All Nbs isolated by the STAR system empower CAR T cells to potently kill AML cells in vitro. (A) Schematic diagram of Nb CAR structure, including signal peptide (SP), IgG4 mutant (IgG4m) hinge, CD8 transmembrane domain (TM), 4-1BB, and CD3z domain. (B-D) Nb CAR T cells showed potent and specific cytotoxicity against THP-1 or HL60 cells, but not K562 or Jurkat cells, in a dose-dependent manner. UTD T cells did not exert obvious killing (n = 3). THP-1 cells stimulated Nb157 or Nb163 CAR T cells, but not UTD T cells, to release cytokines, including IFN-γ (E) and TNF-α (F) (n = 3). (G) Only THP-1 cells induced Nb157 or Nb163 CAR T cells to degranulate (ie, CD107a localization to the cell membrane) after a 4-hour coculture (n = 3). CellTrace Far Red–labeled Nb157 (H-I) or Nb163 (J-K) CAR T cells were coincubated with heat-inactivated THP-1 cells (I-K) or K562 cells (H-J) for 4 days, followed by flow cytometry analysis. CAR T cells were gated on GFP⁺ signals (n = 3). ***P < .001, 1-way analysis of variance. ns, not significant (P > .05).