CAR-T CELL FUNCTION EVALUATION IN LONG-TERM KILLING ASSAYS

INTRODUCTION AND OBJECTIVE: One of the first steps in screening a new design of CAR-T cell is to measure the cancer cell-killing ability of an effector cell with a cytotoxicity assay. The most used tests for evaluating the capacity of cytotoxicity activity of CAR-T cells in vitro are the chromium (51Cr)-release assay and other tests like calcein fluorescent and bioluminescent assays (Kiesgen et al, 2021). However, these tests only evaluate the lysis for a short period (4h-24h). The benefit of a long assay is to understand how the cells are behaving in terms of kinetics, memory, and exhaustion marks which assays in 24 hours do not predict. Therefore, our objective is to standardize a long cytotoxicity assay using different tumor lineages and define whether our CAR construct has a cytotoxicity effect against the CD19+ cells using this method. MATERIAL AND METHOD: Nalm-6-GFP and RS4;11-GFP are tumor cell lineage of B-Cell Acute Lymphoblastic Leukemia that expresses CD19 and were used as target cells. As CAR-T effector cells we used human cells modified with the CAR-encoding pT4-19BBz Sleeping Beauty transposon vector. We used different ratios of CAR-T/tumor cells defined by the total number of T cells and incubated cells at 37ºC for killing evaluation at 0h, 24h, 48h, and 72h time points. To discriminate the living from death cells, we used Fixable Viability Dye - eFluor™ 780 before gating GFP+ target cells. T cells without CAR (mock) were used as a control in the same conditions as CAR-T cells. We evaluated the number of remaining living GFP+ cells to determine the proportion of killed cells. Tests were performed in duplicate. RESULTS AND CONCLUSION: We performed the tests on 3 different donors and we saw a decrease of alive cells in the first 24h to at least 60% in Nalm-6 and RS4;11 in all E:T ratios. In agreement with our previous results (Chicaybam et al., 2020), the lysis of Nalm-6 is lower than the observed for RS4;11 in each time point and ratio. The present data shows we have better results of lysis and the largest difference between CAR-T cells and mock at times later than 24h. CD19BBz positive cells promote at least 15% of leukemia killing even in the lower E:T ratio at 72h. Furthermore, we observed that only 1.7% of RS4;11 cells were alive at the highest ratio and 5.4% at the lowest ratio at 72h. Similar results were also obtained with the Nalm-6 cell line. Thus, we demonstrate that our 19BBz CAR-T cells are capable of killing tumor cells efficiently even in lower doses and this result may be important for in vivo tumor killing dynamics. In the next steps of this study, we will use a modified Nalm-6 cell line expressing low levels of CD19 and evaluate the kinetics using a calcein and caspase assay for flow cytometry and high throughput microscopy-based evaluations of killing dynamics.