Figure 1.

Murine CD8⁺ T cells expressing short spacer L1CAM-specific CARs with CD28/ζ signaling domain show superior anti-tumor activity in vivo. (A) Scheme of retroviral CAR T cell constructs used to generate L1CAM-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 SK-N-BE(2) target cells for 24 h. Levels of secreted cytokines were measured by Ifng and Il2 ELISA. Shown is one representative experiment of three. (C) Tumor growth curves of SK-N-BE(2) tumor-bearing Rag^−/− mice treated with 1 × 10⁶ EGFRt⁺ ChRLuc/OT-1/Rag^−/− CAR T cells transduced with L1CAM-SS-4-1BB/ζ (n = 6), L1CAM-LS-4-1BB/ζ (n = 5), L1CAM-SS-28/ζ (n = 9), L1CAM-LS-28/ζ (n = 6) or with untransduced T cells (n = 5) at indicated time-point. Each line represents change in tumor volume of an individual mouse over the period of the experiment. Data are representative of two independently performed experiments. (D) Kaplan–Meier survival analysis is shown from total mice for each indicated CAR T cell treatment. Combined data from two experiments are shown. Statistical significances are indicated as ***, p ≤ 0.005. ns = not statistically significant, SS = short spacer, LS = long spacer, ATT = adoptive T cell transfer, ms = median survival.