In vivo, combinatorial bispecific and split CD13 and TIM3 CAR T cells eradicate tumor expressing CD13 and TIM3, but not tumor expressing only CD13. (A) Schematic diagram of combinatorial bispecific and split CD13 and TIM3. Nb157 linked with CD3z recognized CD13 on normal HSCs or LSCs. Anti-TIM3 linked with CD28 and 4-1BB recognized TIM3 only on LSCs. Such Biss CAR T cells can be fully activated only by LSCs but not by HSCs. (B) Flow cytometry showing the expression of TIM3, CD90, CD13 on normal donor bone marrow cells (ND-BM) or PD AML cells, which were gated from CD45⁺Lin⁻CD34⁺CD38⁻ subsets. Ten million NB4 (C) or NB4-TIM3 (D) cells were transplanted subcutaneously into NSG mice to form 100-mm³ tumors. Three million combinatorial BissCAR T cells, conventional Nb157 CAR T cells, or UTD T cells were injected IV into each NSG mouse with the tumors. The engraftment volume was monitored by measuring the length and width of the tumor every other day (n = 4). (E) Three weeks after mice with NB4 or NB4-TIM3 tumors were treated with BissCAR T cells or UTD T cells, human T-cell (CD3⁺) numbers in mouse peripheral blood were analyzed by flow cytometry and quantified using CountBright counting beads (n = 3). *P < .05, Student t test.