Onyx 015 clinical trial

ONYX CI , an adenovirus modified selectively to replicate in and kill cells that harbor p53 mutations, is under development by Onyx Pharmaceuticals for the potential treatment of various solid tumors, including head and neck, gastrointestinal and pancreatic tumors.

It is a recombinant adenovirus that carries a loss-of-function mutation at the E1B locus, the product of which is a 55 kDa protein that binds to and inactivates the p53 tumor suppressor protein.

Wild-type adenoviruses must disable this gene before viral replication can occur. The function of p53 is closely linked to the cellular machinery promoting cell cycle progression. This became very clear when a new tumour suppressor protein was identified, p14ARF, which is induced by activation of E2F and acts as an upstream activator of p53 Bates et al , ; Palmero et al , The result of Mdm2 neutralization by p14ARF is that p53 protein levels will rise and subsequently cause cell cycle arrest and apoptosis.

Furthermore, recent studies have demonstrated additional mechanisms for p14ARF's function in controlling p53 activity independent of its ability to sequester Mdm2 in the nucleolus Weber et al , As the cellular p53 protein level rises, p53 binds to the promoter of the Mdm2 gene, stimulating its transcription pMdm2 negative feedback loop; Piette et al , Importantly, the INK4a gene locus is frequently targeted by mutations in a variety of human cancers, making it the second most altered gene locus in tumours Orlow et al , ; Thompson et al , Amplifications of the Mdm2 oncogene and loss of p14ARF have been reported as an alternative mechanism of inactivating pdependent checkpoint control Cordon-Cardo et al , Defects in both pathways Rb pathway and p53 pathway then allow tumour cells to escape protective responses triggered by p53, such as growth arrest and apoptosis, following activation of E2F by oncogenes or DNA-damage.

It has been known for many years that a variety of viruses are capable of altering the cell cycle. Viruses have evolved to utilize molecular factors in the infected cell in a way that allows maximizing their replication and production.

In this respect, viruses and cancer cells share a common goal: to replicate their DNA freely and efficiently. Not surprisingly, viruses interfere with the same signal transduction pathways that are altered in cancer, promoting G1 to S-phase transition of the cell cycle.

In particular, p53 and Rb dependent cell cycle checkpoints are bypassed through virus induced mechanisms. Viruses have evolved gene products that either interact physically with cell cycle regulatory proteins or activate their transcription, thereby mimicking cell cycle activation in quiescent cells. One major function of the early adenoviral gene products, such as E1A and E1B, is to push the infected cell into the S phase of the cell cycle Flint and Shenk, , since it is necessary for the virus in order to use the cellular DNA replication machinery to replicate its own genome efficiently.

On the other hand, E1A also induces the expression of p14ARF and subsequent accumulation of active p53 in the nucleus of the cell de Stanchina et al , This would lead to growth arrest of the infected cell induced via a p21WAF1 triggered mechanism el-Deiry et al , In both cases viral yields would be significantly reduced. The E1B19K protein is a functional homologue of the proto-oncogene Bcl-2 and prevents apoptosis Rao et al , ; Debbas and White, The E1B55K gene product is capable of binding p53 and inactivating it Kao et al , Together with another early viral protein, the E4orf6 protein, E1B55K exports p53 to the cytoplasm and targets it for degradation Moore et al , ; Querido et al , Furthermore, at later times in infection, both proteins act in concert to facilitate the transport of viral late mRNAs while inhibiting the transport of most cellular mRNAs Babiss et al , ; Halbert et al , Lane and Crawford, ; Linzer and Levine, ; zur Hausen, Theses mutations render ONYX incapable of blocking p53's function.

Infection of normal cells with ONYX should evoke a p53 response: either growth arrest or apoptosis, resulting in aberrant viral replication. Replication of ONYX should therefore be restricted to p53 deficient cells resulting in selective destruction of cancer cells.

In the first experiments to test this hypothesis, ONYX's ability to grow was tested in tumour cell lines of known p53 status Bischoff et al , These experiments suggested a correlation between p53 status of the tumour cell lines and susceptibility to ONYX Genetic experiments in tumour cell lines provided further evidence for ONYX's p53 selectivity.

Functionally pdeficient cell lines were derived from tumour cell lines with wild-type p53 protein RKO and A by expression of a dominant negative p53 allele. While ONYX replication is not supported in the parental cell lines, the p53 defective derivative was killed at very low multiplicities of infection MOI; Bischoff et al , ; Rogulski et al , b ; Ganly et al , These experiments provide genetic evidence for an important role of p53 in response to virus infection and underscore the necessity for adenovirus to eliminate p53's function in order to replicate efficiently.

In agreement with the initial hypothesis, investigations in normal cells demonstrated that ONYX does not replicate in normal human mammary epithelial cells and microvascular endothelial cells, while wild-type adenovirus is not restricted in its replication in these cells under identical conditions Heise et al , Subsequent work by several laboratories, however, found that a variety of tumour cell lines with wild-type p53 allow efficient replication of ONYX Goodrum and Ornelles, ; Rothmann et al , ; Turnell et al , In a recent study, we were able to explain some of these observations.

We found that lack of p14ARF expression in tumours with wild-type p53 disrupts the p53 signalling pathway leading to high and uncontrolled Mdm2 protein activities and, therefore, facilitates ONYX replication Ries et al , Through deregulation of Mdm2, p53 is inhibited from exerting its protective effects after adenoviral infection. Re-introduction of functional p14ARF into such tumour cells led to induction of p53 activity and prevented replication of ONYX, but not wild-type adenovirus.

This protective effect of p14ARF is p53 dependent. Re-introduction of p14ARF did not prevent replication of ONYX in an isogenic cell line, in which the wild-type p53 gene has been deleted by homologous recombination Ries et al , Thus, genetic lesions affecting molecular factors within the p53 pathway other than mutations of p53 itself can render cells permissive for ONYX Finally, it should be noted that E1B55K has functions other than neutralizing p53, such as promoting the export of late viral mRNAs Babiss et al , ; Halbert et al , In some cells types, differences of replication efficacy between wild-type adenovirus and ONYX might be explained by these pindependent functions of E1B55K.

Until recently, the prevailing view was that adenovirus had developed strategies to negate pdependent effects in order to circumvent cellular mechanisms for inhibition of viral replication. In recent studies, it was speculated that wild-type p53 might actually be required for efficient adenovirus replication Dix et al , despite the fact that several cell lines with various mutations in the p53 gene including homozygous deletions supported efficient replication of wild-type adenovirus and ONYX The study fails to assess the actual production of a new virus in cell lines with wild-type p53 and did not examine infected cells for evidence of apoptosis.

Therefore, the experiments did not distinguish between cell death due to viral replication or apoptosis.

The debate on the role of p53 as a regulator of adenovirus replication was extended recently by an interesting study in which researchers used a mutant p53 molecule containing sequences from the p53 homologue p73 to analyze p53 dependent effects on virus replication Koch et al , According to the authors, this chimeric molecule cannot be degraded by E1B55K while the function of p53 as a transcription factor remains fully preserved.

Curiously, this mutant p53 molecule had no inhibitory effect on replication of wild-type adenovirus or ONYX in normal or tumour cells Koch et al , Their analysis of transcriptional targets of p53 is restricted to p21WAF1. Of particular importance, they do not examine expression of the proapoptotic gene Bax. Experiments in primary cells have shown that the suppression of ONYX replication by p53 in normal cells is preferentially mediated through Bax L Johnson and C O'Shea, personal communication.

The clinical studies also do not support the view that ONYX replicates in normal cells with functional p As discussed in the following paragraphs, ONYX has no demonstrable toxicity in animals or humans, and in some clinical studies can be shown to replicate in tumours lacking functional p Subsequent to the initial investigations in cell lines, studies in tumour bearing mice were performed to evaluate the anti-tumour efficacy of ONYX, dose-limiting toxicities, effects of scheduling, and the possibility of combining this treatment in a meaningful way with other established therapeutic modalities.

Initial experiments in xenograft models of human tumours harbouring wild-type p53 or mutated p53 demonstrated significant anti-tumour activity of intratumourally administered ONYX in tumours with mutated Heise et al , In contrast, ONYX injection into tumours derived from wild-type p53 glioblastoma cells had no inhibitory effect on tumour growth. While mutated p53 tumours showed signs of virus replication, as demonstrated by in situ hybridization and immunohistochemistry, this was not the case in the wild-type p53 cells Heise et al , In addition, mixing experiments were performed in which only a fraction of subcutaneously injected cells has been pre-incubated with ONYX These data correlate well with in vitro studies and demonstrated significant therapeutic efficacy of ONYX in vivo with evidence for pdependent virus replication.

Subsequent studies examined xenograft tumours derived from a laryngeal cancer cell line, HLaC, that showed an increased survival without achieving complete remissions following ONYX treatment. Although these cells harbour wild-type p53, the pathway is partly inactivated because of a mutation of the p14ARF tumour suppressor gene. A Phase II trial of intralesional ONYX 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. 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 Patients were assessed for response and toxicity. However, it has not been established how the host's p53 genotype might affect the efficacy of this strategy, and neutralizing antibody titers before or after treatment with dl have not been predictive of antitumor activity. Current data from trials suggest that this approach is safe, but there is a difference in efficacy between in vivo animal models and human use.

Although there is clear evidence of some efficacy, the available clinical data suggest that even with multiple intratumoral injections, there is room to improve the efficacy of these treatments for a larger proportion of patients. Lastly, to improve the potency of oncolytic viruses, the dynamics between virus replication, release and spread on one side and immune-mediated viral clearance on the other have to be better understood.

STS are a group of histologically and genetically diverse cancers that predominantly arise from mesenchymal cells. Prognosis and preferred treatment depend on histological grade and tumor stage.

While low-grade tumors are usually curable by surgery alone, high-grade sarcomas are associated with higher local treatment failure rates and increased metastatic potential. Dose escalation was performed to determine the maximum tolerated dose, but the maximum was not reached; the treatment regimen was well tolerated. In vivo viral replication was evaluated by in situ hybridization of tumor biopsy and qPCR of peripheral blood. Furthermore, the authors observed viral replication in some patients.

However, it is unclear whether the chemotherapy regimen inhibited viral DNA replication. One patient achieved a partial response of the virus-inoculated lesion and other metastasis. Further evaluation of this treatment modality is warranted, but establishing another antitumor agent for this tumor type will be most welcome, as in noncurable neoplasms therapeutic agents are often used sequentially to halt progression of the tumor and to achieve the best possible quality of life for patients.

Wildner O. Oncolytic viruses as therapeutic agents. Ann Med ; 33 : — Replication-selective virotherapy for cancer: biological principles, risk management and future directions. Nat Med — Shenk T. Lippincott-Raven: Philadelphia, , pp — Google Scholar.

Bischoff JR et al. An adenovirus mutant that replicates selectively in pdeficient human tumor cells. Science ; : — Heise C et al. ONYX, an E1B gene-attenuated adenovirus, causes tumor-specific cytolysis and antitumoral efficacy that can be augmented by standard chemotherapeutic agents.

Nat Med ; 3 : — Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein.