Era of Hope 2011 Investigator Vignette

Title: Targeted Strategy to Overcome Resistance to Immunotherapy

Investigator: Gayathri Devi, PhD; Duke University Medical Center

My Idea Award is looking at developing a targeted genomics-based strategy to overcome resistance to immunotherapy and enhance cancer vaccine in breast cancer patient therapy.

One of the issues with cancer vaccines and immunotherapy is that there is a subset of a cells called T-regulatory cells that suppress the efficacy of the cancer vaccines. In a normal immune system, they actually help in controlling autoimmunity so they do not allow antigen-specific T-cells to mount an immune response to everything, so they protect us. However, the same mechanism becomes a problem in cancer and in viral infections where they suppress the potency of cancer vaccines. Indeed these T-regulatory cells or T-suppressor cells are actually very high in cancer patients. So if we can specifically target these cells then we can definitely use them as a novel adjuvant for any type of cancer vaccines and immunotherapy.

So we developed a very novel strategy of an antisense-based oligomers that are unique in such that they can target the T-regulatory cells specifically nontoxically and in conjunction with cancer vaccines. So, how do we isolate these T-suppressor cells? This is a big challenge because there are a very small percentage of the T-cells in the body. So we've come up with a flow cytometric analysis by which we can sort for these specific sub-populations of T-cells by looking at certain markers which are the CD4 positive and the CD25 markers as shown here. And this population of T-cells has very high levels of expression of a protein called FoxP3. Now FoxP3 is a transcription factor and it has now been identified to be very specific for T-regulatory development and also it is considered as a marker of T-regulatory function. So our idea now was okay, can we target FoxP3; that way we will specifically eliminate the T-regulatory cells from the patient's system and thereby increasing the potency of the cancer vaccines that are administered.

This slide shows that indeed these markers of FoxP3 expressing T-regulatory cells can suppress the proliferation of normal antigen specific T-cells which is what is needed to mount a robust immune response when a patient is vaccinated.

Immune cells are very difficult to target. It's very difficult to get agents into the immune cells, so in order to be able to do that we need to have a safe and at the same time a nontoxic product that can get in, stay for a long period of time to elicit its response before it gets degraded.

So what we did was we came up with a very novel way of conjugating these oligomers called phosphorodiamidate morpholino oligomers or also called PPMO chemistry. it is basically a derivative of our normally occurring RNA molecule which has the phosphodiester bond, or in some cases a phosphorothioate bond. we substituted the ribose sugar with the morpholine sugar and also substituted the oxygen that amide bond. This allows for this to be resistant to enzymatic cleavage in the body. It also allows them to be more stable and they'll have better binding to the target RNA.

So by adding on peptide conjugates to this basic chemistry, these peptide-conjugated phosphorodiamidate morpholino oligomers, also called PPMO allow us to get very high delivery into monocytes into T-cells including the T-regulatory cell population and also into the B-cells which are all important components of the immune system. And we could do this in a more nontoxic manner, there was no change in viability and these cells were healthy when they had the PPMO delivered to them.

So then we wanted to see does the target T-cells express our target gene which is FoxP3. And we saw indeed that when we activate T-cells these do have high expression of FoxP3. So our next challenge was can we get our PPMO into these activated T-cells that are also expressing high levels of FoxP3. And indeed we had about 89 to 90% increase in PPMO delivery into the activated PBMCs and also even the nonactivated PBMC take up these PPMO so it tells us that we can use them broadly in multiple scenarios.

So our idea now was okay, can these PPMOs inhibit our target gene expression which is FoxP3. We took peripheral blood, mononuclear cells from normal donors and from the patient population and looked at FoxP3 expression before and after treatment for multiple days. We were able to reduce the FoxP3 expression which in a sense means that we are able to target the T-regulatory cells.

However, really the proof lies in showing a functional efficacy, so we decided to look at a model of CMV-induced antigenspecific response. So we took blood from people who are CMV positive and used this as a model to show that when we treat these immune cells with our FoxP3 PPMO we were then able to have a very strong enhanced immune response in this functional assay.

So, in summary, that showed us that this strategy is not only easy to deliver into human cells; this chemistry allows us to target specifically a small subset of these T-cells which causes resistance to various immunotherapeutic strategies and also not only are we reducing the frequency of these T-regulatory cells but modulating them to such that we get a better enhanced immune response. So our idea is that we should target these T-suppressor cells or T-regulatory cells which will then enhance the potency of any cancer vaccine that is being tried in the clinic.