Abstract
Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder haemophilia A and occurs in 20%-30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full-length CTB-FVIII sequence to cover the entire patient population, regardless of individual CD4(+) T-cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15- to 42-fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII-HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB-FVIII-SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long-term storage as revealed by GM1-ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB-FVIII-HC/CTB-FVIII-LC reduced inhibitor titres ~10-fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP-expressing CD4(+) CD25(+) FoxP3(+) Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant-based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.