Fig. 1.

AK002 is a non-fucosylated, humanized antibody that is specific for Siglec-8 and interacts with CD16a on NK cells. a AK002 specificity and cross-reactivity was examined using a recombinant human Siglec cross-reactivity ELISA. Recombinant Siglecs were coated on the plate overnight at 0.2 µg/mL and AK002 was added at 2 µg/mL for 2 h. The binding kinetics of (b) monovalent AK002 Fabs or (c) full-length IgG AK002 (two-fold dilutions from 12.5 to 0.8 nM) to the Siglec-8 ECD antigen was measured using biolayer interferometry. The following kinetic parameters were determined for the Fab and full-length IgG, respectively: k_on = 1.04 × 10⁶ 1/Ms, k_off = 4.82 × 10⁻⁴ 1/s, Kd = 464 pM and k_on = 1.86 × 10⁶ 1/Ms, since no significant dissociation was observed for the measured time of 600 s Kd was assigned to <1 pM. d Binding of 50 nM AK002-G4 (light blue) and AK002 (dark blue) to eosinophils, NK cells, and T cells gated as shown in (c) in human peripheral blood by flow cytometry. e Titration of AK002 binding in human peripheral blood to eosinophils, NK cells, and T cells gated as shown in panel C by flow cytometry. Siglec, sialic acid-binding immunoglobulin-like lectin; NK, natural killer; MFI, median fluorescence intensity; FMO, fluorescence minus one.