Preclinical comparison of non-signaling domain in CD19 CAR T cell with interleukin-7 receptor alpha signaling domain

Chimeric antigen receptor (CAR) T cells have emerged as an effective immunotherapy for hematologic malignancies. The non-signaling domain of CARs, comprising the spacer and transmembrane regions, is a key structural component that can be engineered to influence CAR expression and function. In this study, we evaluated three non-signaling domain configurations—IgG2.CH3/CD28, IgG2/CD28, and CD8/CD8—within a CD19 CAR construct incorporating 4–1BB and interleukin-7 receptor alpha (IL-7Rα) signaling domains. CARs incorporating the IgG2/CD28 domain exhibited reduced surface expression and diminished functional responses compared with IgG2.CH3/CD28 and CD8/CD8 constructs. The CD8/CD8 configuration supported the highest CAR expression and sustained surface density. In contrast, IgG2.CH3/CD28 CAR T cells displayed increased IL-2 and TNF-α secretion and enhanced CD107α upregulation following antigen stimulation. In a serial tumor cell rechallenge assay, IgG2.CH3/CD28 CAR T cells maintained cytotoxic activity and persistence compared with CD8/CD8 CAR T cells. In a NALM-6 xenograft model, IgG2.CH3/CD28 CAR T cells achieved durable tumor control and were associated with improved survival relative to CD8/CD8 CAR T cells. Collectively, these findings support the IgG2.CH3/CD28 non-signaling domain as a suitable structural component for CD19 CARs incorporating IL-7Rα signaling and provide insight into CAR design strategies aimed at improving T cell persistence and anti-leukemic activity. • Non-signaling domain choice affects CAR expression and function in CD19 IL-7Rα CAR T cells. • IgG2.CH3/CD28 CAR T cells maintain cytotoxic activity and persistence during repeated antigen exposure. • IgG2.CH3/CD28 CAR T cells improve tumor control and survival in a NALM-6 xenograft model.