Figure 1.

CAR design and T-cell engineering of HER2-CARs containing CD28 or 4–1BB intracellular costimulatory domains. A, Diagram of the lentiviral expression cassette with HER2-CARs containing the scFv from trastuzumab targeting HER2, with a 229–amino acid human IgG4 Fc linker containing a double mutation in the CH2 domain (EQ), a transmembrane domain (either CD28 or CD8), a cytoplasmic CD28 or 4–1BB costimulatory domain, and a cytolytic CD3z domain. A truncated nonsignaling CD19 (CD19t) is separated from the CAR with a T2A ribosomal skip sequence for tracking CAR-expressing cells. B, Mock (untransduced), HER2–28ζ, or HER2-BBζ CAR T cells expressing CD19 to detect lentiviral transduction of CARs (left histogram), Protein L to detect the scFv (middle histogram), or binding to Alexa Fluor-647–labeled soluble HER2 (right histogram) as determined by flow cytometry. C, Ex vivo expansion kinetics for Mock and HER2-CAR T cells over3 weeks in culture. D, Cell-surface expression of indicated markers on HER2-CAR T cells as determined by flow cytometry. All data are representative of at least two independent experiments.