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Antibody affinity measurement

All scFv-binding affinity measurements were conducted at Y-Biologics (Republic of Korea). The sequences of anti-CD5 scFvs were cloned into an N293F expression vector containing an mIgG2a Fc tag. Each plasmid was transfected into HEK293F cells, and recom binant scFv proteins were purified using size-exclusion chromatog raphy. Binding kinetics was measured using the Octet RH96 (Sarto rius, Germany) according to the manufacturer’s instructions. Briefly, recombinant anti-CD5 scFv-mIgG2a Fc fusion proteins were immo bilized onto anti-mouse IgG Fc capture biosensors as ligands and binding was assessed using recombinant human CD5 extracellular domain protein (ACROBiosystems, USA; catalog no. CD5 H52H58) as analyte. Data were analyzed using Octet BLI Analysis Software v.12.2.