(M1130-05-31) Antigen-Specific Tolerogenic Nanodiscs for the Treatment of Experimental Autoimmune Encephalomyelitis

Purpose: Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system and may impair vision, coordination, and movement. Available treatments include broadly immunosuppressive agents that can help slow disease progression or improve quality of life, but chronic administration may lead to toxicities and various side effects. Regulatory T cell (Treg)-based immunotherapies aim to overcome these problems by restoring immune tolerance with long-term protection using Tregs. One such approach is antigen-specific immunotherapy, which generates antigen-specific Tregs and works to eliminate or inactivate autoantigen-specific Th1 and Th17 cells, allowing the patient to develop immune tolerance towards the autoantigen while maintaining immune reactivity to other antigens. Our lab has engineered synthetic high-density lipoprotein (sHDL) nanodiscs (NDs) as a platform for peptide delivery and has demonstrated efficacious delivery of antigens and adjuvants to antigen-presenting cells (APCs) in draining lymph nodes.¹ Our goal is to utilize this platform to deliver antigens along with immunomodulatory agents to expand antigen-specific Tregs and exert a tolerogenic immune response in the Experimental Autoimmune Encephalomyelitis (EAE) model, an animal model of MS.

Methods: Briefly, sHDL NDs were prepared by thermocycling a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and ApoA1 mimetic peptide 22A mixture in 10 mM phosphate buffer. The NDs were loaded with an antigen peptide conjugated to dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), followed by loading of rapamycin by gentle mixing. Particle size distribution was measured by dynamic light scattering (DLS, Zetasizer Nano ZSP) and loading efficiency was determined by high-performance liquid chromatography (HPLC). For coculture studies, bone-marrow derived dendritic cells (BMDCs) were pulsed with Ag-NDs or rapa-Ag-NDs loaded with ovalbumin 323-339 (OVAII) peptide for 24 h and cocultured with CD4+ T cells harvested from spleens and inguinal lymph nodes of OT-II mice. After 144 h, cells were stained for CD4, CD25, and Foxp3 and analyzed by flow cytometry. To assess therapeutic efficacy in vivo, mice were induced with EAE and administered subcutaneously with Ag-NDs or rapa-Ag-NDs loaded with myelin oligodendrocyte glycoprotein (MOG) peptide. Mice were scored on a scale from 0 to 5 based on severity of paralysis.

Results: sHDL NDs had an average diameter of 10 nm and maintained stable after loading with antigen peptides and rapamycin (Figure 1A). When loading Rapa-Ag-NDs with MOG peptide, there was a 76% loading (33 ug/mg ND) of the peptide and a 28% loading (1.4 ug/mg ND) of rapamycin (Figure 1B). Further loading rapamycin onto OVAII-NDs significantly enhanced Treg induction in vitro (27% compared to 8.9%) (Figure 2). Treatment of EAE mice with Rapa-Ag-NDs loaded with MOG peptide led to significantly lower EAE scores.

Conclusion: Codelivery of antigen peptide along with rapamycin loaded onto sHDL NDs led to antigen-specific Treg induction in vitro and tolerogenic immune responses in vivo. This promising data displays the potential of our NDs to be developed as a platform for treating autoimmune diseases like MS.

References: 1. Nature Mater 16, 489–496 (2017).

Acknowledgements: Cellular Biotechnology Training Program T32-GM008353. J.J.M. and A.S. declares financial interests for board membership, as a paid consultant, for research funding, and/or as equity holder in EVOQ Therapeutics. The University of Michigan has a financial interest in EVOQ Therapeutics, Inc.

Figure 1. Characterization of NDs. A) DLS size measurements of blank NDs, Rapa-NDs, and Rapa-Ag-NDs. B) Stacked HPLC chromatograms of Rapa-MOG-NDs and the individual components.

Figure 2. Frequency of CD25+Foxp3+ Treg cells among CD4+ T cells after coculturing CD4+ T cells with dendritic cells treated with PBS, OVAII-NDs, or Rapa-OVAII-NDs.