Sheila Heitner

Intratumoral Spread of Wild-Type Adenovirus Is Limited After Local Injection of Human Xenograft Tumors: Virus Persists and Spreads Systemically at Late Time Points

Oncolytic replicating adenoviruses are a promising new modality for the treatment of cancer. Despite the assumed biologic advantage of continued viral replication and spread from infected to uninfected cancer cells, early clinical trials demonstrate that the efficacy of current vectors is limited. In xenograft tumor models using immune-incompetent mice, wild-type adenovirus is also rarely able to eradicate established tumors. This suggests that innate immune mechanisms may clear the virus or that barriers within the tumor prevent viral spread. The aim of this study was to evaluate the kinetics of viral distribution and spread after intratumoral injection of virus in a human tumor xenograft model. After intratumoral injection of wild-type virus, high levels of titratable virus persisted within the xenograft tumors for at least 8 weeks. Virus distribution within the tumors as determined by immunohistochemistry was patchy, and virus-infected cells appeared to be flanked by tumor necrosis and connective tissue. The close proximity of virus-infected cells to the tumor-supporting structure, which is of murine origin, was clearly demonstrated using a DNA probe that specifically hybridizes to the B1 murine DNA repeat. Importantly, although virus was cleared from the circulation 6 hr after intratumoral injection, after 4 weeks systemic spread of virus was detected. In addition, vessels of infected tumors were surrounded by necrosis and an advancing rim of virus-infected tumor cells, suggesting reinfection of the xenograft tumor through the vasculature. These data suggest that human adenoviral spread within tumor xenografts is impaired by murine tumor-supporting structures. In addition, there is evidence for continued viral replication within the tumor, with subsequent systemic dissemination and reinfection of tumors via the tumor vasculature. Despite the limitations of immune-incompetent models, an understanding of the interactions between the virus and the tumor-bearing host is important in the design of effective therapies.

Wild-Type Adenovirus Decreases Tumor Xenograft Growth, but Despite Viral Persistence Complete Tumor Responses Are Rarely Achieved— Deletion of the Viral E1b-19-kD Gene Increases the Viral Oncolytic Effect

Strategies to target viral replication to tumor cells hold great promise for the treatment of cancer, but even with replicating adenoviruses complete tumor responses are rarely achieved. To evaluate replicating adenoviral vectors, we have used A549 human lung cancer nude mouse xenografts as a model system. Intratumoral injection of wild-type adenovirus (Ad309) significantly reduced tumor growth from day 14 (p = 0.04) onward; however, tumor volumes reached a plateau at day 50. At 100 days, high levels of titratable virus were present within persistent viable tumors. In contrast to viral injection into established tumors, when tumor cells were infected in vitro with wild-type virus and then mixed with uninfected tumor cells, 1% of infected cells was sufficient to prevent tumor establishment. An E1b-19kD-deleted viral mutant (Ad337) was more efficient than Ad309 in this cell-mixing model. Just 1 cell in 1000 infected with Ad337 prevented tumor growth. However, although better than wild-type virus, Ad337 was unable to eradicate established flank tumors. These data suggest that although replicating adenoviruses exhibit significant oncolytic activity, barriers within the established tumor, such as connective tissue and tumor matrix, may limit the spread of virus. Strategies to enhance viral spread through established tumors are therefore likely to greatly improve the therapeutic efficacy of replicating adenoviruses.

Deletion of the Adenoviral E1b-19kD Gene Enhances Tumor Cell Killing of a Replicating Adenoviral Vector

Replicating adenoviral vectors are a promising new modality for cancer treatment and clinical trials with such vectors are ongoing. Targeting these vectors to cancer cells has been the focus of research. However, even if perfect targeting were to be achieved, a vector still must effectively kill cancer cells and spread throughout the bulk of the tumor. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis and may therefore compromise the therapeutic efficacy of an adenoviral vector. In this study we have investigated if an E1b-19kD gene deletion could improve the ability of a replicating adenoviral vector to spread through and kill cancer cells. In several lung cancer cell lines an E1b-19kD-deleted virus (Ad337) induced substantially more apoptosis than did a wild-type virus (Ad309), and tumor cell survival was significantly reduced in three of four cell lines. In addition, the apoptotic effects of cisplatin or paclitaxel were augmented by Ad337, but inhibited by wild-type virus. The number of infectious virus particles in the supernatant of infected cells was increased with Ad337 compared with wild-type virus, indicating enhanced early viral release. Ad337, in contrast to Ad309, induced significantly larger plaques after infection of A549 cells. This well-described large plaque phenotype of an E1b-19kD mutant virus is likely the result of early viral release and enhanced cell-to-cell viral spread. Loss of E1b-19kD function caused only minor cell line-specific increase or decrease in viral yield. We conclude that deletion of the E1b-19kD gene may enhance the tumoricidal effects of a replicating adenoviral vector.

Targeting the Replication of Adenoviral Gene Therapy Vectors to Lung Cancer Cells: The Importance of the Adenoviral E1b-55kD Gene

It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.

Late Expression of p53 from a Replicating Adenovirus Improves Tumor Cell Killing and Is More Tumor Cell Specific than Expression of the Adenoviral Death Protein

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.

Angiopoietin-1 increases survival and reduces the development of lung edema induced by endotoxin administration in a murine model of acute lung injury.

Objective:To evaluate the effect of angiopoietin-1, an angiogenic growth factor, on lung capillary leakage and survival in a murine model of acute lung injury. Design:Laboratory investigation. Setting:Research laboratory at New York University School of Medicine and Department of Veterans Affairs, NY Harbor Healthcare System. Subjects:C57BL/6 mice weighing 18–20 g, susceptible to endotoxin-induced acute lung injury. Interventions:Acute lung injury was induced in C57BL/6 mice by the intraperitoneal administration of endotoxin. The effects of angiopoietin-1, expressed from a nonreplicating E1a-deleted adenovirus containing the angiopoietin-1 complementary DNA (AdAng1), on survival and lung injury were evaluated. An E1a-deleted adenovirus that does not contain a transgene (Ad312) and phosphate-buffered saline were used as controls. Measurements and Main Results:Angiopoietin-1 protein was detected by immunoblotting in the serum of mice that received an intraperitoneal injection of AdAng1 but not in mice that received the control virus Ad312. When compared with control groups, mice that received AdAng1 5 days before endotoxin administration had improved survival and significantly less protein leakage from the circulation into the lungs, as detected by quantitative spectrophotometric measurements of Evans blue dye. Furthermore, when compared with controls, histopathology and immunostaining of lungs against CD31 and smooth muscle actin suggested preservation of vascular integrity and decreased tissue damage in mice pretreated with AdAng1. When endotoxin administration preceded infection with AdAng1 by 3 hrs, no benefit was observed. Conclusions:These data show that adenoviral mediated expression of angiopoietin-1 can protect against the development of lung capillary protein leak and decrease the mortality induced by endotoxin. However, the timing of AdAng1 administration in relation to the onset of lung injury may be critical.

292. Modification of the p53 Transgene of a Replication-Competent Adenovirus Improves p53 Stability and Cancer Cell Killing

Top of pageAbstract Replication-competent adenoviruses have oncolytic properties, but this oncolytic effect is insufficient for successful cancer therapy. In order to improve the anti-tumor activity, a replication-competent adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle was previously constructed. p53 expression from this vector significantly improved tumor cell killing and viral spread (Hum Gen Ther 2002: 13, 859). However, the full extent of this effect may be limited by the expression of inhibitory adenoviral and cellular proteins. The goal of the current work is to further enhance the anti-tumor activity of the p53-expressing replicating vector. The adenoviral E1b-55kD protein and cellular mdm2 have been shown to bind and inactivate p53. E1b-55kD is an early viral protein and p53 expression from Adp53rc has been engineered to peak late in the viral life cycle. However, the effect of p53 may still be limited by an interaction with E1b-55kD and mdm2. We hypothesized that modification of the p53 transgene to prevent binding to E1b-55kD and mdm2 would further improve the oncolytic effect of the vector. Therefore, a new vector (Adp53 Δ23) that is identical to Adp53rc except for a change of amino acid 23 (from tryptophan to serine) within the E1b-55kD/mdm2 binding region of the p53 transgene was constructed. In contrast to wild-type p53, the modified p53 protein expressed from this vector did not co-precipitate with E1b-55kD. In addition, the half-life of the modified p53 protein was prolonged. After viral infection with Adp53rc and Adp53 Δ23 for 24 hours, new protein production was inhibited with cycloheximide and p53 levels were analyzed by immunoblotting. Wild-type p53 was rapidly degraded and almost undetectable at 1 hour. In contrast, the levels of the modified p53 did not substantially change for at least 24 hours. Expression of the modified p53 protein did not impair effective virus propagation. Replication of Adp53 Δ23, measured by a modified plaque assay, was similar to replication of Adp53rc and an identical p53-negative control virus (Ad-co). Survival of three cancer cell lines (H1299, A549, H116), evaluated by WST-1 assay, was substantially decreased after infection with Adp53 Δ23 compared to Adp53rc and Ad-co. In conclusion, modification of the p53 transgene of a replication-competent adenovirus improves p53 stability and cancer cell killing without impairment of viral replication.