Developing a screening platform for genetic and small molecule targets of cancer immunotherapy.

Abstract

66 Background: The success of checkpoint blockade therapy is often dependent on CD8 T cell activation against tumor antigens. However, clinical benefit is only seen in a subset of patients, suggesting that there are other possibly targetable immunosuppressive pathways that are allowing the tumor to escape immune surveillance. Methods: A CD8 T cell that recognizes the EGFP200-208 peptide epitope allowed for the use of EGFP as a model tumor antigen while monitoring expression levels at the single cell resolution. Using a lymphoma line expressing EGFP or mCherry as our antigen-positive and -negative cancer models, we employed 3 screening strategies: 1) a forward genetics approach in which we selected for tumor cells that had naturally developed resistance to killing by CD8 T cells; 2) a reverse genetics approach that involved the use of pooled CRISPR libraries to identify knockout clones with a selection advantage or disadvantage when pressured by activated T cells; and 3) small molecule libraries, including FDA-approved drugs, to identify compounds that increased antigen-specific CD8 T cell killing. Results: Despite antigen recognition and early activation in response to the resistant tumor line, T cells failed to produce effector cytokines and underwent apoptosis in a PD-L1 and CTLA-4 independent manner. Candidate genes mediating this phenotype were derived from expression differences between the original susceptible tumor line and the immunoedited resistant tumor line. The pooled CRISPR approach was validated in a curated library by identifying genes with known roles in T cell-mediated killing and antigen presentation, such as Fas, B2m, and Tap1, as well as known suppressive molecules such as BTLA and PD-L1. LDL receptor expressed on the cancer cell emerged as a possible novel suppressor of T cells. Decitabine and 4-cinnolinethiol, among other small molecules, emerged as possible enhancers of CD8 T cell activity. Conclusions: We have identified several gene candidates as potentially novel and targetable checkpoint-like molecules, as well as small molecule compounds that may be able to enhance the anti-tumor activity of CD8 T cells. Efforts are ongoing in order to validate these targets and to screen larger libraries.