Neoantigen prediction and reactivity of cerebrospinal fluid–human-derived tumor reactive T cells (CSF-TRT cells) in leptomeningeal disease (LMD) from solid tumors.

2030 Background: Leptomeningeal disease (LMD) is a devastating complication of metastases and its incidence is increasing. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) showed complete responses in patients with metastatic melanoma and other solid tumors. To obtain CSF-TRT cells as a source of T cells for ACT, we aim 1) to identify the expansion method that allows higher expansion of tumor reactive TIL; 2) to enrich tumor neoantigen reactive TILs and determine their in-vivo and ex-vivo functional capabilities. Methods: Cells were isolated from 64 CSF collections from melanoma (M-LMD), 9 CSF from breast cancer (B-LMD), and 9 CSF from lung adenocarcinoma (L-LMD) from patients with LMD. Cells were plated following established TIL culture protocols (high-dose IL-2): 1) OKT3, 2) anti-4-1BB-agonist, 3) IL-7+IL-15+IL-21, 4) anti-CD3/CD28-T-activator, 5) anti-CD3/CD28/CD137-T-activator, 6) anti-CD3/CD28-T-expander. After 4-6 weeks, reactivity was assessed with HLA-matched or autologous tumor cells. Additionally, Whole Exome Sequencing (WES) and RNA sequencing were performed in T cells expanded from CSF and pair-matched extracranial tumors from 12 patients with M-LMD for epitope prediction to identify neoantigens that may be targets of immunoediting using computational algorithms. Results: CSF yielded an average of 1.204e5 viable cells for expansion. After culture in IL-2, 53.4% of samples showed increased cell yield with average 165.29-fold expansion. PreREP resulted in 37.6% CD8+ T cells. REP had a 96.8% success rate (mean 589.87-fold expansion). Post-REP flow cytometry revealed expansion of CD4+ T cells. Additional cultures produced similar yields with reduced T cell input requirement and demonstrated the potential to enrich for CD8+ T cells. Out of five samples tested for reactivity to HLA-matched melanoma cell lines, three produced varying levels of IFN-y. T cells expanded from CSF samples from L-LMD and B-LMD had less successful results. L-LMD had a success expansion rate of 11.1% with IL-2 only, 83.3% with T-Activator, 50% with CD127 T Activator, and 50% with T-Expander. B-LMD had a success rate of 22.2% with IL-2 only, 0% with OKT3, 83.3% with T-Activator, 50% with CD137 T-Activator, and 85.7% with T-Expander. One of the CSF samples from L-LMD tested showed reactivity to autologous HLA-matched tumor cells, whereas none of the CSF T cells from B-LMD showed a successful reactivity. Preliminary studies demonstrated CSF TIL reactivity against tumor reactive neoantigens. Ongoing studies will confirm our results. Conclusions: Results demonstrate successful expansion of T cells ex vivo from CSF in M-LMD, raising the potential to use CSF-derived T-cells as therapeutic strategy for LMD. There was a variable success rate with expansion of T cells from CSF from B-LMD and L-LMD and supplementary studies are needed.