Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors

production of TNF-related apoptosis-inducing ligand (TRAIL), an inducer of apoptosis and a cancer therapeutic (Figure 4B) (Johnstone et al., 2008; Lemke et al., 2014). T cells normally do not produce TRAIL upon TCR stimulation, therefore, if synthetically expressed in a controlledmanner, this could aid in their cytotoxic activity (Figure S4A). Soluble forms of TRAIL are effective at killing the highly susceptible colon cancer cell line HCT116, but for other cancer lines like K562 cells, soluble TRAIL does not induce apoptosis even at high doses (Figures S5A–S5D) (Kim et al., 2000; Park et al., 2009). However, a recent study showed that if TRAIL is delivered in a membrane anchored form (e.g., a supported lipid bilayer or liposome), it is more effective at inducing apoptosis, even for resistant cancer cells such as K562 cells (Nair et al., 2015). Therefore, we engineered the CD4 T cells to produce one of two TRAIL variants: (1) a secreted form of TRAIL fused to the GCN4 trimeric leucine zipper (LZTRAIL) known to be more potent than soluble monomeric TRAIL, or (2) a natural surface displayed TRAIL (Figures S4E–S4H) (Walczak et al., 1999). SynNotch T cells driving TRAIL production were co-cultured with TRAIL-resistant K562 cells to determine if T cells were an effective delivery platform that enhanced the apoptotic effects of TRAIL. SynNotch T cells drove cell surface TRAIL expression (Figure 4C) and LZ-TRAIL secretion within 24 hr of co-culture (Figures S4F–S4H), but only cell surface TRAIL initiated K562 A