Analysis of 20D9-ADC and control IgG1-ADC cytotoxicity in leukemia and lymphoma cell lines. (A) Correlation of MFI of CD64 cell surface expression and MFI of FLT3 cell surface expression of myeloid human cell lines measure in flow cytometry. Expression data presented in supplemental Figure 5A-B. Black lines indicates simple linear regression with error interval. R = Pearson correlation coefficient; r² = coefficient of determination; P value from 2-tailed test with confidence interval of 95%. (B,C,E,F) Assessment of cytotoxicity of ADCs in different human cell lines. Viability was determined after 96-hour treatment with different concentrations of ADCs by resazurin fluorescence and normalized to untreated control. Dashed line indicates 100 ng/mL drug concentration. Mean ± SD of n = 3 biological replicates. (B) Treatment of FLT3⁺ human cell lines with 20D9-ADC. (C) Treatment of FLT3⁻ human cell lines with 20D9-ADC. (D) Correlation of IC₅₀ values of 20D9-ADC and sum of MFI of FLT3 and CD64 cell surface expression of myeloid human cell lines measured in flow cytometry. Expression data presented in supplemental Figure 5A-B and IC₅₀ values of 20D9-ADC in supplemental Table 1. Black lines indicates simple linear regression with error interval. (E) Treatment of FLT3⁺ human cell lines with IgG1-ADC. (F) Treatment of MOLM-13 cells with either native, buffer-incubated control (buffer control) or deglycosylated (deglyc.) 20D9-ADC or IgG1-ADC.