Analysis of cytotoxicity of FLT3-specific 20D9-ADC to different FLT3 variants. (A) Schematic process of P5 conjugation technology39 via disulfite bond reduction and Staudinger-induced Michael addition. Final ADC consists of monoclonal antibody (20D9 or IgG1 antibody) coupled to monomethyl auristatin F toxin. Figure was created with BioRender.com. (B-E,G) Assessment of cytotoxicity of ADCs in different Ba/F3 cell lines. Viability was determined after 72-hour treatment with different ADC concentrations by trypan blue exclusion count and compared with untreated control. Mean ± SD of n = 3 biological replicates. (B) Treatment of Ba/F3-pMIY ev, hFLT3wt, and hFLT3ITD cells with 20D9-ADC. (C) Treatment of Ba/F3-pMIY ev, hFLT3wt, mFLT3wt, and cynoFLT3wt cells with 20D9-ADC. (D) Treatment of Ba/F3-pMIY ev, hFLT3wt, and hFLT3S50P/P54R cells with 20D9-ADC. (E) Treatment of Ba/F3-pMIY ev and hCD64 with either 20D9-ADC or IgG1-ADC. (F) IC50 values of 20D9-ADC and IgG1-ADC in Ba/F3-pMIY hCD64 and hCD64-hFLT3 cells. Calculation based on data from Figure 2E and G calculated by GraphPad Prism. Each dot represents a replicate; horizontal line indicates mean. (G) Treatment of Ba/F3-pMIY ev and hCD64-hFLT3 with different concentrations of either 20D9-ADC or IgG1-ADC. ∗P < .05 (unpaired, 2-tailed t test). ns, not significant.
