Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

Abstract

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.

Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts

“…Oral delivery of plant cells expressing FIX was reported to induce immune tolerance during protein replacement therapy [34••]. Oral administration of lyophilized transplastomic plant cells expressing domains of blood coagulation factor VIII or full length FIX in chloroplasts successfully eliminated inhibitor formation in mice and dog models [4••,35•,36]. Besides hemophilia, the oral administration of the acid alpha glucosidase (GAA) epitopes expressed in plant chloroplasts [37] also eliminated antibody response in Pompe disease mouse model.…”

“…Biopharmaceuticals produced in edible plants eliminate expensive fermentation, purification processes and cold storage/transportation [1,3]. Bioencapsulated protein drugs in freeze-dried plant cells are stable after several years of storage at ambient temperature [4••]. …”

Immune Tolerance for Autoimmune Disease and Cell Transplantation

“…This method overcomes the need for cold storage and transport, does not require expensive purification, and does not have the short shelf life of current protein drugs (72). Additionally, it should eliminate injections, increase patient compliance/convenience, and significantly lower costs.…”

In vivo induction of regulatory T cells for immune tolerance in hemophilia

“…The plant-based system overcomes this barrier. Moreover, successful generation of an edible crop plant (lettuce) transplastomic for CTB-FIX, along with suppression of inhibitor formation over a wide dose range and scale up using an industrial production system, will move the approach forward toward clinical translation [185]. …”