Fig. 5: Off-target cytotoxic interactions of MuSK-CAART were not identified in mouse tissue or using human MPAs.
a, Representative bioluminescence images from the MuSK-CAART biodistribution study in mice injected with 3-28/anti-Ig2 Nalm-6 cells, then treated with vehicle only (n = 6), NTD-T (n = 8), CART-19 (n = 24) or MuSK-CAART (high- and low-dose, n = 24 per each dose). Graph indicates total bioluminescence flux for all mice in each treatment group. Error bars indicate mean ± s.e.m. One-way ANOVA with the Holm–Sidak test for multiple comparisons, day 14. b, Example images from liver and lung on day 36 after treatment (MuSK-CAART, n = 8; CART-19, n = 8). Liver and lung sections from high-dose MuSK-CAART-treated mice show lymphocytic infiltration without cytotoxic effect (black arrows). CART-19-treated mice demonstrated focal hepatocellular necrosis and focal pulmonary thrombus (black arrows). c, Human MPA screened with MuSK-Fc protein identified a potential binding signal with MMP16. d, MuSK-Fc binding to MMP16 demonstrated low mean fluorescence intensity (MFI) relative to protein A and anti-MuSK 4A3 positive controls in validation screening (MMP16 curve overlaps with vector control). A representative graph from two validation screens is shown in e. e, Positive controls are removed in validation screens shown in d and the y axis is rescaled. One of two validation screens confirmed MuSK-Fc binding to MMP16, defined as MFI at least twofold higher than isotype (PD-1-Fc) control at two or more concentrations. f–i, Cytotoxicity of MuSK-CAART from two donor T cell batches was measured after coincubation for 24 hours (5:1 E:T ratio) with Nalm-6 wild-type (negative control), Nalm-6 3-28/anti-Ig2 (positive control) or seven primary human cell types from each of two different donors. Representative results are shown. IFNγ production was not detected in MuSK-CAART cocultures with primary human cells (f). Viability was analyzed using high-content imaging analysis (HCA) for Nalm-6 control and anti-MuSK 3-28 cells (g) and human-derived cells (h) or by flow cytometry (i) at 24 hours, using staurosporin or bortezomib as toxicity controls. Error bars indicate mean ± s.d. of triplicates (f–i). Multiple t-test (two-tailed), Holm–Sidak correction for multiple comparisons. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
