Potent degradation and inhibition of mutated BTK in vitro and in vivo with UBX-382 treatment. (A) Schematic domain structure of BTK with known mutant sites (B) Transient-expressing WT or various mutant BTK in HEK293 cells were treated with UBX-382, ARQ-531, or MT-802 for 24 hours in a concentration-dependent manner (0.1, 1, and 10 μM). BTK levels and Y223 phosphorylation were visualized by immunoblotting. GAPDH was used as an internal loading control. Data were obtained from 2 independent experiments (n = 2). (C) Parental TMD-8 cells, WT-, or C481S BTK–overexpressed TMD-8 cells were treated with the indicated concentration of ibrutinib or UBX-382 for 5 days to measure the inhibitory effect on proliferation. These experiments were performed in duplicates by Cell Titer-Glo 2.0 Assay. Data are presented as the mean ± SEM. (D) UBX-382 showed antitumor effects in TMD-8 BTK C481S in vivo models and induced tumor remission. TMD-8 BTK C481S cells were inoculated subcutaneously into the right flank of male CB17/severe combined immunodeficient mice. When tumors had reached over 180 to 200 mm3, the mice were divided into 7 groups and subjected to oral treatment with a control vehicle (n = 7), ibrutinib (30 mg/kg, n = 7), Binder (30 mg/kg, n = 7), ARQ-531 (30 mg/kg, n = 7), and UBX-382 (3, 10, 30 mg/kg, n = 7), respectively, each day for 21 days. Tumor volume (mean ± SEM) was observed for 84 days.
