Anti-neoplastic viral agents

Details Anti-neoplastic viral agents Anti-neoplastic viral agents

2001-07-30

2003-04-08

Mosher, Mary E. (Department: 1648)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C435S320100, C435S235100, C435S471000, C435S475000, C435S091400, C435S091330, C514S04400A

Reexamination Certificate

active

06544507

ABSTRACT:

The present invention provides viral agents that have application in the treatment of neoplasms such as tumours, particularly tumours derived from colon cells, more particularly liver tumours that are metastases of colon cell primary tumours. Still more particularly are provided replication efficient adenovirus constructs that selectively replicate in response to transcription activators present in tumour cells, these factors being present either exclusively or at elevated levels in tumour cells as compared to other cells, and thus which lead to tumour cell death and cell lysis.
By injecting these viral agents locally into the liver it is possible to treat liver metastases; which are a major cause of morbidity in colon cancer patients. Applications beyond this, e.g. to other sites and other tumours, such as colorectal cancers and melanomas, are also provided.
Colon cancer presents with locally advanced or metastatic disease in the majority of patients. Most patients are left with liver metastases as the only site of disease after resection of the primary tumour. Partial liver resection only cures about 10% of patients, while in patients with multiple metastases in both liver lobes resection is not feasible and loco-regional or systemic treatment with chemotherapy is indicated (Labianca et al., 1997). Systemic chemotherapy with 5-fluorouracil and leucovorin or irinotecan will produce response rates of only 20% (Cunningham et al., 1998; Stupp et al., 1998).
Locoregional chemotherapy of the liver has been explored for over 15 years. Most liver metastases are supplied with blood by the hepatic artery, so intra-arterial hepatic chemotherapy (IAHC) allows for much higher exposure of the metastases to cytotoxic drugs. The high extraction rate of normal liver decreases the systemic drug concentration resulting in less toxicity with IAHC having been shown repeatedly to give response rates over 60% (Kemeny et al., 1987; Kemeny et al., 1992; Patt and Mavligit, 1991).
Specific defects in tumour cells make it possible to devise rational strategies for targeting tumour cells without harming normal cells. With the exception of anti-angiogenic therapy, this usually requires introduction of exogenous DNA into tumour cells, and the most efficient way to do this is with viruses. For example, U.S. Pat. No. 5,698,443 describes a tumour specific adenovirus that is targeted at prostate cancer cells. This virus utilises a prostate specific enhancer sequence driving the viral E1 genes and the patent suggests its use to express toxins specifically in the target cell.
Viruses which replicate selectively in tumour cells have great potential for gene therapy for cancer. In principle, selectively replicating cytotoxic viruses can spread progressively through a tumour until all of its cells are destroyed. This overcomes the need to infect all tumour cells at the time the virus is injected, which is a major limitation to conventional replacement gene therapy, because in principle virus goes on being produced, lysing cells on release of new virus, until no tumour cells remain. An important fundamental distinction in cancer gene therapy is thus between single hit approaches, using non-replicating viruses, and multiple hit approaches, using replicating viruses.
Single hit approaches work by directly transducing tumour cells with toxic genes; ignoring bystander effects, one virus particle kills one cell. Examples include restoration of tumour suppressor gene expression and conditional expression of toxins using tumour-specific promoters. For single hit approaches the amount of virus injected is an important limiting factor. Multiple hit approaches circumvent this limitation either by provoking an immune reaction against tumour cells, or by using viruses that replicate within the tumour. Since the majority of tumour cells are not killed by the injected virus itself, the amount of virus injected should not be an important factor limiting the therapeutic response.
Classic gene replacement therapy has been performed with retroviruses expressing p53 (Roth et al., 1996). Since p53 can be converted to its oncogenic form by mutations at over 500 sites in the open reading frame (Flaman et al., 1994), retroviral replication will convert at least 1% of the transduced p53 to such undesired mutant form. This means that each patient received around 25 million infectious units of virus expressing mutant p53 (Estreicher and Iggo, 1996).
A more promising approach expressing wild type p53 using an adenovirus, Ad-CMV-p53, has been demonstrated clinically in head and neck cancer and is currently under investigation in lung, colon and liver cancer (Clayman et al., 1998). Adenoviruses are relatively stable, can be produced at high titres, and can infect both quiescent and dividing cells of many different types. Overall, Ad-CMV-p53 appears exceptionally non-toxic but probably ineffective as a single agent; hence, there is a place for more aggressive second generation viruses.
Further target specific defects are mutations of p16, cdk4, cyclin D or Rb (Bartek et al., 1997) in the retinoblastoma pathway which cause loss of G1/S control and essentially all tumours have these. The only significant exception is colon cancer, where mutations in the Rb pathway itself are rare. The net result of these defects is increased E2F activity, which means that tumours can be selectively targeted by viruses expressing toxic genes from E2F-regulated promoters. This has been demonstrated using an adenovirus expressing the HSV thymidine kinase gene from such a promoter (Parr et al., 1997); cells containing Rb-pathway mutations express tk and can be killed by ganciclovir. Such an approach relies on an increase in the activity of specific transcription factors in tumour cells.
The rational basis for tumour targeting is better understood for non-replicating E2F-targeting viruses than it is for p53, but both are still single hit approaches and it is very difficult to see how they can ever be used for more than treatment of local disease. The tumour burden in late stage disease is around 10
12
cells, so while at an effective multiplicity of infection of one treatment would be feasible, in practice biodistribution and receptor problems mean that many orders of magnitude higher multiplicities are required.
One elegant way to circumvent this limitation is to recruit the immune system to kill the tumour cells. The role of the gene therapy virus is simply to provoke or reinforce the immune response. There is abundant evidence that tumours express new antigens, but in cancer patients the immune system has clearly failed to prevent tumour formation. Many currently attempted techniques target single antigens, eg production of cytotoxic T cells against MAGE antigens in melanoma, but the goal for therapy must be to induce a simultaneous response against multiple different antigens, because genetic instability in tumours means targeting of single antigens is unlikely to produce lasting responses because the number of tumour cells exceeds the mutation rate. Hence acquired resistance to immunotherapy is common.
An attractive solution to the single hit problem is to produce virus within the tumour. This can be achieved by injecting retroviral producer cell lines into the tumour bed, a strategy currently being tested in a clinical trial for glioblastoma (reviewed by Roth and Cristiano, 1997). This is an elegant but limited approach as it relies on immune privilege in the CNS to avoid immediate rejection of the grafted cells, and tumour targeting depends on the fact that retroviruses do not infect non-dividing normal brain cells. It falls short of the goal of tumour cell-specific viral replication because the viruses produced are not themselves replication competent and virus production is dependent on survival of the packaging cells rather than the presence of tumour cells.
The prototype tumour selective virus is a defective adenovirus lacking the E1B 55K gene (d1 1520ONYX 015, Bischoff et al., 1996). In normal adenoviruses 55K inactivates p53, hence it should not be requi

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