Removing IL-2 in-process promotes metabolic fitness and reduced background killing
Over-activation of T cells prior to transduction and expansion can lead to pre-exhaustion of EOP TCR-T cells with compromised persistence and potentially contribute to cytokine release syndrome (CRS) due to high background reactivity(Alizadeh et al., 2019; Gett, Sallusto, Lanzavecchia, & Geginat, 2003; Schluns & Lefrançois, 2003). We hypothesized that in-process IL-2 withdrawal from complete culture media together with semi-continuous perfusion would attenuate activation signaling and generate T cells with lower background activity and enhanced fitness. When evaluated with two donor samples, we found that less than 5% T cells were positive for CD25 upon harvest when IL-2 was withdrawn on day5 or day6 while cells cultured with IL-2 until harvest had 50-80% CD25+ T cells (Fig. 5A) . The delay in CD25 downregulation post IL-2 withdrawal is expected with a semi-continuous perfusion system where IL-2 containing media was slowly perfused out of the bioreactor resulting a gradual decline in IL-2 concentration. Importantly, cells with lower CD25 expression exhibited reduced nonspecific killing while maintaining specific target cell lysis when E:T ratio was increased (Fig. 5B) . In the two donors tested, withdrawing IL-2 on day6 or day5 differentially affected TCR expression compared to the control condition (with IL-2), although the differences were not significant. (Fig. 5C) .
MMP, ROS, and apoptosis at both resting state and effector state against target cells were measured based on fluorescent intensity (MFI) of DilC1(5), CellROS Orange, and Annexin of viable T cells using flow cytometry. When encountering target cells, MMP became more polarized, and cells generated more ROS (Fig. 5D & 5E) . Spontaneous apoptosis associated with effector function measured by annexin level was elevated upon target cell activation regardless of IL-2 conditions (Fig. 5F) . However, T cells from the IL-2 withdrawal condition had lower levels of MMP and ROS at both resting state and effector state compared to T cells that were supplemented with IL-2 throughout the bioprocess (Fig. 5D & 5E) .