Elimination of factor VIII VIII ‐specific B cells by immunotoxins composed of a single factor VIII VIII domain fused to Pseudomonas Pseudomonas exotoxin A

摘要

Essentials There is still a need for novel therapeutic approaches for hemophilia A patients with inhibitors. A factor VIII domain was used as the targeting moiety for elimination of FVIII ‐specific B cells. The immunodominant C2 domain was fused to exotoxin A from Pseudomonas aeruginosa ( hC 2‐ ETA ). Murine C2 domain‐specific B cells were selectively and efficiently eliminated by hC 2‐ ETA ex?vivo . Summary Background Today, the most serious complication for patients with hemophilia A undergoing factor VIII ( FVIII ) replacement therapy is the development of neutralizing antibodies (inhibitors). Although inhibitors can be eradicated by application of high doses of FVIII , the immune tolerance induction therapy fails in up to 30% of patients. Hence, there is still an urgent need for novel therapeutic approaches for patients with persisting inhibitors. Objectives In the present study, the potential use of immunotoxins containing exotoxin A ( ETA ) from Pseudomonas aeruginosa for selective elimination of FVIII ‐specific B cells was explored. Methods The immunodominant C2 domain of human FVIII was used as a targeting moiety instead of the full‐length FVIII protein and the resulting human C2 domain‐ ETA fusion protein ( hC 2‐ ETA ) was produced in Escherichia coli . Results Binding studies with monoclonal C2 domain‐specific antibodies confirmed the conformational integrity of the C2 domain in hC 2‐ ETA . The functionality of hC 2‐ ETA was tested ex?vivo by incubation of splenocytes from inhibitor‐positive FVIII knockout mice with hC 2‐ ETA and controls. FVIII ‐specific memory B cells from splenocytes were differentiated by FVIII stimulation in antibody‐secreting cells ( ASC ) and detected by an enzyme‐linked immunospot assay. Although the controls showed no effect, incubation of splenocytes with hC 2‐ ETA reduced the number of C2‐specific ASC in a dose‐dependent fashion, indicating specific and efficient elimination of C2‐specific memory B cells. Conclusions Overall, the results of the study support the fact that FVIII domain immunotoxins might be a potential new tool for the elimination of FVIII ‐specific B cells in patients with hemophilia A and persisting inhibitors.