Figure 4.

HER2-specific CAR T cells with CD28 signaling outperform 4-1BB harboring CAR T cells. (A) Scheme of retroviral CAR T cell constructs used to generate HER2-specific second-generation CAR T cells harboring a short or long spacer with 4-1BB or CD28 signaling domain. L: leader sequence; TM: transmembrane domain; T2A: virus 2A self-cleaving sequence, EGFRt, truncated epidermal growth factor receptor. (B) CD8⁺ T cells derived from ChRLuc/OT-1/Rag^−/− transduced with indicated CAR constructs were co-cultured with titrated numbers of SKOV3 target cells for 24 h. Levels of secreted cytokines were measured by Ifng and Il2 ELISA. Shown is one representative experiment of three. (C) Rag^−/− mice were subcutaneously injected with SKOV3 tumor cells and treated with 1 × 10⁶ CAR T cells equipped with the indicated HER2-specific CAR constructs or with untransduced T cells (n = 5 for each treatment group). Tumor volume was determined by caliper measurement. A single experiment is shown with each line representing change in tumor volume of an individual mouse over time.