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. 2017 Feb 1;25(2):512-522.
doi: 10.1016/j.ymthe.2016.11.009.

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Roland W Herzog  1 , Timothy C Nichols  2 , Jin Su  3 , Bei Zhang  3 , Alexandra Sherman  1 , Elizabeth P Merricks  2 , Robin Raymer  2 , George Q Perrin  1 , Mattias Häger  4 , Bo Wiinberg  4 , Henry Daniell  5
Affiliations

Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic Lettuce

Roland W Herzog et al. Mol Ther. .

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.

Keywords: factor IX; hemophilia; inhibitor; oral tolerance; transgenic plant.

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Figures

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Graphical abstract
Figure 1
Figure 1
Characterization of CTB-FIX Protein in Different Batches of Lettuce Lyophilized Leaf Powder Used for Dog Studies (A–C) Western blot analysis and quantification of CTB-FIX lettuce total leaf protein (TLP) (A) Fed-B1: batch 1, 2.0 μg TLP per lane; (B) Fed-B2 and Fed-B3: batches 2 and 3, 1 μg TLP per lane; and (C) Fed-B4: batch 4 2.0 μg TLP per lane. (D) Concentration of CTB-FIX (mg/g DW) in four batches of leaf powder is shown. Data shown are means ± SD of two independent experiments. (E) GM1 binding assay is shown—200 μg total soluble protein (TSP) per assay, 20 ng CTB standard, 1% BSA, and 200 μg TSP from lyophilized untransformed lettuce were used as negative controls. Data shown are means ± SD of triplicates. (F) Western blot analysis of CTB-FIX protein in lyophilized leaves after long-term storage for 4, 12, and 30 months is shown. Equal protein loading is confirmed by re-probing with anti-Rubisco large subunit antibody on the same blot (RbcL). Anti-CTB rabbit polyclonal antibody (titer: 1:10,000) was used to detect CTB-FIX protein in all the western blots.
Figure 2
Figure 2
cGMP Hydroponic Production of CTB-FIX Transplastomic lettuce plants expressing CTB-FIX were grown in Fraunhofer cGMP hydroponic system. Leaves were harvested after 32, 51, 78, 101, and 121 days of plant growth. (A) Western blot quantification of CTB-FIX amounts in lettuce leaves harvested at different ages is shown. (B) Concentrations of CTB-FIX in lyophilized lettuce leaves at different stages of growth are shown. Data shown are means ± SD of two independent experiments.
Figure 3
Figure 3
Antibody Formation against Human FIX as a Function of Time (A) Experimental timeline. (B) FIX-specific IgG1 and IgG2 in control dogs are shown. (C) FIX-specific IgG1 and IgG2 in dogs that received oral CTB-FIX in lettuce twice per week for 13 weeks (FIX-fed) are shown. (D) Inhibitor formation (in BU/mL) in control dogs is shown. (E) Inhibitor formation in orally fed dogs (FIX-fed) is shown. Arrows indicate first and last of weekly intravenous injections of recombinant FIX (Benefix; 10 IU/kg; once/week). These i.v. challenges were performed for 8 weeks (except for P08 and P10: these two control dogs received only four weekly injections).
Figure 4
Figure 4
Specific IgE Formation against Human FIX as a Function of Time in Dogs that Received Eight Weekly i.v. Challenges with Recombinant FIX (A and B) Control dogs. (C–F) Dogs orally fed with CTB-FIX lettuce twice per week for 13 weeks (FIX fed) are shown. Asterisk indicates visible anaphylactic reaction to FIX i.v. delivery. Arrows indicate the first and the last intravenous challenge of recombinant FIX (Benefix; 10 IU/kg; once/week).
Figure 5
Figure 5
Coagulation Times and Hematological Evaluations after Intravenous Recombinant Human FIX Injection into Control and CTB-FIX-Fed Hemophilia B Dogs (A) Whole blood clotting time (WBCT) and (B) thromboelastography were measured weekly over the entire duration (13 weeks) of this study. (C) Weekly platelet counts are shown. (D) Weekly measurements of white blood cell counts are shown. (E) Percentage of hematocrit is shown. (F) Hemoglobin content is shown. (A–F) Arrows indicate i.v. challenges of recombinant human FIX (10 IU/kg of BeneFIX).
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