Figure 1.

Binding of bispecific BAR-bodies to effector cells. (A) T cells were isolated from PBMCs using the Pan T Cell Isolation Kit, human by Miltenyi Biotech GmbH. Isolated T cells were stained with FITC-coupled anti-CD4 and anti-CD8 antibodies and with the secondary system of LRPAP1 bispecific BAR-bodies (anti-His AB → biotinylated anti-mouse AB → Strep-PE) only (upper dot plots), αCD3/LRPAP1 bispecific BAR-bodies (middle dot plots) and αCD16/LRPAP1 bispecific BAR-bodies (lower dot plots). αCD3/LRPAP1 bispecific BAR-bodies but not αCD16/LRPAP1 bispecific BAR-bodies show binding to CD4⁺ and CD8⁺ T cells. (B) NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells using the CD56⁺/CD16⁺ human NK-Cell Isolation Kit (Miltenyi Biotech GmbH). αCD16 antibodies were FITC-coupled and αCD56 antibodies were PE-coupled. The viability of the NK cells after isolation averaged 95% and the CD16⁺ fraction was between 90 and 98% (dot plot on the left). αCD16/LRPAP1 bispecific BAR-bodies (histogram on the right) but not αCD3/LRPAP1 bispecific BAR-bodies (histogram in the middle) showed binding to isolated NK cells. The secondary system for detection was used as in (A). BAR=B-cell receptor antigens for reverse targeting; PBMC=peripheral blood mononuclear cell.