Figure 1.

Organisation of hemagglutinin (H)-designed ankyrin repeat proteins (DARPins) and cell surface expression. (a) Schematic drawing of the modified MV-H used for retargeting, derived from strain Edmonston B (SwissProt P08362).³¹ Four point mutations in the ectodomain (ED) ablating binding to CD46 and signaling lymphocyte-activation molecule (the natural receptors) are indicated.⁹ An N-terminal cytoplasmic tail truncation by 18 amino acids (CTΔ18) allows stable incorporation into lentiviral particles.⁹ The C-terminus of H is fused via a SfiI site to the DARPin, resulting in the sequence EDGTNAAQPADLGKKL where the first underlined sequence is the C-terminus of H and the second is the N-cap of the DARPin. The DARPin terminates in the sequence EILQKLNAAARGSHHHHHH (DARPin C-cap is underlined). The transmembrane domain (TM) and the His-tag are indicated. (b) Expression plasmids for six different H-DARPin and three H-scFv fusion proteins were transfected into HEK-293T cells. Surface expression was analyzed after 48 hours using a His-tag-specific antibody (PE-labeled). H-scFvCD20 and H-scFv7A5 were used as positive and negative controls.