Phase II trial of intratumoral administration of ONYX-015, a replication-selective adenovirus, in patients with refractory head and neck cancer.

PURPOSE: To determine the safety, humoral immune response replication, and activity of multiple intratumoral injections of ONYX-015 (replication selective adenovirus) in patients with recurrent squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: This phase II trial enrolled patients with SCCHN who had recurrence/relapse after prior conventional treatment. Patients received ONYX-015 at a dose of 2 x 10(11) particles via intratumoral injection for either 5 consecutive days (standard) or twice daily for 2 consecutive weeks (hyperfractionated) during a 21-day cycle. Patients were monitored for tumor response, toxicity, and antibody formation. RESULTS: Forty patients (30 standard and 10 hyperfractionated) received 533 injections of ONYX-015. Standard treatment resulted in 14% partial to complete regression, 41% stable disease, and 45% progressive disease rates. Hyperfractionated treatment resulted in 10% complete response, 62% stable disease, and 29% progressive disease rates. Treatment-related toxicity included mild to moderate fever (67% overall) and injection site pain (47% on the standard regimen, 80% on the hyperfractionated regimen). Detectable circulating ONYX-015 genome suggestive of intratumoral replication was identified in 41% of tested patients on days 5 and 6 of cycle 1; 9% of patients had evidence of viral replication 10 days after injection during cycle 1, and no patients had evidence of replication > or = 22 days after injection. CONCLUSION: ONYX-015 can be safely administered via intratumoral injection to patients with recurrent/refractory SCCHN. ONYX-015 viremia is transient. Evidence of modest antitumoral activity is suggested.     

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.     

Phase I trial of intraperitoneal injection of the E1B-55-kd-gene-deleted adenovirus ONYX-015 (dl1520) given on days 1 through 5 every 3 weeks in patients with recurrent/refractory epithelial ovarian cancer.

PURPOSE: Resistance to chemotherapy in ovarian cancer is frequently associated with mutations in the p53 gene. The adenovirus dl1520 (ONYX-015) with the E1B 55-kd gene deleted, allowing selective replication in and lysis of p53-deficient tumor cells, has shown preclinical efficacy against p53-deficient nude mouse-human ovarian carcinomatosis xenografts. PATIENTS AND METHODS: We undertook a phase I trial of intraperitoneal dl1520 in patients with recurrent ovarian cancer. Sixteen women with recurrent/refractory ovarian cancer received 35 cycles (median, two cycles) of dl1520 delivered on days 1 through 5 in four dose cohorts: 1 x 10(9) plaque forming units (pfu), 1 x 10(10) pfu, 3 x 10(10) pfu, and 1 x 10(11) pfu. RESULTS: The most common significant toxicities related to virus administration were flu-like symptoms, emesis, and abdominal pain. One patient receiving 1 x 10(10) pfu developed common toxicity criteria grade 3 abdominal pain and diarrhea, which was dose-limiting. The maximum-tolerated dose was not reached at 10(11) pfu, and at this dose level patients did not experience significant toxicity. There was no clear-cut evidence of clinical or radiologic response in any patient. Blood samples were taken for adenovirus DNA and neutralizing antibodies. Polymerase chain reaction data indicating presence of virus up to 10 days after the final (day 5) infusion of dl1520 are suggestive of continuing viral replication. CONCLUSION: This article therefore describes the first clinical experience with the intraperitoneal delivery of any replication-competent/-selective virus in cancer patients.     

Efficacy with a replication-selective adenovirus plus cisplatin-based chemotherapy: dependence on sequencing but not p53 functional status or route of administration.

Replication-selective adenoviruses are being developed as novel anticancer therapeutics. Clinical trials with dl 1520, an E1B-Mr 55,000 gene-deleted adenovirus (ONYX-015), have demonstrated selective viral replication and biological activity in head and neck and ovarian carcinomas, but durable objective responses were not demonstrated. However, clinical results suggested potentially synergistic interactions with platinum-containing chemotherapy. To better characterize and optimize this interaction, we carried out combined modality treatment with ONYX-015 and cisplatin-based chemotherapy in three nude mouse-human tumor xenograft models with differing tumor locations or p53 functional status. Superior efficacy was demonstrated with combination therapy over either agent alone in all three models, independent of the route of ONYX-015 administration (intratumoral or i.p.). Virus replication was not demonstrably inhibited by cisplatin plus 5-fluorouracil chemotherapy. To assess the role of p53 function or cisplatin resistance in this interaction, we treated ovarian carcinomas that were matched except for p53 functional status (A2780, A2780/CP70). Combination therapy led to improved survival over either agent alone in both the p53(-) and the p53(+) carcinomatosis models. Efficacy was highly dependent on the sequencing of the agents; treatment with ONYX-015 prior to, or simultaneously with, chemotherapy was significantly superior to chemotherapy followed by ONYX-015. These results support further evaluation of replication-selective adenoviruses and cisplatin-based chemotherapy in clinical trials.     

Pilot trial of intravenous infusion of a replication-selective adenovirus (ONYX-015) in combination with chemotherapy or IL-2 treatment in refractory cancer patients

ONYX-015 is an adenovirus that selectively replicates in p53 dysfunctional or mutated malignant cells. We performed a pilot trial to determine the safety and feasibility of treatment with ONYX-015 delivered intravenously in patients with advanced malignancy. One cohort of five patients received ONYX-015 once a week for 6 weeks at a dose of 2 x 10(12) particles per infusion in combination with weekly infusions of irinotecan (CPT11, 125 mg per week) and 5-fluorouracil (5FU, 500 mg per week). A second cohort of five patients received the combination of ONYX-015 at a dose of 2 x 10(11) particles per week for 6 weeks in combination with interleukin 2 (IL 2, 1.1 x 10(6) units daily via subcutaneous injection for 5 days each week for 4 weeks). Toxicity attributable to ONYX-015 was limited to transient fever. All patients demonstrated elevations in neutralizing antibody titers within 4 weeks of the infusion of ONYX-015. Serum levels of IL-6, IL-10, tumor necrosis factor-alpha, and interferon-gamma increased within 6 hours of viral infusion, suggesting immune activation. This response was more pronounced in the cohort of patients who received 2 x 10(12) particles per infusion. Two patients demonstrated uptake of viral particles in malignant tissue by quantitative PCR. Electron microscopy confirmed selective cytoplasmic viral particles within malignant cells but not within adjacent normal tissue in a third patient. In conclusion ONYX-015 can be administered safely in combination with CPT11, 5FU or low-dose IL 2 and is able to access malignant tissue following intravenous infusion. Further investigation of ONYX-015, possibly with agents that may modulate replication activity, or duration of virus survival, is indicated.     

53基因及CAR受体对ONYX-015肿瘤杀伤作用的影响

目的:利用肿瘤特异性增殖型腺病毒ONYX-015分别感染具有柯萨奇病毒和腺病毒联合受体(CAR)水平正常、p53正常或突变的,以及CAR水平低下、p53突变的肿瘤细胞株,研究ONYX 015对这些肿瘤细胞的特异性增殖及杀伤能力。方法:以正常的肝细胞株L02作为对照,用细胞病变效应(CPE)实验观察ONYX-015对细胞的选择性杀伤效应;病毒增殖实验检测野生型腺病毒(Ad5)、ONYX 015在多种肿瘤细胞中的增殖能力。结果:ONYX 015对正常的肝细胞L02无杀伤性,但能够有效地杀伤p53突变的肝癌细胞Hep3B、p53正常的肝癌细胞HepG2及肺癌细胞A549,不能杀伤p53突变的人乳腺癌细胞株MDA-MB 231。在CAR受体水平正常的癌细胞株Hep3B、HepG2和A549中,Ad5和ONYX-015均可增殖。在CAR受体水平低下、p53突变的人乳腺癌细胞株MDA-MB 231中,两种病毒均不增殖。结论:CAR受体对ONYX-015的增殖力起着至关重要的作用。在CAR受体水平正常的前提下,无论肿瘤细胞的p53基因正常与否,ONYX-015均可以有效增殖并杀伤细胞;相反,如果CAR受体水平低下,即使该种肿瘤细胞p53基因突变,ONYX-015在该细胞中的增殖力也会受到限制。ONYX-015不杀伤CAR受体及p53基因均正常的正常肝细胞。     

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.     

In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy

The E1B-deleted, replication-competent ONYX-015 (dl1520) adenovirus was originally described as being able to selectively kill p53-deficient cells due to a requirement of p53 inactivation for efficient viral replication. This hypothesis has become controversial because subsequent in vitro studies have demonstrated that the host range specificity of ONYX-015 is independent of p53 gene status. Using a pair of isogenic cell lines that differ only in their p53 status, we demonstrate here that although ONYX-015 can replicate in both p53 wild-type and mutant cells in vitro, the virus demonstrates significantly greater antitumor activity against mutant p53 tumors in vivo. Moreover, ONYX-015 viral therapy can be combined with radiation to improve tumor control beyond that of either monotherapy. The results demonstrate that ONYX-015 can discern in vivo between tumors having a different p53 status and that it may be an effective neoadjuvant to radiation therapy.     

ONYX-015 works synergistically with chemotherapy in lung cancer cell lines and primary cultures freshly made from lung cancer patients

p53 mutations and loss of heterozygosity (LOH) have been detected in >50% of lung cancers. Wild-type p53 can prevent replication of damaged DNA and promote apoptosis of cells with abnormal DNA. A human adenovirus, ONYX-015, which has a deletion in the E1B region, has shown tumor-specific cytolytic effect in tumor cells with nonfunctional p53 and antitumor efficacy that can be augmented by chemotherapeutic agents. A recent report from an independent group, however, indicates that wild-type p53 is necessary for the infection of this replicating virus, and it is in direct contradiction to previous observations of the ONYX group. In this study, we carried out cytopathic effect (CPE) assays using ONYX-015 on five human lung cancer cell lines with known p53 status. Two of these cell lines, NCI-H522 and NCI-H1703, have mutations and LOH in their p53 gene. Both lines were lysed in a dose-dependent manner and showed 100% cytolysis at a multiplicity of infection of 0.1. Two additional cell lines, NCI-H2347 and NCI-H838, both of which have wild-type p53 gene, showed near complete lysis at a multiplicity of infection of 1. We demonstrate here that the lung cancer cells with nonfunctional p53 are at least 10 times more sensitive to ONYX-015 cytolysis than the lung cancer cells with wild-type p53. In addition, standard chemotherapeutic agents (paclitaxol and cisplatin) showed a synergistic effect when combined with ONYX-015, and this effect was p53 mutant dependent. Furthermore, we tested the cytolytic effect of ONYX-015 on a panel (n = 7) of primary first-passage cultures made from freshly resected lung cancers. ONYX-015 lysed primary lung cancer cells in six of seven (86 %) primary cultures. Two of four primary cultures treated with chemotherapeutic agents had a synergistic effect with ONYX-015. Our data indicate that wild-type p53 is not required for the infection of this replicating virus, and also we demonstrate that ONYX-015 is effective alone and works synergistically with chemotherapeutic agents in lung cancer cell lines and primary cultures. This study suggests that ONYX-015 may be effective, especially in combination with conventional chemotherapy, in the treatment of patients with lung cancer.     

Late viral RNA export, rather than p53 inactivation, determines ONYX-015 tumor selectivity

ONYX-015 is an adenovirus that lacks the E1B-55K gene product for p53 degradation. Thus, ONYX-015 was conceived as an oncolytic virus that would selectively replicate in p53-defective tumor cells. Here we show that loss of E1B-55K leads to the induction, but not the activation, of p53 in ONYX-015-infected primary cells. We use a novel adenovirus mutant, ONYX-053, to demonstrate that loss of E1B-55K-mediated late viral RNA export, rather than p53 degradation, restricts ONYX-015 replication in primary cells. In contrast, we show that tumor cells that support ONYX-015 replication provide the RNA export function of E1B-55K. These data reveal that tumor cells have altered mechanisms for RNA export and resolve the controversial role of p53 in governing ONYX-015 oncolytic selectivity.     

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.     

A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer.

An E1B 55 kDa gene-deleted adenovirus, Onyx-015, which reportedly selectively replicates in and lyses p53-deficient cells, was administered by a single intratumoral injection to a total of 22 patients with recurrent head and neck cancer. The objectives of this Phase I study were to determine the safety, feasibility, and efficacy of this therapy and determine any correlation to p53 status. Six cohorts were investigated with a dose escalation from 10(7)-10(11) plaque-forming units. Toxicity was assessed using NCIC criteria. Tumor response was assessed by clinical and radiological measurement. Blood samples were taken to detect adenovirus DNA and neutralizing antibody to adenovirus. Tumor biopsies were taken to detect adenovirus by in situ hybridization. Treatment was well tolerated, with the main toxicity being grade 1/2 flu-like symptoms. Dose-limiting toxicity was not reached at the highest dose of 10(11) plaque-forming units. Twenty-one of the 22 patients treated showed an increase in neutralizing antibody to adenovirus. In situ hybridization showed viral replication in 4 of 22 patients treated, all of whom had mutant p53 tumors. Using conventional response criteria, no objective responses were observed. However, magnetic resonance imaging scans were suggestive of tumor necrosis at the site of viral injection in five patients, three of whom were classified using nonconventional criteria as partial responders, and two of whom were classified using nonconventional criteria as minor responders. Of these five cases, four had mutant p53 tumors. The response duration for the three partial responders was 4, 8, and 12 weeks. An additional eight patients had stable disease in the injected tumors lasting from 4-8 weeks. These preliminary results show that intratumoral administration of Onyx-015 is feasible, well tolerated, and associated with biological activity. Further investigation of Onyx-015, particularly with a more frequent injection protocol and in combination with systemic chemotherapy, is warranted.     

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.     

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.     

p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53

ONYX-015 has been reported to kill selectively tumor cells lacking functional p53. Genetic alterations of INK4a/ARF locus, which is a predominant event in malignant pleural mesothelioma, may result in loss of p14(ARF) and subsequent disruption of p53 pathway in cancer cells. In the present study, ONYX-015 was able to kill three mesothelioma cell lines (H28, H513, and 211H) with wild-type p53 but lacking p14(ARF). In contrast, MS-1 mesothelioma cells, which expressed both p53 and p14(ARF), were resistant to ONYX-015. Introducing p14(ARF) gene into the H28 cell, a mesothelioma cell without p14(ARF) expression, significantly increased the resistance of this cell line to the cytolytic effect of ONYX-015. Our results suggest that human mesotheliomas with wild-type p53 yet lacking p14(ARF) are potential candidates for ONYX-015 therapy.     

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.     

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.     

Efficient induction of cell death by adenoviruses requires binding of E1B55k and p53

The use of an Elb55k-deficient adenovirus, ONYX-015, to selectively target tumor cells containing a mutated p53 gene has produced promising results. However, recent reports have questioned the selectivity of this virus, showing that ONYX-015 can replicate in cells containing a wild-type p53 and that p53 may actually be required for cell death. To address these apparent contradictions in the literature, we infected a number of mutant and wild-type p53-containing cell lines with ONYX-015 and wild-type adenovirus and observed their death profiles up to 10 days postinfection. We demonstrate that two distinct cell death phenotypes exist, one of which is rapid and dependent on the presence of p53 and one of which is p53 independent. Using adenoviruses expressing E1b55k proteins deficient in their ability to bind p53, we show that formation of a complex between p53 and the adenoviral Elb55k protein is necessary for the activation of the rapid cell death pathway. In the absence of p53 or the absence of complex formation between p53 and Elb55k, cell death is delayed considerably. These data suggest three things: that the selectivity of killing appears to be dependent on the presence of the E1b55k/p53 complex; that viruses lacking Elb55k (such as ONYX-015) kill cells in a delayed manner independent of p53; and that binding of E1b55k to p53 does not merely serve to inactivate p53, but rather is required for the induction of rapid cell death. The components of this complex that lead to rapid cell death remain to be determined.