Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Abstract

Anti-drug antibodies in hemophilia patients substantially complicate treatment. Their elimination through immune tolerance induction (ITI) protocols poses enormous costs, and ITI is often ineffective for factor IX (FIX) inhibitors. Moreover, there is no prophylactic ITI protocol to prevent anti-drug antibody (ADA) formation. Using general immune suppression is problematic. To address this urgent unmet medical need, we delivered antigen bioencapsulated in plant cells to hemophilia B dogs. Commercial-scale production of CTB-FIX fusion expressed in lettuce chloroplasts was done in a hydroponic facility. CTB-FIX (∼1 mg/g) in lyophilized cells was stable with proper folding, disulfide bonds, and pentamer assembly after 30-month storage at ambient temperature. Robust suppression of immunoglobulin G (IgG)/inhibitor and IgE formation against intravenous FIX was observed in three of four hemophilia B dogs fed with lyophilized lettuce cells expressing CTB-FIX. No side effects were detected after feeding CTB-FIX-lyophilized plant cells for >300 days. Coagulation times were markedly shortened by intravenous FIX in orally tolerized treated dogs, in contrast to control dogs that formed high-titer antibodies to FIX. Commercial-scale production, stability, prolonged storage of lyophilized cells, and efficacy in tolerance induction in a large, non-rodent model of human disease offer a novel concept for oral tolerance and low-cost production and delivery of biopharmaceuticals.

Long-term evaluation of mucosal and systemic immunity and protection conferred by different polio booster vaccines

“…Lyophilized plant cells expressing VP1 can be stored without losing efficacy, eliminating cold chain. Long-term (>2 years) studies in dogs showed lack of toxicity of CTB fusion proteins [28]. Although vaccine studies in this investigation used lyophilized tobacco cells expressing VP1 in chloroplasts, high level expression has been achieved in lettuce chloroplasts and large biomass production is in progress [29,30].…”

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

“…These plants achieve expression of effective antigen doses, while also providing natural bioencapsulation by the cell wall and avoiding the need for expensive protein purification (Daniell et al ., ,b; Kwon and Daniell, ; Kwon et al ., ). Importantly, this concept has been successfully applied in murine models of haemophilia A and B and Pompe disease, and even in a large (canine) model of haemophilia B, illustrating translatability to other species (Herzog et al ., ; Sherman et al ., ; Su et al ., ,b; Verma et al ., ).…”

“…Application of this system has been evaluated in animal models of various human diseases including Alzheimer's (Kohli et al ., ), pulmonary hypertension (Shenoy et al ., ), diabetes (Boyhan and Daniell, ; Kwon et al ., ; Ruhlman et al ., ) and retinopathy (Shil et al ., ). Most importantly, this approach is suitable for induction of oral tolerance to therapeutic proteins used in replacement therapy of genetic diseases, as demonstrated in Pompe disease (Su et al ., ) and haemophilia (Herzog et al ., ; Sherman et al ., ; Wang et al ., ) by suppression of immunoglobulin formation.…”

Complexity of immune responses to AAV transgene products – Example of factor IX

“…Approaches to antigen-specific tolerance induction without the use of immune suppressive drugs include oral tolerance induction, as shown by a recent proof-of-concept study [61]. Oral tolerance is mediated by the induction of multiple subsets of Treg, including FoxP3 + Treg and CD4 + CD25 − LAP + Treg that suppress via a TGF-β dependent mechanism and also express the immune suppressive cytokine IL-10 [62–66]. …”