Antigen-specific immunotherapy to restore antigen-specific tolerance in Type 1 diabetes and Graves’ disease

Summary

Type 1 diabetes and Graves’ disease are chronic autoimmune conditions, characterized by a dysregulated immune response. In Type 1 diabetes, there is beta cell destruction and subsequent insulin deficiency whereas in Graves’ disease, there is unregulated excessive thyroid hormone production. Both diseases result in significant psychosocial, physiological, and emotional burden. There are associated risks of diabetic ketoacidosis and hypoglycaemia in Type 1 diabetes and risks of thyrotoxicosis and orbitopathy in Graves’ disease. Advances in the understanding of the immunopathogenesis and response to immunotherapy in Type 1 diabetes and Graves’ disease have facilitated the introduction of targeted therapies to induce self-tolerance, and subsequently, the potential to induce long-term remission if effective. We explore current research surrounding the use of antigen-specific immunotherapies, with a focus on human studies, in Type 1 diabetes and Graves’ disease including protein-based, peptide-based, dendritic-cell-based, and nanoparticle-based immunotherapies, including discussion of factors to be considered when translating immunotherapies to clinical practice.

This article explores current research on antigen-specific immunotherapies for the autoimmune conditions Type 1 diabetes and Graves’ disease. The review covers protein-based, peptide-based, dendritic-cell-based, and nanoparticle-based immunotherapies. We focus on human studies, including factors to be considered when translating immunotherapies to clinical practice.

Graphical Abstract

Graphical Abstract.

Introduction

Autoimmune diseases of endocrine organs, such as Type 1 diabetes (T1D) and Graves’ disease (GD), develop as a result of dysregulated immune responses and impaired self-tolerance. Antigen-specific immunotherapy (ASI) refers to the administration of autoantigens or antigen-specific T cells in order to functionally inactivate, deplete, or regulate pathogenic effector T cells (Teff). In cancer, immunotherapy based on relevant tumour molecular or immune biomarkers is increasingly employed to enhance antigen-specific immunity with few adverse effects. In immune tolerance, allergen desensitization immunotherapy has shown that escalating doses of allergen changes the T cell and antibody immune repertoire, with an increase in regulatory T cells (Treg), decrease in allergen-specific T helper (Th)2:Th1 ratio, reduction in IL-3, IL-4, IL-5, IL-9, and IL-13 and increase in TGF-β and IL-10 [1].

In endocrine autoimmune diseases such as T1D and GD, effective ASI immunomodulating autoreactive T cells would offer the possibility of disease prevention or long-lasting drug-free remission with infrequent re-treatment, without global immune suppression. In this review, we explore progress of ASI in these autoimmune diseases, with a particular focus on nanoparticles (NP).

Pathogenesis of T1D

T1D is a metabolic disease caused by the autoimmune destruction of the insulin-producing pancreatic β cells, leading to dysregulated glucose homeostasis. This chronic disease, most often presenting in childhood, has significant long-term physiological and psychosocial impacts. With complications such as neuropathy, nephropathy, and retinopathy, T1D can reduce life expectancy by an average of 10 years [2]. Glycaemic excursions and the burden of daily injections have a significant impact on quality of life. Incidence is increasing about 4% per year, and autoantibody screening can identify cases in pre-symptomatic cases [3]. There is a need for new treatment options, including preventative strategies.

Immune-mediated decline in β cells commences before and continues after onset of T1D. Early stages are marked by the presence of two or more islet autoantibodies (Stage 1). This progresses to glucose intolerance (Stage 2) and then clinically symptomatic disease (Stage 3). At this stage, it is estimated that more than 70% of the β cells are destroyed [4]. The pancreatic islets are infiltrated by CD4+ and CD8+ T lymphocytes, macrophages, B cells, and other immune cells, resulting in immune-mediated inflammation, stress, and loss of functional β cells. Pathogenetic pathways include proinflammatory cytokine secretion, islet autoantibody production, and compromised regulation by Foxp3+CD25hi Treg and IL-10-producing T regulatory Type 1 (Tr1) cells. However, cytotoxic activity of β-cell specific CD8+ T cells plays a key role in disease pathogenesis [5–7].

Considerations for antigen-specific immunotherapy in T1D

There are two general approaches to immunotherapy—manipulation of immune response independent of β-cell specificity (antigen-independent) or modulation with β-cell ASI (antigen-dependent). In antigen-independent therapy, there has been a recent focus on immunotherapy with the humanized FcR-non-binding anti-CD3 monoclonal antibody teplizumab in T1D. While a Phase 3 trial in recent-onset disease failed to meet its primary endpoint, a randomized double-blind Phase 2 trial of teplizumab in 76 individuals with Stage 2 T1D demonstrated annual rates of diabetes of 14.9% in the teplizumab group and 35.9% per year in the placebo group [8, 9]. The drug was recently approved by the United States Food and Drug Administration for use in Stage 2 T1D. Treatment with teplizumab was associated with CD8+ but not CD4+ memory T-cell anergy (increased expression of KLRG1 and TIGIT and reduced IFN-γ and TNF production) [8], and the frequency of these cells correlated with improved C-peptide for up to 18 months [9]. Appropriate timing of teplizumab is a challenge. Consistent with a mechanism of anergizing CD8+ Teff, preclinical and clinical studies both suggest that efficacy is best achieved with an active autoimmune response, i.e. Stage 2 and early Stage 3 disease [10].

In contrast to anti-CD3, ASI aims to modulate the immune system and dampen the response of specific autoreactive Teff (reviewed in [11]). Using relevant T-cell antigens, ASI therapy aims to upregulate inducible regulatory T (Treg) cell types and inactive/delete antigen-specific Teff. Particular focus is on upregulation of autoantigen-specific Treg cells in diseased tissues and in tissue-draining lymph nodes in order to dampen recruitment, activation, and retention of multiple disease-relevant Teff (bystander tolerance). In contrast, deletion of T cells with cognate autoreactivity may be insufficient to cover the multiple relevant epitope-specific T cells that may concomitantly—or in future—perpetuate autoimmune inflammation.

The target specificity of ASIs promises a high safety profile that does not compromise the host response to infection or malignancy. Table 1 highlights some of the current tolerizing immune approaches for autoimmune diseases. For the development of ASI, several key questions must be answered. Which antigens contribute to the development of a specific autoimmune disease and how can they be delivered to induce tolerance? Will one antigen be tolerogenic across a heterogenous patient population? Will induction of tolerance to a single antigen provide long-lasting protection, or should HLA genotype-specific epitopes or multiple epitopes be employed? What is the optimal timing, dose and dosing frequency for ASI therapy and what biomarkers can be used to determine treatment response?

Table 1:

Antigen-specific tolerizing immunotherapy approaches and their stage of development

Scientific approach	Company/university	Disease	Stage	Mechanism of action
Tolerogenic DCs with peptides	UQ U Newcastle	RA RA, PsA	Phase 1 [105] Phase 1 [106]	Tolerogenic DCs + peptides adoptive transfer
	LUMC	T1D	Phase 1 [107]
Liposomes with calcitriol and peptide	UQ, Dendright	RA	Phase 1 [75]	DCs take up liposomes and the NF-kB suppressive drug
NPs and Rapamycin	Selecta	Gout	[108] Phase 3 (NCT04513366)	NPs deliver Rapamycin to APCs
NPs with rapamycin and antigen	Selecta	PBC	Preclinical	NPs deliver Rapamycin and antigen to APCs
NPs and AHR ligands with antigen	ANTolRx	T1D, MS	Preclinical [73, 109–111]	NPs deliver AHR ligands and antigen to DCs
NPs with antigen	Cour	Coeliac	Phase 2 [112]	NPs target MARCO +ve macrophages and DCs
	Epidermal ASI	T1D	Preclinical (NCT02837094)	Unconjugated AuNPs reduce DCs maturation
Antigen processing independent epitope	Apitope	GD MS	Phase 1 [101] Phase 2 [113]	Soluble peptides bind steady-state DCs
Peptide epitope	UCB	T1D	Phase 1 [56]	Soluble peptides without adjuvant
	KCL/UCB	T1D	Phase 1 [55]
	Neurocrine Biosciences	T1D	Phase 2 [53, 54]
Adoptive Treg transfer	Prometheus Laboratories UCSF	T1D	Phase 1 [63].	Treg adoptive transfer
Protein ASI	Diamyd Medical	T1D	Phase 2 [48]	GAD-alum s.c.
		T1D	Phase 1 [49, 50]	GAD-alum intranodal, oral vitamin D
Liver targeted NPs with peptide	Topas	Pemphigus	Phase 1 (EudraCT 2019-001727-12)	Ferromagnetic NPs target LSECs in liver
Liver targeted glycosylated antigen	ANOKION	Coeliac disease	Phase 1 [114]	Glycosylated antigens taken up by liver cells
Peptide-coupled RBC	Cellerys	MS	Phase 1 [115]	RBC taken up by liver cells leading to tolerance to antigens
NPs coupled to pMHC	Parvus	T1D, RA	Preclinical [116]	NPs target antigen-specific TCR
DNA	Tolerion	T1D	Phase 2 [58]	Reduced CpG plasmid expressing proinsulin
Redox modified epitope	Imcyse	T1D	Phase 2 (NCT04524949)	Induce antigen-specific cytolytic CD4+ cells
mRNA vaccine	BioNTech SE	EAE	Preclinical [117]	mRNA antigen targeting DCs with low adjuvanticity

AHR, aryl-hydrocarbon receptor; ASI, Antigen-specific immunotherapy; AuNP, carbohydrate-coated gold nanoparticles; CpG, cytosine-phosphate-guanine; DCs, dendritic cells; KCL, Kings College London; LSECs, liver sinusoidal endothelial cells; mRNA, messenger ribonucleic acid; MHC, major histocompatibility complex; MS, multiple sclerosis; NP, nanoparticles; PsA, psoriatic arthritis; RA, rheumatoid arthritis; RBC, red blood cells; T1D, Type 1 diabetes; Treg, T regulatory cells; UCSF, University of California San Francisco; U New, University of Newcastle; UQ, University of Queensland.

Disease-specific antigens will be considered below. Preclinical models show that a regulatory response to one epitope can be extended to other epitopes if these epitopes are linked by the same antigen-presenting cell (APC), known as bystander tolerance [12]. Optimal timing depends on various factors, but ongoing capacity for presentation of antigen seems to be critical to maintain functional antigen-specific Treg in the repertoire. Thus in T1D, islet-infiltrating or circulating proinsulin-specific T cells are common, but as the autoimmune target β cells are lost or become stressed, the autoimmune response may spread towards neo-epitopes such as hybrid insulin peptides [13]. Heterogeneity across individuals creates challenges for ASI. For example, in certain individuals, loss of β cells may progress so fast that ASIs are ineffective and the Stage 2 window may also be missed. To try to overcome this, ASI might be feasible in combination with other immune-modulatory approaches to restore and—with prolonged use—sustain self-tolerance. In GD, ASI has been underexplored but has significant potential because unlike T1D, the relevant functional endocrine tissue is preserved, suggesting use of ASI to prevent the development of chronic, relapsing GD.

Clinical biomarkers in endocrine autoimmune diseases include autoantibody levels, biochemical hormone profile, and evidence of organ failure. Autoreactive T-cell-specific biomarkers suitable to predict efficacy of ASI are generally needed in autoimmune diseases, to speed up the implementation of prevention and mechanistic trials. Predictive biomarkers illustrating phenotypic changes in disease-related peripheral T cells can reflect meaningful clinical outcomes such as delayed disease onset or progression, high rates of remission or reduced risk of complications [14]. For example, in Graves’ orbitopathy, clonally expanded CD4+ KLRG1+ cytotoxic T-cell lymphocytes (CD4+ CTL) represent a potential biomarker, as these terminal effector CTLs may mount inflammatory cytokine and chemokine responses to TSHR antigens [15]. Biomarkers are increasingly being explored using high dimensional mass cytometry, single-cell RNA sequencing, and T-cell receptor profiling [16].

We now explore current research on clinical ASI implementation as it relates to the pathogenesis of T1D and GD.

Antigen tolerizing immunotherapy in T1D

Immunomodulatory therapies repurposed from other indications have been trialled with varying success in T1D, including TNF [17] and IL-6 blockade [18], CTLA4-Ig inhibition of T-cell co-stimulation [19], selective depletion of B cells or T cells [20], and expansion of polyclonal Treg with low dose IL-2 [21]. While these strategies target features of T1D-related autoimmunity, they do not target the loss of antigen-specific tolerance. In contrast, ASI in Stage 2 or recent-onset Stage 3 T1D may prevent disease progression through induction of tolerance. ASI could also play a role in individuals with longer duration Stage 3 disease who have residual β-cell function [4, 22].

The premise of ASI is to either inactivate, delete, or regulate pathogenic effector CD4+ or CD8+ T cells. There are multiple approaches, including unencapsulated, protein- or epitope-based vaccines, DNA-based vaccines, antigen loaded into dendritic cells or NP (Fig. 1). Most of the available evidence consists of preclinical studies, but donor islet programs, organoids, and intensive study of peripheral blood have identified autoantigenic hotspots and some bioassays for human antigen-specific T cells. ASI using individual CD8 epitopes to delete antigen-specific CD8+ T cells is unlikely to keep up with the development of new autoreactive CD8 specificities as epitope spreading progresses [23]. However, induction of CD4+ Treg or even CD8+ Treg could regulate multiple antigen-specific T cells through bystander tolerance. Mechanisms for Treg-mediated suppression include immunosuppressive adenosine or transfer of cAMP; suppression of T-cell receptor TCR signalling-induced Ca2+, nuclear factor of activated T cells (NFAT), and NF-κB signalling; production of immunosuppressive cytokines (IL-10, TGF-β, IL-35); consumption of IL-2; downregulation of costimulatory molecules on or killing of APCs; induction of effector cell death; cell–cell suppression through membrane-bound TGF-β, and upregulation of coinhibitory molecules, such as lymphocyte activation gene 3 (LAG3), programmed death-1 (PD-1), and galectin-1 [24–32]. In particular, Tr1 cells or antigen-specific Treg induced with prolonged or repeated antigen exposure in vitro or in vivo, are believed to control autoimmune responses primarily by interacting with peptide-MHC on APCs in inflamed tissues or draining lymph nodes. Their secretion of IL-10 and/or TGF-β suppresses APCs and other T cells [24, 33, 34]. Effector Treg can also suppress conventional T cells independently of antigen, by consumption of IL-2 or CTLA4 depletion of CD80/CD86 with enhanced PDL1 expression by APC [33–35]. CD8+ Treg are also increasingly appreciated. For example, in multiple sclerosis, glatiramer acetate was shown to induce CD8+ T cells that suppressed pathogenic CD4+ T cells in an HLA-E-dependent manner [36]. CD8+ Tregs were also shown to regulate GAD65-specific CD4+ T-cell responses in healthy people through contact-dependent and IL-10-dependent mechanisms, but this mechanism was dysregulated in T1D [37]. While CD8+CD25+Foxp3+ T cells that were generated in vitro with teplizumab suppressed CD4+ T-cell proliferation [38], CD8+KLRG1+TIGIT+ T cells identified in patients treated with teplizumab expressed low levels of Foxp3 [39].

Figure 1:

Targets of antigen-specific immunotherapy. There are multiple approaches to peptide presentation in ASI including unencapsulated, protein- or epitope-based vaccines, DNA-based vaccines, antigen loaded into dendritic cells and nanoparticles. These different approaches target and modulate different aspects of the immune response. Promotion of activity denoted by black arrows and suppressive activity denoted by black solid line.

Protein-based ASI

Insulin as an unencapsulated protein antigen has been trialled as ASI, aiming to regulate or inactivate insulin-specific T cells. While effective in rodent models, multiple human trials using intranasal, subcutaneous, and intravenous insulin have not replicated this in Stage 3 T1D or in early at-risk stages [40–43]. Potential reasons include dosing strategy, delivery method, antigen choice (the major antigenic hotspot is in pre-proinsulin), subject heterogeneity, including HLA genotype, or a requirement for long-term antigen re-exposure once β cells are lost.

Insulin-based tolerance is being further explored in three ongoing human studies. The GPPAD-POInT Study is a randomized, placebo-controlled, double blind, multicentre, multinational primary prevention Phase IIb study in which oral insulin is administered for a period of 29–32 months to children with an elevated genetic risk for T1D (NCT03364868). The PINIT study explores whether intranasal insulin in children with high genetic risk for T1D will induce protective IgG or IgA antibody responses or T cell responses to insulin or proinsulin (NCT03182322). Intranasal insulin administered daily for the first 7 intervention days, is followed thereafter by once weekly dosing for 6 months. The Fr1da Insulin Intervention is a preventative mechanistic study exploring tolerance, insulin immunity, and development of dysglycemia after oral insulin for 12 months in multiple islet autoantibody-positive children (NCT02620072).

Three clinical trials explored GAD-alum in recent onset Stage 3 disease [44–47]. While the primary endpoint of preservation of C-peptide/β-cell function was not met, post-hoc analysis showed that the HLA-DR3-DQ2 haplotype was associated with C-peptide preservation, and this was dose-dependent across the three trials combined. GAD-alum has also been administered to children with Stage 1 T1D. Two doses of s.c. GAD-alum did not affect progression to T1D [48]. More recently, GAD-alum was administered via an intranodal route monthly for 3 months, in combination with daily oral vitamin D (2000 IU) in an open pilot trial of 12 patients, aged 12–24 years with recent-onset T1D [49, 50]. Stimulated C-peptide was stable at 6 months but declined after 15 months relative to baseline. HbA1c declined but there was no change in insulin dose. At 15 months there were a reduction of CD8+ T cell activation, decreased memory T cells and increased CD4+ and CD8+ Teff. Further stratification demonstrated that those with a poor clinical response had higher baseline levels of GAD_65-induced cytokines such as IL-2, IL-10, IFN-γ, TNF, lower levels of IL-10 at 15 months and an increase of non-suppressive FOXP3^lowCD45RA⁻ Tregs compared to good clinical responders. This is being further explored in a clinical trial in latent autoimmune diabetes in which 3 intranodal injections of GAD-alum are administered with oral vitamin D supplementation (NCT04262479).

Another trial is exploring 2 injections of GAD-alum (or placebo) with daily 250 mg Gamma-Amino Butyric Acid (GABA) in T1D patients with any HLA haplotype (NCT02002130). GABA is locally produced by pancreatic β cells, promotes β-cell growth and survival, reduces glucagon production and increases Treg.

Peptide-based ASI

MHC class II-restricted peptide-based ASI are suitable for patients carrying specific HLA alleles. Peptide antigens do not need additional processing by APC and, unlike proteins, are less likely to be targeted by neutralizing antibodies to form immunogenic immune complexes. In T1D, pre-proinsulin and the insulin A chain contain multiple immunoreactive epitopes [51, 52]. James et al. [52] reviewed T-cell epitopes for human β-cell autoantigens.

Clinical trials of peptide ASI have had a mixed outcome. In a Phase 1 trial of 32 patients with recent onset T1D receiving an altered peptide ligand derived from insulin B_9-23 (NBI-6024) [53], IL-5 response to NBI-6024 increased. However, in a placebo-controlled Phase 2 trial, there was no improvement or maintenance of β-cell function and no difference in CD4+ or CD8+ T cells [54]. In a placebo-controlled trial of intradermal proinsulin C19-A3 peptide 2 or 4 weekly over 6 months in 27 recent-onset HLA-DR4+ T1D patients, C-peptide decline and insulin requirements were reduced in the combined treatment groups compared with placebo [55]. Proinsulin-specific IL-10 production was increased ex vivo in patients receiving the high frequency regimen, highlighting potential issues related to longevity of tolerance with the short half-life of naked peptide. In the MultiPepT1De Phase 1 dose escalation trial, 6 islet-derived peptides were administered s.c. monthly for 6 months in 24 HLA-DRB1*04:01+ adults with detectable stimulated C-peptide [56]. The peptides were safe, but there were no significant changes in stimulated C-peptide, HbA1c or insulin requirement. No immunological analyses were reported. A current Phase 2a trial is exploring s.c. C19-A3 linked to a thioreductase motif (Imotope IMCY-0098) with alum in HLA-DR4+ adults with newly diagnosed T1D (NCT04524949). In preclinical studies with a GAD65 epitope containing the thioreductase motif injected into 4-week old NOD mice, GAD65-specific cytotoxic CD4+ T cells expanded, which could kill APCs presenting GAD65 and bystander T cells, and diabetes development was reduced [57].

DNA

DNA ASI has the advantage that multiple antigens and immunomodulatory molecules can be included to broaden the impact on immune responses. The hypothesis is that while only few cells are transfected, the cells surrounding the site where the plasmid is injected will express the encoded proteins. In a trial of 80 adults administered a DNA plasmid encoding proinsulin or placebo intramuscularly for 12 weeks within 5 years of T1D diagnosis, some patients had preservation of C-peptide associated with reduction in proinsulin-specific CD8+ T cells for up to 15 weeks [58]. However, this C-peptide response was not persistent and HbA1c and insulin usage increased. Antigen-specific CD4+ T cells, IFN-γ, IL-4, and IL-10 production did not change. In mouse models, a DNA vaccine encoding GAD65_190-315 (consisting of four known epitopes) combined with IL-2 and IL-10 genes, delayed disease development and islet inflammation and was more protective than full-length GAD65 [59]. This may reflect an advantage in presenting smaller tolerogenic epitopes without co-delivering dominant CD8 epitopes. On the other hand, DNA vaccines encoding mixed CD4 and CD8 native epitopes (insulin B9-23, ChgA, GAD65, IGRP) or mimetopes (ChgA mimetope p79, insulin R22E) might enhance longevity of the response by targeting a larger number of diabetogenic T-cell clones [60].

TOPPLE T1D is a current placebo-controlled, double-blinded trial exploring safety outcomes and stimulated C-peptide response to a recombinant supercoiled plasmid encoding four human proteins: pre-proinsulin, TGF-β1, IL-10, and IL-2 (NCT04279613). The DNA is administered s.c. weekly over 12 weeks with gradual dose escalation. Another trial is exploring safety and efficacy of a proinsulin-expressing DNA plasmid administered intramuscularly to new-onset and established patients with T1D (NCT03895437).

Antigen-loaded tolerogenic dendritic cell immunotherapy

This approach involves adoptive transfer of tolerogenic dendritic cells (DCs) loaded with peptide or protein ex vivo. Tolerogenic DCs have been produced using dexamethasone, vitamin D3, IL-10, TGF-β, or NF-κB inhibitors [61]. Vitamin D3-induced tolerogenic DCs loaded with C19-A3 peptide modulated IL-10 in a HLA-DR4-transgenic NOD mouse model [62]. A Phase I clinical trial of intradermal C19-A3-loaded tolerogenic DCs in 9 patients with longstanding T1D, demonstrated safety but no change in glycaemic control after 6 months of treatment [63].

Nanoparticle-based approaches

NP offer the opportunity for immunomodulation and are comprised of various materials including lipids, silica, carbohydrates, or metals [64]. NP-based ASI alter multiple immune cell functions (including antigen-specific Teff and Treg) by selectively inhibiting disease-associated T-cell immunity and not compromising systemic immunity [65]. They confer multiple delivery advantages for immunotherapy. First, entrapped peptide, protein, or mRNA antigen is both protected from degradation and presented in a favourable format for uptake by myeloid APCs, thus enhancing efficacy and durability of the antigen-specific immune response. Second, antigen can be combined with modifiers of co-stimulation or immune-response, such as immunomodulatory small molecules, cytokines, or other biologics to promote simultaneous immune regulation. Lipid-based or liposome NP have the capacity to entrap lipophilic immunomodulatory drugs. Third, NP can be functionalized for delivery to a tolerogenic environment such as liver, or to specific APC using antibodies or receptor ligands.

Biodegradable drug delivery particles are preferable as they enable localized or targeted drug delivery with high safety. NP made of metals, such as gold or iron, may be associated with allergy or other toxicities. Key parameters to be considered in the design of tolerogenic NP include size, charge, delivery route, and cargo capacity [66, 67]. Depots of larger NP or microparticles are usually engulfed over time by migratory DCs or macrophages, with contents transported to draining lymph nodes (LN). Smaller NP may be passively phagocytized by LN DCs, e.g. in subcapsular sinus after drainage through afferent lymphatics, and ultra-small NPs (≤25 nm) may drain directly through lymphatics to be taken up by lymphocytes through pinocytosis. Phagocytosis of negatively charged particles is preferred by DCs, while positively charged particles may be cytotoxic [66, 68]. A variety of natural and synthetic biomaterials can be chosen to stabilize and protect the antigenic cargo—such as negatively charged protein or positively charged mRNA—from degradation or release before it reaches its target APCs. Biocompatible formats include liposomes, polylactic-glycolic-acid (PLGA), and polylactide-coglycolide (PLG) polymer-based NPs, lipid NPs, and other NP comprised of lipid/lipoprotein [67]. Liposome NPs are made of biocompatible phospholipids that auto-assemble into bilayered vesicles and can encapsulate hydrophilic peptides in the core and entrap lipophilic drugs in the lipid shell. mRNA vaccines consist of antigen-encoding RNA encapsulated in NP.

Immunomodulatory agents include inhibitors of NF-κB and/or mTOR, such as calcitriol, rapamycin, curcumin, the aryl-hydrocarbon receptor (AhR) ligand ITE, and TGF-β1, or coinhibitory molecules designed to stimulate regulatory T cells, such as PD-L1.

In an alternative NP approach, ultra-small iron oxide NP coated with BDC2.5 mimetope-MHC-II complexes ligated the TCRs of autoantigen-specific T cells after administration to NOD mice, inducing antigen-specific Tr1 cells, which tolerized multiple specificities through bystander regulation [69]. Similarly, NP coated with IGRP_206-214-MHC I promoted memory differentiation of IGRP-specific CD8+ T cells. These T cells regulated other specificities and controlled diabetes through depletion of DCs presenting multiple islet epitopes [70]. So far, this approach has not been translated to T1D clinical trials.

NP co-entrapping antigen and inhibitory compounds

NPs loaded with the aryl-hydrocarbon receptor (AhR) ligand 2-(1ʹH-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) and an autoantigenic peptide were shown to induce antigen-specific Foxp3+ Tregs in preclinical models [71]. Proinsulin/ITE NPs suppressed the development of autoimmune diabetes and induced a tolerogenic profile in DCs through a SOCS2-dependent mechanism [72]. This concept is in preclinical development for translation to trial in T1D [73].

Hyperglycaemic NOD mice treated s.c. twice weekly for 4 weeks with liposomes encapsulating BDC2.5 mimotope or BDC2.5 mimotope and NF-κB inhibitor calcitriol enhanced survival for up to 6 months [12]. The liposomes were taken up by inguinal draining lymph node DCs and induced PD-1⁺Foxp3⁺ and IL-10⁺ Foxp3⁻ Treg that exerted bystander tolerance of IGRP_206-214-specific CD8+ T cells in the pancreatic draining LN. IGRP_206-214/calcitriol but not IGRP_206-214 liposomes also reduced the development of diabetes and increased survival of hyperglycaemic NOD mice, again through suppression of the effector function of IGRP_206-214-specific CD8+ T cells. Pancreatic LN DCs expressed lower levels of CD40, CD80, and CD86 after IGRP/calcitriol liposomes. Of interest, when IGRP_206-214/calcitriol and BDC2.5 mimotope/calcitriol liposomes were co-delivered, survival of hyperglycaemic mice was still prolonged but suppression of diabetes development by IGRP_206-214/calcitriol liposomes was lost, associated with expansion of effector IGRP_206-214-specific T cells in spleen [74]. These data highlight the critical importance of the pancreatic LN for Treg-mediated islet immune tolerance and the potential consequences of creating an additional depot of class I-restricted autoantigen that could expand memory CTL.

While not yet translated to clinical trials in T1D, a liposome product (DEN-181) co-encapsulating calcitriol and Type II collagen_259-273 (CII) was trialled in a single ascending dose Phase 1b trial in 17 anti-citrullinated protein antibody-positive rheumatoid arthritis patients with either HLA-DRB1*04:01 or *01:01. DEN-181 was well tolerated. CII-specific T cells in peripheral blood upregulated PD1 within 1 month of dosing. Improved RA disease activity was associated with expansion of CII-specific and bystander citrullinated vimentin-specific T cells and other immune correlates of a tolerogenic effect of DEN-181 [75].

Combining antigen, immunomodulation, and cytokines may be advantageous to sustain peripheral tolerance. For example, intravenous administration of BDC2.5 mimetope, IL-2/anti-IL-2 complexes and rapamycin suppressed IGRP_206-214-specific TCR-transgenic T cell-induced diabetes development in NOD mice by expanding suppressive Foxp3+ Treg [76]. However, in an open-label mechanistic study, in adults with T1D and residual detectable C-peptide who received low-dose s.c. IL-2 for 1 month and oral rapamycin for 3 months, Treg, eosinophils, and NK cells increased after 1 month but stimulated C-peptide declined after 3 months [77]. While subsequent trials of IL-2 have been able to fine-tune dosing for a more specific increase in Treg in T1D, this particular combination appeared to be β-cell toxic. Furthermore, in a clinical trial that combined low dose IL-2 and adoptive transfer of polyclonal Treg, the transferred Treg expanded with IL-2 but the cytotoxic phenotype of circulating CD8+ T cells increased simultaneously [78].

NP entrapping antigen alone

In mouse models, i.v. administration of BDC2.5 mimotope-poly(dl-lactide-co-glycolic acid) PLG-NPs induced tolerance, with prevention of diabetes onset in recipient NOD mice for up to 2 months and increased intra-islet Treg [79]. There was a decrease in number of Teff expressing IFN-g, IL-2, and GM-CSF in the pancreas and an increase in Foxp3+ Treg and IL-10. IGRP-PLG-NP inhibited CD8+ T-cell-mediated β-cell destruction [79].

An adoptive transfer model using 2.5HIP neo-antigen coupled to PLG-NPs delayed disease onset for at least 2 months in 80% of animals [80]. There was a reduction in IFN-γ and TNF production by 2.5HIP-specific T cells in tolerized animals, an increase in 2.5HIP-specific Foxp3+ Treg and a reduced ratio of Teff/Treg in islets and spleen. High expression of CD103 integrin in the pancreatic Treg suggested their retention in islets. CD4+Foxp3- T cells overexpressed anergic and exhaustion markers, such as CD73, as also observed after BDC2.5mim/calcitriol liposomes [12].

In another study, carboxylated polystyrene beads coated with a mixture of immunodominant HLA-A*02:01-restricted epitopes administered to HLA-A*02:01-transgenic β2m^−/− HHD mice induced tolerance, with activation and accumulation of Treg and CD11c+ DCs, and T-cell anergy [81]. Delay in progression of diabetes was confirmed in ‘humanized’ NOD.β2m null HHD mice. Suppression of HLA-A*02:01-restricted autoreactive peripheral blood CD8+ T cells co cultured with ZnT8 or IGRP polystyrene beads was demonstrated in mice with new-onset T1D [81].

Also in preclinical studies, negatively charged ultra-small (5 nm) carbohydrate-coated gold nanoparticles (AuNPs) conjugated to C19-A3 and extruded through microneedles for i.d. administration to human skin ex vivo, were taken up predominantly by resident Langerhans cells. Unconjugated AuNPs reduced the maturation of DCs and the proliferation of allogeneic T cells in vitro [82]. An open-label, uncontrolled single group clinical trial is exploring the response to C19-A3 AuNPs in T1D (NCT02837094).

While not translated to T1D clinical trials, PLGA NP immunotherapy has been trialled in coeliac disease in a Phase 2a study involving 33 patients randomized to receive TAK-101, a gliadin antigen encapsulated in PLGA NP [83]. Gliadin-specific IFN-g production upon challenge was significantly reduced in patients treated with TAK-101 vs placebo. Changes in circulating α4β7+CD4+, αEβ7+CD8+, and γδ effector memory T cells also supported a tolerogenic effect of TAK-101. However, patients remained symptomatic, and further trials of NP are needed to determine their capacity to induce immune tolerance against T1D.

GD pathogenesis

GD is an antibody-mediated autoimmune thyroid disorder, characterized by stimulating thyroid hormone receptor antibodies (TSHRabs) or immunoglobulins (TSI), which bind to the thyrotropin receptor (TSHR), resulting in uninhibited iodine uptake and iodination, thyroid hormone release, and clinical thyrotoxicosis. Symptoms include weight loss, diaphoresis, tremors, memory changes, cardiovascular, and gastrointestinal symptoms and can result in a significant physiological and psychological toll. Ophthalmopathy, which is marked by T-cell infiltration in the orbital tissue, is a major complication. Unlike T1D, the clinical symptoms of thyrotoxicosis signal the presence of autoimmunity, well before significant end-organ destruction.

As with most other autoimmune disease, environmental factors prime the immune system in genetically predisposed individuals [84] to break thyroid autoantigen ‘self-tolerance’. For example, smoking enhances thyrocyte apoptosis, while bacterial and viral pathogens and certain drugs may stimulate immune responses towards the TSHR. Damaged TSHR may induce thyroid follicle release of cytokines or their differentiation into APCs. During the early phase, TSHR epitopes are taken up by APCs, and epitopes are presented to T helper cells in the cervical draining LN, while tertiary lymphoid tissue later develops in the thyroid, with unregulated autoantibody production. Dysregulated antigen presentation in GD includes fewer tolerogenic DCs and aberrant MHC class II expression by non-professional follicular cells [85–87]. The immune response varies during the course of the disease. An early Th1-like phase may precede a Th2-like chronic phase but this is debated [88].

Thyrotropin receptor

The TSHR is a 744 amino acid glycoprotein-coupled hormone receptor that consists of an extracellular domain (ECD: amino acids 1–418), a seven transmembrane domain (7TMD: 418–683) and an intracellular domain [89]. The TSHR holoreceptor undergoes spontaneous intra-molecular cleavage with loss of a 50 amino acid ectodomain region (C domain) to form the α and β subunits. Reduction in the disulphide bonds holding the subunits together may result in extracellular α subunit shedding [90]. The α subunit appears to contain the primary autoantigens driving the pathogenesis of GD, as it is easily shed, has higher glycan content and thus higher likelihood of endocytosis by DCs, to incite a pathological immunogenic response [91, 92].

As a result of post-translational processing, including glycosylation, phosphorylation, cleavage and multimerization, the TSHR can have multiple receptor configurations, resulting in signal variability [93]. Multiple antigenic epitopes of the TSHR associated with GD include peptides 52–71, 142–161, 146–165, 145–163, 158–176, 160–179, 202–221, 207–222, 247–266, 248–263, 272–291 and 343–362 [94–98]. However, data have been inconsistent, potentially reflecting disease heterogeneity and/or inconsistencies in assays and samples used.

Tolerizing ASI in GD

The current pharmacological, radioiodine treatment and surgical treatment options for GD all focus on the modulation and release of thyroid hormones. These approaches do not target the underlying immunological mechanism of the disease. Inadequacies of non-invasive medical management include inadequate remission and subsequent relapses with chronic pill burden, and serious side effects, such as agranulocytosis.

While immunosuppressive therapies—glucocorticoids, rituximab, azathioprine, tocilizumab, and cyclosporin—have been explored in GD, their associated adverse effects limit their use [99]. GD is a particularly attractive model for tolerizing ASI. If the autoimmune response could be regulated, the underlying thyroid endocrine function should remain intact, associated with long-term remission. Limitations to ASI in GD include an inadequate understanding of the antigenic epitopes of the TSHR responsible for the development of the disease. To explore this, HLA-DR3 and HLA-DR4 transgenic mice were immunized with 30 overlapping peptides from the TSHR extracellular domain, to identify 5 immunodominant regions-based on T-cell proliferation in draining LN and spleen, and proliferative response of PBMC from individuals with GD [100]. After ascertaining solubility and predicting binding to multiple HLA-DR molecules, two suitable peptide ‘apitopes’ were combined and tested as ASI in HLA-DR3 transgenic mice immunized with adenoviral TSHR. Repeated administration of apitopes subcutaneously to HLA-DR3 transgenic mice prevented the induction of anti-TSHR antibodies upon adenoviral TSHR immunization.

This approach was translated into a Phase 1 trial of a 15 amino-acid TSHR α chain sequence and a second 21 amino-acid peptide that replaced charged amino acids to increase solubility (ATX-GD-59). This open-label, uncontrolled trial included 12 individuals with HLA-DR3 or DR4 and mild to moderate GD not previously treated with anti-thyroid medication, who received escalating i.d. doses of ATX-GD-59 [101]. Treatment was well-tolerated. Although 7/10 participants had improved thyroid function and 5/10 had reduced antibodies at the end of the study, 8/10 still had subclinical hyperthyroidism with TSH < 0.02, and two required anti-thyroid medication. In the absence of a control group, the clinical significance of this small trial is unclear, as spontaneous remission can occur in 10–25% of such cases [102]. Nevertheless, this is an interesting proof-of-concept trial of ASI in Grave’s disease and paves the way for consideration of future antigen-specific immunotherapy.

Exploration of NP immunotherapy in GD has been scarce. Nanoliposomes combined with thyroid-stimulating hormone were shown to accumulate in the thyroid, at 3.5-fold higher levels in comparison to untargeted nanoliposomes [103]. One study explored 60 nm gold particles coupled to recombinant human TSHR α-subunit protein and endogenous aryl-hydrocarbon receptor agonist, injected i.p. four times at weekly into human-TSHR/NOD.H2h4 mice. Instead of inducing tolerance, there was an accelerated and enhanced development of pathogenic TSHRabs measured by inhibition of TSH binding to the TSHR [104]. As such, more research is required in this area.

Conclusion

Tolerizing ASI demonstrates promise in preclinical and early clinical studies and has the potential to regulate autoimmunity to sustain disease remission, without significant immunosuppression. While various platforms exist for ASI, nanotechnologies are of particular interest because of their flexibility, widespread applicability, and efficient delivery of active pharmaceutical ingredients to APCs as compared to cell-based systems or unencapsulated antigens. However, more work is needed to map the timelines of antigen autoreactivity and immunogenicity in across T1D and GD and to create robust biomarkers for mechanistic assessment of trial outcomes. Because of good preclinical tools, comprehensive definition of autoantigens and promising evidence that a T-cell immunotherapy is effective in Stage 2 T1D, many ASI strategies with potential across multiple autoimmune diseases are being trialled for proof-of-concept/mechanism in T1D. If effective, this should lead to better outcomes in T1D and further translation to other autoimmune diseases.

Abbreviations

7TMD

7 transmembrane domain

AhR

aryl-hydrocarbon receptor

APC

antigen-presenting cell

ASI

antigen-specific immunotherapy

AuNPs

gold nanoparticles

ChgA

chromogranin A

CTL

cytotoxic T-cell lymphocytes

CTLA4

cytotoxic T-lymphocyte-associated protein 4

DCs

dendritic cell

ECD

extracellular domain

Foxp3

Forkhead box P3

GABA

gamma-aminobutyric acid

GAD

glutamate acid decarboxylase

GD

Graves’ disease

GM-CSF

granulocyte-macrophage colony-stimulating factor

GO

Graves orbitopathy

HIP

hybrid insulin peptides

HLA

human leukocyte antigens

i.d.

intradermal

i.v.

intravenous

IAb

islet autoantibodies

IFNγ

interferon gamma

IGF1R

insulin-like growth factor I receptor

IGRP

islet glucose-6-phosphatase catalytic subunit related protein

IL

interleukin

ITE

ligand 2-(1ʹH-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester

LAG3

lymphocyte activation gene 3

LN

lymph nodes

mAb

monoclonal antibody

MHC

major histocompatibility complex

mRNA

messenger RNA

NFAT

nuclear factor of activated T cells

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NOD

nonobese diabetic

NP

nanoparticles

PBMC

peripheral blood mononuclear cells

PD-1

programmed death-1

PLG

polylactide-coglycolide

PLGA

poly-lactic-glycolic-acid

s.c.

subcutaenous

SOCS2

suppressor of cytokine signalling 2

T1D

Type 1 diabetes

TCR

T-cell receptor

Teff

effector T cells

Tfh

T follicular helper T cells

TGF-β

transforming growth factor beta

Th1

T helper 1

Th2

T helper 2

TNF

tumour necrosis factor

Tr1

T regulatory Type 1 cells

Treg

inducible regulatory T cell

TSHR

thyroid stimulating hormone receptor/thyrotropin receptor

TSHRab

thyroid stimulating hormone receptor antibodies

TSI

thyroid stimulating immunoglobulins.

Conflict of interest

R.T. reports issued patent 9,017,697 B2: 2006 surrounding technology for targeting DCs for antigen-specific tolerance, and funding from Janssen Biotech Inc. to UniQuest resulting in development and trial of a liposome product in rheumatoid arthritis.

Funding

R.T. was supported by Arthritis Queensland and NHMRC grant 2008287.

Data availability

Not applicable.

Author contributions

Conceptualization A.Z. and R.T. Funding acquisition R.T. Supervision R.T. Writing—original draft A.Z. Writing—review and editing R.T. and A.Z.

Permission to reproduce

Not applicable.