T-cells play a critical role in cancer immunosurveillance and control. As such, there has been a recent emergence of T cell based immunotherapies with durable clinical outcomes. These include the immune checkpoint inhibitors Ipilimumab (anti-CTLA-4) and Nivolumab (anti-PD-1), and adoptive cell therapy, which have conferred clinical response rates of up to 50%. Despite these encouraging outcomes, there is still a large cohort of patients for which these treatments are currently not applicable. They are either inherently resistant to or acquire resistance to immunotherapy. As such, understanding mechanisms of resistance to immune response and ascertaining efficacious therapy combinations is important in overcoming immune-resistance and developing novel treatment regimens.
To address this need we performed preliminary screening of about 850 different bioactive compounds (Selleckem) to find candidate drugs that could modulate the sensitivity of tumor cells to T- cell mediated killing, using an established protocol for an in vitro T-cell mediated cytotoxicity screen. We hypothesized that either tumor cells treated with different compounds will increase or decrease their sensitivity to T cell mediated killing, and compounds that increase T cell killing can be used in combination with immunotherapy. Using patient-derived melanoma tumor cells and their autologous tumor infiltrating T cells (TILs), we assessed the increase or decrease of T cell killing of tumor cells following treatment with the test compounds via intracellular detection of active caspase-3 by flow cytometry in a 96-well format. This initial screen identified Heat shock protein 90 (Hsp90), Aurora Kinase and Topoisomerase I inhibitors as enhancers of T cell mediated killing, and studies to understand their mechanisms of action are currently underway.
However, as this screening process is very time-intensive, we have increased the efficiency and feasibility of this screen by miniaturizing the workflow. This work highlights the development and initial findings of the screening method that is performed in 384-well plates. This format allows for a larger library of compounds, with a greater drug concentration range, and for more autologous tumor and T-cell pairs to be screened within a shorter time. The miniaturized assay also utilises robotics to streamline and increase output. Additionally, by understanding the signaling pathways and molecular factors that regulate tumor response to T cell mediated killing, we can translate these findings applicable to other cancer. This project aims to discover novel compounds that work synergistically with T-cell mediated tumor cell cytotoxicity, understand their mechanism of action and how the inhibited pathways contribute to resistance to immunotherapy and as a result better inform combination strategies with immunotherapy for clinical use.
Citation Format: Leila Williams, Shruti Malu, Jodi McKenzie, Rina Mbofung, Jason Roszik, Patrick Hwu. Using a high throughput T-cell cytotoxicity assay to develop combination strategies for immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B118.
- ©2016 American Association for Cancer Research.