8-DAY VERSUS 12-DAY MANUFACTURING OF LV20.19 CAR T-CELLS IMPACTS SINGLE CELL CYTOKINE PROFILES WITHOUT INCREASING SEVERITY OF TOXICITIES

Background & Aim: Bispecific lentiviral transduced anti-CD19, anti- CD20 (LV20.19) CAR T-cells may improve outcomes in B-cell NHL by mitigating CD19 antigen loss as a mechanism of relapse (Shah et al. Nature Med. 2020). To optimize the LV20.19 product, we designed a Phase I/II trial of LV20.19 cells in relapsed, refractory B-cell NHL with CAR T-cells manufactured under variable lengths of time (8 vs 12-days) (NCT04186520). We hypothesize that shorter manufacturing may result in a more naive CAR product improving functionality. Here we describe the single cell cytokine analysis & toxicities associated with LV20.19 CAR T-cells manufactured for 8- vs 12-days. Methods, Results & Conclusion: LV20.19 CAR T-cells were manufactured in the CliniMACS Prodigy device using IL-7 & IL-15 for expansion & patients were sequentially assigned to 8 vs 12-day arms (n=3, each arm). To assess the cytokine profile of each product, LV20.19 cells were thawed, CD4 & CD8 cells isolated, stimulated with CD19+ K562 cells, loaded onto single-cell Isoplexis Adaptive Immune Isocode chips & read in an IsoLight instrument. The single cell production of 32 cytokines was assessed and a polyfunctional strength index (PSI) was generated using Isospeak software. Polyfunctionality (PFA) was defined as a single stimulated cell secreting ≥2 cytokines. Single cell cytokine profiles for the LV20.19 CAR-T cells were dominated by effector cytokines independent of cell subset (CD4 vs CD8) or manufacturing length (Fig. 1). There was a trend towards higher PSI in the 8 vs 12-day arms (1448 vs 1192, CD4; 1578 vs 1143, CD8) (Fig. 1), in addition to a trend towards a higher PFA (55 vs 47.8%, CD4; 55.9 vs 42.8%, CD8) (Fig. 2). CD4 cells in the 8-day arm had a higher secretion frequency of IL-8 (p=0.031) & IFN-γ (p=0.053) & CD8 cells in the 8-day arm had a higher secretion frequency of MCP-1 (p=0.059) (Fig. 3). For the 8 vs 12-day arms, cytokine release syndrome occurred in 100% vs 66.7% (all grade 1) and neurotoxicity in 0% vs 33.3% (grade 2). [Figure presented] [Figure presented] CD8 LV20.19 CAR T-cells manufactured for a shorter duration (8-days) had a trend towards higher PSI and greater PFA than 12-day arm cells without increasing toxicity. PSI has been shown to correlate with treatment response (Rossi et al. Blood 2018). This data suggests that timeline of expansion modifies the functional phenotype of CAR T- cells which could be exploited in manufacturing to improve outcomes. Additional patients are enrolling to further elucidate differences in these products.