Fig. 6: High avidity drives the elimination of antigen-dim targets.
a Constructs for generation of B7H3 and GFP/luciferase sublines of SupT1 cells. b Expression of B7H3 in the unsorted “SupT1-B7H3range“ and flow-sorted “SupT1-B7H3hi“ cells. c Rechallenge assay experimental schematic. d Cytotoxicity was measured by luciferase signal as relative to tumor luminescence in the presence of donor-matched non-transduced (NTD) T cells (mean ± SEM, two-way ANOVA). e T cell numbers in the same assay following either a range of challenges with tumor targets or f target-free (mean ± SEM, two-way ANOVA). g Cytokine measurements in co-culture supernatants following a single challenge with SupT1-B7H3range targets at a 1:1 E:T ratio (mean ± SEM, two-way ANOVA). h B7H3 expression on targets measured using flow cytometry following the fifth re-challenge. Shown are concatenated histograms that were generated by combining flow data adjusted for 10,000 SupT1 cells per sample. i The same SupT1-B7H3range positivity for antigen as analyzed in each sample separately (mean represents geometric mean fluorescence intensity (gMFI) ± SEM, two-way ANOVA). j Schematic of leukemia model using SupT1-B7H3range T cell lymphoma cells. k, l Concatenated histograms (k) and graphical representation (l) showing B7H3 expression in gated SupT1 tumor cells in bone marrow at day 14 following CAR-T cell infusion. The samples with 500 or fewer gated tumor cell events were uniformly excluded from quantitation, as shown: error bars show SEM: two-way ANOVA. m Quantification of CAR-T cells in bone marrow at day 14 post-T cells; N = 5, error bars show SEM, two-way ANOVA. n Respective CAR fold-proliferation in response to five challenges with SupT1-B7H3range targets plotted against IL-2 production following the initial challenge. o IL-2 production after first challenge plotted against the mean avidity for SupT1-B7H3hi targets at 1000 pN. p Proliferation after five challenges with targets was plotted against the mean avidity that the respective CARs achieved when combined with SupT1-B7H3hi targets at 1000 pN. q SupT1-B7H3range cell B7H3 gMFI after five challenges with targets was plotted against the mean avidity that the respective CARs achieved when combined with SupT1-B7H3hi targets at 1000 pN. In n–q each symbol represents an individual donor experimental replicate, the lines are simple linear regressions. For all data in the panels: N = 4 independent donors across four experimental replicates.
