Cancer selective adenoviruses

Molecular Aspects of Medicine 28 (2007) 42–58 www.elsevier.com/locate/mam

Review

Cancer selective adenoviruses Ramon Alemany

*

Virus Therapy Group, Translational Research Laboratory, Institut Catala` d’Oncologia, Barcelona, Spain Hospital Duran i Reynals, Gran Via s/n Km 2.7, L’Hospitalet, 08907 Barcelona, Spain Received 15 December 2006; accepted 18 December 2006

Abstract Ten years ago Frank McCormick proposed dl1520 as an oncolytic adenovirus. Although great as an inspiration for better oncolytic viruses it was far from a good product. As Onyx-015, it underwent a wish-fulfilling clinical development program seizing the opportunity left by its p53-targeted non-replicative counterpart Ad-p53. Now, facing a skeptical environment, more selective and potent oncolytic adenoviruses await their clinical opportunity. However, advance in key issues remains elusive, such as, selectivity or retargeting at the level of cell receptors to improve pharmacokinetics. Preclinical models and a few clinical data on biodistribution show that only a minimal proportion of the injected dose reaches the tumors after systemic administration. Once in the tumor, the virus must overcome barriers to efficient spread imposed by stroma and immune responses. Arming the oncolytic virus with transgenes is a natural combination of virotherapy and gene therapy strategies. Transgenes that increase virus production or cellular spread may help to overcome these barriers. Cytotoxic transgenes can help to eliminate tumor cells but need to be compatible with efficient virus replication. These challenges require a careful approach to clinical development and a great deal of collaboration to launch clinical tests with a virus backbone that contains intellectual property from multiple sources.  2007 Elsevier Ltd. All rights reserved. Keywords: Oncolytic viruses; Adenovirus; Virotherapy; Gene therapy

* Address: Hospital Duran i Reynals, Gran Via s/n Km 2.7, L’Hospitalet, 08907 Barcelona, Spain. Tel.: +34 932607462. E-mail address: [email protected]

0098-2997/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2006.12.002

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Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . The case of dl1520 or Onyx-015 The case of CV706. . . . . . . . . . A current model backbone . . . . Arming the backbone. . . . . . . . Working together. . . . . . . . . . . Acknowledgements . . . . . . . . . References. . . . . . . . . . . . . . . .

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1. Introduction For oncologists viruses do not have the appeal of drugs. Viruses cause diseases and drugs cure them. More selective drugs are available in oncology with prospects of high efficacy and low toxicity. A drug is smaller and much more simple than a virus and intuitively it appears less toxic. However there are two things that drugs can not do. First drugs can inhibit an active target but cannot detect or replace the missing function of a protein. That is, they do not act upon the absence of a target. In cancer, tumor suppressors are missing targets central to the progression of the disease. Inactivation of oncogenes is seldom enough to stop cancer progression. Second, a drug does not amplify itself. This means that a very high concentration is needed to reach every tumor cell. At such concentration toxicity to tissues that proliferate such as bone marrow is difficult to avoid. This shortcoming of conventional drugs have prompted a few oncologists to seek for the magic bullet of immunotherapy. The immune response effector cells should amplify themselves until all the target tumor cells are destroyed. However tumors evolve in the presence of these cells and hence they are selected to evade them. Despite the complexity of a virus, virotherapy is a rational approach to circumvent these limitations. Virus therapy or virotherapy of cancer is an old theme. Almost every virus has been injected into humans in an attempt to treat cancer since early clinical observations of spontaneous cures after viral infections. However a new age of virotherapy was initiated by Frank McCormick proposing the use of dl1520 for cancer treatment. Previously virotherapy went through different phases. Initially all viruses that propagated in tumors were used. Then research focused on a few viruses that have a natural tropism for cancer cells. This tropism was associated mainly to the property of tumor cells to allow the continuous propagation of IFN-sensitive viruses, such as Newcastle Disease Virus. Genetic engineering techniques and a better understanding of how viruses use cell metabolism, allowed the rational design of oncolytic viruses. Pioneers crippled nucleotide metabolism genes of herpes simplex virus to achieve a certain level of tumor-selective replication (Martuza et al., 1991). However, McCormick’s proposal improved the prospects of selectivity and efficacy because the virus modifications targeted the genetic defects that cause cancer (Bischoff et al., 1996). Unfortunately, dl1520 was not only taken as a great conceptual leap to inspire more

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selective oncolytic viruses but as a product itself, with high expectations of clinical success backed by daring investments.

2. The case of dl1520 or Onyx-015 Adenovirus early proteins have several functions aimed to activate the S-phase of the cell cycle (Fig. 1). The main control is at the level of E2F release from the E2FpRb complex that occurs when E1a 12s and 13s proteins bind to pRB. The released E2F can activate the transcription of cyclin E, cyclin A and other cell cycle progression genes. p53 could counterbalance this activation inducing the expression of p21, and inhibitor of cyclin E and cyclin A dependent kinases. To avoid this cellular counterbalance mechanism, E1B-55K forms a complex with E4orf6 and p53 that leads to the ubiquitin-mediated proteolysis of p53. dl1520 is an adenovirus mutant lacking functional E1b-55K described by Berk (Barker and Berk, 1987). Frank McCormick proposed that this mutant could be used as an oncolytic virus as it should depend on the absence of p53 to propagate. Leaving aside the geniality of the idea, from a virology standpoint the theoretical selectivity of dl1520 for cells without p53 function was a simplification. During the late phase E1B-55K forms a different complex with E4orf6 that shuts off host mRNA nuclear export and host protein synthesis. It also ML E1a 0

E3 1

2

3

4

5 E2

E1a-12s E1a-13s

E1b19

8

E2A

9

10

m

E4

p5 p21

Cell Cycle

7

E1b55K E4-orf6

pRB Free

6

Bax Apoptosis

Fig. 1. Adenovirus transcription units and main functions related to cell cycle. Each unit is transcribed from a different promoter and yields a pre-mRNA that is processed by differential splicing to produce several proteins. Major late unit transcription is activated after the replication of the genome. The conventional map of adenovirus is divided in 100 map units rightwards (1 mu = 0.36 Kb). Below main virus proteins relevant to cell cycle control are shown. Further below the interaction with cellular proteins and their effect is shown. Adenovirus inhibition of pRB and p53 leads to release of free E2F, induction of cell cycle and inhibition of apoptosis.

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facilitates the nuclear localization of transcription factor YB1 to activate the E2 late promoter (Holm et al., 2002). Tumor cells often have inactivated p53, counteract protein shut off and contain nuclear YB1. Defects of 55K are then complemented accordingly. In general, however, the replication efficiency of 55 K mutant seldom reaches the level of Adwt (Heise et al., 2000; Howe et al., 2000). Nevertheless the idea of using dl1520 (soon after known with the product name of Onyx-015) as a selective replicating adenovirus has to be seen in the context of other p53-targeted adenoviruses that do not replicate at all. Cancer gene therapy with adenovirus vectors containing p53, as well as with vectors with prodrug activating genes, clearly showed a limitation at the level of the amount of cells that could be reached even when injecting tumors directly and repeatedly. In cancer patients, those vectors had shown a good safety track and the real problem was efficacy. With this scenario Onyx-015 moved rapidly to the clinic. Eighteen phase I and II clinical trials have been done where the administration route evolve from intratumoral to systemic and the severity of the disease from terminal to premalignant lesions (Aghi and Martuza, 2005). Initial trials included patients with head and neck tumors with bad prognosis and accessible lesions. Dose escalated to 2 · 1012 vp (1011 pfu) daily for five consecutive days (Ganly et al., 2000). Replication was demonstrated in tumor biopsies at day 8 but not at day 22 after the last injection. It was well tolerated with only mild flu-like symptoms (fever, nausea and chills) without hematological and biochemical alterations. No objective responses were observed. Next, intratumoral Onyx-015 was combined with systemic chemotherapy for head an neck tumors in a phase II trial (Khuri et al., 2000; Lamont et al., 2000). The response rate was very encouraging, with 19 out of 30 injected tumors decreasing 50% or more its size, 8 of them to complete response but long-term survival did not seem to improve. Then other tumor types of bad prognosis as pancreatic carcinoma and glioblastoma were injected with similar doses of Onyx015 (Chiocca et al., 2004; Hecht et al., 2003; Mulvihill et al., 2001). In pancreatic tumors injection up to 2 · 1012 vp gave the characteristic flu-like symptoms but no objective responses were observed. No toxicity and no efficacy was concluded as well from another phase I/II intratumoral injection in pancreatic tumors combined with gemcitabine with doses up to 2 · 1011 vp. In glioblastoma up to 1010 vp were injected at the margins of the cavity left at surgery. No adverse events were reported and the low toxicity did not allow to find a maximum tolerated dose but no efficacy was found. After intratumoral administration, trials of intracavital administration began. Vasey et al. used intraperitoneal injection to target peritoneal metastases of ovarian carcinoma (Vasey et al., 2002). At the highest dose of 1011 pfu daily for five consecutive days, more toxicity compared to intratumoral administration was noted with transient fevers, nauseas, emesis, head and abdominal pains. No objective responses were observed. Mouth wash with up to 1011 pfu for five consecutive days of Onyx015 were used to treat oral tumors (Rudin et al., 2003). Occasional transient responses were observed with no toxicity. The lack of toxicity led to intravascular administration. The first trial was performed in patients with hepatic metastases of colorectal carcinoma (Reid et al., 2001). The hepatic artery was infused with up to 2 · 1012 vp at days 1, 8, 22, 50 and 78 of treatment (accumulated dose of 1013 vp). Even then, a maximum

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tolerated dose was not reached. Common dose-dependent adverse events were flu-like symptoms (fever, myalgia, asthenia, fatigue, and chills). Careful attention to hepatotoxicity and coagulopathy revealed no changes. Efficacy was occasional and transient. Later, similar results were found in a phase II with the same dose (1013 vp) combined with 5FU and leucovorin (Reid et al., 2002). Neumunaits et al. injected Onyx015 iv in patients with lung metastases (Nemunaitis et al., 2001). This has been the trial in which a highest dose has been injected with one patient receiving up to 2 · 1013 vp at day 1 followed by 11 weekly doses of 2 · 1012 vp (accumulated dose of 4.2 · 1013 vp). Dose limiting toxicity was not reached but there were dose dependent fevers, vomits, nauseas, and chills. Following doses above 2 · 1012 vp transaminitis was detected. No objective responses were seen. In a phase II, Habib et al. injected three doses of 3 · 1011 pfu in three consecutive days via hepatic artery in patients with colon cancer metastases to the liver (Habib et al., 2002). Liver function and hematology were normal. Responses were negative. In a similar phase II with 18 colon cancer patients with liver metastases Hamid et al. injected iv 2 · 1012 vp at days 1 and 15 of 28-days cycles for six cycles (Hamid et al., 2003). Most patients (83%) had flu-like symptoms (fever, fatigue, chills). No responses were seen. A death related to disease progression allowed a biodistribution study of the virus. Most virus at 56 h post-injection was found in spleen and liver hepatocytes, somewhat confirming the biodistribution seen in murine models. The amount of virus in the tumors was 20-fold lower than in the liver. In general, clinical data with Onyx015 point to the need of more potent and selective viruses. In this regard two phase I trials have been finished with a derivative of Ony015 that expresses cytosine deaminase and thymidine kinase. Up to 1012 vp of this virus, Ad5-CD/Tkrep, were injected intratumorally in prostate tumors followed by systemic prodrug administration (Freytag et al., 2002, 2003). Dose limiting toxicity was not found. The most common adverse events were a rise in creatine phophokinase (83%), lymphopenia (53%) haematuria (56%), anemia (44%), and flu symptoms (38%). However, despite a transient drop in PSA levels no objective long term responses were observed. Despite the minor efficacy observed in all the phase I/II trials with Onyx-015 it may surprise that in China Sunway Biotech is ready to market its Onyx-015 version named H101. Besides lacking E1b-55K, H101 also lacks all E3 proteins, including ADP, which renders it less potent and more immunogenic, traits that may skew the therapeutic mechanism towards immunotherapy. H101 is intended for intratumoral injection of head and neck and other types of accessible solid tumors in combination with chemotherapy. In a phase II trial with 50 patients and a phase III with 123 patients the response rate doubled when H101 was added to the chemotherapy (Lu et al., 2004; Xia et al., 2004). The virus dose, chemotherapy regimen (cisplatin and 5FU), and injection procedure were very similar to the trials performed in USA (Khuri et al., 2000; Lamont et al., 2000). The results were also very similar with 83% of injected tumors responding in the virus plus chemotherapy group compared to a historical 30–40% tumor response rates with chemotherapy. Time to tumor progression also improved but survival rate have not been clearly assessed. With these similar results, the approval of H101 by the China’s State Food and Drug Administration and the rejection of Onyx-015 by the FDA is a matter of policy. Lowering

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the bar to get approval will allow to test more products in more patients but these patients and the public health systems should understand the proven risk-benefit of the product they are buying. A similar case can be drawn with Ad-p53 vector, trademarked Advexin in USA and Gendicine in China.

3. The case of CV706 The trend initiated with Onyx-015 was soon spurred with CV706. CV706 is an adenovirus 5 (Ad5) that contains the enhancer and promoter of the prostate-specific antigen (PSA) gene upstream of the E1a adenovirus gene (Rodriguez et al., 1997). It is the first adenovirus designed for virotherapy of cancer. E1a is the first gene expressed from the adenovirus genome and controls the expression of other virus genes. The control of E1a under the PSA promoter results in a virus that is only turned on in PSA-expressing cells. The idea of using tissue or tumor-selective promoters to target the expression of toxic genes or prodrug converting genes had been established in replication defective adenovirus vectors. The shortcoming was clear: a few cells became transduced and eliminated in vivo. With CV706, Daniel Henderson at Calydon Inc. pioneered the transfer of this concept to oncolytic adenoviruses. CV706 has a much narrower application than Onyx-015 but it has an important conceptual advantage: no virus gene should be expressed in normal cells. This is crucial for a virus intended for systemic administration because many hepatocytes will be transduced and E1a is toxic for them. Soon followed several improved versions of CV706 and oncolytic adenoviruses with other tumor-selective promoters regulating E1a and other early virus genes such as E1b, E4 and E2. The translation to the clinic of CV706 was also very quick. Intratumoral injection of up to 1013 pfu was well tolerated with mild fever and pain at the injection site (DeWeese et al., 2001). PSA level decreased 50% in 25% of patients, all of them in the highest dose groups but the response was transient. CV716 was an improved, more selective version of CV706 where the PSA enhancer and promoter controlled E1a and E1b. Likewise, CV740 used another two androgen-dependent prostate-specific promoters from the rat probasin gene. However, the repetition of these promoter sequences proved unstable upon propagation. To solve it, two different promoters were used. CV787 has E1a under the probasin promoter and E1b under the PSA promoter. In addition, the E3 genes, deleted in previous Calydon viruses, were included as their role in virus release became clear. This virus, renamed CG7870 after Calydon was acquired by Cell Genesys, has the proper potency and selectivity to test the clinical benefit of the new age of virotherapy. The phase I clinical trial of systemic administration of CG7870 had a very good design to begin this path (Small et al., 2006). An escalating dose of 1010 to 6 · 1012 vp was given in a single injection. Flu-like toxicity was common. A thorough analysis of hepatic and hematological toxicity showed grade I–II hypotension at doses over 6 · 1011 vp. Grade 1–2 transaminitis was noted over 1012 vp. Lymphopenia (grade 2–3) at all doses and thrombocytopenia (grade 1) over a 1011 vp. Although no coagulopathy was observed, D dimmer level increased 20% without dose correlation

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Table 1 Insertions and deletions to be considered in a current adenovirus backbone for optimal oncolysis. The effect of the different modifications on the selectivity and potency are explained. Examples are given for each modification Deletions E1a –CR1 single mutants are barely selective (e.g. dl1101). –CR2 single mutants are not very selective in normal dividing cells (e.g. Add24TyrE4). Potency preserved. Good for combination with E2F promoter. –CR1/2 is more selective and less hepatotoxic but 5-fold less potent (e.g. dl01/07). –Nterm/CR2 seems most selective (e.g. dl4-25/121-128), potency? E1b –d19K:Increased potency (e.g. CG8840). Conditional to apoptosis resistance? E4orf6/7: Did not increased the selectivity (e.g. Onyx411 with this deletion). VA-RNAs: Complemented by translational enhancement (e.g. AdVAdel). Better for RNAi? Insertions Promoters Range of tumor types Narrow: AFP (HCC), DF3/MUc1 and pS2 (Breast), Tcf and CEA (Colon), Tyr (Melanoma), OC, hK2, probasin and PSA (Prostate), hUP II and L-plastin (Bladder), SLPI (Lung) Medium:Cox2, Midkine Wide: E2F1, hTert (T255-4DEB) , HIF (hypoxic cells). Modular Small, defined regulation, preserve virus genome (HIF sites, Tcf sites, Ebox). Tightness Controlled by activation, repression or activation and repression: (E2F1, hTERT) Insulation: Relocation of Packaging signal, Insulators: polyA, HS4, DM1 Orientation: E1 cassette: Left to right better than reverse (Cox2). Location In E4 can reduce replication in permissive cells (e.g. CV757). In E2 there is interference from E3 promoter. Combinations E1a and E1b: Same promoter: PSA (e.g. CV716), AFP (e.g. 733) and PB (e.g. CV740) . 100fold better than one promoter but unstable. Linked by IRES: AFP (e.g.CG8900), SI 1E5. uPII (e.g. CG8840) Different Promoters: PB and PSE (e.g. CV739) and PSE and hK2 (e.g. CV764). Stable. SI: 1E4. E1a and E4: E2F1 (e.g. Onyx411) more selective but unstable, Ad2xTyr (more selective but less potent), tcf (e.g vH6), E2F/hTert (e.g. OVA002), E2F1/hTert, W at right (e.g. OAS403) 100-fold more selective than E2F-E1a but less potent and unstable. Tumor targeting/liver detargeting Detargeting: Fiber shaft KKTK or Y477A + TAYT + HI-loop-extended knob Targeting: K20, RGD Replacement: Ad5/3, 5/35, 5/40s Transgenes Pro-apoptotic genes: Adenovirus ADP , p53 Prodrug-activating genes: TK, CD. Proteases: Relaxin Fusogenic proteins: GALV. Immune stimulatory genes: TNF, GMCSF, MCP3, IL24, etc.

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indicating that this is a symptom to be monitored as well. Cytokine levels in blood indicated a rise of IL6 and IL10 at highest doses. With regard to efficacy, PSA levels transiently dropped (25–49%) in 3 out of 8 highest-dosed patients. A neutralizing antibody response in all patients could explain the short duration of the response, and transient immune suppression was recommended (Table 1).

4. A current model backbone Onyx-015 and CV716 have inspired a myriad of novel oncolytic adenoviruses. The former represents the idea of deleting virus functions that are dispensable in tumors. The latter represents the idea of inserting exogenous DNA sequences to make an oncolytic vector. A classification of these alternatives as type 1 and type 2 conditionally replicative viruses has been proposed but it is clear that a proper oncolytic virus will embody a combination of deletions and insertions (Fig. 2). Among deletions, I have already mentioned that the deletion of E1b-55K found in Onyx-015 has the caveat that many tumors will not complement efficiently the defect in RNA transport and the nuclear translocation of YB1. A mutant that only affects the binding site of p53 would preserve the potency in p53-defective tumors but the overlap of these functions in E1b-55K hampers this task. Another deletion of interest is the E1b-19K. This protein inhibits apoptosis at the early phase of adenovirus infection. As apoptosis resistance is a common trait in cancer, E1B-19Kd can be phenotypically complemented in cancer cells and E1b-19K defective mutants can be used as oncolytic viruses (Duque et al., 1998). E1b-19K mutants were first analyzed

Relocate

Ψ E1a E1b

VA E2

E3

Fiber

E4

PolyA Insulator Add

Promoter Promoter

Promoter Overexpression ADP

Promoter Trans gene

Tumor targeting Liver-detargeting Delete

Nterm CR1 CR2

19K

(VAdel)

Orf6/7

Fig. 2. Insertions and deletions that can be used to construct an oncolytic adenovirus. Critical modifications for clinical efficacy by systemic administration are indicated in bold.

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before apoptosis was described and they were characterized by inducing cellular toxicity (cyt phenotype) and degradation of the cellular genome (deg phenotype) with the ladder pattern later associated to apoptosis. These mutants were found to be greatly defective in non-complementing cell lines and lack of progeny was attributed to cyt and deg effects on the infected cells. More recent studies with E1b-19K mutants have described that, in certain cell lines, these mutants are not defective but grow to wild type levels and sometimes even more. It is conceivable that this different outcome is related to the apoptotic-inhibitory functions of tumors. The oncolytic potency increase seen in several tumor models seem to result from the earlier release of virions from the infected cells causing an accelerated cell-to-cell spread (Sauthoff et al., 2000). E1a has become the preferred target for deletions in oncolytic adenoviruses. E1a encode the first viral proteins synthesized from the adenovirus genome. They activate the cell cycle and the transcription of other virus genes (Fig. 1). CR1 and CR2 are two conserved regions of E1a essential to bind pRB, p130 and p107 family protein members. pRB binding releases free E2F from the E2F-pRB complex to activate the transcription of viral genes and cellular genes involved in the control of the cell cycle (e.g. cdk2, cdk4, cyclins a, D, and E) or DNA synthesis (e.g. PCNA, DNA polymerase, ribonucleotide reductase). The carboxy-terminal half of CR1 and amino-terminal domain of E1A bind p300 to stimulate E2F transcriptional activity. Either p300 or pRb binding is enough to induce cellular DNA synthesis but binding to both is necessary to pass G2/M. With regard to oncolytic adenoviruses, single CR1 mutants such as dl1101 are barely selective and have an undesired attenuation in tumor cells (Heise et al., 2000). In contrast, single CR2 mutants preserve the oncolytic potency in tumor cells and they are attenuated in normal cells as long as they are arrested (by serum deprivation or by pRb gene transfer) (Fueyo et al., 2000; Heise et al., 2000). The CR1 mutation may have value to further increase the selectivity of certain CR2 mutants (Howe et al., 2000). dl01/07, a virus with a CR1 and partial CR2 deletion that only affects pRB binding (residues 111–123), is selective for tumor cells as opposed to viruses that contain either mutation. Most interestingly this doubleablated E1A is attenuated 1 log in proliferating normal lung fibroblasts. Other efforts using dl01/07 as the basis for further oncolytic virus development show the same 1 log attenuation compared to wild type in proliferating lung fibroblasts and an additional 2 logs upon arrest (Doronin et al., 2000). Nevertheless the selectivity gained with the CR1 and CR2 deletion may also reduce the potency in several tumor cell lines (dl0701). So far the most selective combination of mutations seems the amino-terminal and CR2 deletion (dl4-25/121-128) but careful studies on potency in tumor cells have not been done with this double mutant. Finally, as E4-orf6/7 binds also to pRB, the combination of this deletion with E1a-CR2 deletion seemed necessary to achieve a better selectivity. However so far this has not been the case (Working et al., 2005). Another deletion that has been used to confer cancer selectivity to adenovirus is the deletion of virus-associated (VA) RNA genes. VAI an VA-II RNA genes of adenovirus encode 160-nucleotide-long non-translated RNAs transcribed by DNA polymerase III at the late phase of the virus replication cycle. These small RNAs bind to

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protein Kinase R (PKR) and act as competitive inhibitors of the activation of this enzyme by other double-stranded RNAs produced during infection. PKR is a double–stranded RNA-activated serine/threonine protein kinase induced by interferon that plays a major role in the IFN antiviral cell defense by phosphorylating the eIF-2-alpha translation factor, leading to shut-off of protein synthesis in infected cells (Williams, 1999). By binding to PKR, VA-RNAs counteract this IFN response to adenovirus and, conversely, adenoviruses defective in VA-RNAs are blocked by IFN via protein translation inhibition. However, besides antiviral functions, IFNs also inhibit cell proliferation by inducing p21 and p202 expression and downregulating c-myc expression, inhibit caspases, and enhance antigen presentation by inducing MHC expression. Hence, it is not surprising that tumor cells present a truncated IFN pathway (Grander and Einhorn, 1998). In such tumor cells the function of VARNAs becomes dispensable and their deletion can confer oncotropism (Cascallo et al., 2003, 2006). Similarly, the deletion of viral genes that counteract IFN has been used to design oncolytic herpes and influenza viruses (Bergmann et al., 2001; Farassati et al., 2001) and, in general terms, viruses sensitive to IFN been used as oncolytic viruses. The tumor selective replication of these viruses unable to block the IFN pathway correlates broadly to an enhanced initiation of protein translation found in tumor cells. Several pathways, Ras and PI3K among them, contribute to this phenotype and therefore the correlation of virus replication and specific molecular traits is difficult. For Epstein Barr Virus-derived tumors this correlation is much more clear to establish as they express EBER RNAs that have the same function of VA-RNAs (Wang et al., 2005). Finally, the deletion of VA-RNAs can be important when an oncolytic adenovirus is designed to carry a siRNA as it has been reported that VA-RNAs suppress Dicer (Andersson et al., 2005). However, whether it is necessary to delete VA RNAs to allow for the proper function of siRNAs remains controversial as oncolytic adenoviruses that express VA RNAs and functional siRNAs have been described (Carette et al., 2004; Zhang et al., 2006). Other deletions important to achieve cancer selective replication involve the entry pathway of adenovirus in the cell. For systemic delivery of adenovirus it is important to avoid its hepatotropism. In terms of hepatotropism one has to distinguish the uptake of the virus by liver macrophages (Kupffer cells) and by hepatocytes. The former capture most of the circulating virus and degrade it. Hepatocytes, on the other hand, capture less virus but contribute more to virus gene expression (transduction) and liver toxicity. It is known that the deletion of the residues of the fiber and penton base responsible for binding the natural receptors of adenovirus, the coxsackie-adenovirus receptor (CAR) and integrins, does not reduce the hepatocyte transduction. This has recently been explained by the role of blood factors (FIX and complement C4) in hepatocyte transduction. These blood factors bridge the fiber knob to heparan sulfates of the hepatocyte. This key role of the knob implies that mutations that affect the proper function of the knob for infectivity will reduce liver transduction. One of these mutations is located near the hinge of the shaft (Bayo-Puxan et al., 2006). However this mutation is not compatible with the insertion of tumor-targeting ligands in the fiber knob. A combination of two CAR-ablating mutations with the extension of the HI-loop also

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reduces liver transduction preserving the knob function and the mutation seems to affect specifically the binding of blood factors (Shayakhmetov et al., 2005). This modified knob should be able to present small tumor-targeting ligands. However, taking into consideration the key role of the knob in liver detargeting and the structural constrains of the knob to include large turmor-targeting ligands, ideally it should be deleted. In this case the fiber trimerization function of the knob has to be provided by other trimerization domains such as the phage T4 fibritin (Krasnykh et al., 2001). Recently the structure of this fiber-fibritin fusion has been optimized to allow the production of retargeted viruses with wild type yields (Noureddini et al., 2006). We expect that soon this will allow the insertion of targeting ligands of high affinity and selectivity such as single chain antibodies. Among insertions, promoters are a key element in oncolytic adenovirus design. There are several parameters to consider when inserting them in oncolytic adenoviruses. First, the broader the tropism the better in terms of application to different tumor types. In this regard, those regulated with E2F1 or HIF2alpha binding sites are very attractive. Second, artificial modular promoters offer the advantage over natural promoters that they are shorter, may allow higher level of transcriptional activation and inhibition, and may preserve better the adenovirus genome. Third, the promoter should be tight. Those promoters that are regulated by repression in normal cells and activation in tumor cells such as the E2F1 and the hTERT promoters are ideal. Promoter insulation with insulators, polyadenylation signals or relocation of the enhancers of the packaging signal far away from the inserted promoter may help to improve the tightness of the promoter. Another parameter to consider is the gene or genes to control. E1a is a key gene that has to be under control. For other early genes one has to take into account that the E3 promoter can interfere with control of E2 (Fuerer and Iggo, 2002), and that location of promoters in E4 can reduce replication in permissive cells (Working et al., 2005). It has been well documented that the control of two early transcription units is better than one. The use of the same promoter for this double regulation should be avoided to improve the stability of the resulting genome (Working et al., 2005). The combined control of E1a and E1b with the same promoter in viruses such as CV716, 733 and 740 has improved the selectivity but resulted in genome instability (Yu et al., 2002). A solution is to link E1a and E1b with an IRES such in viruses like CG8840 and CG8900 that show a very high selectivity index (105-fold better replication in tumor permissive cells than in non-permissive cells). Another solution is to use two different promoters. CV739 (E1a under probasin promoter and E1b under the PSE promoter) and CV764 (PSE and kallikrein promoters) are two examples of this strategy that show high selectivity (104 selectivity index). However the use of two different promoters restricts the potency to the few cell types able to overexpress them. The transcriptional control of E1a has also been combined with the control of E4. Promoter repetition has also led to genome instability in viruses such as Onyx-411 with two E2F1 promoters. However, even when two different promoters have been used this problem has been observed, leading to the possibility that any E4 modification can decrease the stability of the oncolytic adenovirus (Working et al., 2005).

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5. Arming the backbone Among the insertions that can be done in the tumor-selective adenovirus backbone, transgenes deserve special mention. An oncolytic virus with a transgene embodies the fusion of virotherapy and gene therapy. Cancer gene therapy has used non-replicative adenoviruses to deliver transgenes in different therapeutic approaches. Clinical failure with tumor suppressors and prodrug-converting genes has been attributed to poor transduction. It is true that a replicating vector can multiply the transfer of a cytotoxic gene but more appealing is the idea that gene transfer can amplify virotherapy. These two concepts are not the same, as the kind of genes used to improve virus spread may not have any antitumoral potential per se. The goal of a transgene in an oncolytic virus is not to kill the transduced cell (eventually the cell dies by virus replication), specially if this death compromises virus replication, but to eliminate surrounding cells (bystander effects) and overall to favor virus spread. The E3 adenovirus death protein (ADP) can be inserted as an ‘‘endogenous’’ transgene. Adenovirus release from infected cells is a rather inefficient process aided by the late expression of ADP from the major late promoter. Overexpression of ADP enhances the oncolytic potential of replicating adenoviruses (Doronin et al., 2000). Although it is not established that ADP promotes apoptosis, it is known that this type of cell lysis can favor virus release. Other ‘‘exogenous’’ transgenes that promote apoptosis can be used with the same purpose. Expression of p53 at the late phase of the viral cycle improved virus release and spread of a replication-competent adenovirus (Sauthoff et al., 2002). p53 expressed in this way can substitute the function of ADP and, because the induction of apoptosis by p53 is more tumor-selective, this strategy increases the selectivity of the replicating adenovirus. Apoptosis can also be used as a means of oncolytic virus spread throughout the tumor. Mi et al. have demonstrated that induction of apoptosis stimulates viral spread through apoptotic bodies (Mi et al., 2001). TNF has a pro-apoptotic function inducing activation of caspases and an antiapoptotic function via activation of NF-kB, a transactivator of antiapoptotic genes. Most tumor cells are resistant to TNF-induced apoptosis because the latter function predominates. An adenovirus expressing a mutated form of IKB that avoids NF-kB activation sensitizes tumor cells to TNF-induced apoptosis. If apoptosis is induced before virion assembly it has a negative effect on virus production. In contrast when it is induced at a late stage (40 h post-infection) it does not affect the production of viruses but increases the release of virus to the supernatant and the virus spreads faster. Virions are present in the lumen or membrane apoptotic bodies suggesting a cell-to-cell spread through phagocytosis of apoptotic bodies. Interestingly an electron microscopy study of Ad-p53 gene therapy revealed that tumor cells can phagocyte apoptotic bodies containing the vector (Mitry et al., 2000). The utility of this kind of approach is underscored considering that the main shortcoming of oncolytic adenoviruses is their neutralization by antibodies and that this could be an antibody-resistant cell-to-cell spread. The role of the immune response as a friend or foe of virotherapy remains to be elucidated. The antiviral effects of the immune system can be already detected in nude mice models where adoptive transfer of anti-adenovirus antibodies or a further

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suppression of the immune system, inhibit or enhance oncolysis, respectively (Chen et al., 2000). The rediscovery of hamsters as permissive hosts for adenovirus and its use on adenovirus virotherapy will help to define which immunoregulatory approaches can benefit virotherapy (Khoobyarian et al., 1975; Thomas et al., 2006). If virotherapy, even with transient immunosuppression by mieloablative chemotherapy for example, proves insufficient for therapy, then it may become a great adjuvant for immunotherapy by debulking the tumor, releasing tumor antigens, and providing the appropriate danger signal that sparks the response. As a clear example of this trend, recently an oncolytic adenovirus expressing IL24 has been shown very effective to treat tumors in mice (Zhao et al., 2006). On the other hand the presence of immune dominant viral antigens could mask responses to tumor antigens. These hypotheses could now be tested in hamsters. In addition to the neutralization by the immune system, oncolytic adenoviruses face another major problem: the intratumoral barriers formed by connective tissue or fibrosis. Many tumor types are characterized by small groups of tumor cells surrounded by large areas of tumor-associated fibroblasts and connective tissue. In this environment, intratumoral spread is very limited. Recently, expression of proteases to digest the connective tissue has been proposed to solve this problem (Kim et al., 2006). Expression of relaxin improves the oncolytic potential of a tumor-selective adenovirus. Bystanter effects could also help the spread and cytotoxicity of oncolytic adenoviruses. Transgenes, as TK and CD, have proved to boost the potency of the viruses. The expression of these genes at the late phase of the virus replication cycle diminish the antivirus effects of this cytotoxicity. Among the different strategies that assure a late phase expression, the use of additional splicing signals along the major late transcript unit may be more efficient than the use of IRES and also it avoids the deletion of any late gene and the repetition of promoters.

6. Working together Even if the best armed backbone is assembled, gene-virotherapy will benefit from the help of other cancer treatments. Adenovirotherapy has demonstrated synergy with chemotherapy and radiotherapy, for example. However, one of the obstacles to assemble a good adenovirus backbone is just commercial. It is very expensive to produce the GLP preclinical studies and the GMP virus for clinical development. These steps need industry investment, and industry invests when intellectual property is clear. The model backbone of a tumor-targeted, highly selective and potent oncolytic adenovirus loaded with transgenes that can be used for a broad spectrum of tumor types is a nightmare in terms of patents. Assembling this multi-owner virus seems an academic exercise far away from any commercial interest. The improvement in oncolytic virus design is a slow stepwise process that needs to rest on the contribution on many researchers. Unfortunately, the parameter that determines if a virus is worth for clinical test is whether the intellectual property involved can be brought together by the investor source. For many years the promise of good clinical results has been enough to foster a reasonable level of research in gene therapy

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and virotherapy of cancer but this promise needs to translate into facts. What we really need now is to deliver the goods of cancer gene-virotherapy before the skepticism generated by poor clinical results obtained with ill conceived vector backbones becomes irreversible.

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