Abstract 547: Generation of synthetic cytokine receptor-induced cytotoxic innate lymphocytes (iCILs) from iPSCs as off-the-shelf cancer therapeutics

Off-the-shelf immune cell products derived from induced pluripotent stem cells (iPSC) have the potential to address many of the current limitations of engineered autologous and allogeneic blood-derived cell therapies. IPSC-based platforms offer a scalable and renewable source of allogeneic cells that can be engineered to augment cell phenotype and function. Current approaches to generating iPSC-derived immune effector cells have focused on recapitulating natural differentiation pathways to defined effector cell types (e.g., CD8 T cells or NK cells) utilizing feeder cells and exogenous cytokines. By engineering cells to express an artificial cytokine receptor, termed rapamycin activated cytokine receptor (RACR), we can control immune effector cell differentiation and expansion through synthetic signals independent of endogenous receptor expression, reducing the complexity and variability of cell differentiation while deriving cells with unique phenotypic and functional features not found in conventionally defined native immune cells. Here we demonstrate the ability to differentiate and expand synthetic cytotoxic innate lymphoid cells through engineering progenitor cells to express an artificial cytokine receptor, the RACR system, that is activated by a non-native small molecule ligand, rapamycin. This approach involves a period of feeder cell free hematopoietic and lymphoid progenitor specification followed by rapamycin-mediated activation of the artificial cytokine receptor (RACR), inducing an IL-2/IL-15-like signal to drive cytotoxic innate lymphocyte differentiation and feeder cell free expansion resulting in approximately 3000-fold expansion from the iPSC starting material. The protocol has demonstrated robustness in the small-scale setting with multiple research-grade iPSC lines and enables extensive effector cell expansion at low cost, enabled by the stability of the rapamycin ligand and elimination of complex raw materials such as exogenous cytokines. The resulting RACR-induced cytotoxic innate lymphocytes (RACR-iCILs) exhibit potent polyfunctional anti-tumor activity driven by the synergistic activity of innate immune receptors and the engineered expression of a chimeric antigen receptor (CAR), including cytolytic activity and the secretion of IFNγ and TNFα. The artificial cytokine receptor system can be used ex vivo to generate cells but also has the potential to enable rapamycin-controlled and selective expansion and survival of the engineered cel