For the study on binding characteristics of anti–B7-H3 mAb or B7-H3×4-1BB bsAb by ELISA, 0.1 μg of the following B7 family antigens [diluted in phosphate-buffered saline (PBS)] was coated on Nunc Maxisorp plates overnight at 4°C: hB7-H3/CD276/His tag (11188-H08H), human B7-1/CD80/His tag (10698-H08H), human B7-2/CD86/His tag (10699-H08H), human B7-DC/PD-L2/CD273/His tag (10292-H08H), human B7-H1/PD-L1/CD274/His tag (10084-H08H), human ICOS-L/B7-H2/His tag (11559-H08H), human B7-H4/B7S1/B7x/VCTN1/His tag (10738-H08H), human B7-H5/Gi24/VISTA/His tag (13482-H08H), human B7-H6/His tag (16140-H08H), human B7-H7(ECD)/HHLA2/Fc tag (16139-H02H), cynomolgus B7-H3(ECD)/Fc tag (90806-C02H), mB7-H3/His tag (50973-M08H), and m4-1BB/His tag (50811-M08H) (all from Sinobio). After washing with 0.05% Tween 20 in PBS and blocking with 1% bovine serum albumin (Gibco, 30063) in PBS, purified antibodies were added and incubated for 2 hours at 37°C. The wells were washed and incubated for 1 hour at 37°C with horseradish peroxidase (HRP)–conjugated goat anti-human IgG F(ab′)2 cross-adsorbed secondary antibody (Pierce, 31414) and developed with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, T0440). Absorbance at 450 nm was measured by SPECTROstar Nano (BMG Labtech).

SPR affinity assay was performed on a Biacore T200 (Cytiva). Anti–B7-H3 mAb was captured on Sensor Chip Protein A (Cytiva, 20127). Determination of hB7-H3 binding affinity was performed by applying dilutions of hB7-H3 protein (ranging from 3.125 to 100 nM) to the chip surface with a flow rate of 30 μl/min for 60 s; HBS-EP Buffer (Cytiva, BR100188) was passed over the surface for 180 s while monitoring hB7-H3 dissociation. Kinetics was analyzed under the 1:1 binding model (Langmuir model) by Biacore Insight Evaluation Software (Cytiva).

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