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[Cancer Research 59, 2623-2628, June 1, 1999]
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[Cancer Research 59, 2623-2628, June 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Intravenous Administration of ONYX-015, a Selectively Replicating Adenovirus, Induces Antitumoral Efficacy

Carla C. Heise, Angelica M. Williams, Shirley Xue, Meisa Propst and David H. Kirn1

ONYX Pharmaceuticals, Richmond, California 94806


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Replication-incompetent viral vectors are being developed for the gene therapy of cancer. Although some of these may eventually be proven to have significant localized antitumoral activity, none to date have been shown to infect and cause regression of established tumors following i.v. administration. Because cancer is a systemic disease in almost all fatal cases, the lack of i.v. efficacy is a major limitation to treatment with replication-incompetent viral vectors. ONYX-015 (dl1520) is an attenuated adenovirus that replicates in and causes selective lysis of cancer cells. We carried out i.v. efficacy and distribution studies in nude mice with s.c. and intraparenchymal tumor xenografts. ONYX-015 infected and replicated efficiently within tumors following i.v. administration. Viral titers in livers were relatively high 3 h after administration but decreased rapidly, becoming undetectable after 24 h. Effective antitumor doses were not associated with hepatic toxicity. Viral replication within tumors was associated with regressions in several tumor models. Selectively replicating viruses like ONYX-015 hold promise as agents to treat metastatic cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene therapy approaches hold promise for the treatment of cancer. Replication-incompetent adenoviruses are frequently used as vectors for gene delivery to tumor cells (1, 2, 3) . Significant preclinical efficacy has been demonstrated with direct intratumoral injection of localized tumor masses with selectively replicating (4, 5, 6) and nonreplicating (3 , 7 , 8) adenoviral vectors. Tumor growth within the peritoneal cavity has also been significantly inhibited by i.p. injection of nonreplicating (9, 10, 11, 12) and selectively replicating (13) adenoviral agents. Unfortunately, cancer patients die due to systemic metastases in the vast majority of cases. i.v. efficacy against established tumors has not been shown with nonreplicating viral vectors to date. Therefore, one of the major limitations of nonreplicating viral vectors is their apparent inability to effectively treat systemic tumor metastases (14) . Here, we report data documenting i.v. efficacy with a selectively replicating adenovirus (ONYX-015) against established tumors.

The efficacy of i.v. administration of nonreplicating adenoviral vectors may be limited by several factors. Adenovirus constructs are rapidly cleared from the systemic circulation predominantly by the liver and spleen (15) . As a result, their half-life in the blood is short. Consequently, deposition within tumor masses would be expected to be minimal, and relatively few cells within the tumor would be infected. In addition, i.v. administration might also cause toxicity because of the exposure of normal cells to virus. Liver toxicity caused by high systemic concentrations of adenovirus has been documented in several studies (16 , 17) . Strategies are, therefore, needed to selectively amplify the expression of viral vectors in tumor tissue following i.v. administration.

ONYX-015 (dl1520) is an E1B gene-deleted adenovirus that replicates in and selectively lyses cancer cells (4 , 5) . We have previously reported antitumoral efficacy and increased survival following intratumoral injection of ONYX-015 in human tumor xenografts grown in nude mice. In contrast to replication-incompetent viruses, selective replication in tumor tissue leads to amplification of the virus. In this report, we examined the ability of ONYX-015 to infect and replicate in s.c. tumors following either (a) direct injection into and replication within a distant tumor or (b) i.v. administration. Infection and replication was documented in both cases. i.v. efficacy studies were then performed using s.c. or intraparenchymal tumor models in nude mice. The results demonstrate that ONYX-015 localized and replicated efficiently within tumors following i.v. administration and that viral replication was associated with significant antitumoral efficacy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Virus
ONYX-015 is an E1B 55 kDa gene-deleted adenovirus (also referred to as dl1520; Ref. 18 ). The virus contains a deletion between nucleotides 2496 and 3323 in the E1B region encoding the 55 kDa protein. In addition, a C to T transition at position 2022 in E1B generates a stop codon at the third codon position of the protein. These alterations eliminate expression of the E1B 55 kDa gene in ONYX-015-infected cells. ONYX-015 was grown in the human embryonic kidney cell line HEK293 and was purified by CsCl gradient ultracentrifugation as described previously (5) .

In Situ Hybridization of Adenovirus DNA
In situ hybridization was performed on formalin-fixed, paraffin-embedded tissue, cut into 5-µm sections. Slides were deparaffinized in xylenes, hydrated through ethanols, digested with proteinase K, and postfixed in 4% paraformaldehyde. Hybridization was carried out overnight at 37°C with 0.5 µg/ml biotinylated adenovirus DNA probe (Enzo Diagnostics, Inc., Farmingdale, NY). After three successive washes in 1x SSC at 55°C, an alkaline phosphatase conjugated-antibiotin antibody (Vector Laboratories) was applied. Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate was used as the chromagen, and slides were counterstained with nuclear fast red.

Biodistribution Studies
Distribution of ONYX-015 following i.v. Injection
For evaluation of the relative uptake of virus into liver and tumor after i.v. injection, female athymic nu/nu mice (Harlan Sprague Dawley, Inc., Indianapolis, IN) with bilateral s.c. C33A tumors (mean size, 250 mm3) were given 109 pfu2 of ONYX-015 in a single tail vein injection and euthanized at 3, 6, 24, or 72 h postinoculation (n = 6–8 per time point). Livers and tumors were excised, divided, and processed for viral titer or in situ hybridization.

Virus Titers in Tumor and Liver Tissue
Tissues were weighed, flash-frozen in liquid nitrogen, and stored at -70°C until they were titered. To prepare tissue homogenates, we minced thawed specimens in 0.5 ml of medium using sterile scalpels and further dissociated the samples through a mesh screen. Virus was extracted from the homogenate by three consecutive freeze/thaw cycles. The homogenate was centrifuged (2500 x g), and the virus titer in the supernatant was determined by a plaque assay using HEK293 cells.

i.v. Dosing and Liver Toxicity Studies
Several regimens were evaluated to assess a maximum tolerated i.v. dose of ONYX-015 in tumor-bearing nude mice (Table 1)Citation . Virus was diluted in PBS and injected into the tail vein in a volume of 0.1 ml. Mice were weighed and observed daily; if signs of acute toxicities (such as anorexia or lethargy) were noted, the mice were immediately euthanized.


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  		Table 1 i.v. dosing studies of ONYX-015 administered to nude mice

 
A single i.v. dose of ONYX-015 was injected into C57BL/6 mice to evaluate acute liver toxicity. The dose was chosen with approximate equivalence to the LD50 defined in the nude mouse dosing studies (5 x 109 pfu). When signs of acute toxicities were observed, mice were euthanized. Surviving animals were euthanized 14 days after virus injections. Gross pathological changes were recorded at necropsy, and livers were preserved in formalin for histopathological analysis.

Antitumor Efficacy Studies
s.c. Human Tumor Xenografts in Nude Mice
Intratumoral Injection Studies.
To study virus dissemination from an infected tumor to an uninfected tumor, C33A p53(-) cervical carcinoma cells (107 cells) were injected s.c. into each flank of five athymic, female nu/nu mice. When tumors reached 5–6 mm in diameter, one tumor was directly injected with 109 pfu of ONYX-015. Animals were euthanized 3 weeks after treatment, and both tumors were removed and analyzed for the presence of viral infection by immunohistochemistry for adenoviral hexon protein, as described previously (5) .

Suppression of new tumor outgrowth was evaluated following direct intratumoral injection of unilateral, s.c. C33A tumors with 108 pfu of ONYX-015 daily for 5 days (n = 13); controls (n = 10) received vehicle injections in identical fashion. Seven days after the first injection, 106 C33A cells were injected into the contralateral flank; previous studies with untreated mice showed an ~75% tumor formation rate following inoculation of 106 C33A cells. Mice were followed for 14 weeks for tumor development in the contralateral flank. Comparison of tumor-free survival was analyzed using the log-rank statistical test.

i.v. Administration Studies.
Dose-response relationships and injection schedules were studied using C33A human cervical cancer xenografts. Mice were pair-matched into control and treatment groups of 10 animals each. Two injection schedules were studied; a 109 pfu total dose was divided into either 2 doses of 5 x 108 pfu or 10 doses of 108 pfu. The 10-injection regimen was repeated using two different initial tumor volumes (20 and 40 mm3). Dose response was evaluated using a 109 or 108 pfu total dose, given in two injections. ONYX-015 or vehicle was administered i.v. into the tail vein in a volume of 0.2 ml.

For efficacy studies, three carcinoma cell lines that were sensitive to the cytopathic effects of ONYX-015 in vitro were used: HCT-116 and SW620 (human colon; n = 10 per group) and C33A (human cervix; n = 10 per group). One additional tumor type (HLaC laryngeal carcinoma; n = 5 per group) with relative resistance to ONYX-015 in vitro was also tested for in vivo sensitivity. Once tumors grew to 20–60 mm3, i.v. administration was initiated. A total dose of 109 pfu of ONYX-015 or 0.2 ml of vehicle (Tris-buffered saline with 10% glycerol) was given by tail vein (108 pfu daily for 10 days or 2 x 108 pfu daily for 5 days). Length and width of tumors were measured twice weekly. Mice were euthanized when tumors reached 1000 mm3. At necropsy, tumors and livers were excised and fixed in formalin for histopathology and in situ hybridization to detect adenovirus DNA.

Statistical comparison between tumor volumes in the treatment and control groups was analyzed using a two-tailed Student’s t test. Comparisons were made using the last measurements taken before any mice were euthanized due to tumor size. Mouse survival was compared using the log-rank test (P < 0.05 was considered significant).

Liver Metastases Model
Male CD-1 athymic nu/nu mice (Charles River Laboratories, Houston, TX) were used when they were 7–8 weeks old. HT29 human colon carcinoma cells (5 x 106 cells; American Type Culture Collection, Manassas, VA) were injected in 0.05 ml of culture medium into the posterior splenic parenchyma of each mouse. ONYX-015 was injected i.v. into the tail vein in five daily doses of 108 pfu each starting either 3 or 5 weeks after tumor cell inoculations. Control mice were injected with 0.1 ml of vehicle (Tris-buffered saline with 10% glycerol) in an identical manner (n = 10 mice per group). All animals were euthanized 7 weeks after tumor cells were given (i.e., 2 or 4 weeks after treatment). Livers were collected, examined macroscopically for tumors, then fixed in formalin for histology. The number of macroscopic hepatic metastases was recorded for each liver lobe and scored based on the following scale: 0, no tumor; 1, single tumor focus; 2, 2–4 tumor foci; 3, 5–10 tumor foci or confluent tumor involving less than one-third of the lobe; and 4, >10 tumor foci or confluent tumor involving more than one-third of the liver lobe. The maximum score assigned to a liver lobe was 4, and the maximum score possible for the whole liver was 16.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intravascular Virus Spread and Inhibition of Tumor Development at Distant Sites after Intratumoral Injection
Five nude mice with bilateral s.c. C33A tumors were used to evaluate ONYX-015 dissemination from a tumor directly injected with virus to an untreated tumor in the contralateral flank. When tumors were ~150 mm3, one tumor on each animal was directly injected with 109 pfu of ONYX-015. Three weeks later, four of the five untreated contralateral tumors had positive immunohistochemical staining for adenovirus hexon protein. Therefore, virus had spread from the treated tumor and initiated replication in the tumor localized in the contralateral flank. A longer-term study showed there was not significant growth inhibition of established 60–70 mm3 tumors after ONYX-015 injection (108 pfu for 5 consecutive days) into the tumor in the opposite flank (data not shown).

Although virus spread had no effect on the growth of distant established tumors, we performed one study to examine the effect on outgrowth of new tumors (microscopic tumor foci) following ONYX-015 injection into established tumors. Seven days after the first intratumoral injection of ONYX-015, 106 C33A carcinoma cells were injected into the contralateral flank, and mice were followed for up to 14 weeks for evidence of tumor development. Mice were sacrificed once they became moribund due to primary tumor progression or a contralateral tumor developed. Whereas 6 of 10 control mice developed contralateral tumors over the course of the study, only 2 of the 13 mice pretreated with ONYX-015 developed contralateral tumors (P = 0.04; Fig. 1Citation ). By 10 weeks, all remaining control mice had to be sacrificed due to the size of the primary tumor; four of these mice had not developed contralateral tumors.



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  		Fig. 1. ONYX-015 treatment in one s.c. tumor inhibited the outgrowth of tumors at a distant site of tumor cell inoculation. Data points, percentage of mice free of distant tumors following injection of a contralateral tumor with 109 pfu of ONYX-015 (•) or vehicle ({square}). Curve ends at 10 weeks for controls because all mice had been euthanized due to morbidity from progression of the primary vehicle-injected tumors.

 
Evaluation of Maximum Tolerated i.v. Doses of ONYX-015
Several different doses and regimens were explored to determine the maximum tolerated i.v. doses of ONYX-015 in nude mice with human tumor xenografts (Table 1)Citation . The highest dose evaluated that resulted in 100% survival was 1.7 x 109 pfu divided into five daily doses of 3.3 x 108 pfu. A dosing regimen of 5 x 109 pfu given as five daily injections of 109 pfu resulted in 50% lethality. Livers from the mice given the lethal dose of 5 x 1010 total pfu all exhibited severe hepatic necrosis (data not shown).

A single i.v. injection of 5 x 109 pfu of ONYX-015 was administered to C57BL/6 mice to evaluate liver pathology associated with acute adenovirus toxicity. Although syngeneic tumor models are not possible for efficacy studies using a replicating human adenovirus, we wanted to evaluate toxicity in an immunocompetent animal. Histopathological changes in livers from mice that died acutely (4–6 days after injection) included severe, diffuse hepatic necrosis and hepatocyte degeneration (Fig. 2)Citation . Mice that survived the treatment were sacrificed 2 weeks after virus injection; liver histology was normal in these animals.



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  		Fig. 2. Liver histology in C57BL/6 mice following a single i.v. injection of 5 x 109 pfu of ONYX-015. A and B, representative photomicrographs of H&E-stained liver sections from mice that died acutely (4–6 days after injection). A, multifocal hepatic necrosis and vacuolated, degenerating hepatocytes. B, higher magnification of A shows necrotic cells (long arrow) and hepatocyte vacuolation (short arrow). C and D, liver histology of surviving mice (sacrificed 2 weeks after injection). C, liver histology appears normal. D, higher magnification of C shows typical hepatocyte arrangement adjacent to terminal hepatic venule.

 
Distribution and Replication of ONYX-015 in Liver and Tumors after i.v. Injection
Studies were done to evaluate the uptake and replication of ONYX-015 in both liver (the main site of virus deposition following i.v. injection) and s.c. tumors after i.v. injection. Nude mice with bilateral s.c. C33A tumors were given 109 pfu of ONYX-015 in a single tail vein injection and euthanized 3, 6, 24, or 72 h later. Livers and tumors were excised and processed for viral titer or in situ hybridization. Tissue titers demonstrated that ONYX-015 is rapidly taken up by the liver (Fig. 3)Citation . Virus titer was highest in liver tissue (1 x 105 pfu/g) 6 h after injection. However, by 24 h, the titers of ONYX-015 in the liver had decreased by ~1000-fold, and by 72 h, the virus was undetectable. In contrast, virus titers in tumors increased by ~150-fold between 3 and 72 h after injection (Fig. 3)Citation .



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  		Fig. 3. Virus titer in liver ({blacksquare}) and C33A human tumor xenografts () in nude mice following a single i.v. injection of ONYX-015. By 72 h, virus was undetectable in liver (*), whereas virus titers in tumors increased over time.

 
In situ hybridization for adenovirus DNA was used to visualize virus-infected cells in tissue sections. No evidence of viral replication was detected within the livers at any time point. In contrast, virus replication was confirmed within increasing numbers of tumor cells over the time course of the study (data not shown). At 24 h, a few scattered infected cells were seen. By 72 h, infection was more widespread in focal areas near blood vessels at the periphery of the tumor. These results suggest that, although a small proportion of tumor cells were initially infected by the i.v. inoculation, this was followed by virus replication, release, and spread allowing infection of adjacent tumor cells.

Efficacy of i.v. ONYX-015 Treatment on s.c. Human Tumor Xenografts in Nude Mice
Dosing Studies in C33A Tumor Xenografts.
Inhibition of tumor growth and increased survival was observed when either of the 109 pfu dose groups (given in either 2 or 10 injections) were compared to vehicle-treated control mice. There were no significant differences in tumor growth or survival whether the dose was given as 108 pfu daily for 10 days or 5 x 108 pfu for 2 days. The mean tumor volume was reduced by 47% following ONYX-015 treatment compared to vehicle-treated mice (P = 0.004; Fig. 4ACitation ). Median survival was also significantly increased from 29 days for controls to 36 days with i.v. ONYX-015 treatment (P = 0.003). A 10-fold lower total dose (two doses of 5 x 107 pfu) did not result in significant reduction in tumor growth compared to control treatment.



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  		Fig. 4. Tumor growth inhibition after i.v. administration of ONYX-015. A, C33A tumor xenografts. Mice were treated i.v. with 10 doses of vehicle solution ({square}) or 109 pfu ONYX-015 divided into ten daily injections of 108 pfu (•). ONYX-015 significantly inhibited growth compared to control (P = 0.004). B, 10 daily injections of vehicle ({square}) or 108 pfu ONYX-015 (•) inhibited HCT116 tumor growth and resulted in two complete regressions (P = 0.02 versus control). C, five daily i.v. injections of 2 x 108 pfu ONYX-015 (•) inhibited SW620 tumor growth compared to vehicle ({square}; P = 0.002).

 
We also examined antitumoral effects of i.v. ONYX-015 treatment on HCT116 and SW620 colon carcinoma xenografts. Three of 10 ONYX-015-treated HCT116 tumors underwent complete regression, and the mean tumor volume of the treated tumors was significantly inhibited compared to vehicle-treated control mice (339 ± 98 versus 815 ± 161 mm3; P = 0.02; Fig. 4BCitation ). SW620 tumor xenografts were inhibited by 50% compared to controls (361 ± 40 versus 734 ± 93 mm3; P = 0.002; Fig. 4CCitation ).

Virus replication was documented by in situ hybridization in 100% of the tumors removed 3–4 weeks after i.v. injection. Viral replication-associated cytopathology and necrosis was observed in tumors from ONYX-015-treated mice (Fig. 5)Citation .



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  		Fig. 5. Viral replication and virus-associated cytopathology in HCT116 tumor xenografts after i.v. treatment with ONYX-015. Tumor was removed on study day 30 (i.v. injections were given on days 1–10). A, in situ hybridization for adenoviral DNA shows ONYX-015-infected tumor cells (dark blue stain) localized at the interface between viable and necrotic tissue. B, higher magnification of boxed area in A showing virus-infected cells. C, H&E-stained tissue section demonstrates cellular degeneration and nuclear condensation in regions of virus infection shown in B.

 
These results demonstrate that three cell lines with sensitivity to ONYX-015 in vitro are sensitive in vivo to i.v. ONYX-015. In contrast, HLaC tumor cells were less sensitive in vitro and in vivo (following intratumoral injection compared to C33A tumors), and these tumors were relatively resistant to i.v. ONYX-015 in vivo. No responses were documented and the tumor growth rates were not significantly different from vehicle-treated control tumors (data not shown).

Inhibition of Liver Metastases
Intrasplenic inoculation of nude mice with HT29 human colon carcinoma cells led to metastatic tumor development in the liver as well as primary tumor growth in the spleen. Initial studies showed grossly visible liver metastases in ~20–30% of animals at 3 weeks and 75–85% at 5 weeks after tumor cell inoculation (data not shown). At 7 weeks postinoculation, nine of nine vehicle-treated animals had developed numerous liver tumors (Table 2)Citation .


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  		Table 2 i.v. administration of ONYX-015 reduces liver metastases and splenic tumors in nude micea

 
Treatment with i.v. injections of ONYX-015 resulted in significantly reduced tumor burdens and tumor incidence. Treatment at either 3 or 5 weeks after injection of tumor cells caused a reduction in the incidence of liver tumors versus vehicle (50 and 40% of animals, respectively, versus 100%). Additionally, ONYX-015-treated animals showed a decreased number of liver metastases, decreased overall hepatic tumor burden and reduced number of splenic tumors compared to controls (Table 2)Citation . These results demonstrate that i.v. dosing of ONYX-015 has significant antitumoral activity against intraparenchymal HT29 tumors.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report extends our previous work with ONYX-015, a replication-competent adenovirus (4 , 5 , 19) , and demonstrates antitumoral efficacy following i.v. administration of the virus. Following intratumoral delivery, replication of ONYX-015 resulted in the release of virions into the bloodstream and subsequent infection of a distant tumor site. As a result, the growth of microscopic tumor deposits was inhibited. When the virus was administered by direct i.v. injection, tumor cell infection, intratumoral viral replication, and antitumor efficacy were observed. Although i.v. dosing results in relatively minute viral titers within the tumor compared with intratumoral injection, efficacy was seen following i.v. treatment. This may be a result of the enhanced intratumoral virus distribution documented following i.v. administration. Intratumoral virus distribution and localization at the tumor periphery (where tumor cell growth and invasion are greatest) have been shown to be critical determinants of efficacy with a replication-competent adenovirus (19) .

It is well documented that the host immune response prevents persistent expression of transgenes delivered by nonreplicating adenoviral vectors (15 , 20 , 21) . Although the T-cell response is an important factor in the long-term, 90% of the adenoviral vector is eliminated from the mouse liver within 24 h after i.v. administration (15) . This occurred in both immunocompetent and athymic nude mice, indicating the involvement of innate immune mechanisms. Because the innate immunity of athymic nude mice is intact, nude mouse-human tumor xenograft models have value for the study of replicating adenoviral constructs. It is encouraging that, despite rapid clearance of the entire input dose from the liver, ONYX-015 replication within the tumor was sustained and resulted in tumor growth inhibition. Replicating virus was detected as long as 4 weeks after i.v. administration. Therefore, the tumor microenvironment may be a sanctuary against innate immune system-mediated clearance of ONYX-015 or other selectively replicating viruses; further studies in syngeneic tumor models may help to clarify this issue.

Unfortunately, because mouse and rat tumors do not support efficient replication of human adenoviruses (22 , 23) , syngeneic immunocompetent rodent tumor models are not available to evaluate T lymphocyte-dependent immunity in the context of replication-dependent tumor destruction. In addition, the effect of neutralizing antibodies on the efficacy of i.v. ONYX-015 needs to be studied. Mice have no neutralizing antibodies to adenovirus at baseline, whereas adult humans have positive (albeit usually low titer) neutralizing antibody titers to Ad5 in ~80% of cases (24) . If future studies show neutralizing antibodies limit the efficacy of i.v. therapy, approaches to minimize antibody production or decrease antibody binding to the virus will need to be evaluated (25, 26, 27) .

Similarly, the replication and toxicity of ONYX-015 in normal tissues of the mouse may not necessarily predict what will be encountered in patients. For example, wild-type (nonattenuated) adenovirus replicates inefficiently and undergoes an abortive infection in mouse liver and lung after i.v. administration (16 , 22) . Definitive proof of safety and efficacy will have to await results from clinical trials with ONYX-015. To date, no liver toxicity has been reported following intratumoral or i.p. injections of ONYX-015 (1010 pfu daily for 5 consecutive days) into cancer patients in Phase I and II trials (28) ; systemic dissemination of the virus has been documented in some cases. In addition, direct intratumoral injection of masses within the liver did not result in ONYX-015-induced toxicity (29) . No damage to normal tissue was observed after direct injections of tumor margins in head and neck cancer patients. Therefore, ONYX-015 appears to be tumor selective in human cancer patients. However, to reach widely disseminated tumors, a systemic form of therapy will be necessary.

Selectively replicating adenoviruses may be powerful tools to specifically lyse tumor cells or to increase the amount and distribution of gene expression within tumors (30) . Other selectively replicating infectious agents such as herpesviruses or Salmonella may also have these properties (31, 32, 33) . For the i.v. efficacy of these agents to be optimized, however, improvements in delivery to tumors and intratumoral spread may be beneficial (34 , 35) .


   ACKNOWLEDGMENTS
 
We acknowledge the contributions of the Institute of Drug Development and ILEX Oncology (San Antonio, TX); Suzette Weber of ONYX Pharmaceuticals for animal studies; and Patrick Trown, Ali Fattaey, and Allan Balmain for insightful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at ONYX Pharmaceuticals, 3031 Research Drive, Richmond, CA 94806. Phone: (510) 222-9700; Fax: (510) 222-9758; E-mail: dkirn{at}onyx-pharm.com Back

2 The abbreviation used is: pfu, plaque-forming unit(s). Back

Received 1/ 7/99. Accepted 4/ 2/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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