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 pH 2 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.

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.

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ABSTRACT. Presentation of antigen with immune stimulating "signal" has been a cornerstone of vaccine design for decades. Here, the antigen plus immune "signal" of vaccines is modified to produce antigen-specific immunotherapies (antigen-SITs) that can potentially reprogram the immune response toward tolerance of an autoantigen. The codelivery of antigen with a cell adhesion inhibitor using Soluble Antigen Arrays (SAgAs) was previously shown to slow or halt experimental autoimmune encephalomyelitis (EAE), a murine form of multiple sclerosis (MS). SAgAs are comprised of a hyaluronic acid backbone with cografted intercellular cell adhesion molecule-1 ligand derived from α L -integrin (CD11a 237-246 , "LABL") and an encephalitogenic epitope peptide of proteolipid protein (PLP 139-151 , "PLP"). Here, the physical characteristics of the carrier were investigated to evaluate how structure, size, and solubility drive the immune response when treating EAE. A bifunctional peptide (small, soluble), SAgAs (large, soluble), and PLGA nanoparticles (large, insoluble) all displaying PLP and LABL in equimolar ratios were compared. Maximum EAE suppression was achieved with coincident display of both peptides on a soluble construct.

Abstract. 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 antigenspecific immunotherapy using SAgAs can successfully suppress EAE through co-delivery of autoantigen and peptides targeting with the B7 signaling pathway.KEY WORDS: antigen-specific immunotherapy; B7/CD28:CTLA-4 co-stimulatory pathway; experimental autoimmune encephalomyelitis (EAE); proteolipid peptide; soluble antigen array.