IMMU-03. UTILIZING IN SILICO SINGLE-CELL TRANSCRIPTOMIC AND TCR PROFILING FOR THE IDENTIFICATION AND ISOLATION OF TUMOR-REACTIVE TCR CLONOTYPES IN RECURRENT GLIOBLASTOMA PATIENTS

Abstract

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive form of primary brain cancer with no current cure. Immunotherapies, such as checkpoint blockade and adoptive T cell therapy, have effectively treated other cancers, but in GBM, these therapies have shown limited efficacy. This may be partially due to a lack of infiltrating tumor-reactive T cell populations capable of eliminating the tumor. METHODS & RESULTS We used single-cell RNA and TCRsequencing to examine the transcriptome and TCR repertoire of tumor-infiltrating T cells from a cohort of 32 recurrent GBM patients, 18 of whom had received neoadjuvant anti-PD-1 therapy. After dimensional reduction, we found that the cluster defined by T cell activation and exhaustion gene expression had the greatest number of expanded T cell clones and a transcriptomic profile similar to a previously published signature of tumor-reactive T cells. Focusing on a patient treated with neoadjuvant anti-PD-1 and a EGFRvIII Bispecific T cell engager therapy, we selected TCRs from T cells within the activated and exhausted cluster. Utilizing the PathFinder DNA Assembly technology developed by Rootpath, we generated a TCR library containing the selected TCRs and transduced the library into a fluorescent-reporter Jurkat line that measures T cell activation. We co-cultured the transduced Jurkats with the patient-derived gliomasphere, FACs sorted for activated Jurkats and performed TCR sequencing on the sorted cells. Six TCRs within the sorted population were over-represented relative to sorted T cells from control co-cultures, indicating their tumor reactivity. CONCLUSIONS In this study we provide a proof-of-concept framework for how incorporating silico single-cell analysis of a T cell’s transcriptome and TCR can be used to identity tumor-reactive T cells for potential downstream adoptive T cell therapies for patients with recurrent GBM.