CD19/CD3-scFv-Fc demonstrates activity against CLL cells from ibrutinib-treated patients. PBMCs from patients receiving ibrutinib for 12 months and from treatment-naïve CLL patients were cultured with CD19/CD3-scFv-Fc, HER2/CD3-scFv-Fc, or medium alone. Percent CD25⁺/CD69⁺ cells (A); percent granzyme B⁺ cells (B), both after 48 hours (n = 9, ibrutinib-treated; n = 10, treatment-naïve). (C) CLL specific killing by treatment group after 3 and 7 days, respectively (n = 12, ibrutinib-treated; n = 14, treatment-naïve). The average (range) viability of medium-only control samples from ibrutinib treated patients was 65% (48% to 85%) on day 3 and 68% (49% to 84%) on day 7, and from treatment-naïve patients 65% (46% to 86%) on day 3 and 68% (52% to 89%) on day 7. (D) Spearman’s correlation between initial E:T ratios in PBMC samples used and CLL cell specific killing by CD19/CD3-scFv-Fc after 3 days in culture. Solid and dotted lines show regressions for ibrutinib-treated and treatment-naïve samples, respectively. (E) CLL specific killing at 3 days for samples with initial E:T ratios of >1:10 (n = 8, ibrutinib-treated; n = 8, treatment-naïve). Median and IQR are shown. Asterisks in panels A-C denote significance using Wilcoxon matched-pairs signed rank test for comparisons within cohorts or Mann-Whitney U test for comparisons across cohorts. Mann-Whitney U test was used for panel E. *P < .05; **P < .01; ***P < .001; ****P < .0001; ns, P > .05. IB, ibrutinib-treated; TN, treatment naïve.