Mechanism Of Oral Tolerance Induced By Bioencapsulated Coagulation Factor IX In Hemophilia B Mice
Authors: Wang Xiaomei , Sherman Alexandra , Su Jin , Daniell Henry , Herzog Roland W 
Published: October 2019

Abstract
Currently, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) against the therapeutic coagulation factor. Inhibitor formation in treatment of hemophilia B (deficiency in factor IX, FIX) is less frequent than in hemophilia A. However, FIX inhibitors tend to be high-titer, and 25-50% of such patients develop anaphylactic reactions against FIX. Clinical immune tolerance induction (ITI) protocols (to eliminate the inhibitors) are lengthy, expensive, and are often terminated in hemophilia B due to anaphylaxis or nephrotic syndrome. In order to establish a protocol for prevention of FIX inhibitors in high-risk patients (i.e. those with gene deletions or other severe mutations) while avoiding immune suppressive drugs or genetic manipulation, we have developed a source of bioencapsulated human FIX. Oral delivery of plant leaf material, expressing CTB-FIX in chloroplasts, effectively suppresses inhibitor formation and prevents anaphylaxis in FIX replacement therapy in hemophilia B mice of the C3H/HeJ F9-/- strain (PNAS 107:7101, 2010). This is accomplished by delivery of the fusion molecule to the epithelium of Peyer’s Patches (PP), which expresses high levels of GM1, the receptor for the CTB transmucosal carrier. Subsequently, FIX antigen is transferred to dendritic cells (DCs) in the PP and also into the circulation. Mice fed twice per week for 2 months fail to form inhibitors during a 2-month treatment phase (1 IU rFIX, IV, once/week), lack IgE formation and fatal anaphylactic reactions (which occur in control mice), but still form non-inhibitory IgG1 at reduced titers. An oral dose of FIX antigen equivalent to ∼1.5 IU rFIX/kg is sufficient to achieve tolerance. To define the underlying mechanism, we analyzed DC and lymphocyte populations in different compartments (n=4-6 for all experimental groups and data sets). Compared to control mice, which received FIX only or were fed with WT plant material and treated with FIX, tolerized mice had a marked increase in the frequencies of CD103+ DC and pDC in mesenteric lymph nodes (MLN) and PP. Both DC subsets are known to play crucial roles in oral tolerance and Treg induction. A minor, non-significant increase pDCs was also seen in the spleen. Interestingly, tolerized mice also showed a significant increase in the frequencies of CD4+ T cells expressing gut homing receptors (CCR9+ and α4β7), including in the spleen and thus indicating interaction between the gut and systemic immune systems. The spleen is a major site for systemic immune surveillance and critical for responses to intravenous FIX. Splenocytes re-stimulated in vitro with hF.IX were anayzed by RT-PCR array. We found that Th1 and Th2 cytokines (IL2, IL-4, IL-13, IFN-γ) were downregulated by oral FIX delivery, whereas immune suppressive cytokines (IL-10, TGF-β), and the Treg marker Foxp3 were upregulated, suggesting a switch from an effector to a suppressive response. Moreover, the frequency of LAP+CD4+CD25-FoxP3- T cells (which suppress responses via TGF-β) was dramatically increased in FIX-fed mice in PP (from <5% to 10-20%), and to lesser extent in spleen and MLN. Although the overall frequency of CD4+CD25+FoxP3+ Treg was not altered, tolerized mice had a significant increase in LAP+ cells in that population, indicating Treg activation. Interestingly, we failed to prevent anaphylaxis or inhibitor formation in C3H/HeJ [F9-/- x IL-10-/-] mice (n=7 per group, 42% mortality after 4th FIX injection, >5 BU in surviving animals). Therefore, both IL-10 and TGF-βare integral components of the tolerance mechanism. In summary, oral delivery of CTB-FIX antigen bioencapsulated in plant cells prevents anaphylaxis and inhibitor formation against FIX via induction of a complex immune regulatory network, involving increases in Treg-promoting subsets of DC, induction/activation of LAP+ Treg, and active cytokine-mediated suppression. Preliminary data suggest that the regimen can also be used to desensitize mice from FIX. While animal experiments performed thus far have utilized transgenic tobacco cells, we have now generated and characterized FIX-transplastomic lettuce expressing CTB-FIX, which will be tested towards translation of this approach.

Disclosures:
Daniell: Bayer: Research Funding. Herzog:Genzyme: AAV technology, AAV technology Patents & Royalties.