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REVIEW

Cancer Scene Investigation: how a cold virus became a tumor killer Dawn E Post†, Hyunsuk Shim, Esra Toussaint-Smith & Erwin G Van Meir †Author

for correspondence Laboratory of Molecular Neuro-Oncology, Emory University, Atlanta, GA 30322, USA Tel.: +1 404 778 2267; Fax: +1 404 778 5240; [email protected]

Oncolytic therapy is a novel anticancer treatment with attenuated lytic viruses such as adenovirus (Ad). These viruses kill the host cells through their lytic replication cycle and are thus distinct from classical gene therapy viruses, which serve as gene delivery agents and do not replicate. Oncolytic Ads are genetically engineered so as to replicate only in cancer cells. Their replication cycle leads to viral multiplication, the killing of the host cells and spreading of the infection throughout the tumor. Following success in preclinical studies, their anti-tumor potential is now being evaluated in the clinic. Three oncolytic Ads (dl1520, Ad5-CD/TKrep, and CV706) have completed Phase I and II clinical trials in cancer patients where their administration via multiple routes and in combination with chemo- or radiotherapies, has demonstrated overall safety. These viruses are being re-engineered to arm them with additional therapeutic genes, bolstering their oncolytic activity with a bystander effect. For example, Ad5-CD/TKrep delivers a therapeutic prodrug-activating (suicide) gene. These data indicate that oncolytic Ads are a promising novel cancer treatment approach that can be combined with other modalities, such as gene therapy and classical chemo- and radiotherapies. Further improvements to enhance their specificity, targeting and oncolytic activity are needed however, as these first-generation viruses showed modest anti-tumor activity. To improve their efficacy in the clinic, it will be important to devise and incorporate novel monitoring techniques in the clinical trials, such as analysis of viral replication in biopsies and through the use of creative noninvasive imaging technologies.

Adenovirus as an anti-tumor therapy

Keywords: adenovirus, gene therapy, tumors, oncolytic therapy, oncolytic virus, virotherapy

Genetically modified adenoviruses (Ads) are being evaluated as biological agents for antitumor therapy with serotypes 2 and 5 being the most frequently used. Human Ads are nonenveloped, double-stranded DNA viruses with a linear genome approximately 36 kb in length that primarily infect the epithelial tissue lining the respiratory tract. Ads can convert both dividing and quiescent cells into virus-producing factories, culminating in the release of progeny virus by host cell cytolysis and death. Current strategies aim to target the lytic properties of the virus specifically to neoplastic cells while avoiding toxicity to normal cells. The use of viruses, such as Ads, for this purpose is known as oncolytic virotherapy. The rationale underlying this therapy is that viral replication in permissive tumor cells will lead to exponential amplification of the initial viral inoculum and repeated cycles of cell death and viral progeny dispersal to additional tumor cells until tumor eradication. Many features of the Ad make it suitable for use as a tumor-cell-killing agent. Ads are endemic in the human population and cause only mild respiratory infections and conjunctivitis. Genetic modification of the Ad genome is easily performed using standard recombinant

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DNA techniques. The viral replication cycle and the functions of many viral proteins are well characterized. The virus can be produced on a large scale and purified to relatively high titers (1012 pfu/ml) for its use in the clinic. The viral DNA does not integrate into the cell genome, but remains episomal, thereby removing the risks for disruption of critical endogenous genes. Moreover, the DNA packaging capacity (105% of the wild-type genome size) allows for the insertion of small foreign genes that have anti-tumor activity. The cloning capacity can be further increased by the deletion of viral genes. There are also several drawbacks to using Ads as an anti-tumor therapy. The Ad has a relatively high immunogenicity. In addition, safety concerns of viral therapy were increased following the death of a patient on a gene therapy trial in September 1999 [1]. This patient received hepatic intra-arterial infusion of 4 × 1013 particles of a replication-deficient Ad for the treatment of ornithine transcarbamylase deficiency. It is believed that this extremely high viral dose was responsible for this tragic death. In contrast, it is important to note that no deaths or serious side effects have been reported in clinical trials of oncolytic replication-competent Ads. It is also unclear Future Oncol. (2005) 1(2), 247–258

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REVIEW – Post, Shim, Toussaint-Smith & Van Meir

whether the presence of Ad-specific neutralizing antibodies in patients interferes with the efficacy of these viral agents as an antitumor treatment. Furthermore, the expression of the cellular coxsackie–adenovirus receptor (CAR), which mediates Ad infection, is a limiting factor in the efficient transduction of tumor cells due to its aberrant or highly variable expression in these cells [2,3]. Finally, Ads do not inherently possess a mechanism for inactivation. Clearly, there is a need for continued research to alleviate these current disadvantages. Targeting Ad replication to tumor cells

Two main strategies have been utilized to target Ad replication specifically to tumor cells: viral gene mutation (Figure 1A); and transcriptional regulation of viral replication genes (Figure 1B) [4,5]. The function of several Ad proteins is to modify specific cellular pathways to create an environment that supports efficient viral replication and progeny production. Several of these cellular pathways play a direct role in tumorigenesis and are already constitutively activated in neoplastic cells. An intellectual leap occurred when it was realized that this rendered these viral proteins superfluous for viral replication in cancer cells, and that deletion of the corresponding genes from the viral genome would restrict Ad replication to tumor cells while prohibiting viral replication in normal cells. In support of this, Ads that contain mutations of the Ad E1A, E1B, or viral-associated (VA) RNA coding sequences have been generated and shown to selectively replicate in cells that contain respectively, disrupted pRb or p53 tumor suppressor pathways or an activated Ras pathway. The second strategy involves placing genes that function in Ad replication (E1A and E4) under the transcriptional control of promoters that are preferentially active in tumor cells [4]. The exogenous promoters that have been used fall into two categories. The first class contains promoters that are primarily active in only one tumor type and have been used to restrict Ad replication to breast, prostate, liver, melanoma, lung, colorectal, neuroblastoma, ovarian and bladder cancers. The second class of promoters are active in a wide range of tumor types and include the tetracycline, telomerase reverse transcriptase (TERT), hypoxiainducible factor (HIF) [6,7], and E2F-1 responsive promoters. The use of these latter promoters overcomes tumor type restrictions and genetic alterations. The continued investigation of the viral replication cycle, the functional interactions 248

between Ad and host cell proteins, and identification of promoters that are overactive in tumor cells relative to normal cells, will undoubtedly reveal additional means to regulate Ad replication. For example, the Ad E1B-55kD protein is involved in the nuclear accumulation of the host cell Y-box protein (YB)-1. YB-1 transcriptionally activates the Ad E2 genes needed for viral DNA synthesis and the cellular P-glycoprotein (gp) multifunctional drug transporter gene, mdr1, which plays a role in the development of the multidrug resistant cancer phenotype [8]. Nuclear overexpression of YB-1 has been found in numerous tumor types. Consistent with these findings, replication-defective Ads that lack the entire E1A gene (Ad312), or contain a mutated E1A gene that does not express the larger 289 amino acid E1A protein (Ad520), can undergo viral replication and specifically kill multidrug-resistant (MDR) tumor cells that overexpress YB-1 [8,9]. This suggests that YB-1 can compensate for the critical viral replication functions of E1A and that all conditionally replicative oncolytic Ads will be able to effectively kill this clinically relevant subclass of tumor cells. Therefore, this is an alternative strategy to design an oncolytic Ad. In parallel with approaches to restrict Ad replication, there has been a major effort to develop strategies to enhance or specifically target Ad infection to tumor cells [2,3]. This is known as transductional targeting, which seeks to overcome problems associated with: • Sequestration of the virus by nontargeted cells (liver or non-neoplastic cells); • Inefficient infection of tumor cells due to aberrant or absent/low expression of the cellular CAR Ad serotype 2 or 5 infection involves binding of the viral fiber protein to the CAR and interaction of the Arg-Gly-Asp (RGD) peptide sequence of the viral penton protein with the cellular αvβ3 and αvβ5 integrin receptors. Genetic strategies that have been used to modify the tropism of oncolytic Ads include: • Pseudotyping which is the substitution of the Ad2 or Ad5 fiber with one from another Ad serotype, such as Ad3 [10]; • Incorporation of targeting ligands into the fiber protein, such as polylysine residues [11] or RGD peptides [12] that allow binding to the more broadly expressed cellular heparin sulfate or αv integrins receptors, respectively. Future Oncol. (2005) 1(2)

Cold virus – a cancer killer – REVIEW

Figure 1. Strategies used to target Ad replication specifically to tumor cells.

A Rb+

Normal cell: no viral replication

Rb-

Mutated viral gene, such as E1A

Tumor cell: viral replication

Cytolysis and viral spread

B

Normal cell: promoter not active

E1A replication gene regulated by a promoter that is preferentially active in tumor cells

Tumor cell: promoter active

Cytolysis and viral spread

A. Targeting Ad replication to tumor cells by viral gene mutation. Specific mutations (circles) within the Ad E1A, E1B, or viral-associated RNA coding sequences that abrogate protein function have been shown to result in the selective replication of the mutant Ad in tumor cells that contain dysfunctional pRb, p53, or Ras pathways respectively. For example, the dl922-947 [42] and delta-24 [43] Ads contain a small deletion in a conserved region (CR)-2 of E1A that abrogates its interaction with the pRb family of proteins. While these E1A-mutant Ads can infect both normal (pRb-functional) and tumor (pRb-deficient) cells, their replication is restricted to cells with a dysregulated Rb pathway. A single Ad infected cell can generate up to 1000 viral progeny which after host cell lysis can infect nearby tumor cells, thereby disseminating the viral infection and host cell lysis cycle throughout the tumor mass. In contrast, normal cells do not support replication of the E1A mutant Ad. B. Targeting Ad replication to tumor cells by transcriptional regulation of viral replication genes. The Ad E1A gene (arrow) encodes a protein that is essential for viral replication and its absence leads to a replicationdeficient phenotype. Therefore, the transcriptional regulation of this gene by a promoter (yellow rectangle) that is preferentially overactive in tumor cells relative to normal cells will result in tumor-selective Ad replication. For example, hypoxia is a distinctive attribute of solid tumors that results in the activation of the hypoxia-inducible factor (HIF) pathway [44]. HIF is a transcription factor that activates target genes needed for cell survival and proliferation under hypoxic stress. In the HYPR-Ad #1 virus, a hypoxia/HIF-responsive promoter regulates the transcription of the E1A gene resulting in selective viral replication and host cell lysis in hypoxic/HIF-active tumor cells but not normoxic cells [6,7]. (Figure adapted and modified from [4]).

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Additional genetic modifications for targeting infection of replication-deficient Ads have been described and these could potentially be applied to oncolytic Ads [13,14]. An alternative retargeting approach involves coating the virus with a polymer, such as polyethylene glycol (PEG) and poly [N-2(hydroxypropyl)methylacrylamide (HPMA)[15], which have the added advantage of providing protection from Ad-specific neutralizing antibodies. Further modifications of the polymers to incorporate targeting ligands, such as basic fibroblast growth factor (bFGF), vascular epidermal growth factor (VEGF) or RGD, have resulted in viruses with redirected tropism and CAR-independent infection [15,16]. The ‘polymer coating’ strategy has not been applied to an oncolytic Ad. In addition, this strategy is limited to the initial viral inoculum and will not confer an advantage to progeny virus produced in situ. In summary, these preclinical studies provide critical proof that the tropism of an Ad can be successfully modified. It will be important to evaluate these various transductional targeting strategies in the context of oncolytic Ads in clinical trials to determine if they lead to augmented anti-tumor activity. Preclinical studies

Over the last 8 years more than 50 genetically altered replication-competent Ads have been generated for cancer therapy purposes [4,5]. Some of these oncolytic Ads have been engineered to function as therapeutic gene delivery vehicles for various prodrug activating (suicide) and cytokine Figure 2. Schematic of Ad E3 gene region (A) and protein function (B). A. 12.5K

6.7K

gp19K

ADP

RID α

RID β

14.7K

B. Protein Function 12.5K 6.7K gp19K ADP RID α, β 14.7K

Unknown Inhibits TRAIL-induced apoptosis in conjunction with RID Inhibits host cell killing by CTL Promotes lysis and progeny virus release from infected cells Inhibits TNF, FasL and TRAIL-induced apoptosis, degrades EGFR Inhibits TNF, FasL and TRAIL-induced apoptosis

The Ad E3 gene region encodes seven proteins that function to protect Adinfected cells from the host immune response and mediate efficient host cell lysis and viral progeny release [19]. Ad: Adenovirus; ADP: Adenovirus death protein; CTL: Cytotoxic T-lymphocytes; EGFR: Epidermal growth factor receptor; FasL: Fas ligand; gp; Glycoprotein; RID: Receptor internalization and degradation complex; TNF: Tumor necrosis factor; TRAIL: TNF-related apoptosis-inducing ligand.

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genes. All of these Ads display preferential lysis of targeted tumor versus normal cells. Their antitumor activity was promising with evidence of tumor growth reduction, including a small number of complete responses that result in long-term tumor eradication. These preclinical studies also have some limitations that interfere with the ability to fully evaluate the anti-tumor capabilities of these viruses prior to entering the clinic. Some of these limitations are due to the viruses themselves. First, the inclusion of appropriate controls, replication-deficient and wild-type Ads, are lacking in many studies and are essential to determine to what degree, if any, replication of the recombinant Ad is attenuated. The importance of this issue is best exemplified by studies with dl1520, which does not express the E1B-55kD protein due to mutations within its coding sequence, and was predicted to replicate only in cells lacking functional p53 [17]. The function of E1B55kD is to bind and target the p53 tumor suppressor protein for degradation and it is also involved in nuclear export of viral transcripts. The abrogation of this latter function dramatically reduces the efficiency of dl1520 replication even in permissive p53-deficient tumor cells when compared with a wild-type Ad [18]. Second, most of the exogenous promoters used to target viral replication to tumor cells are also active in a subset of normal cells, which may result in some degree of toxicity. Most normal mouse tissues do not support efficient human Ad replication, thereby making it difficult to evaluate viral replication at unwanted sites and potential toxicity to normal tissues. Many of the oncolytic Ads also contain deletions within the E3 gene region. This region encodes seven proteins that function to protect Ad infected cells from the host immune response, and mediates efficient host cell lysis and viral progeny release (Figure 2) [19]. While the E3 gene region is nonessential for viral replication in vitro, its inclusion in oncolytic Ads leads to enhanced oncolytic potency in vitro and anti-tumor activity in athymic mice [20,21]. The adenovirus death protein (ADP) is believed to be primarily responsible for the enhanced effect [22,23]. Another set of limitations is with the preclinical tumor models, which are human tumor cell lines xenografted in immunocompromised mice. Current preclinical tumor models utilize human cancer cell lines that are genetically homogenous, and hence, not representative of the tumor heterogeneity present in patients. One would Future Oncol. (2005) 1(2)


expect that tumor cells lacking the targeted phenotype (i.e., p53-deficient, prostate-specific antigen [PSA]-negative) would not be susceptible to viral-mediated death. There is also evidence that murine tumor-supporting structures, such as connective tissue cells and noncellular matrix components, are a barrier to human Ad spread within tumor xenografts [24]. Additionally, the use of immunodeficient rodent tumor models has made it difficult to completely evaluate whether the host immune response will reduce: • Viral delivery to tumor cells following systemic administration; • Dispersion of progeny virus to additional cells within the tumor mass; and • Anti-tumor efficacy This limitation was recently overcome by the identification of several mouse carcinoma cell lines that can support human Ad replication and undergo Ad-mediated cytolysis, thereby allowing tumor studies to be carried out in immunocompetent mice [25]. Using these model systems it was demonstrated that a replicative Ad containing a deletion of the E3B genes (receptor internalization and degradation [RID]-α, -β, and 14.7K), exhibits reduced viral gene expression and replication, rapid viral clearance, and reduced anti-tumor activity. In contrast, dl704 Ad, which lacks the E3 gp19K gene, exhibits increased replication and anti-tumor efficacy by an unknown mechanism. The function of gp19K during the viral lytic cycle is to bind major histocompatability (MHC) class I molecules and inhibit their transport to the cell surface, thereby preventing the detection and clearance of infected cells by Ad specific cytotoxic T-lymphocytes (CTLs). Since tumor cells have already evolved various mechanisms for eluding CTLs, gp19K may be dispensable for Ad replication in these cells [26]. These immunocompetent tumor models that support human Ad replication are valuable novel tools to study interactions between the host immune system and viral proteins. They will also allow investigators to dissect the importance of the individual genes within the E3 region with the aim of developing new oncolytic Ads that have enhanced anti-tumor activity. Clinical trials

The safety and anti-tumor activity of three oncolytic Ads (dl1520, Ad5-CD/TKrep and CV706) either alone or in combination with chemo- or radiotherapies, have been evaluated in www.futuremedicine.com

393 cancer patients in 21 clinical trials (Tables 1 & 2). dl1520 is a mutant virus that does not express E1B-55kD and is expected to conditionally replicate in p53-deficient cells [17]. It should be noted that its selective replication has been controversial with some studies supporting [17,27], and others contradicting [28,29] the proposed mechanism. This disagreement can be explained, in part, by findings that the loss of p14ARF [30] and differential RNA export [31] between tumor and normal cells, play a role in the selective replication of this virus in neoplastic cells. Ad5-CD/TKrep is E1B-55kD null and in addition, contains a cytosine deaminase (CD)– thymidine kinase (TK) fusion gene [32]. TK is an enzyme that, in conjunction with other cellular kinases, converts the prodrug gancyclovir into gancyclovir triphosphate, an inhibitor of both viral and cellular DNA synthesis that can spread from cell-to-cell via gap junctions. CD converts the prodrug 5-fluorocytosine (FC) into 5-fluorouracil (FU) which inhibits tumor growth by interfering with RNA and DNA synthesis. The transcriptional targeting virus, CV706, was designed for the treatment of prostate cancer by using the minimal enhancer/promoter of the PSA gene to drive viral replication [33]. dl1520 has been widely evaluated as an anti-tumor agent for numerous tumor types using multiple routes of administration. CV706 and Ad5-CD/TKrep have undergone more limited testing in prostate cancer patients using intraprostatic delivery. Several important findings have come from these trials. First, and most important, the overall safety of treating cancer patients with oncolytic Ads was demonstrated. There were no deaths associated with virus treatment and no maximum tolerated dose (MTD) was reached. The highest dose that can be administered is based on current manufacturing capabilities. In general, oncolytic viral therapy was well tolerated with all routes of delivery and the most common adverse events were fever and flu-like symptoms of Grade 1–3 toxicity. In addition, in patients receiving hepatic artery or intravenous viral infusions, there was no evidence of treatment-emergent or progressive clinical hepatotoxicity (Tables 1 & 2). Only three of 393 patients, each on a separate study, experienced a dose limiting toxicity (DLT). Clinical trials evaluating the safety of combining oncolytic Ad treatment with chemo-, radio-, or prodrug therapies showed similar safety profiles (Tables 1 & 2). In almost all of the clinical studies, oncolytic Ad therapy led to a measurable anti-tumor response with multiple routes of viral 251

252

Intratumoral

Hepatic artery

Intratumoral Hepatic artery, or intravenous

Intravenous

Intravenous

Intraperitoneal

Intracerebral

Mouthwash

Intraprostatic

Intraprostatic

Intraprostatic

dl1520

dl1520

dl1520

dl1520

dl1520

dl1520

dl1520

dl1520

CV706

Ad5-CD/TKrep

Ad5-CD/TKrep

5-FC valganciclovir radiation

5-FC ganciclovir

Irinotecan + 5-FU, or IL-2

Carboplatin paclitaxel

5-FU leucovorin

Additional treatment

Prostate

Prostate

Prostate

Oral dysplasia

Resected cavity of malignant glioma

Ovarian

Advanced metastatic of various sites

Carcinoma metastatic to the lung

Hepatocellular carcinoma or colorectal liver and metastases

Metastatic gastrointestinal carcinoma to the liver

Pancreatic

Head and neck

Tumor

15

16

20

19

24

16

10

10

9

11

23

22

Pts (n)

Blood

Blood

7/16 ≥ 25% decrease in serum PSA; 3/16 ≥ 50% decrease in serum PSA; transgene expression 2 weeks postinjection 15/15 decrease in serum PSA; transgene expression up to 3 weeks postinject

Tumor normal (+)

13/20 ≥ 30% decrease in serum PSA

Not examined

Peritoneal Washings normal (+)

Blood tumor (+)

Blood tumor (+)

Not examined

Blood

Blood tumor

Tumor (+)

Viral replication§§§

Tumor (+)

1

1

DL T

7/19 resolution of dysplasia; 1/19 grade of dysplasia improved

No responses

2/16 SD

4/10 SD; 6/10 PD

1/10 mixed; 8/10 SD; 1/10 PD

Not reported

1/11 PR; 2/11 SD

11/22 SD; 6/22 MR; 4/22 PD

3/22 PR; 2/22 MR; 8/22 SD; 9/22 PD§

Therapeutic response§§

Measured not reported

Pre: 50% Post: 100% no correlation


Post:1/7 increased

Pre: 8% Post: 17%

Pre: 40% Post: 92%

Pre: 20% Post: 100%

Pre: 20% Post: 100%

Not measured

Pre: 55% Post: 100%



Neutralizing Ab§§§§

[56]

[55]

[54]

[53

[52]

[51]

[50]

[49]

[48]

[47]

[46]

[45]

Refs

(blood, tumor tissue, normal tissue) used to assess viral replication is listed. (+) indicates detection of viral replication in tumor and normal samples. §§§§ The percentage of patients with Ad neutralizing antibody (AdNAb) levels pre and post viral treatment is indicated. Those studies that demonstrated no correlation between baseline AdNAb levels and tumor response are indicated. Ab: Antibody; AdNAb; Adenovirus neutralizing antibodies; DLT: Dose-limiting toxicity; FC; Fluorocytosine; FU: Fluorouracil; IL: Interleukin; PSA: Prostate-specific antigen.

Measured using nonconventional criteria by subtracting the central necrosis §§ CR: Complete response; MR: Minor response; PD: Progressive disease; PR: Partial response; SD: Stable disease §§§ Sample

Intratumoral

dl1520

§

Route of viral delivery

Virus

Table 1. Phase I trials of oncolytic Ads.


Future Oncol. (2005) 1(2)


administration (Tables 1 & 2). Consistent with preclinical studies [34], there are also indications that combining oncolytic Ad treatment with chemotherapy leads to improved therapeutic outcomes in patients. This is most evident when comparing Phase II trials involving intratumoral delivery of dl1520 alone and in combination with 5-FU and cisplatin to patients with head and neck cancer. The Ad5-CD/TKrep trials also demonstrated the ability of oncolytic Ads to deliver a therapeutic transgene to the tumor environment with transgene expression being detected up to 3 weeks post treatment. Induction of anti-Ad neutralizing antibodies (AdNAb) and in situ viral replication were also examined. AdNAb titers were measured at baseline and at specified intervals following virus administration, to evaluate the potential impact on the anti-tumor efficacy of oncolytic Ads. The percentage of patients with pre-existing AdNAb at baseline varied among the studies from 8 to 76%. Following viral administration, AdNAb titers increased in most patients with the rate and magnitude of change being highly variable. Exceptions to this AdNAb increase were noted in two dl1520 studies, the intracerebral injection of virus into the resected cavity of patients with malignant glioma, and as a mouthwash therapy in patients with oral dysplasia. Unexpectedly, there was no correlation between baseline AdNAb levels and treatment response irrespective of the mode of administration (Tables 1 & 2). This indicates that patients with pre-existing AdNAb may still be candidates for oncolytic Ad therapy. Multiple rounds of treatment could be achieved with various pseudotyped versions of the same virus to avoid immune response. Viral replication following administration was measured by two independent methods: • In blood samples using polymerase chain reaction (PCR); • In tissue by either immunohistochemistry (IHC) or in situ hybridization (ISH) The detection of viral genome in blood samples, which is interpreted as evidence of viral replication, is controversial, as it can also be seen with a replicative-defective Ad [35]. Direct evidence for viral replication in tumors tissues was found following intratumoral (Tables 1 & 2) [36], intravenous and mouthwash administration. However, viral replication has also been detected in normal cells (Tables 1 & 2) [36]. The assessment of viral replication in tumors of patients has been hampered by the inability to obtain sufficient www.futuremedicine.com

numbers or quality tissue samples. This limitation could be overcome using noninvasive imaging technologies for examining viral spread and replication. Noninvasive imaging technology

There is currently a strong need for noninvasive imaging technology to assess the safety and efficacy of oncolytic Ad therapy in both preclinical and clinical studies. Noninvasive evaluation of therapeutic response and correlation of the location, magnitude and duration of viral replication in vivo would be particularly useful for the optimization of virus delivery protocols and development of second-generation oncolytic Ads. Noninvasive imaging will complement established ex vivo assays that require tissue sampling and would be of considerable value in cases of inaccessible tumors or multi-lesion disease. The major molecular imaging modalities that are readily translatable to the clinic are nuclear imaging (positron emission tomography [PET], single photon emission tomography [SPECT]) and magnetic resonance (MR) imaging/MR spectroscopic (MRS) imaging. There are two genes that are most commonly used as reporters for noninvasive imaging; cytosine deaminase (CD) and herpes simplex virus type 1 thymidine kinase (HSV1-tk). CD deaminates 5-fluorocytosine to 5-fluorouracil and this reaction can be monitored using 19F-MRS [37]. In vivo monitoring of HSV1-tk activity can be imaged by SPECT or PET using reporter probes 123I-labeled FIAU (2´fluoro-2´-deoxy-1-β-Darabinofuranosyl-5-iodo-uracil) or 18F-labeled FHBG [9-(4-fluoro-3-hydroxymethylbutyl) guanine], respectively. The CD and HSV1-tk genes have been used as imaging reporters in preclinical studies with great success [38]. In addition, the feasibility of using this technology in patients was demonstrated in a gene therapy trial in which HSV1-tk expression was monitored using PET and 124I-FIAU [39]. However, these reporters have not yet been evaluated as imaging agents in clinical trials of oncolytic Ads. New imaging modalities are being produced in animal models that have the potential for tracking viral replication. These are based upon the development of single-fusion proteins with multiimaging capabilities. A single-fusion protein with three functional subunits (enhanced green fluorescence protein [eGFP], HSV1-tk, and firefly luciferase) was recently designed that allows detection by fluorescence, nuclear imaging, and bioluminescence (BLI) techniques [40]. This 253


Table 2. Phase II trials of oncolytic Ads. Virus

Route of viral delivery

dl1520

Intratumoral

dl1520

Intratumoral

dl1520

Additional treatment

Tumor

Patients (no.)

Therapeutic response§

DL T

Viral replication

Neutralizing Ab§§§§

Refs

§§§

Head and neck

40

3/36 CR; 2/36 PR; 3/36 MR; 13/36 SD; 15/36 PD§

Blood Tumor (+)


[57,58]

5-FU cisplatin

Head and neck

14

3/9 CR; 3/9 PR; 1/9 MR; 2/9 SD

Tumor (+)

Measured but not reported

[59]

Intratumoral

5-FU cisplatin

Head and neck

37

8/30 CR; 11/30 PR

Tumor (+)


[60]

dl1520

Intratumoral

Gemcitabine

Pancreatic

21

2/21 PR; 2/21 MR; 6/21 SD; 11/21 PD

Aspirates

Pre: 76% Post: 100%

[61]

dl1520

Intratumoral

Hepatobilia ry

19

1/16 PR; 1/16 SD; 8/16 reduction in serum tumor markers, 3/16-PD

Not examined

Pre:100% Post: 100% no correlation

[62]

dl1520

Hepatic artery

5-FU

Colorectal liver metastases

7

6/7 50% reduction in CEA levels 6 SD, 1 PD

Not examined

Not measured

[48]

dl1520

Hepatic artery

5-FU leucovorin

Metastatic gastro intestinal carcinoma to the liver

27

3/27 PR; 4/27 MR; 9/27 SD; 11/27 PD

Blood

Pre: 50% Post: 100%

[63]

dl1520

Intravenous

Metastatic colorectal

18

7/18 SD for 1118 weeks, 2/18 SD for 4 months

Blood Tumor (+) Normal (+)


[64]

dl1520

Intratumoral

Oral carcinoma

15

No responses

Tumor (+) Normal (+)

Not measured

[65]

§ Measured

using nonconventional criteria by substracting the central necrosis

§§

1

CR: Complete response; MR: Minor response; PR: Partial response;

SD: Stable disease; PD: Progressive disease §§§ Sample (blood, tumor tissue, normal tissue) used to assess viral replication is listed. (+) indicates detection of viral replication in tumor and normal samples. §§§§ The percentage of patients with neutralizing antibody (NA) levels pre and post viral treatment is indicated. Those studies that demonstrated no correlation between baseline NA levels and tumor response are indicated. CEA: Carcinoembryonal antigen; FU: Fluorouracil.

triple-fusion gene could potentially be incorporated into the genome of an oncolytic virus, thereby providing assessment of efficacy in preclinical studies in small rodents using whole-body BLI imaging and nuclear imaging, which can be further validated using in situ fluorescence image analysis upon tissue collection. A drawback of optical imaging is that it is currently restricted to small animal studies due to the limited signal penetration depth. A bifunctional fusion gene has also been created between CD and uracil phosphoribosyltransferase (UPRT) [41]. UPRT 254

converts 5-fluorouracil to fluorouridine monophosphate. Both CD and UPRT activity can be monitored using 19F-MRS. In addition, diffusion-weighted MRI was used in parallel to measure early changes in cellular water mobility, which reflects cellular death. Both 19F-MRS and diffusion-weighted MRI can be used as tools to assess therapeutic efficacy. The recent development of these reporters enhances the feasibility of using multimodality noninvasive imaging for the quantitative assessment of overall therapeutic efficacy and in vivo replication of oncolytic Ads. Future Oncol. (2005) 1(2)


However, it is still uncertain how many cells need to be infected in one area to produce a noninvasive image. Conclusions & future perspective

Replication-competent Ads, whose cytolytic replicative cycle has been engineered to be selectively active in tumor cells, are showing promise as anti-tumor agents in clinical trials. Administration of these viruses via multiple routes has proven relatively safe both alone or in combination with chemo-, radio-, or prodrug therapies. It needs to be emphasized that oncolytic Ads are not a form of ‘gene therapy’ and should not be classified as such. Rather these replication-competent viruses are a biologic agent that can directly kill tumor cells via their cytolytic life cycle. In contrast, replication-defective Ads exert their antitumor effect by delivering therapeutic genes. The latter viruses are limited

by their low in vivo transduction efficiency, resulting in poor therapeutic gene delivery and the inability to specifically target diseased versus normal tissue. This lack of distinction, as well as the poor replication ability of dl1520 and discontinuation of the therapeutic virus program, that includes dl1520 (ONYX-015) in June 2003 by ONYX [101], have dampened initial enthusiasm making it more difficult to obtain venture capital funds for new trials to test second-generation oncolytic Ads in cancer patients. At present, there is a strong need to keep the field of oncolytic Ad therapy progressing forward by generating new viruses with improved specificity, targeting, and oncolytic activity, as well as evaluating the anti-tumor efficacy of additional viruses in the clinic. It will also be important to develop better preclinical models and standardization of preclinical studies. This will aid prioritization of oncolytic Ads intended for

Executive summary Adenovirus as an anti-tumor therapy • Replication of an adenovirus (Ad) in a host cell culminates in cell death by cytolysis with the release of > 1000 progeny virus. These features make the Ad attractive for use as a tumor cell-killing agent. Targeting adenoviral replication to tumor cells • Two strategies are used to target Ad replication and cytolytic cell death, specifically to tumor cells: (i) viral gene mutation and (ii) transcriptional regulation of viral replication genes using promoters that are preferentially active in only one tumor type or a wide range of tumors. • There has also been a major effort to develop strategies to enhance or specifically target Ad infection to tumor cells. These strategies include pseudotyping and incorporation of targeting ligands into the fiber protein. Preclinical studies • Over 50 Ads with a lytic replicative cycle have been generated for cancer therapy purposes that display preferential lysis of targeted tumor versus normal cells and anti-tumor activity. • However, the viruses and the preclinical tumor models have limitations that interfere with the ability to fully evaluate the anti-tumor activity prior to entering the clinic. Clinical trials • 393 cancer patients in 21 clinical trials have been treated with oncolytic Ads by intratumoral, intraarterial, intravenous, intracerebral and mouthwash administration. • The safety and anti-tumor activity of three oncolytic Ads (dl-1520, Ad5-CD/TKrep, and CV706) either alone or in combination with chemo- or radiotherapies was demonstrated. • There is no correlation between baseline anti-Ad neutralizing antibodies and treatment response in those studies where a conclusion could be drawn. • Evidence of viral replication in tumors following intratumoral, intravenous and mouthwash delivery was demonstrated. However, viral replication was also detected in normal cells. Noninvasive imaging technology for monitoring virotherapy • Noninvasive imaging is a means to assess viral distribution, replication, safety and efficacy in both preclinical and clinical studies. • Nuclear imaging and magnetic resonance (MR) imaging are readily translatable to the clinic for viral imaging. • Optical imaging (fluorescence and bioluminescence) is restricted to small animal studies due to limited signal penetration depth.


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cancer patient treatment and allow improved virus-to-virus comparisons. Another limitation that currently exists is targeting infection by Ads to tumor cells and away from normal cells. This is particularly important for systemic administration where the majority of virus is bound by normal liver cells. Several oncolytic Ads with altered tropism have been generated, but have not entered clinical trials [4]. The continued measurement of AdNAb levels is important in all future studies as it is unclear whether the route of viral administration influences the magnitude of AdNAb response and if there is a correlation between baseline AdNAb levels and treatment Bibliography Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers. 1. Raper SE, Chirmule N, Lee FS et al.: Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab. 80, 148–158 (2003). 2. Kanerva A, Hemminki A: Modified adenoviruses for cancer gene therapy. Int. J. Cancer 110, 475–480 (2004). 3. Everts M, Curiel DT: Transductional targeting of adenoviral cancer gene therapy. Curr. Gene Ther. 4, 337–346 (2004). 4. Chu RL, Post DE, Khuri FR, Van Meir EG: Use of replicating oncolytic adenoviruses in combination therapy for cancer. Clin. Cancer Res. 10, 5299–5312 (2004). • Comprehensive review on oncolytic adenoviruses. 5. Dobbelstein M: Replicating adenoviruses in cancer therapy. Curr. Top. Microbiol. Immunol. 273291–27334 (2004). • Comprehensive review on oncolytic adenoviruses. 6. Post DE, Van Meir EG: A novel hypoxiainducible factor (HIF) activated oncolytic adenovirus for cancer therapy. Oncogene 22, 2065–2072 (2003). 7. Post DE, Devi NS, Li Z et al.: Cancer therapy with a replicating oncolytic adenovirus targeting the hypoxic microenvironment of tumors. Clin. Cancer Res. 10, 8603–8612 (2004). 8. Holm PS, Bergmann S, Jurchott K et al.: YB1 relocates to the nucleus in adenovirusinfected cells and facilitates viral replication by inducing E2 gene expression through the E2 late promoter. J. Biol. Chem. 277, 10427–10434 (2002). 9. Holm PS, Lage H, Bergmann S et al.: Multidrug-resistant cancer cells facilitate

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The authors regret that due to limits imposed by the journal on the number of references, they could not cite all original references. The authors wish to acknowledge support by NIH grants CA87830 and NS41403 (EGVM), NS49300 (DEP), and GM00680 (ETS).

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Website 101 www.onyx-pharm.com

• Dawn E Post Laboratory of Molecular Neuro-Oncology,, Department of Neurosurgery, Emory University School of Medicine, Emory University, 1365C Clifton Rd NE, Room C5068, Atlanta, GA 30322, USA Tel.: +1 404 778 2267; Fax: +1 404 778 5240; [email protected] • Hyunsuk Shim Departments of Hematology/Oncology,/Radiology, Emory University School of Medicine, Winship Cancer Institute, Atlanta, GA 30322, USA • Esra Toussaint-Smith Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA 30322, USA • Erwin G Van Meir Laboratory of Molecular Neuro-Oncology, Departments of Neurology, Hematology/Oncology, and, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA

(Accessed March 2005)

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