Optimizing Chimeric Antigen Receptor Endodomains for Effector Function and Cell Expansion in iPSC-Derived T-Cell Therapies

Autologous chimeric antigen receptor (CAR) T-cell therapies are demonstrating success in the treatment of some hematological malignancies. Building on these clinical successes, we are developing engineered, allogeneic, iPSC-derived, iNK- and iT-cells to complement autologous cell therapies. In both hematological and solid tumor settings, antigen-driven expansion and maintenance of cytotoxic potential are important functional phenotypes to achieve therapeutic responses. CAR-mediated signaling is a primary and direct mechanism impacting these functionalities. To optimize CAR-driven function in our iPSC-derived platform cells, we generated a diverse panel of 45 different CAR formats that will be fully described within the data set and represents combinations of key immune effector cell signaling modalities, including: costimulatory domain families, cytokine support, ITAM multiplicity, hinge, transmembrane domain variants, and CD3 family signaling. This CAR panel was transduced via lentivirus into iPSC-derived T cells and assessed in vitro for their impact on the cell differentiation process, cytotoxic persistence in serial killing assays, and antigen-driven expansion. Through this, we identified a family of signaling components that mediate target-driven expansion and improved, persistent, cytotoxic function. This allowed for subsequent rational design of lead CAR constructs that combine signaling domain hits to further improve CAR-mediated expansion. The lead designs were characterized in a mouse tumor inhibition model and selected endodomains are incorporated into therapeutic designs for preclinical programs.