from possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies can be produced by the method first described by Kohler and Milstein, Nature, 256:495, 1975 or may be made by recombinant methods, see Cabilly et al, US Patent No. 4,816,567, or Mage and Lamoyi in Monoclonal Antibody Production Techniques and Applications, pages 79-97, Marcel Dekker Inc, New York, 1987.
  • a mouse or other appropriate host animal is immunised with the antigen by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the HectH9 polypeptide used for immunisation.
  • lymphocytes may be immunised in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell, see Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986) .
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium.
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the HectH9 polypeptide.
  • the binding specificity is determined by enzyme-linked immunoabsorbance assay (ELISA) .
  • ELISA enzyme-linked immunoabsorbance assay
  • the monoclonal antibodies of the invention are those that specifically bind to a HectH9 polypeptide.
  • the monoclonal antibody will have an affinity which is greater than micromolar or greater affinity (i.e. an affinity greater than 10 ⁇ 6 mol) as determined, for example, by Scatchard analysis, see Munson & Pollard, Anal. Biochem., 107:220, 1980.
  • hybridoma cells After hybridoma cells are identified that produce antibodies of the desired specificity and affinity, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include Dulbecco's Modified Eagle's Medium or RPM1-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumours in an animal .
  • Nucleic acid encoding the monoclonal antibodies of the invention is readily isolated and sequenced using procedures well known in the art, e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies.
  • the hybridoma cells of the invention are a preferred source of nucleic acid encoding the antibodies or fragments thereof. Once isolated, the nucleic acid is ligated into expression or cloning vectors, which are then transfected into host cells, which can be cultured so that the monoclonal antibodies are produced in the recombinant host cell culture.
  • Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” should be construed as covering any binding substance having a binding domain with the required specificity. Thus, the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope, here a HectH9 polypeptide.
  • antibody fragments capable of binding an antigen or other binding partner
  • Fab fragments consisting of the VL, VH, Cl and CHl domains
  • Fd fragment consisting of the VH and CHl domains
  • Fv fragment consisting of the VL and VH domains of a single arm of an antibody
  • dAb fragment which consists of a VH domain
  • isolated CDR regions and F(ab')2 fragments a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region.
  • Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies, humanised antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2 188 638 A or EP 0 239 400 A. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.
  • Nucleic acid may of be double- or single-stranded, cDNA or genomic DNA, or RNA.
  • the nucleic acid may be wholly or partially synthetic, depending on design.
  • the skilled person will understand that where the nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.
  • the present invention also encompasses the expression product of any of the nucleic acid sequences disclosed and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions in suitable host cells.
  • GAP Genetics Computer Group, Madison, WI
  • GAP GAP binding protein
  • Particular amino acid sequence variants may differ from a known HectH9 polypeptide sequence as described herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20, 20-30, 30-50, or more than 50 amino acids .
  • Sequence comparison may be made over the full-length of the relevant sequence described herein, or may more preferably be over a contiguous sequence of about or greater than 20, 25, 30, 33, 40, 50, 70, 120, 150, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000 or more nucleotide triplets, compared with the relevant nucleotide sequence, or over a contiguous sequence of about or greater than 20, 25, 30, 33, 40, 50, 70, 120, 150, 200, 500, 1000, 2000, 3000, 4000 or more amino acids, compared with the relevant amino acid sequence.
  • a nucleic acid of the invention may hybridise with the nucleic acid sequence shown in Fig 7 under stringent conditions, or may have a complement which hybridises to the nucleic acid sequence shown in Fig 7 under stringent conditions.
  • Suitable conditions include, e.g. for sequences that are about 80-90% identical, hybridisation overnight at 42°C in 0.25M Na 2 HPO 4 , pH 7.2, 6.5% SDS, 10% dextran sulphate and a final wash at 55- C in 0.1 X SSC, 0.1% SDS.
  • suitable conditions include hybridisation overnight at 65°C in 0.25M Na 2 HPO 4 , pH 7.2, 6.5% SDS, 10% dextran sulphate and a final wash at 60- C in 0. IX SSC, 0.1% SDS.
  • a nucleic acid encodes a polypeptide with HectH9 activity, as described above.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Suitable vector systems include vectors based on the Moloney murine leukaemia virus (Ram, Z et al., Cancer Research (1993) 53; 83-88; Dalton and Triesman, Cell (1992) 68; 597-612. These vectors contain the murine leukaemia virus (MLV) enhancer cloned upstream at a ⁇ - globin minimal promoter. The ⁇ -globin 5' untranslated region up to the initiation ATG is supplied to direct efficient translation of the cloned protein. The initiator ATG straddles an Ncol restriction site and thus can be used to clone a protein coding sequence into the vector.
  • MLV murine leukaemia virus
  • This vector further contains a polylinker to facilitate cloning, followed by the ⁇ -globin 5' untranslated region and polyadenylation sites.
  • the MLV enhancer is of particular use since it is a strong enhancer and is active in most murine and human cells.
  • Suitable viral vectors further include those which are based upon a baculovirus .
  • Recombinant baculovirus may be produced using a transposon-based system described by Luckow (Luckow, V. A, et al . , J Virol 67:4566-79, 1993) .
  • This system which uses transfer vectors, is sold in the Bac-to-Bac®. kit (Life Technologies, Rockville, Md.) .
  • the transfer vector, pFastBacl (Life Technologies) contains a Tn7 transposon to move the DNA encoding the HectH9 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid".
  • the pFastBacl transfer vector has a AcNPV polyhedrin promoter to drive the expression of the HectH9 gene.
  • pFastBacl can be modified, for example by replacing the polyhedrin promoter can be removed with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection.
  • baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
  • the transfer vector containing HectH9 is transformed into E. coli, and screened for bacmids which contain an interrupted lacZ gene indicating recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g. Sf9 cells.
  • Recombinant virus that expresses HectH9 is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art.
  • viral vectors include those which are based upon a retrovirus. Such vectors are widely available in the art. Huber et al . , (Proc. Natl. Acad. Sci. USA (1991) 88, 8039) report the use of amphotropic retroviruses for the transformation of hepatoma, breast, colon or skin cells. Culver et al (Science (1992) 256/ 1550-1552) also describe the use of retroviral vectors in GDEPT. Such vectors or vectors derived from such vectors may also be used. Other retroviruses may also be used to make vectors suitable for use in the present invention. Such retroviruses include rous sarcoma virus (RSV) . The promoters from such viruses may be used in vectors in a manner analagous to that described above for MLV.
  • RSV rous sarcoma virus
  • Englehardt et al (Nature Genetics (1993)4/ 27-34) describes the use of adenovirus based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells, and such adenovirus based vectors may also be used.
  • Vectors utilising the adenovirus promoter and other control sequences may be of use in delivering a system according to the invention to cells, in particular the cells of the lung, and hence useful in treating lung tumours.
  • operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.
  • there may be elements such as 5 ' non-coding sequence between the promoter and coding sequence which is not native to either the promoter nor the coding sequence. Such sequences can be included in the vector if they do not impair the correct control of the coding sequence by the promoter.
  • Suitable promoters include viral promoters such as mammalian retrovirus or DNA virus promoters.
  • Suitable promoters include those used in vectors described above, e.g. MLV, CMV, RSV and adenovirus promoters.
  • Preferred adenovirus promoters are early gene promoters. Strong mammmalian promoters may also be suitable.
  • An example of such a promoter is the EF-l ⁇ promoter which may be obtained by reference to Mizushima and Nagata (1990) Nucl. Acids Res. 18; 5322. Variants of such promoters retaining substantially similar transcriptional activities may also be used.
  • the promoter may be a polyhedrin promoter.
  • the baculovirus basic protein promoter also known as Pcor, p6.9 or MP promoter
  • Pcor baculovirus basic protein promoter
  • MP MP promoter
  • the promoter may be heterologous to the nucleic acid sequence.
  • heterologous is used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering or recombinant means, i.e. by human intervention.
  • a regulatory sequence which is heterologous (i.e. exogenous or foreign) to a coding sequence is not associated with that coding sequence in nature i.e. it does not direct the expression of the coding sequence in natural systems .
  • Host cells may be any cell capable of reproducing exogenous DNA contained therein. Preferable, the host cell will also express protein from exogenous protein- coding DNA. Host cells include bacterial, mammalian and insect cells.
  • Insect cells may be used where a baculovirus expression system is used.
  • the recombinant virus is used to infect host cells, typically a cell line derived from Spodoptera frugiperda (Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C, 1994) .
  • Another suitable cell line is the High FiveO® cell line (Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435) .
  • Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 II (Life Technologies) or ESF 921 (Expression Systems) for Sf9 cells, and Express FiveO® (Life Technologies) for the T.
  • the cells are grown up from an inoculation density of approximately 2-5.times .10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Standard methods for purifying proteins are described in, for example, Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3 rd edn; Brent et al. (2003) Current Protocols in Molecular Biology, Wiley; Ausubel, et al . (2002) Short Protocols in Molecular Biology, Current Protocols, 5 th edn, New York.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others.
  • treatment includes any measure taken by the physician to alleviate the effect of the tumour on a patient.
  • effective treatment will also include any measures capable of achieving partial remission of the tumour as well as a slowing down in the rate of growth of a tumour including metastases. Such measures can be effective in prolonging and/or enhancing the quality of life and relieving the symptoms of the disease .
  • compositions according to the present invention may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous .
  • Oligonucleotides, proteins or other therapeutic molecules may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, liposomes or lipid formulations and the like in addition to the oligonucleotide.
  • Pharmaceutical compositions may also include one or more active ingredients such as interferons, antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, liposomes, diluents and other suitable additives.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, a fusion protein of the invention optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like.
  • the composition to be administered may also contain auxiliary substances such as pH buffering agents and the like.
  • auxiliary substances such as pH buffering agents and the like.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration.
  • composition as described herein is to be administered to an individual, administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • Administration may be, for example, daily, weekly or monthly.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. It will also depend upon toxicity of the therapeutic agent, as determined by pre-clinical and clinical trials. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .
  • Dosing is dependent on severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a reduction in disease state is achieved. Optimal dosing schedules are easily calculated from measurements of drug accumulation in the body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Therapeutically or prophylactically effective amounts (dosages) may vary depending on the relative potency of individual compositions, and can generally be routinely calculated based on molecular weight and EC50s in in vitro and/or animal studies.
  • a dose in mg/kg is routinely calculated.
  • dosage is from 0.001 ⁇ g to 100 g and may be administered once or several times daily, weekly, monthly or yearly, or even every 2 to 20 years.
  • a yeast two-hybrid screening was performed to identify novel potential regulator of hCDC ⁇ , a protein essential for DNA replication.
  • the screening identified of part of KIAA0312, also called URE-Bl, (Gu et al. , 1995; Gu et al., 1997; Gu et al . , 1994) corresponding to the HECT- domain of a putative E3 ubiquitin ligase, HectH9 (Sylvia E. Schwarz, 1998) .
  • the predicted gene maps on chromosome X in a contig NT_011630 and it is split in 82 exons giving rise to a transcript of 13653 bases.
  • the putative ATG start codon is at position 403 and an in- frame stop codon is located 30 bp upstream, making this ATG the translation start site.
  • the 3' untranslated region (3'UTR) of 126 bp ends in poly (A) tail.
  • HectH9 ORF encodes a protein of 4374 amino acids with an expected molecular mass of 482 kDa (Fig IA) .
  • Three distinct domains can be distinguished: an ubiquitin- a_ssociated domain (UBA) (Bertolaet et al., 2001; Chen et • al., 2001), a WE domain (Aravind, 2001) and a Homologous to E6-AP Carboxy Terminal (HECT) domain (Gu et al., 1997; Huang et al. , 1999; Huibregtse et al. , 1995; Schwarz et al., 1998) .
  • the full-length cDNA was cloned in a HA-tagged eukaryotic expression vector. Carboxy-terminal and amino-terminal truncation mutants were also generated. Furthermore, two sets of specific antibodies were generated. A rabbit polyclonal (Rl) and a mouse monoclonal (M2) recognizing the carboxy-terminal region (HECT domain) were obtained, as well as a mouse monoclonal (Ml) raised against the central region of the protein (Fig. IA) . All the antibodies recognize a single band corresponding to the expected molecular weight of the endogenous protein. Furthermore the band disappears after interfering specifically for HectH9 RNA using double stranded RNA interfering oligonucleotides (siRNA oligos) (see below) .
  • siRNA oligos double stranded RNA interfering oligonucleotides
  • HA-HectH9 and HA-delta2473 mutant were overexpressed in U2OS cells, 48 hours after transfection cells were collected and analysed by western blot. Filters were probed with anti-HA antibody first and then with Ml monoclonal antibody. The size of overexpressed HectH9 corresponds to the size of the endogenous protein (Fig. IB, compare panel a lane 2 to panel b lanel) .
  • HectH9 was primarily localized in the cytoplasm.
  • the cytoplasmic fraction was subfractionated to investigate if the protein was associated with a specific organelle, but HectH9 always localized in the soluble fraction (data not shown) .
  • HectH9 mRNA is highly expressed in a subset of human cancer
  • ISH mRNA in situ hybridisation
  • HectH9 staining was seen in a subset of cancers: in particular, in 43% (67/153) of colon carcinomas, 49% (17/35) of lung adeno-carcinomas and in 46% (25/54) of breast carcinomas analysed (Fig2A) . Therefore we consider HectH ⁇ in these tumours to be
  • HectH9 is overxpressed in human cancer
  • the copy number of HectH ⁇ in primary tumours was evaluated.
  • a BAC clone containing HectH9 located on chromosome XpIl.22 was isolated.
  • the fluorescent-labeled BAC clone was used for FISH analysis on tissue microarrays together with a probe specific for the centromeric region of chromosome X.
  • one (in male samples) or two copies of both the centromeric region of chromosome X and HectH ⁇ locus was observed (not shown) .
  • HectH9 is overexpressed in cancer cell lines both at the mRNA and protein level .
  • Fig.3A HectH ⁇ is expressed in all cell lines analysed but the level of expression varied significantly from one sample to another. Comparison of HectH ⁇ expression in cancer and primary cells shows that a subset of the analysed cancer cells have an increased amount of HectH9 (>3 fold) .
  • cell lysates from the same cell lines were used for Western blot (WB) analysis. As shown in Figure 3B, HectH9 was highly expressed when the corresponding mRNA was high, in particular in some breast cancer cells (T47D, MDAMB468 and MCF7) .
  • HectH9 is required for proliferation of a subset of tumours
  • RNA interfering oligonucleotides were designed (Tuschl, 2001; Tuschl, 2002) . As shown in Figure 4A three different oligos were tested but only two of them (oligol and 3) significantly- inhibited HectH9 synthesis in HeLa cancer cell.
  • HectH9 is involved in cytoskeleton organization
  • HCT116 and HCT116p53KO colon carcinoma cell lines (Bunz et al, 1998, Science 282, 1497-1501) were grown in DMEM medium containing 10% fetal bovine serum
  • HCT116 and HCTll6p53 negative were infected with a retrovirus expressing a short hairpin RNA directed against HectH9 or control empty virus and selected with l ⁇ g/ml puromycin. Bulk populations of infected cells were obtained 4 days later and HectH9 levels were evaluated by Western blotting (Fig 9A) .
  • Colony assays were set up in parallel to assess the long- term effect of HectH9 depletion on drug response. Briefly, after 12 hours 5FU treatment cells were harvested, counted and reseeded in triplicates in 6 well plates (cnts: 1000 cells/well; 5FU: 5000 cells/well) . After 12 days cells were fixed and stained with crystal violet to evaluate colony outgrowth. As shown in Fig 9C, HectH9 depletion in the drug-resistant HCTll ⁇ p53 negative cell line dramatically reduces the number of colonies grown after 5FU exposure (compare HCT116p53 negative and HCT116p53 negative HH9i-labelled plates) .
  • Apoptosis was confirmed microscopic inspection and by Western blotting of cell extracts and probing with an anti-caspase 3 antibody to detect caspase-3 cleavage. Upregulation of Bax protein expression when HectH9 expression was inhibited was also detected by Western blotting (see Fig 10) The same experiments were performed on a different colon carcinoma cell line, the adenocarcinoma cell line SW480. Similar results were seen in the short term (Fig HA) and long term (Fig 11B) survival assays. Knockdown of HectH9 induced cell death and markedly increased the level of apoptosis occurring on treatment with 5-FU.
  • HCT116 cells were also treated with the chemotherapeutic drug oxaliplatinum to determine the effect of HectH9 knockdown.
  • HectH9 increased the level of apoptosis occurring on oxaliplatinum treatment in both p53 positive and p53 negative cells. This demonstrates that knockdown of HectH9 can increase cell death in combination with chemotherapeutic drugs with different modes of action.
  • Knockdown of HectH9 had similar results when treating HCT116 cells with oxaliplatinum. Cells were treated with 25 ⁇ M or 50 ⁇ M oxaliplatinum.
  • HectH9 cDNA was obtained by the assembly of KIAA0312,
  • the cDNA was cloned in a pCMV HA-tagged expression vector.
  • the region of HectH9 encoding the UBA domain was amplified by PCR and cloned in pGEX4T .
  • a polyclonal rabbit (Ab3) antibody was generated against the HECT domain region.
  • monoclonal antibody 1 abl
  • the region from nt 7591 to nt 7790 was cloned in pGEX4T.
  • Transfection and immunoblotting 1 ug of pCMVGFP and 19 ug of pCMVHa empty vector, pCMVHAHectH9 or pCMVHA-delta2473HectH9 were transfected in 10 cm plates using the calcium phosphate method. 48 hours after transfection cells were lysated in ElA buffer, (25OmM NaCl, 5OmM Hepes pH7.5, 0.1% NP40, 5mM EDTA plus protease inhibitors), cell lysates were incubated 30 minutes on ice, sonicated twice for 30 seconds and spun at 14000 rpm in a refrigerated centrifuge. Supernatants were collected and equal amount of protein were analysed by Western blot.
  • ElA buffer 25OmM NaCl, 5OmM Hepes pH7.5, 0.1% NP40, 5mM EDTA plus protease inhibitors
  • Indicated cell lines were grown in DMEM medium supplemented with 10% South America serum. Cells were washed twice with IX PBS and incubated in hypotonic buffer (2OmM K-Hepes pH7, 5mM K-acetate 0.5 mM MgC12, 0.5mM DTT) for 10 minutes. Cells were collected in 500 ⁇ l of the same buffer and broken using a Dounce homogenizer. Cells were then spin at 4000 rpm for 5 minutes to separate the nuclear pellet from cytoplasm.
  • hypotonic buffer 2OmM K-Hepes pH7, 5mM K-acetate 0.5 mM MgC12, 0.5mM DTT
  • HeLa cells were plated on glass coverslips. After transfection with the indicated constructs cells were washed twice in IX PBS, fixed for 10 minutes in 4% paraformaldehyde and permeabilized with 0,1% Triton for 5 minutes . Cells were incubated with the indicated antibody for 1 hour, washed three times in IX PBS and incubated with a cy3-conjugated secondary antibody.
  • HectH9 mRNA expression was assessed by ISH using [35S] UTP-labeled sense and antisense riboprobes .
  • the ISH was performed as previously described.
  • the slides were lightly H&E counterstained and analysed using a darkfield condenser for the silver grains.
  • siRNA oligos targeting HectH9 mRNA were designed as indicated by Dharmacon Research. The most efficient oligos were: oligol AAGTACAGGCCATGCAGAGCTTT oligo3 AAGGGCAAAATGCAGAGCAGGTT. A non specific oligo (GL3) targeting the luciferase gene was also synthesized. Cells were transfected using Oligofectamine (Invitrogen) and samples for FACS and Western blot were taken at the indicated time points .
  • Quantitative PCR cDNA was generated by RT-PCR using the PE Applied Biosystems TaqMan Reverse Tarnscription Reagents . Reactions were determined using the SYBR Green I detection chemistry system (Applied Biosystems Foster bCity, CA) using an ABI PrismWOO Sequence Detection
  • GAPDH was used as a control for normalization.
  • the WE domain a common interaction module in protein ubiquitination and ADP ribosylation. Trends Biochem Sci 26, 273-275.
  • UREBl a tyrosine phosphorylated nuclear protein, inhibits p53 transactivation. Oncogene 11, 2175- 2178.
  • Plectin a high-molecular-weight cytoskeletal polypeptide component that copurifies with intermediate filaments of the vimentin type. Cold Spring Harb Symp Quant Biol 46 Pt 1, 475-482.

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Abstract

L’invention se rapporte à des matériaux et méthodes de production de protéines impliquées dans la prolifération et l’apoptose de cellules, en particulier de cellules tumorales. Des analyses pour identifier des inhibiteurs des protéines et des méthodes de production de ces inhibiteurs sont également prévues, ainsi que des méthodes de traitement utilisant ces inhibiteurs.
PCT/GB2005/003247 2004-08-20 2005-08-19 Régulation de la prolifération et de la mort de cellules WO2006018654A1 (fr)

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