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Clinical Cancer Research Vol. 12, 3137-3144, May 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Infectivity-Enhanced Adenoviruses Deliver Efficacy in Clinical Samples and Orthotopic Models of Disseminated Gastric Cancer

Lotta Kangasniemi1,2, Tuula Kiviluoto3, Anna Kanerva1,2,4, Mari Raki1,2, Tuuli Ranki1,2, Merja Sarkioja1,2, Hongju Wu7, Frank Marini8, Krister Höckerstedt5, Helena Isoniemi5, Henrik Alfthan6, Ulf-Håkan Stenman6, David T. Curiel7 and Akseli Hemminki1,2

Authors' Affiliations: 1 Rational Drug Design Program, University of Helsinki; Departments of 2 Oncology, 3 Surgery, and 4 Obstetrics and Gynecology, 5 Transplantation and Liver Surgery Unit, Department of Surgery, and 6 Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland; 7 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, Alabama; and 8 Department of Bone and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Akseli Hemminki, Rational Drug Design Program, University of Helsinki, P.O. Box 63, 00014 Helsinki, Finland. Phone: 358-9-1912-5464; Fax: 358-9-1912-5465; E-mail: akseli.hemminki{at}helsinki.fi.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous.

Experimental Design: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans.

Results: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer.

Conclusions: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.

Gastric cancer currently ranks second in global cancer mortality (1). Most patients are either diagnosed at an advanced stage, or develop relapse after apparently curative operation (2). When metastatic, gastric cancer remains incurable, and despite some recent advances, chemotherapy provides only a modest increase in survival, often with significant side effects (3). Therefore, new approaches are critical for the treatment of this common and deadly disease. Gene therapy with adenoviruses is a promising modality for the treatment of advanced cancers refractory to other therapies. Importantly, the safety and efficacy of the approach has been recently validated in landmark randomized trials (4–6). However, a limiting factor for the most frequently used serotype 5 adenoviruses (Ad5) is dependence on the coxsackie-adenovirus receptor (CAR), which is variably expressed in most advanced cancers (7). This has also been studied in the context of gastric cancer cell lines, which were shown to have low to moderate expression of CAR (8). Native Ad5 tropism can be modified to circumvent CAR deficiency in cancer cells. Transductional targeting of adenoviruses aims at enhanced transduction of the target cell, e.g., by incorporating targeting moieties into the fiber knob region. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide in the HI loop of the fiber knob allows the virus to utilize {alpha}vß-class integrins for binding and internalization (9). These integrins are frequently highly expressed in advanced gastric cancers (10). Adenoviruses with a COOH-terminal polylysine tail (11, 12) bind to heparan sulfates, often expressed to a high degree in gastric cancer (13). Substitution of the entire fiber knob was used in the construction of Ad5/3, an Ad5 vector that features a chimeric fiber with the adenovirus serotype 3 (Ad3) knob domain (14). The receptor for Ad3 is currently unknown, although some candidates have been proposed and CD46 seems to be emerging as a likely main receptor candidate (15–17). However, it is unknown if CD46 would also be the receptor for 5/3 chimeric viruses. Nevertheless, it is intriguing that CD46 has been reported to be highly expressed in advanced gastric cancers (18).

Replication-deficient vectors may be of use when tumor burden is small or when combined with another treatment modality (4, 5). However, when faced with advanced metastatic masses, tumor penetration is key to efficacy and cannot be easily achieved with replication-deficient viruses due to poor intratumoral diffusion (19). To overcome this obstacle, conditionally replicative oncolytic adenoviruses (CRAd) were constructed. Ad5-{Delta}24 (also called dl922-947) contains a 24-bp deletion in the constant region 2 of E1A. Thus, the expressed protein is unable to bind the retinoblastoma protein for the induction of S phase (20, 21). Therefore, the virus replicates selectively in cells deficient in the retinoblastoma/p16 pathway. Most advanced human tumors, including gastric cancers, are deficient in this crucial pathway (22–24). Importantly, in contrast with earlier CRAds such as the prototype dl1520 (25), the constant region 2 deletion does not reduce replication of the agent in tumor cells (21).

S.c. tumors have been widely used in cancer research due to the ease of inoculation and tumor measurement. Nevertheless, human cancers are rarely located s.c. and it is well-established that the tumor environment has an effect on growth and behavior, including response to antitumor agents. Furthermore, it has been shown that orthotopic tumors, growing in the correct organ or region, resemble the primary clinical tumor more accurately with regard to treatment sensitivity, than the same cells grown s.c. (26). Gastric cancer typically spreads i.p., which rationalizes i.p. delivery for reduction of systemic exposure. Therefore, we developed an orthotopic model of peritoneally disseminated gastric cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissues. A549 human lung adenocarcinoma cells and 293 embryonal kidney cells were from the American Type Culture Collection (Borås, Sweden). Six gastric cancer cell lines, comprising three intestinal (MKN-7, MKN-28, and MKN-74), two diffuse (KatoIII and MKN-45), and an adenosquamous type (MKN-1) were used (from Dr. Hiroshi Yokozaki). KatoIII is from American Type Culture Collection. All lines were cultured as recommended. Fresh gastric cancer samples were obtained with signed informed consent and ethical committee permission from patients undergoing surgery at Helsinki University Central Hospital. For transportation, the tissue was kept on ice in RPMI 1640 with 20% FCS, 200 IU/mL penicillin, 200 µL/mL streptomycin, and 2 mmol/L L-glutamine. Sample processing was started within an hour from surgery. The size of the available sample usually allowed only one dose of virus to be investigated.

Adenoviruses. Replication-competent and -incompetent viruses (Table 1 ) were propagated on A549 and 293 cells, respectively, and purified on cesium chloride gradients. The viral particle concentration was determined at 260 nm, and standard plaque assay on 293 cells was done for infectious particles.


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  		Table 1. Viruses used in the experiments

 
Gene transfer assays. Cells were infected with replication-deficient viruses for 30 minutes at room temperature in 200 µL of growth medium with 2% FCS. Cells were washed once and complete medium was added. After 24 hours, incubation at 37°C, luciferase (Luciferase Assay System, Promega, Madison, WI) or ß-gal (Galacto Light Plus, Tropix, Bedford, MA) assays were done.

Human gastric cancer samples were cut into 200 mg pieces and homogenized. Samples were washed twice with RPMI 1640 containing 2% FCS and infected with 2.5 x 108 viral particles in 500 µL of growth medium. After 1 hour of infection at 37°C, the medium was replaced with 2 mL of growth medium. Tissue homogenates were lysed by adding 200 µL of Cell Culture Lysis Buffer (Promega), or with lysis buffer for luminescent ß-galactosidase reporter system 3 (BD Biosciences, Palo Alto, CA). Samples were freeze-thawed thrice and supernatant was analyzed for luciferase or ß-gal as above. Protein concentration was determined by using a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA).

Biodistribution. Female 3- to 4-week-old NMRI nude mice were purchased from Taconic (Ejby, Denmark) and injected with 1 x 107 MKN-45 cells i.p. after quarantine. Eighteen days later, mice were randomized (n = 4-5/group) and injected i.p. with transductionally targeted replication-deficient viruses at 1 x 109 viral particles in 500 µL of MEM. Forty-eight hours later, selected organs were collected and snap-frozen. The tissue samples were homogenized and lysed with Cell Culture Lysis Buffer, freeze-thawed, and supernatant was analyzed for transgene expression and protein content as with the clinical samples. Animal experiments were approved by the Experimental Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland.

Cell viability. Gastric cancer cells were seeded at 1 x 104 cells/well on 96-well plates and cultured overnight. Cells in triplicate were infected in growth medium with 2% FCS. After 1 hour, medium was changed to 5% FCS which was replaced every other day. Ten to 14 days after infection, cell viability was measured with MTS assay (Promega).

In vivo survival. Female Fox Chase severe combined immunodeficiency mice (Taconic) were purchased at 4 to 5 weeks of age. MKN-28 cells (5 x 106) were injected i.p. into mice randomized into five treatment groups (n = 15). Ten days later, mice received a single i.p. injection of 1 x 109 viral particles in 500 µL of MEM. Serum samples for ßhCG were collected from the tail vein 50 days after injecting the cells (n = 2-6). ßhCG concentration was quantitated with a time-resolved immunofluorometric assay based on free ßhCG-specific monoclonal antibodies with a 2 pmol/L cutoff (27). Levels in non–tumor-bearing severe combined immunodeficiency mice were 11.8 pmol/L (n = 3, SE = 2.5).

Liver explants. Fresh normal human liver samples were obtained and transported as tumor samples above. Precision-cut, 200-µm-thick slices (1000 Plus Sectioning System, Vibratome, St. Louis, MO) were infected with CRAds at 107 viral particles and incubated at 37°C. At the indicated time points, tissue was homogenized, freeze-thawed thrice and infectious particles were determined by TCID50. During slicing, tissue was kept on ice in William's medium E (Sigma, St. Louis, MO) containing 25 mmol/L D-glucose and 50 µg/mL gentamicin.

Statistical analysis. Two-tailed Student's t test was used to compare the differences between CRAds and control viruses. Survival was analyzed according to Kaplan-Meier with SPSS 11.5 for Windows.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Capsid modifications increase gene transfer to gastric cancer cell lines. We compared a panel of adenoviruses in six gastric cancer cell lines representing three different histologic groups (Fig. 1A ). In CAR-positive nonmalignant 293 cells, differences in gene transfer were mostly <2-fold (Fig. 1B). With the intestinal gastric cancer cell lines, gene transfer with Ad5.pK7 was increased up to 24-fold in MKN-7 and 5-fold in MKN-28, compared with the wild-type Ad5 capsid virus. Transductional efficacy with Ad5pK21LacZ was increased up to 479-fold in MKN-7, 35-fold in MKN-28, and 52-fold in MKN-74; with Ad5lucRGD, the respective values in these cell lines were 34-, 8-, and 3-fold, and with Ad5/3luc1 it was 5-, 9-, and 3-fold.


Figure 1
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  		Fig. 1. A, capsid modifications increase the infectivity of gastric cancer cell lines. Cells were infected with transductionally modified replication-deficient viruses at the indicated viral particles/cell. Luciferase or lacZ activity was measured as relative light units (RLU) 24 hours after infection. Results represent RLU compared to the isogenic control virus with an unmodified serotype 5 capsid, which was given the value of 1. Mean background transgene activity was subtracted from the data. B, to investigate infectivity of nonmalignant cells, 293 human embryonal kidney cells were also analyzed. Columns, mean of triplicates; bars, ±SE (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
In the diffuse-type cell lines, gene transfer with Ad5.pK7 was increased 17-fold in KatoIII and 75-fold in MKN-45 compared with the virus with the Ad5 capsid. With Ad5pK21LacZ, relative increases in transduction were up to 15-fold in KatoIII and 71-fold in MKN-45. With Ad5lucRGD, the respective values in these cell lines were 8- and 10-fold, and with Ad5/3luc1, 5- and 7-fold. Ad5RGD.pK7 increased adenovirus gene level in MKN-45 up to 3-fold, but not in KatoIII cells. In adenosquamous MKN-1 cells, gene transfer was increased up to 52-fold with Ad5pK21LacZ and 29-fold with Ad5.pK7 compared with Ad5. Ad5/3luc1 and Ad5lucRGD increased gene transfer up to 3- and 6-fold, respectively.

Capsid modifications increase gene transfer to primary gastric cancer specimens. Ad5.pK7 increased gene transfer in six out of the seven samples up to 32-fold (Fig. 2 ). Ad5/3luc1 and Ad5lucRGD increased transduction in all the samples up to 256-fold and 198-fold, respectively. Ad5RGD.pK7 increased gene transfer in three out of seven samples, and up to 10-fold compared with Ad5. Ad5pK21LacZ was tested on two samples, and it was 11- and 15-fold more effective than the respective control Ad5LacZ.


Figure 2
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  		Fig. 2. A-G, capsid modifications increase the infectivity of fresh primary gastric cancer tissue from patients. Tissue samples were infected with transductionally targeted replication-deficient viruses at 2.5 x 108 viral particles. Luciferase or lacZ activity was measured as RLU 24 hours after infection. Results represent RLU compared to the isogenic control virus, which was given a value of 1. Mean background transgene activity was subtracted from the data. Columns, mean of quadruplicate readings; bars, ±SE. Ad5pK21LacZ was included when sufficient material was available for LacZ analysis (A and E). All samples were diffuse adenocarcinomas (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
Capsid modifications enhance gene transfer in vivo. Relative to Ad5, Ad5/3luc1 increased gene transfer to tumors, spleen, heart, and lungs, but not to liver or kidneys (Fig. 3A ). Ad5.pK7 augmented the transduction of tumors but not of other organs. Ad5pK21LacZ or Ad5RGD.pK7 did not affect gene transfer significantly. Reduced transduction of tumor was seen with Ad5lucRGD. When comparing marker gene level in the tumors versus the liver, Ad5RGD.pK7 resulted in 11 times more activity in tumor tissue than in the liver (Fig. 3B).


Figure 3
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  		Fig. 3. Biodistribution of capsid-modified viruses in nude mice with i.p. disseminated MKN-45 carcinomatosis. Transductionally targeted replication-deficient viruses were injected i.p. at 1 x 109 viral particles. Tumors and normal organs were collected 48 hours after injection and luciferase or lacZ activity was measured from tissue homogenates as RLU. A, biodistribution of viruses as measured by luciferase analysis of organs. B, tumor to liver ratios of gene transfer. Mean background transgene activity was subtracted from the data. Columns, mean of triplicates; bars, ±SE (*, P < 0.05).

 
Capsid modifications increase the oncolytic potential of CRAds in vitro. Three isogenic CRAds were available, with capsids identical to the replication-deficient viruses. In all of the cell lines, Ad5/3-{Delta}24 was the most oncolytic (Fig. 4 ); with well-differentiated MKN-28 and MKN-7 cells, as well as with poorly differentiated MKN-45 cells. Wild-type virus was the next best and Ad5-{Delta}24E3 was third. With well-differentiated MKN-74 cells, Ad5-{Delta}24E3 displayed nearly the same cell-killing capacity as Ad5/3-{Delta}24, and wild-type virus was the third most effective. In poorly differentiated KatoIII and adenosquamous MKN-1, there was no significant difference between Ad5/3-{Delta}24 and wild-type, which was the second most effective after Ad5/3-{Delta}24. With MKN-7, MKN-45, MKN-74, and MKN-1 cells, Ad5-{Delta}24RGD displayed only modest oncolytic activity. A CRAd with a polylysine modification of the fiber and a 24 bp deletion in E1A would have been of interest for comparison, but unfortunately has not been described.


Figure 4
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  		Fig. 4. Oncolysis of gastric cancer cells following infection with CRAds. Cells were infected with viruses at the indicated viral particles/cell and analyzed for cell viability. Bars, SE. MKN-28, MKN-7 and MKN-74 are intestinal, KatoIII and MKN-45 are diffuse, whereas MKN-1 is adenosquamous.

 
Capsid-modified CRAds increase survival of severe combined immunodeficiency mice bearing orthotopic gastric cancer. Median survival was 37 days in the nontreated (mock) group and 40 days in the group treated with replication-deficient Ad5luc1 (Fig. 5A ). With the CRAds, median survival was 64 days with both Ad5-{Delta}24E3 and Ad5-{Delta}24RGD, and 67 days in the group receiving Ad5/3-{Delta}24. There was no significant difference in survival between the mock mice versus the Ad5luc1-treated mice (P > 0.25). All CRAds prolonged the survival significantly both compared with the mock (P < 0.0005) as well as with the Ad5luc1-treated mice (P < 0.05). Despite a trend favoring Ad5/3-{Delta}24, there was no significant difference between the mice treated with Ad5-{Delta}24E3, Ad5/3-{Delta}24, or Ad5-{Delta}24RGD. Serum ßhCG concentration is a prognostic factor in patients with gastric cancer (28). Mean ßhCG values were lower in CRAd-treated mice (not significant, Fig. 5B).


Figure 5
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  		Fig. 5. Survival and tumor burden of mice bearing peritoneally disseminated gastric cancer. MKN-28 cells were injected i.p. into severe combined immunodeficiency mice, and tumor was allowed to develop for 10 days. Mice were treated i.p. with CRAds or replication-deficient control virus and followed for survival. A, all oncolytic viruses significantly improved the survival of mice over mock (P < 0.0005) and Ad5luc1 (P < 0.05) injected mice. B, 50 days after injecting the cells, serum samples were collected from the tail vein to measure tumor marker ßhCG.

 
Liver replication assay. Human liver explants were infected with CRAds and control viruses (Fig. 5). For most viruses, no replication was seen. However, functional titers of Ad5/3-{Delta}24 increased ~3-fold, which suggests a low but detectable level of virus replication.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
There is an urgent need for new approaches for the treatment of metastatic gastric cancer. Using adenoviruses that enter through non-CAR receptors, we were able to increase gene transfer to gastric cancer cell lines (Fig. 1A). There was no obvious systematic relationship between histologic tumor classification and the best capsid modification. However, Ad5.pK7 seemed to be rather effective in the poorly differentiated samples. Also, Ad5pK21LacZ increased gene transfer to all gastric cancer cell lines studied and Ad5/3luc1 was more effective than Ad5 in most lines.

An important part of the study was the analysis of clinical samples fresh from patients (Fig. 2). As it is increasingly accepted that established cell lines differ from the initial clinical tumors (29), it was not completely unexpected to see a different profile of gene transfer in comparison with the cell lines. Although many of the capsid modifications were more effective than Ad5, Ad5/3luc1 emerged as the "virus of choice." Moreover, given the variation between samples, the results suggest that for clinical intervention with adenovirus gene therapy, it might be useful to analyze the tumor before selecting a virus for treatment.

In vivo, in a model that closely resembles human metastatic disease, Ad5/3luc1 and Ad5.pK7 increased gene transfer to i.p. disseminated tumors (Fig. 3A). Ad5/3luc1 also showed increased gene transfer to the spleen, heart, and lungs. Importantly, as the liver is the most important organ with regard to side effects (30), neither virus increased hepatic gene transfer. More importantly, none of the CRAds replicated effectively in liver explants (Fig. 6 ). Although Ad5/3-{Delta}24 levels increased to ~3-fold, this is a rather ineffective replication compared with permissive cells, which can amplify the input dose up to 1010-fold (31). Nevertheless, liver toxicity should be monitored carefully in human trials.


Figure 6
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  		Fig. 6. A and B, human liver explants for analysis of virus replication. Precision-cut liver slices were infected with CRAds and the number of infectious particles was determined by the TCID50 at the indicated time points.

 
Promising gene transfer and biodistribution data rationalized analysis of the respective CRAds for oncolytic activity (Fig. 4). Three isogenic {Delta}24-type CRAds were available. Each has an identical 24-bp deletion in the constant region 2 of E1A, which allows selective replication in retinoblastoma/p16 pathway–deficient tumor cells. Also, each virus has an intact E3 region, which is important for effective oncolysis (32). The viruses only differ with regard to the capsid. Ad5/3-{Delta}24 was consistently the most efficient. Moreover, these data suggest that it might be of interest to create CRAds featuring pK7 or pK21.

Most patients who die of gastric cancer have i.p. disseminated disease. Therefore, we developed orthotopic murine models to allow the analysis of CRAds (Figs. 3 and 5). Treatment was given i.p. to maximize local activity whereas limiting systemic exposure. I.p. adjuvant chemotherapy has been found to increase survival for patients undergoing resection of stage III gastric cancer (33). The survival of mice treated with the CRAds was significantly improved compared with controls (P < 0.05; Fig. 5A). Although there seemed to be a trend favoring Ad5/3-{Delta}24 over the other CRAds, the difference was not statistically significant. All mice in the control groups died due to rapid disease progression. Analysis of ßhCG secreted from the tumor cells suggested a smaller tumor load in animals treated with the CRAds (Fig. 5B). Analysis of markers could allow sensitive and noninvasive detection of antitumor activity in this model as it requires only a small blood sample. However, this approach needs further optimization and would benefit from more frequent sampling at various time points.

These data set the stage for clinical testing of the agents. Gastric cancer may be a good disease for intervention with CRAds, due to the low effect and often significant toxicity of currently available agents. Also, frequent confinement of the disease to the peritoneal cavity creates a rationale for i.p. treatment. If confirmed, it would be useful that a number of CRAds have activity in human gastric cancer. Treatment with adenoviruses induces a neutralizing antibody response (34). Although this does not prevent readministration, particularly in the context of locoregional delivery, it could reduce the efficacy of subsequent cycles of therapy (35). However, neutralizing antibodies are conformation-sensitive, and it has been shown that even slight changes in the fiber allow escape from preexisting neutralizing antibodies (36–39). Thus, subsequent treatments could be given with viruses featuring different capsids.

A recent randomized phase III clinical trial validated the utility of CRAds for the treatment of squamous cell cancer of the head and neck or esophagus (6). Adding H101 to cisplatin and 5-fluorouracil chemotherapy increased the overall response rate from 39.6% to 78.8% (P < 0.0001). H101 is a CRAd that lacks the E1B55K protein, which may confer selectivity to tumor cells dysregulated in the p53/p14ARF pathway, and is closely related to dl1520, which has been previously evaluated in a number of phase I and II trials (40). In particular, promising activity was seen in a phase II trial when dl1520 was combined with cisplatin and 5-fluorouracil (41). Therefore, the phase III results with H101 are well in accord with earlier data with dl1520. Nevertheless, dl1520 is a prototype agent which is rather attenuated even in tumor cells, when compared with wild-type adenovirus (25, 40). In contrast, the 24-bp deletion in {Delta}24-based agents does not attenuate but may in fact increase replication of the virus (21), which could predict superior efficacy versus H101/dl1520, as already shown preclinically (21, 42).

The oncolytic potency of the viruses used here is further increased by capsid modification–mediated infectivity enhancement (40, 43). If preclinical data has predictive power, it is possible that this generation of CRAds could yield further improvements in efficacy (43–46). Moreover, it is important to note that the side effect profiles of agents currently used for treatment of metastatic cancers, including radiation therapy, chemotherapy, hormonal therapies, small molecular inhibitors, and monoclonal antibodies, are mostly different from the side effect profiles of oncolytic viruses. This could facilitate combination treatments with increased efficacy without increases in side effects (6, 31).

In summary, we have done a preclinical evaluation of various capsid-modified adenoviruses for the treatment of gastric cancer. Promising activity and favorable biodistribution was seen. Next, clinical evaluation is needed for the analysis of the safety and efficacy of these agents in humans. Eventually, such developments could translate into increased treatment options for patients with gastric cancer.


   Acknowledgments
 
We thank Dr. Hiroshi Yokozaki (Division of Surgical Pathology, Kobe University, Kobe, Japan) for gastric cancer cell lines and Prof. Esa Korpi (Institute of Biomedicine, University of Helsinki, Finland) for providing the facilities for the preparation of liver explants.


   Footnotes
 
Grant support: Helsinki University Central Hospital Research Funds (EVO), Academy of Finland, Emil Aaltonen Foundation, Finnish Cancer Society, University of Helsinki, Sigrid Juselius Foundation, Sohlberg Foundation, Biocentrum Helsinki, Instrumentarium Research Fund, Research and Science Foundation of Farmos and Finnish Oncology Association (L. Kangasniemi, A. Kanerva, M. Raki, T. Ranki, M. Sarkioja, and A Hemminki), and NIH grants R01CA083821, R01CA094081, and 1R01CA111569-01A1 (H. Wu and D.T. Curiel).

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.

Received 11/28/05; revised 2/28/06; accepted 3/10/06.


   References
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 Abstract
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 Results
 Discussion
 References
 

  1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74–108.[Abstract/Free Full Text]
  2. Ikeguchi M, Kaibara N. Detection of circulating cancer cells after a gastrectomy for gastric cancer. Surg Today 2005;35:436–41.[CrossRef][Medline]
  3. Ohtsu A. Current status and future prospects of chemotherapy for metastatic gastric cancer: a review. Gastric Cancer 2005;8:95–102.[CrossRef][Medline]
  4. Immonen A, Vapalahti M, Tyynela K, et al. AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study. Mol Ther 2004;10:967–72.[CrossRef][Medline]
  5. Pearson S, Jia H, Kandachi K. China approves first gene therapy. Nat Biotechnol 2004;22:3–4.[CrossRef][Medline]
  6. Xia ZJ, Chang JH, Zhang L, et al. Phase III randomized clinical trial of intratumoral injection of E1B gene-deleted adenovirus (H101) combined with cisplatin-based chemotherapy in treating squamous cell cancer of head and neck or esophagus. Ai Zheng 2004;23:1666–70.[Medline]
  7. Bauerschmitz GJ, Barker SD, Hemminki A. Adenoviral gene therapy for cancer: from vectors to targeted and replication competent agents [review]. Int J Oncol 2002;21:1161–74.[Medline]
  8. Ono HA, Davydova JG, Adachi Y, et al. Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer. J Gastroenterol 2005;40:31–42.[CrossRef][Medline]
  9. Dmitriev I, Krasnykh V, Miller CR, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998;72:9706–13.[Abstract/Free Full Text]
  10. Kawashima A, Tsugawa S, Boku A, et al. Expression of {alpha}v integrin family in gastric carcinomas: increased {alpha}vß6 is associated with lymph node metastasis. Pathol Res Pract 2003;199:57–64.[CrossRef][Medline]
  11. Wu H, Seki T, Dmitriev I, et al. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency. Hum Gene Ther 2002;13:1647–53.[CrossRef][Medline]
  12. Yotnda P, Zompeta C, Heslop HE, Andreeff M, Brenner MK, Marini F. Comparison of the efficiency of transduction of leukemic cells by fiber-modified adenoviruses. Hum Gene Ther 2004;15:1229–42.[CrossRef][Medline]
  13. Theocharis AD, Vynios DH, Papageorgakopoulou N, Skandalis SS, Theocharis DA. Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma. Int J Biochem Cell Biol 2003;35:376–90.[CrossRef][Medline]
  14. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res 2002;8:275–80.[Abstract/Free Full Text]
  15. Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med 2003;9:1408–12.[CrossRef][Medline]
  16. Short JJ, Pereboev AV, Kawakami Y, Vasu C, Holterman MJ, Curiel DT. Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. Virology 2004;322:349–59.[CrossRef][Medline]
  17. Sirena D, Lilienfeld B, Eisenhut M, et al. The human membrane cofactor CD46 is a receptor for species B adenovirus serotype 3. J Virol 2004;78:4454–62.[Abstract/Free Full Text]
  18. Juhl H, Helmig F, Baltzer K, Kalthoff H, Henne-Bruns D, Kremer B. Frequent expression of complement resistance factors CD46, CD55, and CD59 on gastrointestinal cancer cells limits the therapeutic potential of monoclonal antibody 17–1A. J Surg Oncol 1997;64:222–30.[CrossRef][Medline]
  19. Puumalainen AM, Vapalahti M, Agrawal RS, et al. ß-Galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses. Hum Gene Ther 1998;9:1769–74.[Medline]
  20. Fueyo J, Gomez-Manzano C, Alemany R, et al. A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo. Oncogene 2000;19:2–12.[CrossRef][Medline]
  21. Heise C, Hermiston T, Johnson L, et al. An adenovirus E1A mutant that demonstrates potent and selective systemic anti-tumoral efficacy. Nat Med 2000;6:1134–9.[CrossRef][Medline]
  22. Sherr CJ. Cancer cell cycles. Science 1996;274:1672–7.[Abstract/Free Full Text]
  23. Hernando E, Nahle Z, Juan G, et al. Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control. Nature 2004;430:797–802.[CrossRef][Medline]
  24. Rogoff HA, Kowalik TF. Life, death and E2F: linking proliferation control and DNA damage signaling via E2F1. Cell Cycle 2004;3:845–6.[Medline]
  25. Barker DD, Berk AJ. Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection. Virology 1987;156:107–21.[CrossRef][Medline]
  26. Hoffman RM. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 1999;17:343–59.[CrossRef][Medline]
  27. Alfthan H, Schroder J, Fraser R, Koskimies A, Halila H, Stenman UH. Choriogonadotropin and its ß subunit separated by hydrophobic-interaction chromatography and quantified in serum during pregnancy by time-resolved immunofluorometric assays. Clin Chem 1988;34:1758–62.[Abstract/Free Full Text]
  28. Louhimo J, Kokkola A, Alfthan H, Stenman UH, Haglund C. Preoperative hCGß and CA 72–4 are prognostic factors in gastric cancer. Int J Cancer 2004;111:929–33.[CrossRef][Medline]
  29. Lam JT, Kanerva A, Bauerschmitz GJ, et al. Inter-patient variation in efficacy of five oncolytic adenovirus candidates for ovarian cancer therapy. J Gene Med 2004;6:1333–42.[CrossRef][Medline]
  30. 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 2003;80:148–58.[CrossRef][Medline]
  31. Raki M, Kanerva A, Ristimaki A, et al. Combination of gemcitabine and Ad5/3-{Delta}24, a tropism modified conditionally replicating adenovirus, for the treatment of ovarian cancer. Gene Ther 2005;12:1198–205.[CrossRef][Medline]
  32. Suzuki K, Alemany R, Yamamoto M, Curiel DT. The presence of the adenovirus E3 region improves the oncolytic potency of conditionally replicative adenoviruses. Clin Cancer Res 2002;8:3348–59.[Abstract/Free Full Text]
  33. Yu W, Whang I, Suh I, Averbach A, Chang D, Sugarbaker PH. Prospective randomized trial of early postoperative intraperitoneal chemotherapy as an adjuvant to resectable gastric cancer. Ann Surg 1998;228:347–54.[CrossRef][Medline]
  34. Hemminki A, Wang M, Desmond RA, Strong TV, Alvarez RD, Curiel DT. Serum and ascites neutralizing antibodies in ovarian cancer patients treated with intraperitoneal adenoviral gene therapy. Hum Gene Ther 2002;13:1505–14.[CrossRef][Medline]
  35. Tsai V, Johnson DE, Rahman A, et al. Impact of human neutralizing antibodies on antitumor efficacy of an oncolytic adenovirus in a murine model. Clin Cancer Res 2004;10:7199–206.[Abstract/Free Full Text]
  36. Mastrangeli A, Harvey BG, Yao J, et al. "Sero-switch" adenovirus-mediated in vivo gene transfer: circumvention of anti-adenovirus humoral immune defenses against repeat adenovirus vector administration by changing the adenovirus serotype. Hum Gene Ther 1996;7:79–87.[CrossRef][Medline]
  37. Hemminki A, Belousova N, Zinn KR, et al. An adenovirus with enhanced infectivity mediates molecular chemotherapy of ovarian cancer cells and allows imaging of gene expression. Mol Ther 2001;4:223–31.[CrossRef][Medline]
  38. Hemminki A, Zinn KR, Liu B, et al. In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst 2002;94:741–9.[Abstract/Free Full Text]
  39. Kanerva A, Wang M, Bauerschmitz GJ, et al. Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses. Mol Ther 2002;5:695–704.[CrossRef][Medline]
  40. Kirn D. Clinical research results with dl1520 (Onyx-015), a replication-selective adenovirus for the treatment of cancer: what have we learned? Gene Ther 2001;8:89–98.[CrossRef][Medline]
  41. Khuri FR, Nemunaitis J, Ganly I, et al. a controlled trial of intratumoral ONYX-015, a selectively-replicating adenovirus, in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer. Nat Med 2000;6:879–85.[CrossRef][Medline]
  42. Jiang H, Gomez-Manzano C, Alemany R, et al. Comparative effect of oncolytic adenoviruses with E1A-55 kDa or E1B-55 kDa deletions in malignant gliomas. Neoplasia 2005;7:48–56.[CrossRef][Medline]
  43. Kanerva A, Zinn KR, Chaudhuri TR, et al. Enhanced therapeutic efficacy for ovarian cancer with a serotype 3 receptor-targeted oncolytic adenovirus. Mol Ther 2003;8:449–58.[CrossRef][Medline]
  44. Bauerschmitz GJ, Lam JT, Kanerva A, et al. Treatment of ovarian cancer with a tropism modified oncolytic adenovirus. Cancer Res 2002;62:1266–70.[Abstract/Free Full Text]
  45. Douglas JT, Kim M, Sumerel LA, Carey DE, Curiel DT. Efficient oncolysis by a replicating adenovirus (ad) in vivo is critically dependent on tumor expression of primary ad receptors. Cancer Res 2001;61:813–7.[Abstract/Free Full Text]
  46. Hemminki A, Dmitriev I, Liu B, Desmond RA, Alemany R, Curiel DT. Targeting oncolytic adenoviral agents to the epidermal growth factor pathway with a secretory fusion molecule. Cancer Res 2001;61:6377–81.[Abstract/Free Full Text]
  47. Suzuki K, Fueyo J, Krasnykh V, Reynolds PN, Curiel DT, Alemany R. A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency. Clin Cancer Res 2001;7:120–6.[Abstract/Free Full Text]



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