Viral oncolysis

Surg Oncol Clin N Am 11 (2002) 661–680

Viral oncolysis James M. Donahue, MD, John T. Mullen, MD, Kenneth K. Tanabe, MD* Massachusetts General Hospital, Harvard Medical School, Cox 626, 100 Blossom Street, Boston, MA 02114-2696, USA

Until recently, the use of viruses as antineoplastic agents has been limited to non-replicating viruses serving as delivery vectors for gene therapy strategies. Recent advances in the understanding of the interaction between the life cycles of viruses and tumor cells have enabled replication-competent viruses with tumor cell specificity to be engineered. These viruses possess the ability to infect and destroy tumor cells. This process has been termed viral oncolysis. A number of viruses, including adenovirus, herpes simplex virus, reovirus, Newcastle disease virus, and vaccinia virus, are currently being studied. Furthermore, this antineoplastic effect can be amplified by the release of progeny virion to infect and destroy adjacent tumor cells. Although these viruses differ in their biology, they have all demonstrated oncolytic properties. Based on exciting preclinical studies detailing their safety and efficacy, several clinical trials examining the use of replicationcompetent viruses as therapy for advanced malignancies are underway [1]. In this article, we review the historical precedents for using replicationcompetent viruses in cancer therapy. We also briefly describe some of the basic biologic differences among the viruses being studied (Table 1). Next, we examine the preclinical work documenting the tumor specificity and efficacy of these viruses. Finally, we comment on the early results of their use in clinical trials. Historical background Although recent advances in virology and molecular biology have propelled viral oncolysis into the clinical setting, the concept of treating tumors with replicating viruses is not a new one. Early in the twentieth century, it was noted that patients with various malignancies experienced spontaneous tumor regression after rabies vaccination or a bout with a viral illness [2]. * Corresponding author. E-mail address: [email protected] (K.K. Tanabe). 1055-3207/02/$ - see front matter Ó 2002, Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 3 2 0 7 ( 0 2 ) 0 0 0 2 5 - X

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Table 1 Oncolytic viruses for cancer gene therapy Vector (size)

Advantages

Disadvantages

Insert size

Adenovirus (36 kb)

Infects dividing and nondividing cells Efficient gene transfer Nontoxic to host cells High viral titers possible

Transient expression Small insert capacity Antigenicity generates immune response

7.5 kb

HSV-1 (152 kb)

Infects dividing and nondividing cells Potential for prolonged gene expression Large transgene capacity High viral titers possible Sensitive to acyclovir/ ganciclovir Potentiates immune responses to tumor antigens

Possibility of herpes encephalitis Antigenicity generates immune response

40–50 kb

Vaccinia virus (187 kb) Transient gene expression Efficient gene transfer and expression Large transgene capacity Antigenicity generates immune response Infects most mammalian cell types

Strongly immunogenic Safety concerns in immunosuppressed patients

25 kb

Reovirus (?)

Mild pathogenicity Unable to infect normal cells Potentiates immune responses to tumor antigens

Infects only cells with an activated Ras pathway

?

NDV (?)

Not pathogenic in humans Does not establish a permanent infection in host High potency

Mechanism of selective tumor cell lysis unclear Transgene insertion reduces viral replication

?

Later, preclinical studies showed that viruses were capable of replicating in and lysing experimental murine tumors [3]. Beginning in the late 1940s, studies of oncolytic viruses in the treatment of cancer patients were initiated. Perhaps the most recognized of these studies was one performed at the National Cancer Institute in 1956, in which wild-type adenoviruses of different serotypes were injected into patients with cervical carcinomas [4]. More than half of the patients treated with live virus exhibited tumor regression without evidence of toxicity, whereas the control patients treated with inactivated virus showed no response. Unfortunately, the initial tumor regression was soon followed by disease progression in all patients. This

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apparent lack of antitumor efficacy was mirrored in other human trials of that day, thereby leading investigators to abandon this mode of therapy. Despite these early setbacks, advances in the fields of tumor biology, molecular biology, and virology have provided investigators with the tools necessary to further develop oncolytic viruses for cancer therapy. Basic viral biology Adenovirus Adenovirus is a non-enveloped, linear, double-stranded DNA virus with a genome size of approximately 38 kb. Following infection, adenoviruses coax the infected cell to enter the cell cycle through the actions of the E1 gene complex, and as such, adenoviruses are capable of replication in dividing and non-dividing cells [5]. E1A binds to and inhibits the action of the cellular tumor suppressor retinoblastoma protein (pRB). This serves to release pRB from the transcription factor E2F and facilitate entry into the G1 phase of the cell cycle. The ultimate effect of immediate E1A expression is to create a cellular environment favorable for the synthesis of multiple copies of the adenoviral genome. The adenoviral E1B 55 kD protein is also expressed and binds to and inhibits the function of the cellular tumor suppressor protein p53. In response to adenoviral infection, cellular levels of p53 rise, resulting in either apoptosis or cell cycle arrest. This cellular defense mechanism would attenuate adenoviral replication were it not for E1B expression. Herpes simplex virus Herpes simplex virus 1 (HSV-1) is an enveloped, double-stranded DNA virus with a genome size of approximately 152 kb [6]. Several features of this virus make it attractive for gene therapy. First, as much as 30 kb of this genetic material is dispensable in replication-conditional HSV-1 mutants, allowing delivery of multiple transgenes and incorporation of heterologous promoters. This represents an advantage over adenovirus, whose much smaller genome limits the size and number of transgenes. HSV-1 rarely produces severe medical illness in immune-competent adults. Moreover, antiherpetic agents such as acyclovir and ganciclovir are available that provide a safety mechanism to shut off viral replication should systemic toxicity ensue. Finally, HSV-1 does not integrate into the cellular genome, as does the retrovirus, and so insertional mutagenesis is not a concern. The genes of HSV-1 are organized into three groups according to their temporal expression after viral entry into the cell: (1) immediate-early genes, such as ICP0, ICP4, ICP8, and ICP27, are generally involved in regulatory functions such as transcriptional control; (2) early genes are then expressed and encode enzymes necessary for DNA synthesis, such as ribonucleotide reductase and thymidine kinase (TK); and (3) late genes are finally expressed and encode mainly structural proteins.

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Vaccinia virus Vaccinia virus is best known as the first widely used vaccine that resulted in eradication of smallpox, and as such has the longest track record of use in humans [7]. Despite this, vaccinia virus has not been widely recognized as a suitable vector for gene therapy in humans until more recently [8]. Perhaps the observation that vaccinia virus induces a vigorous immune response and can be fatal to immunocompromised hosts explains the modest pace of its development. Recent enthusiasm for vaccinia virus as an antineoplastic agent is based on several observations. First, vaccinia virus exhibits tropism for a wide range of mammalian cell types. In addition, its nearly 200 kb genome allows the insertion of large DNA fragments for gene therapy applications. Also, its immunogenicity can be exploited to augment host immunity against tumor cells. This potential enhancement of a therapeutic immune response is the principle behind the development of poxvirus vaccines for melanoma, some of which are now in clinical trials. Reovirus Reovirus is an interesting virus in that it is selectively oncolytic for many tumor cell types and this selectivity is inherent in the biology of the virus [9,10]. It is a ubiquitous, non-enveloped double-stranded RNA virus with minimal pathogenicity in humans. Further details of reovirus biology will be discussed in the section on preclinical studies. Newcastle disease virus Newcastle disease virus (NDV) is a chicken paramyxovirus with oncolytic properties. It is an enveloped, single-stranded RNA virus that contains an RNA polymerase in its virion. The experience using NDV as an antineoplastic agent will be discussed in the section on clinical studies. Preclinical studies In addition to cell lysis resulting from replication, oncolytic viruses can mediate the destruction of tumor cells by a variety of potential mechanisms. As a research endeavor, the field of viral oncolysis has been concerned with both optimizing the antineoplastic efficacy of these viruses as well as improving their specificity for neoplastic cells. Towards these ends, numerous discoveries have been made to help propel viral oncolysis to the realm of clinical trials. Optimizing tumor-specific replication Improved understanding of the relationship between viral and cellular proteins After a virus infects a cell, its proteins interact in a complex way with host cellular proteins. The goal of this interaction is to allow viral replication to

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proceed and ultimately to liberate progeny virion. Some viral proteins function to hasten cellular destruction, while others serve to promote viral replication. It has been observed that viral-host protein interactions differ in some neoplastic cells as compared with normal cells. This difference has allowed manipulations of the viral genome to target viral replication more specifically to cancer cells, and to enhance their efficacy in destroying those cells. Certain oncolytic viruses generate proteins during their replicative cycles that are directly cytotoxic to cells. Adenoviruses, for example, express the E3 11.6 kD death protein and the E4ORF4 protein late in the cell cycle. Both of these proteins are toxic to cells. Furthermore, both adenoviruses and herpes viruses produce proteins that inhibit apoptosis. In the adenoviral system, the E1B-19kD gene product is a potent inhibitor of apoptosis. Although this gene likely serves an important role in maintaining viral replication in normal cells, this is not true for some cancer cells. Deletion of the E1B-19kD gene resulted in an adenoviral mutant that is more effective in tumor cell killing than wild-type virus [11]. Similarly, in the herpes virus system, the gene product of the US3 locus encodes a protein kinase that inhibits host cell apoptosis. Efforts to examine the antitumor efficacy of a US3 herpes virus mutant are underway. The deletion of certain gene products from the viral genome that are critical for viral replication in normal cells but are dispensable upon infection of neoplastic cells has greatly improved viral tumor specificity. This strategy seizes upon the unique biology of tumor cells, in which there has often been loss of cell cycle control mechanisms. Some of these gene products are listed in Table 2. In the adenoviral system, the oncolytic virus termed d11520 (ONYX-015) was created by engineering two mutations in the E1B-55 kD gene [12]. When adenovirus infects a normal cell, p53 levels are upregulated and the cell undergoes either cell cycle arrest or apoptosis, thereby preventing viral replication. Wild-type adenovirus evades this cellular defense mechanism by expressing the E1B-55kD gene, which encodes a protein that binds to and inactivates p53, thus allowing viral replication to proceed. ONYX-015, however, lacks this gene and so viral replication is markedly attenuated in cells with normal p53 function. Many tumor cell types lack functional p53, and ONYX-015 replicates within and lyses these cells preferentially. It is the loss of p53 (or p14ARF) function that accounts for the tumor-selective replication of ONYX-015 [13]. ONYX-015 may be useful then in the treatment of tumors with mutations within the p53 pathway as a whole. The deletion of several genes in the herpes virus genome has similarly improved the tumor specificity of these viruses. The first herpes virus mutant studied for tumor-selective replication, dlsptk, contains a 360-base pair deletion within the gene for thymidine kinase (TK) and was studied in a malignant rat glioma model [14]. Malignant glioma cells are actively dividing and so have high endogenous levels of TK. In contrast, the surrounding normal brain is composed of quiescent neurons and glia. Accordingly, dlsptk

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Table 2 Viral proteins necessary for viral replication and complementing/interacting cellular proteins Virus

Viral protein

Cellular factor

Adenovirus

E1A E1B-55 kD RR (UL39, UL40) TK (UL23) c134.5 TK SPI-1/SPI-2 ? ?

pRb p53 intracellular nucleotide pools intracellular nucleotide pools eIF2a dephosphorylation intracellular nucleotide pools ? Ras/PKR N-ras

Herpes simplex virus

Vaccinia virus Reovirus Newcastle disease virus

replicates well in cultured tumor cells and induces significant growth inhibition of human U87 gliomas growing in the brains of nude mice. However, this HSV-1 mutant was not examined in clinical trials because it produces neurotoxicity at higher titers and it is resistant to the antiviral agents acyclovir and ganciclovir by virtue of its disrupted TK gene. A second herpes virus mutant, hrR3, maintains sensitivity to acyclovir and ganciclovir and also exhibits tumor-specific replication. This virus was created by inserting the Escherichia coli b
galactosidase gene into the ICP-6 locus, which inactivates the gene for the large subunit of HSV-1 ribonucleotide reductase [15,16]. This mutation attenuates pathologic virulence while permitting lytic infection of cells that are actively dividing and have high levels of cellular ribonucleotide reductase and nucleotide precursors that can complement the absence of viral ribonucleotide reductase. In our laboratory, we have demonstrated that cellular ribonucleotide reductase levels are extremely low in normal hepatocytes and high in liver metastases [17]. Accordingly, titers of infectious hrR3 recovered after infection of colon carcinoma cells are three log orders greater than those recovered after infection of hepatocytes [18,19]. In contrast, wild-type HSV-1 strains replicate equally well in hepatocytes and colon carcinoma cells. A second line of investigation to improve tumor-specific replication in the herpes virus system has involved the herpes c134.5 gene product. In the herpes virus mutant Myb34.5, both endogenous copies of the c134.5 gene are deleted, and this gene has been reinserted by homologous recombination into the ICP6 locus under the regulation of the B-myb promoter [20]. HSV-1 expression of c134.5 is required for robust viral replication, as interaction between this viral protein and the cellular protein phosphatase 1a is required to dephosphorylate eIF2a [21–23]. In response to viral infection, cells normally phosphorylate eIF2a, which leads to a shutdown of host protein synthesis. Without ongoing host protein synthesis, the virus is unable to replicate. Expression of c134.5 during HSV-1 infection leads to eIF2a dephosphorylation, thereby overcoming this cellular defense against viral infection and replication. HSV-1 mutants that are completely defective in

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c134.5 expression are significantly attenuated in their ability to replicate in normal cells. Myb34.5 is similar to hrR3 in its preferential replication in mitotically active cells by virtue of its defective ribonucleotide reductase expression. Moreover, its replication is further regulated by the B-myb promoter. This promoter upregulates gene expression in E2F-deregulated cells and cycling cells. Studies in our laboratory have shown that Myb34.5 replication in colon carcinoma cells is as robust as that of hrR3, whereas its replication in normal human hepatocytes is significantly more attenuated than hrR3 [24]. As would be expected, toxicity following intravascular administration of Myb34.5 is lower than that of hrR3. Similar genetic modifications are being applied in the vaccinia virus system to augment tumor-specific replication. Mutant vaccinia viruses with improved tumor selectivity have been generated by the deletion of the SPI-1 and SPI-2 genes [25]. These viral genes encode serine protease inhibitors that are homologous to human proteins known to be upregulated in tumor cells and so are dispensable upon infection of tumor cells but are required for successful viral replication in normal cells. Another means of improving specificity is by insertional activation of the TK gene, again with the aim of limiting viral replication to cells with large intracellular nucleotide pools, such as tumor cells [26]. Studies at the NCI with TK-deleted vaccinia virus mutants have shown that these viruses are able to target tumor tissue after systemic delivery in mouse models [27]. In contrast to the other viruses mentioned, reovirus demonstrates impressive tumor specificity without manipulation of its genome. The basis for this specificity has been partially elucidated in recent years. For several decades it was known that reovirus exhibited preferential cytotoxicity for transformed cells compared to normal cells [9,10]. Normal mouse fibroblasts (NIH 3T3 cells), which are resistant to reovirus infection, become susceptible to infection following transformation with activated ras or with any activated element of the ras pathway, such as the EGF receptor or the v-erbB oncogene [28–30]. When reovirus infects normal mouse fibroblasts, early viral transcripts activate double-stranded, RNA-activated protein kinase (PKR). PKR then inhibits protein translation by phosphorylation of EIF2a, as described above. This cellular response inhibits reovirus replication. However, in cells with an activated ras pathway, PKR phosphorylation and activity are impaired, thereby allowing viral protein synthesis and the lytic cycle to proceed. The mechanism by which ras activation leads to PKR inactivation is unclear; nevertheless, it is clear that a common mechanism by which viruses evade the cellular antiviral defense system is through inhibition of PKR. Because 30% or more of all human tumors possess an activating mutation of ras, reovirus appears to be an ideal oncolytic agent. Coffey et al examined the efficacy of reovirus against flank tumors established from v-erb-B-transformed NIH 3T3 cells and human U87 glioblastoma cells, which overexpress the platelet-derived growth factor receptor and thus have an

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activated ras pathway. A single intratumoral injection of reovirus resulted in tumor regression in 65% to 80% of mice [28]. The virus also proved effective in treatment of ras-transformed C3H-10T1/2 tumors in immune-competent C3H mice. Pre-existing immunity to reovirus did not abrogate the oncolytic effect. Intravenous administration of reovirus is effective in reducing tumor burden and prolonging survival of mice bearing Lewis lung carcinoma metastases [31]. Clinical trials of reovirus will soon be initiated. Use of tumor-specific promoters to drive viral replication Deletion of viral genes that are critical for viral replication in normal cells but dispensable in neoplastic cells is an elegant but challenging strategy to improve viral targeting to tumor cells. Another strategy involves insertion of tumor-specific or tumor-associated promoters upstream of viral genes that are essential for efficient viral replication. This approach has already been touched on in the discussion of the herpes virus mutant Myb34.5. In the adenovirus system, a mutant, termed AvE1a04i, has been created by placing expression of the critical E1A gene under the transcriptional control of the tumor-specific a-fetoprotein (AFP) promoter [32]. AvE1a04i replicates preferentially in AFP-expressing cells such as hepatocellular carcinoma (HCC) cells, but not in non-AFP-producing cells. AvE1a04I infection of AFP-expressing flank tumors resulted in a greater than 50% long-term survival rate in treated animals. Another example of this strategy is the adenoviral vector Ad.DF3-E1, in which the DF3/MUC1 promoter drives expression of E1A, thereby resulting in preferential replication of this virus in MUC1-positive breast cancer cells [33]. These studies demonstrate the successes of targeting viral replication to tumor cells by cell-specific transcriptional regulation of adenoviral genes. Current studies in our laboratory are focusing on placing HSV-1 immediate early genes under the transcriptional control of tumor-associated promoters. We have also demonstrated improved tumor specificity by placing the DF3/MUC1 promoter upstream of the c34.5 gene. This virus has demonstrated improved in vitro and in vivo antitumor efficacy compared with viruses that are completely defective in c34.5. Optimizing antitumor efficacy Use of oncolytic viruses in multimodality therapy Combining viral oncolysis with chemotherapy or radiation therapy offers a promising enhancement of the antineoplastic effect achieved by oncolysis alone. Oncolytic viruses may themselves help to sensitize tumor cells to chemotherapy or radiation therapy. These viruses may also serve as vectors for the intratumoral delivery of prodrug activating transgenes. Combined modality treatment with therapies using different mechanisms of resistance dramatically reduces the risk of the emergence of resistant clones of tumor cells. In support of this notion, patients with solid tumor metastases are presently treated with multiple agent chemotherapy regimens, but are rarely

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cured, in part due to the eventual development of drug-resistant tumor cells [34,35]. In contrast, a strategy that combines two completely different mechanisms of antitumor activity, such as intratumoral prodrug activation and HSV-1-mediated lytic replication, may reduce the risk of tumor cell resistance. Both adenovirus and herpes virus have been shown to increase the sensitivity of cancer cells to chemotherapy and radiation therapy. In the adenovirus system, the E1A gene product is a potent chemosensitizer, particularly in cells with functional p53 [36]. In these cells, E1A can induce high levels of p53 and render the cells susceptible to DNA damage from chemotherapy and radiation. Normal, nontransformed cells appear to be unaffected by E1A [37]. Interestingly, E1A can sensitize tumor cells to chemotherapeutic agents even in the absence of functional p53 [38]. Data in the herpes virus system suggest that oncolytic viral therapy may be synergistic with radiation therapy. Treatment of malignant gliomas with the combination of a replication-competent herpes virus mutant and radiation demonstrated greater antitumor efficacy than either modality alone [39]. Our laboratory group is interested in incorporation of transgenes into the HSV-1 genome that encode proteins which convert metabolically inactive prodrugs into chemotherapeutically active metabolites. Antitumor activity observed in prodrug activation models has been partially attributed to bystander killing. Bystander killing refers to the death of untransduced tumor cells that are adjacent to transduced tumor cells [40–42]. The importance of bystander killing lies in the realization that it is unlikely that any gene delivery vector will be able to transduce 100% of targeted tumor cells. Bystander killing may result in complete destruction of a tumor despite transduction of only a fraction of the tumor itself. Furthermore, it has been shown that transgene expression is better distributed throughout a tumor following direct intratumoral inoculation of a replication-conditional HSV-1 mutant than following inoculation with a replication-defective HSV-1 mutant [43]. As one example of such a strategy, we have integrated the yeast cytosine deaminase gene into the HSV-1 genome [44]. This gene product efficiently converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), which is one of the most active chemotherapy agents used in the treatment of gastrointestinal malignancies. We have inserted this gene into the ICP6 locus of HSV-1 to create the herpes virus mutant termed HSV1yCD. HSVyCD-infected cells are able to convert 5-FC to 5-FU. The combination of HSV-1 replication with the intratumoral conversion of 5-FC to 5-FU is more effective than either modality alone when treating mice with diffuse liver metastases. We have also studied an HSV-1 mutant containing the rat cytochrome P450 2B1 gene. The HSV-1 mutant rRp450 was created by inserting this gene into the ICP6 locus [45]. Rat cytochrome P450 2B1 can be used as a suicide gene because it encodes an enzyme responsible for bioactivation of prodrugs, including cyclophosphamide, ifosfamide, and procarbazine. This provides a means for intratumoral generation of alkylating metabolites

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[45,46]. Treatment of mice harboring diffuse hepatocellular carcinoma with the combination of rRp450 and systemic cyclophosphamide leads to marked tumor reduction and prolongation of survival [47]. In the vaccinia virus system, Alexander and Bartlett and colleagues at the NCI have shown that systemic administration of a TK-deleted vaccinia virus expressing the suicide gene purine nucleoside phosphorylase in combination with 6-methylpurine deoxyriboside treatment leads to a 50% cure in a mouse model of hepataic metastases [48]. Likewise, systemic administration of a TK-deleted vaccinia virus expressing the cytosine deaminase gene in combination with 5-FC leads to tumor specific gene expression and cure rates of up to 30% in mice with established liver metastases [49]. When using this strategy of incorporating prodrug-activating transgenes into the viral genome, the choice of the transgene is critical because expression of transgenes whose products abrogate viral replication is counterproductive. For example, HSV-1 vectors with a wild-type TK locus confer sensitivity to ganciclovir to infected cells or those that are in the vicinity of an infected cell (bystander effect). Yet because ganciclovir inhibits herpes viral replication, the combination of HSV-1 oncolysis and ganciclovir is no better than HSV-1 oncolysis alone [45,47,50]. Therefore, therapeutic transgenes that do not attenuate viral replication are more likely to enhance overall antineoplastic efficacy. Augmentation of the host antitumor immune response Although in humans the host immune response to a tumor is largely ineffective, a great deal of recent work has demonstrated that this response can be rendered more vigorous. The relationship between viral oncolysis and the host immune system is quite complex, and is just beginning to be thoroughly investigated. Certainly, immune-mediated clearance of the virus could pose a cumbersome obstacle to effective oncolytic therapy. Theoretically, however, enhanced tumor cell destruction by oncolytic viruses may make a greater number of tumor cell antigens available for presentation to the immune system. This offers the tantalizing possibility of combining oncolysis with immunotherapy. One of the great challenges of oncolytic viral therapy, and of gene therapy in general, is the efficient delivery of the viral vector to the tumor. Ikeda and colleagues have demonstrated that much of this inefficiency of viral delivery is related to inactivation of the virus by the immune system [51]. Interestingly, they found evidence of both innate immunity as well as an elicited humoral response. The innate activity against HSV-1 was seen in both rat and human plasma and was due in part to preimmune IgM as well as complement. This activity was present in both naive and previously infected mice. A single dose of the B-cell immunosuppressive agent cyclophosphamide suppressed this early innate activity, and later,the specific neutralizing antibody response. Rats with intracerebral tumors treated with both cycluphosphanide and the oncolytic herpes virus by intravascular injection had

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increased viral survival and replication within the tumors, leading to greater tumor regression and improved survival. Other investigators have found that pre-existing immunity to HSV has minimal effects on viral oncolytic therapy. In our laboratory, we have found that the presence of neutralizing antibodies to HSV-1 in vaccinated mice neither enhanced nor reduced the efficacy of an oncolytic HSV-1 mutant after intraportal administration to mice with diffuse liver metastases [19]. Similarly, studies by Delman et al also demonstrated in a liver metastasis model using immunocompetent mice that the tumor response to an oncolytic herpes virus was minimally affected by immunity to HSV [52]. Recent studies support the notion that the immune response directed against the oncolytic virus enhances antitumor efficacy by generating antitumor immunity. Toda et al demonstrated in a highly immunogenic mouse colon cancer model that oncolytic HSV replicating within a flank tumor elicits an immune response to specific tumor antigens and to the virus itself [53]. In this study, identical mouse colon tumors were established bilaterally in the flanks of syngeneic, immunocompetent mice, and one of the flank tumors was subsequently infected with an oncolytic HSV-mutant. The injected tumor promptly regressed, as did the tumor on the contralateral flank, despite the fact that the virus had not spread to this tumor. CD8þ cytotoxic T lymphocyte (CTL) activity against a specific tumor cell antigen on the colon cancer cells was observed in these mice. The somewhat contradictory findings in all of the aforementioned studies can be reconciled by the fact that the mode of viral administration and the endpoints examined are different in each study. Oncolytic viruses can be administered locally by direct intratumoral inoculation or systemically by intravascular (ie, tail vein, portal venous, etc) administration. It is possible that the immune system serves to antagonize the effectiveness of oncolytic viruses administered intravascularly by limiting viral delivery to the tumor by virtue of both innate and acquired immunity. On the other hand, once the virus has reached its target and begins replicating within and destroying tumor cells, the immune response can theoretically augment tumor reduction by redirecting the CTL response from viral antigens to tumor antigens. Based on this possibility, the role of the immune system in oncolytic viral therapy is an area of intense research. Clinical studies Based on the breadth of preclinical data on oncolytic viruses, and the exciting nature of the results, several clinical trials have been initiated (Table 3). These translational investigations mirror the strategies used in preclinical studies to optimize both tumor specificity and antitumor efficacy. In this section, we will briefly describe these studies, with emphasis on the similarity with which the viruses have been manipulated as compared with preclinical strategies.

Strain

Adv 2/5 chimera

Adv 5

Adv 5

HSV-1

HSV-1/HSV-2

Vaccinia

NDV

Name

ONYX-015

CV706

CV787

G207

NV1020

Vaccinia-GM-CSF

PV701

Regulation of E1A under the PSA promoter; E3 deletion Regulation of E1A under the rat probasin promoter and E1B under the human PSA promoter; wild-type E3 lacZ insertion into ICP6 gene; deletion of both copies of c134.5 gene 700bp TK deletion+15kb deletion across the joint region, which contains an exogenous copy of TK gene under control of HSV-1 a4 promoter and a 5.2kb fragment of HSV-2 DNA Insertion of GM-CSF and lac Z genes into viral TK locus Naturally attenuated

E1B-55 kD deletion

Genetic alterations

Table 3 Partial list of oncolytic viruses in clinical trials

Advanced solid cancers

I

I–II

I

Colorectal carcinoma liver metastases

Malignant melanoma

I–II

Malignant glioma

I–II

Prostate cancer (organ-confined and metastatic)

Phase II–III I I–II I–II

Disease Head and neck cancer Ovarian cancer Primary and secondary liver tumors Prostate cancer (organ-confined)

Ref.

[5,53,64]

[59]

[7,14,36,62,63,65]

[35,51,60]

[10]

[32,41,54] [16] [15] [11]

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Adenovirus As previous described, ONYX-015 contains a deletion of the E1B-55kD gene, and this mutant virus exhibits marked cytopathic effects in p53 mutant cancer cells, but only limited cytotoxicity in normal human fibroblasts and endothelial cells with normal p53 function [12]. As a result of promising in vitro and in vivo studies, ONYX-015 is now under study for the treatment of a number of p53-deficient malignancies in phase I and II clinical trials. A phase I study in patients with unresectable primary and secondary liver tumors showed that ONYX-015 was well tolerated when administered intratumorally, intravenously, or intra-arterially up to a dose of 31011 pfu [54]. Moreover, in another phase I/II dose escalation trial of intra-arterial ONYX-015 administration to patients with colorectal carcinoma liver metastases, patients who received the highest doses of ONYX-015 experienced better survival than those patients treated with the lower doses [55]. In addition, a phase I trial of intraperitoneal ONYX-015 is under way in the treatment of patients with cisplatin-resistant ovarian carcinoma [56]. A second approach to achieve tumor-selective adenoviral replication is the use of tumor or tissue-specific promoters to drive the expression of an adenoviral gene that is critical for efficient viral replication, such as E1A. By replacing the endogenous viral E1A promoter with a human promoter sequence that is more transcriptionally active in tumor cells, one can restrict viral replication to these tumor cells, compared with normal cells that lack the proteins necessary to activate this promoter. One example of this strategy is exemplified by the virus CV706 (formerly CN706, Calydon, Inc., Sunnyvale, CA), in which the prostate-specific antigen (PSA) gene promoter-enhancer element is inserted upstream of the E1A gene. Because of this modification, CV706 replication is greatest in tissues with high-level PSA expression [57]. This virus is currently in a phase I/II dose escalation trial of intraprostatic injection in patients with locally recurrent prostate carcinoma following definitive radiotherapy. Calydon has also developed a more potent oncolytic adenovirus, CV787, which contains the prostate-specific rat probasin promoter driving E1A expression and the human prostate-specific enhancer/promoter driving the E1B gene. This virus, unlike CV706, maintains a wild-type E3 region, which encodes proteins that play a role in assisting virus release and in evading host immune responses to the virus. This virus eliminates distant prostate tumor xenografts in athymic mice after a single intravenous tail vein injection [57]. CV787 is being studied in phase I and II clinical trials in the treatment of patients with either organ-confined prostate carcinoma or hormone-refractory metastatic prostate cancer. To date, efforts aimed at improving the antitumor efficacy of oncolytic adenoviruses in the clinical setting have involved combining viral therapy and chemotherapy. A phase II study of patients with unresectable primary and secondary liver tumors showed that the combination of ONYX-015 and 5-fluorouracil (5-FU), when infused into the hepatic artery, was well-tolerated

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but did not induce significant liver tumor regression as judged by serial CT scan measurements [54]. Phase II trials of intratumoral ONYX-015 with or without systemic chemotherapy in patients with recurrent squamous cell carcinoma of the head and neck have demonstrated limited treatmentrelated toxicity and significant antitumor activity (10%–33% complete responses) [58,59]. A phase II clinical trial of ONYX-015 injection combined with cisplatin and 5-FU in patients with recurrent squamous cell cancer of the head and neck was recently completed [60]. Nearly two-thirds of the patients had objective responses, and 27% of the study population had complete responses. Toxicities from this combination therapy were acceptable. This study and others demonstrate that the addition of chemotherapeutic agents, many of which might be expected to curb viral replication, augments the antitumor efficacy obtained with replicating adenoviruses. HSV-1 The herpes virus mutant G207 (MediGene, Inc., San Diego, CA) harbors deletions of both copies of c134.5 and contains an insertional inactivation of the ICP6 gene. The presence of two mutations makes spontaneous reversion to a wild-type strain exceedingly unlikely and so provides an added level of safety. Mammalian ribonucleotide reductase (and thus the level of intracellular nucleotides) is elevated in tumor cells relative to normal cells, so HSV-1 mutants defective in ribonucleotide reductase replicate preferentially in tumor cells [17,18]. Furthermore, ribonucleotide-reductase-negative HSV-1 mutants retain sensitivity to acyclovir and ganciclovir, augmenting this built-in, all-important safety mechanism of HSV-1. Preclinical toxicology evaluation of G207 was undertaken by intracerebral inoculation into owl monkeys, which are exquisitely sensitive to HSV-1 infection [61]. Inoculation of G207 at doses as high as 1109 pfu, well above the efficacious dose in mice, was well tolerated, whereas monkeys that received an inoculation of only 1103 pfu of wild-type HSV-1 died rapidly. These encouraging safety data prompted a phase I clinical study of G207 in the treatment of recurrent malignant glioma in which cohorts of patients were treated with increasing doses of G207. The highest dose examined was 3109 pfu inoculated into five sites [62]. No toxicity related to G207 was seen at any dose. Preparations for phase II clinical trials designed to examine the efficacy of G207 alone and in combination with irradiation in this group of patients are now underway. NV1020 (formerly R7020, MediGene, Inc.) is another genetically engineered oncolytic herpes virus that is being actively studied in clinical trials. NV1020 was originally designed as a candidate for human immunization against infections with HSV-1 and HSV-2 [63]. It contains a 700 bp deletion in the endogenous HSV-TK gene as well as a 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 NV1020 contains a 5.2 kb fragment of HSV-2 DNA inserted

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for previous vaccine studies and an exogenous copy of the HSV-TK gene under the control of the powerful HSV-1 a4 promoter. NV1020 has only one copy of c134.5 deleted and maintains sensitivity to acyclovir and ganciclovir. Based on encouraging preclinical results, a phase I trial of intrahepatic arterial injection of NV1020 in patients with colorectal carcinoma liver metastases is under way. A clinical study of rRp450 in patients with unresectable liver tumors is being planned. Vaccinia viruses In a phase III randomized, double-blind trial, Wallack and colleagues examined the use of a vaccinia-melanoma oncolysate as an active specific immunotherapeutic agent. In this study, patients with stage III melanoma were treated in an adjuvant setting [64]. In specific subsets of patients, the vaccinia-melanoma oncolysate conferred a survival advantage, but there was no difference in survival when all patients were considered together. Other investigators have generated vaccinia virus mutants that are replication-conditional such that they destroy cancer cells as a byproduct of viral replication. The strategy most commonly employed is analogous to one used in HSV-1 mutants, in which insertional inactivation of the vaccinia virus TK gene limits viral replication to cells with large intracellular nucleotide pools, such as tumor cells. Mastrangelo and colleagues inserted the gene for GMCSF into the vaccinia virus TK gene locus of a wild-type vaccinia virus to generate an oncolytic virus that induces antitumor immunity following infection of malignant melanoma. This virus is currently under study in a phase I clinical trial of intralesional administration to patients with refractory, recurrent melanoma. In the first seven patients studied, two had a complete response and three other patients had partial responses [65]. Injected lesions showed evidence of viral replication and GM-CSF production with concomitant immune infiltrates. Newcastle disease virus Newcastle disease virus (NDV) is a chicken paramyxovirus that was first noted to replicate in and destroy tumor cells in 1955 [66]. The virus is not pathogenic to humans and has been extensively studied as an oncolytic agent in several different human tumor cell lines and tumor models [67,68]. These studies characterized the most widely known strain of NDV as 73-T, so named because it was passaged through mouse ascites tumor cells 73 times in vitro. It was chosen for trials in humans as both a viral oncolysate, which is a suspension of virus and tumor cells, and as free virus, because this strain demonstrated potent oncolysis and limited toxicity to normal cells. A recent 15-year follow-up of patients with stage III malignant melanoma treated post-surgically with an NDV oncolysate in 1975 as part of a phase II clinical study reveals a 55% overall 15-year survival. The oncolysate,

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composed of both allogeneic and autologous human melanoma cells infected with live NDV, presumably acts as a tumor vaccine, because the treated patients show evidence of increased numbers of CD8þ T cells [69]. Investigators at Pro-Virus, Inc. (Gaithersburg, MD) have isolated a naturally attenuated strain of NDV, cloned by non-recombinant methods, that exhibits a broad range of oncolytic activity against human tumors. This strain, PV701, is characterized by its tumor-selectivity, because 80% of human cancer cell lines are two to four log orders more sensitive than normal human cells to PV701-mediated killing [70]. Intratumoral treatment of a variety of human tumors growing in the flanks of nude mice, including fibrosarcoma, ovarian carcinoma, and melanoma, caused high rates of tumor regression and minimal toxicity. Intravenous administration of PV701 in a dose-escalation study produced partial flank tumor regressions at doses as low as 6105 pfu and complete tumor regressions in greater than 80% of the mice at doses up to 6108 pfu. The antitumor response was associated with evidence of viral replication, and although large amounts of virus were recovered from tumor tissue, no virus was isolated from the heart, lung, liver, kidney, or brain tissue [71]. These encouraging preclinical data have led to the initiation of a phase I clinical trial of PV701 administered intravenously to patients with advanced solid cancers who failed conventional therapy. Two partial responses in patients with colon carcinoma and mesothelioma were observed at higher doses of PV701, and six patients with diverse malignancies, including melanoma, colon carcinoma, and pancreatic carcinoma, exhibited measurable tumor reduction [72]. More clinical studies of this novel oncolytic virus are now in the planning stages. Summary Although the concept of using viruses as antineoplastic agents dates back nearly a century, recent advances in the fields of molecular biology, genetics, and virology have enabled investigators to engineer viruses with greater potency and tumor specificity. Further enhancements involve arming these viruses with therapeutic transgenes, and combining the traditional modalities of chemotherapy and radiation therapy with oncolytic viral therapy in hopes of reducing the chance of developing resistant tumor cell clones. Another means of augmenting the antineoplastic effect of these viruses involves modulating the immune response to minimize antiviral immunity, while at the same time maximizing antitumor immunity. A better understanding of mechanisms that viruses use to overcome cellular defenses to achieve robust replication within the cell will lead to development of oncolytic viruses with better tumor specificity and reduced toxicity. Initial clinical studies have shown that oncolytic viral therapy for metastatic disease is safe and well tolerated. In addition, using similar genetic modification strategies, these viruses have demonstrated antineoplastic effects in humans similar to those seen in preclinical animal models.

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