WO2013038066A1

WO2013038066A1 - Modified oncolytic vaccinia virus

Info

Publication number: WO2013038066A1
Application number: PCT/FI2012/050895
Authority: WO
Inventors: Akseli Hemminki; Sari Pesonen; Marko Ahonen; Markus VÄHÄ-KOSKELA; Suvi Parviainen; Vincenzo Cerullo; Julia DIACONU; Eerika Karli
Original assignee: Oncos Therapeutics Ltd.
Priority date: 2011-09-16
Filing date: 2012-09-14
Publication date: 2013-03-21
Also published as: FI20115914A0; FI20115914L

Abstract

The invention relates to modified oncolytic vaccinia virus vectors and their uses in cancer therapy. The vaccinia virus vectors comprise inactivating mutations in the thymidine kinase gene and in the vaccinia growth factor gene in addition to genes encoding marker or adjuvant proteins. A production method for the inventive vaccinia construct is also disclosed. The vaccinia virus vectors are useful for systemic and sequential therapeutic use for cancer.

Description

MODIFIED ONCOLYTIC VACCINIA VIRUS

Field of the invention

The present invention relates to virus vectors for cancer therapy. Further, the present invention relates to a modified vaccinia virus vector, cells containing said vectors, modified vaccinia virus particles containing said vectors, methods for producing said vectors, pharmaceutical compositions and kits containing said vectors and methods for producing said vectors, methods for inhibiting malignant cell proliferation in a subject, methods for detecting the presence of a modified vaccinia virus vector in a subject, and use of said vectors for medicaments and cancer therapy.

Background of the invention

Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. An imbalance in either of these processes may develop into a cancerous state. Lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, renal cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphomas, pre-neoplastic lesions, pre-neoplastic lesions in the lung, colon cancer, melanoma, bladder cancer and any other cancer or tumor that may be treated, including metastatic or systemically distributed cancers are examples of cancers that can result. In fact, the occurrence of cancer is so high that over 500,000 deaths per year are attributed to cancer in the United States alone.

Currently, there are a few effective options for treating common cancer types. The course of treatment for a given individual depends on the diagnosis, the stage to which the disease has developed and factors such as age, sex, and general health of the patient. The most conventional options of cancer treatment are surgery, radiation therapy and chemotherapy. Surgery plays a central role in the diagnosis and treatment of cancer. Typically, a surgical approach is required for biopsy and to remove cancerous growth. However, if the cancer has metastasized and is widespread, surgery is unlikely to result in a cure and an alternate approach must be ta ken. Oncolytic viruses are an example of recently developed novel approaches for the treatment of cancer. These viruses can cause tumor cell death through direct replication-dependent and/or viral gene expression-dependent oncolytic effects. In addition, oncolytic viruses are able to enhance the induction of cell-mediated antitumoral immunity within the host. These viruses can also be engineered to express therapeutic transgenes within the tumor to enhance antitumoral efficacy. However, major limitations exist that prevent a more wide-spread use of these therapeutic approaches.

Adenovirus therapy has been used previously for the treatment of various tumor types with excellent safety record and local responses have also been reported. However, none of the existing clinical approaches have been able to cure metastatic advanced disease.

Vaccinia virus (vv) is a genetically complex DNA virus encoding a large number of genes, some of which have immune evading properties allowing the virus to establish local pockets of infection within an infected host.

Preliminary results (published in abstract form) from phase I clinical trial (first-in-man study) showed that intralesional injection of WR strain of vaccinia virus for cancer patients with metastatic breast, colorectal, or pancreatic cancer was safe and no dose limiting toxicity was seen (46). This virus was armed with a prodrug converting enzyme but no prodrug was administered . No immunostimulatory genes or fluorescent genes were incorporated in this virus, nor was any part of the thymidine kinase deleted, but merely subjected to insertion. Injection of subcutaneous lesions showed no viral shedding and the normal skin was spared. 2 out of 4 analyzed patients showed evidence of viral spreading from the injected lesion to other metastatic sites. 2 out of 16 patients showed complete resolution of injected tumors and one patient showed response in uninjected distant metastases. Many cases of tumor necrosis were seen. However, also adverse events were reported. One patient died 21d after treatment and association to treatment could not be excluded. Also, a serious adverse event was reported on d7. A higher rate of adverse events was seen with higher virus doses. These data suggest that WR vaccinia is a promising approach for anti-cancer use in humans but several improvements are needed e.g. in safety and efficacy of the therapy before this approach may be taken into common usage.

Immunotherapy of cancer has resulted in recent clinical successes validating the potential of the approach. A key realization has been that in addition to induction of an anti-tumor immune response, reduction of tumor immune suppressiveness is also required. Oncolytic vaccinia virus seems a promising platform for immunotherapy. However given its expression of anti-inflammatory molecules[ l-3], an "arming" strategy with immunostimulatory molecules is useful to maximize the immunotherapeutic effect. Antigen-presenting cells (APCs) such as a dendritic cells (DCs) present antigens to T cells and have the ability to determine between immune response and tolerance. Normally, peptides derived from endogenously expressed proteins are presented by APC in the context of MHC class I (MHC I) to CD8+ T cells, whereas peptides obtained from exogenously derived proteins are normally loaded onto MHC class II (MHC II) for presentation to CD4+ T cells. However, exogenous antigens can be also loaded onto MHC I for "cross-presentation" to CD8+ T cells [6] .

In tumor-draining lymph nodes both cross-priming and cross-tolerization have been reported, tumor antigen-specific T-cell proliferation has been detected, but the numbers of T cells proliferating are often too low, and therefore the overall effect of CD8+ T-cell activation does not always result in inhibition of tumor growth [7] . Thus, additional stimulatory or anti-suppressive measures would be useful.

High expression of co-stimulatory factors that act directly on T cells has been proposed to enhance T cell activation. CD154, also known as CD40L, is one such molecule. Normally it binds to CD40 on APC, which can trigger various signaling cascades on the target cell. In general, CD40L functions as a co-stimulatory molecule and induces activation in APC in association with T cell receptor stimulation by MHC molecules[8]. In addition to its effect on the immune system, CD40L also promotes direct apoptosis of CD40+ cells[9-l l] . Recombinant CD40L has been used in trials, with some efficacy, but systemic adverse events limited the dose that could be achieved locally, resulting in suboptimal efficacy[ 12] . Monoclonal antibodies against CD40 have also provided exciting proof-of-concept data[ 12] .

Although CD40L as an arming device has been explored in the context of other viral platforms such as adenoviruses[ 13-17], or other gene therapy approaches[ 18-20] the combination of the oncolytic efficacy of vvdd and the immunological effects of CD40L have not been fully studied and the technology is not commonly used in cancer therapy. Previous preclinical studies with non-replicative adenoviruses armed with CD40 have not provided oncolytic viruses that could have been adopted into wide clinical use. Recent observations have also underlined the importance of the type of death tumor cells undergo. The immunogenicity of cell death can significantly influence subsequent anti-tumor immune response and the overall efficacy of a drug[21-23] . Specifically it has been suggested that the translocation of the endoplasmic reticulum resident calreticulin-ERp57 complex to the plasma membrane is useful for immunogenicity cell death[24] . Subsequently, it was shown that the nuclear alarmin HMGB1 has to be released into the tumor microenvironment to engage TLR4 on host DCs to facilitate antigen processing and presentation [25] . Finally, it was reported that ATP release from dying cells stimulates DCs for T cell priming [26] . This emerging theory applies also to biological drugs including oncolytic viruses[ 17] .

Therefore, despite advances in the field, there remains a great need for novel modalities for safely treating advanced cancer.

Object of the invention

An object of the present invention is to provide novel modified replicative oncolytic vaccinia virus vectors and methods for producing such vectors. Another object of the present invention is to provide novel uses of the inventive vaccinia virus vectors for cancer therapy, including gene therapy. Another object of the invention is to provide novel cancer therapies for treating mammalian subjects, in particular human, cat or dog subjects. Another object of the invention is to provide methods for tracking a vaccinia virus in subjects receiving therapy with, or being exposed to, oncolytic vaccinia virus according to the invention. Summary of the invention

The present invention provides modified oncolytic vaccinia virus vectors for cancer gene therapies for humans, dogs, and cats. More specifically, the invention provides construction of oncolytic vaccinia virus recombinants and cells and pharmaceutical compositions comprising said vectors which preferentially replicate in tumor cells and express at least one transgene to facilitate antitumor efficacy and apoptosis induction and to modulate host immune responses in a human, dog, or cat. The present invention also provides said viruses for treating cancer in a subject and a method of treating cancer in humans, dogs, or cats. The invention provides increased safety of oncolytic vaccinia virus due to a new arming device and incorporation of a fluorescent marker allowing tracking of the virus in subjects and the environment. It also provides use of oncolytic adenoviruses and oncolytic vaccinia viruses in combination to improve overall antitumor efficacy of treatment and to modulate host immune responses favorably towards efficient induction and long term maintenance of antitumor immunity. Oncolytic vaccinia virus is an attractive platform for immunotherapy. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, arming the virus can improve efficacy further. CD40 ligand (CD40L, CD154) is known to induce apoptosis of tumor cells and it also triggers several immune mechanisms. One of these is a T-helper type 1 (Thl) response that leads to activation of cytotoxic T- cells and reduction of immune suppression. The inventors have constructed a vaccinia oncolytic virus expressing human CD40L (vvdd-hCD40L-tdTomato), which in addition features a cDNA for the tdTomato fluorochrome for detection of virus, useful for biosafety evaluation and for detecting cells infected by the virus in living subjects and in samples derived from subjects. In contrast to previous Western Reserve strain based vaccinia designs featuring a mere insertion into the vaccinia thymidine kinase (TK) gene, a deletion of TK region was engineered to completely avoid the possibility of back-recombination which would result in a wild type TK gene.

In an aspect the invention provides a modified vaccinia virus vector, a virus particle, a host cell, a pharmaceutical composition and a kit comprising vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein. In another aspect is provided the modified vaccinia virus vector, the virus particle, the pharmaceutical composition or the kit above for use in cancer therapy, for eliciting immune response in a subject, for use in a method of inhibiting malignant cell proliferation in a mammal, for use in a therapy or prophylaxis of cancer, for detecting the presence of the modified vaccinia virus in a subject, and as an in situ cancer vaccine, optionally in combination with adenovirus. In another aspect is provided a method of producing a modified vaccinia virus comprising vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein, comprising the steps of providing producer cells capable of sustaining production of vaccinia virus particles and carrying the modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the virus particles.

The inventors show effective expression of functional CD40L both in vitro and in vivo. In a xenograft model of bladder carcinoma sensitive to CD40L treatment, growth of tumors was significantly inhibited by oncolysis and apoptosis following both intravenous and intratumoral administration. In a CD40-negative model CD40L expression did not add potency to vaccinia oncolysis. vvdd-hCD40L-tdTomato oncolysis resulted in signs of immunogenic cell death in the presence and absence of human lymphocytes. The inventive modified oncolytic vaccinia virus coding for CD40L mediates multiple anti-tumor effects including oncolysis, apoptosis and induction of T-cell responses through upregulation of Th l cytokines.

Description of the drawings The following figures are included to further demonstrate certain aspects and features of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments, including examples, presented herein.

Figure 1 depicts schematically the cloning plasmids used in the present invention. Figure 2: 2.1a-c: The expression of the hCD40L transgene does not abrogate oncolytic efficacy. Human lung adenocarcinoma cells (A549, CD40-) (a), CD40+ bladder cancer cells (EJ) (b), and breast cancer cells (M4A4-LM3, CD40-) (c) were infected with different concentrations of replication-competent vaccinia viruses either coding for CD40L or not. Three days later, cell viability was measured using an MTS assay. 2.2a-c: vvdd-hCD40L-tdTomato promotes immunogenic cell death. A549, PC3MM2 and EJ cells were infected 1 pfu/cell of virus and cultured for 12 hours. After incubation markers for immunogenic cell death were assessed, (a) HMGB1 (b) ATP release (c) Calreticulin exposure.

Figure 3: In vitro and in vivo expression of hCD40L. (a) A549 cells were infected with different pfu/cell of vvdd-hCD40L. Supernatant was collected at different time points and analyzed by FACSarray for expression of hCD40L. Nude mice bearing M4A4-LM3 breast tumors were intratumorally injected with vvdd-tdTomato or vvdd- hCD40L-tdTomato. (b) Mice were imaged by IVIS. (c) Blood was collected on days 3 and 13 and tumors were harvested on day 13, both were analyzed for hCD40l concentration with FACSarray.

Figure 4: vvdd-hCD40L-tdTomato showed increased anti-tumor efficacy in CD40-positive tumors following intratumoral administration. Nude mice bearing human tumors were treated with PBS, vvdd-tdTomato and vvdd-hCD40L- tdTomato and tumor growth was measured over time. Administration of the virus was performed intratumorally. (a) hCD40L-sensitive bladder tumors (EJ cell line), (b) hCD40 negative lung tumors (A549 cell line).

Figure 5: Efficacy of vvdd-hCD40L-tdTomato in CD40-negative tumors following intravenous administration. Nude mice bearing human tumors were treated with PBS, vvdd-tdTomato and vvdd-hCD40L-tdTomato and tumor growth was measured over time. Administration of the virus was performed intravenously, (a) hCD40L-sensitive bladder tumors (EJ cell line), (b) hCD40 negative tumors (A549 cell line).

Figure 6 vvdd-hCD40L-tdTomato activates human lymphocytes and boosts their cytokine production, (a) Human derived lymphocytes are activated by the vaccinia virus expressed hCD40L. Filtered supernatant from A549 cells infected with different concentrations of vvdd-tdTomato or vvdd-hCD40L-tdTomato were used to stimulate Ramos-blue cells. After 48h immunological activation, as measured by NFkB activation, was determined using QUANTI-Blue. (b) Human PBMCs were stimulated with oncolysate from vvdd-hCD40L-tdTomato infected A549 cells. Media was collected and cytokines were assessed by FACSARRAY.

Figure 7 depicts that systemically injected vvdd-tdtomato (SEQ ID NO: 7) enters into mammary fat pad tumors and expresses transgene in tumors.

Figure 8 depicts that Vaccinia virus infected cancer cells produce GMCSF (A, ELISA assay) and the virus produced GMCSF is fully functionally active (B, TF1 cell assay).

Figure 9 depicts that Vaccinia viruses are able to kill hamster cancer cell lines in vitro.

Figure 10 depicts that Vaccinia viruses are able to eradicate HapTl tumors in immunocompetent Syrian Hamsters. Figure 11 depicts that splenocytes collected from virus treated HapTl tumor bearing hamsters are able to kill HapTl cells ex vivo.

Figure 12 depicts that SCCF1 cells produce dominantly EEV form of vvdd-luc while A549 cells produce both EEV and IMV forms of viral particles.

Figure 13 depicts that 100% confluent feline squamous cell carcinoma SCCF1 cells are resistant to vaccinia virus oncolysis but are able to continuously produce EEV particles (A). EEV particles produced by SCCF1 cells and IMV produced by A549 cells. EEV are useful stealth vehicles for intravenous delivery as they are not neutralized by antibodies.

Figure 14 depicts the concept that Vaccinia virus shows increased tropism towards the producer cell line in comparison to non-parental cell types (A). Also, there might be tropism towards cells of the same animal. Virus produced on human cancer cells transduces human cancer cells better than monkey cells (B). Figure 15 depicts enhancing the release of EEV particles by silencing A34R gene.

Figure 16 depicts a summary of advantages from vaccinia virus - adenovirus combination therapy.

Figure 17 depicts that Vaccinia virus and human adenovirus type 5 combine to kill cancer cells.

Figure 18 depicts that Vaccinia virus is able to replicate and spread in cells already infected with human adenovirus.

Figure 19 depicts molecular mechanisms for autocrine and paracrine synergy mechanisms between adenovirus and vaccinia virus. Figure 20 depicts that Adenovirus + vaccinia virus combination enhances therapeutic efficacy in an in vivo model of induced resistance towards oncolytic virotherapy

Figure 21 depicts Tumor destruction in immunocompetent mouse melanoma model and demonstrates the oncolytic potency of the vaccinia virus + adenovirus combination. Vaccinia followed by adenovirus was the optimal schedule. Figure 22 depicts that splenocytes from adenovirus (lower line) or vaccinia virus (upper line) treated B16.0VA-tumor-bearing mice are able to destroy B16.0VA cells grown in culture.

Figure 23 depicts that combination immunovirotherapy with vaccinia virus and adenovirus generates antigen-specific cytotoxic T cells. Figure 24 depicts that after Vaccinia virus + adenovirus combination treatment antitumor immune response dominates over antiviral responses.

Figure 25 depicts that feline (SCCF1) and canine (others) cancer cell lines can be transduced by vaccinia virus vvdd-luc.

Figure 26 depicts that subconfluent feline and canine cancer cell lines can be killed by vaccinia virus vvdd-tdtomato (SEQ ID NO: 7).

Figure 27 depicts that Vaccinia virus vvdd-luc (SEQ ID NO : 6) reduces the growth of canine ACE1 prostate cancer tumors in mice.

Figure 28 depicts that Vaccinia virus is able to infect feline fibrosarcoma tumor tissue ex vivo. Figure 29 depicts that VV and Ad can transduce cells in canine osteosarcoma tissue even when combined.

Figure 30 depicts that hCD40L is active in canine PBMCs.

Figure 31 shows that both adenovirus and vaccinia virus replicate productively in co- infected human SKOV3 ovarian cancer cells.

Figure 32 shows the proportion of NK cells in tumors, as analysed by FACS. Vaccinia followed by adenovirus resulted in highest NK cell percentages.

Figure 33 shows virus load and levels of neutralizing antibodies with different virus dosings. Figure 34 shows tumor volumes following Ad+VV dosing. At the end of the experiment, tumor sizes were smallest in the vaccinia followed by adenovirus group.

Figure 35 shows the results from Adenovirus (Ad5/3-D24-TK/GFP, 10 TU/cell) and VV (VV-tdTomato, 0.1 PFU/cell) infections. Antiviral effects of interferon gamma and beta were attenuated in Ad+VV combination treated cells. Figure 36 shows the quantitation of virus titers with Vaccinia and Adenovirus in A549 cells. Even in the presence of antiviral cytokines TN Fa I pha and interferons gamma and beta, high titer of virus could be produced following co-infection.

Figure 37 shows that oncolytic vaccinia virus and adenovirus are able to co-infect primary surgical human ovarian cancer tissue. Figure 38 shows the levels of infectious virus in primary ovarian cancer tumor tissue following infection with single viruses or their combination.

Figure 39 shows the results in vaccinia virus production on SCCF1 cells which preferentially produce EEV (the most appealing form of vaccinia for intravenous injection). Sucrose gradient ultracentrifugation of cell pellets results in the IMV form of vaccinia while centrifugation of the supernatant reveals the EEV band.

Figure 40: Vvdd-hCD40L-tdTomato targeting and replication in vivo following intratumoral and intravenous administration.

Detailed description of the invention

Definitions Unless defined otherwise, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Standard one-letter notations for nucleic acids and three-letter and one-letter notations for amino acids are used interchangeably herein. The term "heterologous nucleic acid sequence" as used herein in relation to a specific virus refers to a nucleic acid sequence that originates from a source other than the specified virus. Similarly, as used in relation to host cells, "heterologous nucleic acid sequence" refers to a nucleic acid sequence that originates from a source other than the specified host cell. The term "mutation" as used herein refers to a deletion, an insertion of heterologous nucleic acid, an inversion, or a substitution, including an open reading frame ablating mutations as commonly understood in the art.

The term "gene" as used herein refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a "coding sequence" or "coding region"), optionally together with associated regulatory regions such as promoters, operators, terminators and the like, that may be located upstream or downstream of the coding sequence.

The terms "mutant virus", "modified virus" and "modified virus vector" as used herein refer to a virus comprising one or more mutations in its genome, including but not limited to deletions, insertions of heterologous nucleic acids, inversions, substitutions or combinations thereof.

The term "naturally-occurring" as used herein with reference to a virus indicates that the virus can be found in nature, i.e. it can be isolated from a source in nature and has not been intentionally modified. The term "wild-type virus" as used herein refers to the most frequent genotype of a virus found in nature and against which mutants are defined.

The term "anti-viral response" as used herein refers to a cell's response to viral infection and includes, for example, production of interferons, cytokine release, production of chemokines, production of lymphokines or a combination thereof. The term "normal host cell" as used herein refers to a non-cancerous, non-infected cell with an intact anti-viral response.

The term "oncolytic agent" as used herein refers to an agent capable of inhibiting the growth of and/or killing tumour cells. The term "adjuvant" as used herein refers to a substance which, when added to a vaccine, is capable of enhancing the immune response stimulated by the vaccine in a subject.

The term "marker" as used herein refers to a marker that would confer an identifiable change to the cell permitting identification of cells containing the vector.

The term "subject" as used herein refers to any living organism, including an animal, animal tissue, animal cell, human, human tissue, and human cell.

Vaccinia virus

Vaccinia virus is a member of the Orthopoxvirus genus of the Poxviridae. It has large double-stranded DNA genome (~200 kb, ~200 genes) and a complex morphogenic pathway produces distinct forms of infectious virions from each infected cell. Viral particles contain lipid membranes(s) around a core. Virus core contains viral structural proteins, tightly compacted viral DNA genome, and transcriptional enzymes. Dimensions of vaccinia virus are ~ 360 x 270 x 250 nm, and weight of ~ 5-10 fg . Genes are tightly packed with little non-coding DNA and open-reading frames (ORFs) lack introns. Three classes of genes (early, intermediate, late) exists. Early genes (~ 100 genes; immediate and delayed) code for proteins mainly related to immune modulation and virus DNA replication. Intermediate genes code for regulatory proteins which are required for the expression of late genes (e.g. transcription factors) and late genes code for proteins required to make virus particles and enzymes that are packaged within new virions to initiate the next round of infection. Vaccinia virus replicates in the cell cytoplasm.

Different strains of vaccinia viruses has been identified (as an example: Copenhagen, modified virus Ankara (MVA), Lister, Tian Tan, Wyeth ( = New York City Board of Health), Western Reserve (WR)). All approaches described in this application utilize the WR strain of vaccinia virus. The genome of WR vaccinia has been sequenced (Accession number AY243312).

Different forms of viral particles have different roles in the virus life cycle Several forms of viral particles exist: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), extracellular enveloped virus (EEV). EEV particles have an extra membrane derived from the trans-Golgi network. This outer membrane has two important roles: a) it protects the internal IMV from immune aggression and, b) it mediates the binding of the virus onto the cell surface.

CEVs and EEVs help virus to evade host antibody and complement by being wrapped in a host-derived membrane. IMV and EEV particles have several differences in their biological properties and they play different roles in the virus life cycle. EEV and IMV bind to different (unknown) receptors (1) and they enter cells by different mechanisms (2). EEV particles enter the cell via endocytosis and the process is pH sensitive. After internalization, the outer membrane of EEV is ruptured within an acidified endosome and the exposed IMV is fused with the endosomal membrane and the virus core is released into the cytoplasm. IMV, on the other hand, enters the cell by fusion of cell membrane and virus membrane and this process is pH-independent. In addition to this, CEV induces the formation of actin tails from the cell surface that drive virions towards uninfected neighboring cells.

Furthermore, EEV is resistant to neutralization by antibodies (NAb) (3, 4) and complement toxicity (5), while IMV is not. Therefore, EEV mediates long range dissemination in vitro and in vivo (6). Comet-inhibition test has become one way of measuring EEV-specific antibodies since even if free EEV cannot be neutralized by EEV NAb, the release of EEV from infected cells is blocked by EEV NAb and comet shaped plaques cannot be seen (4, 7). EEV has higher specific infectivity in comparison to IMV particles (lower particle/pfu ratio) (1) which makes EEV an interesting candidate for therapeutic use. However, the outer membrane of EEV is an extremely fragile structure and EEV particles need to be handled with caution which makes it difficult to obtain EEV particles in quantities required for therapeutic applications. EEV outer membrane is ruptured in low pH (pH ~6) (2). Once EEV outer membrane is ruptured, the virus particles inside the envelope retain full infectivity as an IMV.

Some host-cell derived proteins co-localize with EEV preparations, but not with IMV, and the amount of cell-derived proteins is dependent on the host cell line (8) and the virus strain. For instance, WR EEV contains more cell-derived proteins in comparison to VV IHD-J strain (9). Host cell derived proteins can modify biological effects of EEV particles. As an example, incorporation of the host membrane protein CD55 in the surface of EEV makes it resistance to complement toxicity (5). In the present invention it is shown that human A549 cell derived proteins in the surface of EEV particles may target virus towards human cancer cells. Similar phenomenon has been demonstrated in the study with human immunodeficiency virus type 1, where host- derived ICAM-1 glycoproteins increased viral infectivity (10). IEV membrane contains at least 9 proteins, two of those not existing in CEV/EEV. F12L and A36R proteins are involved in IEV transport to the cell surface where they are left behind and are not part of CEV/EEV (9, 11). 7 proteins are common in (IEV)/CEV/EEV: F13L, A33R, A34R, A56R, B5R, E2, (K2L). For Western Reserve strain of vaccinia virus, a maximum of 1% of virus particles are normally EEV and released into the culture supernatant before oncolysis of the producer cell. 50-fold more EEV particles are released from International Health Department (IHD)-J strain of vaccinia (12, 13). IHD has not been studied for use in cancer therapy of humans however. The IHD-W phenotype was attributed largely to a point mutation within the A34R EEV lectin-like protein (12, 13). Also, deletion of A34R increases the number of EEVs released (13, 14). EEV particles can be first detected on cell surface 6 hours post-infection (as CEV) and 5 hours later in the supernatant (IHD-J strain). Infection with a low multiplicity of infection (MOI) results in higher rate of EEV in comparison to high viral dose. The balance between CEV and EEV is influenced by the host cell (15) and strain of virus.

Oncolytic virus induced immune responses

To better understand the various aspects of the present invention, some mechanisms of eliciting an immune response by oncolytic adenovirus and vaccinia virus is briefly discussed below. Immune responses elicited by oncolytic adenovirus

According to mouse data, adenoviruses activate innate responses (characterized by IL-6 and IL-12 secretion) through both MyD88 and TLR9 dependent and independent mechanisms (16). Whereas MyD88/TLR9 knockout DCs showed reduced secretion of these cytokines upon challenge with Ad5 virus, there was no difference in cytokine induction in peritoneal macrophages from MyD88/TLR9-negative mice compared to those from WT mice. In vivo in mice, adenovirus induces strong type I interferon mainly in splenic mDCs (IFN-beta) via a RIG-I/MDA5-independent and TLR- independent mechanisms associated with endosomal release and subsequent IRF-7 activation, and in pDCs (IFN-alpha) via endosomal TLR-dependent recognition (17). Besides IFN induction by DCs, adenovirus elicits IL-6 by non-DC cells in the spleen. Also, in Kuppfer cells, adenovirus infection triggers a biphasic response including tumor necrosis factor alpha, macrophage inflammatory protein 2 (MIP-2), and interferon gamma-inducible protein 10 (IP-10) at 6 hr and 5 days post i.v. administration. Adenovirus dsDNA may be recognized by TLR9 and NLRP3 inflammasome, leading to robust inflammatory cytokine secretion by infected macrophages (18), but also components of the virion activate macrophages independently of these sensors (19).

Adenoviruses do not efficiently activate macrophages in vitro unless cocultured with epithelial cells (20). Adenoviruses do not directly activate NK cells but instead do so via macrophages or DCs, via NKG2D upregulation (21). At the same time, adenovirus encoded E1A upregulates NKG2D also on tumor cells, rendering them targets for NK mediated destruction (22). Adenovirally transduced DCs are susceptible to Treg- mediated immunosuppression, regardless of maturing conditions (23). Immune responses elicited by oncolytic vaccinia virus

Systemic VV infection elicits a rapid chemokine response involving Mig and Crg-2 in several organs (24). In contrast to adenovirus, serum levels of TNF-alpha, IL-6, IFN- gamma or MCP-1 do not increase notably over background at 24, 48 or 72 hours post infection (25). Murine plasmacytoid dendritic cells also do not produce type I IFN or TNF-alpha upon vaccinia virus infection, unlike infection with myxoma virus (26). This is in contrast to MVA, which in addition to these chemo/cytokines induces noticeable blood serum levels of MIP-lalpha, IP-10 and IL-lbeta, also in humans (27).

VV gene product H I blocks phosphorylation of STAT-1, whereas E3L prevents induction of IFN-beta by blocking detection of VV dsDNA or RNA intermediates (26). VV gene product A46 protein, on the other hand, interferes with TLR signaling by binding to both MyD88 and TRIF (28, 29). While type I IFN is important for clearance of VV from mice, PKR-independent mechanisms are likely at play since E3L is a potent inhibitor of PKR (30). These and several other VV components may contribute to enhancement of otherwise sensitive viruses (31). Clinical use of vaccinia viruses

Safety of vaccinia viruses

Vaccinia has been used for eradication of smallpox and later, as an expression vector for foreign genes and as a live recombinant vaccine for infectious diseases and cancer. Vaccinia virus is the most widely used pox virus in humans and therefore safety data for human use is extensive. During worldwide smallpox vaccination programs, hundreds of thousands humans have been vaccinated safety with modified vaccinia virus strains and only very rare severe adverse events have been reported . Those are generalized vaccinia (systemic spread of vaccinia in the body), erythema multiforme (toxic/allergic reaction), eczema vaccinatum (widespread infection of the skin), progressive vaccinia (tissue destruction), and postvaccinia! encephalitis.

Cancer applications

All together 44 melanoma patients have been treated in early clinical trials with wild type vaccinia virus in 1960s - 1990s (32-37) and the overall objective response rate of injected tumors was 50% (38). Also some beneficial immunological responses were seen (36). Wild type vaccinia virus has been used also for treatment of bladder cancer (39), lung and kidney cancer (40), and myeloma (41) and only mild adverse events were seen. JX-594, an oncolytic Wyeth strain vaccinia virus coding for GM-CSF, has been successfully evaluated in three phase I studies (42-44) and preliminary results from randomized phase II trial has been presented in the scientific meeting (45).

CD40L - Accession code: P29965

CD40L is a type II transmembrane protein belonging to the tumor necrosis factor family. CD40L is also known as CD154 or gp39 and is predominately expressed on CD4+ T-cells and binds to the CD40 receptor on the membrane of antigen-presenting cells (APCs) [ 1, 2] . CD40 is expressed on macrophages and dendritic cells (DCs) where its activation by CD40L leads to antigen presentation and cytokine production followed by T-cell priming and a strong innate immune response [3] . Interactions between CD40L and its receptor CD40 provide critical costimulatory signals that trigger T-lymphocyte expansion [ 1], and increase IL-12 production which is required for the engagement of cytotoxic T lymphocytes (CTL) in the anti-tumor immune response [4, 5] . Previous observations demonstrate that recombinant soluble protein CD40L (rsCD40L) has direct effects in suppression of tumor cell proliferation in vitro [6, 7] and in vivo [8, 9] . Other direct effects of rsCD40L are stimulation of survival signaling pathways (including PI-3-kinase and ERK/MAPK) and induction of apoptosis in carcinoma cells [8, 10] . Some recent reports showed that adenoviruses armed with CD40L can induce inhibition of tumor growth together with an increase of apoptotic events at the tumor site [4, 11, 12] . Also, there is some evidence with regard to anti- tumor immune response through increased lymphocyte infiltration and presence of cytotoxic T-cells-CD8+ [ 13, 14] .

Importantly, preclinical data has been translated into human trials with recombinant CD40L and also an adenoviral vector coding for CD40L [ 15] . The former approach has demonstrated the safety of CD40L in humans, and while there are many examples of patients benefiting from treatment, the overall level of activity has not been high enough to result in successful phase 3 studies heretofore. Side effects at non-target site may limit the efficacy of systemic recombinant CD40L. The vector approach is an improvement in this regard as it can yield higher local CD40L concentrations while reducing systemic exposure. hGMCSF - Accession code: BC108724

Granulocyte-macrophage colony stimulating factor (GMCSF) is among the most potent inducers of anti-tumor immunity (Dranoff, G. GM-CSF-based cancer vaccines. Immunol Rev 188, 147-154 (2002)). It acts through several mechanisms, including direct recruitment of Natural Killers (NK) and APCs such as dendritic cells (DC) (Degli- Esposti, M.A. & Smyth, MJ. Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nat Rev Immunol 5, 112-124 (2005); Andrews, D. M., et al. Cross-talk between dendritic cells and natural killer cells in viral infection. Mol Immunol 42, 547-555 (2005)). GMCSF can also specifically activate DCs at the tumor site to increase their expression of co-stimulatory molecules to enhance cross-priming and T cell activation rather than cross-tolerance.

TK (Accession code: P68563) and (Accession code: P01136) deletions

Vaccinia virus is appealing for cancer gene therapy due to several characteristics. It has natural tropism towards cancer cells [ 16] and the selectivity can be significantly enhanced by deleting some of the viral genes. The present invention relates to the use of double deleted vaccinia virus (vvdd) in which two viral genes, viral thymidine kinase (TK) and vaccinia growth factor (VGF), are at least partially deleted. TK and VGF genes are needed for virus to replicate in normal but not in cancer cells [ 17] . The partial TK deletion may be engineered in the TK region conferring activity.

TK deleted vaccinia viruses are dependent on cellular nucleotide pool present in dividing cells for DNA synthesis and replication. Therefore TK deletion limits virus replication significantly in resting cells allowing efficient virus replication to occur only in actively dividing cells (e.g. cancer cells). VGF is secreted from infected cells and has a paracrine priming effect on surrounding cells by acting as a mitogen [ 18] . Replication of VGF deleted vaccinia viruses is highly attenuated in resting (non- cancer) cells [ 19] . The effects of TK and VGF deletions have been shown to be synergistic.

The oncolytic vaccinia virus vector according to the present invention may comprise further transgenes depending on the intended application of the virus. Examples of transgenes suitable for including in the inventive vaccinia vectors alone or in combination with others are TNF-alpha, hNIS, interferon alpha, interferon beta and interferon gamma, and monoclonal antibodies directed against various targets such as CTLA-4 or TGF-beta

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that specific examples, while indicating specific embodiments of the invention, are given by a way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Cloning of new virus constructs Previous vaccinia constructs have been usually made by inserting transgenes into the middle of vaccinia virus thymidine kinase (TK) region without actually deleting any part of TK region, only disrupting it. This facilitates back-recombination for an intact TK region, and since such a construct would have a replicative selection advantage over TK deleted virus, it would quickly out-grow the original strain. Thus, with the conventional way of engineering transgenes into TK, there may be a risk of generation of TK-wild-type escape mutants lacking transgenes or tumor selectivity. In the present invention, however, the plasmid (pMA) has been modified so that after transgene insertion into the insetion site in the TK region, there is no possible way for virus to gain intact TK gene (Example 3). Ensuring that the transgene is present makes the virus safer since an immunogenic virus is more rapidly cleared from normal tissues.

Tdtomato (Accession code: AY678269) is used as a transgene in several constructs of the present invention. It is an ideal fluorescent protein for live imaging studies due to its excellent brightness and photostability (Example 4). It can be detected as deep as 1 cm below the surface and extremely small lesions can be visualized. This feature gives a possibility to follow virus spread in the animal (normal vs. malignant tissue) and helps to optimize the treatment schedule (new injection when no virus is left in the body). No adverse effects are seen in a safety evaluation of vaccinia viruses coding for tdtomato and luciferase. Also, tdTomato allows the virus to be detected in organs, tissues, secretions, excretions and environment. These aspects are relevant from the point of view of understanding possible adverse events (their association with treatment) and biosafety. No other oncolytic viruses with tdTomato have been reported previously.

The VV recombinants of the present invention carry in their VGF region an enzymatically inactive form of beta galactosidase, which nevertheless is recognized by antibodies. Because beta galactosidase is foreign to humans, it may be recognized by the immune system and function as an adjuvant in therapeutic settings (Example 5). Therefore, this novel aspect increases the immunogenicity of the virus making it safer (faster clearance from normal tissues) and more effective (enhanced anti-tumor immunity). However, to avoid metabolic issues in treated patients, in the present invention enzymatically inactive variant is used while retaining the immunogenicity and capacity for detection with antibodies.

Production and purification of enriched EEV preparations

Enhancing the release of EEV-particles by silencing the A34R gene The vaccinia virus strain Western Reserve (WR) A34R gene encodes a lectin-like glycoprotein, which is expressed in the outer membrane of extracellular enveloped virus (EEV). The glycoprotein binds EEV-particles to the host cell membrane and inhibits the release of the particles. It has been shown that WR A34R deletion mutant virus is capable of releasing up to 24-fold more EEVs from infected cells than normal WR virus. Also vaccinia virus strain International Health Department-J (IHD-J) is able to produce large amounts of EEV particles because of a mutation within the A34R protein. Our novel approach is to silence the expression of A34R gene in virus production cells by using designed siRNA constructs against this gene (Example 19). This way EEV release, EEV infectivity and virulence are enhanced. Production of EEV particles by using the feline SCCF1 cell line

We show in Example 16 that feline squamous cell carcinoma cell line (SCCF1) produces almost exclusively EEV form of vaccinia virus particles if cells are 100% confluent at the time of infection. EEV particles are released from the host cells via exocytosis. Host cells are not lysed in this process which allows continuous production of EEV from infected cells. EEV particles can be concentrated from cell culture medium by using Optiprep gradient purification (see below).

Purification of EEV by using Optiprep gradients

EEV particles can be purified from the cell culture medium with Optiprep (Sigma) system, which is based on the use of continuous gradient of iodixanol solution (47). Both IMV and EEV particles can be collected separately from the cell lysate by using the same continuous gradient (48). Iodixanol purification method offers many advantages in comparison to CsCI or sucrose purification methods. Virus infectivity is retained well during iodixanol purification (49) and also particle: infectivity ratio is lower in comparison to CsCI purification methods (50). Viscosity of iodixanol is lower if compared to sucrose solution of the same density and this may help retaining glycoprotein structures on the viral surface (51). In contrast to sucrose and CsCI purification methods, iodixanol purified virus prep can be used directly without any further processing (such as dialysis). Additional processing steps often decrease the infectivity of the virus and potentially damage the outer membrane of EEV particles. Optiprep is available as a sterile solution which is critically important for clinical applications.

Human lung adenocarcinoma A549 cell line as a host cell for enhanced tumor cell targeting Oncolytic vaccinia virus has previously been produced in non-tumor monkey cells. For human therapy, virus produced in foreign organisms causes several problems, including regulatory issues related to injecting non-human material in humans. A virus produced in non-human system is also rapidly cleared from the subject receiving therapy due to recognition of foreign surface structures of the virus by the subject's immune system. This may result in poor therapeutic effect when a foreign host is used as a production host.

To enhance production and targeting of oncolytic vaccinia meant for human cancer therapy human lung adenocarcinoma A549 cells to produce human cancer targeted viral preparations is used in the present invention. As host cell proteins exist in the EEV outer membrane, these proteins may help virus to be targeted for the same cell type. Surprisingly, increased tumor targeting for viral preparations produced in human cancer cells is observed, as is readily seen in Example 18.

The profile of proteins associated with EEV varies with cell type, indicating involvement of host factors (6, 15). The association of cell-derived antigens is also influenced by the virus strain. Several host membrane proteins that are present in the trans-Golgi network (TGN), early endosomes or plasma membrane fractions have been found in EEV preparations e.g. CD46, CD55, CD59, MHC class I and others (5). In electron microscopy studies, low levels of these proteins have been found in EEV particles as well. Host cell proteins from the ER, intermediate compartment (IC), and early Golgi membranes are not found in EEV or IMV preparations, suggesting these membranes are not utilized for EEV formation. Vaccinia virus infects a single type of vertebrate host cell during its life cycle (ICTVdB Virus Descriptions 00.058.1.01.001. Vaccinia virus).

Administration In treating a tumor, the methods of the present invention administer an oncolytic vaccinia virus, optionally followed or preceded by administration of a composition comprising an agent used in cancer therapy, including oncolytic adenovirus. The routes of administration vary, naturally, with the location and nature of the tumor, and include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, regional (e.g., in the proximity of a tumor, particularly with the vasculature or adjacent vasculature of a tumor), percutaneous, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration. Compositions are formulated relative to the particular administration route.

In one aspect of the present invention, the methods of the present invention comprise administering an oncolytic vaccinia virus, followed or preceded by one or more traditional cancer therapy, such as chemotherapy, radiotherapy, surgery or immunotherapy. Suitable treatments in therapeutic applications may include various "unit doses." Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered and the particular administration route and formulation are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terms of TCID50 units (median tissue culture infective dose, AdEasy Vector System) for a viral construct. In one embodiment of the present invention suitable unit doses in therapy may range from 10³ to 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹² TCID50 units. In another embodiment the lower limit of the therapeutic unit dose range may be 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹² TCID50 units and the upper limit of the therapeutic unit dose range may be 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ TCID50 units. As is apparent to persons working in the field of cancer therapy, variation in dosage will necessarily occur depending for example on the condition of the subject being treated, route of administration and the subject's response to the therapy. The person responsible for administration will in any event determine the appropriate dose for the individual subject.

Immune responses and therapeutic benefit elicited by combination of adenovirus with vaccinia virus

There are several key issues that spur the exploration of this matter; first, both adenovirus and VV have an extensive history of use in humans, and they both have excellent safety profiles. Second, both viruses are potent vaccine vectors and have been used successfully in vaccination, particularly in heterologous prime-boost settings (52). Whereas prime-boost with the same virus biases the immune response against the vector rather than the immunogen, vaccination using immunologically different viruses has been shown to circumvent this problem and to provide superior vaccination efficacy against the target antigen (53). Third, enhancement of one or the other virus may occur. Notably, despite an intact p300-binding domain in the E1A protein, responsible for conferring resistance to type I IFN in vitro (54), oncolytic Ad appears to be restricted by type I IFN-inducible mechanisms in vivo (55). In this regard, it has been proposed that VV is able to facilitate replication of Ad in a manner similar to VSV (56). Moreover, Ad is also sensitive to antiviral effects mediated by IFN-gamma (57), a cytokine to which VV carries countermeasures (soluble B8R protein). Conversely, vaccinia virus expressing adenovirus 14.7K protein has been reported to exhibit reduced sensitivity to TNF-alpha (58) and thus vaccinia could benefit from the presence of adenovirus (Example 23). Finally, since each virus genome occupies its own subcellular compartment (adenovirus in the nucleus, vaccinia in the cytoplasm) there is minimal risk of recombination.

In one study, VV activated mouse mDCs and conferred antigen (OVA)-specific therapeutic potential with similar efficacy to Ad (59). In another study, plasmacytoid dendritic cells were infected by both viruses, but whereas Ad infection resulted in pDC activation, it also reduced CTL activation compared to VV or saline (60). Vaccinia, on the other hand, induced CTL activation but did so without activating and maturing the pDCs. pDCs from IFNAR KO mice also fail to undergo maturation upon Ad challenge, yet are able to instigate specific CTL responses. Nevertheless, in both cases infected pDCs directed activation of antigen-specific CTLs when the cells were simultaneously loaded with antigen peptides, demonstrating cross-presentation. In a study by Maeda et al. heterologous prime-boost in C57 mice with Ad and VV vectors expressing Sin Nombre virus antigens was superior in inducing antigen-specific CTLs compared to single-virus regimens, with Ad followed by VV emerging the best (61). On its own, adenovirus invoked significantly stronger target cell-killing capacity in splenocytes as compared VV, which is in line with our own results shown in Example 26 and has been seen also with other antigens (62).

When co-administered in a mouse HIV antigen vaccination model, adenovirus type 5 and modified vaccinia Ankara (MVA) vectors displayed reduced vaccine efficacy compared to either vector alone (63). In vitro, MVA was found to suppress adenovirus gene transcription partly through soluble factors. Since MVA was obtained by serial passage of an ancestral vaccinia virus strain, resulting in multiple gene deletions including the type I and type II interferon (IFN) scavengers B18R and B8R, respectively, it is likely the combined interferon and/or other cytokines released into the culture supernatant by MVA infection could potentially interfere with adenovirus replication. As was recently shown by the present inventiors, adenovirus may be partially sensitive to these antiviral mechanisms (55). However, oncolytic vaccinia virus deleted for the vaccinia virus growth factor (VGF) as well as thymidine kinase (TK) do not hamper the IFN-neutralizing activity of VV, and VV has been demonstrated to enhance replication and spread of IFN-sensitive VSV by antagonizing antiviral responses (56). Therefore the VV vector of the present invention does not inhibit oncolytic Ad - and in fact enhances Ad instead -, through soluble components. Indeed, in contrast to MVA and Ad which did not co-infect A549 cells in culture, we constantly detected double-infected cells at many different ratios (Examples 21-22).

In a comparison of several replication-defective virus vectors, Ad5 alone in comparison to all other vectors was able to generate antigen-specific protective responses in mice against lethal VV challenge (64), underscoring the oft superior immunogenic property of Ad compared to most other viruses used in gene therapy today. In a head-to-head comparison, Ad5 was superior to VV in inducing hCMV- specific immune responses in mice (65). Thus, we were not surprised to see greater target killing capacity of splenocytes isolated from Ad-treated mice as compared to VV-treated mice (Example 26). In contrast to many reports, it was unexpectedly found in the present invention that administration of VV followed by Ad is the best regimen (as measured by induction of OVA-specific CTLs) in the B16.0VA model. Without being bound to any theory, this was likely because VV enhanced replication of Ad via its many inhibitors of type I interferon responses (31), which Ad is known to elicit (references above). This also explains why Ad appears better at activating splenocytes to target cancer cells, as type I interferon is critical for activation of NK cells. In other heterologous prime- boost reports mentioned above, the type I IFN-inducing agent has been placed last in the treatment regimen (e.g. MVA after Ad) (63), and MVA interferes with Ad replication via induction of type I IFN. As our oncolytic vaccinia virus instead suppresses type I IFN, for the duration of its own replication it should enhance replication of Ad, which once VV has been cleared would elicit the immunotherapeutically critical IFN responses enhancing heterologous prime-boost.

We report the first human CD40 ligand expressing Western Reserve strain double deleted vaccinia virus. The virus presents several attractive features compared with a similar virus that has been used in several clinical trials (JX-594)[29] . First, our virus bears a double deletion, Thymidine Kinase (TK) and Vaccinia Growth Factor (VGF) whereas the previous virus only has a deletion of TK and therefore our virus is more tumor selective. In addition we have disrupted the TK gene not only by insertion of a cDNA as in earlier constructs, but also by engineering a partial deletion into the open reading frame of the TK DNA. The deletion in the TK is preferably an open reading frame ablating deletion in the TK region responsible for TK activity. However, viral TK activity may also be controlled effectively in the construct by an open reading frame ablating mutation outside the active region, e.g. to the 5' direction from the region conferring TK activity. The precise size of the TK deletion is not crucial as long as it provides a partially deleted TK which is not able reconstitute an active TK. The partial deletion of TK comprises deletion of as few as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, but may also comprise deletion of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350 or 400 nucleotides as long as enough wild type TK remains for recombination and inserting the transgene. As is obvious to a skilled artisan, the length of the deletion may depend on the method used to insert the transgene and it may be longer or shorter than what is practical when inserting the transgene with homologous recombination. This completely avoids any possibility of reconstitution of the wild type TK gene in the event of expulsion of the transgene, which has been reported to occur and shown also in this application. It is logical that vaccinia without a transgene that disrupts TK gene would have an advantage over the recombinant virus and therefore there is selective pressure for back-recombinants. Indicating backrecombinations with earlier generation agents, we have noticed that the purification of vvdd starting with our shuttle plasmids requires less passage and it is significantly more rapid than the purification of vvdd based on the other shuttle plasmid where TK is only disrupted but not deleted [4] . The possibility of back-recombination could pose a biosafety concern and therefore deletion of a part of TK could be preferable over previous approaches featuring only an insertion. This would be especially true in the presence of an immune system, since back-recombinants lacking an immunostimulatory transgene would be less immunogenic. Another interesting new characteristic of our new virus is the use of tdTomato for virus identification. tdTomato bears excellent features such as an increased photostability and better penetrance in the tissue[35] . This allows better visualization of the virus in vitro and in wVo[30, 31] .

In a recent trial featuring another vaccinia strain, GFP was utilized to demonstrate that pox-lesions in the skin and mucous membranes of patients had virus and were not just reactive (Harrington K. et al,; oral communication at Oncolytic Virus Meeting, Las Vegas NV 2011). Without the fluorophore the biosafety implications of the lesions might have been missed. Therefore, to understand the safety and shedding of oncolytic viruses it could be quite important to have a non-invasively imageable transgene incorporated. The superior tissue penetration of tdTomato could be useful in case of lesions or adverse events occurring deeper than the skin. The imaging device we developed for imaging mice can be used in a human trial featuring a tdTomato coding virus, to evaluate presence, persistence and amplification in tumor versus normal tissues. While formal toxicity studies are needed, tdTomato is not expected to be toxic[30, 31, 35] .

In addition to the effect of human CD40L on the immune system, which are difficult to test in mouse experiments due to the species-specificity of CD40L, we observed a certain degree of advantage of the armed virus in CD40-expressing tumors. This is in accordance with previous suggestions of the interaction of CD40L with CD40 resulting in apoptosis of tumor cells[ l l] .

Vaccinia virus has legendary immunogenicity due to its use in eradication of small pox. However, this was achieved mainly through antibody induction and in fact vaccinia per se is not very potent in inducing cellular immunity[ l, 36] . It has a complex genome that encodes for several immunomodulatory proteins, including B18R which naturally antagonizes innate cellular and antiviral responses initiated by type I interferons[ l] . Its immunomodulatory properties make this virus an intriguing platform to express immune stimulatory molecules by rational design.

To boost cellular anti-tumor immune responses we armed vaccinia virus with the human soluble ligand of CD40 (hCD40L). We hypothesized that stimulating co- stimulatory molecules on CD40-expressing cells such as APCs (mainly macrophages and dendritic cells) would increase cross-priming of T cells. To our knowledge, this is the first report on oncolytic (tumor selective) vaccinia virus expressing hCD40L. The only similar approach was taken by Ruby et al. where they have studied the antiviral activity of CD40L in the context of a replication-competent but not tumor selective vaccinia virus [32] . They demonstrate that IL4-CD40L-expressing VV was attenuated to the extent that even immunocompromised mice could survive lethal infections[32] . If it is true that CD40L restricts virus replication (in normal tissues), this could provide an additional safety aspect to our construct. Obviously, additional safety is only useful if efficacy is retained, as suggested by our results here.

A similar approach has also been taken by Bereta et al. ; who generated a non- oncolytic vaccinia virus expressing CD40L to boost the immune system in the context of vaccination[37] . In their work they show that the oncolysate from tumor cells infected with the CD40L-expressing vaccine was capable of activating DCs. Similar results have also been presented by Feder-Mengus et al. who describe a CD40L coding replication-deficient vaccinia virus, which allowed APC activation, thereby enhancing a tumor-specific T cell response, bypassing the requirement of activated T helper cells[38] . All these data, taken together, support the rationale behind our approach and give some insights into the mechanism of action of our virus.

In summary, we have generated and tested a new double deleted vaccinia virus expressing human CD40L. This virus has increased safety and immunogenicity in addition to have a more pronounced capability to kill CD40-expressing tumors. These preclinical work set the stage for clinical translation. In one aspect is provided a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein.

In another aspect the modified vaccinia virus vector comprises a thymidine kinase gene which comprises at least one insertion site for inserting the heterologous protein and thymidine kinase inactivation is carried out by a deletion in the thymidine kinase region conferring activity. In another aspect the in the modified vaccinia virus vector the first heterologous protein is an enzymatically inactive beta-galactosidase, capable of producing an immune response, inserted in the place of the deleted vaccinia growth factor gene and another heterologous protein is at least one of CD40L, a fluorescent marker protein, and GMCSF inserted in the insertion site of the partially deleted thymidine kinase gene.

In another aspect in the modified vaccinia virus vector the modified vaccinia virus is Western Reserve (WR) strain vaccinia virus and the modified vaccinia virus vector comprises the genes for expressing human CD40L (hCD40L) and tdTomato as heterologous proteins. In another aspect human CD40L (hCD40L) gene and tdTomato gene are inserted in the in the partially deleted thymidine kinase gene of the modified vaccinia virus vector.

In another aspect is provided a host cell carrying a modified vaccinia virus vector. In another aspect is provided the host cell in which the host cell is a cancer cell originating from feline squamous cell carcinoma cell line SCCF1 or from human lung adenocarcinoma A549 cell line.

In another aspect is provided a modified vaccinia virus particle containing a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and in which the virus particle is of the type intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), or extracellular enveloped virus (EEV), more preferably the virus particle is of the type EEV or IMV, and most preferably the virus particle is of the type EEV.

In another aspect is provided the modified vaccinia virus particle in which the modified vaccinia virus particle comprises inactivation of the viral A34R protein by gene silencing, by post-translational gene silencing, by RNA interference, or by small interfering RNA (si RNA).

In another aspect is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; and the virus particle comprising the modified vaccinia vector above.

In another aspect is provided the pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; and the virus particle comprising the modified vaccinia vector above; and a therapeutically active amount of an oncolytic adenovirus. In another aspect is provided a kit comprising one or more containers and one or more of the following : the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; the host cell comprising the modified vaccinia vector above; the virus particle comprising the modified vaccinia virus vector above; the pharmaceutical composition according to the invention; and the pharmaceutical composition according to the invention wherein the oncolytic adenovirus is provided in the same or a separate container.

In another aspect is provided an in vitro method for producing a modified vaccinia virus wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and comprising the steps of providing producer cells capable of sustaining production of vaccinia virus particles and carrying a modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the virus particles.

In another aspect is provided an in vitro method for producing a modified vaccinia virus wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein and comprising the steps of providing SCCF1 or A549 producer cells capable of sustaining production of vaccinia virus particles and carrying a modified vaccinia vector; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the EEV virus particles.

In another aspect is provided a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in cancer therapy.

In another aspect is provided a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use as a medicament to elicit immune response in a subject.

In another aspect is provided an in situ cancer vaccine comprising the modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above.

In another aspect is provided a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in a method of inhibiting malignant cell proliferation in a mammal wherein the method comprises administering to the mammal the modified vaccinia virus vector, the composition or the kit an amount sufficient to inhibit malignant cell proliferation compared to the malignant cell proliferation that would occur in the absence of the said modified vaccinia virus vector or the composition. In another aspect is provided a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; a virus particle comprising the modified virus vector above, a pharmaceutical composition comprising the modified virus vector above, or the kit comprising the modified virus vector above for use in combination therapy or prophylaxis of cancer, wherein in the combination therapy or prophylaxis comprises administering the modified vaccinia virus vector and adenovirus. In another aspect is provided an in vitro or in vivo method for detecting the presence of a modified vaccinia virus vector wherein the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein; a host cell comprising the modified virus vector above; or a virus particle comprising the modified virus vector above wherein the method comprises the step of detecting the presence of a marker present in the virus.

Material and Methods Cell lines and viruses

Cancer cell lines used in this study included human breast cancer cell line M4A4- LM,[39], EJ cells, lung adenocarcinoma cells A549 and CV-1 cells (African green monkey kidney fibroblasts) (ATCC, Manassas, VA USA). All cell lines were maintained in the recommended conditions. Viruses

All vaccinia viruses used in this study are of the Western Reserve strain with disrupted TK and VGF genes for enhanced cancer cell specificity. For generation of vvdd-tdtomato, the tdTomato gene[35] was cloned into pSC65 (a kind gift from Bernie Moss, National Institutes of Health, Bethesda, MD) under the control of the P7.5 promoter to create pSC65-tdTomato. hCD40L cDNA was inserted under the control of the pE/L promoter to create pSC65-tdTomato-hCD40L. These shuttle plasmids were co-transfected with vvdd-luc in CV-1 cells. Successfully recombined viruses with tdTomato or with tdTomato-hCD40l replaced luciferase from vvdd-luc. vvdd-tdTomato and vvdd-tdTomato-hCD40L were selected by picking plaques positive for red fluorescence and negative for luciferase. Viruses were amplified on A549 cells and purified over a sucrose cushion, and titers were determined with a standard plaque assay on Vero cells as described previously[4] . PFU virus titers (PFU/ml) were determined by plaque assay. The presence of the inserted genes was verified by PCR, with fluorescence microscope and with FACSarray. UV light inactivation of viruses.

Briefly, viruses were suspended in lOug/ml psoralen in Hanks balanced salt solution containing 0.1% bovine serum albumin. The suspension was incubated for 10 min at room temperature and then irradiated in a CL-1000 UV cross-linker (UVP, Cambridge, United Kingdom) with UV-A light (365 nm) for 3 min. A 5-day plaque assay was used to confirm lack of replication competent virus.

In vitro cytotoxicity assay

Cells on 96-well plates were infected with different concentrations of virus suspended in growth medium containing 2% FCS. One hour later, cells were washed and incubated in growth medium containing 5% FCS for 72 h. Cell viability was then analyzed using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium] (Cell Titer 96 AQueous One Solution proliferation assay; Promega).

Marker gene transfer assay

Cells on 24-well plates were infected with different concentrations of virus suspended in growth medium containing 2% FCS. Thirty minutes later, cells were washed and incubated in complete growth medium. Supernatants were collected and analysed according to the manual (FACSarray for soluble hCD40l).

Animal experiments

Animal experiments were approved by the Experimental Animal Committee of the University of Helsinki, Finland. Mice were purchased from Taconic (Ejby, Denmark, and Hudson, NY) at the age of 4 to 5 weeks and housed under standard conditions with food and water ad libitum. M4A4-LM3 cells were injected subcutaneously into flanks of nude Naval Medical Research Institute (NMRI) mice. When tumors reached the size of approximately 5 by 5 mm, virus was injected either intratumorally or intravenously. Bioluminescence images were captured using the IVIS imaging series 100 system (Xenogen, Alameda, CA). hCD40L concentration in mouse serum and from tumor lysate was determined with FACSarray. To assess hCD40L concentration in blood or in tumor, mice were injected intravenously with virus, and bioluminescent imaging and blood samples were taken 3 and 13 days post injection. On day 13 tumors were collected and lysed with ultrasonification. Samples were analysed with FACSarray for hCD40L quantification. Human derived lymphocyte stimulation

A549 cells were infected with viruses and supernatants were collected at 32h and filtered and added on top of Ramos Blue cells for 24h. Ramos-Blue cells are B lymphocyte cell lines that stably expresses an NFkB/AP-l-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. When stimulated, they produce SEAP in the supernatant. The levels of SEAP can be readily monitored using QUANTI-Blue™, a medium that turns purple/blue in the presence of SEAP. Levels of activation were read with microplate reader at the wave length of 450nm.

Isolated human peripheral blood mononuclear cell stimulation

Confluent 24-well plates of A549 cells were infected with vvdd-tdtomato or vvdd- tdtomato-hCD40L for 96h. Samples and controls without virus were freeze-thawed two times to completely lyse the cells. Isolated human PBMC (Tebu-bio) were grown as instructed. Lysates were added on top of PBMC's for 16h, Id or 3day stimulation; supernatants were analyzed by FACSarray for stimulation markers human TNF alpha, IL-lalpha, IL-6, IL-10 and RANTES. Statistical analysis

Tumor sizes as a function of time were compared by Mann-Whitney test (SPSS 16.0; SPSS Inc., Chicago, IL), and P values of <0.05 were considered statistically significant. A single preplanned comparison of mean tumor volume was done by using a two-tailed t test. Examples

The following examples are given solely for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned above, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1 Generation of human CD40L-expressing vaccinia virus CD40 ligand (CD40L, CD154) can induce apoptosis of tumor cells and it also able to trigger several immune mechanisms. One of these is a T-helper type 1 (Th l) response that leads to activation of cytotoxic T-cells and reduction of immune suppression. The advantages of CD40L-expressing oncolytic viruses [ 16, 17] together with encouraging results with oncolytic vaccinia virus[27-29] lead us to generate novel oncolytic vaccinia virus expressing hCD40L (vvdd-CD40L-tdTomato). The virus construct also includes a cDNA expressing the tdTomato fluorochrome in order to facilitate identification of the virus in vitro and in vivo[30] . Compared to prior commonly used fluorophores, such as green fluorescent protein, tdTomato possesses greater tissue penetration [31] and is superior to the more frequently used EGFP. In contrast to previous Western Reserve strain based vaccinia designs which have only featured a transgene insertion into TK, we engineered a deletion of the TK region (in addition to the transgene insert) to completely avoid the possibility of back- recombination which could result in a wild type TK gene. Example 2 (depicted in Figure 1) : Schematic presentation of cloning plasmids. Shuttle vector was cloned as follows: TK disruption was changed to TK deletion by PCR with primers F (SEQ ID NO: 1) : TAATC TAGAG CCGTG GGTCA TTG and R (SEQ ID NO : 2) : TAAGG TACCC ATGCG TCCAT AGTCC on shuttle plasmid pSC65 to create insert which was inserted back to pSC65 creating pMA. Tdtomato gene from pTdTomato was cut with Asel and EcoRI and was inserted to pMA (cut with Ndel and EcoRI) to create pMA-tdtomato. pMA-tdtomato-hCD40L (SEQ ID NO: 3) was cloned from pShuttle/CMV-hCD40l (cut with accl and hinpll) and inserted to pMA-tdtomato (cut with Accl). pMA-tdtomato-hGMCSF (SEQ ID NO : 4) was cloned from pSC65- hGMCSF-hNIS (SEQ ID NO: 5) (cut with Taql) and inserted to pMA-tdtomato (cut with Accl). Compared to the wild type TK, 117 bases are missing in the active region of the partially deleted TK in the construct.

Example 3: Partial deletion in TK region prevents formation of functional TK in recombinant viruses resulting in increased safety. Partial deletion in the vaccinia virus thymidine kinase (TK) region prevents back-recombination for an intact TK increasing the safety of the virus, vvdd-luc virus with a disrupted TK region (SEQ ID NO: 6) (without a partial deletion) was serially passaged in A549 cells and luc- negative plaques were collected for PCR analysis. Back-recombination for an intact TK region was seen in virus extracted from plaque 5 (p5) (TK region identical in size with wild type WR; wt). Example 4: Described vaccinia virus expresses enzymatically inactive but antigenic beta galactosidase. Human A549 lung cancer cells cultured in 12-well plates were infected with indicated vaccinia recombinants at MOI 1 and 24 hours later fixed with 3.7% paraformaldehyde for 30 minutes at room temperature. Parallel wells were stained using X-gal solution, which is converted into a blue dye by enzymatically active beta galactosidase encoded in the VGF region of the vaccinia virus genome, and with polyclonal anti-beta galactosidase antibody, which does not discriminate between enzymatically active or inactive forms of beta galactosidase. Thus, described VV recombinant VV-tdTomato (SEQ ID NO: 7) carries in its VGF region an enzymatically inactive form of beta galactosidase, which nevertheless is recognized by antibodies. Because beta galactosidase is foreign to humans, it can be recognized by the immune system and function as an adjuvant in therapeutic settings. Lack of enzymatic activity is important from a safety point-of-view as there is no risk of adverse events related to enzymatic activity, while the benefits of enhanced immunity are retained, and immunostaining can also be used for detection of infected cells.

Example 5 (depicted in Figure 2) : Expression of the hCD40L transgene does not compromise the oncolytic activity of vvdd-hCD40L-tdTomato.

It was assessed whether the oncolytic activity of the newly generated virus was retained. To this end, different cell lines were infected with vvdd-hCD40L-tdTomato and its unarmed counterpart expressing only tdTomato (Figure 2.1a-c). Interestingly, we did not observe any significant difference between the two viruses in any of the tested cell line indicating that the expression of this transgene did not compromise the lytic potential of the virus. vvdd-hCD40L-tdTomato promotes a more immunogenic form of cell death compared with mock infection or vvdd-tdTomato Calreticulin exposure as well as ATP and HMGB1 release have been recently proposed as in vitro measurable indicators of immunogenic cell death[33] . We have previously shown that an oncolytic adenovirus expressing hCD40L is able to stimulate immunogenic cell death as measured through the release of these markers[34] . We found that cells expressing CD40 showed higher levels of these markers following oncolysis (Figure 2.2a-c).

Example 7 (depicted in Figure 3) : vvdd-hCD40L-tdTomato displays tumor- restricted expression of the transgene

One of the most important advantages of using armed oncolytic viruses, in comparison to injection of recombinant peptides, is their capability to replicate and express the transgene locally at the tumor site for increased local efficacy while reducing side effects due to high systemic concentrations. We performed several in vitro and in vivo experiments to study the expression of hCD40L from vvdd-hCD40L- tdTomato (Figure 3a). vvdd-tomato was used as control to exclude that A549 could produce CD40L after infection per se. A dose-dependent increase of hCD40L was observed in infected cells (Figure 3a). Next we sought to study the pharmacokinetics of transgene expression in vivo in tumor bearing mice. To this end nude mice were implanted with M4A4-LM3 breast tumors, which were then injected with vvdd-hCD40L-tdTomato or control (Figure 3b-c, 40) . As expected, no difference in tdTomato expression was observed between the group of mice treated with vvdd-tdTomato and vvdd-hCD40L-tdTomato, confirming comparable transgene expression (Figure 3b). Simultaneously, ELISA for hCD40L was performed to quantify the protein in the serum and in the tumor. Interestingly, while hCD40L was undetectable in the serum at any assessed time points, high levels of hCD40L were found in the tumor at day 13 (Figure 3c). This suggests that expression of the transgene wass tumor-restricted, highlighting the advantage of using oncolytic viruses as a delivery system when tumor restricted expression is desired.

Example 8 (depicted in Figure 4) : vvdd-hCD40L-tdTomato results in increased anti-tumor activity in CD40-positive tumors following intratumoral and intravenous administration

We next sought to investigate the anti-tumor activity of vvdd-hCD40L-tdTomato in vivo. To this end, nude mice bearing bladder cancer xenografts (EJ cell line, CD40 positive) were injected intratumorally. The CD40L-expressing virus showed significantly (p=0.031) increased anti-tumor activity compared with the unarmed virus and all tumors were eventually cured (Figure 4a). In addition, since both viruses express tdTomato, virus replication could be followed by IVIS (Figure 40a). Interestingly, when the experiment was repeated in a CD40 negative cell line, vvdd- hCD40L-tdTomato lost its advantage over its paternal unarmed virus (Figure 4b). We next wanted to assess the efficacy of the armed virus in the more challenging situation of intravenous administration. Tumor growth in CD40-sensitive (Figure 5a) and non-sensitive (Figure 5b) tumor models were assessed . In addition, virus targeting and replication in the tumor was also measured (Figure 40b). Both viruses seemed to have anti-tumor effect. Example 10 (depicted in Figure 6) : vvdd-hCD40L-tdTomato activates human lymphocytes and boosts their cytokine production

Finally, in order to investigate the effect of vvdd-hCD40L-tdTomato on human immunological cells we performed a set of experiments using human lymphocytes. In the first experiment we used human a B cell derived cell line (Burkitt's lymphoma) stably expressing an NF-KB/AP-l-inducible secreted embryonic alkaline phosphate reporter construct (Ramos-Blue™ cells). This cell line is responsive to human CD40L and expresses alkaline phosphatase when activated. We cultured these cells with media filtered from A549 oncolysates (cells infected with oncolytic viruses). We found significantly enhanced activation when used media derived from vvdd-hCD40L- tdTomato oncolysate (Figure 6a).

In order to translate these findings in an even more clinically relevant experiment, we repeated the experiment with human primary lymphocytes and followed their cytokine profile over time. Interestingly PBMCs treated with lysate from vvdd- hCD40L-tdTomato showed a more immunogenic profile with characteristics of Th l- type of response (Figure 6b) .

Example 11 (depicted in Figure 7) : Systemically injected vvdd-tdtomato (SEQ ID NO: 7) enters into mammary fat pad tumors and expresses transgene in tumors. 5xl0e6 pfu of vvdd-tdtomato-hCD40L (SEQ ID NO: 8) virus was injected systemically into nude mice carrying GFP-positive M4A4-LM3 mammary fat pad tumors. Noninvasive imaging was done on indicated days to assess the expression of transgene (tdtomato) in animals (upper panel). Lower panel shows tumor cells that stably express GFP.

Example 12 (depicted in Figure 8) : Vaccinia virus infected cancer cells produce active form of hGMCSF. A549 cells were infected with vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) at given viral doses and hGMCSF concentration in the supernatant was assessed at different time points with FACSArray (A). Human erythroleukemic TF1 cells, which are dependent on fully functional human GMCSF for viability, were incubated with supernatant from virus (vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) or vvdd-tdtomato (SEQ ID NO : 7) ) infected A549 cells. Negative and positive controls were incubated with growth medium and commercial hGMCSF (2 ng/ml; Invitrogen), respectively. Growth medium was changed every other day and cell viability was assessed 7 days later (B).

Example 13 (depicted in Figure 9) : Vaccinia viruses are able to kill hamster cancer cell lines HapTl and Hak in vitro. Hamster cancer cell lines were infected with vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato-hGMCSF (SEQ ID NO : 9) at viral doses of 0.01, 0.1, 1, or 1 pfu/cell. Cell viability was assessed 48 hours later.

Example 14 (depicted in Figure 10) : Vaccinia viruses are able to eradicate HapTl tumors in immunocompetent Syrian Hamsters. HapTl cells (7* 106 cells/tumor) were injected subcutaneously into flanks of Syrian hamsters (8 animals; 2 tumors/animal). Tumors were allowed to grow until average diameter of 5mm. Tumors were injected i.t. with 1* 10^Λ6 pfu / tumor of vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato- hGMCSF (SEQ ID NO : 9). Mock animals received only growth media. Tumor size was followed.

Example 15 (depicted in Figure 11) : Splenocytes collected from virus treated HapTl tumor bearing hamsters are able to kill HapTl cells ex vivo. Spleens were collected from Hamsters previously cured from HapTl tumors with vvdd-tdtomato (SEQ ID NO : 7) or vvdd-tdtomato-hGMCSF (SEQ ID NO: 9) viruses (data presented in Example 14). Splenocytes were isolated and cultured in 10% RMPI growth media for two days. HapTl and Hak cells were seeded 50000 cells/well on 96-well-plates and splenocytes were added next day (in ratios splenocytes to cancer cells were 1 : 1, 10: 1, or 20: 1). The cell viability for HapTl and Hak cells was assessed 24 hours later with MTS assay (490nm). Data show specific cell killing of HapTl cells by splenocytes collected from virus treated HapTl tumor bearing hamsters. As a control, HaK cells (foreign tumor cell type) are spared.

Example 16 (depicted in Figure 12) : SCCF1 cells produce dominantly the EEV form of vvdd-luc while A549 cells produce both EEV and IMV forms of viral particles. Immunofluorescence visualization of EEV and IMV particles in SCCFland A549 cells 48h after infection with vv-tdtomato at 0.1 MOI.

Example 17 (depicted in Figure 13) : 100% confluent feline squamous cell carcinoma SCCF1 cells are resistant to vaccinia virus oncolysis but are able to continuously produce EEV particles. SCCF1 cells were infected with vvdd-luc at 0.1 pfu/cell and cell viability was assessed 3, 4, and 11 days later with MTS-assay. A549 cells were used as a control cell line (A). To visualize EEV particles, 100% confluent SCCF1 cells were infected with vvdd-luc at a viral dose of 0.1 pfu/cell and supernatant was collected 10 days later. Cellular debris was spinned down and clarified supernatant was stored in 50 ml Falcon tube at +4 °C for 3 days. Visible sediment formed into the bottom of Falcon tube was visualized with electron microscopy after negative staining. EEV form of viral particle isolated from supernatant of vvdd-luc infected SCCF1 cells. EEV particle is enveloped by two lipid bilayers and the measured size is 436 x 327 nm. Purified and negative stained vvdd-luc preparation was used as a control for visualization of IMV form of vaccinia particle. IMV particle is smaller in size (270 x 220) since it lacks the outer most lipid bilayer present only in EEV (B).

Example 18 (depicted in Figure 14) : Vaccinia virus shows increased tropism towards producer cell line in comparison to non-parental cell types. Vaccinia virus (vvdd-luc) was produced in 11 different cancer cell lines from 5 different species (A). Producer cell line and other cell lines were infected with vvdd-luc at 1 pfu/cell and luciferase expression was assessed 6 hours later. Purified viral preparations were used for viral transduction assay in parental and other cancer cell lines. Transduction efficiency of viral preparations was enhanced in parental cell line in comparison to other cancer cell lines (B). The data shows that production in A549 human tumor cells results in higher transduction of the same cells in comparison to monkey cells.

Example 19 (depicted in Figure 15) : Enhancing the release of EEV particles by silencing A34R gene. Schematic presentation of silencing viral gene A34R by using designed siRNA constructs against the gene to enhance the release of EEV particles.

Example 20 (depicted in Figure 16) : Summary of advantages from vaccinia virus - adenovirus combination therapy.

Example 21 (depicted in Figure 17) : Vaccinia virus and human adenovirus type 5 combine to kill cancer cells. When vaccinia virus and adenovirus were mixed at varying ratios and used to infect 786-0 human renal cell carcinoma cells in culture (where the immune system is not present) at indicated MOIs, additive cell killing was seen. A complex pattern of cell killing was observed 72 hours post infection : at some MOIs, VV and Ad are antagonistic (where the graph bulges outward compared to single infection as shown on the far left and right walls), at others synergistic (where graph bends inward compared to singly infected cells).

Example 22 (depicted in Figure 18) : Vaccinia virus is able to replicate and spread in cells already infected with human adenovirus. Human A549 cells in culture were infected with Ad5/3-D24-TK/GFP at MOI 10 and incubated for 12 hours. Subsequently, VV-tdTomato was added at varying concentrations to parallel wells and washed out 1 hour later. Agarose overlay was added and plaques (VV in red) were visualized under fluorescence microscope 72 hours later [left panel] . Singly VV infected cells were used as comparison [right panel] . Results show VV is able to form plaques on cells expressing late adenovirus genes indicative of full adenovirus replication cycle. Plaque size is similar to singly VV infected cells, further suggesting that the presence of adenovirus does not adversely affect VV replication cycle and that both viruses can therefore be combined to target the same cancer.

Example 23 (depicted in Figure 19) : Molecular mechanisms for autocrine and paracrine synergy mechanisms between adenovirus and vaccinia virus. Example 24 (depicted in Figure 20) : Adenovirus + vaccinia virus combination enhances therapeutic efficacy in an in vivo model of induced resistance towards oncolytic virotherapy. 5-to-7-week old SCID mice (groups of five mice each) were injected intraperitoneally with 3e5 SKOV3Luc cells in 100 ul PBS. Three days later, mice received an i.p. injection of either PBS (Vehicle) or le9 VP adenovirus (Ad5/3- D24) [triangles] . Two days after that, mice received i. p. either PBS or le8 PFU VV- tdTomato [squares] . The schedule was maintained weekly until study termination. Tumor burden was quantitated by IVIS (shown are means + SD of group tumors). Compared to saline, adenovirus alone is able to excert initial tumor control which diminishes over time and leads to growth of virus-resistant tumors as published (66). Vaccinia alone also gave initial anti-tumor activity which was later lost indicating that the model allows induction of resistance also against vaccinia. When combined, early tumor destruction was more emphatic than with single treatments and some mice were cured of their tumors. The combination resulted in a statistically significant improvement in efficacy over either virus alone. Example 25 (depicted in Figure 21) : Tumor destruction in immunocompetent mouse melanoma model demonstrates oncolytic potency of the vaccinia virus + adenovirus combination. Mouse B16.0VA cells were implanted subcutaneously in C57/BL6 mice (3e5 cells per mouse) and allowed to form palpable tumors (~5mm in diameter). Groups of mice received an intratumoral injection of either PBS or virus (for adenovirus, we used Ad5/3-D24 at 2el0 VPs and for vaccinia, le8 PFUs). Six days later, a separate set of mice (4 each, see Figure 2) were sacrificed for immunological analysis and another set of mice received another intratumoral injection, forming the indicated treatment groups (5-6 mice each) in the left panel), and six days after that the mice were sacrificed and organs and tumor extracted for analysis. Upper panel shows the size of the extracted tumors from each treatment group at study endpoint. Lower panel shows the quantitated data of tumor sizes, demonstrating that vaccinia virus followed by adenovirus provides greatest tumor control.

Example 26 (depicted in Figure 22) : Splenocytes from adenovirus or vaccinia virus-treated B16.0VA-tumor-bearing mice are able to destroy B16.0VA cells grown in culture. Singly treated mice from the experiment described in Figure 6, having received a single intratumoral injection of either adenovirus (2el0 VP) or vaccinia virus (le8 PFU), were sacrificed 6 days after virus injection for analysis. Spleens were harvested and pooled (from 4 mice) and single-cell suspensions of splenocytes generated by gentle needle aspiration. Twenty-four hours later, splenocytes (effectors) were mixed with trypsinized B16.0VA cells (targets) at indicated ratios and plated out in 96-well plates, keeping the amount of B16 cells constant (10 000 cells per well). Cell death was quantitated by MTS assay 72 hours later and shown is the viability compared to B16 cells plated without splenocytes. Results reveal that splenocytes from adenovirus-treated animals carry a larger tumor-destroying capacity compared to splenocytes from vaccinia-treated animals.

Example 27 (depicted in Figure 23) : Combination immunovirotherapy with vaccinia virus and adenovirus generates antigen-specific cytotoxic T cells. Tumors from B16.0VA tumor bearing mice treated with adenovirus or vaccinia virus were extracted and single cell suspensions generated by trituration and passing through a 40 um nylon mesh. Cells were stained with fluorescent antibodies against CD8+ T cells as well as with pentamer against mouse MHC loaded with the ovalbumin immunodominant peptide epitope SIINFEKL and analysed by flow cytometry (A). Results reveal a significantly greater antigen-specific CD8+ T cell accumulation in tumors treated with vaccinia virus followed by adenovirus (VV+Ad), 0.119% vs <0.005% in all other groups as indicated in the upper right quadrant of the rightmost panel (APC-A = pentamer vs. CD8+) where plotted are the cells in the region defined to the left. Tumors in this group were also the smallest at study endpoint (Example 18), reinforcing the finding that in this model, VV+Ad provides the greatest therapeutic benefit. (B) FACS data plotted into a bar graph. Example 28 (depicted in Figure 24) : Vaccinia virus - adenovirus combination treatment: anti-tumor immune response dominates over antiviral responses. The best therapy effect is achieved when vaccinia virus is followed by adenovirus by a novel mechanism : On one hand, vaccinia virus reduces antiviral effects in tumors and increases adenovirus replication. On the other hand, vaccinia virus induces central memory T cells against the tumor, which receive a strong inflammatory signal to proliferate and attack the tumor when adenovirus is injected. No other combination yields this effect; vaccinia followed by vaccinia lacks robust inflammation and the immune response recognizes the virus rather than the tumor. Adenovirus-adenovirus regimen also gears the immune response against the virus rather than the tumor, and while there is inflammation, adenovirus has not stimulated central memory T cells. In the adenovirus-vaccinia virus regimen inflammation after vaccinia infection is poor, and there are no central memory T cells to stimulate. Example 29 (depicted in Figure 25) : Feline and canine cancer cell lines can be transduced by vaccinia virus. Feline (SCCF1) and canine (Abrams, D17, ACE1, MDCK) cell lines were infected with vvdd-luc at viral doses of 0.04, 0.2, 1, or 5 pfu/cell. Luciferase expression was measured 24 hours later. ACE1 (prostatic carcinoma), Abrams (osteosarcoma), D17 (osteosarcoma), MDCK (kidney cell line), SCCF1 (squamous cell carcinoma).

Example 30 (depicted in Figure 26) : Feline and canine cancer cell lines can be killed by vaccinia virus vvdd-tdtomato (SEQ ID NO: 7). Subconfluent feline and canine cell lines were infected with vvdd-tdTOM at viral doses of 0.01, 0.1, 1, or 1 pfu/cell. Cell viability was assessed 4 days later. ACE1 (Canine prostatic carcinoma), Abrams (Canine osteosarcoma), D17 (Canine osteosarcoma), MDCK (Canine kidney cell line), SCCF1 (Feline squamous cell carcinoma).

Example 31 (depicted in Figure 27) : Vaccinia virus vvdd-luc reduces the growth of canine ACE1 prostate cancer tumors, lx 107 ACE1 (canine prostatic carcinoma) cells were injected subcutaneously into nude mice. Two intratumor injections of vvdd-luc at a dose of 1x105 pfu were done at indicated time points (arrows) and tumor growth was followed.

Example 32 (depicted in Figure 28) : Vaccinia virus is able to infect feline fibrosarcoma tumor tissue ex vivo. Cat fibrosarcoma tumor tissue was obtained from tumor surgery following owner informed consent and manually dissected into multiple, roughly equally sized fragments. These were split into 24-well plates containing 0.5 ml fresh growth media supplemented with antibiotics (P/S), serum (10%) and L-glutamine. 24 hours later, tissue pieces were either left untreteated or infected with le5 PFU VV-tdTomato virus. Fluorescence micrographs taken 24 hours later show evidence of VV replication [red] . Importantly, data shows that clinical tumor specimen can be infected with our vaccinia constructs.

Example 33 (depicted in Figure 29) : VV and Ad can transduce cells in canine osteosarcoma tissue even when combined. Primary cancer tissue was obtained during surgery of a male dog with solid osteosarcoma. Cultured slices were infected at le6 PFU VV-tdTomato and/or le9 TU Ad5/3-D24-TK/GFP virus and followed under microscope. Results show both VV and Ad can transduce cells in dog osteosarcoma tissue even when combined.

Example 34 (depicted in Figure 30) : hCD40L is active in canine PBMCs. PBMC's were isolated from blood donor dogs. PBMCs were cultured with the supernatant collected from virus infected A549 cells (control virus Ad5/3hTERTE3 or CD40L coding virus Ad5/3hTERT-CD40L) and supernatant was filtered through 2um filter to remove virus particles before adding to the PBMCs. Growth media was collected at 24, 48, 72, and 96 hours post adding the supernatant and ELISA for canine IL-8 was performed. Values are presented after subtraction of mock values (=stimulation with supernatant from non-infected A549 cells).

Example 35 (Figure 31) : Both adenovirus and vaccinia virus replicate productively in co-infected human SKOV3 ovarian cancer cells (adenovirus Ad5/3-D24 100 TU/cell, vaccinia virus VV-tdTomato 1 PFU/cell) as seen by EM 72 hours post infection. Example adenovirus particles are indicated by black arrows, vaccinia particles by white arrows. Magnification ~40 OOOx.

Example 37 (Figure 32):Tumor tissue was analyzed by FACS : increased infiltration of NK cells was observed in tumors treated with vaccinia virus (either VV+Ad or VV+VV). Heterologous combination with vaccinia virus followed by adenovirus yielded the greatest responses, demonstrating that the effect is not dependent on either virus alone but likely is dependent on vaccinia virus.

Example 38 (Figure 33): Virus load and levels of neutralizing antibodies. B16.0VA tumor tissue was extracted at study endpoint and snap-frozen. After rotor homogenization in 1000 ul PBS, samples were freeze-thawed three times to release infectious virus. Then tumor homogenate was titered for adenovirus by TCID50 and vaccinia virus by plaque assay on A549 cells. Results show repeated dosing of adenovirus reduces virus load in the tumors at study end below detection level. Switching the latter dosing to vaccinia virus rescues adenovirus presence in the tumors. On the other hand, vaccinia virus is able to persist in mouse B16 tumors for longer than adenovirus. Bottom panels: analysis by neutralizing antibodies in serum from treated tumor-bearing mice at study end reveals induction of antibodies against adenovirus in mice receving repeated injections, whereas vaccinia virus alone induces neutralizing antibodies in 12 days irrespective of whether adenovirus is injected in the same tumors 6 days after vaccinia virus or not. This suggests vaccinia virus replicates productively in B16 tumors and therefore elicits a qualitatively more robust antibody response in mice compared to adenovirus. This antibody response does not manifest within 6 days after virus inoculation. On the other hand, coupled with the virus levels in the tumors at study end, it appears adenovirus persists as long as only one dose is given (Ad-VV group, top row), and vaccinia virus persists despite appeareance of neutralizing antibodies (titers seen even in the VV-VV group, top row) . Example 39 (Figure 34): Evaluation of the role of NK cells in vaccinia/adeno combination therapy 6.67χ10^Λ9 VP of adenovirus was given intratumorally per injection. For vaccinia virus, the dose was 3.33χ10^Λ7 per injection. Also, all mice were given intraperitoneal injections of 33 ul polyclonal rabbit anti-mouse Asialo GM 1 antibody (to deplete NK cells) on days indicated by yellow circles. Animals were sacrificed and sampled 14 days post first virus injection. The results show NK cells are not critical for the anti-tumor effect of vaccinia virus, despite induction of such cells (Example 37, Figure 32).

Example 41 (Figure 35): Adenovirus (Ad5/3-D24-TK/GFP, 10 TU/cell) and VV (VV- tdTomato, 0.1 PFU/cell) were used to infect human SKOV3Luc ovarian cancer cells either pre-treated or not for 6 hours with putative antiviral recombinant human cytokines; interferon (IFN) beta, IFN gamma and/or tumor necrosis factor alpha. Fluorescence micrographs taken 72 hours post infection suggest a reduction in replication of both viruses in response to the antiviral cytokines.

Figure 42 (Figure 36): Quantitation of virus titers in the experiment described in the previous figure. To determine vaccinia virus titers in infected SKOV3Luc cells 72 hours post infection, plaque assay on A549 cells was conducted using whole well freeze-thaw lysate (top panel), and for adenovirus, TCID50 assay in A549 cells was performed (bottom panel). Results show increased vaccinia virus titers in cells pretreated with IFN gamma and co-infected with vaccinia and adenovirus compared to vaccinia virus alone (*, p<0.05, studen't t test on log-transformed titers, duplicate wells per virus/regimen). This suggests that adenovirus may be antagonizing the antiviral activity of IFN gamma against vaccinia virus. In general, IFN beta and IFN gamma appear to mediate a more potent antiviral effect against both adenovirus and vaccinia virus than TNF alpha. Co-infection of cells does not markedly reduce (or increase) replication of either virus, despite pretreatement with antiviral cytokines, suggesting heterologous virus interference is minimal and does not involve antiviral signaling mechanisms.

Example 43 (Figure 37): Oncolytic vaccinia virus and adenovirus are able to co- infect primary surgical human cancer tissue. Shown are fluorescence micrographs of double-infected human ovarian cancer tissue (adenovirus Ad5/3-D24-TK/GFP, 1χ10^Λ8 TU/well, vaccinia virus VV-tdTomato 1x107 PFU/well in a 24-well plate in 0.5ml standard growth medium) 72 hours post infection. Double-infected cells appear yellow in the overlay micrograph.

Example 44 (Figure 38): Analysis of levels of infectious virus in primary ovarian cancer tumor tissue at experiment end (previous figure) shows productive replication of vaccinia virus over time, irrespective of whether adenovirus was co-infecting the tissue or not, and productive replication of adenovirus when combined with vaccinia virus, suggesting enhancement of adenovirus replication by vaccinia virus.

Example 45 (Figure 39): Vaccinia virus production on SCCF1 cells which preferentially produce EEV (the most appealing form of vaccinia for intravenous injection). Sucrose gradient ultracentrifugation of cell pellets results in the IMV form of vaccinia while centrifugation of the supernatant reveals the EEV band.

Tube 1 : Collected cell pellet (containing IMV)

Tube 2: harvested supernatant (containing mostly EEV and only a small amount of IMV) Tube 3 : uninfected cell supernatant (no virus) Both bands of the middle tube are infectious.

Example 46 (Figure 40): Vvdd-hCD40L-tdTomato targeting and replication in vivo following intratumoral and intravenous administration. Mice bearing EJ and A549 tumors were injected with vvdd-tdTomato and vvdd-hCD40L-tdTomato (a) intratumorally and (b) intravenously. tdTomato expression was visualized by IVIS.

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Claims

1. A modified vaccinia virus vector, characterized in that the vector comprises vaccinia virus genome wherein the thymidine kinase gene is inactivated by an open reading frame ablating deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a heterologous protein.

2. The modified vaccinia virus vector according to claim 1, characterized in that the thymidine kinase gene comprises at least one insertion site for inserting the heterologous protein and thymidine kinase inactivation is carried out by a deletion in the thymidine kinase region conferring activity.

3. The modified vaccinia virus vector according to any one of claims 1 - 2, characterized in that the first heterologous protein is an enzymatically inactive beta- galactosidase, capable of producing an immune response, inserted in the place of the deleted vaccinia growth factor gene and another heterologous protein is at least one of CD40L, a fluorescent marker protein, and GMCSF inserted in the insertion site of the partially deleted thymidine kinase gene.

4. The modified vaccinia virus vector according to any one of claims 1 - 3, characterized in that the modified vaccinia virus is Western Reserve (WR) strain vaccinia virus and the modified vaccinia virus vector comprises the genes for expressing human CD40L (hCD40L) and tdTomato as heterologous proteins.

5. The modified vaccinia virus vector according to claim 4, characterized in that the human CD40L (hCD40L) gene and tdTomato gene are inserted in the partially deleted thymidine kinase gene.

6. A host cell carrying a modified vaccinia virus vector according to any one of claims 1 - 5.

7. The host cell according to claim 6, characterized in that the host cell is a cancer cell originating from feline squamous cell carcinoma cell line SCCF1 or from human lung adenocarcinoma A549 cell line.

8. A modified vaccinia virus particle containing a modified vaccinia virus vector according to any one of claims 1 - 5, characterized in that the virus particle is of the type intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell- associated enveloped virus (CEV), or extracellular enveloped virus (EEV), more preferably the virus particle is of the type EEV or IMV, and most preferably the virus particle is of the type EEV.

9. The modified vaccinia virus particle according to claim 8, characterized in that the modified vaccinia virus particle comprises inactivation of the viral A34R protein by gene silencing, by post-translational gene silencing, by RNA interference, or by small interfering RNA (siRNA).

10. A pharmaceutical composition characterized in that the pharmaceutical composition comprises a pharmaceutically acceptable carrier and one or more of the following : the modified vaccinia virus vector according to any one of claims 1 - 5, the host cell according to any one of claims 6 - 7, and the virus particle according to any one of claims 8 - 9.

11. The pharmaceutical composition according to claim 10 comprising a therapeutically active amount of an oncolytic adenovirus.

12. A kit characterized in that the kit comprises one or more containers and one or more of the following : the modified vaccinia virus vector according to any one of claims 1 - 5; the host cell according to any one of claims 6 - 7; the virus particle according to any one of claims 8 - 9; the pharmaceutical composition according to claim 10; and the pharmaceutical composition according to claim 11 wherein the oncolytic adenovirus is provided in the same or a separate container.

13. An in vitro method for producing a modified vaccinia virus, characterized in that the method comprises the steps of providing producer cells capable of sustaining production of vaccinia virus particles and carrying a modified vaccinia vector according to any one of claims 1 - 5; culturing the producer cells in conditions suitable for virus replication and production; and harvesting the virus particles.

14. The method according to claim 13, characterized in that the producer cells comprise SCCFl cells or A549 cells and the harvesting step comprises harvesting EEV particles.

15. The modified vaccinia virus vector according to any one of claims 1 - 5, the host cell according to any one of claims 6 - 7, the virus particle according to any one of claims 8 - 9, the pharmaceutical composition according to claim 10 or 11, or the kit according to claim 12 for use in cancer therapy.

16. The modified vaccinia virus vector according to any one of claims 1 - 5, the host cell according to any one of claims 6 - 7, the virus particle according to any one of claims 8 - 9, the pharmaceutical composition according to claim 10 or 11, or the kit according to claim 12 for use as a medicament to elicit immune response in a subject.

17. An in situ cancer vaccine comprising the modified vaccinia virus vector according to any one of claims 1 - 5, the host cell according to any one of claims 6 - 7, the virus particle according to any one of claims 8 - 9, the pharmaceutical composition according to claim 10 or 11, or the kit according to claim 12.

18. The modified vaccinia virus vector according to any one of claims 1-5 or the composition according to claim 10 or 11 for use in a method of inhibiting malignant cell proliferation in a mammal, characterized in that the method comprises administering to the mammal the modified vaccinia virus vector or the composition an amount sufficient to inhibit malignant cell proliferation compared to the malignant cell proliferation that would occur in the absence of the said modified vaccinia virus vector or the composition.

19. The modified vaccinia virus vector according to any one of claims 1-5, the host cell according to claim 6 or 7, the virus particle according to claim 8 or 9, the pharmaceutical composition according to claim 10 or 11, or the kit according to claim 12 for use in combination therapy or prophylaxis of cancer, wherein in the combination therapy or prophylaxis comprises administering the modified vaccinia virus vector and adenovirus.

20. An in vitro or in vivo method for detecting the presence of the modified vaccinia virus vector according to any one of claims 1 - 5 in a subject, characterized in that the method comprises the step of detecting the presence of a marker present in the virus.