Figure S5.
Effects of CAR affinity, antigen density, and monomerization on CAR T cell activity. (A) Low HER2-expressing MCF7 (open circle) and high HER2-expressing SKBR3 (solid circle) cell lines were used to assess differences between HER2 levels in cell-cell interactions in vitro. (B) MCF7 and SKBR3 cells expressing nuclear localization signal (NLS)-tagged mKate were incubated with anti-CD45-Alexa647-labeled T cells for 30 min. Coupling analysis was performed by gating inclusively for singlets and doublets. Cells were then gated on DAPI−, CD45+, and CAR+ (or CAR− for untransduced controls). Percentage of these cells that are mKate+ was reported as T cells in doublets with tumor cells (right). Representative dot plot for the HA monomeric CAR T cell/SKBR3 co-culture is shown overlaying the CD45− and CD45+CAR+ populations (left). Representative of three independent experiments is shown. (C) Gating strategy for coupling assay in B. Example data here is from a co-culture of LA dimeric CAR T cells with MCF7 target cells. (D) The entire DAPI- population in E is overlaid with the doublets of CAR+ T cells population. Plotting by FSC-H vs. FSC-W shows that the doublets of CAR+ T cells population falls in the expected region (above the singlets). (E) CAR gMFIs are shown for each receptor type. While there is some variability in expression, differences are within ∼2/3rds of the highest gMFI. Data shown are from a representative experiment (n = 6). (F) Early activation is similar across all CAR+ conditions with low and high HER2 at 18 h following co-incubation, as seen by anti-CD69-BUV395 positivity. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). (G) Percentage of anti-Ki67-PE-eFluor 610 positive cells at 96 h following co-incubation, indicating entry into cell cycle, is similar across all CAR+ conditions with low and high HER2. Replicates from two independent experiments of different donors are pooled (n = 6). Error bars represent SD. (H) Gating strategy used for defining human CAR+ (and CAR−) T cells in flow cytometry experiments is shown. Example data shown here is from a co-culture of LA dimeric CAR T cells with SKBR3 target cells. (I) LA (blue) and HA (red) CARs were retrovirally expressed in primary mouse CD8+ T cells. HER2-expressing MC38 cells were sorted into 5 bins of expression levels. MC38-HER2 (1-5) and parental MC38 (n/a) were cultured with CAR T cells for 18 h and then stained for IFN-γ. LA and HA CARs performed similarly. Isotype control, untransduced T cell, MCF7 (open circle), SKBR3 (filled circle), and PMA/ionomycin controls are shown below. n = 3 replicates per group. Data shown is representative of three independent experiments. (J) Gating strategy used for defining mouse CAR+ T cells in flow cytometry experiments following in vitro co-culture is shown. Example data shown here is from a co-culture of LA dimeric CAR T cells with MC38-HER2-high targets in normoxia.

Effects of CAR affinity, antigen density, and monomerization on CAR T cell activity. (A) Low HER2-expressing MCF7 (open circle) and high HER2-expressing SKBR3 (solid circle) cell lines were used to assess differences between HER2 levels in cell-cell interactions in vitro. (B) MCF7 and SKBR3 cells expressing nuclear localization signal (NLS)-tagged mKate were incubated with anti-CD45-Alexa647-labeled T cells for 30 min. Coupling analysis was performed by gating inclusively for singlets and doublets. Cells were then gated on DAPI−, CD45+, and CAR+ (or CAR− for untransduced controls). Percentage of these cells that are mKate+ was reported as T cells in doublets with tumor cells (right). Representative dot plot for the HA monomeric CAR T cell/SKBR3 co-culture is shown overlaying the CD45− and CD45+CAR+ populations (left). Representative of three independent experiments is shown. (C) Gating strategy for coupling assay in B. Example data here is from a co-culture of LA dimeric CAR T cells with MCF7 target cells. (D) The entire DAPI- population in E is overlaid with the doublets of CAR+ T cells population. Plotting by FSC-H vs. FSC-W shows that the doublets of CAR+ T cells population falls in the expected region (above the singlets). (E) CAR gMFIs are shown for each receptor type. While there is some variability in expression, differences are within ∼2/3rds of the highest gMFI. Data shown are from a representative experiment (n = 6). (F) Early activation is similar across all CAR+ conditions with low and high HER2 at 18 h following co-incubation, as seen by anti-CD69-BUV395 positivity. Box and whiskers plot error bars showing minimum and maximum values (n = 12 samples per group pooled from four independent experiments and two donors). (G) Percentage of anti-Ki67-PE-eFluor 610 positive cells at 96 h following co-incubation, indicating entry into cell cycle, is similar across all CAR+ conditions with low and high HER2. Replicates from two independent experiments of different donors are pooled (n = 6). Error bars represent SD. (H) Gating strategy used for defining human CAR+ (and CAR−) T cells in flow cytometry experiments is shown. Example data shown here is from a co-culture of LA dimeric CAR T cells with SKBR3 target cells. (I) LA (blue) and HA (red) CARs were retrovirally expressed in primary mouse CD8+ T cells. HER2-expressing MC38 cells were sorted into 5 bins of expression levels. MC38-HER2 (1-5) and parental MC38 (n/a) were cultured with CAR T cells for 18 h and then stained for IFN-γ. LA and HA CARs performed similarly. Isotype control, untransduced T cell, MCF7 (open circle), SKBR3 (filled circle), and PMA/ionomycin controls are shown below. n = 3 replicates per group. Data shown is representative of three independent experiments. (J) Gating strategy used for defining mouse CAR+ T cells in flow cytometry experiments following in vitro co-culture is shown. Example data shown here is from a co-culture of LA dimeric CAR T cells with MC38-HER2-high targets in normoxia.

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