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

Abstract

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 (hC2-ETA). Murine C2 domain-specific B cells were selectively and efficiently eliminated by hC2-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 (hC2-ETA) was produced in Escherichia coli. Results Binding studies with monoclonal C2 domain-specific antibodies confirmed the conformational integrity of the C2 domain in hC2-ETA. The functionality of hC2-ETA was tested ex vivo by incubation of splenocytes from inhibitor-positive FVIII knockout mice with hC2-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 hC2-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.

• Authors: K Brettschneider, A Schmidt, J Kahle, A Orlowski, D Stichel, D Schwabe, C Königs
• Journal: Journal of thrombosis and haemostasis : JTH (J Thromb Haemost) Vol. 16 Issue 11 Pg. 2223-2232 (11 2018) ISSN: 1538-7836 [Electronic] England
• PMID: 30152083 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2018 International Society on Thrombosis and Haemostasis.

Chemical References

• Antibodies, Monoclonal
• Antibodies, Neutralizing
• Bacterial Toxins
• Exotoxins
• Immunotoxins
• Recombinant Fusion Proteins
• Virulence Factors
• F8 protein, human
• Factor VIII
• ADP Ribose Transferases
• Pseudomonas aeruginosa exotoxin A
• Serum Albumin, Human

Topics

• ADP Ribose Transferases (pharmacology)
• Animals
• Antibodies, Monoclonal (immunology)
• Antibodies, Neutralizing (immunology)
• B-Lymphocytes (immunology)
• Bacterial Toxins (pharmacology)
• Escherichia coli
• Exotoxins (pharmacology)
• Factor VIII (pharmacology)
• Hemophilia A (therapy)
• Humans
• Immune Tolerance
• Immunotoxins (pharmacology)
• Mice
• Mice, Knockout
• Protein Binding
• Protein Domains
• Recombinant Fusion Proteins (pharmacology)
• Serum Albumin, Human (pharmacology)
• Spleen (cytology)
• Virulence Factors (pharmacology)