Emerging new therapies for chemotherapy—resistant cancer using adenoviral vectors

Drug Resistance Updates 5 (2002) 34–46

Emerging new therapies for chemotherapy—resistant cancer using adenoviral vectors John Nemunaitis, Casey Cunningham∗ US Oncology, Mary Crowley Medical Research Center, Baylor University Medical Center, 3535 Worth Street, Collins Building, 5th Floor, Dallas, TX 75246, USA Received 21 March 2002; received in revised form 14 May 2002; accepted 16 May 2002

Abstract The treatment of cancer by genetic manipulation of either the tumor itself or the patient as a whole offers new avenues for the treatment of otherwise refractory cancers. Gene therapy seeks to correct underlying genetic defects in malignant tissue or to augment the host defense response or to promote selectivity of other therapies. Many innovative and exciting genetic targets have been recently identified. However, the field as a whole is still constrained by limitations of gene delivery. The most common vector for gene delivery is modified adenovirus. In this review, we survey a sampling of current therapeutic approaches that depend upon adenoviral delivery vehicles and outline the advantages and disadvantages of this vector system. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Adenovirus; Viral vector; Gene therapy; Malignancy; p53; TNF; E1A; GMCSF

1. Introduction In the past 50 years, the development of cytotoxic chemotherapy has brought new hope to the treatment of cancer as once inaccessible sites of disease could now be addressed. For some patients, this has indeed meant the possibility of cure, but for still too many, chemotherapy remains palliative at best. One reason for this ultimately disappointing situation is the quick emergence of chemoresistant tumor cells. This resistance can take several forms, but commonly occurs because the cytotoxic is an external agent that depends upon cellular exposure to achieve its effects. Therefore, decreases in uptake or retention by the cell result in suboptimal exposure and lack of effect. Further, once mechanisms of resistance emerge, they are usually applicable to a wide variety of agents, resulting in highly refractory tumors. However, in the last several decades, better understanding of basic cellular processes of normal cells, and how those processes go awry in cancer, has yielded new therapeutic approaches to malignancy that may escape the limitations of chemotherapy virus (Borst et al., 2001; Schmitt and Lowe, 2001; Brown and Wouters, 2001; Roninson et al., 2001). To understand the new developments, recall cellular biology is built, for the most part, on the interactions of proteins ∗

Corresponding author. Tel.: +1-214-370-1870; fax: +1-214-370-1886. E-mail address: [email protected] (C. Cunningham).

with each other or with other cellular constituents. Genetic mutations can produce abnormally functioning proteins that allow the development of malignancy, as is the case in a large percentage of human cancers. The idea naturally arose then, that replacement of abnormal or missing proteins by introduction of normal copies of the affected gene might correct the malignant state. This was subsequently expanded to include the delivery of new genes that are not normally found in the tumor cells but produce proteins lethal to the malignant cells specifically. The goal of gene therapy—simple to state but difficult to achieve—is to introduce new genetic material into cancer cells that will selectively kill them with no toxicity to the surrounding non-malignant tissue. In the past decade, investigations into possible gene therapy approaches to cancer treatment have grown exponentially. There is no lack of available genes that affect cancer cell proliferation, apoptosis, immune surveillance or direct cytotoxicity to use as therapeutics. Clinical success has been limited by difficulties in accurately delivering these genes to malignant cells. The most common vehicles for gene delivery are viruses. Viruses can be viewed simply as a small number of genes (usually little more than is necessary to produce a new virus) surrounded by a protective protein coat. The protein coat allows transport into the cell where the viral genetic information can be produced using the host cell’s own translational machinery. In essence, viruses are constructed to introduce new genetic material into cells and hence should be suitable

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gene delivery vehicles. This has been indeed the case: herpes simplex virus (Geller et al., 1990), adeno-associated viruses (Kay et al., 2000), vaccinia virus (Hodge et al., 1994), lentivirus (Trono, 2000), Newcastle disease virus (Pecora et al., 2001), Reovirus (Coffey et al., 1998) and vesicular stomatitis virus (Stojdl et al., 2000) have been investigated, and all offer some advantages. However, the greatest clinical experience has been achieved with adenovirus and this review will concentrate primarily on this virus as a gene delivery vehicle.

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2.1. Advantages of adenoviruses as vectors Adenoviruses offer several advantages as gene delivery vehicles. In contrast to other viruses such as retrovirus, adenoviral DNA is not integrated into host genome and thus, does not affect germ lineage transmission. Further, since infection is not dependent on the cell cycle phase, both dormant and cycling cells can be infected with adenoviruses, thereby enhancing transduction. However, the choice of adenovirus as a vector brings with it several problems. 2.2. Limitations of adenoviruses as vectors

2. Adenoviral vectors Adenoviruses are double-stranded DNA viruses surrounded by an icosahedral protein capsid composed of hexon, penton and fiber components. Infection of a target cell begins by binding of the fiber knob region to a specific receptor. This is the coxsackie-adenovirus receptor (CAR) in the case of the serotypes most commonly used for gene delivery (Russell, 2000). After bindingof the knob receptor to CAR, an arginine–glycine–aspartic acid (RGD) motif of the penton base binds to particular ␣5 cellular integrins and leads to endosome formation and internalization. Adenoviral DNA is then transported to the nucleus where early viral protein synthesis begins (Table 1). If infection has occurred with wild-type, non-genetically altered, adenoviral particles, these early proteins take control of host cellular protein production and initiate viral replication, ultimately leading to host cell lysis and death. However, for gene delivery, most adenoviral delivery vehicles have been genetically crippled, usually by deletion of early E1 genes plus partial deletion of the E3 region, to ablate replicative capacity. Second generation vectors also include deletions of E2 and E4 regions and recently, ‘‘gutless’’ adenoviral vectors have been described which contain more dramatic E region deletions (Hammerschmidt, 1999). In these vectors, transgene expression follows nuclear transport.

2.2.1. Targeting of adenovirus First, although, CAR is expressed on most epithelial tissues, the expression of CAR on primary human tumor cells is variable (Dmitriev et al., 1998; Hemmi et al., 1998; Miller et al., 1998; Kasono et al., 1999; Li et al., 1999; Mori et al., 1999; Van der Kwaak et al., 1999; Dodson, 2000; Fechner et al., 2000; Heinicke, 2000; Kelly et al., 2000; Cripe et al., 2001) and there is some suggestion that CAR expression is actually down-modulated in advanced cancers (Okegawa et al., 2000), limiting delivery of the transgene to the tumor. To circumvent this problem, investigators have attempted several different strategies. One method is to alter the fiber knob portion of the capsid by inserting motifs with known binding to other cellular receptors, resulting in CAR-independent infection. Alternatively, the viral particles can be pre-treated with bivalent targeting complexes that connect adenoviral components to specific cancer receptors. Such bivalent complexes have already been developed to receptors for folate (Douglas et al., 1996), epidermal growth factor (Miller et al., 1998; Blackwell et al., 1999), EpCAM (Haisma et al., 1999), and fibroblast growth factor (FGF2) (Goldman et al., 1997). Preclinical trials using an anti-fiber-Fab FGF2 retargeting molecule which links adenovirus components to the FGF2 receptor on the malignant cell to facilitate delivery of a thymidine kinase gene did

Table 1 Adenoviral delivered genes in clinical development Gene

Stage

Reference

Immune modulatory IL-2 HLA-B7 GM-CSF

Clinical Clinical Clinical

Toloza et al., 1996 Gleich et al., 1998; Gleich et al., 2001 Simons et al., 1997; Soiffer et al., 1998; Nemunaitis et al., 2001

Prodrug HSV-TK Cytosine deaminase

Pre-clinical Pre-clinical

Wilson et al., 1996 Rogulski et al., 2000

Others TGF␣ Superoxide dismutase Bcl-2 E1A p53

Pre-clinical Pre-clinical Pre-clinical Pre-clinical/clinical Clinical

Laird et al., 1994; Grandis et al., 1998 Yan et al., 1996; Liu et al., 1997; Lam et al., 2000 Gibson et al., 2000 Deng et al., 1998/in progress Clayman et al., 1998; Clayman and Dreiling, 1999; Clayman et al., 1999; Nemunaitis et al., 2000

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demonstrate some activity with this approach (Rogers et al., 1997; Gu et al., 1999; Printz et al., 2000). This is particularly exciting as EGFR is highly expressed in most cancers (Kawamoto et al., 1991; Flotte et al., 1992; Stanton et al., 1994). Significantly, non-specific binding to normal organs was reduced in both approaches, thereby potentially minimizing any toxic effects (Gu et al., 1999; Printz et al., 2000). A related approach is to transduce unique tumor receptor genes, capable of binding and internalizing tumor toxic proteins. In a preclinical study, transfer of the IL-13 R␣2 chain gene was used to modulate this receptor’s binding of IL-13 (Blackwell et al., 1999). Animals were then treated with a linked IL-13 carrying a mutant form of pseudomonas endotoxin. Survival advantage was demonstrated in the IL-13 R␣2 receptor gene transduced cells (Blackwell et al., 1999). 2.2.2. Adenoviral clearance A second problem in adenoviral delivery of genes is that much of the clearance of adenoviruses involves non-specific uptake by Kupffer cells of the liver (Tao et al., 2001), creating a strong ‘‘first-pass’’effect that reduces the amount of virus exposure to tumor tissue. Transient blocking or depletion of Kupffer cells can improve uptake beyond the liver following intravenous administration (Alemany et al., 2000), a finding with potential clinical importance. 2.2.3. Antibody response Finally, infection with adenoviruses creates a strong humoral and cellular response against the virus and infected cells (Gahery-Segard et al., 1997; Molinier-Frenkel et al., 2000; Molinier-Frenkel et al., 2000). Although this may be associated with immune recognition against tumor antigens as a potential benefit, it also leads to enhanced viral clearance. 2.3. Safety Attention to the use of adenovirus as a gene delivery vehicle was intensified after the death of a patient at the University of Pennsylvania in a trial to correct ornithine-carboxylase deficiency syndrome by delivery of the OTC gene with an adenoviral vector (Balter, 2000). Since then, there has been assiduous study of adenoviral safety and likely toxicities. However, safety analysis of adenovirus dates back several decades. There are over 43 different serotypes of adenovirus of varying pathogenicity (Brandt et al., 1969). Serotypes 2 and particularly 5 are most frequently used as viral vectors due to their safety profile. Serotype 5 infection must occur commonly in nature as 80% of adults have existing antibodies to this type (Nicholson, 1993). However, less than 15% of exposed patients become clinically symptomatic. The most common symptoms are cough, gastroenteritis, conjunctivitis, cystitis, and rarely, pneumonia. Note that the infrequency of these symptoms includes even exposed immune-compromised patients (Hierholzer, 1992), reassuring when contemplating

use of adenoviral vectors in patients with advanced cancer. Finally, if serious viral infection does develop, there are possible therapeutic approaches. Wildtype adenovirus dissemination has been seen in organ transplant recipients but, in most cases, the viremia has been eliminated with the use of intravenous Ribavirine (Liles et al., 1993). Long- and short-term safety of adenoviral injection has also been shown in several animal models (Le Gal La Salle et al., 1993; Simon et al., 1993; Xu et al., 1997). A unique strain of cotton rats (Gigmodon hispidus) can develop pulmonary infection in response to inoculation with adenovirus serotype 5 (Pacini et al., 1984) with pathogenicity related to the dose of the viral inoculum. Additional safety testing has been conducted in mice and cotton rats in which high doses of adenovirus were injected locally and systemically. These animals developed minor histopathologic changes in several organs, but no pulmonary toxicity (Pacini et al., 1984; Ginsberg et al., 1991). However, inflammatory infiltrates related to the adenoviral vector have been observed in the lungs of animals given high doses of Adp53 directly to the bronchial airway (Ghosh-Choudhury et al., 1986; Engelhardt et al., 1993; Zhang et al., 1995). The resulting inflammatory responses were characterized by interstitial infiltration of neutrophils and monocytes within 1–2 days of exposure. This early inflammatory process may be mediated by local elaboration of various cytokines such as tumor necrosis factor (TNF), IL-1, and IL-6 (Printz et al., 2000). An additional inflammatory response can occur within 3– 7 days and consists of a peribronchial infiltration of lymphocytes. However, direct exposure of the lung to low concentrations of the adenovirus vector does not appear to be associated with pulmonary toxicity (Simon et al., 1993; Yei et al., 1994). The possibility of adenoviral replication competency developing after vector injection also appears to be negligible, given the construction design of the vector (Zhang et al., 1995). In humans, ␤-gal vector was injected into patients with endobronchial lung cancer. Evidence of replication competent adenovirus was studied in caretaker staff samples. No replication competent adenovirus was detected, and elevated antibody formation did not inhibit gene expression with repeat injections (Tursz et al., 1996). Nevertheless, complete inhibition of DNA replication solely from E1 deletion has not been 100% successful (Engelhardt et al., 1993; Rich et al., 1993), necessitating monitoring of the delivered expression cassette for replication competency. Theoretical concerns regarding oncogenicity of adenoviruses are unlikely to be realized in actual practice. The life cycle of an adenovirus does not require integration into the host genome, thus, foreign genes delivered by adenoviral vectors are expressed episomally and have low genotoxicity (Zhang et al., 1995). DNA samples from thousands of human tumors have also been analyzed for the presence of adenovirus DNA and no integrated viral DNA has been isolated from any human tumor (Green et al., 1979).

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3. Control of transgene expression Once delivered to malignant tissue, expression of the therapeutic gene is under the control of whatever DNA promoter was selected during contruction of the expression cassette. There are many different promoters and the choice of which to utilize to maximize gene expression often depends upon the target tissue and specific gene being delivered. Promoters can be broadly divided into those that continually encourage gene expression (constitutive promoters) and those that limit expression to particular situations. The latter limitation can be either that the promoter is only activated within certain malignant cells, or in the presence of unique gene abnormalities, or only in the presence of a particular agent (inducible promoters). For example, the MMTV promoter has increased activity when transfected cells are exposed to steroids (Lee et al., 1981; Shillitoe and Noonan, 2000). Reports have shown that the MMTV promoter can be used to control anti-bcl-2 ribozyme expression and p53 gene expression in cancer cells (Lee et al., 1981; Gibson et al., 2000). The MDR1 promoter has also been shown to have upregulated expression of downstream genes when combined with taxol/doxorubicin (Walther et al., 1997) and may be more active in malignant cells containing p53 mutations (Nguyen et al., 1994). Other promoters that have been tested include the promoter of Rous sarcoma virus promoter (Tursz et al., 1996), EF1␣ promoter (Thompson et al., 1990), promoters of keratin genes (Leask et al., 1990; Liu et al., 1999) and the p53 promoter of the adeno-associated virus (Flotte et al., 1992). The Egr-1 promoter, which is inducible by ionizing radiation can enhance release of downstream expression of the TNF gene following irradiation to the injected area and is currently undergoing clinical investigation.

4. Therapeutic genes 4.1. Immune modulatory approaches Almost by definition, harnessing the body’s innate immune system, with its ability to finely discriminate between normal and abnormal cells and destroy the latter, would fulfill the goal stated for gene therapy at the beginning of this review. Unfortunately, the very existence of patients with clinically significant tumor burden means that this system often fails. Interestingly, there are adequate tumor-specific antigens on most tumors, yet a decreased immune response to those antigens. This decreased immune recognition can occur for several reasons. For example, MHC class I and II antigens expressed by malignant and nonmalignant cells serve as restriction elements for T cell-mediated cytotoxicity (Houck et al., 1990). If downregulation of MHC class I or II antigen expression occurs, as it does in some tumor states (Prime et al., 1987; Esteban et al., 1990), the cancer cells can avoid T cell-mediated cytotoxicity (Mattijssen et al., 1991; Arosarena et al., 1999).

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Measures to increase immune recognition can be provided by the administration of additional ‘‘signals’’to alert the major immune recognition cells, the antigen-presenting cells (APCs) (Matzinger, 1994). These signals (called immunostimulants) include adjuvants, cytokines, and co-stimulatory molecules. While immunostimulants can be given directly, sustained administration is best achieved via transfer of the corresponding gene and thus are clearly an area of potential for gene therapy approaches. In the situation described above where downregulation of MHC class I or II antigens has occurred, gene transduction with IFN␥, known to upregulate MHC class I and II antigens in a variety of malignancies (Prime et al., 1987; Esteban et al., 1990), should allow greater immune recognition. This concept has been tested in early clinical trials where the IFN␥ gene was delivered in patients with melanoma via a retroviral vector and some antitumor activity noted (Nemunaitis et al., 1999). The delivery of immunostimulatory molecules can be (a) via direct injection into the tumor, (b) injection into immunologically important non-tumor sites, or (c) by removing tumor cells from the patient, transducing them in the lab, and then re-administering the transduced cells to the patient. 4.1.1. Direct injection One of the earliest approaches tested involved intratumoral injection of the adenoviral IL-2 gene (Toloza et al., 1996). Results were variable, possibly due to the finding that the IL-2 transgene product expression was dependent on CAR receptor density (Li et al., 1999). Other gene therapy approaches utilizing an HLA B7 (major histocompatibility complex) plasmid as an alloantigen to stimulate an antitumor response have also been completed. In one trial, nine patients with advanced HNSCC (HLA B7 non-expressive), intratumoral injection of an HLA B7 plasmid (Allovectin-7) was explored (Gleich et al., 1998). No toxic effects were observed. Four of nine patients achieved a partial local regional response and induction of HLA-B7 expression was confirmed in two of four patients who responded. These encouraging results were followed by two trials where a total of 60 patients with unresectable HNSCC who failed conventional treatment were treated with two intratumoral injections of 10 ␮g of the HLA B7 plasmid or 100 ␮g of the plasmid. Patients with stable or responsive disease during the first cycle of treatment were eligible for a second cycle. Thirty-three percent of the patients achieved stable disease or a partial response with the first cycle (6 weeks interval) (Gleich et al., 2001). After the second cycle, six patients had stable disease and four had a partial response, and one had a complete response. Responses persisted for 21–106 weeks. 4.1.2. Transduction of genes ex vivo Granulocyte monocyte-colony stimulating factor (GMCSF) is a particularly potent immune stimulatory molecule and even shows some activity when administered systemically. However, pre-clinical data clearly demonstrates that the best immune effects are seen when the GM-CSF protein

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is present adjacent to the target tumor antigens, is present in high concentrations, and is present for a prolonged period. One way of accomplishing these requirements is to transduce tumor cells with an adenoviral vector carrying the gene for GM-CSF. When done on autologous tumor cells ex vivo, the resultant modified tumor cells, now secreting GM-CSF, can then be irradiated for safety and used to vaccinate patients to stimulate an immune response against their tumor. The first human study of GM-CSF gene-transduced tumor vaccines was conducted in renal cell carcinoma. The vaccine was well tolerated and there were no dose-limiting toxicities (Simons et al., 1997). A phase I trial in melanoma also found good tolerability and some antitumor activity (Soiffer et al., 1998). In both trials, the injection sites of patients were infiltrated with T-cells, dendritic cells and macrophages, indicating the initiation of an immune response. A follow-up multicenter phase I/II trial of this approach in patients with either early or advanced stage non-small cell lung cancer (NSCLC) is also finding activity. Of the initial three patients who completed the whole course of six vaccinations and underwent radiologic restaging at week 12, one had a complete response, one had a mixed response and the third had stable disease (Nemunaitis et al., 2001). This trial has now completed and the data are being analyzed, however, the preliminary encouraging results have led to plans to expand this approach into several other tumor types. 4.2. Epidermal growth factor receptor (EGFR) Transforming growth factor alpha (TGF␣) is a polypeptide growth factor that binds exclusively to the epidermal growth factor receptor (EGFR) (Todaro and De Larco, 1976; Todaro et al., 1980). If either TGF␣, or particularly EGFR, is overexpressed in cells, an autocrine growth loop can be set up that promotes malignant transformation (Ghanem et al., 2001). This autocrine loop is dependent upon continued elevated levels of EGFR and the presence of TGF␣ (Di Marco et al., 1989). Overexpression of TGF␣ and EGFR is common in the malignant state (Grandis et al., 1996) where it is associated with poor prognosis (Kawamoto et al., 1991). One obvious strategy, then, is to down-modulate TGF␣ expression with an antisense expression construct targeting the TGF␣ gene. Such an approach is successful in cell line models (Laird et al., 1994; Grandis et al., 1998). In nude mice xenograft models, direct innoculation of the TGF␣ antisense construct into established HNSCC tumors also resulted in inhibition of tumor growth with antitumor activity observed for up to 1 year after treatments were discontinued. Down-modulation of TGF␣ was accompanied by increased apoptosis in vivo. These experiments suggested that interference with the TGF␣/EGFR autocrine signaling pathway may be an effective therapeutic strategy for cancers which overexpress this ligand receptor (Grandis et al., 1998).

4.3. Superoxide dismutase (SOD) gene Superoxide radicals are produced in a variety of intracellular reactions but are normally dispersed by the intracellular enzyme, superoxide dismutase (SOD) (Cerutti, 1985). A wide variety of human tumor cell lines display decreased expression of SOD (Oberly, 1982) that, if corrected, suppresses tumor cell proliferation (Church et al., 1993; Yan et al., 1996; Zhong et al., 1997). The exact mechanism of MnSOD growth suppression remains unclear but is apparently independent of necrosis or apoptosis. There is some suggestion that MnSOD transfectants accumulate in the G1 phase of the cell cycle (Lam et al., 2000), however, these results are preliminary. H2 O2 produced during dismutation of O2 , may also alter signal transduction pathways in cancer cells (Lo et al., 1996). In any event, the effects of increased SOD may also explain at least a part of the anti-tumor effect of cytokines such as tumor necrosis factor (TNF), IL-1 and interferon-␥ as these all induce MnSOD (Manna et al., 1998; Takahashi et al., 1998). In preclinical work, increases in SOD activity can be accomplished by the introduction of MnSOD cDNA into the cells. This approach has been tested in several cell lines, including simian virus-40 transformed human lung fibroblast cells (Yan et al., 1996) and human SCC-25 tumor cells (Liu et al., 1997). In xenografts models, MnSOD gene has been safely delivered by direct injection using an adenoviral delivery vehicle. In this study, expression of MnSOD gene in the injected tumors persisted for 10 days following delivery with evident tumor regression compared to untreated controls (Lam et al., 2000). Further exploration of MnSOD as a potential gene therapeutic certainly appears warranted. 4.4. Suicide gene approach Several cytotoxic drugs are available in an innocuous pro-drug form that can be readily converted into a toxic metabolite by specific metabolic enzymes. Suicide gene therapy involves the delivery of a gene encoding one of these enzymes into malignant tissue. The pro-drug is then given systemically but only metabolized to its active form in locations with adequate concentrations of the converting enzyme, i.e. the tumor. The result should be a high concentration of toxic product intratumorally, but little systemic toxicity. More than 20 suicide gene systems have been identified and tested in a variety of cancers (reviewed in Coukos and Rubin (2001)). Two of the most advanced towards clinical investigation are herpes simplex type I thymidine kinase (HSV-1 TK) using the antiviral prodrug ganciclovir and the Escherichia coli enzyme, cytosine deaminase (CD) that converts 5-fluorocytosine (5-FC) into the active 5-fluorouracil (5-FU). Preclinical studies with HSV-TK gene followed by Ganciclovir administration in HNSCC animal models confirm expression of the TK gene product with subsequent tumor

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regression (Cheng et al., 1983; Sewell et al., 1997; Thomas et al., 1998). Functionally, the HSV-TK gene product phosphorylates monophosphate ganciclovir to a triphosphate ganciclovir. Triphosphate ganciclovir causes cell death within the transfected cell and adjacent non-transfected cells (bystander effect) by interfering with DNA synthesis (Cheng et al., 1983). Among the vectors used to deliver the HSV-TK gene are non-replicating adenovirus, adeno-associated virus and conditionally replicating adenovirus (Wilson et al., 1996; Morris et al., 2000; Rogulski et al., 2000; Fukui et al., 2001). In preclinical assessment, the replicating adenoviral delivery vehicles show greater activity than non-replicating vectors (Wilson et al., 1996; Freytag et al., 1998; Morris et al., 2000). However, non-replicating adenovirus and adeno-associated virus are the only vectors that have been used in the clinic (Singh et al., 2001). Unfortunately, only limited activity has been seen to date, so further investigation is needed. Cytosine deaminase converts 5-FC to the toxic metabolite 5-FU (Calabresi and Chabner, 2002). Use of the CD/5-FC suicide gene system may be more relevant when using replicating adenoviral vectors since ganciclovir has antiviral activity that may adversely affect viral replication (Wilson et al., 1996). The CD/5-FC suicide gene system has been explored in a variety of cancers (Hirschowitz et al., 1995; Dong et al., 1996). Further, the CD/5-FC system is able to discriminate between normal and neoplastic squamous epithelial cells in a preclinical model, suggesting that it might have potential for the treatment of premalignant lesions (Sandalon et al., 2001). Fusion vectors combining both CD and TK genes demonstrate greater anti-tumor activity than either suicide gene alone in murine models (Rogulski et al., 2000), opening up a new area of exploration.

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4.6. E1A The interaction of E1A, a nuclear phosphoprotein with a wide range of cellular proteins in multiple signal transduction pathways (cell cycle, DNA damage, histone deacetylation) results in multiple biological activities. E1A was initially appreciated for its ability to repress transcription, leading to down regulation of HER-2/neu protein (Yu et al., 1990; Yan et al., 1991; Yu et al., 1991; Frisch and Dolter, 1995; Zhang et al., 1995) and the loss of malignant phenotypes. The anti-oncogenic activity of the E1A gene, however, is not limited to tumors that overexpress HER-2/neu. E1A also modulates expression of other genes, resulting in differentiation of certain cancer cells and enhancing the antitumor activity of VP16, cisplatin, paclitaxel and adriamycin (Frisch, 1991; Lowe et al., 1993; Ueno et al., 1997; Ueno et al., 2000). Preclinical studies also suggest a significant tumor radiosensitization response to E1A therapy (Sanchez-Prieto et al., 1996; Deng et al., 1998; Duque et al., 1999). Clinical investigations using cationic liposomes mixed with plasmid DNA encoding for E1A has shown safety and efficacy in animal models (Xing et al., 1998), as well as preliminary safety and activity in clinical trials. In one trial, 18 patients with either breast or ovarian cancer were treated with the E1A gene administered using a lipid complex (Hortobagyi et al., 2001). Fever, nausea and vomiting, and injection site pain were the major toxicities. and minimal toxicity observed. E1A transfection was confirmed and down regulation of HER-2/neu demonstrated in several patients who overexpressed HER-2/neu at baseline. Although clinical response was modest in this single agent trial, the combination of E1A gene with chemotherapy or radiation therapy is currently being investigated further. 4.7. p53 (Adp53)

4.5. Bcl-2 targeting In recent years, better understanding of the normal pathways of programmed cell death (apoptosis) has led to new therapeutic targets. Within cells are cascades of proor anti-apoptotic signals that tightly regulate normal cell turnover. At the center of the anti-apoptotic control is the protein Bcl-2 (Fujita and Tsuruo, 2000). Not surprisingly, overexpression of Bcl-2 is a risk factor for the development of malignancy and such overexpression occurs in many cancers (Jordan et al., 1996; Valassiadou et al., 1997; Stefanaki et al., 1998; Yao et al., 1999). However, the malignant phenotype can be reversed with inhibition of Bcl-2 expression (Cotter et al., 1994). One way to accomplish this is by use of a hammer-head ribozyme designed to cleave the Bcl-2 transcript. Such a ribozyme, cleaving after nucleotide 279, was recently tested and shown to have activity that correlated with reduction in Bcl-2 protein 24 h after infection with a ribozyme-expressing adenovirus vector in several oral cancer cell lines (Gibson et al., 2000).

The most frequent mutation in human cancer involves the coding sequence for the p53 tumor suppressor gene (Baker et al., 1990). The majority of tumors contain a mutant or dysfunctional p53 gene product. Functions of the p53 gene product include upregulation of p21, a protein that inhibits cyclin-dependent kinase (CDK), and is necessary for G1 to S phase transition. The p53 protein also upregulates Bax (a positive regulator of apoptosis), MDM2 (a negative regulator of p53 function), thrombospondin-1 (inhibitor of angiogenesis), GADD45 (facilitates DNA repair) and IGF-BP3 (growth regulator)(Harper et al., 1993; Dameron et al., 1994; Miyashita and Reed, 1995). Extensive analysis of tumors showing p53 dysfunction indicates that abnormal function correlates with poor prognosis (Horio et al., 1993; Thorlacius et al., 1993; Lai et al., 1995; Preudhomme and Fenaux, 1997). The p53 is not essential for normal development, however, p53 ‘‘knock out’’mice are susceptible to tumors earlier in life. Preclinical studies have reported the introduction of wild type p53 gene into human tumor cells with a mutant p53 genotype using a variety of delivery

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mechanisms, including retroviral vectors, lipid complexes, and adenoviral vectors (Wills et al., 1994; Zhang et al., 1995; Blagosklonny and El-Deiry, 1996; Nguyen et al., 1996). Results demonstrate that expression of the transgene product provides a normal functioning wild type p53 protein that induces tumor regression and improve survival in animal models. Preclinical studies also reveal enhanced efficacy when combined with chemotherapy or radiation therapy (Nguyen et al., 1994; Nielsen et al., 1998). The first published trial to explore gene transduction of the p53 gene via intratumor injection in humans utilized a retroviral vector (Roth et al., 1996). In this trial, nine patients with NSCLC containing a p53 mutation were treated with four patients receiving the p53 gene via bronchoscopic injection, and five patients treated via a percutaneous injection with CT guidance. Vector transduction was confirmed in eight patients by PCR analysis, and six patients showed induction of apoptosis by TUNEL assay. Three of the patients who received endobronchial injections had tumor regression of ≥50%. No toxic effects were attributed to the vector. Retroviral sequences were not detected in non-injected pulmonary tissue analyzed by PCR, and there was no evidence of replication competent retrovirus. However, the low transduction efficiency associated with retroviral vectors was a major limiting factor. Several studies with adenoviral-delivered p53 (Adp53) were subsequently initiated in NSCLC and HNSCC (Clayman et al., 1998; Nemunaitis et al., 2000). One Phase I trial investigating the tolerability of Adp53 in advanced NSCLC studied 52 patients who had previously failed conventional treatment (Nemunaitis et al., 2000). Adp53 doses were escalated from 106 to 1011 PFU and injected monthly into a single primary or metastatic tumor via bronchoscopy (12 patients) or computed tomographic (CT) guidance (40 patients). Patients were then randomized to combination with or without cisplatin (80 mg/m2 ) given IV over 2 h prior to Adp53 injection. Vector-specific deoxyribonucleic acid (DNA) was detected and p53 transgene expression confirmed by RT-PCR in tumor biopsies collected 3 days post-treatment from 43% of evaluable patients overall. However, persistent p53 transgene expression was dose-dependent, being seen in 10 of 17 (58%) patients receiving vector dose levels ≥3 × 1010 PFU, but only 8 of 27 (30%) patients who received the lower dose level. In 11 of 15 patients whose tumors could be assayed, there was in increased number of apoptotic cells. Despite being intratumorally injected, there was clearly systemic vector exposure. Anti-adenoviral type 5 IgG antibody response (≥2-fold increase) was shown in 19 of 20 evaluable patients following course 1 and cytopathic effect assays revealed the presence of Adp53 vector in plasma within 30 min of intratumor injection in all 16 patients tested. Nevertheless, toxicity attributed specifically to the vector was limited to transient fever. Cisplatin-related toxicity was also not increased. Finally, four patients fulfilled a definition of partial response (PR) (8%), 33 patients (64%) experienced stable

disease, 11 patients (20%) had progressive disease, and 4 patients (8%) were not evaluable for response. The above results then supported phase I investigation in HNSCC (Clayman et al., 1998; Clayman and Dreiling, 1999; Clayman et al., 1999) in which patients with recurrent or refractory HNSCC who had either surgically resectable lesion or unresectable lesions were analyzed as two cohorts. Results were that repeated intratumoral injections of up 1011 PFU were safe and well tolerated and transgene expression occurred despite evidence of adenovirus antibody response. Peri- and post-operative Adp53 injection had no adverse effect on surgical morbidity and/or wound healing. In the unresectable patient group, 2 of 17 achieved a partial response to Adp53 intratumoral injection, 6 achieved stable disease for 1–3.5 months, and 9 had progressive disease. Fifteen patients with recurrent disease were in group one, and underwent surgical resection. Historically, patients with recurrent disease who undergo a second surgical resection have a poor survival but 4 of the 15 Adp53 injected patients (27%) remained disease free with a median follow up time of 18.25 months. This approach is now in phase III trials. 4.8. When the vector is the therapy: Onyx-015 Adenoviruses have evolved sophisticated mechanisms for evading normal cellular defenses such as the p53 tumor suppressor system. One of the main viral protections is afforded by the 55 kDa product of the adenoviral E1B gene that inhibits normal p53 function. Therefore, viruses deficient in E1B product are at severe disadvantage in normal cells but should replicate normally in p53-defective cells, which describes about 50% of all human tumor cells. The ONYX-015 (dl1520) virus is a DNA adenovirus constructed with an E1B gene deleted region that indeed demonstrates replicative capacity leading to necrosis in malignant tissue with abnormal p53 function (Bischoff et al., 1996). ONYX-015 does not deliver anticancer genes, but is being discussed as a potential gene therapy because it may one day be used as a cancer selective gene delivery vehicle for intravenous delivery (Nemunaitis et al., 2001). ONYX-015 has cytopathic effects in a wide variety of malignant cells that have abnormal p53 function (Heise et al., 1997), although there continues to be controversy over the mechanism of selectivity (Oliner et al., 1992; Goodrum and Ornelles, 1997; Hall et al., 1998) as even some malignant cells with wildtype p53 genotype are sensitive to Onyx 015, particularly when combined with cytotoxic chemotherapy (Heise et al., 1997; Khuri et al., 2000; You et al., 2000). More basic research is clearly needed in this area. In animal human xenografts studies, intratumoral injection of ONYX-015 virus suppressed tumor growth of HLaC laryngeal carcinoma cells, which have a p53 gene defect (Heise et al., 1997) but did not affect normal cell growth. This translated into improved median survival in the injected animals, an effect that was enhanced by the addition of chemotherapy (Heise et al., 1997; You et al., 2000).

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Such results encouraged several clinical trials with ONYX-015 among which are direct injection studies in patients with HNSCC both as a single agent (Ganly et al., 2000; Nemunaitis et al., 2000; Nemunaitis et al., 2001) and in conjunction with chemotherapy in recurrent HNSCC patients (Nemunaitis et al., 2000). In the first study, 30 refractory HNSCC patients who had unresectable tumor, which had progressed on chemotherapy or radiation therapy were treated with a dose of 1 × 1010 PFU of ONYX-015 daily × 5 days every 3 weeks via intratumor injection. Results were compared to 10 similar patients who received a more prolonged schedule (2 weeks) at the same dose administered twice a day rather than once a day. Pain at the injection site and fever were common, occurring more frequently in patients receiving the more frequent dose schedule. Overall, a total of 533 intratumoral viral injections were administered to 40 patients. Thirty-six were evaluable for response. Five patients achieved a 50% or greater reduction in bidimensional measurements (2 PR, 3 CR) of the injected lesion. Quantitative PCR was used to assess systemic distribution following intratumoral injection. Twelve of 29 assesssed patients had detectable viral genome in the blood 24 h after the fifth injection in cycle 1. Two (9%) patients showed persistent genome detected at 10 days. No samples were positive at 22 days. Correlation of response was linked to abnormal p53 status (Nemunaitis et al., 2000, 2001). A subsequent study explored ONYX-015 virus (1 × 1010 PFU daily × 5 days every 3 weeks) combined with chemotherapy (cisplatin 100 mg/m2 , IV on day 1; and 5-FU 800–1000 mg/m2 by continuous infusion per day on days 1–5 every 3 weeks) (Khuri et al., 2000). Thirty patients with recurrent HNSCC who had not previously been exposed to chemotherapy or radiotherapy in the recurrent tumor setting were entered into trial. The toxicity profile for ONYX-015 was similar to phase II results with ONYX-015 alone. No additional toxicity to chemotherapy was observed. Twenty-six patients were evaluable for response, 16 (63%) achieved a partial or complete response of the injected lesion. Historically, the expected activity of similar patients receiving the same chemotherapy regimen is a 30–40% partial or complete response rate (Paredes et al., 1988; Forastiere et al., 1992; Jacobs et al., 1992; Clavel et al., 1994). Interestingly, both p53 normal and mutant genotypes were found to be responsive to the combined ONYX-015/chemotherapy approach.

5. Conclusions and future directions One of the goals of cancer therapy is to maximally affect the tumor tissue while minimally affecting the normal tissue. The approaches outlined in this review attempt to do this generally by one of two approaches. The first is that the therapy can be generally toxic to cells but constructed to have selective delivery to the tumor. The other is that the therapy has general delivery but is selectively toxic to malignant

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cells. Each of these approaches has merit and a history of some success. However, the future may lie in a ‘‘combined’’ attack where selectively toxic agents are selectively delivered to the tumor. The use of one of the conditionally replicating adenoviruses as a delivery vehicle for a gene under the control of a cancer-specific promoter would be a powerful weapon. As these modalities approach clinical investigation, and our experience and confidence builds with current gene/vector constructs for local regional approaches, it is likely that investigation of systemic gene delivery will become a reality. The gene therapy field is severely limited by the inability to deliver sufficient concentrations of gene vectors to malignant tissues via a systemic route. However, despite limited experience, an enticing argument can be made for the use of conditional replicating viruses to deliver genes which have already demonstrated efficacy through local regional (intratumoral injection) approaches in HNSCC. Furthermore, safety, and access to metastatic disease sites in refractory cancer patients has been demonstrated with intravenous administration. Conditional replicating adenoviral delivery vehicles could theoretically be further enhanced in activity with the addition of tumor-specific promoters within the gene construct and engineering of surface ligands independent of CAR which would have selective binding to malignant tissue. For the immediate future, we remain hopeful that one or several gene therapy modalities for local regional management will demonstrate activity justifying approval by the FDA in the next 3–5 years. Specific approaches furthest along in clinical development in advanced cancer include Adp53, HLA-B7 genes and ONYX-015 virus. References Alemany, R., Suzuki, K., Curiel, D.T., 2000. Blood clearance rates of adenovirus type 5 in mice. J. Gen. Virol. 81, 2605–2609. Arosarena, O.A., Baranwal, S., Strome, S., Wolf, G.T., Krauss, J.C., Bradford, C.R., Carey, T.E., 1999. Expression of major histocompatibility complex antigens in squamous cell carcinomas of the head and neck: effects of interferon gene transfer. Otolaryngol. Head Neck Surg. 120, 665–671. Baker, S.J., Markowitz, S., Fearon, E.R., Willson, J.K., Vogelstein, B., 1990. Suppression of human colorectal carcinoma cell growth by wildtype p53. Science 249, 912–915. Balter, M., 2000. Gene therapy on trial. Science 288, 951–957. Bischoff, J.R., Kirn, D.H., Williams, A., Heise, C., Horn, S., Muna, M., Ng, L., Nye, J.A., Sampson-Johannes, A., Fattaey, A., McCormick, F., 1996. An adenovirus mutant that replicates selectively in p53-deficient human tumor cells. Science 274, 373–376. Blackwell, J.L., Miller, C.R., Douglas, J.T., Li, H., Reynolds, P.N., Carroll, W.R., Peters, G.E., Strong, T.V., Curiel, D.T., 1999. Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma. Arch. Otolaryngol. Head Neck Surg. 125, 856–863. Blagosklonny, M.V., El-Deiry, W.S., 1996. In vitro evaluation of a p53expressing adenovirus as an anti-cancer drug. Int. J. Cancer 67, 386– 392. Borst, P., Borst, J., Smets, L.A., 2001. Does resistance to apoptosis affect clinical response to antitumor drugs? Drug Resist. Updates 4, 129–131. Brandt, C.D., Kim, H.W., Vargosko, A.J., Jeffries, B.C., Arrobio, J.O., Rindge, B., Parrott, R.H., Chanock, R.M., 1969. Infections in 18,000 infants and children in a controlled study of respiratory tract disease.

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