Figure 4.

Selection of sdAbs by in vivo phage display and NGS. (A) In order to elect the most promising sdAbs for brain targeting and BBB transposition, the rabbit derived immunized phage displayed library was intravenously injected into the tail vein of CD1 mice. Phages were allowed to circulate for 2 and 60 min and the mice were perfused, sacrificed and phages extracted from the brain were re-amplified. Three rounds of in vivo biopanning were performed, in which the phages recovered from the brain were reamplified and re-injected into the mice for a new round of selection. For each selection round, quantification of the injected phages (input) and the phages recovered from the brain (output) was determined. (B) Following in vivo selection, the selected phage library was re-amplified and analysed by next-generation sequencing and data analysed by an in-house bioinformatic script. Sequences with more than 20 representatives in at least one time point are presented. The clones selected for in vivo biodistribution, following expression and stability assays, are depicted.