ACAR-H mediated tumor clearance in vivo. (A) Twenty-two NSG mice were injected IV with 10 × 10⁶ HA+Fluc+MM.1s cells at D0 and monitored by BLI for tumor burden at different time points (dorsal views shown). On D+13 there was clear evidence of intramedullary tumor in all animals, at which point 6 animals were left untreated, and 8 animals were intravenously injected with T cells transduced with a control EGFRvIII targeting CAR (EGFRvIII CAR) or ACAR-H (5 × 10⁶ CAR cells/animal). (B) Average radiance (p/s/cm²/sr) of whole mice in the 3 groups at different timepoints. (C) At termination of experiment (D+25 posttumor, D+12 post-CAR) by FACS, there was significant reduction of tumor in the bone marrow of ACAR-treated mice in comparison with EGFRvIII-treated and untreated animals. Tumor cells were identified as live/single/muCD11b⁻/HA⁺ with numbers normalized to Flow-Check beads to calculate relative engraftment. (D) Eradication of CD138⁺ tumor cells by ACAR was confirmed in bone marrow by IHC of femur. Hematoxylin and eosin staining shown at ×12.5 and ×400 (left and central panels) and immunostaining for CD138 (right panels) at ×200 original magnification. Mean ± SEM shown. *P < .05, **P < .01, ***P < .001, by t test.