Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

• Main Authors: Kento Fujiwara, Masaki Kitaura, Ayaka Tsunei, Hotaka Kusabuka, Erika Ogaki, Naoki Okada
• Format: Article
• Language: English
• Published: MDPI AG 2021-03-01
• Series: International Journal of Molecular Sciences
• Subjects: chimeric antigen receptor; signal transduction domain; CAR structure-activity relationship
• Online Access: https://www.mdpi.com/1422-0067/22/5/2476

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.