Effective termination of CART123 allows subsequent engraftment of human bone marrow cells. (A) Experimental schema: NSGS mice were conditioned with busulfan 30 mg/kg IP prior to IV injection of 2.5 × 106 T-cell–depleted (TCD) BMMCs from a healthy normal male donor (ND). Human hematopoietic engraftment was confirmed 2 weeks later by FC analysis of murine peripheral blood (PB). CD123-redirected CAR T cells were generated from the same donor. Xenografted mice were treated with 1 dose of saline, UTD, CART123, or CART123-CD20 IV (n = 5 mice per cohort). Two weeks following T cells, mice were bled to confirm reduction/ablation of myeloid cells by CART123-CD20. Mice subsequently received 1 dose of 10 μg rituximab (RTX) IP to deplete CAR T cells and were assessed by serial peripheral blood analysis to confirm eradication of T cells. Mice were then injected with 2.5 × 106 T-cell–depleted BMMCs IV from a healthy female donor. Four weeks after infusion of the female-origin bone marrow, mice were euthanized and bone marrow was harvested for analysis of tissues. (B) Analysis of murine peripheral blood 2 weeks after T-cell treatment demonstrates significant expansion of CD3+ T cells in mice treated with CART123, CART123-CD20, or UTD, but not with saline. (C) Analysis of murine peripheral blood 2 weeks after T-cell treatment demonstrates significant reduction of human CD3-PE-Cy7/CD19-APC double-negative myeloid cells in mice treated with CART123-CD20 or CART123 versus UTD or saline controls. (D) At study end point (3 weeks following injection of female BMMCs), xenograft mice treated with CART123-CD20 and rituximab ablation demonstrate peripheral blood engraftment of myeloid cells and differentiation into monocytes (E), whereas mice treated with CART123 cells rejected engraftment of female donor BMMCs. (F) At study end point, FISH analysis of bone marrow from xenograft mice treated with CART123-CD20 shows engraftment of second donor female-origin (XX) hematopoietic engraftment. Conversely, mixed male-origin (XY) and female-origin hematopoietic engraftment is observed in saline-treated control mice, and previously, CART123-treated animals rejected second transplantation with female BMMCs. Data are representative of 2 independent experiments.
