Taking a leaf from the book of oral tolerance

In this issue of Blood, Sherman et al demonstrate that mucosal delivery of engineered tobacco leaves can lead to tolerance to factor VIII (FVIII) inhibitor production. Eat your veggies for tolerance!¹ 

The notion that mucosal delivery of antigen can lead to unresponsiveness came from seminal experiments a century ago by Wells and Osborne.²  These workers demonstrated that prior oral feeding of antigens could prevent the immune response to a number of proteins in guinea pigs. This was followed up by Sulzberger³  and Chase,⁴  who pointed out its immunologic specificity, and was pioneered later by Weiner’s group in applying oral tolerance to treat autoimmune diseases.⁵  Indeed, there is evidence for oral tolerance in humans based on anecdotal epidemiologic data in that the incidence of peanut allergy is 10-fold lower in Israel, where peanut snacks are widely given to infants, than in the United Kingdom.⁶  A formal clinical study is now being done in the United Kingdom (http://www.leapstudy.co.uk/).

This use of mucosal administration of antigen to induce tolerance for inhibitor formation to clotting factors was first described by Alpan et al at the American Society of Hematology (ASH),⁷  who persuaded mice to drink milk loaded with factor IX (FIX). Rawle et al in Lillicrap’s group tested the effect of oral and nasal delivery of the FVIII C2 domain in FVIII^−/− mice with hemophilia A.⁸  They found that such mucosal administration of the FVIII C2 domain, a major target of inhibitors, not only blocked the response to C2 but also led to partial reduction of total anti-FVIII antibodies. However, relatively large amounts of protein were necessary for efficacy so that this approach would not seem to be feasible due to the high cost of therapeutic FVIII.

In this issue, Sherman et al, in a collaboration between the Herzog and Daniell groups,¹  report that oral gavage of plant cell extracts expressing FVIII antigens (heavy chain = A1, A2, and B domains; C2 domain) led to significant unresponsiveness to FVIII challenge in terms of both total antibody and inhibitor titers in 2 different hemophilia mouse strains. In this study, transplastomic (using chloroplasts)⁹  lines were created in tobacco to express these FVIII domains fused to the cholera toxin B subunit in order to promote transmucosal delivery. The expressed proteins assembled into pentameric forms, to facilitate binding to GM1 ganglioside receptors, and were shown to be protected and delivered intact to the mucosal immune system.¹  This extends the results of these collaborators who used oral delivery of transgenic plant extracts for tolerance to FIX (presented at the 2012 ASH annual meeting, Atlanta, GA, December 8-11, 2012). Oral tolerance to plant-expressed FVIII led to an increase in immunomodulatory cytokines, transforming growth factor-β/latency associate protein, and interleukin-10; in addition, regulatory T cells were induced that could transfer tolerance to naïve murine recipients. Tolerance did require multiple injections over an 8-week period, with FVIII challenge commencing after 4 weeks. Importantly, oral delivery into previously immunized recipients, as a model for subjects with preexisting inhibitors, also led to a reduction in antibody and inhibitor titers (see figure).

This approach is an important advance because it does not require large amounts of FVIII. It is not known, however, how long the tolerogenic effect will last. Additional administration may just be necessary to maintain tolerance. Thus, transgenic plants may offer less costly alternative therapies for tolerance, not only for hemophilia inhibitor formation, but also for autoimmune diseases. Indeed, there is precedent for the latter in a diabetes model.¹⁰ 

Further development of edible plants, such as lettuce, and efforts for scaled-up low-cost production will clearly be needed to move this therapy into the clinic. Then, there will be good reason to say, “Eat your veggies and become tolerant!”