Hemophilia A (affecting 1 in 5000 male births) is inherited as an X-linked recessive genetic disorder, resulting in a deficiency in coagulation factor VIII (FVIII) [1]. Severe disease (<1% FVIII activity) typically results in frequent, spontaneous and potentially life-threatening bleeds, causing disability, pain, and reduced quality of life [2]. The most common treatment consists of intravenous (i.v.) infusions of replacement FVIII protein, administered either on demand or preferably prophylactically, which allows ∼70% of severe patients to manage their condition [3], [4]. Although the development of FVIII mimetics has replaced the requirement for replacement therapy in a number of patients [5], FVIII is still used to control bleeding in these patients, particularly during and after surgery [6].

The most serious complication of FVIII replacement therapy is the development of anti-drug antibodies (ADAs, called inhibitors), which can strongly diminish or abolish the effect of the therapeutic protein and can occur in greater than 30% of severe patients [7] Antigen-specific immune tolerance (ITI) protocols to eradicate inhibitors involves the frequent administration of escalated doses of functional recombinant FVIII and are effective in ∼70% of “good prognosis patients”, although the protocol itself is highly intensive, protracted and prohibitively expensive, restricting access to low-income and lower-middle-income countries [8], [9], [10].

Preventative tolerance protocols to protect hemophilia patients receiving clotting factor replacement therapy are still lacking. Since inhibitors are identified within the first 50-60 exposure days to FVIII replacement therapy [11], [12], this would indicate that prophylactic treatments should be applied to very young patients. There are important safety considerations for using non-specific immune suppressive treatment protocols that can lead to secondary infections or thwart growth. Therefore, there is increased interest in developing innovative antigen specific treatment strategies that can enable long-lived tolerance without generalized immune suppression or depletion of immune cell subsets. In hemophilia A, tolerance to inhibitor formation has been shown to critically depend on the induction of CD4+CD25+FoxP3+ regulatory T cells (Tregs) [13], [14], with analogous strategies developed in pre-clinical models either via adoptive Treg cellular therapy [15], [16], [17], [18] or the in vivo induction of antigen specific Tregs [19], [20], [21], [22].

Humanized monoclonal anti-CD3 antibodies (α-CD3) such as teplizumab, otelixizumab, foralumab, visilizumab, etc. have been shown to be clinically effective in patients with autoimmune disease and in acute graft rejection [23], [24], [25], [26]. These antibodies have specificity to CD3ε, and are humanized to reduce immunogenicity, differing only in the species from which the antibody is derived (murine, rat, and human), or the antibody isotype used [27]. Further, they have been modified to abrogate binding of Fc receptors to diminish complement activation or “cytokine storms”, thus improving safety and tolerability [28], [29]. Intravenous (i.v.) short course α-CD3 treatment preferentially depletes activated conventional T cells (Tconv), resulting in a transiently decreased ratio of Tconv cells to FoxP3+ Tregs [30], [31]. This window can be exploited to deliver potentially long-lived suppression in an antigen specific manner.

Weiner, Herold, and colleagues hypothesized that oral administration of α-CD3 would replace the need for a cognate antigen to trigger the TCR and lead to induction of Tregs [32], [33]. They and others showed in various animal models of autoimmunity that orally administered α-CD3 is biologically active in the gut and can act systemically to alleviate immune responses by a mechanism that is not Tconv depletion dependent [34], [35]. Oral α-CD3 has also been shown to be suppressive in non-autoimmune inflammation models of artherosclerosis, non‐alcoholic steatohepatitis (NASH) and to prevent transplant rejection [33], [36], [37], [38]. Suppression appears to involve induction of FoxP3 expressing Tregs and CD4+CD25- or CD4+CD25+ Tregs that express latency-associated peptide (LAP) on their surface and function in vitro and in vivo through IL-10 and TGF-β dependent mechanisms [32], [34], [37], [39]. LAP forms the distal domain of the TGF-β precursor peptide, which remains noncovalently associated with TGFβ-1 after translational processing to confer latency to the TGF-B complex [40].

To our knowledge, the direct or indirect role of oral α-CD3 therapy in preventing ADA formation has not been well examined. We hypothesized that oral delivery of α-CD3 should modulate CD4+ T helper cell or T follicular helper cell responses, which are critically important for the generation of class switched, high affinity inhibitor producing B cells in a germinal center dependent or independent manner [41], [42], [43]. Here, we evaluated the capacity of oral α-CD3 therapy to prevent inhibitor formation to FVIII protein replacement therapy in hemophilia A. We compared the effect of full length and F(ab’)2 formats of α-mouse CD3, dose and schedule of therapy on long term inhibitor suppression in a murine model of hemophilia A. We further assessed whether combining oral α-CD3 therapy with oral administration of FVIII bioencapsulated in lettuce cells leads to a synergistic improvement in inhibitor suppression. We report that oral α-CD3 therapy has a modest effect in preventing inhibitor responses in response to continuous i.v. FVIII exposures. Suppression is associated with the early induction of FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+ Tregs, with upregulation of activation and co-inhibitory markers CD69, CTLA-4, and PD1. We further observe that oral α-CD3 and oral encapsulated FVIII lettuce plant cell therapy exhibits distinct mechanisms of suppression that are not enhanced upon combining both approaches.