Era of Hope 2011 Investigator Vignette

Title: Novel Enzyme Prodrug Therapy for Breast Cancer

Investigator: Roger Harrison, PhD, University of Oklahoma

The goal of my Idea Award is to develop a new therapy based on the novel enzyme prodrug that will selectively eradicate metastatic breast cancer.

And so we use the idea of targeting the tumor vasculature in order to very selectively and specifically deliver a drug in the tumor but to minimize or completely negate any side effects. And this is one of the significant things we feel about this new research is that it—it will attack metastatic breast cancer, which up to now there is no cure for.

And so this therapy then will find that tumor wherever it occurs in the body and eradicate it. And so we have a fusion protein consisting of—of methioninase and Annexin V. The reason we chose Annexin V this is a human protein that binds to the tumor vasculature selectively and to the cancer cells. And it does not bind in the normal vasculature. OK, so the idea would be to administer this fusion protein into the bloodstream, let it then pass out of the bloodstream whatever is not bound and we have found in tests that this takes about 6 hours in animals. And then after that to introduce the prodrug selenomethionine that then gets converted by the enzyme to the toxic drug methylselenol. This methylselenol acts on the endothelial cells and it’s pushed through the artery wall and here would be tumor cells and the tumor, and it not only affects the endothelial cells but it also can get to the breast cancer cells.

So we don’t have to have a large molecule like this protein going through; all we have to do is create the drug right here and then it will go in and affect these vascular cells and affect these breast cancer cells.

We have two fusion proteins—one, where we have the methioninase; there and also cytosine deaminase.

Cytosine deaminase is actually a more common cancer drug and has been used for breast cancer treatment. And so we’ve gotten these disassociation constants in the range of about two nanomolars, which is indicative of very strong binding. So this is going to bind very strongly here to the wall of the arteries.

We’ve also done some imaging where we have made our fusion protein fluorescent, and what we see is the fluorescence is around the perimeter of the cell. It’s around the outside of the cell. And we anticipated that it would stay on the surface. And here we have some proof that it’s staying on the surface of the cells. Here is our data for in vitro testing of cells in the laboratory, and we have actually only shown data in this poster of two cells, the endothelial cells, and then one cell line of the breast cancer cells, these MDA-MB-231 cells.

Here we’re measuring basically viability of the cells for different levels of our prodrug. So we’re plotting our prodrug concentration here and we’re seeing what the viability is. And basically what we’re finding is that we’re not seeing a change in viability when we just have the prodrug.

And—but then when we have the protein bound and our enzyme bound we’re showing a large killing of the cells and in some cases almost complete killing of the cells.

In vivo we have this test is really we consider a preliminary test for this methioninase enzyme and we’ve used nude mice. We have three other controls: untreated mice, mice treated with just the fusion protein, and mice treated with just the prodrug. And these are pretty much staying the same, and they’re not seeing much difference. And this bottom curve is the one that the mice were treated. And so we’re getting this going down to practically zero here and we’re getting a very large—we’re getting significant—starting fairly early in the test

The methioninase Annexin V fusion protein we get cell killing in 1 day. The cytosine deaminase and 5-fluorocytosine we get cell killing in 3 days, but we don’t get any significant effect with the prodrug present but no enzyme present, okay; so that’s a very positive result.

And so it looks like that—that is getting complete killing, which we’re very pleased about and it validates our original hypothesis.