To improve the persistence of CAR T cells in vivo, Guedan et al. modified specific intracellular signaling domains (ICDs) to induce preferred phenotypes. The ICOS ICD in CD4+ CAR T cells drove them towards a Th/Th17 phenotype and enhanced in vivo persistence, which in turn improved the persistence of co-transferred CD8+ CAR T cells with CD28 or 4-1BB ICDs. Third generation CARs incorporating ICOS and 4-1BB ICDs showed enhanced persistence and efficacy, but only when the ICOS ICD was proximal to the ICOS transmembrane domain.
Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.