Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells

Abstract

• Coadministering FIX orally and systemically induces tolerance via complex immune regulation, involving tolerogenic dendritic and T-cell subsets.• Induced CD4 Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed.

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

“…Fusion of the (nontoxic) Cholera toxin B subunit (CTB) to a reporter protein (GFP) expressed in chloroplasts and bioencapsulated in plant cells was orally delivered across the gut epithelium through GM1 receptors and furin cleavage site engineered between CTB-GFP released GFP into circulation or different tissues [9]. Fusion of CTB to therapeutic proteins facilitate their effective oral delivery for induction of oral tolerance [10–13] or deliver functional proteins to sera [5,14,15] or across blood brain or retinal barriers [15,16]. …”

“…Such a protocol should ideally avoid immune suppressive drugs because inhibitors tend to form at a very young age (during the first 50 days of exposure) [18]. To address this unmet medical need, we are developing an oral tolerance protocol using clotting factors expressed in plant cells [11–13,21]. …”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Likewise, oral delivery of myelin basic protein reduced Ab plaques in advanced mouse and human Alzheimer's brains (Kohli et al, 2014). Delivery of coagulation factors to hemophilic mice induced oral tolerance and suppressed inhibitor formation and anaphylaxis Sherman et al, 2014;Wang et al, 2015a). The aforementioned examples illustrate the significance of this novel, cost-effective protein drug-delivery concept.…”

Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease

“…Peptides, proteins, or conjugated peptides (e.g., peptides conjugated to anti-DEC205) delivered systemically do not benefit from specific targeting, as in the case of our sortase-modified RBCs. Oral tolerance likewise requires administration of large amounts of antigen and multiple doses (35)(36)(37)(38), but orally administered peptide treatments have so far failed in human clinical trials (39).…”