Fig. 1

Construction and in vitro evaluation of HER2 CAR T cells. a To create anti-HER2 CAR T cells, the 4D5 anti-HER2 scFv was ligated into a retroviral backbone vector containing a CD3 zeta chain signaling domain and 4-1BB costimulatory domain. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were activated with anti-CD3/anti-CD28 beads on Day 0 and serially transduced with retrovirus containing the CAR construct on days 2 and 3. Beads were removed on day 4 and T cells were allowed to expand until day 9–11, at which point CAR T cells were harvested. Anti-HER2 CAR expression was assessed by Protein L staining and flow cytometry on Day 4. Representative Protein L staining results are shown for mock transduced T cells, CD19 CAR T cells, and HER2 CAR T cells. b HER2 Expression was assessed for the DAOY, D283, and D425 human medulloblastoma cell lines via either qPCR (left) or flow cytometry using the trastuzumab-APC antibody (right). The 928 cell line was used as a positive control for flow cytometry. c In a 24-h Incucyte Zoom assay, tumor cells were co-cultured with CD19 CAR or HER2 CAR transduced T cells at the indicated ratios or with trastuzumab. Percent total live tumor cells was measured compared to 100% at time zero. The percentage of live tumor cells at the 24 h time point was compared for each condition using a one-way ANOVA with Sidak’s correction for multiple hypothesis testing. Statistics are shown for the comparison of HER2 CAR 2.5:1 and CD19 CAR 10:1 for all three lines, as well as the comparison of trastuzumab and HER2 CAR 2.5:1 for D425. Denotion of “n.s” indicates a p value greater than 0.95. d Tumor cells were co-cultured for 24 h with CD19 CAR or HER2 CAR transduced T cells at a 1:1 ratio. IFNγ, IL-2, and TNFα production was measured by a Meso Scale Discovery immunoassay kit, and compared for each condition using multiple T tests with the Holm-Sidak correction