Preventive treatment with ATX-F8-117 reduces FVIII inhibitor formation in an FVIII antibody model. (A) Groups of 18 or 19 HLA-DR2tg mice (FVIII^+/+) were treated by dose escalation with ATX-F8-117 or PAP 133-152 as a peptide control. Four days after the peptide pretreatment, mice were primed 8 times, at weekly intervals, via subcutaneous (s.c.) flank injections with 1 µg of rhFVIII. Treatment with ATX-F8-117 or PAP 133-152 control was continued once weekly for 8 additional weeks starting 3 days after the initial FVIII priming. Plasma samples were collected from both treatment groups as indicated (B = bleed). (B) Plasma was collected at days 28, 42, and 56, and total anti-FVIII IgG levels were determined by ELISA. Graphs show end point anti-FVIII IgG titers on a log-scale; data are mean ± standard error of the mean (SEM). Each circle represents 1 mouse. Open blue circles, ATX-F8-117; filled red circles, PAP 133-152. (C) FVIII inhibitors from plasma collected at the indicated time points were analyzed using a modified Bethesda assay. Data are mean ± SEM; each circle represents 1 mouse. (D) Plasma was collected at day 56, and anti-FVIII IgG subclass distribution was determined by ELISA. Data are mean ± SEM; each circle represents 1 mouse. *P < .05, **P < .01, ***P < .001, 2-tailed Mann-Whitney U test (B-D). (E) Correlation between anti-FVIII IgG1 subclass antibodies and FVIII inhibitors in plasma samples collected at day 56 from animals treated with PAP 133-152 (upper panel) or ATX-F8-117 (lower panel) using 2-tailed nonparametric Spearman correlation analysis (r); 95% confidence intervals are also shown. Data are representative of 2 experiments performed. ***P < .001, ****P < .0001. ns, not significant; Open blue circles, ATX-F8-117; filled red circles, PAP 133-152.