Functional validation of 161519 TriKE. (A) Simplified schema of 161519 TriKE construct, cloned into the pET28c vector via NcoI and XhoI restriction sites. The construct consists of 3 arms: an anti-CD16 scFv arm, IL-15 arm, and anti-CD19 scFv arm joined by 2 linkers (HMA and EASGGPE). Frozen PBMCs from healthy donors (n = 6) were incubated with noted treatments (30 nM for everything but IL-15 [used at 0.3 nM]) to evaluate background CD107a expression (degranulation) (B) or intracellular IFNγ production (C) on CD56⁺/CD3⁻ NK cells in a 4-hour assay. CD107a expression (D) and intracellular IFNγ (E) were also evaluated under the same conditions as stated before on NK cells within the PBMCs but in the presence of Raji targets at a 2:1 effector/target ratio. To evaluate the priming effects of the molecules on NK cells before target encounter, noted treatments (30 nM for everything but IL-15 [used at 0.3 nM]) were incubated overnight with enriched NK cells, and then CD107a (F) or IFNγ (G) was assessed after a 4-hour coculture with Raji targets on the next day (n = 5). One-way analysis of variance (ANOVA) with repeated measures was used to calculate differences against the 161519 group. Error bars indicate the mean ± standard error of the mean. Statistical significance are determined as *P < .05, **P < .01, ***P < .001, and ****P < .0001. NT, no treatment; Ritux, rituximab.