Schematic representation for obtaining scFvs that bind and block FcγRIIIA. BALB/c mice were immunized with the recombinant human FcγRIIIA, total splenic RNA was isolated, and genes encoding the V_H and V_L chains were amplified. A second polymerase chain reaction stitched V_H and V_L with a linker, and the products were cloned into a phagemid vector via Gibson assembly. E coli was then transformed with the constructs and a scFv-phage display library obtained. Five rounds of selection (R1, R2A, R2B, R3A, and R3B) were performed to select phages bound to FcγRIIIA with minimal cross-reactivity with FcγRIIA (supplemental Figure 1). Selection of scFv was based on binding to NK cells by flow cytometry and inhibition of hIgG-FcγRIIIA interaction by homogeneous time-resolved fluorescence. Purified scFv were analyzed for binding to FcγRIIIA by ELISA and Octet; minimal cross-reactivity with the other human receptors and inhibition of hIgG-FcγRIIIA interaction (ie, FcγRIIIA blockers) were part of the selection process. The final antibody fragment, 17C02-scFv, was selected from 10 candidates based on these assessments as well as sequencing analysis to screen for glycosylation, oxidation, aggregation, deamidation/isomerization, and proteolytic sites to exclude scFv molecules with low biochemical stability.