Figure 3.

Combination with conjugator enhances recognition of multiple antigens by α-Cot-NK92 cells. (A) Additive cytotoxic effects of α-Cot-NK92 cells in multi-targeting through co-treatment with α-HER2-Cot and ZEGFR-Cot. α-Cot-NK92 cells were co-cultured with calcein-stained AU565 cells with/without α-HER2-Cot (50 or 500 μg/mL), ZEGFR-Cot (5 or 50 ng/mL), or both α-HER2-Cot and ZEGFR-Cot at a 1:1 E:T ratio for 4 h, or were co-cultured with calcein-stained A549-Red-Fluc cells with/without α-HER2-Cot (5 or 500 μg/mL) and/or ZEGFR-Cot (2.5 or 50 ng/mL) at a 5:1 E:T ratio for 4 h. (B) To mimic the heterogeneity of the recurrent tumor, the EGFR⁺HER2⁻ MDA-MB-231 and EGFR⁻HER2⁺ MDA-MB-453 cells were mixed. On day 1, one group was not treated with α-Cot-NK92 cells (none) and the other groups were co-cultured with α-Cot-NK92 cells with/without ZEGFR-Cot at a 1:1 E:T ratio for 4 h. After removing α-Cot-NK92 cells, the remaining target cells were cultured for 2 d and then co-cultured with the α-Cot-NK92 cells with ZEGFR-cot or α-HER2-cot. (C) Cytotoxicity of α-Cot-NK92 cells to tumor cells on day 1 and (D) after 2 d in a model mimicking a recurrent tumor. Population change was measured using a FACS. All cytotoxicity data are presented as the mean ± S.D. of triplicates. Statistical significance of differences between groups was evaluated using paired Student’s t-test. ***P < 0.001; **P < 0.01; *P < 0.05. CAR, chimeric antigen receptor; Cot, cotinine; NK, natural killer. α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92α-Cot-NK92.