Figure 7.
BissCAR T cells targeting CD13 and TIM3 eradicate AML PDXs, but with reduced toxicity to human HSCs in vivo. (A) Schematic diagram of AML PDX mice treated with control or BissCAR T cells. Twenty million PD AML cells were injected into NSG mice, followed by injection of 5 million BissCAR T cells or UTD T cells 2 weeks later. Next, human peripheral blood CD3+ cells were analyzed by serial bleeding weekly. (B-C) PD AML cells or T cells in mouse peripheral blood were monitored weekly by flow staining with anti-human CD33 or anti-human CD3 antibodies. Blood volume was normalized and quantified using CountBright counting beads (n = 3). (D) Mice survival was monitored and recorded (n = 6 per group). (E) Schematic diagram of HIS mice for evaluation of human HSC toxicity. A total of 1.5 million normal donor bone marrow (BM) CD34+ cells was injected into each NSG mouse. Four weeks later, 3 million Nb157 anti-TIM3 BissCAR T cells, conventional Nb157 CAR T cells, or UTD T cells were injected IV, followed by flow cytometry analysis of peripheral blood and bone marrow (n = 5 per group for BissCAR and UTD T cells; n = 3 per group for Nb157 T cells). (F) Bone marrow of HIS mice, which were treated with T cells for 3 weeks, was analyzed by flow cytometry after staining with CD45/Lin/CD34/CD38/7-AAD. Representative fluorescence-activate cell sorting plots were used to identify HSC (CD34+CD38−) and myeloid progenitors (CD34+CD38+). (G) HSCs (CD45+Lin−CD34+CD38−) in the bone marrow of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment. (H) Myeloid progenitors (CD45+Lin−CD34+CD38−) in the bone marrow of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment. (I) Monocytes (human CD45+CD33+) from peripheral blood of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment; cell number and blood volume were quantified using CountBright counting beads. In (G-I), n = 5 per group for BissCAR T cells and UTD T cells, n = 3 per group for Nb157 T cells. *P < .05, **P < .01, ***P < .001, Student t test.

BissCAR T cells targeting CD13 and TIM3 eradicate AML PDXs, but with reduced toxicity to human HSCs in vivo. (A) Schematic diagram of AML PDX mice treated with control or BissCAR T cells. Twenty million PD AML cells were injected into NSG mice, followed by injection of 5 million BissCAR T cells or UTD T cells 2 weeks later. Next, human peripheral blood CD3+ cells were analyzed by serial bleeding weekly. (B-C) PD AML cells or T cells in mouse peripheral blood were monitored weekly by flow staining with anti-human CD33 or anti-human CD3 antibodies. Blood volume was normalized and quantified using CountBright counting beads (n = 3). (D) Mice survival was monitored and recorded (n = 6 per group). (E) Schematic diagram of HIS mice for evaluation of human HSC toxicity. A total of 1.5 million normal donor bone marrow (BM) CD34+ cells was injected into each NSG mouse. Four weeks later, 3 million Nb157 anti-TIM3 BissCAR T cells, conventional Nb157 CAR T cells, or UTD T cells were injected IV, followed by flow cytometry analysis of peripheral blood and bone marrow (n = 5 per group for BissCAR and UTD T cells; n = 3 per group for Nb157 T cells). (F) Bone marrow of HIS mice, which were treated with T cells for 3 weeks, was analyzed by flow cytometry after staining with CD45/Lin/CD34/CD38/7-AAD. Representative fluorescence-activate cell sorting plots were used to identify HSC (CD34+CD38−) and myeloid progenitors (CD34+CD38+). (G) HSCs (CD45+Lin−CD34+CD38−) in the bone marrow of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment. (H) Myeloid progenitors (CD45+Lin−CD34+CD38−) in the bone marrow of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment. (I) Monocytes (human CD45+CD33+) from peripheral blood of HIS mice were analyzed by flow cytometry 3 weeks after the initial treatment; cell number and blood volume were quantified using CountBright counting beads. In (G-I), n = 5 per group for BissCAR T cells and UTD T cells, n = 3 per group for Nb157 T cells. *P < .05, **P < .01, ***P < .001, Student t test.

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