Fig. 1

Expression of scFv28z and DARPin28z on the surface of murine and human T lymphocytes. a Schematic representation of CAR structures. Each construct was composed of an antigen recognition domain (either scFv or DARPin), specific for HER2, fused to a myc tag, CD8α hinge, CD28 transmembrane and signaling domains, and the signaling portion of CD3ζ. Identical constructs were generated for expression in murine or human T cells, using species specific sequences for CAR components. b Schematic showing orientation of CAR structures in relation to the T cell surface. Ribbon diagrams for the scFv and DARPin domains illustrate differences in tertiary structure and size. c-e CAR expression on the surface of murine (BALB/c or C57BL/6) or human T cells was analyzed by flow cytometry. All plots show virally transduced CD8⁺ lymphocytes (CAR-expressing viruses also expressed transduction markers; Thy1.1 for murine constructs and NGFR for human constructs). CAR negative cells were used as controls; T cells were transduced with constructs expressing transduction markers in the absence of a CAR. CAR expression was measured by staining with an α-myc tag antibody, or a HER2Fc fusion protein, followed by a secondary detection antibody. c. Representative plots are shown. d-e Level of CAR expression by transduced cells; calculated for murine ((d) % CAR⁺/% Thy1.1⁺) and human ((e) % CAR⁺/NGFR⁺) CAR-T cell cultures. Data representative of multiple experiments ((d) murine: CAR –’ve n = 3, scFv28z n = 6, DARPin28z n = 6) ((e) human: two donors, n = 3 each for CAR –’ve, scFv28z, DARPin28z). Error bars = standard deviation (SD). * = p < 0.05 ** = p < 0.005 *** = p < 0.001