Aims/hypothesis We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. Methods Adults (18–42 years) and adolescents (12–17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12–42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA 1c levels, insulin use, and antigen-specific CD4 + and CD8 + T cell responses using an activation-induced marker assay and pooled tetramers, respectively. Results Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA 1c ) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8 + T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4 + and CD8 + T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8 + T cells after treatment with monotherapy or combination therapy. Conclusions/interpretation Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. Trial registration ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 Funding This study was funded by Precigen ActoBio Graphical Abstract

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12–20 or InsB13–2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370–575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional β-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4+Foxp3+CD25+ regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.

Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammatory conditions, with the major types being Crohn’s disease and ulcerative colitis (UC). Both are complicated diseases that mainly affect young adults. Complexity lies both in the unclear, multifactorial origin and the implications thereof on treatment. Better understanding of the mechanism of action has led to more rational approaches for medical intervention. Molecular immunology and genetic analysis have tailored the current array of IBD therapeutics. The expansion on the concept of IBD being an immune disease, which stems from the observation of massive lymphocyte infiltration and therapeutic effect of corticosteroids, has been the major lead for drug design. Since the description of Nod2 [1], a large array of genetic variants, many of which map within immune pathways, have been associated with disease development. Genetic understanding has strengthened our immunologic insight into IBD and along the way led to better clinical target selection. The clinical development of novel anti-inf lammatory therapeutics, small molecules, neutralizing monoclonal antibodies and cytokines has become a bastion of modern biopharmaceutical industry. Since not all carriers of risk genotypes develop IBD, it is obvious that genetics is not the only factor determining the development of IBD. Environmental factors such as food composition and pathogen invasion have long been suspected to drive at least parts of IBD onset and maintenance. The seminal finding that intestinal bacteria are recognized by mucosal antibodies has, however, greatly shaped the state of mind in this field [2]. Although clear-cut evidence remains scarce, most experts now agree that antigens derived from the – omnipresent and personally unique – intestinal microbiota drive IBD development. Multinational consortia such as the human microbiome project [3] and MetaHit [4] were established to portray the human microbiome composition. Adequate methodology and a broadly available species atlas enables crystallization of the relevant differences between microbiota composition of the healthy and the diseased [5]. Using this knowledge to treat IBD – excluding the malign or (re)introducing benign microflora – is obviously a tempting idea. There are many questions that arise regarding potential uses of this knowledge. Will it be possible and useful to re-tailor IBDassociated microbiota towards a ‘healthy’ state? [6] Will this result in a long-lasting cure of IBD? Will microbiota composition sway back or will once ‘healthy’ species now become the driver of the immune reaction? Core to these considerations is the as-of-now unanswered question of whether the stratification of species causes IBD development (i.e., a combination of species that trigger the immune system) or a result of selection by the diseased immune system. How can the above principles be translated into broadly applicable clinical therapy? Development of any drug requires a clear description in terms of Clinical development of lactocepin: a novel bacterial biologic?

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.

The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD.

The taxonomically diverse lactic acid bacteria (LAB) are unified by their capability to produce lactic acid from carbohydrates by fermentation. The LAB Lactococcus (L.) lactis has been characterized into great detail and is increasingly used as a production host for heterologous proteins. L. lactis is a non-pathogenic and non-colonizing LAB species and can be efficiently engineered to produce proteins of viral, bacterial or eukaryotic origin, both intra- or extracellularly. Importantly, orally formulated L. lactis strains (ActoBiotics), engineered to synthesize and secrete therapeutic peptides and proteins in the gastrointestinal tract, are already in advanced stages of preclinical and clinical development. This review focuses on the genetic engineering of LAB in general and L. lactis in specific to secrete high-quality, correctly processed, bioactive molecules derived from a eukaryotic background. The therapeutic applications of these genetically modified strains are discussed, as well as the need for a sound environmental containment strategy, and a detailed review is presented on Lactococcus strains engineered to produce specific antigens, antibodies, cytokines and trefoil factors, with special regards to immunomodulation.

Chemotherapy-induced intestinal injury, referred to as mucositis, is a major side effect of cancer therapy. At the moment, there are no agents available that prevent or heal the intestinal damage and this limits both the dose and duration of cancer treatment. Trefoil factor 3 (TFF3) is present in the small and large intestine and is thought to play a major role in tissue protection and healing after injury. TFF3 may also modulate the response to cytotoxic agents like 5-fluorouracil (5FU). We previously demonstrated that oral administration of TFF3 secreting Lactococcus lactis (LL) strains leads to active delivery of TFF3 at the intestinal mucosa. Such treatment was shown to be effective in both prevention of disease and healing of colitic mucosa. Here, we report on the effect of these strains on 5FU induced mucositis. Mucositis was induced in C57BL/6 mice by daily IP injection of 250 mg/kg 5FU for 3 days. This resulted in extensive weight loss and injury to the small intestine and colon presumably due to stem cell damage. Mice were treated with LL-TFF3 or with control strains during the induction period and for 3 days afterwards. At day 7, the mice were killed and the intestine was removed for histological analysis. Body weight loss was observed in both groups but at day 7, control mice continued to lose weight while the weight in the TFF3 group was stable or increased. Histologically, the trefoil group showed crypt regeneration whereas in the control mice, crypts were small or absent. This suggests that local delivery of TFF3 hastens the recovery of intestinal injury caused by 5FU and may have therapeutic value. This research was funded by IWT.