Transduction of NK-92 cells with retroviral vector pL-scFv(FRP5)-ζ-SN.

(A) Schematic representation of the pL-scFv(FRP5)-ζ-SN construct. The Moloney murine leukemia virus 5′ long terminal repeat (LTR) controls the expression of the chimeric antigen receptor scFv(FRP5)-ζ, which consists of an N-terminal immunoglobulin heavy-chain leader peptide (SP), the ErbB2-specific single-chain antibody scFv(FRP5), a Myc-tag, the hinge region of murine CD8α, and the murine CD3 ζ chain. The neomycin-resistance gene for G418 selection of transduced cells is driven by the SV40 early promoter. (B) Surface expression of chimeric scFv(FRP5)-ζ antigen receptor. NK-92 cells successfully transduced with the retroviral vector (NK-92-scFv(FRP5)-ζ cells) were selected with G418 (left panel). Cells expressing high levels of the chimeric antigen receptor were enriched by sorting with Myc-tag specific mAb 9E10 and goat anti-mouse IgG–coated magnetic beads (right panel). After each selection step surface expression of scFv(FRP5)-ζ was determined by FACS analysis using mAb 9E10 and FITC-labeled goat anti-mouse secondary antibody . Parental NK-92 cells served as a control. (C) Immunoblot analysis of scFv(FRP5)-ζ expression. Lysates of NK-92 (lanes 1 and 3) and transduced NK-92-scFv(FRP5)-ζ cells (lanes 2 and 4) were separated by SDS-PAGE under nonreducing (NR) or reducing (R) conditions. Immunoblot analysis was performed with a CD3 ζ chain–specific mAb followed by horseradish peroxidase (HRP)–conjugated anti-mouse antibody and chemiluminescent detection. The positions of endogenous and chimeric CD3 ζ proteins and of molecular weight standards (kd) are indicated.