Fig. 2: 376.96 and TE9 but not MGA271 CAR-T form high avidity interactions with tumor cells in a B7H3 density-dependent manner. | Nature Communications

Fig. 2: 376.96 and TE9 but not MGA271 CAR-T form high avidity interactions with tumor cells in a B7H3 density-dependent manner.

From: Functional avidity of anti-B7H3 CAR-T constructs predicts antigen density thresholds for triggering effector function

Fig. 2

a, b Cryopreserved, thawed CAR-T cell surface transgene expression was measured using flow cytometry with clone QBEND10 mAb to detect RQR8 marker gene and histidine-x6 tagged B7H3 protein (B7H3-his) + anti-his mAb to detect the CAR (mean ± SEM, N = 3 donors, two-way ANOVA MGA versus 376.98 p = 0.0257 MGA versus TE9 P = 0.0026). Representative dot plots of gated live (CD3+) T cells from one donor shown in (b). c CAR-T cells stained with Protein L (binds κ light chain) or anti-murine Fab. Representative histograms from one donor gated on live (CD3+) T cells. Binder 376.96 (λ-light chain) is noninformative with protein L staining and MGA271 (humanized framework) is noninformative for anti-mouse Fab. d Relative cell surface expression of RQR8 and CAR was compared between TE9 and MGA271 CAR-T cells using Earth Mover’s Distance (EMD) analysis of the flow cytometry data for RQR8, B7H3-his and Protein L. RQR8 was used as a marker of overall transduction whilst B7H3-his is marker of CAR binding to B7H3; Protein L is marker of CAR surface expression, and EMD measured transduced T cell staining compared to donor-matched non-transduced T cells. Marker expression was compared across three independent T cell donors, and statistical comparison done across linear regression curve slopes using Dunnett’s T3 Multiple Comparisons test (TE9 B7-his vs MGA B7-his p = 0.0115, TE9 B7-his vs TE9-pL p = 0.0054, TE9-pL vs MGA-pL p = 0.0235). Individual donor EMD data were in Supplementary Fig. 5c–e. e, f Binder affinity for B7H3 (4-Ig) using whole antibodies in IgG1 format, using Biacore Surface Plasmon Resonance (SPR). g Association (“on-rate”) and dissociation (“off-rate”) rate constants. h Binder affinity plotted against the gMFI of B7H3-his protein staining of three independent donor CAR-T cell products. i Respective geometric mean fluorescence intensities (gMFI) of B7H3-PE staining of a range of target cells. j Principle of Lumicks z-Movi synapse avidity measurement platform. k CAR-T detachment plotted against force in pN applied to the T cell/target interaction. Line indicates mean T cell attachment of four independent donors across three experimental replicates with matched donors compared for each construct: shaded area represents SEM. l Avidity as represented by attachment at 1000 pN applied force: N = 4 donors, each with three experimental replicates, one-way ANOVA. m Bound CAR-T (%) at 1000 pN plotted against QuantiBright defined B7H3 molecules per cell. The lines are linear regressions of the means of four individual donors (two-way ANOVA).

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