Fig. 5: Functional screening of the complex BiTE cell library HEK293_LP^CD19/BiTE spiked with 0.1% HEK293_LP^CD19/Blin using a double-reporter based orthogonal assay chemistry on the single cell screening platform.

a Representative scatterplots from two independent screening runs showing detected droplet signals after 6-h incubation. Jurkat-ZsG and Jurakt-E2C reporter cells were co-encapsulated without the BiTE library cells (i) or with the BiTE library cells (ii). HEK293_LP^CD19/BiTE was pre-labeled with CellTrace^TM Violet dye. Gating zones and activation rates are also shown. b Representative droplet signal (PMT) profiles and images corresponding to a positive cell (i) and negative cell (ii): HEK293_LP^CD19/BiTE cell (CellTrace^TM Violet, blue), an activated Jurkat-ZsG cell (ZsGreen, green), and an activated Jurkat-E2C cell (E2-Crimson, red). c Representative DNA agarose gel image showing single-cell PCR products from a panel of isolated CD19xCD3 BiTE clones. d Representative flow cytometry plots for bulk functional validation of seven recovered BiTE clones (obtained from two screening runs). Jurkat-ZsG reporter cells were co-cultured with CD19+ Raji in the presence of a day-3 condition medium harvested from the 1-mL-scale expression from Expi293F^TM cells transfected with BiTE gene harboring expression plasmids. After 24-h incubation, the Jurkat-ZsG reporter cells were profiled by flow cytometry to measure the activation rate. Blinatumomab-BiTE was also transfected and expressed in parallel as a positive control. A total of 265 BiTE clones were tested for functionality using Jurkat-ZsG reporter activation assay (only seven clones are represented here). e Estimated abundance of select sequence-verified recovered BiTEs. Note that Clones 4 and 5 are extremely rare (~0.001% abundance). The abundance estimation is based on individual clones’ respective counts in the NGS sequences, assuming that these clones are proportionally amplified during the NGS library preparation.