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

Abstract

SummaryInhibitor 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.

Validation of leaf and microbial pectinases: commercial launching of a new platform technology

“…The plant chloroplast system has been advanced to express therapeutic proteins with various advantages including high‐level expression and ease of oral administration through edible crops (Herzog et al ., ; Kwon et al ., , ). Although FDA‐approved GM edible crops have been in use for more than two decades, all of them contain antibiotic resistance genes.…”

“…However, other enzymes are needed to break down lignin, cellulose, hemicellulose and xylan to achieve total clarification and future studies will include the synergistic effect of enzymes. The plant chloroplast system has been advanced to express therapeutic proteins with various advantages including high-level expression and ease of oral administration through edible crops (Herzog et al, 2017;Kwon et al, 2016Kwon et al, , 2018. Although FDAapproved GM edible crops have been in use for more than two decades, all of them contain antibiotic resistance genes.…”

“…Tobacco transplastomic lines were selected through germination of seeds on ½ MS medium containing spectinomycin (500 lg/ mL), acclimatized in the greenhouse and grown under previously described conditions (Kwon et al, 2018). Hydroponically grown tobacco plants were produced at Fraunhofer CMB (Newark, DE) using previously described conditions (Su et al, 2015), with the following changes: sowing was staggered to increase distance to 4 in 2 between plants, growth medium comprised 50 ppm Peters Professional 5-11-26 Hydroponic Special and 100 ppm Yara Liva Calcinit 15.5-0-0 and a14/10-h day/night photoperiod.…”

Advances in knowledge of inhibitor formation in severe haemophilia A

“…Oral administration of FVIII to reduce inhibitor formation has been studied previously in HA mice (Rawle et al , ), but has recently been successfully adapted to incorporate transplastomic biotechnology, enabling large amounts of antigenic protein to be produced for oral delivery (Daniell et al , ). Despite low systemic FVIII concentrations that are insufficient for therapeutic benefit, oral FVIII delivery has been documented to result in efficient induction of FoxP3 + and latency‐associated peptide‐expressing regulatory T cells that secrete immune‐suppressive cytokines such as IL‐10 and reduce FVIII inhibitor formation (Kwon et al , ). FVIII‐specific suppressive lymphocytes produced in this manner could modulate immunity at distant sites like the spleen, potentially through migration and bystander suppression, resulting in FVIII tolerance.…”

The national blueprint for future basic and translational research to understand factor VIII immunogenicity: NHLBI State of the Science Workshop on factor VIII inhibitors

“…Regulatory T cells (Tregs) are able to suppress antibody formation. The role of CD4 + CD25 + FoxP3 + Tregs in tolerance to FVIII has been described in numerous studies, while latency‐associated peptide (LAP) expressing Tregs are more typical in oral tolerance to FVIII, representing various approaches to achieve tolerance . Presumably, innate immune signals that may derive from tissue damage or molecular patterns associated with pathogens trigger initiation of these adaptive immune responses.…”