Research Paper Volume 12, Issue 11 pp 11061—11070

T cell stimulation and expansion by SunTag-based clustering of anti-CD3/CD28 scFv

Figure 4. In vitro antitumor efficacy of B7-H3-targeted CAR-T cells produced with SBCS. (A) Representative images of B7-H3 IHC staining in HNC and cervical cancer tissues. Scale bar, 20 μm. (B) Cell-surface expression of B7-H3 was evaluated by flow cytometry analysis. (C) Upper panel, schematic illustration of the vector for the expression of αB7-H3scFv-hFc. Lower panel, SDS-PAGE analysis of the purified αB7-H3scFv-hFc. (D) Immunofluorescent analysis of the expression of B7-H3 in FaDu and Hela cells using the recombinant αB7-H3scFv-hFc as the primary antibody. (E) Illustration of a B7-H3-targeted CAR-T cell against the tumor cell. (F) Schematic illustration of the vector for the expression of B7-H3 CAR. (G) The representative expression efficiency of B7-H3-redirected CAR on the T cells was evaluated using flow cytometry tracking the mCherry marker gene 10 days post-transfection. (H, I) Four-hour 51Cr release assays of B7-H3 CAR-T cells produced with SBCS or immobilized CD3/CD28 antibodies against FaDu or Hela cells. Scale bars = 50 μm. Data in G–H represent mean ± s.d. of three experimental replicates and are representative of at three experiments.