Focus: Breast Cancer

Cancers are heterogeneous diseases that, in many cases, are not effectively managed with existing treatments. Oncolytic viruses are promising biotherapeutic tools for cancer, whose clinical and commercial development is progressing rapidly. Currently, a major preclinical initiative in the field is to engineer next generation viruses or virus/drug combinations that break through the barriers to successful treatment. 

Our goal is to harness the innate cytokine response of the immune system — normally a major impediment to oncolytic virus therapy — to attack and kill tumours. Our preliminary experiments demonstrate one strategy to accomplish this: antagonizing the Inhibitor of APoptosis (IAP) gene family. In this case, a drug that antagonized the “Inhibitor of APoptosis” (IAP) gene family rewired tumour cells to initiate a self-destruct sequence upon exposure to cytokines, which were strongly induced in tumours infected with an oncolytic virus.  

To build upon these data, and to broaden the underlying concept, we propose a research plan to (1) elucidate the mechanisms responsible for synergy between oncolytic virus therapy and IAP antagonism, and (2) identify new opportunities to reprogram tumour cells to die in response to the cytokine cloud generated by oncolytic virus therapy.

Vaccine-strain measles virus is a promising platform for the development of genetically programmable anti-cancer therapies. Measles vaccine virus has a natural preference for binding and infecting many types of tumor cells. With an impeccable safety record from decades of clinical use, the measles vaccine virus has already entered clinical trials for several cancers. 

Recently, genetic modifications to the measles vaccine virus that enhance its ability to replicate in cells have shown further improvements in tumor killing in preclinical testing. As this therapeutic virus platform is modified to be more lethal to cancer cells, however, it becomes important to be able to limit its replication to particular times and places in order retain its low toxicity to normal cells.  

We propose to develop two mechanisms for improving measles vaccine virus control and specificity. The first mechanism allows administration of an orally available nontoxic drug to rapidly shut off virus protein production. The second mechanism renders virus protein production dependent on a specific molecular abnormality commonly found in cancer cells. Both will be based on encoding in the virus genome rationally engineered protein switches to control the function of viral replication proteins. In preliminary work, we have successfully created prototypical control elements for both mechanisms.  

We propose to test the ability of these control elements to function in the context of the whole virus in order to control replication in time and by tumor type. If successful, the proposed work will establish a generalizable method by which replication of measles vaccine virus, and other RNA-based viruses, can be programmed for increased tumor specificity and safety.

Probably the greatest limitation to the application of gene therapy for the treatment of cancer remains the difficulty in delivering a therapeutic gene efficiently and selectively to its tumor target.  

 
We have used oncolytic viruses, viruses that have been modified so that they selectively replicate in tumors and not normal tissues, to deliver a variety of genes to tumors. These viruses, based on vaccinia virus can be delivered intravenously into the blood stream, and even though they will then infect many different tissues (including the tumor), the engineered selectivity means they are cleared from all non-tumor tissues. The small amount of virus that does make it to the tumor will rapidly and selectively amplify as the virus replicates and spreads through the tumor. We have also used certain immune cell therapies that are attracted to the tumor, as delivery vehicles to carry the viruses to the tumor, so increasing the amount of virus that gets to the tumor and reducing the amount that goes to other organs.  

 
We have found that if we express therapeutic genes from these viruses (such as cytokines, that attract the host immune response to help destroy the tumor), we are able to increase their therapeutic benefit. However, because the expression of the cytokines results in destruction of the infected cells, we also reduce the amount of viral replication that occurs. This means that the virus does not get a chance to replicate properly, so reducing its initial delivery and spread within the tumor, and leading to clearance of the virus before it is capable of achieving its own therapeutic potential.  

 
We have therefore taken an approach that was developed by our collaborators that allows us to control the stability, and so the function, of any protein, and applied this to our oncolytic viral therapies.  

By attaching a small sequence onto any protein, we can flag the protein for rapid destruction by the cell’s degradation pathways. However, if we apply a small molecule that binds to this sequence and shields it, then we stabilize the protein and its function is restored. This allows us to rapidly, reversibly and safely control the levels of functional protein. In this way we can block cytokine function for a period of time (while the virus is cleared from normal cells and amplifies itself to high levels within the tumor; or while the virus is being delivered within an immune cell to the tumor), and so enhance initial delivery and establishment within the tumor. If we then stabilize the cytokine, there is increased production exclusively within the tumor, and so we can safely increase anti-tumor effects.  

We propose to use this system to control the expression of different genes expressed from oncolytic vaccinia viruses, and will use the system to control the virus replication itself. As a result, we can improve the delivery, safety and effectiveness of these (and other) gene therapy approaches, using a system that can be rapidly moved into the clinic. 

Most chemotherapies in general use are little more than DNA poisons that slow down tumor growth but do not ultimately cure patients. Chemotherapy also has devastating side effects, which is especially tragic in children. There is a desperate need to identify new drugs and therapeutic modalities that conclusively ablate cancer cells but leave normal cells unharmed. The p53 tumor suppressor pathway is inactivated by mutations in almost every cancer and yet we have no targeted drugs to treat p53 defective tumors.  

The overall objective of this proposal is to develop viruses that act as p53-mutation guided missiles, which specifically replicate within p53 mutant tumor cells to implode them from the inside-out. Currently there is no new therapy on the horizon that has more potential than such ‘oncolytic viruses.’ To develop these viral cancer therapies, we will exploit the striking similarities that exist between virally infected cells and tumor cells.  

Adenovirus is a small DNA virus-little more than a protein coat protecting its genome-that reproduces courtesy of the host cell. Normally, cellular replication is a tightly controlled process. However, both adenoviruses and tumor cells overcome cellular controls to drive their respective limitless propagation. Not surprisingly, tumor cells and adenoviruses subvert many of the same cellular checkpoints, albeit with one small difference: In tumor cells the key cellular players are targeted via mutations, while adenovirus uses viral proteins to achieve the same end. The p53 checkpoint normally ‘guards’ our cells against the pathological replication that occurs in cancer and adenovirus infection. Therefore, both tumor cells and adenovirus must inactivate p53; all tumor cells inactivate p53 by mutations and adenovirus encodes a protein called E1B-55K that destroys p53.  

Thus, it was proposed that ONYX-015, a mutant adenovirus that does not have the E1B-55K protein, would only replicate in tumor cells with a mutant p53 checkpoint. However, this turned out to be not so simple, but now we know why. We recently discovered that even when E1B-55K is no longer present, adenovirus has yet another viral protein that inactivates p53 functions. This is an exciting discovery, because with this knowledge we can finally create a virus that has no more options for inactivating p53. This will prevent the virus replicating in normal cells and allow it to only replicate in tumor cells with p53 mutations.  

Such a virus would offer a novel and potentially self-perpetuating p53 cancer therapy: Each time a virus homes in on a p53 mutant tumor cell and successfully replicates, the virus ultimately kills the cancer cell by bursting it open to release thousands of viral progenies, which are ready to seek out remaining tumor cells and distant micro-metastases. The goals of this proposal are to develop these next generation p53-tumor selective replicating viruses, which have the potential to help save the lives of many cancer patients.

We propose to test if protein transduction technology can improve the efficacy of adenoviral gene-therapy vectors for the treatment of cancer. In cell culture experiments, cancer cells can easily be eliminated with cancer targeted adenoviral gene-therapy vectors. However, in mice and humans, inefficient gene transfer remains a hurdle that has been extremely difficult to overcome. A p53-expressing adenoviral vector is in clinical use in China and has been placed on the fast track for FDA approval. 

 Published data indicate an extremely favorable safety profile, but overall, the therapeutic effect has been limited. Clearly, gene-transfer efficacy of adenoviral vectors has to be improved for more efficient therapy. The basic domain of HIV-1 Tat (a section of a protein made by the HIV virus, also called the protein transduction domain) can enter intact cells and has been used to deliver a wide variety of proteins to many cell types. Tat-fusion proteins, produced in bacteria, purified and then applied to cells, have been studied widely to manipulate cancer biology.  

This approach is effective in cell culture experiments, but so far there is only limited evidence for efficacy in mouse tumor models. Very few studies have investigated the potential of the delivery of Tat-fusion proteins with gene therapy vectors. We hypothesize that Tat-fusion peptides expressed at high levels with an adenovirus will result in more effective treatment of solid tumors. 

 Furthermore, we hypothesize that smaller unfolded peptides fused to Tat will lead to more efficient distribution within tumors, compared to complexly folded large proteins. The delivery of peptides fused to a protein transduction domain with adenoviral vectors is a new approach that could find broad application.

It is known that cancer cells are often defective in anti-viral pathways and are thus susceptible to virus infection. SV5, also known as PIV5, is not associated with symptoms or diseases in humans. This study will test the hypothesis that SV5 mutant viruses can selectively kill advanced tumors.  

SV5 viruses with mutation in SH or V proteins induce apoptosis in many cell types. Initial results have been promising with late-stage solid tumors, and in preliminary studies, mutant SV5 viruses killed human metastatic breast cancer cells, as well as Lewis lung carcinoma cells. Using cytolytic viruses as anti-tumor agent provides a viable alternative to surgery and chemotherapy. 

Since most human tumors must recruit new blood vessels in order to grow and move, anti-angiogenic therapies have been viewed as a potential complement to more traditional cancer treatments. We have identified a genetic element that allows genes to be expressed in a group of bone marrow derived cells that contribute to cancer-associated blood vessels.  

The cells are called “endothelial cell precursors” (ECPs). ECPs are a special type of stem cell that play a role in the formation of new blood vessels. We propose to create a simple virus designed to carry genes to ECPs and prevent them from helping tumors to grow.