Background Following the remarkable success of CAR T adoptive cell therapy (ACT) in treating relapsed and refractory hematologic malignancies, ACT is now being avidly pursued for treatment of solid tumors. However, solid tumors pose unique barriers to therapeutic potential of ACT efficacy – rapid loss of function (terminal exhaustion), attrition in cell numbers and inability to respond to checkpoint blockade, are the key challenges that limit CAR T cell efficacy in solid tumors. All of these attributes are primarily regulated by TCF-1^Hi stem-like progenitor lineage of T cells (T_PEX) – we hypothesized that transcriptional engineering of CAR T cells to drive a T_PEX lineage, would mediate durable control of solid tumors and further enhance efficacy of CAR T cells via synergy with checkpoint blockade.

Methods To drive a T_PEX-lineage in CAR T cells, we engineered the ectopic expression of pro-memory transcription factor Inhibitor of DNA binding 3 (Id3) in CAR T cells (Id3-CAR). Beyond control of solid tumors following ACT, CAR T cell lineage diversity and responsiveness to checkpoint blockade were used as key indicators of success in modifying the functional fate of CAR T cells.

Results Id3-CAR T cells preferentially differentiated into T_PEX subset compared to WT-CAR T cells, with higher expression of memory-associated markers (such as Bcl-2, IL-7 and CD62L) and lower expression of markers of terminal differentiation (such as Tim3, granzyme B). In addition to superior control of primary tumors (P<0.001) and remarkable responsiveness to checkpoint blockade (p<0.001), the Id3-CAR T cells mediated significantly (p<0.001) greater long-term protection from tumor relapse in mice that cleared primary tumors compared to WT-CAR T cells. Augmented in vivo tumor control by the pro-memory factor Id3 expression in CAR T cells was mechanistically associated with resistance to exhaustion, robust production of effector cytokines (IFN-γ and TNF-α), as well as increased intratumoral localization of T_PEX CAR T cells.

Conclusions We show here that driving a T_PEX subset by transcriptional engineering of CAR T cells, can overcome some of the cardinal barriers of ACT in solid tumors. Given the critical role of multipotent, self-renewing, stem-like TCF-1^Hi exhausted cells in continuously feeding the transient effector T cell pool, persisting long-term and responding to PD-1 checkpoint blockade immunotherapy, these data highlight Id3 and other such pro-memory transcription factors as attractive CAR engineering targets to bypass exhaustion in the TME, enhance checkpoint blockade immunotherapy outcomes, promote durable CAR T cell memory and protection from tumor relapse.