Immune checkpoint blockade antibodies (eg. anti-CTLA-4 and anti-PD-1) enhance T cell anti-tumor activity and have produced exciting and durable results in treatment of a number of cancers including melanoma. Unfortunately, response to first generation checkpoint blockade therapy is limited to a subset of patients. A more comprehensive understanding of the genes and molecules involved in T cell checkpoint control and other aspects of anti-tumor T cell activity may allow a wider patient population to benefit from this exciting and new approach to cancer therapy.
To this end, we performed a forward genetic screen to identify gene pathways that influence intratumoral T cell infiltration using Sleeping Beauty (SB) mutagenesis. More specifically, this genetic T cell screen was designed to identify additional genes and molecules responsible for selection and expansion of intratumoral T cells via a variety of T cell processes including T cell receptor (TCR)-mediated activation, clonal expansion of tumor-specific T cells, T cell trafficking into the tumor, and maintenance of prolonged viability once there. Advantages of using SB in a genetic screen include the ability to cause both gain- and loss-of-function mutations, as well as mutagenesis of the entire genome followed by easy identification of insertion sites. In addition, SB screens are performed in vivo, and thus preserve the complexity of the tumor microenvironment.
We generated a pilot cohort of mice (n = 12) with SB-mutagenized endogenous T cells. These mice were challenged with syngeneic B16F0 melanoma cells subcutaneously. After tumor development (21 days after tumor challenge), tumor-infiltrating CD4+ and CD8+ T cells were harvested. Splenic T cells representing unselected CD4+ and CD8+ populations were also isolated from the same animals at time of tumor harvest. Harvested T cells were evaluated by high throughput sequencing to identify SB insertion sites. T cells harvested from tumors demonstrated a decrease in clonal insertion sites compared to splenic T cells suggesting intratumoral clonal selection. Moreover, clonal insertions in intratumoral T cells were significantly enriched in or near genes, signifying likely selection for insertions impacting gene function (p<0.000003, Fisher's exact test). Clonal insertion sites in T cells from tumors that were absent from spleens, representing potential immunotherapy targets and including insertion sites in genes known to be associated with the T cell response, were identified. We are currently in the process of expanding this experimental cohort using a larger number of mice.
We conclude T cell specific SB mutagenesis has the potential to identify novel molecules that influence intratumoral T cell infiltration and can be used to identify additional genes that may contribute to the anti-tumor T cell response.
Citation Format: Laura M. Rogers, Adam J. Dupuy, George J. Weiner. Identification of novel cancer immunotherapy targets using Sleeping Beauty mutagenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 526.