neoSwitch™

After transformation,12 random clones were selected to measure surface display by flow cytometry with an anti-Myc secondary. In agreement with bulk-measurements indicating ~75% of the library was functional, most clones were properly displayed and expressed at levels comparable to a validated control VHH. It was concluded that this library is sufficient for antigen specific VHH discovery campaigns.

Neo’s naive VHH library was subjected to two rounds of bead-based counterselection to de-enrich off-target binding. Next, the surface-displayed library was subjected to 3 rounds of bead-based positive selections against soluble PD1 antigen, alternating the format of the antigen to ensure specific interaction with the target. The library was subjected to one round of counterselection by FACS to reduce frequency of off-target binding. Finally, neoSwitch cells were sorted by FACS for specific binding to PD1. Single colonies were recovered on agar plates and subjected to downstream characterization including clonal testing of display and secretion.

Eleven his-tagged anti-PD1 clones were picked into 1mL SCD in 96-well deep well plates and grown at 30°C for 24 hours. Next, 50uL of culture was transferred to 950uL of secretion media and grown for 44 hours at 25°C with agitation. After pelleting, 30uL of the VHH-containing supernatant was analyzed by SDS-PAGE and protein concentration was determined by comparison to a 6-point standard curve. On average, anti-PD1 clones from this library yielded ~300nM, enough for most downstream assays.

Production of purified binders is useful to enable screening assays including epitope binning, stability studies, and cell-based activity assays. To validate affinity purification of secreted VHH from neoSwitchTM, we grew a 200mL culture and AKTA purified His-tagged VHH from the supernatant by Ni-NTA affinity