CANCER THERAPY AGENTS AND METHODS OF USE

FIELD OF THE INVENTION

The present invention relates to the field of cancer therapy, agents suitable in the prophylactic or therapeutic treatment of cancer, and corresponding methods. In particular, the invention relates to the use of viral agents as cancer therapeutics.

BACKGROUND TO THE INVENTION

Numerous viral species have been proposed for use as oncolytic viruses and / or platforms for cancer vaccines and anti-cancer gene therapy. First generation oncolytic viral agents, such as retroviruses, employ innate selectivity for replication in cancer cells, while second generation viral agents, such as adenoviruses, are engineered for selectivity. More recently, third generation agents that combine oncolytic properties with the capacity for delivery of therapeutic gene products have been devised.

In recent years, poxviruses have been studied as therapeutic candidates due to several inherent biological properties including: 1) rapid replication in and lysis of infected cells; 2) broad tumour tissue cell tropism due to the lack of the need for specific cell surface moieties for cell entry; 3) non-integrating replication cycle that operates independent of host cell machinery; 4) limited natural antigenicity, and 5) large coding capacity. In one example, United States patent application 10/543,944, published as US 2006/0159706 on July 20, 2006 and which is incorporated herein by reference, provides further information with regard to therapeutic applications of poxviruses, and discloses poxvirus insertion mutants.

Numerous poxvirus genera have been investigated for their potential use in cancer therapy. Of these the orthopoxvirus, Vaccinia virus (VV), has emerged as a primary candidate. (Non-orthopoxviruses, such as leporipoxvirus Myxoma virus (MV) or the avipoxvirus Canary poxvirus (CPV), which have been proposed as anticancer platforms typically lack the broader cell tropism of VV.) However, due to the systemic eradication of smallpox using VV variants, the use of VV based therapies is hampered by pre-existing immunity against the virus in much of the general population. Furthermore, replication competent VV is contraindicated for use in approximately 30% of the general population due to pre- existing health conditions or treatments, such as immune suppression.

Hence there is a continuing need in the art for viral-based anti-cancer agents, and viruses suitable for use as a platform to develop such anti-cancer agents. There is also a need for poxvirus-based anti-cancer agents that exhibit new properties to counter the disadvantages of previous poxvirus agents. For example, there is a desire to develop poxviruses having narrower cell tropism with an improved capacity to target therapies to specific sites.

SUMMARY OF THE INVENTION

It is one object of the present invention, at least in selected embodiments, to provide an agent suitable for use in cancer therapy.

It is another object of the present invention, at least in selected embodiments, to provide a method for treating cancer in a patient.

Certain exemplary embodiments provide an anticancer agent comprising a Yatapoxvirus mutant. In some embodiments the Yatapoxvirus mutant infects and replicates in cancerous cells, with abrogated infection and replication capacity in non-cancerous cells. In some embodiments a capacity of the Yatapoxvirus mutant to replicate in cancerous cells is regulatable by modulation of ERK-I activity in the cancerous cells.

In selected exemplary embodiments the Yatapoxvirus mutant is a Tanapoxvirus mutant. In selected exemplary embodiments the mutant is incapable of expressing a functional 18L gene product. For example, the mutant may comprise a disrupted 18L gene sequence, wherein the 18L gene sequence comprises at least a portion of the 18L promoter sequence, and / or at least a portion of the 18L open reading frame encoding the 18L gene product, and the disruption comprises point mutation, insertion or deletion of nucleotides of the 18L gene sequence.

In certain exemplary embodiments the 18L gene product is a T18L gene product of Tanapoxvirus. Optionally, the 18L gene sequence encodes a 18L gene product that is at least 70% identical, more preferably at least 90% identical, more preferable at least 95% identical, more preferably at least 99% identical to SEQ ID NO: 3.

In certain exemplary embodiments the mutant comprises an expression cassette inserted into the DNA sequence of the mutant, the expression cassette comprising a promoter sequence, and an expression sequence to be expressed from the promoter sequence. In selected embodiments the expression sequence encodes a cytotoxic protein, or a protein that is able to activate cytotoxic properties of a coadministered compound. In selected embodiments the expression cassette is inserted at or adjacent a position of an 18L gene sequence, or replaces at least a portion of an 18L gene sequence of the virus, thereby to disrupt the 18L gene sequence.

In other exemplary embodiments there is provided an anticancer agent comprising a recombinant virus that is incapable of expressing a biologically active 18L gene product. In some embodiments the recombinant virus infects and replicates in cancerous cells, with abrogated infection and replication capacity in non-cancerous cells. In some embodiments a capacity of the recombinant virus to replicate in cancerous cells is regulatable by modulation of ERK-I activity in the cancerous cells.

In further embodiments the recombinant virus is a poxvirus, preferably a Yatapoxvirus, more preferably a Tanapoxvirus. For example, the recombinant poxvirus may optionally comprise a disrupted 18L gene sequence, wherein the 18L gene sequence comprises at least a portion of the 18L promoter sequence, and / or at least a portion of the 18L open reading frame encoding the 18L gene product, and the disruption comprises point mutation, insertion or deletion of nucleotides of the 18L gene sequence. In selected embodiments the 18L gene sequence encodes a 18L gene product that is at least 70% identical, preferably 90% identical, preferably 95% identical, more preferably 99% identical to SEQ ID NO: 3.

In other exemplary embodiments the recombinant virus comprises an expression cassette inserted into the DNA sequence of the virus, the expression cassette comprising a promoter sequence, and an expression sequence to be expressed from the promoter sequence. Optionally, the expression sequence encodes a cytotoxic protein, or a protein that is able to activate cytotoxic properties of a coadministered compound. Optionally the expression cassette is inserted at or adjacent a position of the 18L gene sequence, or replaces at least a portion of the 18L gene sequence, thereby to disrupt the 18L gene sequence.

Further exemplary embodiments provide a construct or expression cassette for use in producing a recombinant virus that is incapable of expressing a functional 18L gene product. Optionally the construct or expression cassette comprises the nucleotide sequence of SEQ ID NO: 4.

Further exemplary embodiments provide pharmaceutical composition comprising an anticancer agent as provided herein, together with at least one pharmaceutically acceptable excipient, carrier, or diluent.

Further exemplary embodiments provide a method of treating cancer, the method comprising administering of an anticancer agent as provided herein to a patient in need of such treatment. Optionally, the cancer is selected from a cancer of the breast, a cancer of the colon and a cancer of the brain.

Further exemplary embodiments provide for a use of an anticancer agent of as provided herein, in the treatment of cancer.

Further exemplary embodiments provide for a use of an anticancer agent as described herein, in the manufacture of a medicament for use in the treatment of cancer. Further exemplary embodiments provide for a use of an 18L or T18L gene sequence or gene product, or a functionally active portion thereof, in the inhibition of ERK-I or ERK-I activation.

Further exemplary embodiments provide for a use of an 18L or T18L gene sequence or gene product, or a functionally active portion thereof, as an immunosuppressive agent.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - Broad cell tropism of TPV in human cancer cells. The indicated human cancer cells were infected with TPV-WT at the indicated multiplicity of infection (MOI) and fluorescence images taken at Day 3 post-infection (PI). The presence of the foci characteristic of TPV replication and spread in culture indicates productive virus infection, while expression of green fluorescence protein (gfP) engineered into the virus indicates delivery and expression of exogenous genes. Images captured at 10OX magnification.

Figure 2 - Replication of wild-type and deletant viruses in representative human tumour cell types. Representative control (OMK), supportive (U373), restrictive (MCF7) and abortive (HeIa) cell types were infected with TPV-WT or TPVΔ18L at a multiplicity of infection (MOI) of 0.1 (input titer of one infectious virion for every ten cells). Both viruses have been engineered to express green fluorescence protein (gfp) as an exogenous gene for ease of tracking the infection. Fluorescence images were taken at Day 5 post-infection (PI) and show the presence or absence of the foci characteristic of TPV replication and spread in culture. Both viruses replicated in control and U373 neuroglioma cells; however, U373 cells supported more rapid and intensive virus replication as indicated by the increase gfp expression and the size of the foci. Only TPV-WT could replicate and spread in MCF7 breast cancer cells, while neither virus could replicate in HeLa cervical cancer cells. Images captured at 10OX magnification.

Figure 3. Single-step viral growth curves for wild-type and deletant viruses in representative human tumour cell lines. Representative control (A, OMK), supportive (B, U373), restrictive (C, MCF7) and abortive (D, HeIa) cell types were infected with TPV-WT (upper line with diamonds) or TPVΔ18L (lower line with squares) at MOI = 5.0 and cells harvested for virus isolation at the indicated time- points PI. Viral titers were determined by serial dilution of infectious virus obtained from the infected cells and titration on OMK cells. Both viruses replicated in control and U373 neuroglioma cells producing similar maximum titers. TPV-WT replicated in MCF7 breast cancer cells to a comparable extent as in permissive cells, whereas TPVΔ18L did not replicate to even the input titer value. Neither virus could replicate in HeLa cervical cancer cells to produce significant progeny virions. Titers are expressed as focus forming units (ffu) of progeny virus per million infected cells.

Figure 4 - Exogenous gene expression following infection of representative human tumour cell types with wild-type and deletant viruses. Representative control (OMK), supportive (U373), restrictive MCF7 and abortive (HeIa) cell types were infected with TPV-WT or TPVΔ18L at the indicated MOI. Fluorescence was measured in a fluorescence plate reader 24 hpi. High levels of expression of the exogenous gfp cassette were evident in the control and U373 neuroglioma cells permissive to infection by both viruses. Of note, significant and comparable levels of gfp expression were also detected following infection of restrictive MCF7 cells by either virus despite the inability of the Tl 8L deletant to productively infect these cells. Thus, the deletant virus could deliver and express exogenous genes in the absence of productive infection. Images captured at 10OX magnification.

Figure 5 - Rescue of deletant virus infection in Type II cells by expressing T18L. Type II MCF-7 breast cancer cells (A-D) and A549 lung cancer cells (E-H) were engineered to stably express either Flag-tagged Tl 8L (C, D, G, H) or vector expressing the Flag epitope alone (A, B, E, F). Cells were infected with TPV-WT or TPVΔ18L at MOI = 0.1 and foci formation evaluated at Day 3 PI. The small foci indicated above reflect the low initial input titer of virus and the relatively early time-point selected for evaluation. TPV-WT was able to replicate in MCF-7 And A549 expressing either the Flag epitope alone or Flag-Tl 8L; however, replication was more rapid when T18L was overexpressed. In comparison, TPVΔ18L was unable to replicate in MCF7 and A549 cells expressing vector alone, but were able to replicate in cells expressing Tl 8L. Images captured at 200X (A-D) or IOOX (E-H) magnification. Similar results were observed in Type II A549 lung cancer and Type II HepG2 hepatic cancer cell lines, indicating that the tumour cell tropism of the knock-out virus was a product of deleting the T18L gene. Images captured at 200X magnification.

Figure 6 - Viral gene expression in representative tumour cell types.

Representative control (OMK), supportive (U373), restrictive (MCF7) and abortive (HeIa) cell types were infected with TPV-WT or TPVΔ18L at MOI = 1.0 and cells harvested at 24 h and 72 h PI for quantitative real-time PCR (QRT-PCR) detection of typical early (Tl OL) and late (T 149L) genes. Gene expression is expressed relative to the housekeeping gene, GAPDH. Poxvirus gene expression occurs in four distinct phases (early, early/late, genome replication, late). In most cases, early gene expression occurs in all cell types but the blockade to permissive replication occurs at the viral genome replication stage. Thus, early gene expression can occur in the absence of productive infection, whereas late gene expression does not. Both early and late gene expression is evident for TPV-WT and TPVΔ18L in control and supportive cell lines at 24 h and 72 h PI indicating productive infection. Delayed gene expression is evident for the wild-type virus in Type II MCF-7 breast cancer cells, but no viral gene expression was detected in Type II and Type III tumour cells with TPVΔ18L. However, expression of the exogenous gfp gene is evident at the single cell level (e.g. no spread, no foci) following TPVΔ18L infection of MCF cells.

Figure 7 - Expression of T18L attenuates Vaccinia virus (VV) replication.

Human HEK293 kidney cells were engineered to stably express either Flag-tagged T18L (FlagT18L) or vector expressing the Flag epitope alone (vector). Cells were infected with VV expressing gfp at MOI = 0.01, 0.1 or 1.0. Plaque formation was valuated at 24 h (top) and 48 h (bottom) PI by fluorescence microscopy as an indicator of replication and spread. HEK293 are fully permissive to VV replication as indicated in the cells expressing vector alone. Expression of T18L, however, markedly attenuated VV replication and spread at 24 and 48 h. VV replication, unlike TPV replication, is dependent on the activation of the signaling kinase, ERK- 1, early in the lifecycle of the virus. Inhibition of ERK-I in infected cells is associated with a delay in VV infection due to poor early spread of the virus. The resulting profile resembles that observed in HEK293 cells expressing T18L. Images captured at IOOX magnification.

Figure 8 - Loss of T18L expression is associated with increase ERK-I expression. Restrictive human A549 lung cancer cells were mock-infected (M) or infected with TPV-WT (W) or TPVΔ18L (K) at MOI = 0.1, 1.0 or 5.0, Cells were harvested at 6, 24 or 72 h PI for protein isolation and immunoblot detection of the active, phosphorylated form of ERK-I . The housekeeping protein, β-actin, served as loading control. ERK-I activation was reduced in cells infected with TPV-WT, but increased or apparent at levels equivalent to mock-infected cells following TPVΔ18L. At 6 h PI. a failure to suppress initial ERK-I activation in response to infection is observed at the lower MOIs. At 24 h PI, a failure to suppress the increase in ERK-I activation relative to mock-infected cells is seen. These findings, like the VV inhibition shown in Fig 5, indicate a role for T18L in inhibiting ERK-I activation. Under normal circumstances, ERK-I is a critical determinant of the host cell immune response to virus infection through its role in mediating anti-viral interferon (IFN) responses. As a result, poxviruses encode immunomodulatory proteins to circumvent this response. Of note, T18L is one of only a few known viral pyrin domain (PYD) proteins. It exhibits high homology with the IFN regulatory factor (IRF) family of innate immune proteins responsible for mediating the IFN response in infected cells.

Figure 9 - Pharmacological inhibition of ERK-I rescues the deletant mutant.

Type II MCF-7 breast cancer cells were infected with TPVΔ18L at MOI = 1 in the presence or absence of the selective ERK-I inhibitor, UO 126 (20 μM). Foci formation was evaluated at 24 and 48 h PI as an indicator of replication and spread. Inhibition of ERK-I by UO 126, replacing the ERK-I inhibiting activity of the deleted T18L gene product, enabled TPVΔ18L to replicate in MCF cells and reversed the transiently restrictive phenotype of the tumour type. Also of note, the low level expression of the exogenous gfp gene cassette at 48 h PI in restrictive cells following TPVΔ18L infection indicates the capacity for the deletant to deliver and express foreign genes in the absence of productive infection or viral gene expression. Images captured at IOOX magnification.

Figure 10 - Suppression of ERK transcription rescues the deletant mutant.

A-D, Type II A549 breast cancer cells were infected at MOI = lwith TPV-WT (A) or TPVΔ18L in the absence of intervention (B), the presence of a vector control (C), or in the presence of vector expressing siRNA specific to ERK (D). Foci formation was evaluated at 72 h PI as an indicator of replication and spread. Inhibition of ERK-I by siRNA suppression of transcription, replacing the ERK inhibiting activity of the deleted T18L gene product, enabled TPVΔ18L to replicate in A549 cells and reversed the transiently restrictive phenotype of the tumour type. Images captured at 10OX magnification. E, Western blot analysis of ERK expression in mock- transfected controls (M) or cells transfected with vector control (-) or vector expressing ERK-I siRNA (+). Expression of siRNA specific to ERK suppressed ERK production relative to control values. Detection of the actin housekeeping gene served as a loading control.

Figure 11 - T18L expression inhibits other signaling pathways subject to pharmacological manipulation. Human HEK293 cells stably expressing T18L were transfected with a NFKB reporter vector or vector lacking the NFKB response element. NFKB activation in response to TNF stimulation (10 ng/ml) for 24 h was evaluated by expression of the luciferase gene cassette located downstream of the NFKB element. Compared to cells lacking Tl 8L expression, NFKB activity in response to TNF was markedly reduced in T18L-expressing cells. A stable HEK293 cell line expressing the NFKB reporter element only is shown for comparison. These results indicate that Tl 8 L expression can regulate NFKB signaling events in addition to ERK-I activity.

Figure 12 - Insertional mutation of the T18L gene. The tanapoxvirus (TPV) Tl 8L gene was disrupted by insertion of an approximately 754 bp green fluorescence protein (gfp) cassette whose expression was under the control of a synthetic early-late poxvirus promoter (E/L). The insertion was designed such that two nucleotides central to the gene (nt 14054 and 14055) were excised and the gene effectively bisected into two equal sections representing nt 13863 to 14053 (left fragment) and nt 14056 to 14246 (right fragment). No other changes were introduced into the sequence of the wild-type. The open reading frame of the T18L gene is orientated in a direction opposite to that of the nucleotide sequence (arrow). As such, transcription initiates at nt 14246 and terminates at nt 13863.

DEFINITIONS:

"construct", as used herein refers to an engineered DNA molecule including one or more nucleotide sequences from different sources. A preferred construct includes at least an RNA-encoding region operably linked to a promoter sequence.

"downstream" refers to nucleotide sequences that follow, e.g., are on the 3' side, of a reference sequence.

'"DNA segment" refers to a linear fragment of single- or double-stranded deoxyribonucleic acid (DNA), which can be derived from any source.

"effective amount", as used herein, is defined as that amount necessary or sufficient to treat or prevent a disorder. The effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular agent being administered. One of ordinary skill in the art would be able to study the aforementioned factors and make the determination regarding the effective amount of the agent without undue experimentation.

"expressed" means the generation of a RNA molecule from a DNA molecule (i.e., a complementary RNA molecule generated from the DNA molecule by the process of transcription) and / or the generation of a polypeptide or protein molecule from a DNA molecule via a RNA intermediate (i.e., by the processes of transcription and translation), "expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context such as in vitro expression.

"gene" includes genomic DNA, cDNA, RNA, or other polynucleotides that encode gene products, and in the case of a polynucleotide may or may not include promoter or enhancer sequences.

"gene product" refers primarily to proteins and polypeptides, or fragments thereof, encoded by other nucleic acids (e.g., non-coding and regulatory RNAs such as tRNA, sRNPs). "involved" in a disorder includes a gene, the normal or aberrant expression or function of which effects or causes a disease or disorder or at least one symptom of said disease or disorder.

"modulation" or "modulate" refers to an influence upon gene expression (including transcription and / or translation) that may include maintenance and / or upregulation and / or down regulation of the gene expression.

"mRNA" or "messenger RNA" refers to a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

"nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. The term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms.

"mutation" refers to a substitution, addition, or deletion of a nucleotide within a gene sequence resulting in aberrant production (e.g., misregulated production) of the protein encoded by the gene sequence. A "gain-of-function" mutation is a mutation that results in production of a protein having aberrant function as compared to the wild-type or normal protein encoded by a gene sequence.

"pharmaceutical composition" as used herein, refers to an agent formulated with one or more compatible solid or liquid filler diluents, excipients, or encapsulating substances which are suitable for administration to a human or other animal.

"preferred" or "preferably" refers to preferred features of the broadest embodiments of the invention, unless otherwise stated. "promoter" refers to a DNA sequence to which RNA polymerase can bind and initiate transcription. An "inducible promoter" is a DNA sequence which, when operably linked with a DNA sequence encoding a specific gene product, causes the gene product to be substantially produced in a cell only when an inducer which corresponds to the promoter is present in the cell. The term "Pol III promoter" refers to an RNA polymerase III promoter. Exemplary Pol III promoters include, but are not limited to, the U6 promoter, the Hl promoter, and the tRNA promoters. The term "Pol II promoter" refers to an RNA polymerase Il promoter. Exemplary Pol II promoters include, but are not limited to, the CMV promoter and the Ubiquitin C promoter. The term "promoter" may also encompass any "bacterial promoter". The expression "bacterial promoter" includes any promoter that exhibits some degree of functional activity in a bacterium of any kind, and therefore encompasses any wild- type or artificially modified promoter of bacterial or viral derivation. Such a promoter may be selected from the following non-limiting group: a T3 RNA polymerase promoter, a T7 RNA polymerase promoter, and an SP6 RNA polymerase promoter.

"regulation of expression" refers to events or molecules that increase or decrease the synthesis, degradation, availability or activity of a given gene product.

"RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides. "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e.. dsRNA and dsDNA, respectively).

"RNA interference" or "RNAi", as used herein, refers generally to a sequence- specific or selective process by which a target molecule (e.g., a target gene, protein or RNA) is modulated. In specific embodiments, the process of "RNA interference" or "RNAi" features degradation of RNA molecules, e.g., RNA molecules within a cell, said degradation being triggered by an RNA agent. Degradation is catalyzed by an enzymatic, RNA-induced silencing complex (RISC). RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi is known to proceed via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated artificially, for example, to modulate or silence the expression of target genes.

"RNA agent", as used herein, refers to an RNA (or analog thereof), comprising a sequence having sufficient complimentary sequence to a target RNA (i.e., the RNA being degraded) to direct RNAi. A sequence having a '"sufficiently complementary sequence to a target RNA sequence to direct RNAi" means that the RNA agent has a sequence sufficient to trigger the destruction of the target RNA by the RNAi machinery (e.g., the RISC complex) or process. siRNA refers to small interfering RNA.

"treatment", as used herein, is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease or disorder, a symptom of a disease or disorder, or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward a disease or disorder. A therapeutic agent includes, but is not limited to a composition comprising at least one of a virus, a recombinant virus, a mutant virus, interfering RNA, small molecules, peptides, antibodies, ribozymes, antisense oligonucleotides, and chemotherapeutic agents.

"transfecting" or "transfection" refers to a process by which polynucleotides such as DNA are caused to enter a eukaryotic cell or organism. Preferably, following entry the polynucleotides reside in a stable or transient manner in the eukaryotic cell or organism, and RNA is expressed from the polynucleotide where and / or when appropriate. Preferably, the process of transfection involves a technique that is well known in the art including but not limited to the use of viral or liposome vector (e.g. Lipofectamine™), and other techniques involving ballistic acceleration of microparticles.

"transforming" or "transformation" refers to a process by which polynucleotides such as DNA or RNA are caused to enter a prokaryotic organism. Preferably, following entry the polynucleotides reside in a stable or transient manner in the prokaryote, and RNA is expressed from the polynucleotide where and / or when appropriate. Preferably, the process of transforming involves a technique that is well known in the art including but not limited to electroporation, heat shock techniques, and other techniques involving ballistic acceleration of microparticles.

"upstream" refers to nucleotide sequences that precede, e.g., are on the 5' side, of a reference sequence.

"18L gene product" - The expression "18L gene product" as defined herein refers to any protein, or a part thereof, that is a member of a recently discovered class of poxviral pyrin domains (PYD) proteins (see for example Johnston et al, Immunity, 23, p.587-598 (2005) incorporated herein by reference). Members of the family include, but are not limited to, Swinepoxvirus (SPV) 14L, Yaba-like disease virus (YLDV) 18L, Shope fibroma virus (SFV) gpl 3L, Myxoma virus (MV) 13L, and Tanapoxvirus (TPV) T18L. In other embodiments, a group of proteins that fall under the definition of "18L gene product include proteins with at least 70%, preferably at least 90%, more preferably at least 95%, most preferably at least 99% identity to at least a portion of the Tl 8L gene product from Tanapoxvirus as shown in SEQ ID NO: 3.

"18L gene sequence" - refers to a nucleotide sequence that encodes an 18L gene product as defined herein, or a part thereof, or a promoter sequence or a part thereof normally associated with an open reading frame of an 18L gene sequence in a wild- type virus.

"Tl 8L gene product" - refers to a protein product encoded by the 18L gene sequence of a member of the Yatapoxvirus family of viruses. Such protein products are sometimes typified by the presence of a pyrin domain. In selected embodiments, a T18L gene product may refer to a protein encoded by the open reading frame of the T18L gene sequence of Tanapoxvirus, as indicated in SEQ ID NO: 3 or a part or variant thereof, or in selected embodiments an 18L gene product encoded by Yaba- like disease virus (YLDV) or a part thereof.

"Tl 8L gene sequence" - refers to a nucleotide sequence that encodes a Tl 8L gene product as defined herein or a part thereof, or a promoter sequence or a part thereof normally associated with an open reading frame of an 18L gene sequence in a wild- type virus.

The terms used herein are not limiting to the invention as described and claimed.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have succeeded in the development of mutant and recombinant viruses that are suitable for use in cancer therapy. Through significant effort and investigations, the inventors have focused their attention upon mutant and recombinant viruses that, in contrast to their wild-type counterparts, exhibit more selective tropism to tumour cells.

The aforementioned investigations have focused in part upon viruses that include an 18L gene product and variants thereof, which in selected embodiments refers to any protein, or a part or variant thereof, that is a member of a recently discovered class of poxviral pyrin domains (PYD) proteins (see Johnston et al, Immunity. 2005). Members of the family include, but are not limited to, Swinepoxvirus (SPV) 14L, Yaba-like disease virus (YLDV) 18L, Shope fibroma virus (SFV) gpl 3L, Myxoma virus (MV) 13L, the yatapoxvirus family of poxviruses, and in particular the use of Tanapoxvirus (TPV). TPV is a member of the Yatapoxvirus family of viruses that naturally infect human and non-human primates, typically causing a benign systemic infection. Example sequence information for such viruses may be located in the art, such as for example via the NIH NCBI database (aka GenBank) at www.ncbi.nih.gov (e.g. Tanapoxvirus information may be retrieved under Accession # NC009888.1).

The inventors have discovered that any disruption of the 18L gene of such viruses can render the viruses less able to productively infect many cell types, thus altering the selectivity of the virus for cancer cells. Experiments have shown selectivity for nonproductive infection of breast and colon cancer cells, although selectivity for other types of cancer cells is also apparent. For example, the inventors have shown that viruses with disrupted 18L gene function replicate well in glioma cells, thus making brain cancers a logical therapeutic target. In many cases the mutant virus exhibits improved selectivity for cancer cells relative to wild-type virus. Moreover, the inventors have found that loss of 18L gene function is often associated with reduced viral replication in non-cancerous cells. Thus, loss of 18L gene function improves the safety of poxviral therapy, because loss of 18L gene function can hinder or prevent viral replication in normal cells, when compared to cancer cells.

Further experimental data presented herein provides clear evidence to illustrate the molecular mechanism or signaling pathway influenced by 18L disruption. Pharmacological or siRNA silencing of the ERK-I pathway can reverse the inability of 18L knockout viruses to replicate in selected tumour types, thus providing a means to modulate mutant viral replication in a clinical setting.

The inventors have succeeded in the development of constructs selectively able to delete or abrogate expression of the 18L gene from a virus such as TPV and, if desired, to replace it with (or otherwise insert) a transgene expression cassette capable of accepting foreign genes and expressing them under the control of a promoter. For example, an early / late poxvirus promoter or a constitutively active promoter sequence of choice may be used. The properties of the TPV deletion mutants stem from the loss of immunomodulatory properties of the missing or disrupted Tl 8L gene product, which is known to be one of just a handful of virus- encoding pyrin-domain (PYD) containing proteins, orthologs of cellular apoptosis, inflammation, and innate immune regulators. Whilst one example construct generated by the inventors knocks out the Tl 8L gene sequence of TPV, a skilled artisan will recognize that any suitable disruption of expression of an 18L gene sequence, suitable to prevent or reduce expression of a fully functional 18L gene product, will also generate a mutant virus with desired properties in accordance with the teachings herein. The invention is therefore not limited to Tl 8L knockout TPV, but encompasses TPV and other viruses in which the expression of a corresponding 18L gene sequence has been disrupted or changed in some way, for example by mutation, homologous recombination, antisense or RNAi expression etc.

Therefore, in certain exemplary embodiments there is provided an anticancer agent comprising any poxvirus mutant that exhibits increased selectivity or tropism for cancer cells. In certain embodiments, the mutant may be a Yatapoxvirus mutant incapable of expressing a functional 18L gene product. For example, a mutation may be introduced into wild-type virus to generate a knockout virus for the 18L gene sequence, or a partial knockout for at least part of the 18L gene sequence (e.g. part of the 18L promoter sequence and / or part of the 18L open reading frame) sufficient to effectively inactivate or abrogate 18L biological function. Antisense technology or RNA interference etc. may also be used as alternative mechanisms to abrogate 18L expression.

In selected embodiments, the anticancer agent comprises a virus modified by the use of a construct or expression cassette comprising a promoter sequence, and an expression sequence to be expressed from the promoter sequence. Optionally, the expression cassette may include a promoter sequence, and an expression sequence to be expressed from the promoter sequence. The sequence to be expressed may take any form, and may encode an open reading frame, or may be complementary to a sequence that encodes an open reading frame. For example, the open reading frame may encode a cytotoxic protein, or a protein that is able to activate cytotoxic properties of a co-administered compound. In this way, the virus may selectively target cancer cells, and once it has infected the cancer cells the expression of the product of the expression cassette may optionally contribute to the demise or destruction of the cancer cell. Optionally, the expression cassette may be inserted at or adjacent a position of the 18L gene sequence, or may replace at least a portion of the 18L gene sequence.

In other exemplary embodiments there are provided anticancer agents comprising any recombinant virus that is incapable of expressing a functional or biologically active 18L gene product. For example, the recombinant virus may lack at least a portion of an 18L gene sequence. Furthermore, the recombinant virus may comprise an expression cassette inserted into the DNA sequence of the mutant, the expression cassette comprising a promoter sequence, and an expression sequence to be expressed from the promoter sequence. The expression sequence may for example encode a cytotoxic protein, or a protein that is able to activate cytotoxic properties of a co-administered compound. Further, the expression cassette may be inserted at or adjacent a position of the 18L gene sequence, or replace at least a portion of the 18L gene sequence. Importantly, the inventors have shown that, in comparison to wild-type Yatapoxvirus which exhibits broader tropism, deletion or disruption of the 18L gene significantly restricts tumour or cancer cell tropism of the virus. Moreover, the inventors demonstrate that the mutant virus can selectively gain entry into cancer cells such as breast cancer cells, and express a foreign gene from an expression cassette. Further, the production of progeny virions is limited or abrogated.

The inventors have further shown that infection of cancer cells by a deletion mutant for 18L is not necessarily associated with broad spectrum cytotoxicity. Indeed, infection of primary human fibroblasts and monocytes, at least in selected embodiments, is limited by an abortive phenotype that restricts spread of the deletion mutant. Moreover, productive infection by the deletion mutant can be regulated (at least partially turned off and on) pharmacologically using established inhibitors of the ERK-I kinase. Loss of 18L function, at least in selected embodiments, appears to be associated with the inability to activate the JNK kinase following infection. This loss may be the molecular basis for the selective replication of the deletion mutant compared to the wild-type virus.

Whilst the invention is not limited to the use of TPV, nor T18L knockouts of TPV, the examples presented herein focus in part upon the use of TPV as an exemplary model to illustrate the potential of the invention. TPV is expected to present some advantages for cancer therapy compared to other viral vectors. For example, TPV is a natural human pathogen adapted for replication in primates that possess an inherently limited infection profile and little pre-existing immunity. Replication of the T18L deletion mutant is selective and self-restricting with limited capacity for undesired spread. Further, replication of the T18L deletion mutant can be pharmacologically modulated, and the selectivity of the Tl 8L deletion mutant has a definable molecular basis. Thus, TPV mutants with disrupted Tl 8L function represent useful embodiments with promising clinical applications.

Other exemplary embodiments include a construct or expression cassette for use in producing a recombinant virus that is incapable of expressing a functional 18L gene product. For example, known mutagenesis or recombination techniques may be used to engineer recombinant poxviruses.

Other exemplary embodiments include a pharmaceutical composition comprising an anticancer agent as disclosed herein together with at least one pharmaceutically acceptable excipient, carrier, or diluent.

Other exemplary embodiments include a method of treating cancer, the method comprising administration of an anticancer agent as disclosed herein to a patient in need of such treatment. In selected embodiments the cancer is selected from a cancer of the breast, a cancer of the colon, and a cancer of the brain.

Other exemplary embodiments include the use of an anticancer agent as disclosed herein, in the treatment of cancer.

Other exemplary embodiments include the use of the anticancer agent as disclosed herein, in the manufacture of a medicament for use in the treatment of cancer.

If, upon disruption of the 18L gene sequence, it is also desired to insert DNA or gene sequence from another source, for example by ligation or homologous recombination, then any DNA of interest may be inserted into the viral genome at an appropriate position. Because poxviruses have a large genome, they can readily be used to deliver a wide range of genetic material including multiple genes (i.e., act as a multivalent vector). The sizes of the poxvirus genomes ranges between about 130- 300 kbp with up to 300 genes, depending on the strain of the virus. Therefore, it is possible to insert large fragments of foreign DNA into these viruses and yet maintain stability of the viral genome.

In one embodiment, at least one nucleic acid fragment encoding a gene is inserted into a poxvirus vector. In another embodiment at least two and up to about ten different nucleic acids encoding different genes are inserted into the poxvirus vector. In one embodiment, the recombinant poxvirus comprises a DNA encoding a disease-related antigen of interest, such as an antigen(s) from a disease causing agent or an antigen associate with a disease state, inserted at an insertion site, for expression of that antigen(s).

In another embodiment, the recombinant poxvirus comprises a DNA encoding a co-stimulatory molecule(s) inserted at an insertion site, for expression of the co-stimulatory molecule(s).

The recombinant vectors, at least in selected embodiments, are particularly useful to generate cell-mediated immune reactions. Cell-mediated immunity is crucial to cancer and diseases such as those caused by pathogenic microorganisms, particularly viruses and other intracellular microorganisms. Accordingly, the present invention provides a composition that has at least a first recombinant virus which has incorporated into its genome or portion thereof a gene encoding an antigen from cells of a disease state. The first recombinant poxvirus may also comprise one or more genes encoding one or more immunostimulatory molecules or genes. In one preferred embodiment, the co-stimulatory molecule is a combination of nucleic acids encoding B7 (e.g. B7-1), ICAM-I , and LFA-3, also known as TRICOM, which include activation of both CD4 and CD8 activators. Another embodiment provides a composition that has a second recombinant virus that comprises one or more genes encoding one or more immunostimulatory molecules or genes. A host cell infected with both recombinant viruses expresses both the antigen(s) from a disease causing agent and expresses the immunostimulatory molecule(s). The antigen may be expressed at the cell surface of the infected host cell. The immunostimulatory molecule may be expressed at the cell surface or may be actively secreted by the host cell. The expression of both the antigen and the immunostimulatory molecule provides the necessary MHC restricted peptide to specific T cells and the appropriate signal to the T cells to aid in antigen recognition and proliferation or clonal expansion of antigen specific T cells. The overall result is an upregulation of the immune system. In a preferred embodiment the upregulation of the immune response is an increase in antigen specific T-helper lymphocytes and/or cytotoxic lymphocytes, which are able to kill or inhibit the growth of a disease causing agent or a cell infected with a disease causing agent.

The disease-related antigen of interest can be an antigen from a pathogenic microorganism or a tumor associated antigen. The genes can be derived from any organism, including bacteria, parasites, normal or transformed cells, viruses or other microorganisms. Preferred genes are derived from transformed cells. For example, any gene for which a poxvirus-based live vaccine is desired.

Such disease causing agents include but are not limited to cancer and pathogenic microorganisms.

Cancers which may be treated using the recombinant poxvirus of the present invention include but are not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, bladder cancer, colon cancer, liver cancer, non- Hodgkins lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer and the like.

The aforementioned cancers can be assessed or treated by methods described in the present application. In the case of cancer, a gene encoding an antigen associated with the cancer may optionally be incorporated into the recombinant poxvirus genome or portion thereof along with a gene encoding one or more immunostimulatory molecules. Alternatively, the gene encoding an antigen associated with the cancer and the gene encoding one or more immunostimulatory molecules are incorporated into separate recombinant poxviruses. The antigen associated with the cancer may be expressed on the surface of a cancer cell or may be an internal antigen. In one embodiment the antigen associated with the cancer is a tumor associated antigen (TAA) or portion thereof. Examples of TAA that may be used in the present invention include but are not limited to melanoma TAAs which include but are not limited to MART-I (Kawakami et al. J. Exp. Med. 180:347-352, 1994), MAGE-I , MAGE-3, GP-100, (Kawakami et al. Proc. Nat'l. Acad. Sci. U.S.A. 91 :6458-6462, 1994), CEA and tyrosinase (Brichard et al. J. Exp. Med. 178:489, 1993). In another embodiment the TAAs are MUC-I, MUC-2, the point mutated ras oncogene and the point mutated p53 oncogenes (pancreatic cancer), CA- 125 (ovarian cancer), PSA (prostate cancer), c-erb/B2 (breast cancer) and the like (Boon et al., Ann. Rev. Immunol. 12:337, 1994). Other antigens associated with cancer include MN antigen, Jade, and BZLF- 1. The present invention is in no way limited to the genes encoding the above listed TAAs. Other TAAs may be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506, which is incorporated herein by reference.

In another preferred embodiment, the target antigen is a tumor associated antigen, a tumor specific antigen, and/or a tissue-specific antigen. In such embodiments, at least one epitope of an antigen may be for example selected from the group consisting of IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL- 1 1, IL-12, IFN-α, IFN-β, IFN-.beta 17 mutants, IFN-65, CD2, CD3, CD4, CD5, CD8, CDl Ia, CDl Ib, CDl Ic, CD16, CD18, CD21 , CD28, CD32, CD34, CD35, CD40, CD44, CD45, CD54, CD56, OX40L, 4-1BBL, K2, Kl, Pβ, Oa, Ma, Mβ2, Mβl, Hepsin, Pim-1 , LMPl , TAP2, LMP7, TAPl, TRP, Oβ, IAβ, IAa, IEβ, IEβ2, IEa, CYP21. C4B. CYP21P, C4A. Bf. C2, HSP, GIsJb, TNF-a, TNF-β, D, L, Qa, TIa, COLl 1A2, DPβ2, DPα2, DPβL DPαl, DNa, DMa, DMβ, LMP2, TAPiI, LMP7, DOβ, DQβ2. DQa2, DQβ3, DQβl, DQaI, DRβ, DRa, G250, HSP-70, HLA- B, HLA-C, HLA-X, HLA-E, HLA-J, HLA-A, HLA-H, HLA-G, HLA-F, nerve growth factor, somatotropin, somatomedins, parathormone, FSH, LH, EGF, TSH THS-releasing factor, HGH, GRHR, PDGF, IGF-I, IGF-II, TGF-β, GM-CSF, M- CSF, G-CSFl , erythropoietin, β-HCG, 4-N-acetylgalactosaminyltransferase, GM2, GD2, GD3, JADE. MART. BAGE, GAGE, MAGE-I, MAGE-2, MAGE-3, XAGE, MUC-I , MUC-2, MUC-3, MUC-4, MUC-18, ICAM-I, C-CAM, V-CAM, ELAM, NM23, EGFR, E-cadherin, N-CAM, LFA-3 (CD58), EpCAM, B7.1, CEA, DCC, PSA, Her2-neu, UTAA, melanoma antigen p75, K 19, HKer 8, pMel 17, TPlO, tyrosinase related proteins 1 and 2, p97, p53, RB, APC, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, BRCAl, VHL, FCC and MCC, ras. myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst, bcl and abil, CIq, CIr, CIs, C4, C2, Factor D, Factor B, properdin, C3, C5, C6, C7, C8, C9, Cllnh, Factor H, C4b-binding protein, DAF, membrane cofactor protein, anaphylatoxin inactivator S protein, HRF, MIRL, CRl, CR2, CR3, CR4, C3a/C4a receptor, C5a receptor, Epstein-Barr Virus antigens (EBNA), BZLF-I , BXLF-I , and Nuclear Matrix Proteins, modified TAAs, splice variants of TAAs, functional epitopes, epitope agonists, and degenerate nucleic acid variations thereof.

Genes encoding an antigen of a disease causing agent in which the agent is a pathogenic microorganism include viruses such as HIV (GP- 120, pi 7, GP- 160, gag, pol , qp41, gpl20, vif, tat, rev, nef, vpr, vpu, vpx antigens), smallpox, influenza (NP, hemagluttinin (HA antigen), neuraminidase, PBl, PB2, PA, NP, Mi, M ₂ , NS,, NS ₂ )), papillomaviruses (El , E2, E3, E4, E5a, E5b, E6, E7, E8, Ll, L2), adenovirus (ElA, El B, E2, E3, E4, E5, Ll , L2, L3, L4, L5), HSV (ribonucleotide reductase, α-TIF, ICP4, ICP8, 1CP35, LAT-related proteins, gB, gC, gD, gE, gH, gl, gJ, and dD antigens), human papilloma virus, equine encephalitis virus, hepatitis (Hep B Surface Antigen (gp27 ^s , gp36 ^s , gp42 ^s , p22 ^c , pol, x)) and the like. Pathogenic bacteria include but are not limited to anthrax, Chlamydia, Mycobacteria,

Legioniella and the like. Pathogenic protozoans include but are not limited to malaria, Babesia, Schistosomiasis and the like. Pathogenic yeast include Aspergillus, invasive Candida, and the like. In a preferred embodiments the pathogenic microorganism is an intracellular organism.

For purposes of a vaccine, genes of interest are those which encode immunogenic proteins of a pathogenic organism. In many cases, these are protein components of surface structures such as the bacterial cell wall or viral envelope. In appropriate instances, immunogenic fragments or subunits of the proteins may be used.

One preferred group of nucleic acids for insertion into the poxvirus include co-stimulatory molecules, accessory molecules, and/or genes encoding a cytokine and/or growth factor. Examples of costimulatory molecules include but are not limited to B7-1 , B7-2, ICAM-I , CD40, CD40L, LFA-3, CD72, OX40L (with or without OX40), and the like. Examples of cytokines and growth factors encompassed by the present invention include but are not limited to: granulocyte macrophage-colony stimulating factor (GM-CSF), granulocyte-colony stimulating factor (G-CSF), macrophage- colony stimulating factor (M-CSF), tumor necrosis factors (TNFα and TNFβ), transforming growth factors (TGFα and TGFβ), epidermal growth factors (EGF), stem cell factor (SCF), platelet-derived growth factors (PDGF), platelet-derived endothelial cell growth factor, nerve growth factor (NGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF-I and IGF-II), growth hormone, interleukins 1 to 15 (IL-I to IL- 15), interferons α, β and gamma. (IFN-α, IFN-β and IFN -γ), brain-derived neurotrophic factor, neurotrophins 3 and 4, hepatocyte growth factor, erythropoietin, EGF-like mitogens, TGF-like growth factors, PDGF-like growth factors, melanocyte growth factor, mammary-derived growth factor 1 , prostate growth factors, cartilage-derived growth factor, chondrocyte growth factor, bone- derived growth factor, osteosarcoma-derived growth factor, glial growth-promoting factor, colostrum basic growth factor, endothelial cell growth factor, tumor angiogenesis factor, hematopoietic stem cell growth factor, B-cell stimulating factor 2. B-cell differentiation factor, leukemia-derived growth factor, myelomonocytic growth factor, macrophage-derived growth factor, macrophage-activating factor, erythroid-potentiating activity, keratinocyte growth factor, ciliary neurotrophic growth factor. Schwann cell-derived growth factor, vaccinia virus growth factor, bombyxin. neu differentiation factor, v-Sis, glial growth factor/acetylcholine receptor-inducing activity, transferrin, bombesin and bombesin-like peptides, angiotensin II, endothelin, atrial natriuretic factor (ANF) and ANF-like peptides, vasoactive intestinal peptide, Bradykinin and related growth factors. Preferred cytokines and growth factors include but are not limited to IL-2, GM-CSF, TNFα, IFN.gamnma., IL- 12, RANTES, and the like.

One does not have to use a gene encoding an entire protein, but rather only the domain desired. For example, if an immune reaction is desired, only the fragment necessary to stimulate the immune reaction needs to be encoded. The co- stimulatory molecules, accessory molecules, and cytokines of the present invention are useful as biologic adjuvants, which can be administered systemically to the host via inserting nucleic acids encoding such into the same or different recombinant poxvirus vectors. In one preferred embodiment, one administers a poxvirus vector containing B7, LFA-3 and ICAM-I in conjunction with the tumor associated antigen. In a further preferred embodiment, the poxvirus also contains OX40L. In another embodiment, the poxvirus contains OX40L alone. In yet another embodiment, the poxvirus encodes both OX40L or OX40 intrabody and OX40.

Poxviruses expressing B7-1, ICAM-I, and LFA-3, also known as TRICOM™, induce activation of both CD4+ and CD8+T cells. (U.S. Pat. No. 6,045,802; Hodge et al., J. Natl. Cancer Inst. 92: 1228-39 (2000); Hodge et al, Cancer Research 59: 5800-07 (1999)). OX40 is a primary co-stimulator of T cells that have encountered antigen, rather than naive T cells, and promotes T-cell expansion after T cell tolerance is induced. (Bansal-Pakal et al., Nature Med. 7: 907- 12 (2001)). OX40L plays a role during T cell activation by a) sustaining the long- term proliferation of CD4+ and CD8+T cells, b) enhancing the production of ThI cytokines such as IL-2, IGN-g, and TNF-a from both CD4+ and CD8+T cells without changing IL-4 expression, c) protecting T cells from apoptosis. The combination of B7-1 , ICAM-I, LFA-3, and OX40L enhances initial activation and then further potentiates sustained activation of naϊve and effector T cells.

Another preferred group of nucleic acids for insertion into the poxvirus encode antibodies. Antibodies have long been used in biomedical science as in vitro tools for the identification, purification and functional manipulation of target antigens. Antibodies have been exploited in vivo for both diagnostic and therapeutic applications. Recent advances in antibody engineering have now allowed the gene encoding antibodies to be manipulated so that the antigen biding domain can also be expressed intracellularly. The specific and high-affinity binding properties of antibodies, combined with the ability to create large human immunoglobulin libraries and their ability to be stably expressed in precise intracellular location inside mammalian cells, has provided a powerful new family of molecules for gene therapy applications such as the one including a poxvirus vector in the present application. These intracellular antibodies are called "intrabodies". (Marasco et al. Gene Therapy, 4:1 1-15, 1997; U.S. Pat. Nos. 5,965,371 ; 5,851,829; 6,329,173; and 6,072,036). Preferably nucleic acids encoding angiogenesis modulating intrabodies encode a single chain antibody.

Foreign genes for insertion into the genome of a poxvirus in expressible form can be obtained by any conventional technique for isolating a desired gene.

For organisms which contain a DNA genome, the genes encoding an antigen of interest are isolated from the genomic DNA; for organisms with RNA genomes, the desired gene may be isolated from cDNA copies of the genome. If restriction maps of the genome are available, strategies can be designed for cleaving genomic DNA by restriction endonuclease digestion to yield DNA fragments that contain the gene of interest. In some cases, desired genes may have been previously cloned and thus, the genes can be obtained from the available clones. Alternatively, if the DNA sequence of the gene is known, the gene can be synthesized by any of the conventional techniques for polymerase chain reaction or synthesis of deoxyribonucleic acids (e.g., the phosphate or phosphite triester techniques).

Genes encoding an antigen of interest can be amplified by cloning the gene into a bacterial host. For this purpose, various prokaryotic cloning vectors can be used. Examples are plasmids pBR322 and pEMBL.

The genes encoding the antigen of interest can be prepared for insertion into the poxvirus vectors by standard techniques. In general, the cloned genes can be excised from the prokaryotic cloning vector by restriction enzyme digestion. In most cases, the excised fragment will contain the entire coding region of the gene. The DNA fragment carrying the cloned gene can be modified as needed, for example, to make the ends of the fragment compatible with the insertion sites of the poxvirus vectors, then purified prior to insertion into these vectors at restriction endonuclease cleavage sites (cloning sites) as described below.

The basic techniques of inserting genes into viruses are known to the skilled artisan and involve, for example, recombination between the viral DNA sequences flanking a gene in a donor plasmid and homologous sequences present in the parental virus (Mackett, et al., Proc. Natl. Acad. Sci. USA 79:7415-7419 (1982)). For example, a recombinant virus such as a poxvirus for use in delivering the gene can be constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the fowlpoxvirus described in U.S. Pat. No. 5,093,258, the disclosure of which is incorporated herein by reference. Other techniques include using a unique restriction endonuclease site that is naturally present or artificially inserted in the parental viral vector.

First, the DNA gene sequence to be inserted into the virus can be placed into a plasmid, e.g., an E. coli plasmid construct, into which DNA homologous to a section of DNA such as that of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA which is the desired insertion region. The resulting plasmid construct is then amplified by growth within E. coli bacteria and isolated. Preferably, the plasmid also contains an origin of replication such as the E. coli origin of replication, and a marker such as an antibiotic resistance gene for selection and propagation in E. coli.

Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g., chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively results in a poxvirus modified by the presence of the promoter-gene construct in its genome, at a site which does not affect virus viability.

Where the embodiment of the present invention provides insertion of more than one nucleic acid (e.g. a tumor antigen and a costimulatory molecule), the first nucleic acid is inserted into the novel insertion sites of the present invention, as described above, and additional nucleic acid(s) can be inserted either into the novel insertion sites described here or other insertion sites.

In addition to the requirement that the gene be inserted into an insertion site, successful expression of the inserted gene(s) by the modified poxvirus requires the presence of a promoter operably linked to the desired gene, i.e., in the proper relationship to the inserted gene. The promoter may be placed so that it is located upstream from the gene to be expressed. Promoters are well known in the art and can readily be selected depending on the host and the cell type one wishes to target. For example in poxviruses, poxviral promoters should be used, such as the vaccinia 7.5K, 4OK, fowlpox. Enhancer elements can also be used in combination to increase the level of expression. Furthermore, the use of inducible promoters, which are also well known in the art, in some embodiments are preferred.

Promoters useful according to the present invention include poxvirus promoters such as, e.g., an entomopox promoter, an avipox promoter, or an orthopox promoter such as a vaccinia promoter, e.g., HFI, 1 IK or Pi. For example, the Pi promoter, from the Ava 1 H region of vaccinia, is described in Wachsman et al., J. of Inf. Dis. 155, 1 188-1 197 (1987). More particularly, this promoter is derived from the Ava I H(Xho I G) fragment of the L-variant WR vaccinia strain, in which the promoter directs transcription from right to left. The map location of the promoter is approximately 1.3 Kbp (kilobase pair) from the 5' end of Ava IH, approximately 12.5 Kbp from the 5' end of the vaccinia genome, and about 8.5 Kbp 5' of the Hind III C/N junction. The Hind III H promoter (also "HH" and "H6" herein) sequence is an up-stream of open reading frame H6 by Rosel et al., J. Virol. 60, 436-449 (1986). The HK promoter is as described by Wittek, J. Virol. 49. 371-378 (1984) and Bertholet, C. et al., Proc. Natl. Acad. Sci. USA 82, 2096-2100 (1985). One can take advantage of whether the promoter is an early or late promoter to time expression of particular genes. Additionally, as discussed below, one can use additional promoters.

Another embodiment provides a poxvirus vector in which the promoter is modulated by an external factor or cue, allowing control of the level of polypeptide being produced by the vectors by activating that external factor or cue. For example, heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature. The promoter of the gene which encodes the metal-containing protein metallothionine is responsive to Cd ⁺ ions. Incorporation of this promoter or another promoter influenced by external cues also make it possible to regulate the production of the proteins. In another preferred embodiment, the poxvirus genome is modified to carry a nucleic acid encoding at least one gene of interest which is operably linked to an "inducible" promoter. Such inducible systems allow careful regulation of gene expression. See, Miller and Whelan, Human Gene Therapy, 8:803-815 (1997). The phrase "inducible promoter" or "inducible system" as used herein includes systems wherein promoter activity can be regulated using an externally delivered agent. Such systems include, for example, systems using the lac repressor from E. coli as a transcription modulator to regulate transcription from lac operator-bearing mammalian cell promoters (Brown et al. Cell. 49:603-612, 1987); systems using the tetracycline repressor (tetR)(Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551, 1992; Yao et al.. Human Gene Ther. 9: 1939-1950, 1998; Shokelt et al., Proc. Natl. Acad. Sci. USA 92.6522-6526. 1995). Other such systems include FK506 dimer, VP 16 or p65 using castradiol, RU486/mifepristone, diphenol muristerone or rapamycin (see, Miller and Whelan, supra, at FIG. 2). Yet another example is an ecdysone inducible system (see, e.g. Karns et al, MBC Biotechnology 1 :11, 2001). Inducible systems are available, e.g., from Invitrogen, Clontech, and Ariad. Systems using a repressor with the operon are preferred. One would adapt these promoters by substituting portions of pox promoters for the mammalian promoter.

One embodiment provides the use of a regulatory element such as a transcriptional regulatory element or an enhancer.

In one preferred embodiment of the present invention, a "transcriptional regulatory element ^" ' or ''TRE" is introduced for regulation of the gene of interest. As used herein, a TRE is a polynucleotide sequence, preferably a DNA sequence, that regulates (i.e., controls) transcription of an operably-linked polynucleotide sequence by an RNA polymerase to form RNA. As used herein, a TRE increases transcription of an operably linked polynucleotide sequence in a host cell that allows the TRE to function. The TRE comprises an enhancer element and/or pox promoter element, which may or may not be derived from the same gene. The promoter and enhancer components of a TRE may be in any orientation and/or distance from the coding sequence of interest, and comprise multimers of the foregoing, as long as the desired, transcriptional activity is obtained. As discussed herein, a TRE may or may not lack a silencer element.

Another embodiment provides an "enhancer" for regulation of the gene of interest. An enhancer is a term well understood in the art and is a polynucleotide sequence derived from a gene which increases transcription of a gene which is operably-linked to a promoter to an extent which is greater than the transcription activation effected by the promoter itself when operably-linked to the gene, i.e. it increases transcription from the promoter. Having "enhancer activity" is a term well understood in the art and means what has been stated, i.e., it increases transcription of a gene which is operably linked to a promoter to an extent which is greater than the increase in transcription effected by the promoter itself when operably linked to the gene, i.e., it increases, transcription from the promoter.

The activity of a regulatory element such as a TRE or an enhancer generally depends upon the presence of transcriptional regulatory factors and/or the absence of transcriptional regulatory inhibitors. Transcriptional activation can be measured in a number of ways known in the art (and described in more detail below), but is generally measured by detection and/or quantization of mRNA or the protein product of the coding sequence under control of (i.e., operatively linked to) the regulatory element. As discussed herein, the regulatory element can be of varying lengths, and of varying sequence composition. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by at least about 2-fold, preferably at least about 5 -fold, preferably at least about 10-fold, more preferably at least about 20-fold. More preferably at least about 50-fold, more preferably at least about 100-fold, even more preferably at least about 200-fold, even more preferably at least about 400- to about 500-fold, even more preferably, at least about 1000-fold. Basal levels are generally the level of activity, if any, in a non- target cells, or the level of activity (if any) of a reporter construct lacking the TRE of interest as tested in a target cell type.

A "functionally-preserved" variant of a TRE is a TRE which differs from another TRE, but still retains ability to increase transcription of an operably linked polynucleotide, especially cell-specific transcription activity. The difference in a TRE can be due to differences in linear sequence, arising from, for example, single or multiple base mutation(s), addition(s), deletion(s), and/or modification(s) of the bases. The difference can also arise from changes in the sugar(s), and/or linkage(s) between the bases of a TRE.

Certain point mutations within sequences of TREs have been shown to decrease transcription factor binding and gene activation. One of skill in the art would recognize that some alterations of bases in and around known the transcription factor binding sites are more likely to negatively affect gene activation and cell-specificity, while alterations in bases which are not involved in transcription factor binding are not as likely to have such effects. Certain mutations are also capable of increasing TRE activity. Testing of the effects of altering bases may be performed in vitro or in vivo by any method known in the art, such as mobility shift assays, or transfecting vectors containing these alterations in TRE functional and TRE non-functional cells. Additionally, one of skill in the art would recognize that point mutations and deletions can be made to a TRE sequence without altering the ability of the sequence to regulate transcription.

In the present invention, the poxvirus vectors directed at specific target cells may also be generated with the use of TREs that are preferentially functional in the target tumor cells. Non-limiting examples of tumor cell-specific heterologous TREs, and non-limiting examples of respective potential target cells, include TREs from the following genes: α-fetoprotein (AFP) (liver cancer), mucin-like glycoprotein DF3 (MUCl ) (breast carcinoma), carcinoembryonic antigen (CEA) (colorectal, gastric, pancreatic, breast, and lung cancers), plasminogen activator urokinase (uPA) and its receptor gene (breast, colon, and liver cancers), E2F1 (cell cycle S-phase specific promoter) (tumors with disrupted retinoblastoma gene function), HER-2/neu (c-erbB2/neu) (breast, ovarian, stomach, and lung cancers).

In the present invention, tumor-specific TREs may be used in conjunction with tissue-specific TREs from the following exemplary genes (tissue in which the TREs are specifically functional are in parentheses): hypoxia responsive element, vascular endothelial growth factor receptor (endothelium), albumin (liver), factor VII (liver), fatty acid synthase (liver), Von Willebrand factor (brain endothelium), alpha-actin and myosin heavy chain (both in smooth muscle), synthetast I (small intestine). Na — K — Cl transporter (kidney). Additional tissue specific TREs are known in the art.

Accordingly, in one embodiment, the cell specific, heterologous TRE is tumor cell specific. Preferably, both heterologous TREs are tumor cell specific and functional in the same cell. In another embodiment, one of the first heterologous TREs is tumor cell specific and the second heterologous TRE is tissue specific, whereby both TREs are function in the same cell.

Introduction of the viral vector carrying the gene to be delivered to the target host cell may be effected by any method known to those of skill in the art.

Administration of the recombinant poxvirus of the invention can be either "'prophylactic' ^" or "therapeutic ^" ' depending on the subject. When provided prophylactically, the recombinant poxvirus of the present invention is provided in advance of any symptom, but when one believes the subject is at risk. The prophylactic administration of the recombinant poxvirus serves to prevent or ameliorate any subsequent angiogenic-related condition. When provided therapeutically, the recombinant poxvirus is provided at or after the onset of a symptom of infection or disease. Thus the present invention may be provided either prior to the anticipated exposure to a disease-causing agent or disease state or after the initiation of the infection or disease.

The term "'unit dose" as it pertains to the inoculum refers to physically discrete units suitable as unitary dosages for mammals, each unit containing a predetermined quantity of recombinant poxvirus calculated to produce the desired effect in association with the required diluent. The specifications for the novel unit dose of this invention are dictated by and are dependent upon the unique characteristics of the recombinant virus and the particular effect to be achieved. For administration to a subject, the poxvirus of the present invention may, at least in some embodiments, be prepared as an inoculum. The inoculum is typically prepared as a solution in a tolerable (acceptable) diluent such as saline, phosphate- buffered saline or other physiologically tolerable diluent and the like to form an aqueous pharmaceutical composition.

The route of administration may be scarification, intravenous (I.V.), intramuscular (I. M.), subcutaneous (S. C), intradermal (I.D.), intraperitoneal (I. P.), intratumor and the like, which results in eliciting a protective response against the disease causing agent. The dose is administered at least once. Subsequent doses may be administered as indicated.

In one embodiment, heterologous prime-boost regimens are employed. For example, the host can be immunized at least once with a first vector such as a nucleic acid-based vector. Subsequent immunizations are performed with a poxvirus vector. In another example, the host is first immunized with a first poxvirus vector and then with a second poxvirus vector of a different genus.

In providing a manual with the recombinant poxvirus of the present invention, preferably a human, the dosage of administered recombinant poxvirus will vary depending upon such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, disease progression, tumor burden and the like.

In general, it is desirable to provide the recipient with a dosage of recombinant virus in the range of about l CP to about 10 ¹⁰ plaque forming units, although a lower or higher dose may be administered.

In some embodiments one may administer a sufficient amount of the viral vectors to obtain a serum concentration in the organ of interest of the protein ranging between about 1 pg/ml to 20 μg/ml. More preferably between about 0.1 μg/ml to 10 μg/ml. Still more preferably, between about 0.5 μg/ml to 10 μg/ml. Examples of methods for administering the recombinant poxvirus into mammals include, but are not limited to, exposure of tumor cells to the recombinant virus ex vivo, or injection of the recombinant poxvirus into the affected host by intravenous, S. C, LD. or I. M. administration of the virus. Alternatively the recombinant poxvirus or combination of recombinant vectors may be administered locally by direct injection into the cancerous lesion or tumor or topical application in a pharmaceutically acceptable carrier. The quantity of recombinant poxvirus optionally carrying the nucleic acid sequence of one or more antigens in combination with nucleic acid sequences encoding multiple costimulatory molecules to be administered is based on the titer of virus particles. A preferred range of the immunogen to be administered is 10 ⁵ to 10 virus particles per mammal, preferably a human. If the mammal to be immunized is already afflicted with cancer or metastatic cancer, the vaccine can be administered in conjunction with other therapeutic treatments.

The present invention also provides a pharmaceutical composition comprising a poxvirus, including a recombinant poxvirus, and a pharmaceutically acceptable carrier.

The effect of the genetic material delivered can be carefully monitored and regulated using this system. Some poxvirus vectors such as swinepox may only express the genetic material for about two weeks. Thus, if the condition being treated is alleviated within that time frame, since the vector system is self limiting, no unnecessary material will be produced after that time period. Where additional dosages will be needed, additional administration of the material can be accomplished by repeating the injection. In certain cases, the addition of a second, third, etc. material can also be added with these vectors.

One embodiment of the present invention provides treating a subject in need, for example, a subject having cancer, with a poxvirus of the present invention in combination with a chemotherapeutic agent. Preferably, the poxvirus expresses a gene associated with a cancer. The chemotherapeutic agent can be any anti-cancer drug. Examples of anti-cancer drugs that may be used in the various embodiments of the invention, including pharmaceutical compositions and dosage forms and kits of the invention, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; capsitabine; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleulin II (including recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl ; interferon alfa-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine, mechlorethamine oxide hydrochloride rethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, improsulfan, benzodepa, carboquone, triethylenemelamrine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelainine, chlomaphazine, novembichin, phenesterine, trofosfamide, estermustine, chlorozotocin, gemzar. nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, aclacinomycins, actinomycin F(I), azaserine, bleomycin, carubicin, carzinophilin, chromomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-l -norleucine, doxorubicin, olivomycin, plicamyciri, porfiromycin, puromycin, tubercidin, zorubicin, denopterin, pteropterin, 6-mercaptopurine, ancitabine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, enocitabine, pulmozyme, aceglatone, aldophosphamide glycoside, bestrabucil, defofamide, demecolcine, elfornithine, elliptinium acetate, etoglucid, flutamide, hydroxyurea, lentinan, phenamet, podophyllinic acid, 2-ethylhydrazide, razoxane, spirogermanium, tamoxifen, taxotere, tenuazonic acid, triaziquone, 2,2',2"- trichlorotriethylamine, urethan, vinblastine, vincristine, vindesine and related agents. 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1 ; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin

III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin

A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin

B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine, edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic di saccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopraminde; MIF inhibitor; mifepristone; miltefosine; nirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor- saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1- based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; taxel; taxel analogues; taxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rheniuim Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl ; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division ibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor;. translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil, leucovorin, capsitabine. cyclosphosphamide, and gemcitabine. The magnitude of a prophylactic or therapeutic dose of each active ingredient in the treatment of a patient with a solid tumor will typically vary with the specific active ingredients, the severity and type of tumor, and the route of administration. The dose and the dose frequency may vary according to age, sex, body weight, response, and the past medical history of the patient; the likelihood of mestastic recurrence must also be considered. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example,' dosages reported in the literature and recommended in the Physician's Desk Reference® (54th ed., 2000). Unless otherwise indicated, the magnitude of a prophylactic or therapeutic dose of each pharmaceutical used in an embodiment of the invention will be that which is known to those in the art to be safe and effective, or is regulatory approved.

In one embodiment, a treatment method in accordance with the present invention includes treating a subject in need, for example, a subject having cancer, for example breast cancer, ovarian cancer, prostate cancer or brain cancer, with a first poxvirus vector. In further embodiments the poxvirus may be designed to elicit a cytotoxic T-cell response to a foreign gene introduced by the poxvirus. For a cancer patient, the gene can be a tumor associated antigen, such as PSA, PSAM, BRCAl , ras, CEA or MUC. One can also use a foreign gene encoding a viral antigen or antigens to treat an individual having or susceptible to an infectious disease, preferably a viral envelope protein. Cytotoxic T-cells specific for the desired cancer-associated antigen can be generated by administering between about IOM O ⁹ pfu of a recombinant poxvirus carrying a sequence encoding a tumor- associated antigen to the individual affected with the tumor. For detailed construction of such vectors, see, e.g., U.S. Pat. No. 5,656,465. One could also use an immune modulator such as the use of cytokines, e.g., IL-2, or co-stimulatory molecules, e.g., B7.1 or B7.2, as biologic adjuvants, which can be administered systemically to the host via inserting nucleic acids encoding such into the same or different recombinant poxvirus vectors. In one preferred embodiment, one administers a poxvirus vector containing B7, LFA-3, ICAM-I and Ok40L in conjunction with the foreign gene.

The invention will be further characterized by the following examples which are intended to be exemplary of the invention.

EXAMPLE 1 - Summary of the tumour cell tropism of wild-type and deletant viruses Data comparing the tumour cell tropism of wild-type and mutant viruses, and summarizing the altered viral tropism of the mutant viruses, are summarized in Table 1.

A significant number of tumour types have been studied and evaluated by the inventors. Replication of the wild-type (TPV-WT) and knock-out (TPVΔT18L) strains of tanapoxvirus (TPV) was evaluated in the indicated cell types by microscopy and the generation of single- and multi-step growth curves. Cell lines were classified as permissive (+) or nonpermissive (-) to productive infection and virus replication. Three categories of tumour types were revealed: Type 1 (Supportive), permissive to both viruses; Type II (Restrictive), permissive to only TPV-WT; and Type III (Abortive), nonpermissive for either virus. Two permissive (OMK, HEK293) and non-permissive (BGMK, COS7) tumour cell lines were used as comparators.

EXAMPLE 2 - Broad cell tropism of TPV in human cancer cells

The indicated human cancer cells shown in Figure 1 were infected with TPV-WT at the indicated multiplicity of infection (MOI) and fluorescence images taken at Day 3 post-infection (PI). The presence of the foci characteristic of TPV replication and spread in culture indicates productive virus infection, while expression of green fluorescence protein (gfP) engineered into the virus indicates delivery and expression of exogenous genes. Images captured at IOOX magnification. Thus, the inventors have evaluated a significant cross-section of permissive tumour types with indication of foci structure and characteristics. It follows that the anti-cancer agents disclosed herein have clear therapeutic potential for a significant range of tumour types.

EXAMPLE 3 - Replication of wild-type and deletant viruses in representative human tumour cell types

Figure 2 illustrates representative control (OMK), supportive (U373), restrictive (MCF7) and abortive (HeIa) cell types infected with TPV-WT or TPVΔ18L at a multiplicity of infection (MOI) of 0.1 (input titer of one infectious virion for every ten cells). Both viruses were engineered to express green fluorescence protein (gfp) as an exogenous gene for ease of tracking the infection. Fluorescence images were taken at Day 5 post-infection (PI) and show the presence or absence of the foci characteristic of TPV replication and spread in culture. Both viruses replicated in control and U373 neuroglioma cells; however, U373 cells supported more rapid and intensive virus replication as indicated by the increase gfp expression and the size of the foci. Only TPV-WT could replicate and spread in MCF7 breast cancer cells, while neither virus could replicate in HeLa cervical cancer cells. Images captured at I OOX magnification.

EXAMPLE 4 - Single-step viral growth curves for wild-type and deletant viruses in representative human tumour cell lines

Figure 3 illustrates representative control (A, OMK). supportive (B, U373), restrictive (C, MCF7) and abortive (D, HeIa) cell types were infected with TPV-WT (upper line with diamonds) or TPVΔ18L (lower line with squares) at MOI = 5.0 and cells harvested for virus isolation at the indicated time-points PI. Viral titers were determined by serial dilution of infectious virus obtained from the infected cells and titration on OMK cells. Both viruses replicated in control and U373 neuroglioma cells producing similar maximum titers. TPV-WT replicated in MCF7 breast cancer cells to a comparable extent as in permissive cells, whereas TPVΔ18L did not replicate to even the input titer value. Neither virus could replicate in HeLa cervical cancer cells to produce significant progeny virions. Titers are expressed as focus forming units (ffu) of progeny virus per million infected cells.

EXAMPLE 5 - Exogenous gene expression following infection of representative human tumour cell types with wild-type and deletant viruses

Figure 4 demonstrate a capacity to express exogenous genes from an 18L knockout virus background in both permissive and non-permissive tumour types. Thus, the anticancer agents disclosed herein have clear potential for anti-tumour delivery platforms in which a replicative block for the viral agents is a desired trait, but in which expression of exogenous factors is also desired. Representative control (OMK), supportive (U373), restrictive (A549) and abortive (HeIa) cell types were infected with TPV-WT or TPVΔ18L at the indicated MOI. Fluorescence was measured in a fluorescence plate reader 24 hpi. High levels of expression of the exogenous gfp cassette were evident in the control and U373 neuroglioma cells permissive to infection by both viruses. Of note, significant and comparable levels of gfp expression were also detected following infection of restrictive A549 cells by either virus despite the inability of the T18L deletant to productively infect these cells. Thus, the deletant virus could deliver and express exogenous genes in the absence of productive infection.

EXAMPLE 6 - Rescue of deletant virus infection in Type II cells by expressing TlSL.

Figure 5 shows Type II MCF-7 breast cancer cells (A-D) and A549 lung cancer cells (E-H) engineered to stably express either Flag-tagged Tl 8L (C, D, G, H) or vector expressing the Flag epitope alone (A, B, E, F). Cells were infected with TPV-WT or TPVΔ18L at MOI = 0.1 and foci formation evaluated at Day 3 PI. The small foci indicated above reflect the low initial input titer of virus and the relatively early time-point selected for evaluation. TPV-WT was able to replicate in MCF-7 And A549 expressing either the Flag epitope alone or Flag-T18L; however, replication was more rapid when Tl 8L was overexpressed. In comparison, TPVΔ18L was unable to replicate in MCF7 and A549 cells expressing vector alone, but were able to replicate in cells expressing Tl 8L. Images captured at 200X (A-D) or IOOX (E-H) magnification. Similar results were observed in Type II A549 lung cancer and Type II HepG2 hepatic cancer cell lines, indicating that the tumour cell tropism of the knock-out virus was a product of deleting the Tl 8L gene. Thus, the demonstration of rescue in lung cancer cells complements the observed rescue of breast cancer cells, providing clear evidence that the lack of target gene is the blockade to viral replication (i.e. the blockade of viral replication is not tumour type- specific).

EXAMPLE 7 - Viral gene expression in representative tumour cell types Figure 6 shows representative control (OMK), supportive (U373), restrictive (MCF7) and abortive (HeIa) cell types infected with TPV-WT or TPVΔ18L at MOI = 1.0 and cells harvested at 24 h and 72 h PI for quantitative real-time PCR (QRT- PCR) detection of typical early (TlOL) and late (T149L) genes. Gene expression is expressed relative to the housekeeping gene, GAPDH. Poxvirus gene expression occurs in four distinct phases (early, early/late, genome replication, late). In most cases, early gene expression occurs in all cell types but the blockade to permissive replication occurs at the viral genome replication stage. Thus, early gene expression can occur in the absence of productive infection, whereas late gene expression does not. Both early and late gene expression is evident for TPV-WT and TPVΔ18L in control and supportive cell lines at 24 h and 72 h PI indicating productive infection. Delayed gene expression is evident for the wild-type virus in Type II MCF-7 breast cancer cells, but no viral gene expression was detected Type II and Type III tumour cells with TPVΔ18L. However, expression of the exogenous gfp gene is evident at the single cell level (e.g. no spread, no foci) following TPVΔ18L infection of MCF cells.

EXAMPLE 8 - Expression ofT18L attenuates Vaccinia virus (VV) replication

Figure 7 shows human HEK293 kidney cells engineered to stably express either Flag-tagged T18L (FlagT18L) or vector expressing the Flag epitope alone (vector). Cells were infected with VV expressing gfp at MOI = 0.01, 0.1 or 1.0.

Plaque formation was evaluated at 24 h (top) and 48 h (bottom) PI by fluorescence microscopy as an indicator of replication and spread. HEK293 are fully permissive to VV replication as indicated in the cells expressing vector alone. Expression of T18L, however, markedly attenuated VV replication and spread at 24 and 48 h. VV replication, unlike TPV replication, is dependent on the activation of the signaling kinase, ERK-I, early in the lifecycle of the virus. Inhibition of ERK-I in infected cells is associated with a delay in VV infection due to poor early spread of the virus. The resulting profile resembles that observed in HEK293 cells expressing T18L. Images captured at 10OX magnification. EXAMPLE 9 - Loss ofT18L expression is associated with increase ERK-I expression

Figure 8 shows restrictive human A549 lung cancer cells were mock-infected (M) or infected with TPV-WT (W) or TPVΔ18L (K) at MOI = 0.1 , 1.0 or 5.0, Cells were harvested at 6, 24 or 72 h PI for protein isolation and immunoblot detection of the active, phosphorylated form of ERK-I . The housekeeping protein, β-actin, served as loading control. ERK-I activation was reduced in cells infected with TPV-WT, but increased or apparent at levels equivalent to mock-infected cells following TPVΔ18L. At 6 h PI, a failure to suppress initial ERK-I activation in response to infection is observed at the lower MOIs. At 24 h PI, a failure to suppress the increase in ERK-I activation relative to mock-infected cells is seen. These findings, like the VV inhibition shown in Fig 5, indicate a role for T18L in inhibiting ERK-I activation. Under normal circumstances, ERK-I is a critical determinant of the host cell immune response to virus infection through its role in mediating anti-viral interferon (IFN) responses. As a result, poxviruses encode immunomodulatory proteins to circumvent this response. Of note, Tl 8L is one of only a few known viral pyrin domain (PYD) proteins. It exhibits high homology with the IFN regulatory factor (IRF) family of innate immune proteins responsible for mediating the IFN response in infected cells.

EXAMPLE 10 - Pharmacological inhibition of ERK-I rescues the deletant mutant

Figure 9 shows type II MCF-7 breast cancer cells were infected with TPVΔ18L at MOI = 1 in the presence or absence of the selective ERK-I inhibitor, UOl 26 (20 μM). Foci formation was evaluated at 24 and 48 h PI as an indicator of replication and spread. Inhibition of ERK-I by UO 126, replacing the ERK-I inhibiting activity of the deleted Tl 8L gene product, enabled TPVΔ18L to replicate in MCF cells and reversed the transiently restrictive phenotype of the tumour type. Also of note, the low level expression of the exogenous gfp gene cassette at 48 h PI in restrictive cells following TPVΔ18L infection indicates the capacity for the deletant to deliver and express foreign genes in the absence of productive infection or viral gene expression. Images captured at IOOX magnification. EXAMPLE 1 1 - Suppression of ERK transcription rescues the deletant mutant

As shown in Figure 10 A-D, Type II A549 breast cancer cells were infected at MOI = lwith TPV-WT (A) or TPVΔ18 L in the absence of intervention (B), the presence of a vector control (C), or in the presence of vector expressing siRNA specific to ERK (D). Foci formation was evaluated at 72 h PI as an indicator of replication and spread. Inhibition of ERK-I by siRNA suppression of transcription, replacing the ERK inhibiting activity of the deleted T18L gene product, enabled TPVΔ18L to replicate in A549 cells and reversed the transiently restrictive phenotype of the tumour type. Images captured at IOOX magnification. E, Western blot analysis of ERK expression in mock-transfected controls (M) or cells transfected with vector control (-) or vector expressing ERK-I siRNA (+). Expression of siRNA specific to ERK suppressed ERK production relative to control values. Detection of the actin housekeeping gene served as a loading control. Thus, the siRNA data shown in Figure 10 confirms the molecular basis for tumour selectivity, and the capacity to regulate selectivity by targeting a single signaling molecule.

EXAMPLE 12 - T18L expression inhibits other signaling pathways subject to pharmacological manipulation

Figure 1 1 shows human HEK293 cells stably expressing T18L transfected with a NFKB reporter vector or vector lacking the NFKB response element. NFKB activation in response to TNF stimulation (10 ng/ml) for 24 h was evaluated by expression the luciferase gene cassette located downstream of the NFKB element. Compared to cells lacking T18L expression, NFKB activity in response to TNF was markedly reduced in T18L-expressing cells. A stable HEK293 cell line expressing the NFKB reporter element only is shown for comparison. These results indicate that T18L expression can regulate NFKB signaling events in addition to ERK-I activity.

EXAMPLE 13 - Insert ional mutation of the T18L gene

Figure 12 shows the insertional mutation of the T18L gene. The tanapoxvirus (TPV) T18L gene was disrupted by insertion of an approximately 754 bp green fluorescence protein (gfp) cassette whose expression was under the control of a synthetic early-late poxvirus promoter (E/L). The insertion was designed such that two nucleotides central to the gene (nt 14054 and 14055) were excised and the gene effectively bisected into two equal sections representing nt 13863 to 14053 (left fragment) and nt 14056 to 14246 (right fragment). No other changes were introduced into the sequence of the wild-type. The open reading frame of the T18L gene is orientated in a direction opposite to that of the nucleotide sequence (arrow). As such, transcription initiates at nt 14246 and terminates at nt 13863.

Whilst various embodiments of anticancer agents, recombinant viruses, and methods of medical treatment, are described and illustrated herein, the scope of the appended claims is not limited to such embodiments, and the invention encompasses further embodiments readily obtainable in view the teachings presented herein.

SEQUENCE LISTING FREE TEXT

T18L sequence: Tanapoxvirus (TPV) SEQ ID NO.: 1 - Gene sequence (nt 13863 to 14261 of genome)- atttaaaataactttcttcttactgtactacttatatttttcataacattggaatttcta aatctgtg ttttcgtataatttttttacaaactggttcaaactgaacagatggtactaagtcactaat taatctgt ttatgttacttcttgcgtattttgcatttggaaccatacttattgctttttctaaaaaat ataatgat ttaatacctggataagcttgaaatagtttttcagcaaaatctactctatctagtatttgt ttttcttc atcgtttatatttagttcatcctttgttaaaaatattagtattttaaactggtaatgtgt tacgtcct ctagagaaaaaataatcgctgatttadttctcatattatataacat

SEQ ID NO' 2 - ORF sequence (reverse reading frame): atgttatataatatgagαattaaatcagcgattattttttctctagaggacgtaacaca ttaccagtt taaaatactaatatttttaacaaaggatgaactaaatataaacgatgaagaaaaacaaat actagata gagtagattttgctgaaaaactatttcaagcttatccaggtattaaatcattatattttt tagaaaaa gcaataagtatggttccaaatgcaaaatacgcaagaagtaacataaacagattaattagt gacttagt accatctgttcagtttgaaccagtttgtaaaaaaattatacgaaaacacagatttagaaa ttccaatg ttatgaaaaatataagtagtacagtaagaagaaagttattttaa

SEQ ID NO 3 - Ammo acid sequence (translation of ORF): MLYNMRIKSAI I FSLEDVTHYQFKILIFLTKDELNINDEEKQILDRVDFAEKLFQAYPGIK SLYFLEKAI SMVPNAKYARSNINRLI S DLVPSVQFEPVCKKI IRKHRFRNSNVMKNI SSTV RRKLF

SEQ ID NO. 4 - Sequence of disrupted gene with insertion:

ttttcgtataatttttt tacaaactggttcaaactgaacagatggtactaagtcactaattaatctgt ttatgttacttcttgcgtattttgcatttggaaccatacttattgctttttctaaaaaaa aaattgaa aaactattctaatttattgcacatgagtaaaggagaagaacttttcactggagtggtccc agttcttg ttgaattagatggcgatgttaatgggcaaaaattctctgtcagtggagagggtgaaggtg atgcaaca tacggaaaar ttacccttaattttatttgcactactgggaagctacctgttccatggccaacacttgt cactactttctcttatggtgttcaatgcttctcaagatacccagatcatatgaaacagca tgactttt tcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttacaaagatgacg ggaactac aagacacgtgrtgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaa ggtattga ttttaaagaagatggaaacattcttggacacaaaatggaatacaactataactcacataa tgtataca tcatgggagacaaaccaaagaatggcatcaaagttaacttcaaaattagacacaacatta aagatgga agcgttcaattagcagaccattatcaacaaaatactccaattggcgatggccctgtcctt ttaccaga caaccattacctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatca catgatcc ttcttgagtttgtaacagctgctaggattacacatggcatggatgaactatacaaaataa tgatttaa tttatatttagttcatcctttgttaaaaatattagtattttaaactggtaatgtgttacg tcctctag agaaaaaataatcgctgatttaattctcatattatataacat

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