Why YP-P10?

Meet Virginia Calder…
 

My name is Virginia Calder and I am Professor of Ocular Immunology at UCL Institute of Ophthalmology in London. My interests have always been the role of CD4-T cells in immune-mediated disease. My PhD looked at activated T cells in brain tissues from multiple sclerosis patients. My PhD supervisor – Marc Feldman – and his colleague developed the first immunotherapy for autoimmune disease.

Can you tell us about your research?
 

My research group specializes in isolating immune cells from various ocular fluids. We are really focusing on how we can standardize these fluids for further analysis. I’ve worked alongside various companies who are interested in developing new therapeutics or have therapies they think might be applicable to the eye. Over the years, I’ve developed very strong relationships with a number of different commercial outfits and I’m pleased to say that I met the Yuyu Pharma guys at ARVO last year. I was very keen to see exactly what Yuyu’s YP-P10, a novel synthetic peptide with anti-inflammatory properties, does to human T-cell subsets, because at that point they hadn’t addressed that issue with a view to understanding a mechanism of action.

What are some of the issues with currently available dry eye treatments?
 

Steroids have lots of side effects, particularly in the eye. Steroids are given to dampen down immune responses, but they also raise the possibility of infection taking over. Cyclosporine is a much more specific target, which homes in on the activated T-cell process. I strongly believe, alongside many others who work in dry eye diseases, that T-cells play a very important role in driving the inflammatory response. Over the years, research demonstrated that by blocking T-cell activation you can effectively reverse the inflammation occurring in disease. Despite this, cyclosporine is notoriously difficult to solubilize, so it’s hard to get cyclosporine drops into a solution that can be tolerated by patients with already poor ocular surface problems.

Lately, we are discovering there are many different subsets of T-cells and that the immune system specifically adapts to whatever foreign body or stress comes along. Not all patients will respond to any of these specific treatments, because in their disease a different pathway is driving the process. For example, only 40 percent of anti-TNF treated patients will benefit from the drug. A huge proportion of patients don’t do well with anti-TNF, because TNF is not necessarily the main cytokine driving their disease. On top of that, TNF is a secreted product that causes a lot of peripheral damage to the tissues around it, but by blocking it all you're doing is soaking up the material that is being continuously secreted by these activated cells. What we are looking for are agents that will actually work upstream of that to block activation, switch the cells off, and thereby reverse the disease inflammation.

Can you tell us about the research for your poster at ARVO?
 

The emphasis was to look at human cells and, of course, a lot of studies look at frozen populations of cells that have been stored in liquid nitrogen tanks for a long period of time. I was keen to avoid that because through this extended storage you can lose those precious, activated cells. Instead, we insisted on obtaining fresh blood cell samples. We started with healthy bloods from anonymized donors. Of course, these bloods don't have high levels of circulating disease-associated cells because they don’t have any disease. However, with our sensitive flow cytometry-based detection techniques, we are able to pick up low but significant levels of various subsets – we think these are continuously circulating memory cells that are looking for any new infection. By taking the fresh blood, separating off the immune cells, and then stimulating them in culture, we were able to detect very low levels of T Helper 1 (Th1), Th2, and Th17 cells, which are responsible for cell-mediated immunity and phagocyte-dependent protective responses. That was enough for us to then treat the cells in culture with the YP-P10. We had already worked out from cell viability assays that we could add relatively high concentrations without causing any adverse effects on the cell viability – it is a very kind agent without any preservatives.

We treated the cells for about an hour, before we went in with our stimulation. We had stimulated the cells in two different ways. First was through the very general, PMA/ionomycin stimulation, which activates all cells regardless of type. I was also very interested to look specifically at T-cell activation, so in separate wells we stimulated cells with anti-CD3 and anti-CD28 to mimic an antigen binding to the antigen receptor – the T-cells expanded that way. After overnight incubation, we looked for our different T-cell subsets to see if we could see any differences when they were treated with the YP-P10.

What were your findings?
 

We found that the overall levels of B-cells didn't really change; there was no effect on the activated B-cells, either. We didn’t see much of a difference in CD8+ T-cells and we did look at the different interferon gamma T-cells expressing CD8, called TC1s. However, when we looked at the CD4 population of T-cells and split them down – into Th1s, which expressed interferon gamma, Th2s, which expressed intracellular interleukin 4 (IL-4), and Th17s, which expressed intracellular IL-17 – we did see an effect on the Th2 population. We’re not talking about huge levels, but we nevertheless saw significant decreases in the four blood samples we looked at. The Th17 population within these healthy blood samples was extremely low; not put off by that, we decided to enrich the Th17 cells – a five-day treatment in which extra cytokines are added to the culture mix, promoting Th17 expansion. Through this, we were able to get our levels of Th17 cells up to 20-30 percent in the culture and so, when we pre-treated them with YP-P10, we were able to see a significant drop in Th17 expression in the treated samples.

Since completing this work, which was presented in the poster through a flow cytometry histogram analysis showing the percentage decrease from baseline, we have gone on to purify Th17 cells, a 14-day culture in chemically-defined medium, and we are waiting for results from a single-cell RNA-seq analysis on purified Th17s. My question is – does YP-P10 directly target these subsets, or does it need an intermediary cell to mediate its effects?

What would be a long-term mode of analysis for YP-P10 that meshes with your own ongoing research?
 

We have often used these anti-inflammatory products as tools to understand how the immune system works. Given the results we have so far, we feel there is probably a common receptor expressed by these T-cell subsets that is the target for YP-P10. So, our next move is to work out exactly which receptor that is, by using rescue experiments and blocking experiments to see what YP-P10 is doing. Hopefully we can then get a good insight into a mechanism of action. I’m looking forward to seeing the next chapter of this work and getting a handle on how exactly YP-P10 is affecting these T-cell subsets.