Figure 1. A stabilizing disulfide bond in αCD19ds-scFv is essential for generating CD19 targeting vectors.

(A) Schematic drawing of the cytoplasmic tail-truncated hemagglutinin envelope proteins displaying two αCD19 single-chain antibody (scFv) variants. In the mutated hemagglutinin protein (H_mut) that is derived from the NSe variant of the measles virus (MV) vaccine strain Edmonston B, mutations in the MV receptor recognition regions Y481A, R533A, S548L and F549S (ectodomain) [12] are indicated by asterisks. A glycine-serine linker ((G₄S)₃) is used as linker region between H_mut and scFv. The αCD19-scFv and αCD19ds-scFv differ in the presence of a disulfide bond in αCD19ds-scFv. A histidine tag (H6) is present at the scFv C-terminus. Both hemagglutinin proteins are truncated by 18 amino acids in their cytoplasmic tail (Δ18) to allow incorporation into the lentiviral envelope. (B) Cell surface transport of the two different H variants was investigated upon transfection of plasmids pCG-H_mut-αCD19 (red line) and pCG-H_mut-αCD19ds (blue line) into HEK-293T cells. The empty expression plasmid pCG-1 (black line) was used as control. Forty-eight hours after transfection, the cells were stained with a PE-conjugated anti-His antibody to detect cell surface expression of the H constructs by FACS analysis. The respective histograms are shown. (C) To determine the titer of vector particles pseudotyped with H_mut-αCD19ds (CD19ds-LV), CD19⁺ Raji cells were transduced with serial dilutions of the concentrated vector particles. After 72 h, the percentage of EGFP⁺ cells was determined by FACS analysis. (D) To determine cell surface down-regulation of CD19 after CD19ds-LV transduction, CD19⁺/CD20⁺ Raji cells were transduced with CD19ds-LV vectors at an MOI of 2 or remained untransduced. Forty-eight hours later, untransduced cells were stained with PE-conjugated anti-CD19 (left; blue line) and PE-Cy5-conjugated anti-CD20 (right; blue line) antibody, or with the respective isotype controls (black lines). CD19ds-LV transduced cells were stained with PE-conjugated anti-CD19 (left; red line) and PE-Cy5-conjugated anti-CD20 (right; red line) antibody. Then, CD19 and CD20 cell surface expression was analyzed by FACS.