WO2014041119A1

WO2014041119A1 - Oncolytic viruses expressing immuno-nucleases

Info

Publication number: WO2014041119A1
Application number: PCT/EP2013/069000
Authority: WO
Inventors: Dirk Nettelbeck; Ines FERNANDEZ-ULIBARRI; Michaela Arndt; Patrick KETZER
Original assignee: Deutsches Krebsforschungszentrum; Ruprecht-Karls-Universität Heidelberg
Priority date: 2012-09-14
Filing date: 2013-09-13
Publication date: 2014-03-20

Abstract

The present invention relates to an oncolytic virus comprising an expressible genetic construct encoding a fusion polypeptide, wherein said fusion polypeptide comprises a polypeptide binding to a tumor related cell and a polypeptide having a cytotoxic nuclease activity, and to said oncolytic virus for use as a medicament and for the treatment of cancer. The present invention further relates to a method of killing tumor related cells comprising contacting said tumor related cells with the oncolytic virus of the present invention, incubating said tumor related cells under conditions allowing expression of said expressible genetic construct, and thereby killing said tumor related cells. Further, the present invention relates to the use of the oncolytic virus of the invention for the treatment of cancer and for killing cancer cells, as well as to kits and devices comprising said oncolytic virus.

Description

Oncolytic viruses expressing immuno-nucleases

Cancer is, despite major improvements in therapy options, still a leading cause of death in humans, especially in the developed countries. Recent research has made clear that the various forms of cancer actually form a highly diverse group of diseases and that the magic bullet for curing cancer very probably does not exist. This also indicates that there will be further need for additional types of therapy also in the future.

Viruses have been proposed as potentially useful agents in cancer therapy, due to their intrinsic ability to lyse cells (Russel et al. (2012), Oncolytic virotherapy. Nat Biotechnol. 30(7):658-670). The discovery that some naturally occurring viruses have an intrinsic preference to lyse cancer cells lead to the concept of providing "oncolytic viruses". Besides relying on naturally occurring oncolytic viruses, scientists also have devised several strategies of engineering known viruses to have improved oncolytic properties: viral coat proteins were modified to improve entry into and specificity for cancer cells, a method called transcriptional targeting. In some viruses, an essential viral gene was placed under the control of a tumor- specific promoter, such that replication would only occur in tumor cells, a strategy named transcriptional targeting. Further, in a different setting, viral functions expendable in a cancer cell but not in normal cells, were removed, again leading to viruses specifically replicating in cancer cells (attenuation). Unfortunately, replication of oncolytic viruses frequently proved insufficient to efficiently remove cancer cells from a patient. For this reason, "armed" viruses were conceived, carrying genes mediating additional cytotoxicity (e.g. Haisma et al. (2006), Adenoviral-Mediated Gene Transfer of Single Chain Fv 425:sTRAIL Fusion Protein Induces Target Cell Restricted Apoptosis in EGFR- Positive Tumor Cells and a Potent Bystander Effect,Molecular Therapy 13: S21). Since infected tumor cells are destroyed by viral oncolysis, this additional cytotoxicity should affect especially those tumor-related cells not infected by the virus. Ideally, the said genes should encode a secreted and tumor- targeted molecule with cytotoxic activity.

Usefulness of nucleases for effectively killing unwanted cell populations was realized very early, as exemplified by the use of the gene coding for the EcoRI restriction endonuclease in a positive-selection vector already in 1986 (Kuhn et al., Gene 42(3):253). More recently, RNases, especially those comprised in the RNase A superfamily, were found to be suitable candidates for being used in the elimination of unwanted cell populations. Due to their strong cytotoxic effect, nucleases are used in binary systems, meaning that the nuclease is either absent, and the cell lives, or the nuclease is present, and the cell dies. Also due to the strong cytotoxic effect of nucleases, research has focused on providing cellular specificity to said nucleases, in order to ensure that cytotoxicity occurs only in e.g. cancer cells. One exemplary solution providing specificity for cancer cells is the engineering of so-called immuno -RNases, i.e. fusion proteins comprising an antibody, e.g. a single-chain antibody, specifically binding to a cancer cell and an RNase (Arndt MA, Krauss J, Vu BK, Newton DL, Rybak SM. A dimeric angiogenin immuno fusion protein mediates selective toxicity toward CD22+ tumor cells. J Immunother. 2005;28(3):245-251; Braschoss S, Hirsch B, Dubel S, Stein H, Durkop H. New anti-CD30 human pancreatic ribonuclease-based immunotoxin reveals strong and specific cytotoxicity in vivo. Leuk Lymphoma. 2007;48(6): 1179-1186; Hetzel C, Bachran C, Fischer R, Fuchs H, Barth S, Stocker M. Small cleavable adapters enhance the specific cytotoxicity of a humanized immunotoxin directed against CD64-positive cells. J Immunother. 2008;31(4):370-376; Krauss J, Arndt MA, Dubel S, Rybak SM. Antibody- targeted RNase fusion proteins (immunoRNases) for cancer therapy. Curr Pharm Biotechnol. 2008;9(3):231-234; Krauss J, Arndt MA, Vu BK, Newton DL, Rybak SM. Targeting malignant B-cell lymphoma with a humanized anti-CD22 scFv-angiogenin immunoenzyme. Br J Haematol. 2005;128(5):602-609; Krauss J, Arndt MA, Vu BK, Newton DL, Seeber S, Rybak SM, Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein. Biochem Biophys Res Commun. 2005;331(2):595-602; Krauss J, Arndt MA, Zhu Z, Newton DL, Vu BK, Choudhry V, et al. Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUCl scFv and derived immuno enzyme. Br J Cancer. 2004;90(9): 1863-1870; Menzel C, Schirrrnann T, Konthur Z, Jostock T, Dubel S. Human antibody RNase fusion protein targeting CD30+ lymphomas. Blood. 2008; 1 11(7):3830-3837; Rybak SM, Amdt MA, Schirrrnann T, Dubel S, Krauss J. Ribonucleases and immunoRNases as anticancer drugs. Curr Pharm Des. 2009; 15(23) :2665-2675; Schirrrnann T, Krauss J, Arndt MA, Rybak SM, Dubel S. Targeted therapeutic RNases (ImmunoRNases). Expert Opin Biol Ther. 2009;9(l):79-95; Stocker M, Tur MK, Sasse S, Krussmann A, Barth S, Engert A. Secretion of functional anti-CD30-angiogenin immunotoxins into the supernatant of transfected 293T-cells. Protein Expr Purif. 2003;28(2):21 1-219).

There is, however, still a need in the art for improved viral vectors for use in the depletion or removal of unwanted cells from a patient, e.g. in cancer therapy. The present invention discloses means and method to comply with the aforementioned needs.

Accordingly, the present invention relates to an oncolytic virus comprising an expressible genetic construct encoding a fusion polypeptide, wherein said fusion polypeptide comprises (i) a polypeptide binding to a tumor related cell and (ii) a polypeptide having a cytotoxic nuclease activity.

The term "virus" is known to the skilled person and relates to members of a group of submicroscopic entities, which comprise one or more polynucleotides as genome and a protein coat and which are capable of replication only in a living cell. The term "oncolytic virus", as used herein, relates to a virus preferentially infecting, replicating in, killing, and / or lysing cancer cells. The oncolytic virus may be a naturally oncolytic virus, i.e. a virus having oncolytic properties in its wildtype form, like, e.g. Parvovirus HI or Vesicular Stomatitis Virus. Preferably, the oncolytic virus is an engineered oncolytic virus, i.e. a virus engineered, more preferably genetically engineered, to be oncolytic; most preferably, the oncolytic virus is a an engineered oncolytic virus further engineered to improve clinically relevant parameters like, e.g. specificity, preferably increased specificity for one or more kind of target cells; immunogenicity, preferably decreased immunogenicity of the virus, and / or increased immunogenicity of tumor antigens released by oncolysis; cytotoxicity, preferably increased cytotoxicity to tumor related cells; transmissibility, preferably increased transmissibility in the tumor and / or decreased subject-to-subject transmissibility; and the like. It is understood by the skilled person that the oncolytic virus may also be a naturally oncolytic virus further engineered to improve its oncolytic properties or other clinically relevant parameters. As discussed above, methods of engineering oncolytic viruses are well known in the art and include, e.g., modifying a viral coat protein to improve entry into cancer cells (transductional targeting), placing an essential viral gene under the control of a tumor- specific promoter (transcriptional targeting), removal of viral functions expendable in a cancer cell but not in normal cells (attenuation), mutagenesis for improved lytic activity in tumor-related cells, and the like. Preferably, the oncolytic virus is an oncolytic adenovirus, an oncolytic parvovirus, an oncolytic herpesvirus, an oncolytic reovirus, an oncolytic vesicular stomatitis virus, an oncolytic poliovirus, an oncolytic poxvirus, an oncolytic measles virus, an oncolytic Newcastle disease virus, or an oncolytic coxsackievirus. More preferably, the oncolytic virus is a human adenovirus, most preferably a human adenovirus serotype 5 (SEQ ID NO: 1). The term "expressible genetic construct", as used herein, relates to a polynucleotide comprising a nucleic acid sequence encoding a fusion peptide of the present invention and at least one expression control sequence allowing the expression of the expressible construct in eukaryotic cells or isolated fractions thereof. Expression of said genetic construct comprises transcription of the polynucleotide, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They, preferably, comprise regulatory sequences ensuring initiation of transcription, polycystronic expression, e.g. with IRES elements or 2A peptides, or splice acceptor sites for alternative splicing into a viral transcript and, optionally, poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Examples for regulatory elements permitting expression in eukaryotic host cells are the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Moreover, inducible expression control sequences may be used in an expression vector encompassed by the present invention. Such inducible vectors may comprise sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. Besides elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site, the bovine growth hormone poly-A site, a synthetic poly-A site or the tk-poly-A site, downstream of the polynucleotide. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Preferably, said expressible genetic construct is covalently bound to the genome of the oncolytic virus; more preferably, the expressible genetic construct is inserted into the genome of the oncolytic virus. Preferably, the expressible genetic construct is the construct shown in SEQ ID NO:2 providing for expression of a fusion polypeptide having an amino acid sequence as shown in SEQ ID NO: 3.

Preferably, the oncolytic virus of the present invention is capable of sustained growth on appropriate cells, meaning that, preferably, viability of the oncolytic virus, as measured by burst reduced by the expression of the expressible genetic construct by less than hundredfold, less than tenfold, less than fivefold, or less than twofold as compared to a virus not expressing said expressible genetic construct. Also preferably, the titer of viral particles obtainable from a virus expressing the expressible genetic construct is reduced by less than hundredfold, less than tenfold, less than fivefold, or less than twofold as compared to a virus not expressing said expressible genetic construct. More preferably, the mfectivity of the oncolytic virus, as measured by the ratio of infective virus particles to physical viral particles, is reduced by the expression of the expressible genetic construct by less than hundredfold, less than tenfold, or less than fivefold, as compared to a virus not expressing said expressible genetic construct. More preferably, expression of the expressible genetic construct does not significantly reduce viability of the virus as compared to a virus not expressing said construct. Also preferably, the oncolytic virus is capable of mediating production of said fusion polypeptide at an amount toxic for at least one tumor cell line, meaning that the expressible genetic construct is expressed such that the amount of fusion polypeptide produced is high enough to cause cytotoxicity to occur. As detailed herein, obtaining appropriate expression levels ensuring both viability of the oncolytic virus and cytotoxicity to the target cells can, preferably, be achieved by inserting the expressible genetic construct at appropriate sites in the genome of the oncolytic virus. In a preferred embodiment, the expressible genetic construct is inserted into a viral transcription unit, i.e. using an IRES sequence (Martfnez- Salas, E (1999), Internal ribosome entry site biology and its use in expression vectors. Curr Opin Biotechnol. 1999 Oct;10(5):458-64; Rivera et al. (2004) Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo, Virology. 320(1): 121-34; Rohmer et al. (2009), Transgene expression by oncolytic adenoviruses is modulated by E1B19K deletion in a cell type-dependent manner. Virology. 2009 Dec 20;395(2):243-54), a sequence encoding a 2A peptide sequence (Szymczak et al. (2004), Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol. 2004 May;22(5):589-94), or a splice acceptor sequence (Jin et al. (2005), Identification of novel insertion sites in the Ad5 genome that utilize the Ad splicing machinery for therapeutic gene expression. Mol Ther. 12(6):1052-63; Quirin et al. (2011), Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy. Hum Gene Ther. 22(4):389-404; Carette et al. (2005), Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site. J Gene Med. 7(8): 1053-62) at a position downstream of the major late promoter, most preferably between the E4 genes and the right inverted terminal repeat of an oncolytic adenovirus, preferably derived from human adenovirus serotype 5 (SEQ ID NO:4). Also preferably, expression of the expressible genetic construct is regulated by including a nucleic acid sequence coding for a regulatory RNA construct, e.g. a riboswitch, into the expressible genetic construct. The term "riboswitch" is known to the skilled person and relates to a part of an RNA molecule binding a small target molecule, and whose binding of the target affects the activity of the gene encoding said mRNA (Breaker, RR (2011), Prospects for riboswitch discovery and analysis. Mol Cell. 43(6):867-79; Cheah et al. (2007), Control of alternative RNA splicing and gene expression by eukaryotic riboswitches. Nature. 447(7143):497-500). More preferably, the regulatory RNA construct is regulable by the addition of a chemical compound, i.e. the regulatory RNA construct is an aptazyme (Wieland et al. (2008), Improved aptazyme design and in vivo screening enable riboswitching in bacteria. Angew Chem Int Ed Engl. 47(14):2604-7; Ketzer et al. (2012), Synthetic riboswitches for external regulation of genes transferred by replication-deficient and oncolytic adenoviruses. Nucleic Acids Res. 2012 Aug 9. (Epub ahead of print)). Most preferably, the regulatory RNA is the aptazyme P1-F5, having a nucleotide sequence as shown in SEQ ID NO: (SEQ ID NO:5). The skilled person knows how to apply results shown herein below in the Examples to other oncolytic viruses: the effective amount of fusion polypeptide of the present invention required for cytotoxicity is constant for a given cell line, independent of the kind of virus used for delivery. Thus, the skilled person will compare the expression obtained with a given construct to the expression obtained with the oncolytic virases as detailed herein below to ensure both sustained growth of the virus and a cytotoxic effect is obtained.

The term "fusion polypeptide" is known to the skilled person and relates to a polypeptide comprising at least two polypeptides, e.g. proteins, protein domains, or parts thereof, linked covalently, preferably by a peptide bond.

The term "tumor related cell", as used herein, relates to any cell supporting the growth or sustainment of a tumor in a subject. Preferably, the tumor related cell is a tumor supporting cell like, e.g., a tumor associated fibroblast, a tumor endothelial cell, or a tumor promoting immune cell, like, e.g., a suppressor T-cell or a tumor-promoting monocyte-derived cell. More preferably, the tumor related cell is a cancer cell.

As used herein, the term "polypeptide binding to a tumor related cell" relates to a polypeptide having affinity to at least one molecule presented on the surface of a tumor related cell. Preferably, the molecule presented on the surface of a tumor related cell is a molecule preferentially, selectively, or specifically expressed on at least one tumor related cell, i.e. the molecule presented on the surface of a tumor related cell is a tumor specific surface antigen. More preferably, the molecule presented on the surface of a tumor related cell is specific for one kind of tumor related cell, most preferably, the molecule presented on the surface of a tumor related cell is specific for a cancer cell. Preferably, the molecule presented on the surface of a tumor related cell is EGFR, HER2/neu/ErbB2, EpCAM, PSCA, PSMA, MUC1, TAG-72, GD2, VEGFR, MET, FAP, Mesothelin, CEA, TFR, CD52, CD33, CD20, CD19. In also preferred embodiments, the molecule presented on the surface of a tumor related cell is IL-13 receptor or EphA2. In a more preferred embodiment, the molecule presented on the surface of a tumor related cell is a mutant form of one of the aforesaid molecules presented on the surface of a tumor related cell. In a most preferred embodiment, the molecule presented on the surface of a tumor related cell is a mutant form of one of the aforesaid molecules specifically presented on the surface of a tumor related cell, i.e., presented in said mutant form only on the surface of a tumor related cell. Preferably, the dissociation constant for the binding of the polypeptide binding to a tumor related cell to the molecule presented on the surface of a tumor related cell is 10^" 5 mol/1 or lower, 10^" 6 mol/1 or lower, 10^" 7 mol/1 or lower, 10^"8 mol/1 or lower, 10^~9 mol/1 or lower, or 10^~10 mol/1 or lower. In a preferred embodiment, the polypeptide binding to a tumor related cell is selected from the list consisting of antibodies, darpins, affibodies, and natural ligand proteins or mutants thereof.

Preferably, the polypeptide binding to a tumor related cell is an antibody. As used herein, the term "antibody" relates to a soluble immunoglobulin from any of the classes IgA, IgD, IgE, IgM, or, more preferably, IgG. Antibodies against a molecule presented on the surface of a tumor related cell can be prepared by well-known methods using a tumor related cell, a purified protein or a suitable fragment derived therefrom as an antigen. A fragment which is suitable as an antigen may be identified by antigenicity determining algorithms well known in the art. Such fragments may be obtained either from the molecule presented on the surface of a tumor related cell by proteolytic digestion or may be a synthetic peptide. Preferably, the peptide suitable as an antigen is located at the exterior of the tumor related cell in its natural context. Preferably, the antibody of the present invention is a monoclonal antibody, a human or humanized antibody or primatized, chimerized or fragment thereof. More preferably, the antibody is a single chain antibody, preferably an anti-HER2/neu/ErbB2, anti-EpCAM, anti- PSCA, anti-PSMA, anti-MUCl, anti-TAG-72, anti-GD2, anti-VEGFR, anti-MET, anti-FAP, anti-Mesothelin, anti-CEA, anti-TFR, anti-CD52, anti-CD33, anti-CD20, or anti-CD 19 single chain antibody, most preferably an anti-EGFR single-chain antibody, hi also more preferred embodiments, the antibody is an anti-IL-13 receptor or an anti-EphA2 single chain antibody.

Also comprised as antibodies of the present invention are a bispecific antibody, a synthetic antibody, an antibody fragment, such as Fab, Fv or scFv fragments, or single-chain multimeric antibody fragments (single chain diabody/triabody, tandem scFv) etc., or a chemically modified derivative of any of these. It is understood by the skilled person that scFv fragments can also be constructed in a manner that forces the scFvs to dimerize (diabodies), leading to improved, i.e. decreased, dissociation constants. Preferably, the antibody of the present invention shall specifically bind (i.e. does not cross-react with other polypeptides or peptides) to the molecule presented on the surface of a tumor related cell. Specific binding can be tested by various well known techniques. Antibodies or fragments thereof can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be prepared by the techniques originally described in Kohler and Milstein (1975), Nature 256, 495; and Galfre (1981), Meth. Enzymol. 73, 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. The skilled person knows how to derive the nucleic acid sequence of a specific antibody from cells producing the same, e.g. by PCR amplification and sequencing of the cDNAs encoding said antibody. It is understood by the skilled person that some of the antibodies of the present invention comprise more than one polypeptide chain and that in such case one of these polypeptide chains or both are part of the fusion polypeptide of the invention. It is also understood by the skilled person that in such case the other polypeptide chain(s) of the antibody will have to be expressed concomitantly in order to provide for a functional antibody. In a further preferred embodiment, the antibody is a nanobody. The term "nuclease" relates to a polypeptide having a hydrolytic activity on polynucleotides, wherein the polynucleotides preferably are DNA or, more preferably, RNA. Preferably, the polypeptide having a cytotoxic nuclease activity causes, when present in a cell at the appropriate concentration, a significant reduction of the viability of said cell as compared to a cell not comprising said polypeptide having a cytotoxic nuclease activity. Preferably, the viability of the cell is reduced to zero, i.e. the cell dies, preferably by undergoing apoptosis. Preferably, the polypeptide having a cytotoxic nuclease activity is a DNase, e.g. a non- sequence- specific DNase or a restricton enzyme. More preferably, the polypeptide having a cytotoxic nuclease activity is an RNase, more preferably, a member of the RNase A superfamily, like HP RNase, angiogenin, bovine seminal RNase, bovine pancreatic RNase A, human eosinophil-derived neurotoxin, or the like. Most preferably, the polypeptide having a cytotoxic nuclease activity is ranpirnase from the Northern Leopard Frog (Rana pipiens).

Thus, in a preferred embodiment, the fusion polypeptide of the present invention is a fusion polypeptide having an amino acid sequence as shown in SEQ ID NO: 3. It is understood by the skilled person that the subsequences referred to as linker, Myc-Tag, and His-Tag in SEQ ID NO:3 are optional components of the fusion polypeptide and that a fusion polypeptide lacking one or more of said subsequences are equally preferred embodiments.

Advantageously, it was found during the work underlying the present invention that combining the oncolytic effects of an oncolytic virus with the cytotoxic effects of a polypeptide having a cytotoxic nuclease activity provides for improved killing of tumor cells. It was further found that, contrary to expectations, the expression level of the fusion proteins of the present invention can be adjusted in a way such as to not interfere with replication of oncolytic viruses, but at the same time to contribute to the killing of an unwanted cell population. In a preferred embodiment, said adjusted expression level is obtained by inserting the expressible genetic construct of the present invention into suitable sites of the viral genome. In another preferred embodiment, the expression of the expressible genetic construct is controlled by the presence of an aptazyme in the RNA expressed.

The definitions made above apply mutatis mutandis to the following:

The present invention further relates to an oncolytic virus as specified herein for use as a medicament.

The term "medicament", as used herein, comprises the oncolytic virus of the present invention and optionally one or more pharmaceutically acceptable carrier. The compounds of the present invention can be formulated as pharmaceutically acceptable salts. Acceptable salts comprise acetate, methylester, HC1, sulfate, chloride and the like. Preferably, administration of the medicament is oral, parenteral, or by inhalation. More preferably, the medicament is administered topically, e.g. intratumoral and / or peritumoral, into body cavities, e.g. intraperitoneally or intracystically, or systemically, e.g. by intravenous injection. However, depending on the nature and mode of action of a compound, the medicament may be administered by other routes as well. Moreover, the compounds can be administered in combination with other drugs either in a common pharmaceutical composition or as separated medicaments, wherein said separated medicaments may be provided in form of a kit of parts.

The compounds are, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid, a gel or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The diluent(s) is/are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution, hi addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Preferably, carrier cells are used for oncolytic virus delivery, protecting the virus from neutralizing blood components (e.g. antibodies). More preferably, the carrier cells have tumor-homing properties and thus improve delivery to the tumor. Preferably, carrier cells are cells productively infected with the oncolytic virus of the present invention, more preferably homing to the tumor, and releasing the virus there. Also preferably, carrier cells are cells transporting the oncolytic virus "piggyback" at the cell surface, thus protecting said oncoytic virus and releasing it in the tumor. Tumor-homing cells are known to the skilled person and are, preferably, mesenchymal stroma cells, stem cells, inactivated tumor cells, monocytes, macrophages, T cells, or dendritic cells.

A therapeutically effective dose refers to an amount of the compounds to be used in a pharmaceutical composition of the present invention which prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, mode of administration, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. A typical dose can be, for example, in the range of 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or more than 10¹² viral particles or viral genomes if applied systemically or topically; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. The medicaments and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time, for example from one to four times, from two to ten times, or from two to 20 times.

The present invention also relates to an oncolytic virus as specified herein for use in treating cancer.

The term "treating" refers to ameliorating the diseases or disorders referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of the health with respect to the diseases or disorders referred to herein. It is to be understood that treating as used in accordance with the present invention may not be effective in all subjects to be treated. However, the term shall require that a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., detenriination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. "Cancer", in the context of this invention, refers to a disease of an animal, including man, characterized by uncontrolled growth by a group of body cells ("cancer cells"). This uncontrolled proliferation may be accompanied by intrusion into and destruction of surrounding tissue and possibly spread of cancer cells to other locations in the body. Preferably, the cancer is a tumor or cancer, preferably selected from the list consisting of acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, aids-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, burkitt lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, glioblastoma, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, kaposi sarcoma, laryngeal cancer, medulloblastoma, medulloepithelioma, melanoma, merkel cell carcinoma, mesothelioma, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and wilms tumor.

More preferably, the cancer is a carcinoma, preferably selected from the list consisting of basal cell carcinoma, bile duct cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer, head and neck cancer, hepatocellular cancer, hypoharyngeal cancer, laryngeal cancer, mouth cancer, nasopharyngeal cancer, oral cancer, non-melanoma skin cancer, non- small cell lung cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer pharyngeal cancer, prostate cancer, rectal cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, throat cancer, vaginal cancer, vulva cancer, and neurological cancer like, e.g., astracytoma, brain stem glioma, glioblastoma, or medulloblastoma. Most preferably, the cancer is squamous cell carcinoma or head and neck cancer. In a further most preferred embodiment, the cancer is glioblastoma or colorectal cancer (CRC). The present invention also relates to a method of killing tumor related cells comprising: a) contacting said tumor related cells with the oncolytic virus of the invention, b) incubating said tumor related cells under conditions allowing expression of said expressible genetic construct, and c) thereby killing said tumor related cells.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate, e.g., to obtaining rumor related cells for step a), or adding a compound inducing expression of the expressible genetic construct in step b). Moreover, one or more of said steps may be performed by automated equipment.

The terms "killing" and "lysing" a cell are known in the art. Killing relates to causing the death of a cell or group of cells. Preferably, killing relates to lysing. It is to be understood that, for a method to effectively kill or lyse tumor related cells, not all cells of a population of tumor related cells have to be killed or lysed. However, the term shall require that a statistically significant portion of cells can be successfullykilled and / or lysed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art by the methods described herein above. Preferably, killing and / or lysis shall occur for at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of the cells of a given population of tumor related cells.

The term "contacting" as used in the context of the method of the present invention is understood by the skilled person. Preferably, the term relates to bringing a compound of the present invention into physical contact with a tumor related cell and thereby allowing the compound and the tumor related cell to interact.

The term "incubating", as used herein, relates to maintaining a tumor related cell under conditions suited for the growth of said cell and / or for allowing expression of said expressible genetic construct, which vary with the type of tumor related cell and which are well known in the art. It is to be understood that incubating may also refer to leaving said tumor related cells inside the body of a patient. The present invention further relates to a method of treating cancer in a subject afflicted with cancer, comprising: a) applying the steps of the method of killing tumor related cells in said subject and b) thereby treating cancer in a subject afflicted with cancer. The term "subject", as referred to herein, encompasses animals, preferably mammals, and, more preferably, humans. More preferably, said subject suffers from or is suspected to suffer from cancer. Subjects which suffer from cancer can be identified by the accompanying symptoms known for the respective cancer. The present invention further relates to the use of the oncolytic virus of the present invention for treating cancer.

The present invention also relates to the use of the oncolytic virus of the present invention for killing cancer cells ex vivo

Further, the present invention relates to a kit comprising the oncolytic virus of the pesent invention.

The term "kit" as used herein refers to a collection of the aforementioned means, e.g., the oncolytic virus of the current invention and / or means for contacting a tumor related cell with said oncolytic virus, preferably, provided separately or within a single container. The container, also preferably, comprises instructions for carrying out the method of the present invention. The components of the kit are provided, preferably, in a "ready-to-use" manner, e.g., concentrations are adjusted accordingly, etc.

The present invention also relates to a device comprising the oncolytic virus of the present invention.

The term "device", as used herein, relates to a system of means comprising at least the means operatively linked to each other as to allow administration of the oncolytic virus or of the medicament of the present invention. Preferred means for administering medicaments are well known in the art. How to link the means in an operating manner will depend on the type of means included into the device and on the kind of administration envisaged. Preferably, the means are comprised by a single device in such a case. Said device may accordingly include a delivery unit for the administration of the oncolytic virus or medicament and a storage unit for storing said oncolytic virus or medicament until administration. However, it is also contemplated that the means of the current invention may appear as separate devices in such an embodiment and are, preferably, packaged together as a kit. The person skilled in the art will realize how to link the means without further ado. Preferred devices are those which can be applied without the particular knowledge of a specialized technician. In a preferred embodiment, the device is a syringe, more preferably with a needle, comprising the oncolytic virus or medicament of the invention. In another preferred embodiment, the device is an intravenous infusion (IV) equipment comprising the oncolytic virus or medicament. In another preferred embodiment, the device is an endoscopic device comprising the oncolytic virus or medicament for flushing a site of tumor resection before and / or after surgical resection of a tumor. In still another preferred embodiment the device is an inhaler comprising the oncolytic virus of the present invention, wherein, more preferably, said oncolytic virus is formulated for administration as an aerosol.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. Figure Legends

Fig.l. Schematic outline of genetically engineered DAd and OAd variants used in this study. Linear double strand DNA genomes carry left/right inverted terminal repeat (1TR). El genes are deleted in DAds (dEl); E3 genes are not required for replication and are deleted in both DAds and OAds (dE3). For cancer selective replication, the El A gene of OAds bears a 24 bp deletion (El Ad24). For increased infectivity of OAds, the knob domain of the fiber gene was exchanged with the knob domain of adenovirus serotype 3 (fiber 5/3). The major late promoter (MLP) drives late viral gene expression in dependency of virus DNA replication. The replication deficient DAds carry a constitutively active CMV promoter/transgene/pA cassette replacing the El genes. Transgenes are either the ONC-scFvEGFR gene or the firefly luciferase gene (Luc). The OAds have inserted the ONC-scFvEGFR transgene (ONC- scFvEGFR) downstream of either the HAdV-40 long fiber gene splice acceptor sequence (40SA) or the human beta globin splice acceptor sequence (BPSA). Splicing into the viral late transcript (expressed from the MLP) should ensure replication-dependent expression. The control OAd has inserted the green fluorescent protein (GFP) under control of the human beta globin splice acceptor sequence (BPSA).

Fig.2. Characterization of ONC-scFvEGFR produced by Ads. (A) Expression of ONC- scFvEGFR in the supernatant of A549 cells infected with mock, DAd-CMV-ONC-scFvEGFR at 0.01 TCID50/cell, OAd-40SA-ONC-scFvEGFR at 0.01 TCID50/cell or OAd-BPSA-ONC- scFvEGFR at the indicated TCID50/cell. The fusion protein ONC-scFvEGFR in supernatants was detected using an anti-His antibody and ponceau staining was used as loading control. (B) Expression of the surface marker EGFR was determined via FACS analyses in Mel624 and A431 cells. Anti-EGFR-stained cells (gray line), isotype control (marked solid gray). (C) Binding of ONC-scFvEGFR to cell surface EGFR was determined via FACS analyses in Mel624 and A431 cells using supernatant containing the ONC-scFvEGFR from A549 infected cells with OAd-BPSA-ONC-scFvEGFR at 1 TCID50/cell and anti-His (gray line). Solid gray curve: control using supernatant of mock-infected cells. (D) Bystander effect of ONC-scFvEGFR. A431 cells and Mel624 cells were incubated for 3 days with heat- inactivated supernatant harvested from A549 cells infected with mock, OAd-BPSA-GFP at 1 TCID50/cell, or OAd-BPSA-ONC-scFvEGFR at indicated infectious titers. MTT assay was performed to determine the cell viability of the cells after the treatment. Presented is the cell viability in % of control cells cultured in fresh medium. Columns represent mean values and error bars standard deviations of triplicate incubations. Asterisks indicate statistically significant differences (*p<0.05;**p<0.01).

Fig.3. ONC-scFvEGFR cell binding and cytotoxicity in the presence of cetuximab. (A) Specific binding of ONC-scFvEGFR to cell surface EGFR was analyzed via FACS analyses of A431 cells after pre-incubation with medium or cetuximab at indicated concentrations. Supernatant containing the ONC-scFvEGFR was obtained from A549 cells infected with OAd-BPSA-ONC-scFvEGFR at 1 TCID50/cell. (B) Bystander effect of ONC-scFvEGFR in the absence or presence of cetuximab. A431 cells were pre-incubated with cetuximab or mock at indicated concentrations for lh and subsequently washed and incubated for 3 days with heat-inactivated supernatant harvested from A549 cells infected with mock or OAd-BPSA- ONC-scFvEGFR at 1 TCID50/cell. MTT assay was performed to determine the cell viability. Presented is the cell viability in % of control cells cultured in fresh medium. Columns represent mean values and error bars standard deviations of triplicate incubations. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01). (C) Bystander effect of ONC-scFvEGFR in the absence or presence of cetuximab or rituximab, the negative control antibody. A431 cells were pre-incubated with cetuximab or rituximab (indicated) at concentrations 50 nM and 70 nM, respectively, or with medium alone for lh. Subsequently cells were washed and incubated for 3 days with heat-inactivated supernatant harvested from A549 cells infected with OAd-BPSA-ONC-scFvEGFR at 1 TCID50/cell (indicated) or with heat-inactivated supernatant harvested from mock-infected A549 cells. MTT assay was performed to determine the cell viability. Presented is the cell viability in % of control cells cultured in fresh medium. Columns represent mean values and error bars standard deviations of triplicate incubations. Asterisks indicate statistically significant differences (n.s. - non- significant differences; ***p<0.001).

Fig.4. Replication of oncolytic Ads armed with ONC-scFvEGFR. (A) Mel 624 and A431 cells were infected with OAd-BPSA-ONC-scFvEGFR or control OAd-BPSA-GFP at 1 TCID50/cell. At indicated time points post infection, cells were harvested and genome copy numbers were quantified by qPCR. (B) Mel624 and A431 cells were infected with OAd- BPSA-ONC-scFvEGFR or control OAd-BPSA-GFP at 1 TCID50/cell. At indicated time points post infection, cells were harvested and fiber mRNA was quantified by qRT-qPCR. (C) Infectious particle production and virus release was determined in Mel624 and A431 cells at 2 days after infection. Infectious particles were determined in cells and supernatant separately. Total infectious virus particles are virus particles in cells and supernatant. Virus release is virus particles in supernatant divided by virus particles in cells. Columns represent mean values and error bars standard deviations of triplicate infections, n.s. not significant.

Fig.5. Dependence of ONC-scFvEGFR expression on virus replication. A549 cells were infected with DAd-CMV-ONC-scFvEGFR at 0.01 TCID50/cell or OAd-BPSA-ONC- scFvEGFR at 1 TCID50/cell in the presence (+) or absence (-) of replication inhibitor Ara C. Supernatant was harvested at 3 days after infection, and expression of secreted ONC- scFvEGFR was analyzed by western blot using an anti-His antibody. Ponceau staining was used as control.

Fig.6. Combined oncolysis and ONC-scFvEGFR-mediated cytotoxicity. Mel624, A431, Cal27 and Panc-1 cells were infected with OAd-BPSA-ONC-scFvEGFR, with control OAd- BPSA-GFP, with replication-deficient control virus DAd-CMV-Luc (DAd control) or mock at indicated infectious titers. Cytotoxicity was determined by crystal violet staining of surviving cells at the time when initial cytotoxicity was observed for wells infected at 0.0001 TCID50/celI.

Fig. 7. In vivo efficacy of ONC-scFvEGFR-armed oncolytic adenovirus in mice with A431 xenografts. (A) Experimental setup. Experimental animals were female NOD/SCID mice, which were 5-6 weeks old. The time schedule for injection of A431 cells, of for injection of OAd-BPSA-ONC-scFvEGFR, OAd-BPSA-GFP, or PBS, and for mice sacrification is shown. (B) Tumor growth curve. Significant differences are indicated with *p < 0.05; ** p <0.01 ; ***p<0.001. Upper row for comparison mock versus OAd-BPSA-GFP; middle row for comparison mock versus OAd-BPSA-ONC-scFvEGFR; and lower row for comparison OAd- BPSA-GFP versus OAd-BPSA-ONC-scFvEGFR (C) Kaplan-Meier survival curve. Survival of tumor-bearing animals after injection of OAd-BPSA-ONC-scFvEGFR was significantly longer (p < 0.05) than animals injected with OAd-BPSA-GFP or PBS. Experimental animals dying immediately after injection, thus independent of viral infection and ONC-scFvEGFR expression, were excluded from the analysis.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention. Example 1 : Materials and Methods

Cell culture

Human cell lines A549 (lung adenocarcinoma), A431 (epidermoid carcinoma), Cal27 (oral adenosquamous carcinoma), Panc-1 (pancreatic cancer), 293 (complementing adenoviral El genes) and 293CrmA (stably expressing the anti-apoptotic protein CrmA) were cultivated in Dulbecco's Modified Eagle's Medium (Invitrogen, Karlsruhe, Germany). The human melanoma cell line Mel624 (kindly provided by J. Schlom, Bethesda, MD) was cultivated in RPMI 1640 medium (Invitrogen). Media were supplemented with 10% heat inactivated fetal bovine serum (FBS) (PAA, C51be, Germany), 100 IU/ml penicillin, and 100 mg/ml streptomycin (both from Invitrogen). Cells were grown at 37°C in a humidified atmosphere of 5% C02. Media were pre-warmed to 37°C in a water bath before use. All cell lines were routinely tested for mycoplasma contamination (Venor®GeM mycoplasma test; Minerva Biolabs, Berlin, Germany) Recombinant Adenoviruses

For a schematic outline of the adenovirus (Ad) genomes generated and used in this study, see Fig. 1. DAd-CMV-Luc or DAd-CMV-ONC-scFvEGFR are replication-deficient Ad serotype 5-derived vectors with El and E3 genes deleted and a CMV promoter-Luc-polyA or CMV promoter-ONC-scFvEGFR-polyA cassette inserted in the El region using the pAdEasy system (MP Biomedicals, Solon, OH). OAd-BPSA-GFP, OAd-BPSA-ONC-scFvEGFR and OAd-40SA-ONC-scFvEGFR, are E3-deleted conditionally replication-competent (oncolytic) Ads generated by insertion of GFP-polyA or ONC-scFvEGFR-polyA sequences into an oncolytic Ad Ad5/3A24. This virus has a deletion of 24 bp in the El A gene responsible for tumor-selectivity (Fueyo et al., Oncogene, 2000) and the cell-binding knob domain of the capsid protein fiber is replaced with the corresponding domain of Ad serotype 3 for improved cell binding and entry (Rivera et al., Gene Therapy, 2004). In OAd-BPSA-GFP and OAd- BPSA-ONC-scFvEGFR the GFP-polyA and ONC-scFvEGFR-polyA cassettes, respectively, are inserted with an upstream artificial splice acceptor site derived from the beta globulin gene (BPSA, Jin et al., Molecular Therapy, 2005) between the Ad E4 genes and the right inverted terminal repeat. In OAd-40SA-ONC-scFvEGFR the ONC-scFvEGFR-polyA cassette is inserted with an upstream splice acceptor sequence of the Ad serotype 40 long fiber gene downstream of the Ad fiber gene (following procedures described in Rohmer et al., Virology 2009). Virus genomes were cloned using standard cloning procedures and homologous recombination in bacteria as described in Nettelbeck et al., Cancer Research 2002 and Rivera et al., Virology 2004).

Virus particles were produced by transfection of A549 ceils (OAd viruses) or 293 cells (DAd viruses) with Pac I-digested genome plasmids using Lipofectamine (Invitrogen) following the manufacturer's recommendations. When cytopathic effect was observed, cells were harvested and viruses were released by three rounds of freezing and thawing. Viruses were amplified by repeated rounds of infection of A549, 293 or 293CrmA cells. Viruses were purified by two rounds of CsCl equilibrium density gradient ultracentrifugation. Verification of viral genomes and exclusion of wild-type contamination were performed by PCR. Physical particle concentration (viral particles/ml) was determined by reading the optical density at 260 nm; infectious viral particle titers were determined by 50% tissue culture infective dose (TCID50) assay on A549 cells (OAd viruses) or 293 cells (DAd viruses). For ratios of virus particles to infectious virus particles of virus preparations see Table 1.

Virus-mediated spread and cytotoxicity To determine virus mediated cytotoxicity, 5xl0⁴ cells per well were seeded in 48-well plates and were infected the next day with 100 μΐ of growth medium containing 2% FBS with concentrations from 0.0001 to 10 TCID50/cell in tenfold serial dilutions or were mock infected. Four hours post-infection, 900 μΐ growth medium containing 10% FBS was added. When the color of the medium changed to yellow, medium in all wells was replaced with 1 ml growth medium containing 10% FBS. When cell lysis was observed at the lowest virus titers, cells were fixed and stained with 1% crystal violet in 70% ethanol for 10 min, followed by washing with tap water to remove excess color. Plates were dried, and images were captured with an Epson (Long Beach, CA) Perfection V500 Photo scanner.

Virus infection to produce supernatant containing ONC-scFvEGFR

To produce ONC-scFvEGFR protein in cell supernatants, cells were plated at a density of 2,5x10⁵ cells/well in six-well plates and infected with mock, DAd-Luc or DAd-ONC- scFvEGFR at 0.01 TCID50/cell or with OAd-BPSA-GFP or OAd-BPSA-ONC-scFvEGFR at indicated viruses titers (0.01 ; 0.1 ; or 1 TCID50/cell) in 1 ml of growth medium containing 2% FBS. Three hour post-infection, cells were washed and 2 ml of fresh growth medium containing 10% FBS was added with or without 2 mM Ara C (Sigma, Deisenhofen, Germany). Ara C was replenished every 12 hours. At 72 hours supernatant (2 ml final volume) was harvested and was used for WB, FACS and MTT assays.

Immunoblot analysis

30 μΐ of total supernatant was mixed with western blot loading buffer (Laemmli sample buffer: 62.5 mM Tris-HCL pH 6.8, 2% SDS, 10% glycerol, 0.1 M DTT and 0.01% bromophenol blue) and boiled for 5 ^' at 95°C, run on 12,5% SDS-PAGE gels and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Munchen, Germany). The membrane was blocked with PBS supplemented with 0.1% Tween 20 (T-BST) and 5% nonfat milk for 1 hour at room temperature, and probed with anti-His antibody (Clone 13/45/31-2; Dianova, Hamburg, Germany) diluted in T-BST 3% BSA (1 :1000) for 1 hour at 4°C and followed by anti-mouse-HRP-linked antibody (Cell Signalling Technology, Danvers, USA) diluted in T- BST 5% nonfat milk (1 :5000) for 1 hour at room temperature. Antibody binding was visualized using chemiluminescence (Pierce ECL, Thermo Fisher Scientific, Bonn, Germany) following the manufacturer's recommendation.

Flow cytometry analysis Cells (5x1 Cr each) were trypsinized, washed and diluted in fluorescence-activated cell sorting (FACS) buffer (0.5% fetal bovine serum, 0.05% NaN3 in phosphate-buffered saline). For quantification of cell-surface EGFR, cells were incubated either with anti-EGFR monoclonal antibody (Clone 528, Merck Millipore, Darmstadt, Germany) or with human IgG2a isotype control (Clone MOPC-173, BioCat, Heidelberg, Germany) at dilution 1 :400 in FACS buffer on ice for 30 min. For quantification of ONC-scFvEGFR binding to EGFR, cells were incubated with 100 μΐ of supernatant containing ONC-scFvEGFR harvested from cells infected with OAd-BPSA-ONC-scFvEGFR at 1 TCID50/cell for 1 h. Subsequently, cells were washed and incubated with anti-His monoclonal antibody (Clone 13/45/31-2; Dianova, Hamburg, Germany) at 1 :400 in FACS buffer on ice for 30 min. For competitive binding experiments, cells were pre-incubated with anti-EGFR monoclonal antibody cetuximab (Merck, Darmstadt, Germany) or with anti-CD20 monoclonal antibody control rituximab (Roche, Mannheim, Germany) at indicated concentration (5, 10, 20, 50 nM) in FACS buffer on ice for 30 min before incubation with SN containing ONC-scFvEGFR. After washing, cells were stained with phycoerythrin- conjugated goat anti-mouse IgG (BD Biosciences Pharmingen, Heidelberg, Germany) at 1 :400 dilution in FACS buffer for 15 min on ice. Cells were washed twice and then run on FACS Calibur (BD Biosciences) and evaluated with CellQuest software (Becton Dickinson, Heidelberg, Germany). MTT cytotoxity assay (bystander effect)

For the determination of the bystander effect, cells were plated in 96-well plates at a density of 5,000 cell/well for A431 but of 10,000 cell/well for Mel624 in 100 μΐ of growth medium containing 5% FBS. Next day, cells were incubated for 4 hours with 100 μΐ of supernatant previously inactivated by heating for 15 min at 50°C. Subsequently, fresh growth medium containing 5% FBS was added and incubated for an additional 72 h. For competitive binding assays, cells were pre-incubated with cetuximab or rituximab at indicated concentrations for 1 h at 4°C. Subsequently, the antibody was washed away and cells were incubated with supernatant as indicated above. Cell viability was subsequently assayed using the MTT colorimetric assay following the manufacturer's instructions (Sigma- Aldrich, Taufkirchen, Germany). The plates were read using a Lab Systems Multiscan MS (Thermo Fisher Scientific, Braunschweig, Germany) at 570 nm. Background absorbance for blank wells was substracted from each value. The absorbance of wells with mock-treated cells was defined as 100% viability and was used for normalization of the cell viability of the other conditions. Experiments were performed in triplicates and the results were reproduced in independent experiments.

DNA/RNA quantification by quantitative PCR

For quantification of viral genome copy numbers or RNA expression, 5x10⁴ cells were seeded in 24-well plates in 1 ml growth medium containing 5% FBS. The next day, cells were infected in 250 μΐ of growth medium containing 2% FBS. One hour post-infection, 750 μΐ growth medium containing 10% FBS was added. Samples were harvested at indicated time points, and DNA was purified from cell lysates with the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions; RNA was purified with the RNeasy kit including DNase digest (Qiagen) following the manufacturer's instructions. Oligonucleotides used for quantification of viral genomes, viral E1A, E4, or fiber mRNA, cellular DNA, and cellular RNA were as in Rivera et al. (Virology, 2004). Quantitative PCR was performed with the 7300 Real Time PCR system (Applied Biosystems, Darmstadt, Germany) using MicroAmp 96-well reaction plates (Applied Biosystems) in a total volume of 25 μ1 for each PCR assay. Each probe contained 23 μΐ of lx Power SYBR Green Master Mix (Applied Biosystems), 2 μΐ of template mRNA or DNA, and 10 pmol of each oligonucleotide. For mRNA quantification, reverse transcriptase and RNase inhibitor (both from Applied Biosystems) were added. Quantitative PCR (qPCR) was performed with an initial denaturation step of 10 min at 95°C, followed by 50 cycles of 15 sec of denaturation at 95°C, 10 sec of annealing at 60°C, and 15 sec of elongation at 72°C. At the end of each cycle, the fluorescence emitted by the SYBR Green was measured. After completion of the cycling process, samples were subjected to melting curve analysis. A plasmid containing the Ad serotype 5 genome (10¹⁰, 10⁸, 10⁶, 10⁴, and 10² copies/μΐ) was amplified for each reaction series to generate a standard curve for quantification of the copy numbers of viral genomes or viral mRNA. Data were normalized with cellular genomic DNA or cellular RNA for each sample individually. Cellular RNA was quantified using glyceraldehyde 3-phosphate dehygrogenase oligonucleotides and 200, 20, 2, and 0.2 ng/ml human RNA isolated from A431 or Mel624 cells was the standard. Cellular DNA was quantified using beta-actin oligonucleotides and 200, 20, 2, and 0.2 ng/ml human DNA isolated from A431 or Mel624 cells as the standard. Data were analyzed with 7300 System SDS software (Applied Biosystems). Negative controls with no template were carried out for each reaction series.

Burst Assay Experiments were carried out in triplicates using 5x10⁴ cells per well plated in 24- well plates. The next day, cells were infected at 1 TCID50 per cell in a volume of 250 μΐ of growth medium containing 2% FBS. Two hours post-infection the medium was removed, and cells were washed twice with phosphate-buffered saline to remove unbound viruses. Then 1 ml of growth medium containing 5% FBS was added. Two days post-infection, supernatants and cells were harvested, and viruses were released from cells by three cycles of freezing and thawing. Cell debris was removed by centrifugation and infectious virus particles were determined by TCID50 assay on A549 cells.

Animal experimentation

Tumor cenografts of epidermoid carcinoma cell line A431 were established subcutaneously by implating 5x10⁶ cells into the flanks of 5 to 6 week old female NOD/SCID mice (in house animal breeding facility). When tumors reached a size of 50 mm , animals were randomized and were intratumorally injected with PBS, OAd-BPSA-GFP or OAd-BPSA-ONC- scFvEGFR at lxl 0⁹ TCID₅₀/mouse in 100 μΐ PBS. Every third day, tumor sizes and animal body weights were recorded and tumor volumes were calculated according to the formula [(largest diameter) x (smallest diameter) x 0.5], Animals were sacrificed when tumor volumes reached 1,500 mm .

Statistics

Statistical analyses were performed using GraphPad Prism software (version 5.04; GraphPAd Software, La Jolla, CA, USA). Differences between indicated groups of in vitro assays were analyzed using Student's t test. The two-way analysis of variance (ANOVA) test was used for comparing the tumor progression in mice in the different treatment groups. The log-rank test was used for comparing survival distribution of mice in the different treatment groups. Values of p<0.05 were considered statistically significant.

Example 2: Generation and characterization of adenoviruses armed with ONC-scFvEGFR To investigate whether ONC-scFvEGFR can be expressed by adenoviruses, we generated a replication-deficient (DAd-CMV-ONC-scFvEGFR) and two replication-competent oncolytic (OAd-40SA-ONC-scFvEGFR and OAd-BPSA-ONC-scFvEGFR) Adenoviruses (Ads) containing a gene encoding onconase (ONC) fused to a single-chain antibody fragment (scFv) against EGFR (ONC-scFvEGFR). For the replication-deficient Ad, the essential El genes were replaced with CMV promoter/ONC-scFvEGFR/pA (Fig. 1). In a matching control virus (DAd-CMV-Luc) the essential El genes were replaced with CMV promoter/luciferase/pA. For the replication- competent Ad, considering that the transgene expression efficiency and kinetics are determined by the insertion strategy, ONC-scFvEGFR was expressed under the control of the viral major late promoter. In the first virus that we generated, OAd-40SA-ONC- scFvEGFR, the ONC-scFvEGFR/pA cassette was inserted with an upstream splice acceptor site of the long fiber of HAdV-40 (40SA) immediately downstream of the fiber gene (Fig. 1). After propagation of these two Ad variants expressing ONC-scFvEGFR, it was observed that most of the virus particles produced were not infectious (Table 1), most probably due to interference of ONC-scFvEGFR expression with virus replication. In contrast, the control virus DAd-CMV-Luc showed normal infectivity, as indicated by a viral particle to infectious particle ratio below 100. Importantly, obtaining infectious virus is essential to guarantee the efficient replication, spread and tumor cell lysis of the oncolytic Ad. For this reason, to improve virus production of either DAd-CMV-ONC-scFvEGFR or of an ONC-scFvEGFR- encoding oncolytic Ad, we implemented two different strategies. For the DAd-CMV-ONC- scFvEGFR, instead of propagating Ads in the widely-used 293 cells, we used another cell line, 293 CrmA. CrmA is an anti-apoptotic protein and the rationale was to block possible apoptosis induction by ONC-scFvEGFR and thus rescue vector production. However, in spite of apoptosis inactivation in this cell line, infectivity of the produced virus remained low (Table 1). For oncolytic Ads, in contrast to replication-defective Ad vectors, replication is required not only during production, but also after application to the tumor during therapy. Therefore, we pursued another insertion strategy using the beta globulin splice acceptor site (BPSA) and another position, between the E4 genes and the right inverted terminal repeat of the virus genome (OAd-BPSA-ONC-scFvEGFR). Previous studies in our lab showed that transgene expression at that position was moderate and highly replication-dependent (Quirin et al., 2011). We then hypothesized that this is an ideal alternative for a toxic transgene like ONC-scFvEGFR. Indeed, after propagation of OAd-BPSA-ONC-scFvEGFR, viral particles showed normal infectivity (Table 1) similar to the particles of a matching control virus encoding GFP instead of ONC-scFvEGFR (OAd-BPSA-GFP, Fig. 1). Our results suggest that the expression of ONC-scFvEGFR at high concentrations and/or early during virus replication interferes with the virus replication cycle and, consequently, impede an efficient production of infectious virus particles. Thus, we propose that the insertion of immune-nuclease into OAds requires a strategy that ensures expression at adequate levels and late during the virus replication cycle. Example 3: Characterization of ONC-scFvEGFR expressed by Ads

Next, we analyzed the expression of ONC-scFvEGFR in the supernatant of cells infected with the different Ad variants. DAd-CMV-ONC-scFvEGFR and OAd-40SA-ONC-scFvEGFR expressed a high amount of ONC-scFvEGFR compared to OAd-BPSA-ONC-scFvEGFR, when cells were infected at the same infectious titer (0.01 TClD50/cell; note that this corresponds to much higher virus particles for DAd-CMV-ONC-scFvEGFR and OAd-40SA- ONC-scFvEGFR (Fig. 2A). Nevertheless, the expression of ONC-scFvEGFR by OAd-BPSA- ONC-scFvEGFR was increased when cells were infected at higher infectious virus titer (Fig. 2; 0.1 and 1 TCID50/cell; Note that this still corresponds to lower physical particles due to its higher infectivity than for the other variants). At 1 TCID50/cell of OAd-BPSA-ONC- scFvEGFR similar concentrations of ONC-scFvEGFR than for the other viruses were obtained (Fig. 2A).

To mediate cytotoxicity, ONC-scFvEGFR must first bind to EGFR on target cells before being taken up by these cells. We next determined the surface expression of EGFR via FACS analysis on both the potential target and control cells. As expected, A431 cells showed a high expression level of EGFR at the surface. In contrast, no EGFR expression could be detected on the control melanoma cell line Mel624 (Fig. 2B). Next, to assay whether the secreted ONC-scFvEGFR is able to bind to the EGFR overexpressed in the target cells, mock supernatant (SN) and SNs containing ONC-scFvEGFR were incubated with EGFR+ A431 and EGFR- Mel624 cells. Results showed that binding of ONC-scFvEGFR is effective and specific for EGFR+ cells (Fig. 2C) suggesting that the binding is limited to their native antigen EGFR. To test whether ONC-scFvEGFR has the potential to induce specific bystander killing of EGFR+ cells (i.e. in combined therapy those cells that oncolytic Ads do not reach), we performed a cytotoxicity assay. The experiment consisted in incubating EGFR+ A431 or EGFR- Mel624 cells with SN containing ONC-scFvEGFR from cells infected with OAd-BPSA-ONC-scFvEGFR, or with SN of OAd-BPSA-GFP infected or mock-infected cells. SNs were previously heat-treated to inactivate the viruses. We observed that ONC-scFvEGFR decreased cell viability specifically in EGFR+ A431 cells in a dose- dependent manner (Fig. 2D). In conclusion, infection of tumor cells with OAd-BPSA-ONC- scFvEGFR results in expression and secretion of ONC-scFvEGFR that shows specific . binding to and killing of EGFR+ tumor cells.

Example 4: ONC-scFvEGFR bystander effect relies on its binding to EGFR To further characterize the mechanism of interaction of ONC-scFvEGFR with the target cell, we next studied whether the binding of ONC-scFvEGFR is mediated exclusively by the interaction of scFvEGFR with its receptor or by the unspecific binding of ONC to the cell membrane. Therefore, we pre-incubated EGFR+ A431 cells with the EGFR+ specific antibody cetuximab (from which the scFvEGFR is derived) or the control antibody rituximab with specificity for CD20 at different concentrations. Next, cells were washed and incubated with mock SN and SNs containing ONC-scFvEGFR. Results showed that the binding of ONC-scFvEGFR to A431 cells pre-incubated with cetuximab was reduced in a dose- dependent manner (Fig. 3A) but was not reduced with control antibody rituximab (data not shown). Additionally, cetuximab inhibited cytotoxicity of ONC-scFvEGFR for A431 cells in a dose-dependent manner (Fig. 3B), which was not the case for rituximab (Fig. 3C). Therefore, these findings confirm that binding of ONC-scFvEGFR is restricted to their native antigen EGFR and that the induced cell death depends on EGFR targeting. Example 5: Expression of ONC-scFvEGFR does not interfere with the OAd-BPSA-ONC- scFvEGFR replication cycle

It is well known that insertion and expression of transgenes can interfere with the expression of viral genes, virus replication and, ultimately, with the cell killing induced by oncolysis. This was indeed the case for OAd-40SA-ONC-scFvEGFR resulting in low infectivity (see above). To investigate the influence of ONC- scFvEGFR gene insertion and protein expression on the replication cycle of OAd-40SA-ONC-scFvEGFR, we quantifively analyzed the kinetics of viral gene expression and genome replication. EGFR+ A431 cells and EGFR- Mel624 cells were infected with OAd-BPSA-ONC-scFvEGFR or the control OAd-BPSA- GFP at 1 TCID50/cell and viral genome copies and fiber mRNA copies were quantified at 4, 8, 16, 24, and 32 hours. The kinetics of genome replication and expression of the late fiber mRNA and thus virus replication efficiency were similar in both Ads variants and in both cell lines (Fig. 4 A, B). Likewise, we found that ONC- scFvEGFR did not have any effect on total infectious virus production nor on infectious viral particle release at 48 hours post infection measured by burst assay (Fig. 4C). In conclusion, the replication cycle of OAd-BPSA-ONC- scFvEGFR was maintained after the insertion of the ONC-scFvEGFR transgene into the late transcriptional unit via the BPSA sequence at the specific site used.

Example 6: Expression of ONC-scFvEGFR depends on virus replication We next investigated whether the expression of ONC-scFvEGFR by OAd-BPSA-ONC- scFvEGFR depends on virus replication. Therefore we infected A549 cells in the absence and presence of the viral replication inhibitor Ara C. At 72 hours post infection, supernatant harvested from cells infected with DAd-CMV-ONC-scFvEGFR showed a high ONC- scFvEGFR independent of Ara C treatment. Importantly, supernatant of cells infected with OAd-BPSA-ONC-scFvEGFR showed strongly reduced ONC-scFvEGFR amounts in the presence of Ara C (Fig. 5), thus demonstrating replication-dependent ONC-scFvEGFR expression for OAd-BPSA-ONC-scFvEGFR. Example 7: Combination of oncolysis and ONC-scFvEGFR activity improve tumor cell killing

Our final goal is to determine whether combining oncolysis and immunoRNase toxicity specifically improves cancer cell killing. To assay whether the virus-encoded ONC- scFvEGFR enhances oncolysis activity, A431, Cal27, Panc-1, and Mel624 cells were infected with a serial dilution of OAd-BPSA-ONC-scFvEGFR, of the control virus OAd-BPSA-GFP or were mock-infected. To determine cell killing, living cells were stained with crystal violet. In EGFR- Mel624 cells, OAd-BPSA-ONC-scFvEGFR showed cytotoxicity similar to control OAd-BPSA-GFP (Fig. 6). In contrast, cytotoxicity by OAd-BPSA-ONC-scFvEGFR was about 100-fold superior to OAd-BPSA-GFP in EGFR+ A431 cells (Fig. 6), showing that the expression level of ONC-scFvEGFR are sufficient to achieve this combined effect. This effect was also observed in the EGFR + cells Cal27 and Panc-1 cells. The result of this experiment demonstrates that expression of ONC-scFvEGFR by OAds, using an optimized expression strategy, dramatically increases cytotoxicity specifically in cells targeted by the scFv domain. Example 8: Evaluation of in vivo efficacy.

Mice carrying subcutaneous A431 tumor xenografts on the flank were treated i.t. with OAd- BPSA-GFP or OAd-BPSA-ONC-scFvEGFR at lxlO⁹ TCID₅₀/mouse or with PBS (Fig. 7A). OAd-BPSA-ONC-scFvEGFR showed significantly enhanced antitumor activity (Fig. 7B) and resulted in significantly enhanced animal survival (Fig. 7C) compared with OAd-BPSA-GFP. These data demonstrate that the expression of ONC-scFvEGFR in vivo is sufficient to increase the therapeutic activity of oncolytic adenoviruses after intratumoral injection. References for the examples

- Fueyo et al. (2000). A mutant oncolytic adenovirus targeting the Rb pathway produces anti- glioma effect in vivo. Oncogene. Jan 6;19(1):2-12;

- Jinet al. (2005). Identification of novel insertion sites in the Ad5 genome that utilize the Ad splicing machinery for therapeutic gene expression. Mol Ther. Dec;12(6):1052-63;

- Nettelbeck et al. (2002). Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of El A mutants from the tyrosinase enhancer/promoter. Cancer Res. Aug 15;62(16):4663-70;

- Quirin et al. (2011). Selectivity and efficiency of late transgene expression by transcriptionally targeted oncolytic adenoviruses are dependent on the transgene insertion strategy. Hum Gene Ther. Apr;22(4):389-404.

- Rivera et al. (2004). Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells. Gene Ther. Dec;l l(23):1694-702;

- Rivera et al. (2004). Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo. Virology. Mar 1 ;320(1): 121-34;

- Rohmer et al., (2009). Transgene expression by oncolytic adenoviruses is modulated by E1B19K deletion in a cell type- dependent manner. Virology. Dec 20;395(2):243-54.)

Table 1. Virus production conditions and titers

Unless indicated, DAds were titered in 293 cells and OAds in A549 cells. Vp, virus particles.

Claims

1. An oncolytic virus comprising an expressible genetic construct encoding a fusion polypeptide, wherein said fusion polypeptide comprises (i) a polypeptide binding to a tumor related cell and (ii) a polypeptide having a cytotoxic nuclease activity.

2. The oncolytic virus of claim 1, wherein said oncolytic virus is capable of sustained growth and wherein said oncolytic virus is capable of mediating production of said fusion polypeptide at an amount toxic for at least one tumor cell line.

3. The oncolytic virus of claim 1 or 2, wherein the polypeptide having a cytotoxic

nuclease activity is ranpirnase.

4. The oncolytic virus of any one of claim 1 to 3, wherein the polypeptide binding to a tumor cell is an antibody.

5. The oncolytic virus of claim 4, wherein said antibody is an anti-EGFR antibody.

6. The oncolytic virus of any one of claims 1 to 5, wherein the oncolytic virus is an oncolytic adenovirus.

7. The oncolytic virus of claim 6, wherein the expressible construct is inserted between the viral E4 genes and the right inverted terminal repeat using an upstream splice acceptor sequence.

8. The oncolytic virus of any one of claims 1 to 7, wherein the expressible genetic

construct comprises a polynucleotide sequence coding for a riboswitch.

9. An oncolytic virus according to any one of claims 1 to 8 for use as a medicament.

10. An oncolytic virus according to any one of claims 1 to 8 for use in treating cancer.

11. An in vitro method of killing tumor related cells comprising:

(a) contacting said tumor related cells with the oncolytic virus of any one of claims 1 to 8, (b) incubating said tumor related cells under conditions allowing expression of said expressible genetic construct, and

(c) thereby killing said tumor related cells.

12. Use of the oncolytic virus of any one of claims 1 to 8 for treating cancer.

13. Use of the oncolytic virus of any one of claims 1 to 8 for killing cancer cells ex vivo.

14. A kit comprising the oncolytic virus of any one of claims 1 to 8.

15. A device comprising the oncolytic virus of any one of claims 1 to 8.