Comparison of Fc-engineered anti-CD33 antibody BI 836858 and nonengineered HuM195. (A) Hydrogen-deuterium exchange mass spectrometry (HDXMS) analysis of BI 836858 and HuM195 complexed with CD33. Protection from D₂O exchange when bound to CD33 mapped onto the structure of CD33. Blue region indicates protection of CD33 by BI 836858; red region indicates protection by HuM195. Resolution of the method is determined by the peptides produced by digestion with pepsin. The antibodies bind to nonoverlapping amino acid residues in the N-terminal region of CD33. A structural model was calculated based on Siglec-5²⁶ ; the predicted epitopes for BI 836858 (blue) and Hum195 (red) are depicted. (B) Effect of BI 836858 and HuM195 on CD33 surface retention on HL60 cells. Cell surface–bound IgG was determined at indicated time points with a secondary fluorescence-labeled anti-IgG antibody and data were normalized against time point 0. The means of 6 independent experiments are shown. Incubation of HL60 cell lines with both antibodies results in decreased CD33 surface exposure over time, indicative of internalization of antibody/CD33 complexes. BI 836858 shows significantly higher CD33 levels over time compared with HuM195. (C) ADCC activity on HL60 cells at an E:T ratio of 20:1; peripheral blood mononuclear cells from healthy individuals were used as effectors. (D) Summary of ADCC on leukemia cell lines and primary AML blasts. BI 836858 displays superior efficacy of cytolysis compared with HuM195 under identical experimental conditions in both AML cell lines and 6 primary AML samples. Healthy donor NK cells were used as effectors and the ⁵¹Cr-labeled ADCC assay was done to measure cytotoxicity.