Antibodies (inhibitors) developed by hemophilia B patients against coagulation factor IX (FIX) are challenging to eliminate because of anaphylaxis or nephrotic syndrome after continued infusion. To address this urgent unmet medical need, FIX fused with a transmucosal carrier (CTB) was produced in a commercial lettuce (Simpson Elite) cultivar using species specific chloroplast vectors regulated by endogenous psbA sequences. CTB-FIX (~1mg/g) in lyophilized cells was stable with proper folding, disulfide bonds and pentamer assembly when stored ~2 years at ambient temperature. Feeding lettuce cells to hemophilia B mice delivered CTB-FIX efficiently to the gut immune system, induced LAP+ regulatory T cells and suppressed inhibitor/IgE formation and anaphylaxis against FIX. Lyophilized cells enabled 10-fold dose escalation studies and successful induction of oral tolerance was observed in all tested doses. Induction of tolerance in such a broad dose range should enable oral delivery to patients of different age groups and diverse genetic background. Using Fraunhofer cGMP hydroponic system, ~870 kg fresh or 43.5 kg dry weight can be harvested per 1000 ft2 per annum yielding 24,000–36,000 doses for 20-kg pediatric patients, enabling first commercial development of an oral drug, addressing prohibitively expensive purification, cold storage/transportation and short shelf life of current protein drugs.

Targeted oral delivery of GFP fused with a GM1 receptor binding protein (CTB) or human cell penetrating peptide (PTD) or dendritic cell peptide (DCpep) was investigated. Presence of GFP+ intact plant cells between villi of ileum confirm their protection in the digestive system from acids/enzymes. Efficient delivery of GFP to gut-epithelial cells by PTD or CTB and to M cells by all these fusion tags confirm uptake of GFP in the small intestine. PTD fusion delivered GFP more efficiently to most tissues or organs than other two tags. GFP was efficiently delivered to the liver by all fusion tags, likely through the gut-liver axis. In confocal imaging studies of human cell lines using purified GFP fused with different tags, GFP signal of DCpep-GFP was only detected within dendritic cells. PTD-GFP was only detected within kidney or pancreatic cells but not in immune modulatory cells (macrophages, dendritic, T, B, or mast cells). In contrast, CTB-GFP was detected in all tested cell types, confirming ubiquitous presence of GM1 receptors. Such low-cost oral delivery of protein drugs to sera, immune system or non-immune cells should dramatically lower their cost by elimination of prohibitively expensive fermentation, protein purification cold storage/transportation and increase patient compliance.

Protein drugs (PD) are minimally utilized in dental medicine due to high cost and invasive surgical delivery. There is limited clinical advancement in disrupting virulent oral biofilms, despite their high prevalence in causing dental caries. Poor efficacy of antimicrobials following topical treatments or to penetrate and disrupt formed biofilms is a major challenge. We report an exciting low-cost approach using plant-made antimicrobial peptides (PMAMPs) retrocyclin or protegrin with complex secondary structures (cyclic/hairpin) for topical use to control biofilms. The PMAMPs rapidly killed the pathogen Streptococcus mutans and impaired biofilm formation following a single topical application of tooth-mimetic surface. Furthermore, we developed a synergistic approach using PMAMPs combined with matrix-degrading enzymes to facilitate their access into biofilms and kill the embedded bacteria. In addition, we identified a novel role for PMAMPs in delivering drugs to periodontal and gingival cells, 13–48 folds more efficiently than any other tested cell penetrating peptides. Therefore, PDs fused with protegrin expressed in plant cells could potentially play a dual role in delivering therapeutic proteins to gum tissues while killing pathogenic bacteria when delivered as topical oral formulations or in chewing gums. Recent FDA approval of plant-produced PDs augurs well for clinical advancement of this novel concept.

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.

Inhibitor formation represents a major complication of factor replacement therapy for hemophilia. There are currently no prophylactic protocols against this antibody response. To address this problem, we sought to develop an antigen-specific immune tolerance protocol that does not rely on immune suppressive drugs and that would be feasible in pediatric patients. Oral tolerance fits these requirements but is hampered by vulnerability of the protein antigen to degradation in the stomach, cost of production, and inadequate delivery across the gut epithelium to the immune system (the gut-associated lymphoid tissue or GALT). We had hypothesized that transgenic expression of coagulation factors fused to a transmucosal carrier in the chloroplasts of plant cells would provide high level and cost-effective antigen production, bioencapsulation, and transmucosal delivery. Although we demonstrated this concept by of expression cassettes for factor VIII (FVIII) and factor IX (FIX) antigens fused to cholera toxin B (CTB) subunit in tobacco, further advancement of this system to the clinic is not feasible. Translation of this approach would require generation of transgenic edible plant cells expressing these fusion proteins. Therefore, in this study, we expressed CTB-FIX using species-specific chloroplast vectors regulated by endogenous psbA sequences, a requirement for optimal transgene expression (Plant Physiology 152: 2088-2104). Transplastomic plants were homoplasmic (inserted into all copies of chloroplast genomes in each plant cell) as evidenced by Southern blots. CTB-FIX antigen levels reached ~1mg antigen/g of lyophilized leaf cells and was stable with proper folding, disulfide bond formation, and CTB pentamer assembly when stored at ambient temperature for up to 2 years. Oral gavage of these cells (twice per week for 2 months) to hemophilia B mice (FIX gene deletion, C3H/HeJ background, n=7-11/experimental group) delivered CTB-FIX efficiently to the gut epithelium and immune system (as shown by immunohistochemistry), and induced LAP+ regulatory T cells in response to intravenous challenge with recombinant FIX protein. Moreover, FIX-CTB antigen doses of 1.5 mg, 5 mg, or 15 mg all effectively prevented inhibitor/IgG formation against intravenous FIX and averted anaphylaxis and associated fatal reactions by suppression of FIX-specific IgE production. Average inhibitor titers were 15-fold lower for CTB-FIX fed compared to control mice. Of orally treated mice, >80% had inhibitor titers that were undetectable to <2 BU, while controls formed high-titer inhibitors that were >5BU, with 9/11 showing >10 BU. Induction of tolerance in such a broad dose range should greatly facilitate translation of the approach. Our results support initiation of clinical studies on oral tolerance for hemophilia B using transplastomic lettuce. Disclosures Herzog: Spark Therapeutics: Patents & Royalties: Patent licenses; Novo Nordisk: Research Funding. Daniell:Novo Nordisk: Research Funding.