Replication-selective oncolytic viruses are used for cancer therapy (viral oncolysis). Attenuated viruses with tumor specificity have attracted considerable interest as novel anticancer agents, and clinical testing of several such agents is under way^ref. Tumor selectivity of these viruses has been attributed to various intracellular restrictions to their life cycles that are strongly inhibitory to virus propagation in nontransformed cells but that are overridden by cellular factors present in neoplastic cells^{ref1, ref2, ref3, ref4, ref5}. A pronounced bystander effect caused by self-perpetuating infection of adjacent cells after cytolysis of primary targeted cells is considered a further advantage.

• Parvoviruses
• minute virus of mice (MVM)
• H-1
They have been shown to preferentialy replicate and exert CPEs in various oncogene-transformed cells while sparing their non-transformed counterparts in vitro and in vivo thanks to the strong cell-cycle and oncogene-inducible parvoviral promoter P4. Hence MVM and H-1 virus-based vectors have been constructed for cancer gene therapy purposes, which retain in particular the oncogene-responsive parvoviral expression cassette, including the gene coding for the multifunctional (replicative, transactivating and cytotoxic) nonstructural protein 1 (NS1). Parvoviral vectors proved to be endowed with the capacity of parental viruses for targeting transformed cells on the level of gene expression, yet their use is limited by the relative inefficiency of the system for vector production available to date. Adenovirus vectors, in contrast, lack the oncotropism of parvovirus vectors, but can be produced in very large amounts : chimeric adenovirus vectors that carry the parvoviral expression cassette are amenable to high titer production. The complete suppression of recombinant adenoviruses production as a result of the presence of the parvoviral NS gene can be overcome by transfecting the packaging cells with phosphorothioate-modified (PS) antisense oligodeoxyribonucleotides targeting the translation initiation region of the NS transcript.
• conditionally replicating adenoviruses (CRAD / CRAd)^{ref1, ref2} : the paucity of coxsackie-adenovirus receptors (CARs) on tumor cells is a major stumbling blocks for adenovirus-based treatment. Adenoviruses which have low infectivity on lymphoid cells^ref.
• Ad5 (E3 wild type)
• dl704 (del. E3 gp19 kDa)
• dl309 (del. E3 10.4/14.5, 14.7 kDa) is cleared much more rapidly and/or its activity is lower in 4 mouse carcinoma lines. Intratumoral injection with dl309 results in markedly greater macrophage infiltration and expression of both TNF-a and IFN-g. Adenovirus replication, CD8⁺ lymphocyte infiltration and efficacy are similar upon intratumoral injection with either dl704 or Ad5. E3-dependent differences are not evident in athymic mice
• using tissue-specific (including tumor-specific) promoters to drive adenoviral genes that are essential for replication
• prostate adenocarcinoma-specific viruses
• CG-7870 (therapeutic index : 10,000:1; source : Cell Genesys, Inc.)
• CN-7060 (source : Cell Genesys, Inc.)
• CV-787
• colon cancer-specific viruses
• Tcf binding sites in the ...
• E1A promoter, mutating the p300 binding site in E1A
• E2 promoter, mutating the adjacent E3 enhancer
• E1A and E4 promoters (vCF11), deleting half of the ITR, the E1A enhancer and the packaging signal
• tumor vessels-specific viruses (using a dividing endothelial cells-specific promoter)
• deleting adenoviral genes essential for replication that become dispensable only in cancer cells
• E1B-55K deficient adenovirus dl1520 / CI-1042 (ONYX-015) (source : Onyx Pharmaceuticals Inc), for treatment of recurrent head and neck cancer in combination with cisplatin and 5-fluorouracil^ref, ovarian carcinoma and pancreatic carcinoma with non-functional p53 or p14^ARF pathway
• E1A mutant adenovirus d/922-947 is unable to inactivate the family of retinoblastoma (Rb) proteins and thus lyses only cells with abnormalities in the Rb pathway which occur in a broad range of cancers.
• Delta 24 encompasses an E1A deletion in the Rb protein-binding region
• Delta-24-RGD has an RGD-4C peptide motif inserted into the adenoviral fiber, which allows the adenovirus to anchor directly to integrins.
A major limitation of tumor-targeted CRADs is their relatively poor ability to spread in the tumor mass, thereby requiring repeated viral injections administered at multiple sites and over several days. Targeting the vessels of the tumor instead of the tumor itself potentially offers enhanced viral spread via the tumor's blood supply. In addition, as the target is the genetically normal endothelial cell, resistance to treatment due to somatic mutations in the target cell does not occur.
• replication-selective HHV-1 / HSV-1^ref
• ICP34.5-HSV1716 replicates in and lyses tumour cells (including glioma, melanoma, breast carcinoma, prostate adenocarcinoma, colon cancer, ovarian carcinoma, and pancreatic carcinoma) over a prolonged period of time (up to 31 days), but fails to replicate in growth arrested, terminally differentiated cells. The immune status of the patients with respect to HSV antibody levels pre-virus administration has little impact on the outcome of HSV1716 infection.
• G207
• deletion of both copies of g₁34.5 leading to highly attenuated neurovirulence
• insertion of a lacZ (b-galactosidase) reporter gene to interrupt the ICP6 / U_L39 gene, which encodes for the large subunit of ribonucleotide reductase, an enzyme required for viral DNA synthesis in nondividing cells. It kills human glioma cells^ref. Multiple intratumoral inoculations of G207 and systemic or local rIL-12 administration work synergistically to facilitate tumor regression
• NV1020 is a clonal derivative of R7020 originally constructed for vaccine studies against HSV-1 and HSV-2
• 700 bp deletion in the thymidine kinase (tk) locus
• 15 kb deletion across the joint region of the long (L) and short (S) components of the HSV-1 genome. The L/S junction of the NV1020 contains a 5.2 kb fragment of HSV-2 DNA inserted for previous vaccine studies and an exogenous copy of the thymidine kinase gene under the control of the HSV-1 4 promoter.
• deletion of only one copy of the g₁134.5 gene (haploid for the neurovirulence gene)
• ICP6 gene intact
• replication-selective vaccinia virus
• replication-selective reovirus : RG228 (Reolysin^®; source : Oncolytics Biotech) : there are no particular symptoms associated with it and the body will eliminate reovirus within 2 weeks. It is able to replicate itself in cancer cells with an activated Ras pathway (up to 66% of all human cancers, including many brain cancers for whose stereotactic delivery is practiceable), which turns off the PKR pathway^ref. It is under clinical trials for glioma^ref, breast carcinoma, pancreatic carcinoma^ref, lung carcinoma and T2 prostate adenocarcinoma, ovarian carcinoma and colon cancer^ref. Despite 70-100% of people have been exposed to reovirus in their lifetime, it is usually confined to the gastrointestinal or respiratory tracts in humans, so it would not have access to tumours growing elsewhere in the body, nor in the amount required to impact the tumours.
• conditionally replicating Paramyxoviruses :
• mumps virus : in 1974 it was administered to 90 patients with advanced malignancy^ref, resulting in significant (although mostly short-lived) responses. Toxicity was minimal.
• replication-selective Newcastle disease virus (NDV) : in the 1950s several reports demonstrated potent oncolysis of murine tumors by NDV and influenza virus^{ref1, ref2, ref3}
• live attenuated measles viruses of the Edmonston lineage (MV-Edm) : is naturally strongly lymphotrophic^ref, and occasionally patients with lymphomas who acquired wild-type measles have experienced complete remissions^{ref1, ref2, ref3, ref4}. Despite its profound attenuation as a human pathogen (hence used as a vaccine), MV-Edm replicates more efficiently than nonattenuated measles virus in many primate cell lines, inducing cell cell fusion and the formation of characteristic multinucleated syncytia^ref. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus^ref. Trackable viruses that encode an inert soluble marker peptide in the viral genome have also been generated such that viral gene expression can easily be monitored by following peptide levels in the cell culture media or body fluids^ref.
• the oncolytic potency of MV-Edm in different human tumor xenograft therapy models is highly variable and there is no convenient way to map the distribution of virus-infected tissues in vivo. Myeloma xenografts stably expressing NIS regressed completely after a single dose of ¹³¹I, even when half of the tumor cells did not express the gene^ref. To enhance the oncolytic potency of MV-Edm against radiosensitive malignancies and to facilitate noninvasive imaging of infected tissues, a recombinant MV-Edm encoding the human thyroidal iodide symporter (NIS) was generated. MV-NIS replicated almost as efficiently as unmodified MV-Edm, and human tumor cells efficiently concentrated radioiodine when infected with MV-NIS. Intratumoral spread of MV-NIS was noninvasively demonstrated by serial gamma-camera imaging of iodine-123 (¹²³I) uptake both in MV-sensitive KAS-6/1 myeloma xenografts, which regressed completely after a single intravenous dose of MV-NIS, and in MM1 myeloma xenografts, which were unresponsive to MVNIS therapy. However, MV-resistant MM1 tumors regressed completely when ¹³¹I was administered 9 days after a single intravenous injection of MV-NIS (radiovirotherapy). ¹³¹I alone had no effect on MM1 tumor growth. While the potential hematopoietic toxicity of this new therapy requires further evaluation, image-guided radiovirotherapy is a promising new approach to the treatment of multiple myeloma, an incurable but highly radiosensitive plasma cell malignancy. Testing in other radiosensitive cancers is warranted. Multiple myeloma is one of the most radiosensitive tumors known^ref, and MV-NIS provides us with an opportunity to combine targeted radiation to myeloma deposits with a naturally oncolytic attenuated measles virus. Magnetic resonance imaging of patients with myeloma shows that the distribution of the disease in the bone marrow is frequently multifocal, comprising multiple discrete plasmacytomas.32 Plasmacytomas may provide favorable tumor geometry that maximizes particle cross fire within the tumor exposing noninfected myeloma cells to sufficient radiation to mediate their destruction. This favorable tumor geometry also increases the therapeutic effect of radioiodine by enhancing isotope reuptake and therefore retention time in the tumor^ref (D.D. et al, manuscript in preparation, June 2003). Efficient infection of plasma cells by MV-NIS in vivo requires access of the virus to the tumor. Myeloma is associated with significant angiogenesis in the bone marrow, which may provide access of the virus to infect the cells^ref. One likely toxicity of radiovirotherapy for multiple myeloma is that the accumulation of ¹³¹I in the bone marrow might suppress or eliminate normal hematopoiesis. This is more likely to be a significant concern where the marrow is diffusely infiltrated with malignant plasma cells, as opposed to the more classical focal disease distribution. It is difficult to study the potential impact of radiovirotherapy on hematopoiesis using our current myeloma xenograft model, but we plan to evaluate this problem experimentally in orthotopic models of multiple myeloma that recapitulate either focal or diffuse disease. However, it seems likely that stem cell transplantation will be necessary for some myeloma patients after radiovirotherapy. Our virus is not targeted to infect only myeloma cells, although our in vitro data suggest that the virus preferentially infects and replicates in tumor cells compared with nonmalignant cells^{ref1, ref2}. Measles virus infects primarily lymphoid cells and thus we expect minimal toxicity to additional organs from the virus and/or isotope, since nonlymphoid tissues are not infected significantly by the virus and thus should not express NIS and concentrate the isotope. However, we are working to develop viruses that do not bind to either of the known receptors for MV (CD46 and CD150) and should therefore infect cells with high selectivity and specificity via targeted receptors such as CD38. This should enhance the safety of these vectors for clinical studies and minimize toxicity. Most adults are immune to MV infection either due to prior natural MV infection or vaccination. Preformed antibodies may neutralize a virus that is administered into the bloodstream, thereby blocking access of the virus to the tumor cells. However, patients with myeloma have a profound defect in the humoral arm of the immune system that progressively worsens with advancing disease^ref. Many patients with advanced myeloma have low (nonprotective) levels of antimeasles antibodies. Thus, rapid neutralization of MV-NIS by circulating antibodies is less likely in myeloma patients than in patients with other malignancies. The mechanistic basis for the striking oncolytic specificity of MV-Edm has not yet been determined but is the subject of ongoing investigations. One intriguing property of MV-Edm, acquired during tissue culture adaptation, that distinguishes it from wild-type measles virus is its ability to enter cells efficiently through CD46^{ref1, ref2}. CD46 is a regulator of complement activation that protects cells from complement-mediated lysis and is expressed at higher levels on human tumors (including myeloma) than on nontransformed cells^{ref1, ref2, ref3}. This may, in part, explain the observed tropism of the virus for malignant plasma cells compared with nontransformed peripheral blood lymphocytes^{ref1, ref2}. Also, in an effort to enhance the myeloma specificity of measles virus, we recently demonstrated that MV-Edm could be engineered to enter cells through the myeloma cell surface antigen CD38 by genetically fusing a single-chain antibody to the C-terminus of its H attachment protein^ref. In summary, to enhance the oncolytic potency of MV-Edm against radiosensitive malignancies and to facilitate noninvasive imaging of infected tissues, we generated a recombinant MV-Edm encoding the human thyroidal iodide symporter (NIS). MV-NIS retains the oncolytic activity of the parent virus (MV-Edm), and its spread can be followed noninvasively in vivo using ¹²³I. When combined with ¹³¹I (radiovirotherapy) MV-NIS can eliminate tumors resistant to the virus alone. Image-guided radiovirotherapy is a promising new approach to the treatment of multiple myeloma, and testing in other malignancies is currently under way^ref
• some cutaneous T-cell lymphomas (CTCLs) clonal T cells are deficient in interferon signaling, making them promising targets for viral oncolysis. CTCL cell lines and infiltrating lymphocytes in CTCL expressed MV receptors CD150 and CD46. In a phase 1 dose escalation trial a total of 16 injections of live MV, Edmonston-Zagreb vaccine strain, were given intratumorally to 5 patients with CTCL. Patients had antimeasles-serum antibodies and were pretreated with IFN-a to prevent uncontrolled virus spread. The well-tolerated treatment with MV resulted in clinical responses. Evaluation of biopsies, before and at 11 days after injection, by IHC and RT-PCR demonstrated local viral activity with positive staining for MV nucleoprotein (NP), an increase of the IFN-g/CD4 and IFN-g/CD8 mRNA ratios and a reduced CD4/CD8 ratio. All patients demonstrated an increased antimeasles antibody titer after therapy. The data demonstrate that CTCLs are promising targets for an MV-based oncolytic therapy^ref
• scFv-retargeted MV-Edm : a pseudoreceptor system that allows rescue and propagation of fully retargeted viruses displaying scFv has been developed. Candidate tumour-specific antigens vary for each tumor and are displayed on the surface of MV-Edm as C-terminal extensions of the H glycoprotein :
• multiple myeloma : therapy with replicating MV stimulates a neutrophil anti-Raji tumor response, which can be GM-CSF-enhanced to improve the oncolytic capacity of MV^ref. MV-Edm has potent and selective oncolytic activity against CD138 sorted plasma cells from patients with MM and against myeloma xenografts. MV-Edm caused extensive cytopathic damage through cell-cell fusion and the formation of multinucleated giant cells (syncytia) in myeloma cells but not in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes from healthy volunteers. Single or multiple doses of virus injected intratumorally or intravenously caused growth inhibition or total regression of 2 different subcutaneous human myeloma xenograft models grown in immunocompromised (athymic nude or C-B.17 scid/scid (SCID)) mice : this occurred at a dose of 10⁷ infectious units compared to the recommended dose for immunization of 10³ to 10⁴ infectious units administered subcutaneously, while the therapeutic potency of the virus at doses significantly < 10⁷ infectious units was substantially reduced^ref. The mechanisms underlying the selective antineoplastic activity of MV-Edm have not been elucidated. Possibilities include a relative abundance of measles virus receptors on myeloma cells, higher concentrations of intracellular factors supporting virus replication, and specific defects in the antiviral responses of the myeloma cells. Certain theoretical toxicities of MV-Edm could not be evaluated with the SCID mouse model used in the current study because these mice do not express receptors for the virus. Thus, MV-Edm infection of human monocytes^ref or osteoblasts^ref might lead to the up-regulation of IL-6, a known inhibitor of myeloma cell apoptosis that can also stimulate myeloma cell proliferation^ref. In addition, measles virus can infect and activate human osteoclast precursors and has been implicated in the pathogenesis of Paget disease of bone^{ref1, ref2}. MV-Edm might, therefore, have the potential to exacerbate the osteolytic bone disease characteristic of multiple myeloma. Another potential toxicity is MV-Edm-mediated immune suppression. Measles vaccination is known to be mildly immunosuppressive, leading to transient suppression of delayed-type hypersensitivity responses^ref, and this effect may be greatly amplified after the administration of the large doses of MV-Edm required for antineoplastic therapy. Transgenic mice expressing human CD46 are susceptible to MV-Edm infection and may facilitate the experimental study of some of these potential toxicities^{ref1, ref2}, as may the SCID-Hu model of multiple myeloma in which primary myeloma cells are grown in vivo in a human bone marrow microenvironment, provided by a transplanted fragment of fetal bone^{ref1, ref2}. Another concern associated with the use of replicating viruses as antitumor agents is the theoretical risk that the therapeutic virus might evolve to become highly pathogenic and transmissible, thereby causing disease in the contacts of the patient^ref. For MV-Edm the risk is negligible because most of the patient's contacts will have pre-existing immunity from childhood measles vaccination or infection. It is possible that, in addition to providing a barrier to horizontal virus transmission, the high prevalence of measles virus antibody seropositivity might compromise the value of MV-Edm for cancer therapy. However, considerable evidence suggests that this may not be the case given that asymptomatic measles virus infection has been well documented in vaccinated children^ref. Recently, it was demonstrated that macaques vaccinated with live attenuated virus or DNA can experience subclinical infection when challenged with measles virus^ref. Studies have shown that the therapeutic efficacy of conditionally replicating adenoviruses^ref, herpesviruses^ref, or reoviruses^ref was not negated by pre-existing antiviral antibodies. In addition, the infusion of neutralizing MV-Edm antibodies into immunocompromised mice did not compromise the antitumor efficacy of MV-Edm. It is also worth noting that profound suppression of serum immunoglobulin levels is a characteristic feature of multiple myeloma. Indeed, efficacy from vaccination of myeloma patients against influenza, Streptococcus pneumoniae, and Haemophilus influenza type B is poor, and only a small percentage of patients achieved protective antibodies^ref. Therefore, multiple myeloma may represent the most attractive target for oncolytic virotherapy using MV-Edm. Intracellular restriction of virus replication was the basis for tumor selectivity of all previously studied oncolytic viruses^{ref1, ref2, ref3, ref4, ref5}. Viruses whose host range is restricted at the level of attachment and entry into the target cell have not previously been tested, primarily because it has not been feasible to target viruses in this way. The demonstration that MV-Edm can eliminate CD46⁺ tumors from CD46^- mice when administered intravenously suggests that receptor-mediated targeting is a worthwhile strategy to pursue. Application of MV-Edm for virotherapy of multiple myeloma would require systemic administration of the virus because of the disseminated nature of the disease. They have potent anti-tumor activity but are not entirely tumor-specific owing to widespread distribution of their native receptors, CD46 / MCP and CD150 / signalling lymphocyte activation molecule (SLAM). Thus, to prevent collateral damage to normal tissues, the oncolytic activity of the virus should be targeted exclusively to myeloma cells. MV-Edm was intrinsically oncolytic for tumor cells and caused minimal cytopathic damage on normal nontransformed cells^{ref1, ref2}. To further enhance specificity, virus entry and receptor dependent cell-cell fusion can potentially be targeted specifically to myeloma cells^{ref1, ref2}. The need for targeting entry and infection is readily apparent in biodistribution studies after administration of MV-Edm in MV-susceptible mice. MV-Edm efficiently infects macrophages in the spleen, lymph nodes, and peritoneal cavity^ref. Thus, for systemic therapy of myeloma, we need to generate a retargeted virus, devoid of binding to its natural receptors, that will infect and fuse myeloma cells exclusively through a novel receptor. There are 4 steps to the development of such a retargeted virus: (1) identification of a receptor that is overexpressed in the myeloma cells to allow targeted binding of the virus to this receptor, (2) construction of the ligand (eg, single-chain antibody [scFv]) that will bind to the chosen receptor, (3) display of the ligand as a functional entity on the virus to redirect virus entry through the targeted receptor, and (4) ablation of the original tropism of the virus for its natural receptors. Mutations in the H glycoprotein that inhibit fusion through the CD46 receptor have been reported^{ref1, ref2, ref3, ref4}, and mutagenesis studies are currently in progress to identify mutations in H that ablate SLAM binding. In addition, ablating virus binding to SLAM might interfere with the immunosuppressive properties of the virus, thereby further reducing the dose-limiting toxicity for clinical studies^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11}. Candidate markers :
• CD38 is a type II transmembrane protein (45 kDa) with multiple functions: it is a bifunctional enzyme capable of synthesizing cADP-ribose from NAD and hydrolyzing cADP-ribose to ADP-ribose and is also involved in adhesion and signaling of leukocytes^ref (a counter-receptor of CD31, an Ig superfamily member^ref). CD38 is densely expressed on myeloma cells, and a high-affinity anti-CD38 mAb has been tested in clinical trials for patients with myeloma^ref : display of the anti-CD38 scFv on the C-terminus of the measles viral envelope glycoprotein hemagglutinin (H) through a Factor Xa protease cleavable linker successfully redirected virus entry through CD38 expressed on target cells devoid of the natural MV receptors. Infection of recombinant MVa-CD38 virus in CD46⁺ human HT1080 cells expressing CD38 was not enhanced by the second receptor. Replication of MV-aCD38 was not hindered by the scFv, reaching titers comparable to MV-Edm^ref.
• ovarian carcinoma : CEA, a protein in the immunoglobulin family^ref; MV-Edm displaying an anti-CEA scFv could enter rodent cells through the tumor-associated CEA^ref; the engineered virus enhanced the survival of mice bearing intraperitoneal  tumors by > 250%^ref.
• breast carcinoma and other carcinomas : anti-epidermal growth factor receptor (EGFR)^ref or EGFR mutant vIII (EGFRvIII) scFv-redirected measles viruses efficiently entered cells through their respective targeted receptors in vitro and in vivo,. When administered intratumorally or intravenously to mice bearing EGFR⁺ human tumor xenografts, the targeted viruses demonstrated specific receptor-mediated anti-tumor activity. These data provide an in vivo demonstration of antibody-directed tumor destruction by retargeted oncolytic viruses^ref
• IGF-1-conjugated measles viruses entered cells through IGF1R^ref
• VSV-rp30^ref has increased selectivity and lytic capacity in glioblastoma cells. VSV and Sindbis virus were very effective at replicating, spreading within, and selectively killing human glioblastoma in an in vivo mouse model, whereas PRV and AAV remained at the injection site with minimal spread. Together, these data suggest that 4 (VSV, Sindbis virus, minute virus of mice i-strain (MVMi) and MVMp) of the 9 viruses studied merit further analysis for potential therapeutic actions on glioblastoma^ref

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