WO2014129895A1 - Moyen et méthode d'augmentation de la sensibilité de cancers à la radiothérapie - Google Patents

Moyen et méthode d'augmentation de la sensibilité de cancers à la radiothérapie Download PDF

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WO2014129895A1
WO2014129895A1 PCT/NL2014/050102 NL2014050102W WO2014129895A1 WO 2014129895 A1 WO2014129895 A1 WO 2014129895A1 NL 2014050102 W NL2014050102 W NL 2014050102W WO 2014129895 A1 WO2014129895 A1 WO 2014129895A1
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gene
gpr27
cell
cancer
cells
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Victor Willem Van Beusechem
Jasmina HODZIC
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Stichting Vu-Vumc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to the field of cancer, more in particular to the field of radiotherapy of cancer.
  • Radiotherapy is the most used treatment against cancer, in particular but not exclusively for prostate cancer, lung cancer, head and neck cancer, breast cancer, rectum cancer, oesophageal cancer, pancreatic cancer, cervical cancer, bladder cancer, lymphoma and brain tumors.
  • radiotherapy is combined with other treatment modalities, such as surgery and chemotherapy.
  • the biological basis for the therapeutic effects of RT is that the applied ionizing radiation (IR) causes damage to the cellular DNA. Cancer cells are generally more vulnerable to this damage than healthy cells.
  • Radiotherapy of localized PCa can cure patients from the disease. However, in 30-40% of patients cancer recurs and progresses. In this case, a next cycle of treatment with irradiation is not possible anymore. Advanced prostate cancer has a very poor prognosis,
  • IR-induced DNA damage triggers a multitude of DNA damage response (DDR) signalling pathways in cells. These can result in cell cycle arrest, DNA damage repair and cell death. Differences in the functioning of these processes in different cells or under different conditions determine the final effect of a certain dose of IR.
  • the irradiated cell could repair its damaged DNA and survive, continue to divide despite the damage, or die.
  • the cause of cancer cells surviving irradiation could be intrinsic or acquired resistance of said cancer cells to irradiation. This phenomenon is further referred to as "radioresistance”. Mechanisms underlying radioresistance in various cancers are poorly understood. At present only few genes have been described to play a role in radiation response. These include e.g.
  • ATM and DNA-PKcs (Tannock et al. The Basic Science of Oncology (4th ed.), McGraw- Hill Ltd, New York (2005), pp. 77-99).
  • ATM mutation is associated with high radiosensitivity.
  • the DNA damage more precisely DNA double-strand breaks (DSBs) induced by IR, trigger assembly of the MRN complex.
  • the MRN complex binds to DSBs and activates ATM kinase activity, while ATM on its turn phosphorylates the DNA histone variant H2AX, ⁇ - ⁇ 2 ⁇ .
  • the specific binding of ⁇ - H2AX to the damaged DNA serves as a platform for binding of various proteins, including BRCAl, 53BP1 and MDCl involved in the CDK independent DDR.
  • DNA-PKcs is involved in DNA damage repair, specifically in non-homologous end joining, NHEJ. After DSBs have been induced by IR they are recognized by the Ku70/Ku80 complex. This complex triggers binding of the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs.
  • Ku70/Ku80 and DNA-PKcs form the DNA-PK complex that promotes binding of the Lig4/XRCC4/XLF complex to the damaged DNA ends resulting in the ligation of DNA ends and their repair.
  • Cells deficient in any of the DNA-PK components are radiosensitive (Collis et al., Cancer Res. 63, 1550-1554, 2003;
  • radiosensitizer drug or “radiosensitizer” for short.
  • chemotherapeutic drugs show stronger cancer cell killing effect when combined with IR. In this sense these chemotherapeutic drugs are considered
  • Radiosensitizers include radiosensitizers.
  • radiosensitizing property are 5-FU, platinum analogs such as cisplatin and oxaliplatin, gemcitabine and temozolamide.
  • the mechanisms underlying the radiosensitization brought about by these compounds is usually poorly understood (reviewed in Katz et al., Int. J. Rad. Oncol. Biol. Phys. 73, 988-996, 2009).
  • Many clinical trials have been done combining RT with chemotherapy. Meta-analyses have shown that combination treatment is associated with modest clinical benefit and significantly increased toxicity to healthy tissue (Pignon et al., Lancet 355, 949-955, 2000; Bourhis et al, Semin. Oncol. 31, 822-826, 2004).
  • the colony formation assay is the golden standard assay commonly used to investigate radiosensitivity of cancer cells in vitro.
  • the CFA is a cell survival assay that tests the ability of a single cell to grow into a colony after treatment.
  • the radiosensitizing effect of a radiosensitizer can be described by its "radiosensitization factor" (RF), i.e., the ratio between the fraction surviving cancer cells (i.e., the surviving fraction, SF) after IR alone or IR plus an irrelevant compound or IR plus vehicle and the fraction surviving cancer cells after IR plus radiosensitizer.
  • RF radiosensitization factor
  • the RF can be different at different radiosensitizer concentration and IR dose.
  • Another way to quantify the radiosensitizing effect is by calculating the "dose modifying factor" (DMF).
  • the DMF is the ratio between the IR dose required to kill a certain fraction of cancer cells in the absence of the radiosensitizer and the IR dose required to kill the same fraction of cancer cells in the presence of the radiosensitizer.
  • a DMF of more than 1 indicates that the same fraction of cancer cells is killed with a lower IR dose.
  • the DMF can be different at different radiosensitizer concentration and fraction cancer cell survival.
  • Patients with cancer are usually treated with low dose IR (i.e. up to 4 Gy) to minimize toxicity to healthy tissue. Multiple cycles of low dose IR may be given to increase the total dose IR.
  • a radiosensitizer that exerts a radiosensitizing effect at low dose IR is particularly relevant.
  • the first type of radiosensitizer changes the properties of the irradiated cell to enhance DNA damage.
  • examples of this type of drug include molecules that increase or stabilize free radicals in the nucleus.
  • the second type of radiosensitizer influences pathways that are involved in the response of cancer cells to IR.
  • Non- limiting examples of this type of drug includes molecules that inhibit cell cycle arrest, DNA repair or cell death.
  • This second type of radiosensitizer is referred to as "biological radiosensitizer”.
  • the present invention relates in particular to biological radiosensitizers and to their targets (i.e., the cellular molecules whose function or activity is affected by the biological radiosensitizer) and to the genes encoding said targets.
  • Examples of previously identified biological radiosensitizers include e.g. inhibitors of the EGFR pathway, farnesyl transferase inhibitors, VEGF inhibitors, an ATM inhibitor and a DNA-PKcs inhibitor (reviewed in Katz et al., Int. J. Rad. Oncol. Biol. Phys. 73, 988-996, 2009). The experience with several of these compounds is discussed here.
  • the PI3K inhibitor wortmannin induced sensitivity to IR in mouse tumors (Kim et al., J. Radiat. Res. 48, 187-95, 2007).
  • RT and farnesyl transferase inhibitor FTI-277, L- 744,832 and L-778, 123 compounds resulted in significant reduction of xenograft tumor cell survival (Cohen- Jonathan et al. Radiat Res. 154, 125-32, 2000).
  • the farnesyl transferase inhibitor L-778, 123 entered a phase I clinical trial in combination with RT in HNSCC and NSCLC patients.
  • 5 out of 9 patients had a complete response to RT and L- 778, 123 and no disease progression was observed 7- 12 months after treatment.
  • the results were difficult to interpret since none of the patients had a Ras mutation (Hahn et al., 2002. Clin. Cancer Res.
  • RNA interference RNA interference
  • Cancer cells can be sensitised to IR by silencing expression of genes involved in radioresistance by means of RNAi technology.
  • Other means to accomplish this include the use of antisense RNA, RNA decoys, ribozymes or zinc finger proteins.
  • new chemical drugs can be designed to inhibit the function or activity of the proteins encoded by said genes.
  • Databases can be searched for already available drugs known to inhibit a candidate target protein. In particular drugs that are already approved for use in humans for a different purpose could relatively rapidly be developed as a new radiosensitizer.
  • structural information on candidate target proteins can be retrieved to enable structure-based new drug design.
  • the invention provides the use of a gene of table 3a, or an RNA or protein encoded by the gene as a target for identifying a pharmacologically active compound.
  • the use is preferably for identifying a pharmacological compound active in sensitizing a cancer cell for ionizing radiation.
  • the invention also provides a method for determining whether a compound is capable of sensitizing a cancer cell for ionizing radiation comprising (a)
  • step (a) determining whether a compound is capable of inhibiting the activity of a gene of table 3a or an RNA or protein encoded by the gene, said method further comprising (b) determining whether the compound identified in step (a) is capable of sensitizing a cancer cell for ionizing radiation.
  • a method for increasing the susceptibility of a cell to radiation comprising introducing into the cell and/or contacting the cell with, a compound selected from: - an antisense molecule, in particular an antisense RNA or antisense oligodeoxynucleotide, an RNAi molecule (siRNA or miRNA) or a precursor thereof or a ribozyme capable of binding under stringent hybridization conditions to a gene or an mRNA gene product of the genes selected from the group consisting of genes of table 3a;
  • a (glyco)protein a hormone or other biologically active compound capable of interacting with a gene selected from the group consisting of genes of table 3a, or with a gene product thereof, and
  • the method preferably further comprises irradiating said cell with ionizing radiation.
  • the cell is preferably irradiated with 1-6 Gy.
  • the cell is preferably a cancer cell. In a preferred embodiment the cell is a prostate cell.
  • the invention further provides a compound selected from:
  • an antisense molecule in particular an antisense RNA or antisense oligodeoxynucleotide, an RNAi molecule (siRNA or miRNA) or a precursor thereof or a ribozyme capable of binding under stringent hybridization conditions to a gene or an mRNA gene product of the genes selected from the group consisting of genes of table 3a;
  • a (glyco)protein a hormone or other biologically active compound capable of interacting with a gene selected from the group consisting of genes of table 3a or with a gene product thereof, and
  • the treatment preferably further comprises irradiating said individual or part thereof with ionizing radiation.
  • the invention further provides a compound selected from:
  • an antisense molecule in particular an antisense RNA or antisense oligodeoxynucleotide, an RNAi molecule (siRNA or miRNA) or a precursor thereof or a ribozyme capable of binding under stringent hybridization conditions to a gene or an mRNA gene product of the genes selected from the group consisting of genes of table 3a,
  • a (glyco)protein a hormone or other biologically active compound capable of interacting with a gene selected from the group consisting of genes of table 3a or with a gene product thereof, and
  • the invention further provides a method for determining the radiation sensitivity of a cancer in an individual or predicting the response of a cancer in an individual to radiation therapy, comprising detecting a gene or gene product of a gene of table 3a in the blood and/or a cancer cell of said individual.
  • FIG. 1 Irradiation dose response curves of prostate cancer cell lines PC-3 and DU-145, as measured using with two different assays, i.e. the colony formation assay (CFA; open squares) and the Acumen assay (closed triangles).
  • CFA colony formation assay
  • Acumen assay closed triangles
  • Figure 3 Confirmation screen for PC-3 cell survival with and without 4Gy IR and silencing of 45 primary hit siRNAs.
  • A Cell numbers after gene silencing only (white bars) or after gene silencing followed by IR (black bars).
  • B Relative cell survival after gene silencing and IR compared to gene silencing only.
  • FIG. 1 Target validation by colony formation assay.
  • Each panel shows the irradiation dose response curve for cells transfected with siRNA directed against a different candidate target gene as indicated (closed triangles) and the dose response curve for cells transfected with siRNA directed against an irrelevant control siRNA (open circles). Data are the mean results of two independent experiments.
  • FIG. 5 IR dose response effects on colony formation of PC-3 cells stable transduced with lentiviral vectors expressing shRNAs silencing candidate radiation susceptibility genes.
  • Cells were seeded, irradiated 0-6Gy and allowed to form colonies. Colonies were counted and survival curves were fitted using the linear quadratic equation on the average data of 2 independent experiments.
  • Most panels show the results obtained with cells transduced with a negative control lentiviral vector (scr) and two independent cell cultures transduced with two different lentiviral vectors targeting a distinct mRNA sequence on the same target gene.
  • the lower right panel shows the results obtained with cells transduced with a negative control lentiviral vector (scr) and three cell cultures transduced with different lentiviral vectors targeting a different target gene.
  • FIG. 6 Silencing GPR27 sensitizes prostate cancer cells to fractionated irradiation.
  • PC-3 and DU145 human prostate cancer cells stable transduced with a lentiviral vector expressing a short hairpin silencing GPR27 (black bars) or with a negative control lentiviral vector (white bars) were subjected to 1, 3 or 5 daily fractions of 2Gy IR. Cell survival was assessed using the CFA. Data are means +SD of three (PC-3) or two (DU145) experiments performed in duplicate.
  • FIG. 7 Rescue experiment validating GPR27-dependent radiation protection.
  • PC-3 parental cells WT; left panel
  • PC-3 cells stable expressing a GPR27 mut - coding ORF carrying silent nucleotide mutations in the binding site of siGPR27 D- 005562-02 (ORF; right panel) were transfected with siGPR27 D-005562-02 (open symbols) or non-targeting control siRNA (closed symbols).
  • Cells were irradiated 0- 6Gy and allowed to form colonies. Colonies were counted and survival curves were fitted using the linear quadratic equation on the average data of 2 independent experiments.
  • FIG. 1 Cell cycle distribution of PC-3/LV-shSCR cells and PC-3/LV-shGPR27- El l cells at 4, 16, 24 and 32 hours after 4Gy irradiation, as determined by FACS analysis. Results are the average of two independent experiments.
  • a gene or a product of a gene can be a target for identifying compounds that inhibit the expression of the gene, or that inhibit the activity of a gene product of the gene.
  • Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as e.g. ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • snRNA small nuclear RNA
  • RNAi molecules small nuclear RNA molecules (such as but not limited to miRNAs) that inhibit
  • Exon-skipping can be used to delete specific domains from a protein but it can also be used to inactivate the target gene by skipping an exon that leaves an out of frame coding region and thereby reduce the amount of functional protein being produced.
  • the biological target for the drug or compound is most commonly a protein such as an enzyme, a receptor, transporter or channel.
  • the drug or compound typically interacts with the protein.
  • the interaction can be engaging in a covalent bond. But more often the interaction is through non-covalent interactions.
  • the interaction can also be reversible covalent. -in this case, a chemical reaction occurs between the stimulus and target in which the stimulus becomes chemically bonded to the target, but the reverse reaction also readily occurs in which the bond can be broken.
  • the stimulus is permanently bound to the target when the interaction is through irreversible chemical bond formation.
  • the interaction can lead to a steric hindrance or inactivation of the catalytic site. However, more often the interaction interferes with conformational changes in the biological target thereby acting as an inhibitor (prevention of a conformational change into an active form) or activator (induction of an active conformational form.
  • biological target or drug target is frequently used in pharmaceutical research to describe the native protein in the body whose activity is modified by a drug resulting in a desirable therapeutic effect.
  • biological target is often referred to as a drug target.
  • drug targets of currently marketed drugs include:
  • G protein-coupled receptors target of 50% of drugs
  • enzymes especially protein kinases, proteases, esterases, and phosphatases
  • ion channels ligand-gated ion channels
  • voltage-gated ion channels nuclear hormone receptors
  • structural proteins such as tubulin
  • membrane transport proteins membrane transport proteins
  • Typical nucleic acid targets are (pre-)mRNA, miRNA, promoters and enhancers.
  • the gene or gene product is a gene or gene product of table 3a, more preferably of table 3b, more preferably of table 3c.
  • the gene or gene product is a gene or gene product of table 3d.
  • the gene is GPR27 or CPNE7.
  • the gene product is a gene product of the gene GPR27 or the gene CPNE7.
  • the gene is the GPR27 gene.
  • the gene product is a gene product of the GPR27 gene.
  • the gene as target is preferably a gene of table 3b, or a gene as listed here GPR27; CPNE7; RNF8; RGNEF; HTR1E; CBL; CCNB1; GJB3; KCNQ3; OR2F1; RPL3L; FAM174B; ITPKA; DGAT2; SLC22A10; PDILT; HOXD3; IFNA7; MGC2705;
  • the gene product as target is preferably a gene product of a gene of table 3b, or a gene product of a gene as listed here GPR27; CPNE7; RNF8; RGNEF; HTR1E; CBL; CCNB1; GJB3; KCNQ3; OR2F1; RPL3L; FAM174B; ITPKA; DGAT2; SLC22A10; PDILT; HOXD3; IFNA7; MGC2705; ENTPD5; NLRP9; FBX04;
  • the gene as target is GPR27 or CPNE7.
  • the gene as target is the GPR27 gene.
  • the cancer can be any type of cancer.
  • the cancer is brain cancer, head/neck cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, gastrointestinal cancer, kidney cancer, ovarian cancer, breast cancer, prostate cancer or a cancer originating from the hemopoietic system.
  • the cancer is brain cancer, head/neck cancer, lung cancer,
  • the cancer is a prostate cancer.
  • the radiation is preferably ionizing radiation.
  • Ionizing radiation typically encompasses alpha particles, beta particles, Gamma rays, X-rays, and in general any charged particle moving at relativistic speeds. Neutrons are considered ionizing radiation at any speed.
  • Ionizing radiation includes some portion of the ultraviolet spectrum, depending on context. Radio waves, microwaves, infrared light, and visible light are normally considered non-ionizing radiation, although very high intensity beams of these radiations can produce sufficient heat to exhibit some similar properties to ionizing radiation, by altering chemical bonds and removing electrons from atoms.
  • Ionizing radiation is typically quantified by the absorbed dose indicated in gray.
  • the gray is the SI derived unit of absorbed dose, specific energy (imparted) and of kerma. Such energies are typically associated with ionising radiation such as X-rays or gamma particles or with other nuclear particles. It is defined as the absorption of one joule of such energy by one kilogram of matter.
  • the gray is always defined independently of any target material.
  • a population of cells preferably cancer cells, is said to be sensitized to radiation, i.e. rendered more susceptible to radiation, when the death of (a proportion of) the cells can be induced at a lower radiation dose than prior to the radiosensitizing treatment.
  • the factor by which the dose can be lowered by the radiosensisitzation treatment to achieve the same cell killing effect as is achieved without radiosensisitzation treatment is called the "dose modifying factor" or DMF.
  • a population of cells is also said to be sensitized to radiation if the same dose of radiation induces a larger proportion of cells to die than prior to the
  • radiosensitizing treatment The fold increase in cell killing effect achieved by irradiation following radiosensitization treatment compared to irradiation alone is referred to here as the "radiosensitization factor " or RF.
  • radiosensitization factor or RF.
  • the sensitization or increase in susceptibility is such that the DMF is at least 1.3 and/or the RF is at least 1.1. In a more preferred embodiment the sensitization or increase in susceptibility is such that the DMF is at least 1.6 and/or the RF is at least 1.3, even more preferably the DMF is at least 2.0 and/or the RF is at least 1.5, most preferably the DMF is at least 3.0 and/or the RF is at least 2.0.
  • the sensitization or susceptibility is preferably measured by means of the CFA assay described in the examples.
  • the amount of radiation can be between 1 and 10 gray.
  • the higher doses are typically so high that a lot of damage is done to non-carcinogenic surrounding tissue.
  • the invention provides compounds that induce profound radiosensitization also - and in particular - at low dose IR.
  • the amount of radiation is preferably between 1 and 6 gray. In a particularly preferred embodiment the dose is between 2 and 4 gray.
  • Cells may be irradiated in one single session or in several sessions. In the case of several sessions, it is preferred that the cells are irradiated with the preferred dose at every session. The exception is when the cells are irradiated in several sessions in one day. In that case the individual doses must be added up to arrive at the preferred total dose for the day.
  • affecting the expression and “modulating the expression” of a protein or gene should be understood as regulating, controlling, blocking, inhibiting, stimulating, enhancing, activating, mimicking, bypassing, correcting, removing, and/or substituting said expression, in more general terms, intervening in said expression, for instance by affecting the expression of a gene encoding that protein and/or of the gene product itself.
  • the terms "individual”, “subject” or “patient” are used interchangeably herein and include, but are not limited to, an organism; a mammal, including, e.g., a human, non-human primate, mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; and a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck), an amphibian and a fish, and a non-mammalian invertebrate.
  • a human including, e.g., a human, non-human primate, mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal
  • a non-mammal including, e.g., a non
  • homologous refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared xlOO.
  • homologues preferably have more than 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99,5 or more percent identity with one another.
  • the genes provided herein are the human forms. The skilled person will appreciate that any mammalian and preferably human homologue is expressly intended to be included herein.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents.
  • a therapeutic agent such as antibodies or a polypeptide, genes, and other therapeutic agents.
  • the term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.
  • terapéuticaally effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a disease or condition, in particular cancer, or to exhibit a detectable therapeutic or
  • the effect can be detected by, for example, chemical markers or antigen levels, imaging such as by using MM, PET, SPECT or CT, or molecular, biochemical or histological examination of a tumor (biopsy) sample taken from the body of a patient.
  • Therapeutic effects also include reduction in physical symptoms.
  • the precise effective amount for a subject may depend e.g. upon the subject's size and health, the nature and extent of the condition, other treatments being given previously or simultaneously, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician.
  • an effective dose will be from about
  • the term "functional fragment” refers to a shortened version of a protein, which is a functional variant or functional derivative.
  • a “functional variant” or a “functional derivative” of a protein is a protein the amino acid sequence of which can be derived from the amino acid sequence of the original protein by the substitution, deletion and/or addition of one or more amino acid residues in a way that, in spite of the change in the amino acid sequence, the functional variant or derivative retains at least a part of at least one of the biological activities of the original protein that is detectable for a person skilled in the art.
  • a functional variant is generally at least 50% homologous (preferably the amino acid sequence is at least 50% identical), advantageously at least 70% homologous and even more advantageously at least 90% homologous to the protein from which it can be derived.
  • a functional variant may also be any functional part of a protein; the function in the present case being the capacity to inhibit the activity of a gene of table 3a or an RNA or protein encoded by said gene.
  • the amino acid sequence differs from the native protein sequence mainly or only by conservative substitutions. More preferably the protein comprises an amino acid sequence having 90% or more, still more preferably 95%, sequence identity with the native protein sequence and optimally 100% identity with those sequences.
  • “Functional" as used herein means functional in mammals, preferably human patients.
  • antibody includes reference to antigen-binding peptides and refers to antibodies, monoclonal antibodies, to an entire immunoglobulin or antibody or any functional fragment of an immunoglobulin molecule. Examples of such peptides include complete antibody molecules, antibody fragments, such as Fab, F(ab')2, complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), and any combination of those or any other functional portion of an antibody peptide.
  • antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • antibody fragments can be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody fragments such as single chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies (i.e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e.g., bispecific antibodies); as well as single-domain antibodies, including nanobodies, engineered from e.g. heavy-chain only antibodies found in camelids or cartilaginous fishes.
  • CDR complementarity determining region
  • the invention provides as a therapeutic compound an antibody or derivative thereof (such as an scFv fragment, Fab fragment, chimeric antibody, bifunctional antibody, intrabody, and other antibody- derived molecule) directed against a polypeptide gene product of a gene of table 3a.
  • the antibodies of the present invention have the effect of interfering with the function of the protein such that, for instance, the ligand-receptor interaction, transport or an enzyme function of the protein is blocked.
  • an antibody or derivative thereof that is a receptor antagonist.
  • the invention further provides as a compound an antisense molecule, in particular an antisense RNA or antisense oligodeoxynucleotide, a morpholino, an RNAi molecule or a ribozyme binding under stringent conditions with a gene, or a mRNA of a gene, of table 3a described herein.
  • the antisense molecule inhibits the expression of the respective gene or mRNA of the gene.
  • stringent conditions refers to conditions under which said antisense molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.
  • the term is used to indicate that the nucleotide sequence of the antisense molecule is essentially identical to the complement of a region of a gene, or a mRNA of a gene, of table 3a.
  • the term “essentially identical” is used to indicate that the antisense molecule is more than 90 % identical, more preferred more than 95 % identical, more preferred more than 99 % identical to the complement of a region of a gene, or a mRNA of a gene, of table 3a.
  • the invention further provides a pharmaceutical composition for sensitizing a cancer cell to radiation therapy comprising a compound according to the invention and a suitable excipient, carrier or diluent as explained above.
  • the compound that increases the susceptibility of a cell for radiation is a compound that inhibits the activity of a product of a gene selected from the group of genes of table 3a.
  • the inhibitor is an antibody and/or an antibody derivative directed against an expression product of a gene of table 3a.
  • Therapeutic antibodies are for instance useful against gene expression products located on the cellular membrane and can be comprised in a pharmaceutical composition. Also, antibodies may be targeted to intracellular, e.g. cytoplasmic, gene products such as RNA's, polypeptides or enzymes, in order to modulate the activity of these products.
  • the inhibitor is a small molecule capable of modulating the activity or interfering with the function of a protein expression product of a gene of table 3a.
  • a fragment-based strategy is used to identify low molecular weight chemical fragments (also known as scaffolds or templates) from small compound libraries (Hajduk and Greer, 2007. Nature Reviews Drug Discovery 6: 211-219). Initial fragments are identified by direct binding techniques. Fragments that interact efficiently with a target protein are selected and combined into larger, more complex molecules to provide starting points for further lead optimisation.
  • biophysical screening methods may be used including nuclear magnetic resonance (NMR) and X-ray crystallography to provide structural understanding of the binding of the ligand to the protein expression product of a gene of table 3a.
  • the binding affinity of a compound with an expression product of a gene of table 3a can be measured by methods known in the art, such as by surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), and by using a labeled compound.
  • Biacore surface plasmon resonance biosensors
  • saturation binding analysis with a labeled compound e.g. Scatchard and Lindmo analysis
  • the binding affinity of a compound can be expressed as a dissociation constant (Kd).
  • Kd dissociation constant
  • the dissociation constant, Kd is a measure of how well a compound binds to the expression product and is equivalent to the compound concentration that is required to saturate exactly half of the binding-sites on the expression product.
  • a preferred compound that inhibits a protein expression product of a gene of table 3a exhibits a binding affinity to the protein of at most 1 micromolar, more preferred at most 1
  • a preferred method may comprise a series of assays, each of which is designed to determine whether a drug candidate compound is indeed acting on the protein expression product of a gene of table 3a and to increase the radiation sensitivity of a cancer cell after adding the compound to the cell.
  • an assay designed to determine the binding affinity of a test compound to the protein expression product of a gene of table 3a, or a fragment thereof may be combined with an method to determine whether the test compound interferes with the biological activity of the protein expression product, and/or a cellular assay.
  • an expression product e.g. a G- protein-coupled receptor, a kinase, a phosphatase or a protease
  • the biological activity of a protein expression product of a gene of table 3a may be measured by determining the sensitivity of the cell upon IR in the presence and absence of a candidate compound.
  • a host cell expressing a protein expression product is preferably a cell with endogenous expression of a protein expression product of a gene of table 3a.
  • Compounds that were found active against the selected protein expression product of a gene of table 3a in a cellular assay are preferably tested at different concentrations to determine a dose-response curve.
  • the present invention further relates to a method for identifying a compound that sensitizes a cancer cell for ionizing radiation, comprising:
  • the performance of the compound may be improved by synthesizing analogues with improved potency, reduced off-target activities, and further physiochemical/metabolic properties suggestive of reasonable in vivo pharmacokinetics. This optimization is usually accomplished through chemical modification of the structure of the identified compound with modifications chosen by employing knowledge of the structure -activity relationship as well as structure- based design if structural information about the protein expression product of a gene of table 3a is available.
  • nucleic acids can be used to block the production of proteins by destroying the mRNA transcribed from a gene of table 3a.
  • This can be achieved by antisense drugs, ribozymes or by RNA interference (RNAi).
  • RNAi RNA interference
  • the present invention relates to antisense drugs, such as antisense RNA and antisense oligodeoxynucleotides, ribozymes and RNAi molecules, directed against a gene of table 3a.
  • Trans-cleaving catalytic RNAs are RNA molecules possessing endoribonuclease activity. Ribozymes are specifically designed for a particular target, and the target message must contain a specific nucleotide sequence. They are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression.
  • ribozyme motif is the hammerhead, for which the substrate sequence requirements are minimal.
  • Design of the hammerhead ribozyme is well known in the art, as is the therapeutic uses of ribozymes.
  • Ribozymes can for instance be prepared and used as described in U.S. Pat. No. 5,254,678. Ribozyme cleavage of HIV-I RNA is described in U.S. Pat. No. 5, 144,019; methods of cleaving RNA using ribozymes is described in U.S. Pat. No. 5, 116,742; and methods for increasing the specificity of ribozymes are described in U.S. Pat. No. 5,225,337. Preparation and use of ribozyme fragments in a hammerhead or hairpin structure is also known in the art. Ribozymes can also be made by rolling transcription.
  • the hybridizing region of the ribozyme may be modified or may be prepared as a branched structure.
  • the basic structure of the ribozymes may also be chemically altered in ways familiar to those skilled in the art, and chemically synthesized ribozymes can be administered as synthetic oligonucleotide derivatives modified by monomeric units.
  • liposome mediated delivery of ribozymes improves cellular uptake.
  • ribozymes requires knowledge of a portion of the coding sequence of the gene to be inhibited.
  • a nucleic acid sequence provides adequate sequence for constructing an effective ribozyme.
  • a target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5' and 3' nucleotide sequences that flank the cleavage site.
  • Retroviral vectors are engineered to express monomeric and multimeric hammerhead ribozymes targeting the mRNA of the target coding sequence. These monomeric and multimeric ribozymes are tested in vitro for an ability to cleave the target mRNA.
  • a cell line is stably transduced with the retroviral vectors expressing the ribozymes. The cells are analysed for inactivation of the target mRNA and/or reduction of the gene product of the target mRNA.
  • Antisense polynucleotides are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA- RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected sequence can interfere with expression of the corresponding gene.
  • Antisense polynucleotides are typically generated within the cell by expression from antisense constructs that contain the antisense strand as the transcribed strand. Antisense polynucleotides will bind and/or interfere with the translation of the corresponding mRNA. As such, antisense may be used
  • Antisense RNA or antisense oligodeoxynucleotides can both be used and may also be prepared in vitro synthetically or by means of recombinant DNA techniques. Both methods are well within the reach of the person skilled in the art. ODNs are smaller than complete antisense RNAs and have therefore the advantage that they can more easily enter the target cell. In order to avoid their digestion by DNAse, ODNs and antisense RNAs may be chemically modified. For targeting to the desired target cells, the molecules may be linked to ligands of receptors found on the target cells or to antibodies directed against molecules on the surface of the target cells.
  • Antisense RNA includes reference to locked nucleic acid (LNA). RNAi molecules
  • RNAi is based on the generation of short, double-stranded RNA (dsRNA) which activates a cellular process leading to a highly specific RNA degradation (Zamore et al., 2000. Cell 101: 25-33) and/or suppression of dsRNA.
  • RNAi molecules dsRNA molecules that activate RNAi and their precursors that are processed in a cell to generate dsRNA molecules that activate RNAi are referred to as "RNAi molecules".
  • RNA interference is mediated by the generation of 18-to 23-nucleotide dsRNA molecules with 2 nucleotide -long 3' overhangs termed small interfering RNA (siRNA) duplexes.
  • siRNA duplexes One strand of the siRNA duplex, the antisense or guide strand, is incorporated into a nuclease complex, called the RNA-induced silencing complex (RISC), which acts to destroy mRNAs that are recognized by the guide strand through base -pairing interactions.
  • RISC RNA-induced silencing complex
  • RNAi molecules are thus double stranded RNAs (dsRNAs) that upon processing in a cell and incorporation in RISC are very potent in silencing the expression of their target gene.
  • dsRNAs double stranded RNAs
  • the invention provides RNAi molecules that are potent in silencing the expression of the radioresistance genes of the present invention.
  • an RNAi molecule of the invention is an siRNA duplex that can activate an RNAi process in a cell directly through incorporation of its guide strand into RISC.
  • Said siRNA duplex can also be a miRNA mimic, i.e., an siRNA comprising the guide strand of a miRNA.
  • an RNAi molecule of the invention activates an RNAi process indirectly, because it is a precursor of a molecule that can activate an RNAi process in a cell.
  • Said precursor molecule is preferably a short hairpin RNA (shRNA) or a pre- or pri-miRNA or variant or analogue thereof.
  • a short hairpin RNA typically comprises a 50- 100 nucleotide long RNA molecule comprising two stretches of nucleotides that are complementary and can base-pair, whereby the two stretches are interconnected through a hairpin turn.
  • the shRNA hairpin structure is cleaved by the cellular machinery into one or more 18-23 (typically 19) nucleotide -long double stranded siRNA molecules with 2 nucleotide-long 3' overhangs with one of the strands exhibiting extensive complementary homology to a part of a mRNA transcript from a target gene.
  • Said siRNA molecules activate the RNAi pathway and interfere with the expression of said target gene by specific mRNA degradation.
  • shRNA expression can be driven by a polymerase II or polymerase III enhancer/promoter.
  • Natural miRNA molecules are typically transcribed by polymerase II as pri-miRNA with a cap and poly-A tail and processed to short, 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. These pre-miRNAs are then processed to mature double stranded miRNAs of about 18-25 nucleotides in the cytoplasm which silence gene expression via RNA interference, partly by specific RNA degradation and partly by
  • RNAi molecules can be transcribed from any promoter, for example a polIII promoter, in a format analogous to that of a shRNA. They then differ from an shRNA in that the double-stranded region is not completely complementary.
  • a preferred RNAi molecule according to the invention comprises a double stranded region of between 18 nucleotides and 25 nucleotides per strand.
  • a most preferred RNAi molecule according to the invention comprises a double stranded region that has a length of 19 nucleotides after processing into a mature siRNA.
  • RNAi molecules are prepared by methods well known to the person skilled in the art.
  • an isolated nucleic acid sequence comprising a nucleotide sequence which is substantially homologous to the sequence of at least one of the genes of table 3a and which is capable of forming a partially of fully double stranded (ds) RNA with (part of) the transcription product of said gene will function as an RNAi molecule.
  • the double stranded region may be in the order of between 10-250, preferably 10- 100, more preferably 20-50 nucleotides in length.
  • RNAi strategies rely on RNA-RNA hybrids, it is possible to provide cells with a single stranded antisense oligonucleotide specific for the target mRNA, and have it being broken down via the RNAi pathway.
  • the RNAi molecule of the invention can thus be double or single stranded RNA.
  • the targeted region is preferably located 50-100 nucleotides downstream of the start codon (ATG); is selected from an exon sequence since RNAi only works in the cytoplasm; sequences with > 50% G+C content or sequences with stretches of 4 or more nucleotide repeats are avoided as are sequences that share a certain degree of homology with another related or unrelated gene.
  • the skilled person is able to select one or more targeted regions of a target gene for generating the corresponding RNAi molecule. Testing one or more, preferably at least four, of the selected potential RNAi molecules for reducing expression of the corresponding target gene in a host cell, will provide the skilled person with one or more RNAi molecules that reduce expression of a target gene in a host cell.
  • RNAi molecules examples include siRNAs, shRNAs, miRNAs, pre- miRNAs, pri-miRNAs, and the like.
  • the antibodies used in the present invention may be from any animal origin including birds, fish, and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, llama, dromedary, alpaca, shark, horse, or chicken).
  • the antibodies of the invention are human or humanized monoclonal antibodies.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries (including, but not limited to, synthetic libraries of immunoglobulin sequences homologous to human immunoglobulin sequences) or from mice that express antibodies from human genes.
  • human or chimeric antibodies For some uses, including in vivo therapeutic or diagnostic use of antibodies in humans and in vitro detection assays, it may be preferred to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences or synthetic sequences homologous to human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716, 111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893 and W098/16654, each of which is incorporated herein by reference in its entirety.
  • the antibodies to be used with the methods of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody. Additionally, the derivative may contain one or more non- classical amino acids.
  • the antibodies to be used with the invention have extended half-lives in a mammal, preferably a human, when compared to unmodified antibodies.
  • Antibodies or antigen-binding fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill in the art (see, e.g., PCT Publication No. WO 97/34631).
  • antibodies to be used with the methods of the invention are single-chain antibodies, including scFv and single domain antibodies such as nanobodies.
  • the design and construction of a single-chain antibody is well known in the art.
  • the antibodies to be used with the invention bind to an intracellular epitope, i.e., are intrabodies.
  • An intrabody comprises at least a portion of an antibody that is capable of immuno- specific binding an antigen and preferably does not contain sequences coding for its secretion. Such antibodies will bind its antigen intracellular.
  • the intrabody comprises a single-chain Fv ("sFv").
  • the intrabody preferably does not encode an operable secretory sequence and thus remains within the cell.
  • intrabodies are expressed in the cytoplasm.
  • the intrabodies are localized to various intracellular locations.
  • specific localization sequences can be attached to the intranucleotide polypepetide to direct the intrabody to a specific location.
  • the antibodies to be used with the methods of the invention or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art.
  • phage display methods that can be used to make the antibodies of the present invention include those disclosed in W097/13844; and U.S. Patent Nos. 5,580,717, 5,821,047, 5,571,698, 5,780,225, and 5,969, 108; each of which is incorporated herein by reference in its entirety.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • Techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324.
  • IgE antibodies For a detailed discussion of the technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication No. WO 98/24893. All references cited herein are are incorporated by reference herein in their entirety. In addition, companies such as Medarex, Inc. (Princeton, NJ), Abgenix, Inc. (Freemont, CA) and
  • Genpharm (San Jose, CA) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Recombinant expression used to produce the antibodies, derivatives or analogs thereof requires construction of an expression vector containing a polynucleotide that encodes the antibody and the expression of said vector in a suitable host cell or even in vivo.
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5, 122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • a variety of host-expression vector systems may be utilized to express the antibody molecules as defined herein
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus
  • transcription/translation control complex e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc.
  • an antibody molecule to be used with the methods of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the invention provides an antibody as defined above for use in therapy.
  • antibodies may be produced in vitro and applied to the subject in need thereof.
  • the antibodies may be administered to a subject by any suitable route, preferably in the form of a pharmaceutical composition adapted to such a route and in a dosage which is effective for the intended treatment.
  • Therapeutically effective dosages of the antibodies required for decreasing the rate of progress of the disease or for eliminating the disease condition can easily be determined by the skilled person.
  • antibodies may be produced by the subject itself by using in vivo antibody production methodologies as described above.
  • the vector used for such in vivo production is a viral vector, preferably a viral vector with a target cell selectivity for a specific target cell referred to herein, preferably a brain cancer cell, a head/neck cancer cell, a lung cancer cell, a liver cancer cell, a pancreatic cancer cell, a, skin cancer cell, a gastrointestinal cancer cell, a kidney cancer cell, an ovarian cancer cell, a breast cancer cell, a prostate cancer cell or a cancer cell originating from the hemopoietic system .
  • the invention provides the use of an antibody as defined above in the manufacture of a medicament for use in the treatment of a subject to achieve the said therapeutic effect.
  • the treatment comprises the administration of the medicament in a dose sufficient to achieve the desired therapeutic effect.
  • the treatment may comprise the repeated
  • the invention provides a method of treatment of a human comprising the administration of an antibody as defined above in a dose sufficient to achieve the desired therapeutic effect.
  • the therapeutic effect being the sensitation of a cancer cell in the human to radiation therapy.
  • the diagnostic and therapeutic antibodies are preferably used in their respective application for the targeting of kinases or phosphatases, which are often coupled to receptor molecules on the cell's surface. As such, antibodies capable of binding to these receptor molecules can exert their activity-modulating effect on the kinases or phosphatases by binding to the respective receptors. Also
  • transporter proteins may be targeted with advantage for the same reason that the antibodies will be able to exert their activity-modulating effect when present extracellularly.
  • the above targets, together with signalling molecules, represent preferred targets for the antibody uses of the invention as more effective therapy and easier diagnosis is possible thereby.
  • the diagnostic antibodies can suitably be used for the qualitative and quantitative detection of gene products, preferably proteins in assays for the determination of altered levels of proteins or structural changes therein. Protein levels may for instance be determined in cells, in cell extracts, in tumor biopsies, in supernatants, or body fluids by for instance protein assays such as ELISA or RIA, Western blotting, flow cytometry, immunocytochemistry, immunohistochemistry and imaging technology (e.g., using confocal laser scanning microscopy).
  • protein assays such as ELISA or RIA, Western blotting, flow cytometry, immunocytochemistry, immunohistochemistry and imaging technology (e.g., using confocal laser scanning microscopy).
  • a therapeutic compound such as an anti-sense molecule, a small molecule or a (glyco)protein, a hormone or biologically active compound, or a therapeutic effect as described herein refers to the radiation sensitizing effect of the compound or radiation sensitizing effect of the therapy.
  • the invention does not exclude the possibility that apart from said radiation sensitizing effect, the compound may in the absence of radiation have a beneficial or therapeutic effect on it's own.
  • compositions of the invention can be administered directly to a subject; or delivered to cells in vitro.
  • Direct delivery of the compositions to a subject will generally be accomplished by injection, either subcutaneously, intraperi tone ally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
  • compositions can also be administered into a lesion or site of neoplastic growth.
  • Other modes of administration include topical, oral, catheterized and pulmonary administration, suppositories, and transdermal applications, needles, and particle guns or hyposprays.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, gene transfer using a vector including a virus, dextran-mediated transfection, calcium phosphate precipitation, polybrene® mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
  • a vector including a virus, dextran-mediated transfection, calcium phosphate precipitation, polybrene® mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
  • a target location is located and the therapeutic composition injected in the target directly.
  • arteries which serve target location are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the target location.
  • Image guidance by e.g. ultrasound, X-ray, or MRI imaging can be used to assist in certain of the above delivery methods.
  • Receptor-mediated targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues is also used.
  • Receptor-mediated DNA or RNA delivery techniques are well known in the art.
  • Particularly interesting molecules with high binding affinity and specificity are the so called "aptamers".
  • Aptamers are nuclease- stabilized oligonucleotides with a secondary structure providing binding specificity (Ellington and Szostak, Nature 346, 818-822, 1990). They are considered as nonprotein based alternatives for antibodies. They are manufactured chemically and are therefore attractive reagents for therapeutic use. Many different aptamers with different binding specificities have already been produced.
  • an aptamer with binding specificity for prostate specific membrane antigen was produced and shown to bind tightly to the extracellular part of PSMA on human prostate cancer cells (Lupoid et al., Cancer Res. 62, 4029-4033, 2002).
  • Such aptamers can be covalently coupled to RNA, e.g. siRNA or shRNA, retaining aptamer binding specificity and siRNA gene silencing capacity.
  • RNA e.g. siRNA or shRNA
  • the aptamer-siRNA inhibited tumor growth (McNamara et al., Nat. Biotechnol. 24, 1005- 1015, 2006).
  • compositions containing antisense, ribozyme or RNAi polynucleotides are administered in a range of about 100 ng to about 200 mg of polynucleotides for local administration to a human subject.
  • Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of polynucleotides can also be used.
  • Factors such as method of action and efficacy of uptake into cells and expression are considerations which will affect the dosage required for ultimate efficacy of the polynucleotides.
  • the genes and gene products identified herein are associated with the sensitation of cancer cells to radiation and in particular ionizing radiation.
  • the gene products of the genes of the table 3a and in particular the level of expression of the gene product in the cancer cell is therefore suitable for a diagnostic purpose.
  • the level of expression of the gene product in the cancer cell, and in particular the level of expression of the gene products of two or more of the genes is indicative for the sensitivity of a cancer cell to radiation therapy.
  • the invention thus further provides a method for determining the radiation sensitivity of a cancer cell, comprising determining the level of a gene product of one or more genes of table 3a in said cancer cell and estimating the sensitivity of said cancer cell to said radiation. This test is preferably performed prior to or together with a method for sensitizing a cancer cell to radiation.
  • a method for increasing the sensitivity or susceptibility of a cancer cell to radiation by providing a compound to said cancer cell is preferably combined with a method for determining the expression of a gene of table 3a in said cancer cell.
  • the compound inhibits the expression of a gene and/or the activity of a gene product of said gene, of which it was established that the cancer cell expresses it. It is preferred that the compound for sensitizing the cancer cell to radiation, is directed towards inhibiting the expression of a gene, or the activity of gene product of a gene, of which it was determined that it is expressed in the cancer cell.
  • the invention further provides a method of diagnosis of a cancer and a method for predicting the success of radiation therapy for a specific cancer comprising determining the expression of a gene of table 3a in said cancer cell, or the expression level of a gene product of a gene of table 3a in said cancer cell.
  • the invention further provides a method for the determining the radiation sensitivity of a cancer in an individual or predicting the utility of radiation therapy of a cancer in an individual, comprising detecting a gene or gene product of a gene of table 3a in the blood and/or a cancer cell of said individual.
  • a method may suitably be performed by using quantitative RT-PCR, RNA massive parallel sequencing (RNAseq), a microarray (in particular comprising specific binding partners that bind specifically to at least two biomarkers as defined above bound to a solid support), by using tandem mass spectrometry (MS- MS), by MALDI-FT mass spectrometry, MALDI- FT-ICR mass spectrometry, MALDI Triple-quad mass spectrometry or immunoassay.
  • RNAseq RNA massive parallel sequencing
  • microarray in particular comprising specific binding partners that bind specifically to at least two biomarkers as defined above bound to a solid support
  • MS- MS tandem mass spectrometry
  • Kits of parts for performing a diagnostic method according to the invention are also envisioned herein.
  • Such kits comprise at least one gene product as defined above or a specific binding partner that binds specifically to said gene product, said kit of parts optionally further comprising one or more of the following:
  • Diagnostic reagents that bind specifically to a gene product as defined herein include an antibody or a nucleic acid molecule specifically hybridizing under stringent conditions to said gene product.
  • GJB3 2707 yes ⁇ 5 yes yes 2/4 yes
  • RGNEF 64283 yes ⁇ 10 yes yes 4/4 yes
  • GPR27 CPNE7; MAD2L2; RNF8; RGNEF; HTR1E; CBL; CCNB1; DNA- PKcs; GJB3; KCNQ3; OR2F1; RPL3L; FAM174B; ITPKA; DGAT2;
  • Table 4 Stable gene knockdown in PC-3 cells infected with lentiviral vectors expressing short hairpins.
  • the table lists the shRNA insert sequence as well as the mRNA transcripts targeted by each lentiviral vector used from the TRC or GIPZ library.
  • the percentag knockdown (%KD) obtained in individual stable transduced cell lines is also given.
  • Example 1 Development of a high-throughput assay to identify genes involved in cancer cell resistance to irradiation.
  • the single-cell population includes all objects that are 6- 50um wide and 6- 120um deep, while the multiple-cell population includes all objects that are 50.5- 150um wide and 6- 120um deep.
  • the total cell number was defined as the sum of the single-cell population count plus the multiple-cell population count multiplied by 5. This assay is further referred to herein as the "Acumen assay”.
  • the dose response curves produced by the Acumen assay were quite comparable to the CFA curves ( Figure 1).
  • the curves follow a linear- quadratic equation typical for irradiation dose-effects.
  • the Acumen assay recapitulates irradiation effects on cancer cell lines very well.
  • lOul O. luM siRNA in siRNA buffer (5x siRNA buffer, Dharmacon Cat. No. B-002000-UB-100, 5-times diluted in H20) was incubated for 5 minutes at room temperature; DharmaFECTl transfection reagent was diluted 500-times in RPMI medium (without serum and antibiotics) and incubated for 5 minutes at room temperature.
  • lOul diluted siRNA was added to lOul diluted DharmaFECTl and incubated for at least 20 minutes at room temperature.
  • transfected PC-3 cells were trypsinized, counted and plated 250 and 500 cells per well in 6-well culture plates for the CFA assay or left in the 96-well plates for the Acumen assay. Two days after
  • RNAi screens We used high-throughput RNAi screens to identify modulators of sensitivity to irradiation in cancer cells.
  • PC-3 prostate cancer cells and the Dharmacon siARRAY whole human genome siRNA library.
  • PC-3 cells were grown at 37°C and 5% C02 in a humidified incubator in RPMI 1640 supplemented with L-glutamine , 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin).
  • the siRNA library consists of approximately 21,000 arrayed pools of 4 siRNAs each directed against a different human gene.
  • Figure 3b shows the relative cell survival after gene silencing plus irradiation compared to gene silencing alone.
  • silencing of 33 of the 45 tested genes resulted in substantially more cell growth inhibition upon irradiation than control siRNA transfection plus irradiation (87% cell survival), i.e. they exhibited a survival less than 79% corresponding to a RF >1.1. The radiosensitizing effect of silencing these genes was thus confirmed (see Table 1).
  • silencing of 17 candidate genes enhanced the inhibitory effect of 4Gy irradiation on cell survival and proliferation at least approximately 2-fold (see Table 1).
  • CFA Colony Formation Assay
  • the assay was performed as follows: 1,500 cells were plated in 96-well format plates. The next day, cells were transfected with 20nM siRNA using 0.02ul/well DharmaFECT 1 (Thermo Fisher Scientific) and incubated at 37°C. 24 hours after transfection cells were trypsinized and plated in 6-well plates at 250 or 500 cells/well depending on the irradiation dose (250 cells per well for unirradiated controls and irradiation at 1, 2, or 3 Gy and 500 cells/well for irradiation at 4, 5, 6 and 8Gy) and allowed to attach overnight. Each condition was set up in triplicate. On the next day, cells were irradiated using a Varian linear accelerator as described in Example 1.
  • irradiation dose response curves obtained. They represent the mean results of 2 independent experiments performed. Each panel shows the dose response curve for cells transfected with siRNA directed against a different candidate target gene and the dose response curve for cells transfected with siRNA directed against an irrelevant control siRNA. The latter serves as control for possible siRNA
  • DMF Surviving Fraction
  • the RF at 2Gy was calculated.
  • Table 2 gives the calculated SF2, RF2 and DMF(0.8) values for the 17 tested candidate target genes and DNA- PKcs and irrelevant siRNA controls.
  • the radiosensitizing effect of silencing the 17 candidate target genes differed considerably.
  • the effect of silencing nine genes, i.e. CPNE7, GPR27, MAD2L2, RNF8, RGNEF, HTR1E, CBL, CCNB1 and KCNQ3 appeared stronger than silencing positive control gene DNA-PKcs, as evidenced by a higher RF2 and/or higher DMF(0.8).
  • silencing GJB3 was similar to that of silencing DNA-PKcs. It was particularly interesting to note that silencing either GPR27 or CPNE7 had a profound effect on radiosensitivity of PC-3 cells. Their DMF(0.8), RF2 and SF2 values stood out compared to the other candidate target genes. To inhibit colony formation by 20%, i.e. DMF(0.8), the irradiation dose could be lowered by more than 4-fold; and irradiation at a clinically relevant dose of 2Gy decreased colony formation capacity from 70% to less than 30%. Hence, novel targets for
  • PC-3 cells were stable transduced with short hairpin RNA (shRNA) -expressing lentiviral vectors (LV).
  • shRNA short hairpin RNA
  • LV lentiviral vectors
  • negative control cells were transduced with LV-shSCR expressing a non-targeting shRNA sequence.
  • positive control a cell culture expressing a shRNA against DNA-PKcs was made. Specific gene knockdown efficiencies were determined by quantitative RT-PCR.
  • Example 5 Expression of candidate radiation susceptibility genes in prostate tissue.
  • CPNE7, MAD2L2 and CBL were expressed in PC-3 cells, they were not or only at low levels expressed in human prostate cancer tissue. Hence, the relevance of targeting CPNE7, MAD2L2 or CBL to improve radiotherapy of prostate cancer is questionable.
  • CBL is expressed at higher levels in other cancer types that are being treated with IR, such as glioma, lung cancer, and in particular lymphoma.
  • CPNE7 is expressed in some colorectal cancers and in lymphoma. Thus, CBL and CPNE7 could be useful targets for radiosensitization in these cancers.
  • RGNEF and GPR27 were expressed in many different tissues and tumors, including prostate tumors, at moderate and strong levels, respectively.
  • healthy human prostate tissue was negative for RNF8, whereas this protein was expressed at moderate levels in prostate cancer tissue.
  • Healthy human prostate tissue was also negative for CCNB1, while some prostate cancers expressed this protein at a moderate level.
  • GPR27, RNF8, RGNEF and CCNB1 could all potentially be useful targets to improve radiotherapy of prostate cancer, with the latter two possibly adding tumor specificity.
  • GPR27 clearly stood out.
  • GPR27 the orphan G-protein Coupled Receptor 27, also known as Super
  • SREB1 conserved Receptor Expressed in Brain 1 (SREB1), appears a particularly useful candidate target to improve radiotherapy of prostate cancer, and possibly other cancers as well.
  • Example 6 Silencing GPR27 sensitizes prostate cancer cells to fractionated irradiation.
  • cancer patients are usually treated with IR given at multiple low-dose fractions.
  • Often used treatment schedules consist of daily doses of 2Gy, given 5 days per week (on weekdays) for several weeks, reaching a total cumulative dose of more than 70Gy. Therefore, to further investigate the utility of silencing GPR27 to sensitize prostate cancer cells to IR, we subjected two different human prostate cancer cell lines, PC-3 and DU145, stable transduced with a lentiviral vector silencing GPR27, to fractionated low-dose IR.
  • the lentiviral vector used for this purpose was TRCN0000008871 (see Table 4).
  • GPR27mutORF was synthesized under contract by GeneArt Gene Synthesis (Life Technologies). This ORF carries the human GPR27 ORF sequence with silent nucleotide mutations in the binding site of siGPR27 with catalogue number D-005562-02 (i.e., the most effective individual GPR27-silencing siRNA that we identified).
  • siGPR27 D-005562-02 targets the sequence 5'- CGGAGAAGAGGCTGTGCAA -3' in wild type GPR27.
  • Example 8 Effects of GPR27 silencing on prostate cancer cell cycle after
  • PC-3 cells transduced with a negative control lentiviral vector expressing shSCR; and PC-3 cells transduced with shGPR27- expressing lentiviral vector TRCN0000008871 (cell line Ell) were seeded 100,000 cells per well in 6-well-plates and 4Gy irradiated the next day.
  • PI propidium iodine
  • PI propidium iodine
  • Cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using CellQuest software. Aggregates were gated out on the basis of fluorescence area and width. DNA histograms of single cells were prepared and analyzed for cell cycle distribution using ModFit LT. Two independent experiments were done and the results were averaged.
  • Figure 8 shows that PC- 3/LV-shSCR cells and PC-3/LV-shGPR27 cells had similar cell cycle distribution before irradiation.
  • IR induced a transient loss of cells in Gl-phase and accumulation of cells in the G2-phase of cell cycle. This suggests activation of the G2 M cell cycle checkpoint to allow DNA damage repair.
  • PC-3/LV-shSCR cells normal cell cycle distribution was fully recovered at 32 hours after irradiation.

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Abstract

L'invention concerne l'utilisation d'un gène ou d'un produit génique tel qu'une cible d'identification de composé pharmacologiquement actif, de préférence pour sensibiliser une cellule cancéreuse à un rayonnement ionisant. L'invention concerne également des méthodes permettant de déterminer si un composé peut sensibiliser une cellule cancéreuse à un rayonnement ionisant, ainsi que des méthodes destinées à augmenter la sensibilité d'une cellule à un rayonnement. L'invention concerne encore un composé destiné à être utilisé dans le traitement d'un patient atteint de cancer, ledit traitement consistant de préférence à irradier le patient ou une partie de son corps au moyen d'un rayonnement ionisant. L'invention concerne enfin des méthodes permettant de déterminer la sensibilité au rayonnement d'un cancer chez un patient. La cible préférée de radiosensibilisation est GPR27.
PCT/NL2014/050102 2013-02-19 2014-02-19 Moyen et méthode d'augmentation de la sensibilité de cancers à la radiothérapie WO2014129895A1 (fr)

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WO2016149580A3 (fr) * 2015-03-19 2017-01-05 The Johns Hopkins University Agent de sensibilisation pour la radiothérapie et la chimiothérapie du cancer et utilisations de celui-ci

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