Fig. 3: TE9 and 376.96, but not MGA271 CAR-T, respond to B7H3 ultra-dim tumor targets in serial re-challenge cytotoxicity assays.
a Number of B7H3 molecules/cell (QuantiBright flow cytometry assay) for evaluated target cell lines n = at least 3 technical replicates per cell line. b CAR-T cell cytotoxicity against LAN-1 and Kelly neuroblastoma cell lines at high E:T ratios (4 h 51Cr release assay). “Specific cytotoxicity” denotes cytotoxicity relative to non-transduced (NTD) T cell controls (mean ± SEM, N = 3 donors, two-way ANOVA). c CAR-T cell cytotoxicity against LAN-1 and SK-N-SH neuroblastoma cell lines at low E:T ratios (72 h flow cytometry-based cytotoxicity assay). “Specific cytotoxicity” denotes cytotoxicity relative to non-transduced (NTD) T cell controls (mean ± SEM, N = 3 donors (two technical replicates per donor), Two-way ANOVA for LAN1 376 vs MGA p = 0.003, TE9 vs MGA p = 0.02 for SKNSH 376 vs MGA p = 0.001, TE9 vs MGA p = 0.004). d CAR-T cell cytotoxicity against GFP/Luc-engineered tumoroids by overnight luminescence-based assay. “Specific cytotoxicity” denotes cytotoxicity relative to NTD T cell controls (mean ± SEM, n = 2 biological replicates for MGA and 376, n = 1 biological replicate for TE9: representative of two independent experiments, each with an n = 3 technical replicates; two-way ANOVA 376 vs MGA p = 0.0017, TE9 vs MGA p = 0.0002). e–g Re-challenge assay timecourse (e), against f neuroblastoma tumoroids and g malignant rhabdoid tumoroids (MRT); gray arrows indicate re-challenge with tumor. In (f), data were mean ± SEM of n = 2 biological replicates for MGA and 376, n = 1 biological replicate for TE9: two independent experiments, each with an n = 3 technical replicates; “specific cytotoxicity” denotes cytotoxicity relative to NTD T cell controls; two-way ANOVA; uncorrected Fisher’s LSD with a single pooled variance in (f) 376 vs MGA p = 0.002, TE9 vs MGA p = 0.0055. In g, ”specific cytotoxicity” denotes cytotoxicity relative to tumor alone controls plated in parallel, data were mean ± SEM, N = 1 independent T cell donor across three experimental replicates: two-way ANOVA; Dunnett’s multiple comparisons test, with a single pooled variance 376 vs MGA p = 0.016, TE9 vs MGA p = 0.02. h Representative microscopy images of 103-T Malignant Rhabdoid Tumor tumoroid killing by TE9 CAR-T. The experiment was conducted once at this E:T ratio. i Flow cytometric analysis after CAR-T cell co-culture (691-B and 691-T tumoroids shown in (f)) was pooled across the replicates, dimensionality reduced using PCA, and grouped according to CAR, target and time-point (T0, pre-challenge with targets; T1, 1 week of challenge; T2, 2 weeks of challenge; T3, 3 weeks of challenge). The colored dots represent concatenated individual conditions, whereas the text in green is descriptive of the phenotype each PCA quadrant is enriched for. The arrows indicate the strength (arrow length) and direction of the contribution of each phenotypical marker to the two principal components, indicating that samples on the same side as a certain arrow have a high expression of that phenotypic marker; n = 1 biological replicate from a single experimental run, of three pooled technical replicates. j 1:10 E:T ratio cytotoxicity for the first and 3rd week challenge from the assays showed in panels (f, g) plotted against antigen density (Quantibright); line = mean cytotoxicity, shaded area = SEM:n = 2 biological replicates for MGA and 376, n = 1 biological replicate for TE9: two independent experiments, each with an n = 3 technical replicates.
