CD38 epitope binning

In-tandem epitope binning of CD38-specific nanobodies and Dara scFv was performed on an Octet RED384 instrument (ForteBio). As running buffer HBS-EP + (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20) was used. Experiments were performed at 20 °C. The shake speed during the biosensor preparation and epitope binning was set at 1000 rpm. Amine reactive 2^nd Generation (AR2G) biosensors (ForteBio) were activated for 10 min with EDC(20 mM)/NHS(10 mM) and human CD38 protein was loaded at 10 µg/ml in 10 mM sodium acetate pH6 for 15 min. After immobilization, surfaces were deactivated with 1 M ethanolamine (pH 8.5) for 10 min. In the epitope binning, 100 nM nanobody 1 was loaded during 3 min on immobilized CD38 to saturate all available epitopes. Nanobody 2 was presented after a 10 s dip in running buffer for 3 min followed by a 1 min dissociation. After each cycle the human CD38 surfaces were regenerated via 5 short pulses of 5 s each of 100 mM HCl followed by running buffer. Data was processed with ForteBio Data Analysis Software Version 9.0.0.12. Binding levels of nanobody 2 were determined at the end of the 3 min association and compared to levels at baseline (beginning of association). Irrelevant nanobody controls were included. The binding level of nanobody 2 for each nanobody 2 - nanobody 1 pair was divided by the binding response of nanobody 2 on a CD38 surface saturated with nanobody 2 (self-binning). Normalized data was hierarchically clustered using Ward’s method (distance measure: half square Euclidian distance; scale: logarithmic) and visualized in Spotfire (TIBCO Software Inc.).