Journal Article

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Kwon, Kwang‐Chul, Biswas, Moanaro, Chang, Wan‐Jung, Sherman, Alexandra, Daniell, Henry, Kamesh, Aditya, Herzog, Roland W.

2018

Overview

Summary 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.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc
• Subject: Animals / antibody suppression / Assembly / bioencapsulation / Biomarkers / Bleeding / Blood coagulation factors / CD25 antigen / CD4 antigen / CD4-positive T-lymphocytes / Chains / Chloroplasts / Chloroplasts - genetics / Chloroplasts - metabolism / Cholera Toxin / Clinical trials / Coagulation factors / Cyclic GMP / disulfide bonds / Drug Evaluation, Preclinical / Drug therapy / Epitopes / Escherichia coli / factor VIII / Factor VIII - biosynthesis / Factor VIII - pharmacology / Factor VIII - therapeutic use / Factor VIII deficiency / Foxp3 protein / Freeze drying / Gene expression / genes / Genetic aspects / Hemophilia / Hemophilia A - drug therapy / hemorrhage / human blood clotting factor / humans / Hydroponics / Immune Tolerance - drug effects / Inhibitors / Lactuca / Lactuca sativa / lettuce / Lymphocytes T / Medical research / Mice / Nicotiana tabacum / oral delivery / Patient compliance / Patients / Plant cells / Proteins / protocols / Recombinant Fusion Proteins - metabolism / Recombinant Fusion Proteins - pharmacology / Recombinant Fusion Proteins - therapeutic use / recombinant proteins / storage / therapeutics / Tobacco / transgenic plants