Regulation of antigen-specific response to peptide therapeutics is independent of distribution in lymph nodes

Introduction: : Autoimmune disease occurs when immune cells mistakenly attack specific molecules, termed antigens, on healthy cells. There are no cures for these diseases, and existing treatments do not specifically target dysfunctional cells. Direct lymph node (LN) injections of soluble immune cues are receiving increasing attention in immunotherapy, with several recent trials switching to this route (e.g., Umoja) and intra-lymph node (iLN) allergy shots being offered to accelerate therapy at university medical centers. However, autoimmune disease is complex and often involves reactivity against sets of autoantigens, highlighting the need to deliver multiple antigens in new immunotherapies. We have shown degradable polymer depots encapsulating a single self-antigen and delivered iLN reverse autoimmune paralysis with a single treatment, while preserving healthy immune functions. Here, we used these depots to encapsulate peptide antigens in distinct loading configurations – either with multiple peptides in a single particle or a single peptide in distinct particles, as a flexible approach for autoimmune therapies. Our work demonstrates that these formulations are functionally equivalent, which could enable more facile chemistry and manufacturing control (CMC) processes and simpler regulatory approval of peptide-loaded microparticle (MP) LN therapeutics.

Materials and

Methods: : Polymer MPs were synthesized via double emulsion to encapsulate rapamycin (rapa) with individual peptide antigens—MOG35-55 or OVA323-339—in combination (co-loaded), or as a 1:1 mixture of MPs each containing a single antigen (mixed). These formulations were designated: i) MOG MPs (M MP), ii) OVA MPs (O MP), iii) mixed MPs (M MP + O MP), iv) co-loaded MPs (M/O MP), v) MOG/rapa and OVA/Rapa MPs (M/R+O/R MP), and vi) MOG/OVA/Rapa MPs (M/O/R MP). Bone-marrow derived dendritic cells (BMDCs) were isolated from the femurs of a C57BL/6J mouse and cultured, before treatment with MPs for 3 hours and staining for fluorescent microscopy. T cell proliferation and differentiation was tested in an in vivo mouse model. An iLN injection of MP formulations was administered to the inguinal LNs of mice. One day after treatment, fluorescently-dyed CD4+ T cells specific for MOG and OVA were then transferred i.v. into mice at a 1:1 ratio. LNs were isolated three and seven days after transfer for analysis via flow cytometry. Finally, the maximum peptide loading capacity of MPs with or without the inclusion of rapamycin was established via a microBCA assay.

Results, Conclusions, and Discussions:: We first treated BMDCs with MPs encapsulating fluorescently-labeled peptide cargo, utilizing mixed and co-loaded formulations. Confocal microscopy confirmed MP uptake in cells regardless of MP formulation (Fig. 1A). Next, we delivered these two formulations to mice iLN to test how these treatments affect T cell activity and phenotype in vivo. Both mixed and co-loaded MPs equivalently drove proliferation of cognate, antigen-specific T cells (Fig. 1B). Supported by this data, we hypothesized that both treatments could equivalently polarize T cells towards a regulatory phenotype. To test this, co-loaded and mixed MPs were loaded with equivalent doses of rapa, a known driver of regulatory T cells (Treg). When MPs were delivered iLN to mice, no significant difference in the frequency of Treg was observed among the recovered antigen-specific cells as a function of either MP formulation (Fig. 1C). These data demonstrate the delivery of immunomodulatory cues, such as rapa, alongside one or multiple peptides can equivalently drive polarization of Treg, regardless of the formulation scheme. Finally, we synthesized MPs with increasing doses of peptide to test the hypothesis that MPs can encapsulate greater loading levels of cargo. MP characterization revealed MPs can be loaded with a 5-fold increase in peptide, demonstrating their capability to deliver more peptides than the two model antigens investigated here (Fig. 1D).

New therapies for autoimmune disease will likely require tolerance against multiple self-antigens. The work here demonstrates that a combination of MPs loaded with individual peptides, or with multiple peptides within a single particle, can effectively drive equivalent T cell proliferation and phenotype. Our recent work shows that iLN MP treatments are safe in non-human primates (NHP). Notably, recent clinical trials target the same inguinal lymph nodes as seen in the NHP studies just mentioned, as well as the in vivo studies demonstrated here. This data informs simplified CMC approaches for antigen-specific therapies in which particles with defined antigen content can be simply mixed prior to LN injection as a strategy to tolerize against disease-relevant antigen reactivity profiles.

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