Figure 4.
Figure 4. In vivo termination of CAR T cells with alemtuzumab in human AML xenograft models. (A) MOLM14-bearing NSG mice (n = 10 mice per cohort) were treated IV with saline (gray bar), 1 × 106 UTD (striped bar), or 1 × 106 CART123 (red bars) at week 1 time point and assessed by weekly BLI. CART123 induced AML remission by week 4. One dose of 1 mg/kg alemtuzumab was administered IP at 4 weeks following CART123 (week 5 time point; blue bar) in designated animals for T-cell depletion. MOLM14 rechallenge at week 9 resulted in rapid leukemia progression and animal death only in mice in which CART123 had been previously ablated with alemtuzumab. (B) Representative peripheral blood FC analysis of MOLM14-bearing, CART123-treated mouse before and after alemtuzumab (alem) administration (week 5 time point). No CD3-PacificBlue+ human T cells are detected at 24 hours (h) following a single dose of alemtuzumab. Blue gate denotes human T-cell count in murine blood normalized to quantitative counting beads. Note that fewer T cells remain in peripheral blood at this time point due to prior CART123-mediated AML clearance. (C) Immunohistochemical analysis of CD8+ T cells and CD33+ AML cells in harvested tissues of MOLM14-bearing mice treated with CART123 (week 2, 1 week after CART123) or CART123 with 1 mg/kg alemtuzumab (week 5, 24 hours after alemtuzumab/4 weeks after CART123). Note that animals euthanized at earlier time points in this subexperiment were not included in the main data analysis shown in panel A. (D) FC analyses of human CD45-APC+ CD33-PE+ CD123-PE-Cy7+ AML and CD3-PacificBlue+ CAR T cells in an AML PDX model (juvenile myelomonocytic leukemia [JMML] 117). Rapid T-cell depletion and sustained AML remission were observed in CART123-treated animals following a single dose of alemtuzumab (administered 24 hours prior to week 5 blood sampling). (E) End-study IHC analysis of murine bone marrow from saline and UTD control mice is hypocellular and fibrotic without evidence of residual AML, suggestive of advanced leukemia in these models. Marrow tissues from CART123-treated mice are more cellular and demonstrate AML clearance even after alemtuzumab administration.

In vivo termination of CAR T cells with alemtuzumab in human AML xenograft models. (A) MOLM14-bearing NSG mice (n = 10 mice per cohort) were treated IV with saline (gray bar), 1 × 106 UTD (striped bar), or 1 × 106 CART123 (red bars) at week 1 time point and assessed by weekly BLI. CART123 induced AML remission by week 4. One dose of 1 mg/kg alemtuzumab was administered IP at 4 weeks following CART123 (week 5 time point; blue bar) in designated animals for T-cell depletion. MOLM14 rechallenge at week 9 resulted in rapid leukemia progression and animal death only in mice in which CART123 had been previously ablated with alemtuzumab. (B) Representative peripheral blood FC analysis of MOLM14-bearing, CART123-treated mouse before and after alemtuzumab (alem) administration (week 5 time point). No CD3-PacificBlue+ human T cells are detected at 24 hours (h) following a single dose of alemtuzumab. Blue gate denotes human T-cell count in murine blood normalized to quantitative counting beads. Note that fewer T cells remain in peripheral blood at this time point due to prior CART123-mediated AML clearance. (C) Immunohistochemical analysis of CD8+ T cells and CD33+ AML cells in harvested tissues of MOLM14-bearing mice treated with CART123 (week 2, 1 week after CART123) or CART123 with 1 mg/kg alemtuzumab (week 5, 24 hours after alemtuzumab/4 weeks after CART123). Note that animals euthanized at earlier time points in this subexperiment were not included in the main data analysis shown in panel A. (D) FC analyses of human CD45-APC+ CD33-PE+ CD123-PE-Cy7+ AML and CD3-PacificBlue+ CAR T cells in an AML PDX model (juvenile myelomonocytic leukemia [JMML] 117). Rapid T-cell depletion and sustained AML remission were observed in CART123-treated animals following a single dose of alemtuzumab (administered 24 hours prior to week 5 blood sampling). (E) End-study IHC analysis of murine bone marrow from saline and UTD control mice is hypocellular and fibrotic without evidence of residual AML, suggestive of advanced leukemia in these models. Marrow tissues from CART123-treated mice are more cellular and demonstrate AML clearance even after alemtuzumab administration.

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