Fig. 2

Epitope specificity and pathogenic capacity of MuSK-specific mAbs 2E6 and 6C6. The mAbs 2E6 and 6C6 were tested for domain binding with a CBA expressing MuSK-GFP domain variants. a Illustration of the full-length MuSK domains. b, c The ectodomain of MuSK consists of several different Ig-like domains and a frizzled-like domain. Different mutations of the MuSK protein either consisting of a domain deletion or specific domain-only construct were tested for binding by the mAbs. MuSK-specific human mAbs MuSK1A and MuSK3B were used as positive controls (MuSK1A for the Ig2-like domain and MuSK3B for frizzled-like domain) and the AChR-specific human mAb 637 as the negative control. The mAbs were added at a concentration of 10 µg/ml. Results for each mAb are shown. The ∆MFI was calculated by subtracting the signal acquired from non-transfected cells from the signal of transfected cells. Each data point represents a separate replicate within the same experiment, which was measured in triplicate. Bars represent means and error bars SDs. Values greater than the mean + 4SD of the negative control mAb 637, indicated by horizontal dotted lines, were considered positive. d AChR-clustering assay in C2C12 mouse myotubes demonstrates pathogenic capacity of MuSK mAbs. The presence of agrin in C2C12 myotube cultures leads to dense clustering of AChRs that can be readily visualized with fluorescent α-bungarotoxin and then quantified. Pathogenic MuSK autoantibodies disrupt this clustering. The mAbs 2E6 and 6C6 were tested for their ability to disrupt the AChR clustering. They were tested as divalent mAbs (1µg/mL) and monovalent Fabs (0.3µg/mL). Clonal variant, CVA, of mAb 2E6 was tested with either the mature (mutated) or an unmutated common ancestor (UCA) of the light chain from mAb 2E6, given that the clonal variants were identified with heavy chain-only sequencing. Quantitative measurements of the C2C12 clustering were normalized to the agrin-only effect of each individual experiment. Each data point represents the mean value from 2-8 individual values from a total of 4-10 independent experiments. Bars represent the mean of means and error bars SDs. Multiple comparisons ANOVA (against the pooled results for the three human non- MuSK-specific mAbs), Dunnett’s test; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, only shown when significant