Fig 2: MuSK-CAART reduces anti-MuSK IgG but not total IgG or B cell counts in a syngeneic MuSK EAMG model.CD45.2+ C57BL/6J mice were immunized with MuSK Ig1-2 protein (30 µg in complete Freund’s adjuvant) on day 0 and boosted with MuSK Ig1-Fz protein (30 µg in incomplete Freund’s adjuvant) on day 26. Results of two independent experiments are shown (total numbers NTD-T, n = 12, 1D3-CART, n = 8, MuSK-CAART, n = 12). Equivalent numbers of transduced CD45.1+ T cells or a matching number of NTD CD45.1+ cells were injected on day 35. a,b, Host B cells (CD45.2+CD3-CD19+) were analyzed in spleen and lymph nodes at days 49–63 (2–4 weeks after treatment). Representative flow plot (a) and the frequency of CD45.2+CD19+ B cells in the spleen and lymph nodes (b) are shown. Kruskal–Wallis test with Dunnett’s test for multiple comparisons. c, Anti-MuSK antibody titer was measured in individual mouse blood samples drawn on the day of treatment, normalized to 4A3 mouse antihuman MuSK mAb standard (µg ml-1). Kruskal–Wallis with Dunnett’s test for multiple comparisons. d,e, Anti-MuSK antibody titer and total mouse IgG were measured in mouse blood samples drawn weekly after treatment. Graphs indicate fold change of anti-MuSK antibody titer (d) or total mouse IgG (e) relative to week 1 after treatment (NTD-T, n = 8; 1D3-CART, n = 7 (d) and n = 6 (e); MuSK-CAART, n = 8 to include all mice with longitudinal samples through week 4 after treatment; one 1D3-CART mouse was excluded in e due to low blood sample volume precluding analysis). Error bars indicate mean ± s.e.m. Multiple linear regression-coefficient test for difference between the slopes. f, Frequency of CD45.1+ T cells were analyzed in the spleen and lymph nodes on day 49–63. Statistical analysis was not performed since absolute number of CD45.1+ T cells varied among treatment groups to achieve the same transduced cell dose in set 1. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.Source data

Fig 3: MuSK-CAART off-target effects on muscle are not observed.a, Effects of MuSK-CAART or CART-19 (E:T 10:1) on agrin-induced AChR clustering in C2C12 mouse myotubes were visualized by a-bungarotoxin staining (×25 magnification, top row, plus bottom inset (red boxes); scale bar, 50 µm). Representative images are shown from two individual experiments. b, AChR clustering was quantitated by fluorescence intensity, measured at six different sites in each image (error bars indicate mean ± s.e.m.). One-way ANOVA with the Holm–Sidak test for multiple comparisons. c, Differentiated primary human myotubes were incubated with agrin (5 ng ml-1) or medium alone. Agrin-induced MuSK phosphorylation was confirmed by phospho-MuSK ELISA. OD450/570 value relative to medium-alone control is shown. Mann–Whitney U-test (two-tailed). d, Human myotubes were coincubated with NTD-T, MuSK-CAART or Wise-CAART in the presence or absence of agrin for 24 hours. IFN? production was measured in cell-culture supernatants by ELISA. Error bars indicate mean ± s.d. of triplicates. One-way ANOVA with the Holm–Sidak test for multiple comparisons. NS, P > 0.05; *P < 0.05; ****P < 0.0001.Source data

Fig 4: Generation and validation of 13-3B5* antibody-secreting Nalm-6 cells.a) Nalm-6 13-3B5* anti-Ig1 antibody-secreting cells were generated by transducing soluble 13-3B5/anti-Ig1 antibody heavy chain plasmid into Nalm-6 13-3B5 BCR-expressing cells. Jurkat cells expressing individual MuSK extracellular domains linked with GFP were stained with cell-culture supernatants from Nalm-6 13-3B5* for epitope mapping. Soluble 13-3B5 antibody binding was detected using anti-human IgG4-APC. b, c) Nalm-6 13-3B5 (red) and Nalm-6 13-3B5* (green) were stained with PE-conjugated mouse anti-human IgG to quantify BCR density. The mean fluorescence intensity of BCR expression is shown by histogram (b) and bar graph (c). d) Nalm-6 13-3B5 or Nalm-6 13-3B5* cells were co-incubated with either NTD-T or MuSK-CAART cells at indicated E:T ratios for 8 hours. MuSK-CAART cytotoxicity was measured using a luciferase-based assay. Error bars indicate mean ± standard deviation of triplicates.

Fig 5: Evaluation of soluble anti-MuSK monoclonal antibody effects on MuSK-CAART cytotoxicity, IFN? production, and proliferation.a) Non-transduced T cells (NTD-T) or MuSK-CAART were co-incubated with Nalm-6 control or individual MuSK domain-specific Nalm-6 target cells at an E:T ratio of 10:1 in the absence (0 µg/mL) or presence of matching soluble anti-MuSK IgG4 mAb at 1.25 or 6.25 µg/mL, in a total of 10 mg/mL normal human IgG. Cytotoxicity was evaluated at 24 hours using a luciferase-based assay. Error bars indicate mean ± standard deviation of triplicates. b) NTD-T or MuSK-CAART cells were incubated with each anti-MuSK IgG4 mAb (0.2, 1, 5, or 25 µg/mL) and human IFN? was quantitated by ELISA in cell culture supernatants after 24 hours. Error bars indicate mean ± standard deviation of triplicates. c) Proliferation of NTD-T (top) and MuSK-CAART (bottom) was evaluated 96 hours after the addition of the indicated anti-MuSK mAbs using Cell Trace Violet (CTV) cellular labeling dye dilution by flow cytometry. (a-c) Representative plots from two (b,c) or three (a) individual experiments using different donor T-cells are shown.