An activation-biased 2B4 costimulatory domain enhances IFN-γ production and supports sustained cytotoxic activity in CD5-targeting CAR-NK92 cells

The intracellular domain of 2B4 (CD244) contains immunoreceptor tyrosine-based switch motifs (ITSMs) that mediate both activating and inhibitory signaling in natural killer (NK) cells. Although wild-type 2B4 has been shown to enhance CAR-NK cell activity, whether selective biasing of 2B4 signaling toward activation can further enhance CAR-NK function remains unclear. We engineered an activation-biased 2B4 variant, 2B4(A), by substituting a conserved ITSM tyrosine residue with phenylalanine, a modification designed to potentially attenuate SHP-1/SHIP-1-mediated inhibitory signaling. CD5-targeting CAR constructs incorporating 4-1BB, wild-type 2B4, or 2B4(A) were expressed in NK92 cells. Compared with wild-type aCD5-2B4ζ CAR-NK92 cells, aCD5-2B4(A)ζ CAR-NK92 cells preserved cytotoxicity while exhibiting enhanced interferon-γ production and sustained serial killing. Transcriptomic profiling showed a distinct 307-gene signature in aCD5-2B4(A)ζ CAR-NK92 cells, enriched for programs involving cell-cycle regulation, mTOR1 signaling, cytokine-responsive signaling, and NK-κB-associated activation pathways. In a Jurkat xenograft model, aCD5-2B4(A)ζ CAR-NK92 cells reduced tumor burden and improved survival tendency compared with wild-type aCD5-a2B4ζ CAR-NK92 cells. These findings support activation-biased 2B4 signaling as a rational strategy to enhance CAR-NK functional durability, supporting NK cell-aligned costimulatory engineering for next-generation CAR-NK therapies.