Figure 1.

Expression of first- and second-generation chimeric antigen receptors (CARs) in NK-92 cells. (A) Schematic representation of first- and second-generation CARs for expression in NK-92 cells that consist of an extracellular single chain fragment variable (scFv) antibody domain for target recognition fused via a hinge region derived from CD8α (hinge) to transmembrane and intracellular domains of CD3ζ (left), transmembrane and intracellular domains of CD28, and the intracellular domain of CD3ζ (middle), or transmembrane and intracellular domains of CD137 (4-1BB) and the intracellular domain of CD3ζ (right). (B) To assess endogenous expression of CD28 and CD137, lysates of NK-92 cells were subjected to SDS-PAGE and subsequent immunoblotting with CD28- and CD137-specific antibodies as indicated. Lysates of peripheral blood mononuclear cells (PBMCs) from healthy donors were included for comparison. (C) For analysis of CAR expression, lysates of NK-92 cells transduced with lentiviral vectors that encode CD19-specific CARs containing CD3ζ, composite CD28-CD3ζ, or CD137-CD3ζ signaling domains as represented in (A) were subjected to SDS-PAGE under reducing (R, left panel) or non-reducing conditions (NR, right panel) and subsequent immunoblotting with CD8α-specific antibody, which detects the hinge domain. The positions of CAR monomers, homodimers of the CD3ζ CAR, and heterodimers of the CD3ζ CAR with endogenous CD3ζ are indicated. Data in panel (C) are from Oelsner et al. (74).