Suppression of Inhibitor Formation Against Factor VIII in Hemophilia A Mice by Oral Delivery of Bioencapsulated Antigen
Authors: Herzog Roland W , Verma Dheeraj , Wang Xiaomei , Sherman Alexandra , Lin Shina , Daniell Henry 
Published: October 2019

Abstract 14

Approximately 25% of hemophilia A patients develop inhibitors against factor VIII during replacement therapy by infusion of factor VIII concentrates, rendering this treatment ineffective. Elimination of this antibody response is currently achieved by highly expensive immune tolerance induction (ITI) protocols involving prolonged administration of FVIII. No prophylactic immune tolerance protocols are available. To overcome these limitations, this study seeks to develop a cost-effective approach for tolerance induction by oral delivery of human factor VIII (hF.VIII) immunogenic domains expressed in chloroplasts and bioencapsulated in plant cells. Previously, we have shown that this approach effectively suppresses inhibitor formation and anaphylaxis against factor IX in hemophilia B mice (PNAS 107:7101, 2010). Bioencapsulation protects protein antigens from gastric enzymes and acidic environment of the stomach, resulting in antigen release to the immune system via digestion of plant cell walls by microbes that colonizing the gut. The transplastomic tobacco plants created expressed the heavy chain (HC, A1-A2), A3 and C2 domains fused to the transmucosal carrier cholera toxin B subunit (CTB) to facilitate GM1 receptor mediated delivery. Besides a GPGP hinge, a furin cleavage site was introduced to link CTB with the different domains of hF.VIII coding sequence for proper folding and release of hF.VIII domains into the circulatory or immune system. PCR and/or Southern blot analysis was carried out to confirm site-specific transgene integration. Western blot analysis showed expected size fusion protein band in all four transplastomic lines expressing CTB-HC, CTB-A2, CTB-A3 and CTB-C2 fusion protein. The GM1-ganglioside receptor binding ELISA assay with chloroplast synthesized CTB-C2 and CTB-A2 fusion protein showed equivalent absorbance when compared to the purified CTB, confirming the correct folding and disulfide-bond formation of CTB pentamers within transformed chloroplasts. Transplastomic leaves expressed CTB-HC, CTB-A2, CTB-A3 and CTB-C2 in the range of 0.4–1.4%, 0.1–0.2%, 0.3–0.7% and 3.0–9.1% in the total leaf protein. Leaf materials were ground in liquid nitrogen and orally delivered to male hemophilia A mice (C57BL6/129 F8e16 −/−) for tolerance induction. In a first set of experiments, 125 mg plant material was used per oral dose, representing a mix of an approximately equal amount of HC-CTB and C2-CTB fusion proteins. Gavages were performed twice per week for 8 weeks. Control mice were fed with wild-type plant material. During the last 4 weeks, all mice (n=6 per group) were additionally treated with recombinant B domain deleted human F.VIII (intravenous injection of 1 IU once per week). By the end of the experiment, control mice had formed IgG2a (up to 0.9 μg/ml) and IgG2b (up to 1.7 μg/ml) titers against hFVIII, which were undetectable in hF.VIII-fed mice. Moreover, the control mice formed very high-titer IgG1 against hF.VIII (ranging from 7–24 μg/ml), resulting in an inhibitor titer of up to 400 BU (with an average of 211±126 BU). In contrast, hFVIII-fed mice only developed 1.9±0.6 μg/ml IgG1 and 30±12 BU, representing a highly significant (P=0.006 and P=0.001, respectively) 7–10 fold reduction in antibody formation upon factor replacement therapy. These data demonstrate that hF.VIII antigen can be produced by transplastomic technology and provide first proof-of-principle that oral delivery of bioencapsulated hF.VIII antigen is effective in controlling inhibitor development. Current work focuses on further optimization, and generation of an edible crop plant (lettuce) expressing hFVIII domains in the chloroplast for future translational studies is well on its way.

Disclosures:

Daniell: Bayer: Research Funding.