Prior CLL therapy with a BTKi increases cytotoxicity of CD19/CD3 bsAb in vitro. PBMCs from BTKi-naïve (BN, n = 18), ibrutinib-treated (n = 20), or acalabrutinib-treated (n = 12) CLL patients were cultured with either CD19/CD3 bsAb, HER2/CD3 bsAb (nontargeting control), or medium only. CLL cell viability was analyzed after 3 and 5 days by flow cytometry. (A) Percent specific killing of CLL cells by bsAbs for BTKi-treated patients (BTKi, n = 32) and BN (n = 18) calculated as follows: ([%untreated CLL viability – %treated CLL viability]/[%untreated CLL viability] × 100). (B) Comparison of CLL-specific killing between acalabrutinib-treated (n = 12) and ibrutinib-treated (n = 20) patient samples with CD19/CD3 bsAb. (C) Comparison of baseline E:T ratio in PBMCs from BN (n = 18), acalabrutinib-treated (Aca, n = 12), and ibrutinib-treated (Ibr, n = 20) patients, calculated as follows: ([% CD4⁺ and CD8⁺ T cells]/[% CLL cells]). (D) Spearman’s correlation of baseline E:T ratios and percent specific killing of CLL cells in samples from BN (gray triangles, dashed regression line) and BTKi-treated patients (blue circles, solid regression line) cultured with CD19/CD3 bsAb for 3 and 5 days. Asterisks indicate statistical significance using Wilcoxon matched-pair signed-rank test for comparison of different treatments applied to individual patient samples or Mann-Whitney U test for comparison of different patient groups. *P < .05; **P < .01; ****P < .0001.