Injectable butyrate-prodrug micelles induce long-acting immune modulation and prevent autoimmune arthritis in mice.

Short chain fatty acid (SCFAs), such as butyrate, have shown promising therapeutic potential due to their immunomodulatory effects, particularly in maintaining immune homeostasis. However, the clinical application of SCFAs is limited by the need for frequent and high oral dosages. Rheumatoid arthritis (RA) is characterized by aberrant activation of peripheral T cells and myeloid cells. In this study, we aimed to deliver butyrate directly to the lymphatics using a polymeric micelle-based butyrate prodrug to induce long-lasting immunomodulatory effects. Notably, negatively charged micelles (Neg-ButM) demonstrated superior efficacy in targeting the lymphatics following subcutaneous (s.c.) administration and were retained in the draining lymph nodes, spleen, and liver for over one month. In the collagen antibody-induced arthritis (CAIA) mouse model of RA, only two s.c. injections of Neg-ButM successfully prevented disease onset and promoted tolerogenic phenotypes in T cells and myeloid cells, both locally and systemically. These results underscore the potential of this strategy in managing inflammatory autoimmune diseases by directly modulating immune responses via lymphatic delivery.

A serine-conjugated butyrate prodrug with high oral bioavailability suppresses autoimmune arthritis and neuroinflammation in mice.

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.

Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses.

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.

Masking the immunotoxicity of interleukin-12 by fusing it with a domain of its receptor via a tumour-protease-cleavable linker.

Immune-checkpoint inhibitors have shown modest efficacy against immunologically 'cold' tumours. Interleukin-12 (IL-12)-a cytokine that promotes the recruitment of immune cells into tumours as well as immune cell activation, also in cold tumours-can cause severe immune-related adverse events in patients. Here, by exploiting the preferential overexpression of proteases in tumours, we show that fusing a domain of the IL-12 receptor to IL-12 via a linker cleavable by tumour-associated proteases largely restricts the pro-inflammatory effects of IL-12 to tumour sites. In mouse models of subcutaneous adenocarcinoma and orthotopic melanoma, masked IL-12 delivered intravenously did not cause systemic IL-12 signalling and eliminated systemic immune-related adverse events, led to potent therapeutic effects via the remodelling of the immune-suppressive microenvironment, and rendered cold tumours responsive to immune-checkpoint inhibition. We also show that masked IL-12 is activated in tumour lysates from patients. Protease-sensitive masking of potent yet toxic cytokines may facilitate their clinical translation.

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant.

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

Polymersomes Decorated with the SARS-CoV-2 Spike Protein Receptor-Binding Domain Elicit Robust Humoral and Cellular Immunity.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBDsurf) in mice compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Immunoengineering approaches for cytokine therapy.

Since the discovery of cytokines, much effort has been put forth to achieve therapeutic translation for treatment of various diseases, including cancer and autoimmune diseases. Despite these efforts, very few cytokines have cleared regulatory approval, and those that were approved are not commonly used due to their challenging toxicity profile and/or limited therapeutic efficacy. The main limitation in translation has been that wild-type cytokines have unfavorable pharmacokinetic and pharmacodynamic profiles, either eliciting unwanted systemic side effects or insufficient residence in secondary lymphoid organs. In this review, we address protein-engineering approaches that have been applied to both proinflammatory and anti-inflammatory cytokines to enhance their therapeutic indices, and we highlight diseases in which administration of engineered cytokines is especially relevant.

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity.

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Suppression of Rheumatoid Arthritis by Enhanced Lymph Node Trafficking of Engineered Interleukin-10 in Murine Models.

OBJECTIVE: Rheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials have been performed using interleukin-10 (IL-10), an antiinflammatory cytokine, as a potential treatment of RA, the therapeutic effects of IL-10 have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. This study was undertaken to engineer an IL-10-serum albumin (SA) fusion protein and evaluate its effects in 2 murine models of RA. METHODS: SA-fused IL-10 (SA-IL-10) was recombinantly expressed. Mice with collagen antibody-induced arthritis (n = 4-7 per group) or collagen-induced arthritis (n = 9-15 per group) were injected intravenously with wild-type IL-10 or SA-IL-10, and the retention of SA-IL-10 in the lymph nodes (LNs), immune cell composition in the paws, and therapeutic effect of SA-IL-10 on mice with arthritis were assessed. RESULTS: SA fusion to IL-10 led to enhanced accumulation in the mouse LNs compared with unmodified IL-10. Intravenous SA-IL-10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive alternatively activated macrophages, reduced IL-17A levels in the paw-draining LN, and protected joint morphology. Intravenous SA-IL-10 treatment showed similar efficacy as treatment with an anti-tumor necrosis factor antibody. SA-IL-10 was equally effective when administered intravenously, locally, or subcutaneously, which is a benefit for clinical translation of this molecule. CONCLUSION: SA fusion to IL-10 is a simple but effective engineering strategy for RA therapy and has potential for clinical translation.

Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4+ T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis.

Publisher Correction: Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

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

Conferring extracellular matrix affinity enhances local therapeutic efficacy of anti-TNF-α antibody in a murine model of rheumatoid arthritis.

BACKGROUND: Although disease in a majority of rheumatoid arthritis (RA) patients is often initially limited to one or a few joints, currently approved medications including anti-tumor necrosis factor-α antibody (α-TNF) are injected systemically. Given that α-TNF systemic injection typically does not cure RA and involves risk of treatment-related adverse events, one possible approach to enhance therapeutic efficacy and reduce α-TNF systemic exposure is to retain the antibodies in arthritic joints after local administration. The aim of this study was to evaluate the approach of conferring extracellular matrix (ECM) binding affinity to α-TNF antibodies in a RA model. METHODS: α-TNF was chemically conjugated with a promiscuous ECM-binding peptide derived from placenta growth factor 2 (PlGF-2123-144). The binding activity of PlGF-2123-144-conjugated α-TNF (PlGF-2123-144-α-TNF) against ECM proteins was assessed by ELISA and by immunostaining on human cartilage specimens. The effect of conjugation on antibody function was assessed as a neutralizing activity against osteoclast differentiation. Retention at the injection site and therapeutic efficacy of PlGF-2123-144-α-TNF were tested in a collagen antibody-induced arthritis (CAIA) model in the mouse. RESULTS: PlGF-2123-144 peptide conjugation conferred α-TNF with affinity to ECM proteins without impairment of antigen recognition. PlGF-2123-144-α-TNF locally injected at a paw in the CAIA model was retained for at least 96 h at the injection site, whereas unmodified α-TNF was dispersed rapidly after injection. Local treatment with unmodified α-TNF did not suppress the arthritis score relative to isotype controls. By contrast, local administration of PlGF-2123-144-α-TNF suppressed arthritis development almost completely in the treated paw even at a 1000× lower dose. CONCLUSION: These data demonstrate that retention of α-TNF in arthritic joints can suppress arthritis development and enhance therapeutic efficacy. This simple bioengineering approach of ECM-binding peptide conjugation offers a powerful and clinically translational approach to treat RA.

Combined delivery of VEGF and IGF-1 promotes functional innervation in mice and improves muscle transplantation in rabbits.

Microvascular muscle transfer is the gold standard for reanimation following chronic facial nerve paralysis, however, despite the regenerative capacity of peripheral motor axons, poor reinnervation often results in sub-optimal function. We hypothesized that injection of alginate hydrogels releasing growth factors directly into donor tissue would promote reinnervation, muscle regeneration, and function. A murine model of sciatic nerve ligation and neurorrhaphy was first used to assess the ability of gel delivery of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) to promote functional reinnervation. VEGF + IGF-1 gel delivery to aged mice resulted in prolonged ability to control toe movement, increased toe spreading, and improved static sciatic index score, indicative of improved sciatic nerve and neuromuscular junction function. Further, a 26% increase in muscle fiber area, and 2.8 and 3.0-fold increases in muscle contraction force and velocity, respectively, were found compared to blank alginate in the murine model. This strategy was subsequently tested in a rabbit model of craniofacial gracilis muscle transplantation. Electromyography demonstrated a 71% increase in compound muscle action potential 9 weeks after transplantation following treatment with VEGF + IGF-1 alginate, compared to blank alginate in the rabbit model. Improving functional innervation in transplanted muscle via a hydrogel source of growth factors may enhance the therapeutic outcomes of facial palsy treatments and, more broadly, muscle transplantations.

CD4 T-cells regulate angiogenesis and myogenesis.

Ischemic diseases, such as peripheral artery disease, affect millions of people worldwide. While CD4+ T-cells regulate angiogenesis and myogenesis, it is not understood how the phenotype of these adaptive immune cells regulate these regenerative processes. The secreted factors from different types of CD4+ T-cells (Th1, Th2, Th17, and Treg) were utilized in a series of in vitro assays and delivered from an injectable alginate biomaterial into a murine model of ischemia to study their effects on vascular and skeletal muscle regeneration. Conditioned medium from Th2 and Th17  T-cells enhanced angiogenesis in vitro and in vivo, in part by directly stimulating endothelial sprouting. Th1 conditioned medium induced vascular regression in vitro and provided no benefit to angiogenesis in vivo. Th1, Th2, and Th17 conditioned medium, to varying extents, enhanced muscle precursor cell proliferation and inhibited their differentiation in vitro, and prolonged early stages of muscle regeneration in vivo. Treg conditioned medium had a moderate or no effect on these processes in vitro and no discernible effect in vivo. These findings suggest that Th2 and Th17 T-cells may enhance angiogenesis and myogenesis in ischemic injuries, which may be useful in the design of immunomodulatory biomaterials to treat these diseases.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering.

Solubilized cardiac extracellular matrix (ECM) is being developed as an injectable therapeutic that offers promise for promoting cardiac repair. However, the ECM alone forms a hydrogel that is very soft compared to the native myocardium. As both the stiffness and composition of the ECM are important in regulating cell behavior and can have complex synergistic effects, we sought to develop an ECM-based scaffold with tunable biochemical and mechanical properties. We used solubilized rat cardiac ECM from two developmental stages (neonatal, adult) combined with fibrin hydrogels that were cross-linked with transglutaminase. We show that ECM was retained within the gels and that the Young's modulus could be tuned to span the range of the developing and mature heart. C-kit+ cardiovascular progenitor cells from pediatric patients with congenital heart defects were seeded into the hybrid gels. Both the elastic modulus and composition of the scaffolds impacted the expression of endothelial and smooth muscle cell genes. Furthermore, we demonstrate that the hybrid gels are injectable, and thus have potential for minimally invasive therapies. ECM-fibrin hybrid scaffolds offer new opportunities for exploiting the effects of both composition and mechanical properties in directing cell behavior for tissue engineering.