Soluble antigen arrays disarm antigen-specific B cells to promote lasting immune tolerance in experimental autoimmune encephalomyelitis.

Autoreactive lymphocytes that escape central immune tolerance may be silenced via an endogenous peripheral tolerance mechanism known as anergy. Antigen-specific therapies capable of inducing anergy may restore patients with autoimmune diseases to a healthy phenotype while avoiding deleterious side effects associated with global immunosuppression. Inducing anergy in B cells may be a particularly potent intervention, as B cells can contribute to autoimmune diseases through multiple mechanisms and offer the potential for direct antigen-specific targeting through the B cell receptor (BCR). Our previous results suggested autoreactive B cells may be silenced by multivalent 'soluble antigen arrays' (SAgAs), which are polymer conjugates displaying multiple copies of autoantigen with or without a secondary peptide that blocks intracellular cell-adhesion molecule-1 (ICAM-1). Here, key therapeutic molecular properties of SAgAs were identified and linked to the immunological mechanism through comprehensive cellular and in vivo analyses. We determined non-hydrolyzable 'cSAgAs' displaying multivalent 'click'-conjugated antigen more potently suppressed experimental autoimmune encephalomyelitis (EAE) compared to hydrolyzable SAgAs capable of releasing conjugated antigen. cSAgAs restored a healthy phenotype in disease-specific antigen presenting cells (APCs) by inducing an anergic response in B cells and a subset of B cells called autoimmune-associated B cells (ABCs) that act as potent APCs in autoimmune disease. Accompanied by a cytokine response skewed towards a Th2/regulatory phenotype, this generated an environment of autoantigenic tolerance. By identifying key therapeutic molecular properties and an immunological mechanism that drives SAgA efficacy, this work guides the design of antigen-specific immunotherapies capable of inducing anergy.

Screening Immunomodulators To Skew the Antigen-Specific Autoimmune Response.

Current therapies to treat autoimmune diseases often result in side effects such as nonspecific immunosuppression. Therapies that can induce antigen-specific immune tolerance provide an opportunity to reverse autoimmunity and mitigate the risks associated with global immunosuppression. In an effort to induce antigen-specific immune tolerance, co-administration of immunomodulators with autoantigens has been investigated in an effort to reprogram autoimmunity. To date, identifying immunomodulators that may skew the antigen-specific immune response has been ad hoc at best. To address this need, we utilized splenocytes obtained from mice with experimental autoimmune encephalomyelitis (EAE) in order to determine if certain immunomodulators may induce markers of immune tolerance following antigen rechallenge. Of the immunomodulatory compounds investigated, only dexamethasone modified the antigen-specific immune response by skewing the cytokine response and decreasing T-cell populations at a concentration corresponding to a relevant in vivo dose. Thus, antigen-educated EAE splenocytes provide an ex vivo screen for investigating compounds capable of skewing the antigen-specific immune response, and this approach could be extrapolated to antigen-educated cells from other diseases or human tissues.

Molecular Dynamics of Multivalent Soluble Antigen Arrays Support a Two-Signal Co-delivery Mechanism in the Treatment of Experimental Autoimmune Encephalomyelitis.

Many current therapies for autoimmune diseases such as multiple sclerosis (MS) result in global immunosuppression, rendering insufficient efficacy with increased risk of adverse side effects. Multivalent soluble antigen arrays, nanomaterials presenting both autoantigen and secondary inhibitory signals on a flexible polymer backbone, are hypothesized to shift the immune response toward selective autoantigenic tolerance to repress autoimmune disease. Two-signal co-delivery of both autoantigen and secondary signal were deemed necessary for therapeutic efficacy against experimental autoimmune encephalomyelitis, a murine model of MS. Dynamic light scattering and in silico molecular dynamics simulations complemented these studies to illuminate the role of two-signal co-delivery in determining therapeutic potential. Physicochemical characteristics such as particle size and molecular affinity for intermolecular interactions and chain entanglement likely facilitated cotransport of two signals to produce efficacy. These findings elucidate potential mechanisms whereby soluble antigen arrays enact their therapeutic effect and help to guide the development of future multivalent antigen-specific immunotherapies.

Enhanced delivery of α-glucosidase for Pompe disease by ICAM-1-targeted nanocarriers: comparative performance of a strategy for three distinct lysosomal storage disorders.

Enzyme replacement therapies for lysosomal storage disorders are often hindered by suboptimal biodistribution of recombinant enzymes after systemic injection. This is the case for Pompe disease caused by acid α-glucosidase (GAA) deficiency, leading to excess glycogen storage throughout the body, mainly the liver and striated muscle. Targeting intercellular adhesion molecule-1 (ICAM-1), a protein involved in inflammation and overexpressed on most cells under pathological conditions, provides broad biodistribution and lysosomal transport of therapeutic cargoes. To improve its delivery, we coupled GAA to polymer nanocarriers (NCs; ∼180 nm) coated with an antibody specific to ICAM-1. Fluorescence microscopy showed specific targeting of anti-ICAM/GAA NCs to cells, with efficient internalization and lysosomal transport, enhancing glycogen degradation over nontargeted GAA. Radioisotope tracing in mice demonstrated enhanced GAA accumulation in all organs, including Pompe targets. Along with improved delivery of Niemann-Pick and Fabry enzymes, previously described, these results indicate that ICAM-1 targeting holds promise as a broad platform for lysosomal enzyme delivery. FROM THE CLINICAL EDITOR: In this study, ICAM-1 targeted nanocarriers were used to deliver GAA (acid alpha glucosidase) into cells to address the specific enzyme deficiency in Pompe's disease. The results unequivocally demonstrate enhanced enzyme delivery over nontargeted GAA in a mice model.

Co-delivery of autoantigen and dexamethasone in incomplete Freund's adjuvant ameliorates experimental autoimmune encephalomyelitis.

Current therapies for autoimmune diseases focus on treating the symptoms rather than the underlying disease cause. A major setback in improving current therapeutics for autoimmunity is the lack of antigen specificity. Successful antigen-specific immunotherapy (ASIT) would allow for improved treatment of autoimmune diseases. In this work, dexamethasone was co-delivered with autoantigen (PLP) in vivo to create effective ASIT for the treatment of experimental autoimmune encephalomyelitis (EAE). Using an emulsion of incomplete Freund's adjuvant (IFA) as a co-delivery vehicle, it was discovered that the controlled release of autoantigen was important for the suppression of clinical disease symptoms. Analysis of the immune response via cytokines revealed that dexamethasone was important for shifting the immune response away from inflammation. Co-delivery of both autoantigen and dexamethasone increased B-cell populations and antibody production, signifying an increased humoral immune response. Overall, this data indicated that the co-delivery of PLP and dexamethasone with a water-in-oil emulsion is effective in treating a murine autoimmune model.

Combining antigen and immunomodulators: Emerging trends in antigen-specific immunotherapy for autoimmunity.

A majority of current therapies for autoimmune diseases are general immunosuppressants, which can compromise patient response to opportunistic infection and lead to adverse events. Using antigen-specific immunotherapy (ASIT) to selectively disarm autoimmune diseases, without suppressing the global immune response, would be a transformative therapy for patients. ASIT has been used historically in allergy hyposensitization therapy to induce tolerance to an allergen. Similar strategies to induce immune tolerance toward autoantigens responsible for autoimmune disease have been attempted but have yielded limited clinical success. Recent studies of ASIT for autoimmunity have explored combination therapy, combining the disease-causing autoantigen with an immunomodulatory compound. ASIT combination therapy may direct the immune response in an antigen-specific manner, potentially reversing the root cause of autoimmunity while limiting side effects. This review analyzes recent advances in ASIT applied to autoimmune diseases, emphasizing current combination therapies and future strategies.

Co-delivery of autoantigen and b7 pathway modulators suppresses experimental autoimmune encephalomyelitis.

Autoimmune diseases such as multiple sclerosis (MS) are characterized by the breakdown of immune tolerance to autoantigens. Targeting surface receptors on immune cells offers a unique strategy for reprogramming immune responses in autoimmune diseases. The B7 signaling pathway was targeted using adaptations of soluble antigen array (SAgA) technology achieved by covalently linking B7-binding peptides and disease causing autoantigen (proteolipid peptide (PLP)) to hyaluronic acid (HA). We hypothesized that co-delivery of a B7-binding peptide and autoantigen would suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Three independent B7-targeted SAgAs were created containing peptides to either inhibit or potentially stimulate the B7 signaling pathway. Surprisingly, all SAgAs were found to suppress EAE disease symptoms. Altered cytokine expression was observed in primary splenocytes isolated from SAgA-treated mice, indicating that SAgAs with different B7-binding peptides may suppress EAE through different immunological mechanisms. This antigen-specific immunotherapy using SAgAs can successfully suppress EAE through co-delivery of autoantigen and peptides targeting with the B7 signaling pathway.

Codelivery of antigen and an immune cell adhesion inhibitor is necessary for efficacy of soluble antigen arrays in experimental autoimmune encephalomyelitis.

Autoimmune diseases such as multiple sclerosis (MS) are typified by the misrecognition of self-antigen and the clonal expansion of autoreactive T cells. Antigen-specific immunotherapies (antigen-SITs) have long been explored as a means to desensitize patients to offending self-antigen(s) with the potential to retolerize the immune response. Soluble antigen arrays (SAgAs) are composed of hyaluronic acid (HA) cografted with disease-specific autoantigen (proteolipid protein peptide) and an ICAM-1 inhibitor peptide (LABL). SAgAs were designed as an antigen-SIT that codeliver peptides to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Codelivery of antigen and cell adhesion inhibitor (LABL) conjugated to HA was essential for SAgA treatment of EAE. Individual SAgA components or mixtures thereof reduced proinflammatory cytokines in cultured splenocytes from EAE mice; however, these treatments showed minimal to no in vivo therapeutic effect in EAE mice. Thus, carriers that codeliver antigen and a secondary "context" signal (e.g., LABL) in vivo may be an important design criteria to consider when designing antigen-SIT for autoimmune therapy.

Single-step grafting of aminooxy-peptides to hyaluronan: a simple approach to multifunctional therapeutics for experimental autoimmune encephalomyelitis.

The immune response to antigens is directed in part by the presence or absence of costimulatory signals. The ability to coincidently present both antigen and, for example, a peptide that inhibits or activates the costimulatory pathway, would be a valuable tool for tolerization or immunization, respectively. A simple reaction scheme utilizing oxime chemistry was identified as a means to efficiently conjugate different peptide species to hyaluronan. Peptides synthesized with an aminooxy N-terminus reacted directly to hyaluronan under slightly acidic aqueous conditions without the need for a catalyst. The resulting oxime bond was found to rapidly hydrolyze at pH2 releasing peptide, but was stable at higher pH values (5.5 and 7). Two different peptide species, a multiple sclerosis antigen (PLP) and an ICAM-1 ligand (LABL) known to block immune cell stimulation, were functionalized with the aminooxy end group. These peptides showed similar reactivity to hyaluronan and were conjugated in an equimolar ratio. The resulting hyaluronan with grafted PLP and LABL significantly inhibited disease in mice with experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Aminooxy-peptides facilitate simple synthesis of multifunctional hyaluronan graft polymers, thus enabling novel approaches to antigen-specific immune modulation.