Joseph W. Reda

Summary: Engineering immune tolerance using endogenously expressed autoantigens could enable early intervention in autoimmune disease. Present therapies suppress T cells to slow disease progression. Here, we develop rapamycin-loaded poly(ethylene glycol)- b -poly(propylene sulfide) polymersomes (rPS) to modulate antigen-presenting cells (APCs) during disease-associated antigen presentation. The rPS morphology enables lymphatic drainage following subcutaneous administration to access APCs in draining lymph nodes. Treatment reduced APC costimulatory marker coexpression and shifted CD4⁺ T cells toward an FR4 ^hi CD73 ^hi anergic phenotype. In a type 1 diabetes (T1D) nonobese diabetic (NOD) mouse model, rPS significantly delayed disease onset relative to the standard of care and selectively prevented hyperglycemia. Mediation via APCs, rPS avoids adverse effects including transient lymphopenia. These findings demonstrate how biomaterial-directed drug delivery can reshape APC–T cell interactions, supporting an antigen-agnostic strategy for delaying autoimmune diabetes without systemic lymphocyte depletion. The bigger picture: Type 1 diabetes (T1D) results from a complex autoimmune attack on insulin-producing pancreatic β cells. While the detection of islet autoantibodies before T1D onset enables the development of early intervention strategies, the temporal dynamics of islet antigen presentation and the full repertoire of relevant islet antigens remain unclear. Current immunotherapies primarily target T cells to halt islet injury but can cause transient systemic immune suppression and are not curative. Herein, we identify a potential new therapeutic strategy by which antigen-presenting cells (APCs) are reprogrammed to arrest T1D progression without systemic lymphodepletion. To this end, we utilize rapamycin-loaded polymersome (rPS) nanocarriers which selectively deliver the rapamycin payload to APCs. When given prior to disease onset, rPS treatment delays and prevents hyperglycemia in comparison to a T cell-directed, antibody-mediated systemic therapy. Importantly, our study provides a comprehensive evaluation of therapeutic intervention and disease progression. Through subcutaneous injection, the nanotherapeutic approach leverages lymphatic drainage to reach the draining lymph nodes. Here, rPS-induced costimulation-deficient APCs can prevent T cell activation without supplying a specific antigen in prediabetic mice. Complex immunoprofiling validated APC reprogramming in vivo and characterized T cell activation state in presymptomatic mice. Overall, this novel biomaterial-mediated approach demonstrates the importance of designing early intervention therapies to disrupt the T cell activation pathway and offer protection to functional β cells and an opportunity to achieve long-term insulin independence. Highlights: Rapamycin-loaded polymersomes (rPS) are an APC-directed nanotherapy to slow T1D progression A short course of subcutaneous (SC) rPS therapy delays T1D onset in early prediabetic mice APC-mediated reprogramming halts T1D progression without lymphodepletion Maintenance of effector-memory CD4+ T cell anergy is critical to prevent hyperglycemia Graphical

Atherosclerosis is a chronic inflammatory disease associated with the accumulation of low-density lipoprotein (LDL) in arterial walls. Higher levels of the anti-inflammatory cytokine IL-10 in serum are correlated with reduced plaque burden. However, cytokine therapies have not translated well to the clinic, partially due to their rapid clearance and pleiotropic nature. Here we engineer IL-10 to overcome these challenges by hitchhiking on LDL to atherosclerotic plaques. Specifically, we construct Fab-IL-10 by fusing IL-10 to the antibody fragment (Fab) of four different oxidized LDL-binding antibodies. We show that systemically administered Fab-IL-10 constructs bind circulating LDL and traffic to atherosclerotic plaques in atherosclerosis mouse models. Among them, 2D03-IL-10 significantly reduces aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that 2D03-IL-10 preferentially associates with foamy macrophages and reduces pro-inflammatory activation markers. This modular technology may be applied to a variety of protein therapeutics and shows promise as a potential targeted anti-inflammatory therapy in atherosclerosis.

Butyrate—a metabolite produced by commensal bacteria—has been extensively studied for its immunomodulatory effects on immune cells, including regulatory T cells, macrophages and dendritic cells. However, the development of butyrate as a drug has been hindered by butyrate’s poor oral bioavailability, owing to its rapid metabolism in the gut, its low potency (hence, necessitating high dosing), and its foul smell and taste. Here we report that the oral bioavailability of butyrate can be increased by esterifying it to serine, an amino acid transporter that aids the escape of the resulting odourless and tasteless prodrug (O-butyryl-l-serine, which we named SerBut) from the gut, enhancing its systemic uptake. In mice with collagen-antibody-induced arthritis (a model of rheumatoid arthritis) and with experimental autoimmune encephalomyelitis (a model of multiple sclerosis), we show that SerBut substantially ameliorated disease severity, modulated key immune cell populations systemically and in disease-associated tissues, and reduced inflammatory responses without compromising the global immune response to vaccination. SerBut may become a promising therapeutic for autoimmune and inflammatory diseases.

Atherosclerosis is a chronic inflammatory disease associated with the accumulation of low-density lipoprotein (LDL) in arterial walls. Higher levels of the anti-inflammatory cytokine IL-10 in serum are correlated with reduced plaque burden. However, cytokine therapies have not translated well to the clinic, partially due to their rapid clearance and pleiotropic nature. Here, we engineered IL-10 to overcome these challenges by hitchhiking on LDL to atherosclerotic plaques. Specifically, we constructed fusion proteins in which one domain is IL-10 and the other is an antibody fragment (Fab) that binds to protein epitopes of LDL. In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and traffic to atherosclerotic plaques. One such construct, 2D03-IL-10, significantly reduces aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that 2D03-IL-10 preferentially associates with foamy macrophages and reduces pro-inflammatory activation markers. This platform technology can be applied to a variety of therapeutics and shows promise as a potential targeted anti-inflammatory therapy in atherosclerosis.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal–antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal–antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.

Butyrate, a metabolite produced by commensal bacteria, has been intensively studied for its immunomodulatory effects on various immune cells, including T regulatory cells, macrophages, and dendritic cells. Butyrate’s development as a drug has been limited by its poor oral bioavailability due to its rapid metabolism in the gut, its low potency and thus high dosing, and its foul smell and taste. By simply esterifying butyrate to serine ( O -butyryl -L -serine, SerBut), a design based on the concept of utilizing amino acid transporters to escape the gut and enhance systemic uptake thus increasing bioavailability, we developed an odorless and tasteless compound for oral administration. In the collagen antibody-induced arthritis (CAIA) and experimental autoimmune encephalomyelitis (EAE) murine models of rheumatoid arthritis and multiple sclerosis, we demonstrated that SerBut significantly ameliorated disease severity, modulated key immune cell populations both systemically and in disease-associated tissues, and reduced inflammatory responses without compromising global immune response to vaccination. Our findings highlight SerBut as a promising next-generation therapeutic agent for autoimmune and inflammatory diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.