Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus.     

Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX-015) in human malignant glioma xenografts

In spite of aggressive surgery, irradiation and/or chemotherapy, treatment of malignant gliomas remains a major challenge in adults and children due to high treatment failure. We have demonstrated significant cell lysis and antitumour activity of the E1B-55 kDa-gene-deleted adenovirus ONYX-015 (dl1520, CI-1042; ONYX Pharmaceuticals) in subcutaneous human malignant glioma xenografts deriving from primary tumours. Here, we show the combined efficacy of this oncolytic therapy with radiation therapy. Total body irradiation (5 Gy) of athymic nude mice prior to intratumoral injections of ONYX-015 1 x 10(8) PFU daily for 5 consecutive days yielded additive tumour growth delays in the p53 mutant xenograft IGRG88. Radiation therapy was potentiated in the p53 functional tumour IGRG121 with a 'subtherapeutic' dose of 1 x 10(7) PFU daily for 5 consecutive days, inducing significant tumour growth delay, 90% tumour regression and 50% tumour-free survivors 4 months after treatment. These potentiating effects were not due to increased adenoviral infectivity or replication. Furthermore, cell lysis and induction of apoptosis, the major mechanisms for adenoviral antitumour activity, did not play a major role in the combined treatment strategy. Interestingly, the oncolytic adenovirus seemed to accelerate radiation-induced tumour fibrosis. Potentiating antitumour activity suggests the development of this combined treatment for these highly malignant tumours.     

Phase II clinical trial of intralesional administration of the oncolytic adenovirus ONYX-015 in patients with hepatobiliary tumors with correlative p53 studies.

PURPOSE: ONYX-015 is a genetically modified adenovirus with a deletion of the E1B early gene and is therefore designed to replicate preferentially in p53-mutated cells. A Phase II trial of intralesional ONYX-015 was conducted in patients with hepatobiliary tumors to determine the safety and efficacy of such a treatment. EXPERIMENTAL DESIGN: All patients had biopsy-proven, measurable tumors of the liver, gall bladder, or bile ducts that were beyond the scope of surgical resection. Patients received intralesional injections of ONYX-015 at either 6 x 10(9) or 1 x 10(10) plaque-forming units/lesion up to a total dose of 3 x 10(10) plaque-forming units, and i.p. injections were allowed in patients with malignant ascites. The status of p53 was assessed by immunohistochemistry or Affymetrix GeneChip microarray analysis. Studies were conducted for viral shedding and for the presence of antiadenoviral antibodies before and after the injection of ONYX-015. Patients were assessed for response and toxicity. RESULTS: Twenty patients were enrolled, and 19 patients were eligible. Half of the patients had primary bile duct carcinomas. Serious toxicities (> grade 2) were uncommon and included hepatic toxicity (three patients), anemia (one patient), infection (one patient), and cardiac toxicity (one patient, atrial fibrillation). Sixteen patients were evaluable for response. Among these evaluable patients, 1 of 16 (6.3%) had a partial response, 1 of 16 (6.3%) had prolonged disease stabilization (49 weeks), and 8 of 16 (50%) had a >50% reduction in tumor markers. Of the 19 eligible patients, 18 (94.7%) had specimens available for p53 analysis. Fifteen of these 18 patients (83.3%) had evidence of p53 mutation by one or both methods, although the methods correlated poorly. Viral shedding was confined to bile (two of two patients) and ascites (four of four patients). Pretreatment adenoviral antibodies were present in 14 of 14 patients and increased by 33.2% after ONYX-015 treatment. CONCLUSIONS: Intralesional treatment with ONYX-015 in patients with hepatobiliary tumors is safe and well tolerated, and some patients had evidence of an anticancer effect. The high incidence of p53 mutations in these tumors makes this a logical population in which to test this therapy but precludes definitive evaluation about the necessity of a p53 mutation for ONYX-015 clinical activity.     

ONYX-015: mechanisms of action and clinical potential of a replication-selective adenovirus

Accumulated knowledge in the molecular processes of tumour development combined with the availability of genetically modified viruses resemble the basis for new promising cancer therapeutics. The main advantages of employing replication-competent viruses are achievement of tumour selective killing and amplification of their oncolytic potential within the tumour mass. In this review, we describe the development of ONYX-015, one of the first and most advanced replication-competent viruses for cancer therapy. We discuss the molecular biology of this therapeutic approach and the interesting results obtained with this virus in clinical trials.     

Interactions between adenovirus proteins and the p53 pathway: the development of ONYX-015

dl1520 (ONYX-015) is an adenovirus mutant that lacks the E1b 55K gene. As a result it cannot neutralize p53. Therefore dl1520 should only grow in cells that lack p53, and should replicate selectively in cancer cells. However, there is no correlation between replication and p53 status, in cancer cells. This is for two reasons: (1) E1B 55K has additional functions, that are necessary in some tumor cells and not in others. (2) p53 function can be lost by alternative mechanisms such as loss of p14ARF. In normal cells, dl1520 induces p53, and is generally strongly attenuated for replication. ONYX-015 is currently being tested in clinical trials, and is a promising new therapeutic agent in cancer.     

Intravenous administration of ONYX-015, a selectively replicating adenovirus, induces antitumoral efficacy.

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 (d11520) 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.     

An attenuated adenovirus, ONYX-015, as mouthwash therapy for premalignant oral dysplasia.

PURPOSE: Dysplastic lesions of the oral epithelium are known precursors of oral cancer. A significant proportion of oral dysplastic lesions have functional defects in p53 response pathways. The ONYX-015 adenovirus is selectively cytotoxic to cells carrying defects in p53-dependent signaling pathways. The current study sought to establish the feasibility and activity of ONYX-015 administered topically as a mouthwash to patients with clinically apparent and histologically dysplastic lesions of the oral mucosa. PATIENTS AND METHODS: A total of 22 patients (19 assessable patients) were enrolled onto the study. ONYX-015 was administered on three different schedules to consecutive cohorts. Biopsies of the involved mucosa were performed to evaluate histologic response and changes in expression of putative markers of malignant potential, including p53, cyclin D1, and Ki-67. Serology was performed to measure antiadenoviral titers. RESULTS: Histologic resolution of dysplasia was seen in seven (37%) of 19 patients, and the grade of dysplasia improved in one additional patient. The majority of responses were transient. No toxicity greater than grade 2 (febrile episode in one patient) was observed. Only one of seven patients demonstrated an increase in circulating antiadenoviral antibody titer while on therapy. Although responding and resistant lesions had similar mean p53 staining at baseline, histologic response correlated with a decrease in p53 positivity over time. Significant changes in cyclin D1 or Ki-67 were not observed. Viral replication was confirmed in two of three lesions examined. CONCLUSION: This novel approach to cancer prevention is tolerable, feasible, and has demonstrable activity.     

Efficacy of a replication-competent adenovirus (ONYX-015) following intratumoral injection: intratumoral spread and distribution effects

ONYX-015 is an E1B-deleted adenovirus that replicates in and causes lysis of p53-deficient cancer cells selectively. To study the efficiency of intratumoral (i.t.) spread by ONYX-015, we infected specific fractions of tumor cells (two p53-deficient tumor lines and one p53 functional line) in vitro before subcutaneous inoculation into nude mice. Infection of as few as 5% of p53- tumor cells prevented tumor development in all cases; infection of 1% of p53- tumor cells resulted in significant growth inhibition but did not prevent tumor formation. In contrast, infection with ONYX-015 had no significant effect on p53+ tumor formation. These data suggested that replication-dependent tumor cell lysis and spread was occurring, but that tumor destruction might be improved by increasing i.t. virus distribution. Two treatment parameters were then varied to determine whether virus distribution, and consequently efficacy, could be improved. Divided i.t. injections of virus were more efficacious than a single injection of the same total dose. Likewise, increasing the volume of the viral suspension for i.t. injection allowed better distribution within the tumor mass and increased efficacy. These results have implications for the treatment of cancer patients with viral agents.     

ONYX-015 selectivity and the p14ARF pathway.

ONYX-015, dl1520, is an adenovirus that lacks the E1B 55K gene and therefore lacks the capacity to neutralize p53 during infection. This virus induces high levels of p53 and fails to grow efficiently in primary epithelial cells. However, it does replicate in many tumor cells, including those expressing wild-type p53. In these cells, ONYX-015 fails to induce active p53. This is because the pathway from Ela to p53 is disrupted through loss of p14ARF. We propose that high levels of Mdm2 activity resulting from loss of p14ARF, and high levels of Mdm2 protein resulting from activated Ras prevent accumulation of functional p53 during infection of tumor cells that retain wild-type p53.     

Selective replication and oncolysis in p53 mutant tumors with ONYX-015, an E1B-55kD gene-deleted adenovirus, in patients with advanced head and neck cancer: a phase II trial

ONYX-015 is an E1B-55kDa gene-deleted adenovirus engineered to selectively replicate in and lyse p53-deficient cancer cells. To evaluate the selectivity of ONYX-015 replication and cytopathic effects for the first time in humans, we carried out a Phase II clinical testing of intratumoral and peritumoral ONYX-015 injection in 37 patients with recurrent head and neck carcinoma. Patients received ONYX-015 at a daily dose of 1 x 10(10) plaque-forming units (pfu) via intratumoral injection for 5 days during week 1 of each 3-week cycle (n = 30; cohort A), or 1 x 10(10) pfu twice a day for 10 days during weeks 1 and 2 of each 3-week cycle. Posttreatment biopsies documented selective ONYX-015 presence and/or replication in the tumor tissue of 7 of 11 patients biopsied on days 5-14, but not in immediately adjacent normal tissue (0 of 11 patients; P = 0.01). Tissue destruction was also highly selective; significant tumor regression (>50%) occurred in 21% of evaluable patients, whereas no toxicity to injected normal peritumoral tissues was demonstrated. p53 mutant tumors were significantly more likely to undergo ONYX-015-induced necrosis (7 of 12) than were p53 wild-type tumors (0 of 7; P = 0.017). High neutralizing antibody titers did not prevent infection and/or replication within tumors. ONYX-015 is the first genetically engineered replication-competent virus to demonstrate selective intratumoral replication and necrosis in patients. This agent demonstrates the promise of replication-selective viruses as a novel therapeutic platform against cancer.     

Heat shock phenocopies E1B-55K late functions and selectively sensitizes refractory tumor cells to ONYX-015 oncolytic viral therapy

ONYX-015 is an E1B-55K-deleted adenovirus that has promising clinical activity as a cancer therapy. However, many tumor cells fail to support ONYX-015 oncolytic replication. E1B-55K functions include p53 degradation, RNA export, and host protein shutoff. Here, we show that resistant tumor cell lines fail to provide the RNA export functions of E1B-55K necessary for ONYX-015 replication; viral 100K mRNA export is necessary for host protein shutoff. However, heat shock rescues late viral RNA export and renders refractory tumor cells permissive to ONYX-015. These data indicate that heat shock and late adenoviral RNAs may converge upon a common mechanism for their export. Moreover, these data suggest that the concomitant induction of a heat shock response could significantly improve ONYX-015 cancer therapy.     

A mutant oncolytic adenovirus targeting the Rb pathway produces anti-glioma effect in vivo

Effective anti cancer strategies necessitate the use of agents that target tumor cells rather than normal tissues. In this study, we constructed a tumor-selective adenovirus, Delta24, that carries a 24-bp deletion in the E1A region responsible for binding Rb protein. Immunoprecipitation analyses verified that this deletion rendered Delta24 unable to bind the Rb protein. However, titration experiments in 293 cells demonstrated that the Delta24 adenovirus could replicate in and lyse cancer cells with great efficiency. Lysis of most human glioma cells was observed within 10 - 14 days after infection with Delta24 at 10 PFU/cell. In vivo, a single dose of the Delta24 virus induced a 66.3% inhibition (P<0.005) and multiple injections, an 83.8% inhibition (P<0.01) of tumor growth in nude mice. However, normal fibroblasts or cancer cells with restored Rb activity were resistant to the Delta24 adenovirus. These results suggest that the E1A-mutant Delta24 adenovirus may be clinically and therapeutically useful against gliomas and possibly other cancers with disrupted Rb pathway.     

Temozolomide renders murine cancer cells susceptible to oncolytic adenovirus replication and oncolysis

The preclinical evaluation of oncolytic adenoviruses (OAds) has been limited to cancer xenograft mouse models because OAds replicate poorly in murine cancer cells. The alkylating agent temozolomide (TMZ) has been shown to enhance oncolytic virotherapy in human cancer cells; therefore, we investigated whether TMZ could increase OAd replication and oncolysis in murine cancer cells. To test our hypothesis, three murine cancer cells were infected with OAd (E1b-deleted) alone or in combination with TMZ. TMZ increased OAd-mediated oncolysis in all three murine cancer cells tested. This increased oncolysis was, at least in part, due to productive virus replication, apoptosis, and autophagy induction. Most importantly, murine lung non-cancerous cells were not affected by OAd+TMZ. Moreover, TMZ increased Ad transduction efficiency. However, TMZ did not increase coxsackievirus and adenovirus receptor; therefore, other mechanism could be implicated on the transduction efficiency. These results showed, for the first time, that TMZ could render murine tumor cells more susceptible to oncolytic virotherapy. The proposed combination of OAds with TMZ presents an attractive approach towards the evaluation of OAd potency and safety in syngeneic mouse models using these murine cancer cell-lines in vivo.     

肿瘤增殖腺病毒ONYX-015的实验研究及临床研究进展

肿瘤增殖病毒在肿瘤细胞内的感染、复制及溶解细胞作用增强,而在正常细胞内这种能力减弱甚至消失.E1b55 kD蛋白缺失的肿瘤增殖病毒是第1个用于肿瘤临床的增殖病毒,也是最成功的肿瘤病毒治疗系统,并于1999年进入Ⅲ期临床试验.现综述ONYX-015的实验研究和临床研究进展.     

Oncolytic activity of the E1B-55 kDa-deleted adenovirus ONYX-015 is independent of cellular p53 status in human malignant glioma xenografts

Treatment of malignant gliomas remains a major challenge in adults and children because of high treatment failure. The E1B 55 kDa-gene deleted adenovirus, ONYX-015 (ONYX Pharmaceuticals), was demonstrated to replicate selectively in and lyse tumor cells. Currently ongoing clinical trials of ONYX-015 in head and neck tumors are promising. Here, we demonstrate ONYX-015-mediated cell lysis and antitumor activity in three of four s.c. human malignant glioma xenografts deriving from primary tumors. Intratumoral injections of ONYX-015, 1 x 10(8) plaque-forming units daily for 5 consecutive days, yielded significant tumor growth delay in the p53 mutant xenografts IGRG88 and the p53 wild-type IGRG93 and IGRG121 treated at an advanced tumor stage. The p53 wild-type tumors IGRG93 and IGRG121 experienced 45% and 82% complete tumor regressions. Four and 8 of 11 animals, respectively, survived tumor free 4 months after treatment. Widespread intratumoral adenoviral replication was observed in tumor cells of these two xenografts compared with only scattered replication in the p53-mutant tumors. In addition to a fast tumor growth rate, wild-type p53 status was associated with increased antitumor activity of the E1B-attenuated virus, and induction of functional p53 may therefore determine adenoviral cytolysis in tumor cells. In conclusion, ONYX-015 displayed a major antitumor activity in human xenografts derived from primary malignant glioma supporting its development in the treatment of these highly malignant tumors.     

溶瘤腺病毒的安全性研究进展

溶瘤腺病毒又称条件复制性腺病毒（conditionally replicating adenovirus,CRAD）,是一种用于肿瘤治疗的生物制剂,其安全性备受人们的关注,如何系统化评价其毒性是应用于临床的关键步骤。近年来,对于溶瘤腺病毒安全性的报道越来越多,包括从特异性杀伤肿瘤细胞的细胞实验到动物的毒理实验等。本文对溶瘤腺病毒的细胞实验、荷瘤小鼠治疗的毒性、动物急性毒性实验及长期毒性实验、生殖毒性实验的研究及其评估方法和目前临床研究状况进行综述。