Anti-AML efficacy of RNA-CART123 and of CART123/CD20 with rituximab depletion. (A) MOLM14-bearing NSG mice (n = 10 mice per cohort) were treated IV with saline, mock-RNA T cells × 3 doses, RNA-CART123 × 3 doses, CART123, or CART123-CD20 and assessed by BLI. Some mice were injected IP with 60 mg/kg cyclophosphamide diluted in 200 μL saline at 24 hours prior to second and third doses of mock-RNA T cells or RNA-CART123 to deplete previously administered T cells. RNA-CART123 and CART123-CD20 eradicated leukemia by 4 weeks post-T cells (week 5 time point), with similar kinetics to those of CART123, resulting in long-term animal survival. Ablation of CART123/CD20 with 1 mg/kg rituximab IP administered at week 5 did not alter leukemia remission status. MOLM14 rechallenge at week 9 resulted in rapid disease progression and animal death in mice previously treated with RNA-CART123, confirming lack of longer-term persistence of these T cells in vivo. MOLM14 rechallenge of CART123-CD20–treated animals subsequently treated with rituximab also resulted in rapid AML relapse, confirming successful prior ablation of CART123-CD20 in vivo. (B) Treatment of AML290-PDX mice (established as in Figure 4D; n = 8 mice per cohort) with 3 doses of RNA-CART123, as in panel A, induced human leukemia remission measured in murine tissues harvested at 6 weeks after the third dose of T cells (week 8 time point). (C) Treatment of juvenile myelomonocytic leukemia [JMML] 117 PDX mice with CART123 (123) or CART123-CD20 (123-CD20) at week 1 (n = 5 to 8 mice per cohort) resulted in marked reduction in CD45-APC⁺ CD33-PE⁺ CD123-PE-Cy7⁺ AML burden in murine tissues at 8 weeks post-T cells (week 9 time point). PDX mice treated with 1 dose of 1 mg/kg rituximab at the week 5 time point demonstrated sustained AML remission at the week 9 time point (8 weeks after CART123-CD20, 4 weeks after rituximab). (D) FC analysis of CD45-APC⁺ CD3-PacificBlue⁺ CAR T cells in peripheral blood of JMML117 PDX mice treated with CART123-CD20 without and with subsequent rituximab. Total CAR T-cell counts (blue gate) in murine blood normalized by quantitative counting beads are markedly reduced following rituximab treatment, although T-cell elimination is less rapid than with alemtuzumab treatment, as in Figure 3. Note that detectable CART123-CD20 decreased over time due to ongoing AML clearance from peripheral blood. (E) Immunohistochemical (IHC) staining of harvested murine spleen tissues, as in panel C, for CD33⁺ human AML and CD3⁺ CAR T cells at week 9 time point (8 weeks post-T cells) in JMML117 PDX model. Slides were scanned at ×20 magnification with an Aperio Scanscope CS-O slide scanner with visualization via the Aperio Image Analysis Toolkit (Leica Biosystems).