GB2505237A - Method of screening for therapeutic agents using cell lines including a reference cell line - Google Patents

Abstract

The invention discloses a screening method which identifies therapeutic agents capable of inducing cell death in target cells comprising tumour cells or virus-infected cells but not in reference cells, wherein the agent is selective for the target cells over the reference cells. The present invention further relates to the therapeutic agents identified by the method, pharmaceutical compositions comprising said agents and uses thereof. The cells may be cancer tumour or virus infected cells. Specific cell lines can include HEK293 cells, cancer stem cells, CV-1, human dermal fibroblasts (NHDF), MRC5 fibroblasts, keratinocyes (HKC), MCF10S cells, lung WI-38 cells or BJ-1 cells.

Description

METHOD

Field of the Invention

The present invention relates to a method.

S

In particular, the present invention relates to a screening method which identifies therapeutic agents capable of inducing cell death in target cells comprising tumour cells or virus-infected cells but not in reference cells, i.e. the agent is selective for the target cells over the reference cells. The present invention further relates to the therapeutic agents identified by the method, pharmaceutical compositions comprising said agents and uses thereof.

Background of the Invention

Finding novel therapeutic approaches for the treatment of cancer is an on-going challenge.

Cell death, whether in the form of apoptosis, necropoptosis or necrosis, may be activated by many therapeutic agents. However, due to low specificity, serious side effects in patients can be observed. This may hamper the use of higher and more efficacious doses and hence the therapeutic agents cannot realize their full therapeutic potential.

Recently, distinct genes were discovered that specifically induce cell death in cancer cells.

Upon ectopic expression these factors cause cell death by apoptozis (or alternative forms of cell death) in tumour cells but spare non-cancerous cells. However, thus far these "anti-cancer genes" have been discovered by chance.

The present invention provides for the first time the identification of selective therapeutic agents, such as anti-tumour agents and/or anti-viral agents, by use of a systematic screen.

Summary of the Invention )

In a first aspect, the present invention relates to a method comprising the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell and has no cell death induction effect on said reference cells.

In a second aspect the present invention relates to a therapeutic agent or equivalent thereto identified using the method described herein.

In a third aspect the present invention relates to a pharmaceutical composition comprising a therapeutic agent identified using the method described herein and a pharmaceutically acceptable excipient.

In a fourth aspect the present invention relates to a process for preparing a pharmaceutical composition, said process comprising admixing a therapeutic agent identified using the method described herein or an equivalent thereto, optionally in the form of a vehicle, with a pharmaceutically acceptable carrier, diluent or excipient.

In a fifth aspect the present invention relates to therapeutic agents or pharmaceutical compositions identified using the method described herein for use as a medicament.

In a sixth aspect the present invention relates to anti-tumour agents identified using the method described herein or pharmaceutical compositions comprising anti-tumour agents identified using the method described herein for use in the treatment of cancer.

In a seventh aspect the present invention relates to anti-viral agents identified using the method described herein or pharmaceutical compositions comprising anti-viral agents identified using the method described herein for use in the treatment of viral infection.

The term "equivalent thereto" means that the therapeutic agent has the same composition and/or structure as that identified. The term also includes a bio-similar. The term also includes a bio-isostere.

Brief Description of the Figures

Figure 1 shows an example layout of the screen for the putative agents of the present invention. Aliquots containing single recombinant bacteria clones were inoculated in wells of 96-well blocks, grown up and their plasmids isolated by a robot. The plasmids are then transfected into reference (control) cell(s) and in parallel in a cell line comprising target cells S by a robot. Subsequently, a cellular read-out for cell death is used to identify those genes that cause cell death in the target cells only.

Detailed Description-of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Col d Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James 00. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M J Gait (Editor), 1984, Oligonucleatide Synthesis: A PracticalApproach, Irl Press; 0. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; and F. M Shevach and W. Strober, 1992 and periodic supplements, Current Protocols in Immunology, John Wiley & Sons, New York, NY-Each of these general texts is herein incorporated by reference.

In a first aspect the present invention relates to a method comprising the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cell.

In one embodiment of the first aspect of the invention the method comprises the steps of determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a second tumour cell or second virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell and on said second tumour cell or on said first virusinfected cell and on second virus-infected cell, and has no cell death induction effect on said reference cell.

In another embodiment of the first aspect of the invention the method comprises the steps: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; determining if said putative therapeutic agent has a cell death induction effect on a second reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cells.

In another embodiment the method comprises the steps of determining the cell death induction effect of the putative therapeutic agent in two or more different types of tumour cell or virus-infected cell.

In one embodiment the method comprises the steps of determining the cell death induction effect of the putative therapeutic agent in 2 to 20 different types of tumour cell or virus-infected cell.

Preferably the method comprises steps of determining the cell death induction effect of said putative therapeutic agent in between 2 to 10 different types of tumour cell or virus infected cell, more preferably 2 to 5 different types of tumour cell or virus infected cell.

In another embodiment the method comprises the steps of determining the cell death induction effect of the putative therapeutic agent in two or more different types of reference cell.

In one embodiment the method comprises the steps of determining the cell death induction effect of the putative therapeutic agent in 2 to 20 different types of reference cells.

Preferably the method comprises steps of determining the cell death induction effect of said putative therapeutic agent in between 2 to 10 different types of reference cells, more preferably 2 to 5 different types of reference cell.

In another embodiment of the first aspect ofihe invention the method essentially consists of the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cell.

In the above embodiments the order of each step, with the exception of the final selection step, is not critical to the invention; therefore, with the exception of the final selection step, the steps may be performed in any order. The skilled person would understand that selection step is the final step.

The purpose of this aspect of the invention is to provide a cell based screen to identify therapeutic agents which may be useful in the treatment of cancer or a viral infection.

In one embodiment, the method of the present invention can be performed in high-throughput mode.

Preferably, the method is performed at least 50 times in parallel, more preferably 100 times in parallel, more preferably 200 times in parallel.

In one embodiment the method is performed by robots. These can be custom-made such as the ones that we have described herein as well as commercially known robots.

In one embodiment the therapeutic agent is an anti-tumour agent.

In another embodiment the therapeutic agent is an anti-viral agent.

The therapeutic agent may be selected from nucleic acids, proteins, organic compounds, low molecular weight compounds.

Preferably the therapeutic agent is a nucleic acid. The nucleic acid may be selected from a DNA sequence, or a DNA based sequence, an RNA sequence, or an RNA based sequence.

In one preferred embodiment, the therapeutic agent is a DNA sequence. The DNA sequence may be selected from a library of DNA sequences.

In one embodiment the DNA sequence is selected from a gene library comprising cDNA.

The library may be of various species origin, not only human but other organisms as well, including prokaryotic. Also DNA construct libraries, gene fusion libraries, both random or directed (specific proteins domains) can be used.

The DNA sequences may also be derived from genomic DNA from wild type or mutagenised organisms.

In one embodiment, the DNA sequence may be comprised within a vector. Examples of suitable vectors are commonly known in the art. For example, the DNA sequence may be comprised within plasmids, viral vectors, cosmids, and artificial chromosomes.

In one preferred embodiment, the therapeutic agent is a DNA sequence in plasmid form.

The plasmid may comprise promoters/enhancers in the plasmid backbone which lead to expression of the DNA sequence (transgene).

Treatment via gene therapy may possess distinct advantages over conventional compounds used in therapy (such as anti-tumour therapy or anti-viral therapy) since genes and their encoded proteins are structurally more complex than low molecular weight compounds; hence, they can interact with other targets in the cell and can do so in a more specific way than low molecular weight compounds. In contrast, some low molecular weight compounds are sometimes restricted in their range of activities as in most cases they only inhibit other proteins (such as kinases).

This feature can also be exploited -using established molecular biology methods for protein-protein interactions such as the two-hybrid assay -to uncover the underlying signalling pathways targeted by the nucleotide sequences (e.g. genes), resulting in their optimisation and the facilitation of safety studies.

Moreover, genes can engage a wider range of targets as they do not rely on the inhibition of signalling proteins in the cell, as most low molecular weight compounds do. Thus, with the nucleotide sequences (e.g. genes) not only more refined but also additional interference options may be accessible.

As used herein, the term "cell death induction effect" refers to any effect which leads to the death of the cell. The cell death induction may be a direct effect or an indirect effect.

In one embodiment the cell death induction effect of the method is selected from an apoptotic effect, a necroapoptotic effect, a necrotic effect and an excessive autophagy effect.

Preferably the cell death induction effect of the method is an apoptotic effect.

In one embodiment the cell death induction is detected with the aid of a reporter agent.

Preferably the reporter agent is a reporter enzyme. In one embodiment the reporter enzymes are selected from beta-galactosidase. The integrity of the cell membrane is probed by adding the beta-galactosidase substrate chlorophenol red-beta-D-galactopyranoside (CPRG), which can enter dying cells and is converted into a coloured product by the enzyme. This serves to a) focus the read-out (cell death) on the transfected cells to make it more sensitive and b) normalise for different transfection efficiencies between wells.

As used herein, the term "target cell" refers to cells in which a cell death induction effect is desired. In one embodiment the target cell is a tumour cell; accordingly, in this embodiment the putative therapeutic agent is a putative anti-tumour agent.

Tumour cells interact with other cells such as those of the innate immune response or with stromal cells, infiltrating leukocytes, or cancer-associated fibroblasts in the metastatic niche.

These cells, often in complex interaction with the cancer cells, are activated and sustain the proliferation of the tumour cells though direct interaction with the malignant cells or the paracrine secretion of factors.

Accordingly, in one embodiment the target cells comprise, in addition to at least one tumour cell, cells which are activated and sustain the proliferation of the tumour cells. Preferably the tumour cell is in contact with the cells which are activated and sustain the proliferation of the tumour cells.

In one embodiment the cells which are activated and sustain the proliferation of tumour cells are selected from cells of the innate immune response, stromal cells, infiltrating leukocytes, and cancer-associated fibroblasts in the metastatic niche.

In one embodiment the tumour cell is a naturally occurring tumourigenic cell from a human patient.

In another embodiment the tumour cell is a cell which harbours genetic changes connected with malignant transformation. In particular, the tumour cell may be a cell which has been genetically engineered.

Tumour cells can be distinguished from non-cancerous cells by the acquisition of specific traits, the so-called "hallmarks of cance( (Hanahan and Weinberg, 2000, 2011) (incorporated herein by reference).

These traits can be implemented in the tumour cells by e.g. transfection of genes that mediate these changes such as angiogenic factors, or components of the telomerase for the constitutive telomere activity, anti-senescence genes, or genetic changes that diminish the response to growth inhibiting signals such as TGF-beta. Also, cells can be engineered to mimic other hallmarks of tumour cells such as the epithelial-mesenchymal transition, sustained proliferative signalling, reduced apoptosis and senescence sensitivity, loss of contact inhibition, metastasis formation (by e.g. downregulation of metastasis suppressor genes), genomic instability, avoiding immune attack and inducing tumourpromoting inflammation, and specific metabolic changes such as the switch from respiration to glycolysis (Warburg effect) in metabolic energy reprogramming. Alternatively, genes can be transfected that counteract treatment such as the multiple drug resistance transporter, which is upregulated in treatment-resistant tumour cells.

In one embodiment the tumour cell may be a cell which has been genetically engineered for expressing activated oncogenes, or to express mutated tumour suppressor proteins, or for silencing tumour suppressor genes (e.g. by RNA interference).

In one embodiment the tumour cells are transformed by insertion of an oncogene.

Preferably the oncogene is a human oncogene.

In one embodiment the tumour cells are transformed by insertion of a human oncogene selected from H-ras, K-ras, myc, raf, erbB-2, fos, jun, kit, mdm-2, myb, ret, sis and trk.

In one embodiment the tumour cell expresses mutated suppressor proteins. In one embodiment the mutated suppressor protein is mutated p53. For example, p53 is a tumour suppressor gene and is often mutated (bad subunit") in cancer so that its tetramer is not functional.

In one embodiment, the tumour cells are of human or primate origin in at least one step of the method of the invention. Preferably, the tumour cells are of human or primate origin in at least two steps of the method of the invention. More preferably, the tumour cells are of human or primate origin in at least three steps of the method of the invention. More preferably, the tumour cells are of human or primate origin in all steps of the method of the invention.

In one embodiment the tumour cells are selected from transformed H[K293 cells, cancer stem cells, transformed CV-1 cells, transformed human dermal fibroblasts (NHDF), transformed wild type human fibroblast cells (MRC5), transformed human wild type keratinocytes (HKC), transformed MCF1OA, transformed human lung Wl-38 cells and transformed human Rd-i cells.

in another embodiment the tumour cells are selected from transformed HEK293 cells, transformed CV-1 cells, transformed human dermal fibroblasts (NHDF), transformed wiid type human fibroblast cells (MRCS), transformed human wild type keratinocytes (HKC), transformed MCF1OA, transformed human lung Wl-38 cells and transformed human BJ-1 cells.

In another embodiment the target cell is a virus-infected cell. Accordingly, in this embodiment the putative therapeutic agent is a putative antiviral agent.

Some viruses, for example HIV or Herpes simplex, stay latent in cells for years without leading to virus replication. In such cells, part of the viral genome is still active and ensures that the virus is kept latent. Preferably the virus-infected cell is a latent-virus infected cell.

In one embodiment the virus-infected cell is selected from HIV infected cells, Herpes simplex infected cells, Epstein-Barr virus infected cells, hepatitis B virus infected cells, Cytomegalovirus (CMV) infected cells, Chronic Congenital Rubella infected cells, Herpes simplex virus (HSV) infected cells, Varicella zoster virus (VZV) infected cells, adenovirus infected cells, measles virus infected cells, JC virus infected cells and rubella virus infected cells. Preferably, the preceding viruses are latent in the cell.

As used herein, the term "reference cell" refers to a control cell i.e. cells representing the non-tumourigenic state of the cell, in the situation when the target cells comprise a tumour cell; or cells representing the uninfected state of the cell, in the situation when the target cells comprise a virus-infected cell.

In one embodiment, when the target cells comprise a tumour cell, the reference cells can be selected from primary cells and cells in which the hallmarks of cancer are diminished by genetic or epigenetic means. In particular, reference cells may be cells derived from tumour cells wherein the activating oncogenes are incapacitated or replaced with the respective proto-oncogenes, or mutated oncogenes are replaced with their WT allele, or tumour suppressor genes are re-constituted.

In one embodiment, the reference cells are of human or primate origin in at least one step of the method of the invention. Preferably, the reference cells are of human or primate origin in at least two steps of the method of the invention. More preferably, the reference cells are of human or primate origin in at least three steps of the method of the invention. More preferably, the reference cells are of human or primate origin in all steps of the method of the invention.

In one embodiment the reference cell is a non-tumourigenic, immortalized cell.

In one embodiment the reference cell is a non-tumourigenic, wild type cell.

In one embodiment, when the target cells comprise a tumour cell, the reference cells may be selected from HEK293 cells, CV-1 cells, human dermal fibroblasts (NHDF), human fibroblast cells (MRC5), human keratinocytes (HKC), MCF1OA, human lung Wl-38 cells and human BJ-1 cells.

In one embodiment, at least one of the tumour cells or virus-infected cells is isogenic with at least one of the reference cells. Preferably, at least two of the tumour cells or virus-infected cells are isogenic with at least two of the reference cells. More preferably, at least three of the tumour cells or virus-infected cells are isogenic with at least three of the reference cells.

More preferably, all of the tumour cells or virus-infected cells are isogenic with the reference cells.

As used herein the term "isogenic" means that target cells and reference cells have the same genetic origin and, apart from distinct mutations, the same genetic composition.

Therefore, two cells of the same genetic origin in which one has been transformed and the other not are said to be isogenic.

In one embodiment at least one of the tumour cells is selected from transformed HEK293 cells and at least one of the reference cells is selected from HEK293 cells; or at least one of the tumour cells is selected from transformed CV-1 cells and at least one of the reference cells is selected from wild type CV-1 cells or at least one tumour cells is selected from transformed human dermal fibroblasts and at least one of the reference cells is selected from human dermal fibroblasts or at least one of the tumour cefis is selected from transformed wild type human fibroblast cells (MRC5) and at least one of the reference cells is selected from wild type human fibroblast cells (MRC5) ; or at least one of the tumour cells is selected from transformed MCF1OA and at least one of the reference cells is selected from MCF1OA; or at least one of the tumour cells is selected from transformed human wild type keratinocytes (HKC) and at least one of the reference cells is selected from human wild type keratinocytes (HKC); or at least one of the tumour cells is selected from transformed human lung Wl-38 cells and at least one of the reference cells is selected from human lung Wl-38 cells; or at least one of the tumour cells is selected from transformed human BJ-1 cells and at least one of the reference cells is selected from human BJ-1 cells.

Therapeutic Agents In a second aspect the present invention relates to a therapeutic agent or equivalent thereto identified using the method described herein.

In particular, anti-tumour agents and antivirus agents identified can be developed into therapeutics.

Anti-tumour genes and antkvirus genes can be developed into therapeutics by a range of avenues such as viruses that express them in cells, fusion proteins with cell-penetrating (tat) peptides, or hooked up to cell-penetrating nanoparticles. Consequently, the most important aspect of anti-tumour genes and antivirus genes is the expectation that they can be used directly against tumours and viruses respectively as they constitute therapeutically useful factors.

The present invention also extends to agents which may be derived from such anti-tumour and antivirus genes such as the anti-tumour and antivirus proteins which they express.

Pharmaceutical Compositions In a third aspect the present invention relates to a pharmaceutical composition comprising a therapeutic agent identified using the method described herein and a pharmaceutically acceptable excipient.

In a fourth aspect the present invention relates to a process for preparing a pharmaceutical composition, said process comprising admixing a therapeutic agent identified using the method described herein or an equivalent thereto, optionally in the form of a vehicle, with a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as -or in addition to -the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.

Where the pharmaceutical composition is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose or chalk, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

Administration Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient and severity of the condition. The dosages below are exemplary of the average case.

There can, of course be individual instances where higher or lower dosage ranges are merited.

The compositions (or component parts thereof) of the present invention may be administered orally. In addition or in the alternative the compositions (or component parts thereof) of the present invention may be administered by direct injection. In addition or in the alternative the compositions (or component pads thereof) of the present invention may be administered topically. In addition or in the alternative the compositions (or component parts thereof) of the present invention may be administered by inhalation. In addition or in the alternative the compositions (or component parts thereof) of the present invention may also be administered by one or more of: parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration means, and are formulated for such administration.

By way of further example, the pharmaceutical composition of the present invention may be administered in accordance with a regimen of 1 to 10 times per day, such as once or twice per day. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

The term administered" also includes but is not limited to delivery by a mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution; a parenteral route where delivery is by an injectable form, such as, for example, an intravenous, intramuscular or subcutaneous route.

Hence, the pharmaceutical composition of the present invention may be administered by one or more of the following routes: oral administration, injection (such as direct injection), topical, inhalation, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration or transdermal administration.

Medical uses In a fifth aspect the present invention relates to therapeutic agents or pharmaceutical compositions identified using the method described herein for use as a med icament.

In a sixth aspect the present invention relates to the anti-tumour agents identified using the method described herein or pharmaceutical compositions comprising anti-tumour agents identified using the method described herein for use in the treatment of cancer.

Examples of types of cancer, include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, tumours (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, Iymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, serninoma, embryonal carcinoma, Wilms' tumour, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

In a seventh aspect the present invention relates to the anti-viral agents identified using the method described herein or -pharmaceutical compositions comprising anti-viral agents-identified using the method described herein for use in the treatment of viral infection.

Examples of viral infection, include but are not limited to, HIV, Herpes simplex, Epstein-Barr virus, hepatitis B virus, Cytomegalovirus (CMV), Chronic Congenital Rubella, -Latent Herpes simplex virus (HSV), Varicella zoster virus (VZV), adenovirus, measles virus, John Cunningham (JC) virus and rubella virus.

Material and Methods Screening The various steps of the screening procedure were performed as described using our custom-made robots (Albayrak and Grimm, 2003; Albayrak et al., 2003; Grimm and Kachel, 2002) The DNA isolation robot performed a DNA isolation protocol that yields exceptionally pure DNA that can be transfected efficiently into cells and does not generate background cell death -as described in (Kachel et al., 2006) (incorporated herein by reference).

Screening in the target cells was done in duplicates. Only those genes that are active in both assays were regarded as positive.

In one embodiment the tumour cells are kept under conditions which mimic the tumour environment. For example in low pH, hypoxia and/or high lactate concentration conditions.

An expression vector for luciferase was used as negative control. The integrity of the cell membrane was probed by adding the beta-galactosidase substrate CPRG, which can enter dying cells and is converted into a coloured product by the enzyme. The cells are then lysed by TritonX-100 in order to normalize for the transfection efficiency (Grimm and Kachel, 2002)ncorporated herein by reference). For the read-out we made use of the FLUOROstar microplate reader from BMG Labtech and the data were processed by the Windows-based OPTIMA software on a DELL Dimension 3100 computer.

Plasmid DNA isolation A 96-well block containing 900 p1 of LB medium with bacteria of an 0/N culture in each well was centrifuged for 5 mm at S000xg (SIGMA Zentrifugen, Osterode am Harz, Germany), the supernatant was removed, 170 p1 Fl buffer (50 mM Tris-HCI/ 10 mM EDTA pH 8,0) were added, and the bacteria pellets were completely resuspended. 170 p1 F2 buffer (200 mM NaOH, 1% SOS) were added, mixed and incubated at RI for 5 mm. 170 p1 of 4°C cold buffer F3 (3 M KAc, pH 5,5) terminated the lysis. The 96-well block was shaken to mixe the components. 10 p1 of a RNaseA solution (1,7 mg/mI) were added to digest excessive RNAs, incubated for 5 mm at RT and then at -20°C (10 mm) to efficiently precipitate proteins and genomic DNA. The solution was then again centrifuged for 10 mm at 6000 rpm. The supernatant was transferred in new blodks, the samples received 100 p1 buffer F4 (2,5% SOS in isopropanol) to precipitate LPS. The block was vortexed for 5 mm and incubated first at 4°C for 15 mm and then at -20°C for 15 mm. The block was again centrifuged for 10 mm at 6000 rpm, and the supernatant was added to robust, custom-made 96weII polyoxymethylene (POM) microtiter blocks. 150 p1 silica oxide suspension were added and incubated for 20 mm at RI. The plates were centrifuged for 5 mm at 6000 rpm, the supernatant was carefully decanted, and 400 p1 wash solution (acetone or 75% isopropanol, -20°C) were added. The plates were again vortexed (30 sec) and centrifuged for 3 mm at 6000 rpm. The wash step was repeated once. The pellets were first dried at RT for 5 mm.

They were subsequently resuspended in 75 p1 LPS-free water (60°C) by vortexing 30 sec.

and centrifuged at max. speed (6000 rpm) for 10 mm at 4°C to pellet the silica oxide. The supernatant contained the isolated plasmid DNA.

Transfection of Cells CV-1 cells, a wild type monkey kidney cell line whose transfection efficiency was transfected with a modified PEI protocol or with the calcium phosphate coprecipitation method as described (4). Transfection efficiencies were assayed with the plasmid pGreen Lantern (Gibco BRL Gaithersburg, USA) encoding the green fluorescent protein (1).

Robots Transfection robot To establish a fast and less costly alternative to existing pipetting heads, we developed a 96-fold head that is based on the linear tube-pump principle. A set of parallel silicone tubes is mounted on a linear tube holder. A roller is connected to an up-and-down moveable slide and a pneumatic element that can press the roller onto the tubes until they are closed. Fluid is taken up by moving the pressed roller upward, which allows the tubes to work like syringes with pistons. The upper ends of the tubes are connected to a pressure/suction vessel. The pressure in this vessel is controlled by four valves. For tube-pump pipetting", the vessel is connected to 0.02 bar pressure. By releasing the roller from the tubes, this pressure blows out the content of the pipetting tips into the wells without forming droplets at the tips. Alternatively, in the "suction-pressure" mode, the uptake and release of fluid is controlled by the pressure in the vessel and the releasing time of the roller, which works as a multi-valve. This facilitates the uptake of large volumes up to the millilitre range or the distribution of small or large volumes from the fluid-filled vessel. This supports the use of the head in various protocols (e.g., when the cell culture medium must be changed).

The 96-tube pipetting head was built with four tube-holder plates of 24 tubes in a parallel array together with four simultaneously operating rollers and was mounted on the z-slide of the gantry robot system. The bottom ends of the tubes are connected to commercial pipet tips that are arranged in the format of a 96-well microplate. This pipetting head is fully movable over the complete range of the area that is covered by the microplates. The pipetting volume range in the linear tube-pump mode is 5-100 jiL/channel. The head works above 10 pL with an accuracy of less than 5% standard deviation, as tested in several pipetting trials with distilled water. A very similar result was obtained when cell culture medium with 10% FCS was used.

The gantry robot system has a platform size of 140 x o cm that provides space for 60 96-well microplates. The plates are arranged in a row and column structure that makes the identification of the plates easy and allows one to work without bar codes. The plates are deposited into subplatforms. The first subpiatform is cooled to 4°C and takes up the 20 DNA-containing plates. The second subplatform comprises 20 microplates for preparing, mixing, and incubating the DNA before transfection. The third platform can be heated to 37°C and holds the microplates with the cell cultures. The vessels that contain the transfection fluids and the automatic tip wash station are placed between the first and the second subplatform. The washer consists of 96 vertically mounted plastic tubes arranged in the format of a 96-well microplate. The pipetting tips are immersed into the tubes to clean them inside and outside.

The cell culture plates on the platform are covered by protection lids that have to be removed for transfection. Consequently, a de-lidding device with a vacuum cup was integrated into the pipetting head. The lids are moved with the head and repositioned on their microplates immediately after the transfection.

Commercial robot systems that take and replace microplates from and into an incubator lose a considerable amount of time transporting the plates. In contrast, the platform of our robot system provides enough space for 60 laid-out 96-well microplates. The movable pipetting head has direct access to all these plates at any time. Thus, by optimally interlacing the pipetting and incubation processes of the protocol, the system is nearly in permanent full-speed operation. A platform of 1920 cDNAs can be processed in 45 mm (i.e., a mean transfection throughput of about 2000 samples/h or 40 000-50 000 sam-pIesJ24 h can be reached)-.

DNA isolation robot The robot was specifically tailored to the needs of our particular DNA isolation prOtocol, which uses alkaline lysis of bacteria and binding of plasmid DNA to silica oxide (Grimm and Voss-Neudecker, 2003), but deviates from conventional protocols in that (i) it does not include chaotropic substances for the binding of DNA to silica oxide, (ii) it removes LPS (endotoxin) by co-precipitation with SDS in isopropanol and (iii) it eliminates further residual impurities by an acetone wash. Besides the integration of the computer-controlled centrifuge, a number of additional modifications had to be introduced compared with the original manual protocol to accommodate for the requirements of a robotic system. The transfer of the supernatant, for example, which was formerly accomplished by tilting the plates, is now performed by the pipetting head. The mixing of solutions and the resuspension is done by dedicated mixing platforms instead of a vortex machine. For the drying of the silica pellets we switched from incubations in temperature-controlled incubators to heated platforms and blowing air from the 96-well pipettor. The acetone washing process is now performed by a designated set of pipettors. For cooled incubation, lidded containers were established on the platform, which substitute the refrigerator incubations in the previous protocol. During a single run, incubation-and centrifugation times sum up to about minutes time of robot inactivity. This time is efficiently used by running a second, interlaced process. In this way, the overall processing time of one run is extended from 240 to 270 mm, the throughput, however, is doubled to 768 samples. Prior to the run the platforms A3 and 33 are manually supplied with eight DWP5 containing LB medium with recombinant bacteria. Platforms Al, 31 and A2, B2 are filled with empty DWPs into which the solutions with the plasmid DNAs at various stages of the purification process are transferred. The platforms Ae and Be contain empty 96-well microtitter plates (MTPs) to store the final DNA samples. For simplicity only the process of one group of plates is described here. The run starts with the transport of four DWPs from platform A3 to the centrifuge-After one carrier is loaded the centrifuge automatically turns the rotor to the next position and arrests it by the insertion of a bolt. The gripper can then deposit the next DWP at defined coordinates. After centrifugation the plates are transferred to the pipetting station S1/S2 and the supernatants are removed from the bacteria pellets by a designated pipetting head. The plates are moved to the platform A3 and solution P1 is added by the movable pipettor. The four DWPs are fixed on the platform, which is pneumatically set into linear vibration with pre-selected frequency and duration to re-suspend the bacteria pellet. When the fixing bolts are released, the platform is set to its stop coordinates for access by the pipetting head. After addition of solution P2, gentle shaking and incubation for 5 mm, P3 is added, the solutions are incubated at -20°C for S mm, mixed again and after centrifugation the supernatant is pipetted to a new plate (A2). P4, which contains SDS in isopropanol, is added, mixed and the plates are incubated first at 4°C (15 minutes) on platform A4 and then at -20°C (15 minutes) on platform A-20. After another centrifugation step the supernatants are removed and added to new DWPs on platform Al. Silica oxide is dispensed, mixed and the interaction with the plasmid DNA facilitated by incubation at RT for 20 minutes. The plates are again centrifuged and washed twice with acetone with removal of the supernatant at each step. Drying the silica pellets by blowing air into the 96-well plates through the pipetting head, their re-suspension in water (150 p1) and the subsequent centrifugation yields the pure plasmid DNA in the supernatant, which is pipetted into MPTs on platform Ae.

The gantry system A five axis-gantry robot with stepping motors (working field xl6Ocm y=9Ocm, z=25cm) moves the main pipetting head mounted on the z-sled (Zi, see supplementary information).

The 96-tip pipettor is the same as the one used in our transfection robot (Grimm and Kachel, 2002)(incorporated herein by reference). It has access to aD platform positions. Its movable rollers are controlled by the 4th axis of the gantry system (72). The 5th axis moves the Sl/S2 pipettors up and down (73). The five axes are driven by the gantry robot controller, which is operated by ASCII strings from the PC via the seriall interface. The resolution of the x-and y-axis is 10 steps/mm, that of the spindle-driven is z-axes 80 steps/mm. Maximum speed is 8000 steps/sec. A pneumatically operated gripper for the transport of the 96-well plates between the locations of the working platform is attached to the Zl-sled immediately behind the pipetting head-A two-step z-move is used for the operation of the gripper. The zl sled moves the gripper to the zi base position and a built-in pneumatic cylinder drives it further down a fixed distance of 20cm to the DWP-gripping or -releasing position.

Wash station for pipe tting tips The tips of the 96 main pipetting head are manually plugged onto Gilford type holder cones and arranged in an 8x12 matrix-of 9mm tip distanca The tips are cleaned in a tip washing station in-between pipetting tasks. The washing station consists of 96 vertical tubes through which cleaning water can be pumped upwards. For cleaning, the 96 tips of the head are inserted into the washing tubes. When the water pump is switched on, the up-flowing water cleans the outside of the tips. Simultaneously, cleaning fluid is sucked into the interior of the tips. The contaminated water overflowing at the top of the tubes is drained away into a waste container. The tips are moved out of the washing tubes and positioned over the gaps between the tubes where the contaminated fluid is blown out and drained away. Such cleaning cycles can be programmed as required, each lasting about 5 seconds. As fresh water is used only when the tips are inserted into the washing tubes, it ensures low consumption of cleaning water.

PC-control/ed centrifuge A PC-controlled cooled centrifuge (Sigma 4K15 robotic; Sigma, Osterode, Germany) for the simultaneous centrifugation of four 96-well DWPs was incorporated into the robot. The centrifuge is controlled by ASCII-string commands via the serial 2 interface of the PC.

Automatic loading and unloading is performed through a hatch in the centrifuge lid. At the start of the centrifugation process the centrifuge is checked for imbalance. If an imbalance has been detected the centrifuge stops, an alarm is triggered and the program execution is set to "wait'. The system supervisor may then manually balance the DWPs and continue the process execution.

PipeWng station S1/S2 The z-motion (Z3) of the three pipetting heads of the station 51/52 is controlled by the gantry robot controller. They work in accordance with the "suction-pressure" principle. Suction and pressure are generated in a shared air tube, which branches out into the 96 pipetting tips.

This simple principle allows the dispensing and take-up of equal volumes of fluid and works well with tubes of identical length between the common tube and the pipetting tips. The gripper transport system transfers the DWPs to the pipetting station platforms Si and S2.

Platform 52 serves as suction station independent of the main pipetting head. It is used for removing the high volume of bacteria supernatant after the first centrifugation. On platform Si acetone is dispensed and removed to wash the plasmid DNA5. The 96-fold pipetting head Si a and the joined heads Sib and S2a are swung out when a DWP is delivered by the gripper and placed on Si. The cover is opened, the cooled acetone vessel moves up and the pipetting head Sla is lowered until the tips are immersed into the acetone. By a short suction pulse about 200p1 acetone is taken up by the tips. The head is moved up, swung over the DWP, moved down until the tips enter the top of the wells and the acetone is blown out by a smooth pressure puke. Cooling of the acetone is required in order to reduce the acetone vapour pressure. For acetone removal the DWP is also placed on Si, the head Sib is moved over the DWP, lowered down until the tips are immersed into the acetone supernatant and the supernatant is taken up by a short suction pulse. Sib is moved up, swung over the waste uptake 81w and the content of the pipetting tips is blown out The number of suctions, the depth of immersion and the strength and duration of the suction pulses are experimentally determined. The complete acetone washing process consists of: Transport of the DWPs containing the silica pellets to the Si platform (removal of acetone if acetone has been added in a previous step), addition of 400pl acetone, transport to the A3/B3 platform, shaking and transport to the centrifuge for centrifugation.

iS Fluid supply vessels The six fluid supply vessels P1 -P6 consist of delrin plastic holders and polyetylene insert vessels, which can be filled, emptied and cleaned outside the robot. All vessels are covered by pneumatically operated lids. The functional space (FSP) below the insert vessels allows the insertion of additional elements like stirrers, coolers and heaters. The FSPs of P4 and P5 contain electronic stirrers, P3 is equipped with a cooling element that is supported by the 4°C cooler. P6 contains an electrically driven 60°C heater. The FSPs of P1 and P2 are empty as unstirred fluids at room temperature are provided. The insert vessels of P3 and P6 are made of stainless steel for improved thermal contact.

Safety features of the robot Operator collisions with the moving robot elements are dangerous. Therefore, the work area is enclosed using transparent macrolon safety screens. If these screens are opened during a run, processing is immediately paused and an alarm is triggered until the windows are closed again. The correct program execution is controlled by numerous error detectors on the head, the gripper, the platforms, the thermometers and manometers. Malfunctions immediately trigger an alarm via the parallel I/O bits. The system is set to wait until the operator decides how to proceed. A coordinate test of the movable pipettor ensures correct positioning and prevents collisions. One of the pipetting tips activates a test pin at defined coordinates within the work area. If the pin is not hit an x-y failure alarm is triggered. The accuracy of the z-axis is tested via a micro switch operated via the pin. By stepwise upward movement of the tip the system checks whether the micro switch switches back at the expected z test coordinate. The manual activation of an emergency switch by the operator immediately stops the movement of the gantry axes by disconnecting the power supply.

During acetone washing and evaporation dedicated ventilation at the 311S2 and the Al and Bi platforms minimizes the dangerous concentrations of acetone in the work area.

D

Software for system processing and teaching The PC operating system is Windows 2000 Professional. The programmes for BASY96 system processing and teaching are written in Agilent VEE, a graphical programming language. The program is structured into about 340 functions (subroutines), which are repeatedly called during the process execution. The processing software used is dedicated to the control of the two interlaced high-throughput isolation processes. Programme changes by the user are not supported. A separate teaching programme can be used to define the x, y, zi, z2, z3 coordinates of the fluid vessels and the coordinates of the DWP platforms and the centrifuge.

Cooling platforms and cooling fluid Cooling to temperatures below 0°C requires prevention of water condensation and ice generation. For low temperature processing tasks, a special 4°C cooling box and a -20°C compartment are installed on the work area. Each compartment is constructed as a cooling tray. It is isolated using hard foam and provides space for S DWPs. The tray structure prevents condensation caused by airflow and allows access of the gripper from the top. The top of the -20°C compartment is covered with an automatically operated lid.

Shaking platforms As the centrifuge allows the simultaneous centrifugation of 4 DWPs, the platforms of the work area are likewise designed for the uptake of 4 DWPs. To avoid time-consuming transport and queues by having only one dedicated shaking station for the frequent shaking steps during processing the platforms that require shaking were designed as shaking platforms. Both shaking and locking of the DWPs is performed by pneumatic cylinders.

Each An/Bn shaking platform pair has an electronic hardware controller where the pre-selection of shaking frequencies is set. Via the PC program two pre-defined shaking frequencies can be activated by setting or resetting two control bits. When switched on, the shaking runs independently of the PC until switched off. In between the program can attend to other tasks on the work plafform. The AlIBI shaking platforms are additionally equipped with heating foils for speeding up the evaporation of acetone.

PC and electronic hardware The PC hardware consists of a midi tower standard IBM compatible personal computer with a Pentium 4 CPU. The 5-axis gantry system (ELBAG Electronics, Weisel, Germany) and the communication with the centrifuge are controlled via the serial 1 port and the serial 2 port, respectively. Two additional 48 bit parallel I/O interface boards (ME 14008, Meilhaus Electronic, Puchheim, Germany) are connected to the PCI bus. The 96 control bits programmed as outputs or inputs are used for controlling the function of the pipetting head, the gripper, the platforms, the fluid vessels, the 51/52 station and the pipetting tip washing station. Additional interface boxes between the I/O boards and the work area house general buffer amplifiers, the shaking platform electronics and the 5V to 24V output level converters.

A ten button operation box allows the manual up-and down-motion of the five gantry axes and is used for teaching the system.

Rood-out Because caspases and proapoptotic cysteine proteases are involved in many but not all kinds of apoptosis induction, we wanted to use markers of cell death other than the activation of these proteases. Thus, we explored the use of established reporter enzymes that have a proven record of robustness and the advantage that there are many different substrates described. The apoptosis reporter assay is based on the observation that, while the overall integrity of the plasma membrane is not altered in apoptosis, the membrane becomes permeable for low molecular weight substances. Therefore, we reasoned that substrates for enzymes such as -galactosidase could enter apoptotic cells, be metabolized, and be detected by an optical read-out. To test this, we have transfected 2931 cells with a constitutively active expression plasmid for -galactosidase together with the apoptosis inducer ANT-i or an empty control vector. On addition of the substrate CPRG to the medium, the wells with apoptotic cells displayed a greater than 5-fold increase in the turnover of the substrate in comparison with the control-transfected cells. A similar effect was seen when this experiment was performed in MCF7 cells (data not shown). To verify our hypothesis that the CPRG substrate that entered the dying cells rather than the enzyme was released from these cells, we saved the supernatant. Upon its incubation, we did not observe any further turnover of the substrate. To test whether the cells in the CPRG-negative wells efficiently took up the reporter DNA, the transfection efficiency was controlled by a subsequent Triton® X-100 lysis. This released the enzyme from the cells and led to a metabolism of the substrate even in samples without apoptosis induction. This resulted in an equal increase in the A590 between ANT-1-transfected cells (A0.36) and the control-transfected cells (A0.41) in the same time period.

Test of the Reporter Plasmids with the Robotic System We co-transfected the reporter plasmids together with a control vector or the apoptosis inducing-gene ANT-I using the transfection robot. The f3-galactosidase activity assay allowed the detection of the wells in a 96-well plate in which the proapoptotic ANT-i plasmid was co-transfecte& After the addition of CPRG, the A590 of between 0.81 and 0.98 were clearly distinguishable from the wells without an apoptosis inducer. Triton X-iOO lysis revealed that the cells in the CPRG-negative wells had nevertheless been efficiently transfected with the reporter plasmid.

The invention will now be described by means of the following numbered paragraphs.

1. A method comprising the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference celL 2. The method according to claim 1 wherein said method further comprises the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a second tumour cell or second virus-infected cell; selecting said putative therapeutic agent if it also has a cell death induction effect on said second tumour cell or second virus infected cell.

3. The method according to any one of claims 1 and 2 wherein the method further comprises the steps of: determining if a putative therapeutic agent has a cell death induction effect on a second reference cell; selecting said agent if it also has no cell death induction effect on said second reference cell.

4. A method consisting essentially of the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cell.

5. The method according to any one of the preceding paragraphs wherein the tumour cell harbours genetic changes connected with malignant transformation.

6. The method according to any one of the preceding paragraphs wherein the tumour cells are manipulated to display one or more of the hallmarks of cancer cells such as metabolic energy reprogramming, constitutive telomere activity, sustained proliferative signaling, epithelial-mesenchyrnal transition, reduced apoptosis and senescence sensitivity, reduced responsiveness to growth inhibition factors, loss of contact inhibition, genomic instability, treatment resistance, avoiding immune attack and inducing tumour-promoting inflammation, and secretion of angiogenic factors.

7. The method according to any one of the preceding paragraphs wherein the tumour cell is genetically engineered for expressing activated oncogenes, or to express mutated tumour suppressor proteins or for silencing tumour suppressor genes; preferably the tumour cell expresses mutated suppressor proteins.

8. The method according to any one of the preceding paragraphs wherein the virus-infected cell is a latent virus-infected cell.

9. The method according to any of the preceding paragraphs wherein the at least one of the tumour cells or virus-infected cells is of human or primate origin.

10. The method according to any one of the preceding paragraphs wherein the tumour cells are of human origin, 11. The method according to any one of the preceding paragraphs wherein at least one of the reference cells is of human or primate origin.

12. The method according to any one of the preceding paragraphs wherein the reference cells are of human origin.

13. The method according to any one of the preceding claims wherein the tumour cells are selected from transformed HEK293 cells, cancer stem cells, transformed CV-1 cells, transformed human dermal fibroblasts (NHDF), transformed wild type human fibroblast cells (MRC5), transformed human wild type keratinocytes (HKC), transformed MCF1OA, transformed human lung WI-SO cells and transformed human BJ-1 cells.

14. The method according to any one of the preceding paragraphs wherein the tumour cell is a cancer stem cell.

15. The method according to any one of the preceding. paragraphs wherein the tumour cell is kept in cell culture conditions which mimic the tumour environment.

16. The method according to any one of the preceding paragraphs wherein the tumour is kept in low pH, hypoxia and/or high lactate concentration cell culture conditions.

17. The method according to any one of the preceding paragraphs wherein the virus infected cell is selected from HIV infected cells, Herpes simplex infected cells, Epstein-Barr virus infected cells, hepatitis B virus infected cells, Cytomegalovirus (CMV) infected cells, Chronic Congenital Rubella infected cells, Latent Herpes simplex virus (HSV) infected cells, Varicella zoster virus (VZV) infected cells, adenovirus infected cells, measles virus infected cells, John Cunningham (JC) virus infected cells and rubella virus infected cells.

18. The method according to any one of the preceding paragraphs wherein the reference cell is selected from a wild type cell, a primary cell and a genetically inactivated tumour cell.

19. The method according to any one of the preceding paragraphs wherein the reference cell is an uninfected cell.

20. The method according to any one of the preceding paragraphs wherein the reference cell is an immortalized cell.

21. The method according to any of the preceding paragraphs wherein the reference cells are cells derived from tumour cells wherein the activating oncogenes are incapacitated or replaced with the respective proto-oncogenes or tumour suppressor 22. The method according to any one of the preceding paragraphs wherein at least one of the reference cells is selected from HEK293 cells, CV-1 cells, human dermal fibroblasts (NHDF), human fibroblast cells (MRC5), human wild type keratinocytes (HKC), MCF1OA, human lung Wl-38 cells and human BJ-1 cells.

23. The method according to any one of the preceding paragraphs wherein at least one of the tumour cells is selected from transformed HEK293 cells and at least one of the reference cells is selected from HEK293 cells or wherein at least one of the tumour cells is selected from transformed CV-1 cells and at least one of the reference cells is selected from CV-1 cells or wherein at least one of the tumour cells is selected from transformed human dermal fibroblasts and at least one of the reference cells is selected from human dermal fibroblasts; or wherein at least one of the tumour cells is selected from transformed wild type human fibroblast cells (MRC5) and at least one of the reference cells is selected from wild type human fibroblast cells (MRC5) or wherein at least one of the tumour cells is selected from transformed MCF1OA and at least one of the reference cells is selected from MCF1OA; or wherein at least one of the tumour cells is selected from transformed human wild type keratinocytes (HKC) and at least one of the reference cells is selected from human wild type keratinocytes (l-IKC); or wherein at least one of the tumour cells is selected from transformed human lung Wl-38 cells and at least one of the reference cells is selected from human lung WI-38 cells; or wherein at least one of the tumour cells is selected from transformed human Ri-i cells and at least one of the reference cells is selected from human BJ-1 cells.

24. The method according to any one of the preceding paragraphs wherein at least one of the tumour cells or virus-infected cells is isogenic with at least one of the-reference cel Is.

25. The method according to any one of the preceding paragraphs wherein all of the tumour cells or virus-infected cells are isogenic with the reference cells.

26. The method according to any one of the preceding paragraphs wherein the tumour cells are transformed by insertion of a human oncogene.

27. The method according to any one of the preceding paragraphs wherein the oncogene is selected from H-ras, K-ras, myc, raf, erbB-2, fos, jun, kit, mdm-2, myb, ret, sis and trk.

28. The method according to any one of the preceding paragraphs wherein the target cells comprise cells which are activated and sustain the-proliferation of the tumour cel Is.

29. The method according to any one of the preceding paragraphs wherein the target cells are in contact with cells which are activated and sustain the proliferation of the.* tumour cells.

30. The method according to any one of the preceding paragraphs wherein the target cells comprise cells selected from cells of the innate immune response, activated stromal cells, infiltrating leukocytes and cancer-associated fibroblasts and selection of the putative therapeutic agent is also dependent on cell death induction in these cells.

31. The method according to any one of the preceding paragraphs wherein the target cells are in contact with cells of the innate immune response, activated stromal cells, infiltrating leukocytes and cancer-associated fibroblasts.

32. The method according to any one of the preceding paragraphs wherein the putative therapeutic agent is a nucleic acid sequence.

33. The method according to any one of the preceding paragraphs wherein the putative therapeutic agent is a DNA sequence from a library.

34. The method according to any one of the preceding paragraphs wherein the putative therapeutic agent is in plasmid form.

35. The method according to any one of the preceding paragraphs wherein the plasmid DNA library consists of genomic or cDNA sequences from various species, random or selected DNA fusion constructs.

36. The method according to any one of the preceding paragraphs wherein cell death induction is detected with the aid of a reporter agent.

37. The method according to any one of the preceding paragraphs wherein cell death induction is detected with the aid of a reporter enzyme, preferably beta-galactosidase.

38. The method according to any preceding paragraph which is performed at least 50 times in parallel, preferably at least 75 times, more preferably at least 100 times, more preferably at least 200 times.

39. The method according to any preceding paragraph which is performed by robots.

40. The method according to any preceding paragraph wherein cell death induction is caused by apoptosis, necrosis, necroptosis, or excessive autophagy.

41. A therapeutic agent or equivalent thereto identified using the method according to any one of the preceding paragraphs.

42. A pharmaceutical composition comprising a therapeutic agent according to any one of the preceding paragraphs and a pharmaceutically acceptable excipient.

43. A process for preparing a pharmaceutical composition, said process comprising admixing an therapeutic agent identified using a method according to any one of the preceding paragraphs or an agent equivalent thereto, optionally in the form of a vehicle, with a pharmaceutically acceptable excipient.

44. A therapeutic agent or pharmaceutical composition according any one of the preceding paragraphs for use as a medicament.

45. A therapeutic agent or pharmaceutical composition according to any one of the preceding paragraphs for use in the treatment of cancer.

46. A therapeutic agent or pharmaceutical composition according to any one of the preceding paragraphs for use in the treatment of a viral infection Although the foregoing compositions and methods have been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

REFERENCES

Albayrak, T., and Grimm, S. (2003). A high-throughput screen for single gene activities: isolation of apoptosis inducers. Biochem Biophys Res Commun 304, 772-776.

Albayrak, T., Scherhammer, V., Schoenfeld, N., Braziulis, E, Mund, T., Bauer, MK., Scheffler, I.E., and Grimm, S. (2003). The Tumor Suppressor cybL, a Component of the Respiratory Chain, Mediates Apoptosis Induction. Mol Bid Cell 14, 3082-3096.

Grimm, S., and Kachel, V. (2002). Robotic high-throughput assay for isolating apoptosis-inducing genes. Biotechniques 32, 670-677.

Grimm, S., and Voss-Neudecker, F. (2003). High-Purity Flasmid Isolation Using Silica Oxide.

Methods Mol Biol 235, 83-88.

Hanahan, D., and Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100, 57-70.

Hanahan, 0., and Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. Cell 144, 646-674.

Kachel, V., Sindelar, G., and Grimrri, 5. (2006). High-throughput isolation of ultra-pure plasmid DNA by a robotic system. BMC Biotechnol 6, 9.

Claims (26)

1. CLAIMS1. A method comprising the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cell.
2. 2. The method according to claim 1 wherein said method further comprises the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a second tumour cell or second virus-infected cell; selecting said putative therapeutic agent if it also has a cell death induction effect on said second tumour cell or second virus infected, cell.
3. 3. The method according to any one of claims 1 and 2 wherein the method further comprises the steps of: determining if a putative therapeutic agent has a cell death induction effect on a second reference cell; selecting said agent if it also has no cell death induction effect on said second reference cell.
4. 4. The method according to any one of the preceding claims wherein the tumour cell harbours genetic changes connected with malignant transformation.
5. 5. The method according to any one of the preceding claims wherein the tumour cell is genetically engineered for expressing activated oncogenes, or to express mutated.tumour suppressor proteins or for silencing tumour suppressor genes; preferably the tumour cell expresses mutated suppressor proteins.
6. 6. The method according to any one of the preceding claims wherein at least one of the S tumour cells or virus-infected cells is of human or primate origin.
7. 7. The method according lo any one of the preceding claims wherein at least one of the reference cells is of human or primate origin.
8. 8. The method according to any one of the preceding claims wherein at least one tumour cells or virus-infected cells is isogenic with at least one of the reference cells.
9. 9. The method according to any one of the preceding claims wherein the tumour cells are selected from transformed HEK293 cells, cancer stem cells, transformed CV-1 cells, transformed human dermal fibroblasts (NHDF), transformed wild type human fibroblast cells (MRC5), transformed human wild type keratinocytes (HKC), transformed MCF1OA, transformed human lung Wl-38 cells and transformed human BJ-1 cells.
10. 10. The method according to any preceding claims wherein the target cells are kept in cell culture conditions which mimic the tumour environment.
11. 11. The method according to any one of claims 1 to 3 and 6 to 8 wherein the latent virus infected cell is selected from HIV infected cells, Herpes simplex infected cells, Epstein-Barr Virus infected cells, hepatitis B virus infected cells, Cytomegalovirus (CMV) infected cells, Chronic Congenital Rubella infected cells, Latent Herpes simplex virus (HSV) infected cells, Varicella zoster virus (VZV) infected cells, adenovirus infected cells, measles virus infected cells, John Cunningham (JO) virus infected cells, rubella virus infected cells.
12. 12. The method according to any one of the preceding claims wherein at least one of the reference cells is selected from a wild type cell, primary cell, an immortalized reference cell and an genetically inactivated tumour cell or an uninfected cell.
13. 13. The method according to any of the preceding claims wherein the reference cells are cells derived from tumour cells wherein the activating oncogenes are incapacitated or replaced with the respective proto-oncogenes or tumour suppressor genes are re-
14. 14. The method according to any one of the preceding claims wherein at least one of the reference cells is selected from HEK293 cells, CV-1 cells, human dermal fibroblasts (NHDF), wild type human fibroblast cells (MRCS), human wild type keratinocytes (HKC), MCFI OA, human lung Wl-38 cells and human BJ-1 cells.
15. 15. The method according to any one of the preceding claims wherein at least one of the tumour cells is selected from transformed HEK293 cells and at least one of the reference cells is selected from HEK293 cells; or wherein at least one of the tumour cells is selected from transformed CV-1 cells and at least one of the reference cells is selected from CV-1 cells or wherein at least one of the tumour cells is selected from transformed human dermal fibroblasts and at least one of the reference cells is selected from human dermal fibroblasts or wherein at least one of the tumour cells is selected from transformed wild type human fibroblast cells (MRC5) and at least one of the reference cells is selected from wild type human fibroblast cells (MRC5) or wherein at least one of the tumour cells is selected from transformed MCF1 DA and at least one of the reference cells is selected from MCF1 OA; or wherein at least one of the tumour cells is selected from transformed human wild type keratinocytes (HKC) and at least one of the reference cells is selected from human wild type keratinocytes (HKC); or wherein at least one of-the tumour cells is selected from transformed human lung Wl-38 cells and at least one of the reference cells is selected from human lung Wl-38 cells; or wherein at least one of the tumour cells is selected from transformed human BJ-t cefis and at least one of the reference cells is selected from human BJ-1 cells.
16. 16. The method according to any one of the preceding claims wherein the tumour cells are transformed by insertion of a human oncogene.
17. 17. The method according to any one of the preceding claims wherein the oncogene is selected from H-ras, K-ras, myc, raf, erbB-2, fos, jun. kit, mdm-2, myb, ret, sis and trk.
18. 18. The method according to any one of the preceding claims wherein the target cells comprise cells which are activated and sustain the proliferation of the tumour cells and selection of the putative therapeutic agent is also dependent on cell death induction in these cells.
19. 19. The method according to any one of the preceding claims wherein the target cells comprise cells selected from cells of the innate immune response, activated stromal cells, infiltrating leukocytes and cancer-associated fibroblasts and selection of the putative therapeutic agent is also dependent on cell death induction in these cells.
20. 20. The method. according to any one of the preceding claims wherein the putative therapeutic agent is a nucleic acid sequence.
21. 21. The method according to any one of the preceding claims wherein the putative therapeutic agent is in plasmid form.
22. 22. The method according to any one of the preceding claims wherein cell death induction is detected with the aid of a reporter agent; preferably reporter enzymes.
23. 23. The method according to any one of the preceding claims which is performed by robots.
24. 24. A therapeutic agent or equivalent thereto identified using the method according to any one of claims ito 23.
25. 25. A pharmaceutical composition comprising a therapeutic agent according to claim 24 and a pharmaceutically acceptable excipient.
26. 26. A process for preparing a pharmaceutical composition, said process comprising admixing an anti-tumour agent identified using a method according to any one of claims 1 to 23 or an anti-tumour agent equivalent thereto, optionally in the form of a vehicle, with a pharmaceutically acceptable excipient.Amendments to the claims have been filed as followsCLAIMS1. A method comprising the steps of: determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a first tumour cell or first virus-infected cell; determining if said putative therapeutic agent has a cell death induction effect on a first reference cell; selecting said putative therapeutic agent if it has a cell death induction effect on said first tumour cell or first virus-infected cell, and has no cell death induction effect on said reference cell; wherein said putative therapeutic agent is a nucleic acid sequence.2. The method according to claim I wherein said method further comprises the steps of: o determining if a putative therapeutic agent has a cell death induction effect on target cells comprising a second tumour cell or second virus-infected cell; selecting said putative therapeutic agent if it also has a cell death induction effect on said second tumour cell or second virus infected cell.3. The method according to any one of claims I and 2 wherein the method further comprises the steps of: determining if a putative therapeutic agent has a cell death induction effect on a second reference cell; selecting said agent if it also has no cell death induction effect on said second reference cell.4. The method according to any one of the preceding claims wherein the tumour cell harbours genetic changes connected with malignant transformation.5. The method according to any one of the preceding claims wherein the tumour cell is genetically engineered for expressing activated oncogenes, or to express mutated tumour suppressor proteins or for silencing tumour suppressor genes; preferably the tumour cell expresses mutated suppressor proteins.6. The method according to any one of the preceding claims wherein at least one of the tumour cells or virus-infected cells is of human or primate origin.7. The method according to any one of the preceding claims wherein at least one of the reference cells is of human or primate origin.8. The method according to any one of the preceding claims wherein at least one tumour cells or virus-infected cells is isogenic with at least one of the reference cells.9. The method according to any one of the preceding claims wherein the tumour cells C') are selected from transformed HEK293 cells, cancer stem cells, transformed CV-1 cells, transformed human dermal fibroblasts (NHDF), transformed wild type human C') fibroblast cells (MRC5), transformed human wild type keratinocytes (HKC), 0 transformed MCFIOA, transformed human lung WI-SB cells and transformed human (Q20 BJ-1 cells.10. The method according to any preceding claims wherein the target cells are kept in cell culture conditions which mimic the tumour environment.11. The method according to any one of claims I to 3 and 6 to 8 wherein the latent virus infected cell is selected from HIV infected cells, Herpes simplex infected cells, Epstein-Barr Virus infected cells, hepatitis B virus infected cells, Cytomegalovirus (CMV) infected cells, Chronic Congenital Rubella infected cells, Latent Herpes simplex virus (HSV) infected cells, Varicella zoster virus (VZV) infected cells, adenovirus infected cells, measles virus infected cells, John Cunningham (JO) virus infected cells, rubella virus infected cells.12. The method according to any one of the preceding claims wherein at least one of the reference cells is selected from a wild type cell, primary cell, an immortalized reference cell and an genetically inactivated tumour cell or an uninfected cell.13. The method according to any of the preceding claims wherein the reference cells are cells derived from tumour cells wherein the activating oncogenes are incapacitated or replaced with the respective proto-oncogenes or tumour suppressor genes are re- 14. The method according to any one of the preceding claims wherein at least one of the reference cells is selected from HEK2Q3 cells1 CV-1 cells, human dermal fibroblasts (NHDF), wild type human fibroblast cells (MRCS), human wild type keratinocytes (HKC), MCFIUA, human lung Wl-38 cells and human BJ-1 cells. I015. The method according to any one of the preceding claims wherein at least one of the tumour cells is selected from transformed HEK293 cells and at least one of the reference cells is selected from HEK293 cells or wherein at least one of the tumour cells is selected from transformed CV-1 cells and at least one of the reference cells is selected from CV-1 cells: or wherein at least one of the tumour cells is selected from C') transformed human dermal fibroblasts and at least one of the reference cells is selected from human dermal fibroblasts or wherein at least one of the tumour cells C') is selected from transformed wild type human fibroblast cells (MRC5) and at least 0 one of the reference cells is selected from wild type human fibroblast cells (MRC5) (Q 20 or wherein at least one of the tumour cells is selected from transformed MCFIOA and C\i at least one of the reference cells is selected from MGF1OA; or wherein at least one of the tumour cells is selected from transformed human wild type keratinocytes (HKC) and at least one of the reference cells is selected from human wild type keratinocytes (HKC); or wherein at least one of the tumour cells is selected from transformed human lung Wl-38 cells and at least one of the reference cells is selected from human lung Wl-38 cells; or wherein at least one of the tumour cells is selected from transformed human BJ-1 cells and at least one of the reference cells is selected from human BJ-1 cells.16. The method according to any one of the preceding claims wherein the tumour cells are transformed by insertion of a human oncogene.17. The method according to any one of the preceding claims wherein the oncogene is selected from H-ras, K-ras, myc, raf, erbB-2, fos, jun, kit, mdm-2, myb, ret, sis and trk.18. The method according to any one of the preceding claims wherein the target cells comprise cells which are activated and sustain the proliferation of the tumour cells and selection of the putative therapeutic agent is also dependent on cell death induction in these cells.19. The method according to any one of the preceding claims wherein the target cells comprise cells selected from cells of the innate immune response, activated stromal cells, infiltrating leukocytes and cancer-associated fibroblasts and selection of the putative therapeutic agent is also dependent on cell death induction in these cells.20. The method according to any one of the preceding claims wherein the putative therapeutic agent is in plasmid form.21. The method according to any one of the preceding claims wherein cell death induction is detected with the aid of a reporter agent; preferably reporter enzymes. C')22. The method according to any one of the preceding claims which is performed by C) robots.(Q2O 23. A therapeutic agent or equivalent thereto identified using the method according to (\i any one of claims ito 22.24. A pharmaceutical composition comprising a therapeutic agent according to claim 23 and a pharmaceutically acceptable excipient.25. A process for preparing a pharmaceutical composition, said process comprising admixing an anti-tumour agent identified using a method according to any one of claims I to 22 or an anti-tumour agent equivalent thereto, optionally in the form of a vehicle, with a pharmaceutically acceptable excipient.