Supplementary Figure 1: Mechanistic characterization of CAR macrophage phagocytosis.

a, Constructs utilized in lentiviral vectors to express CAR-19 variants in THP-1 cells (left). Representative FACS plot of CAR19 expression (post-sort) in mRFP+ THP-1 cells (right). FACS plot is representative of at least 3 experiments. b, CAR19ζ+ THP-1 macrophages were pre-treated with media, or the phagocytosis inhibitors cytochalasin-D, blebbistatin, or R406 prior to the phagocytosis assay. Data represent the mean ± SEM of (n) = 3 technical replicates. Statistical significance was calculated via ANOVA with multiple comparisons. c, Phagocytosis of CD19+ K562 target cells by CAR19ζ + or CAR19γ + (a CAR based on the Fc gamma chain) THP-1 macrophages. Data represent the mean ± standard error (SEM) of (n) = 3 technical replicates. Statistical significance was calculated via one-way ANOVA with multiple comparisons. d, Representative in vitro microscopy demonstrating steps in the CAR macrophage phagocytic process. A single macrophage was tracked over 16 hours. Images are representative of at least 3 experiments. e, Imaging cytometry of UTD or CAR19ζ mRFP+ THP-1 macrophages after co-culture with GFP+ CD19+ K562 target cells. Experiment was performed once. f, Representative image of poly-phagocytic CAR19ζ THP-1 macrophages from 4-hour co-culture at a 1:1 effector to target ratio. Experiment was performed at least 3 times. g, Diagram of the anti-HER2 CAR construct engineered into the Ad5f35 vector under the control of a CMV promoter. h, Human macrophages were transduced with CAR-HER2-ζ Ad5f35 at MOIs of 0, 100, 500, or 1000 PFU. CAR expression correlated with MOI (left), in vitro phagocytosis against SKOV3 (middle), and in vitro cytotoxicity against SKOV3 at 48 hours (right). Data are represented as mean ± SEM of (n) = 3 technical replicates. Correlation was determined via linear regression and Pearson correlation. i-j, A panel of 10 human cancer cell lines were tested for surface HER2 expression (isotype and MDA-468 are negative controls). These cell lines were used as targets for CAR-HER2-ζ macrophage phagocytosis. Percent phagocytosis is shown as a heatmap, with each column representing a different donor. Cell lines are ordered by HER2 expression (low-to-high). Experiment was performed twice. k, Luciferase based killing assay of SKOV3 by UTD, empty-vector Ad5f35 transduced (Empty), or anti-HER2 CAR primary human macrophages (CAR) at 48 hours. Data represent the mean ± SEM of (n)=3 technical replicates; statistical significance was calculated with multiple two-sided t-tests. For all panels: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.