Oral Delivery of Bioencapsulated Factor IX Protects From Inhibitor Formation and Anaphylaxis in Protein Replacement Therapy for Hemophilia B.

Abstract

222 While the incidence of inhibitor formation against the infused coagulation factor is overall relatively low in factor IX-deficiency (1–4% of patients), 9–23% of patients with severe hemophilia B form inhibitors, which seriously complicates treatment. Factor IX (F.IX) inhibitors tend to be high titer and not effectively eliminated by immune tolerance induction protocols, and are often associated with serious anaphylactic reactions. Inhibitor formation and anaphylaxis is more typical for patients with F9 gene deletions or early stop codons. We sought to develop a novel approach to induction of antigen-specific immune tolerance that is safe, not based on immune suppression, and does not require knowledge of specific T cell epitopes. Chloroplast transgenic plants expressing human F.IX were generated. Because the plant cell provides bioencapsulation, the recombinant polypeptide is protected from acids and enzymes in the stomach and released in the gut. The hF.IX coding sequences were fused with the transmucosal carrier CTB. The fusion gene cassettes were expressed from the light-regulated psbA 5′ untranslated region/promoter in order to achieve hyper-expression. To prevent steric hindrance, a GPGP hinge was introduced between CTB and FIX. A furin cleavage site was also introduced in one of the constructs. Plasmid DNA was bombarded on nicotine-free tobacco leaves via a particle-mediated method using the gene gun, resulting in transgene integration into the chloroplast genome by homologous recombination. Transformed tissues were repeatedly cultured on selective media, and transplastomic plants were regenerated. Chloroplast transplastomic lines were examined by Southern blot analysis in order to confirm site-specific integration and to test for homoplasmic lines. Transplastomic lines of CTB-GPGP-hFIX (F1) and CTB-GPGP-Furin-hFIX (F2) had expression levels of up to 3.8% and 0.28% fusion protein, respectively, in the total soluble protein. Control and transplastomic plant materials (250 mg) were fed twice per week for 8 weeks by oral gavage to Hemophilia B mice, known to develop strong antibody responses against hF.IX (C3H/HeJ F9−/− mice with targeted F9 gene deletion). For the F2 material, this was equivalent to 2 μg of hF.IX per dose. After 4 weeks, animals received weekly infusions of hF.IX (Benefix) at 1 IU per dose for 4 weeks (1 IP followed by 3 IV doses). As expected, ≥80% of mice control groups had developed significant inhibitors at titers of 4–10 BU (average titers were 5.5 BU for wild-type material fed and 7 BU for unfed controls). In contrast, all F1 and F2 fed mice (n=15 and n=14, respectively) had undetectable or at most very low titer inhibitors (≤2 BU) and showed normal clearance of hF.IX. Some F2 fed mice (n=9) was subjected to 1 more month of weekly IV infusions of hF.IX. At the end of this experiment, inhibitor titers remained undetectable to low (< 3 BU) in 6/9 mice, while 3 mice had formed titers of 4–12 BU, in contrast to on average 80 BU in control mice. Importantly, a rise in inhibitor titers was not seen in another cohort, in which feeding was continued during subsequent treatment. Most importantly, 30–50% of control mice developed allergic reactions, went into respiratory arrest, and died instantly or shortly after the 4th and subsequent injections of hF.IX. This severe anaphylactic response was not observed for up to 12 hF.X injections in any F1/2-fed animals regardless of whether feeding was continued beyond 8 weeks. The underlying mechanism appears to involve a shift to non-inhibitory antibodies and shifts in immunoglobulin subclasses. In summary, repeated feeding of bioencapsulated hF.IX prevents inhibitor formation and life-threatening anaphylactic reactions. Disclosures: Daniell: Chlorogen Inc: Technical founder.

Animal models of inhibitors

“…Studies using mucosal exposure to the immunogenic C2 domain have shown that tolerance to C2 can be achieved through this strategy, but when the mice are exposed to full‐length FVIII, inhibitory antibodies develop [34]. Very recent preliminary evidence suggests that FIX made in transgenic plants can mediate tolerance and prevent fatal anaphylactic episodes after oral administration [35].…”

Special lectures in haemophilia management

“…These investigators have explored pharmacological methods for expanding the Treg population, which could prove to be useful either for gene transfer or ITI protocols [42,43]. Other strategies for inducing tolerance prior to inhibitor development have included the use of oral/nasal tolerance regimens [44,45] and administration of a retroviral vector expressing the transgene to the liver in the neonatal period [46,47].…”