Fig. 8

Cellular targeting of Bruton’s kinase (BTK) by αCD33-mAB-P/P-ibrutinib-Cy3.5 and inhibition of clonal growth of treated AML-cells. A Schematic overview: spontaneous assembly of the αCD33-mAB-P/P-ibrutinib-Cy3.5 nanocarrier. B The αCD33-mAB-P/P conjugate was incubated for 2 h with anionic ibrutinib-Cy3.5 (left panels) or uncharged ibrutinib (trademark: imbruvica™; right panels) in 1:20 ratio and applied to cell-culture treated glass slides for fluorescence microscopy. Only αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes led to the formation of numerous vesicles, where the larger vesicles showed intense Cy3.5 fluorescence (upper left panel) and vesicle formation in phase contrast (PC, lower left panel). No nanocarrier formation in presence of uncharged ibrutinib (upper and lower right panels, bubbles in lower right panel are mounting air inclusion artifacts). C Electromobility shift assays showing the electrostatic loading capacity of ibrutinib-Cy3.5 to conjugates from A in a molar ratio. One mol of αCD33-mAB-P/P can bind at least 20–50 mol ibrutinib-Cy3.5. D CD33-positive OCI-AML2 cells were treated by the respective conjugates shown for 72 h, lysed and subjected to SDS–PAGE and Western blotting for phospho-BTK (pBTK), total BTK (tBTK) and actin as a loading control. Both, free ibrutinib-Cy3.5 and αCD33-mAB-P/P-ibrutinib-Cy3.5 complexes inhibited the phosphorylation of BTK. E-M Fluorescence microscopy of OCI-AML2 cells treated with targeting conjugates and controls showing a marked intracellular enrichment of Cy3.5-signals (I). Fluorescence microscopy of OCI-AML2 cells pre-treated with ibrutinib-bodipy (green, G and M) do not show intracellular enrichment of Cy3.5-signals after αCD33-mAB-P/P-ibrutinib-Cy3.5 treatment (J compared to G). N Upper panels: Photographs of representative colony formation assays as summarized in the lower panel. In colony formation assays, 1200 nM untargeted ibrutinib-Cy3.5 did not reduce colony growth of OCI-AML2 cells, while the specifically targeted αCD33-mAB-P/P-ibrutinib-Cy3.5 (60 nM nanocarrier: 1200 nM ibrutinib-Cy3.5) reduced the colony growth to below 30% of the PBS controls, more than the treatment with 1200 nM uncharged ibrutinib (right-most bar). Significance: *, p < 0.05, 2-tailed T-test. Means plus SD of 3 independent experiments. α, anti