Sweeten the deal: Glycopolymer-based engineering to modulate autoreactive T cell responses

The liver is an important immune sentinel organ responsible for modulating adaptive immune responses to foreign dietary antigens derived from the gut. Mechanistically, this is achieved through maintenance of an anti-inflammatory microenvironment within the liver, which leads to antigen presentation by immature professional and non-professional antigen-presenting cells (APCs) and induction of antigen-specific immune tolerance. Within the liver, major histocompatibility complex (MHC) class I-restricted antigen presentation by hepatocytes and non-professional APCs results in suboptimal activation of antigen-specific CD8⁺ T cells, which can result in exhaustion or even clonal deletion,^1,2 whereas local MHC II-restricted antigen expression in professional and non-professional APCs within the liver induces both CD4⁺ T cell anergy and deletion and drives conversion of T effector cells into regulatory T cells (Tregs).

Now, in a recent issue of Nature Biomedical Engineering, a group from the University of Chicago show that targeted delivery of autoantigens to the liver can lead to suppression of autoimmunity.³ Safe and effective antigen-specific therapies for autoimmune diseases are currently lacking. Autoimmunity is generally treated with systemic immune suppression (IS), which is non-specific and incapable of resetting the immune response to the autoantigen(s). Thus, patients often relapse when they are weaned off their IS regimen. Further, while on IS, patients are at risk for pathogenic infections and have reduced vaccine efficacy (i.e., reduced protective immunity from COVID-19 vaccines).

In their study, Jeffrey A. Hubbell, D. Scott Wilson, and colleagues demonstrate that synthetic glycosylation with polymeric N-acetylgalactosamine (GalNAc) can target an antigen to the liver through the hepatocyte-specific C-type lectin asialoglycoprotein receptor (ASGPR).³ Antigens are conjugated to the side-chain GalNAc glycopolymer (pGal) via a self-immolative linker, which is removed upon endocytosis. Mechanistic evaluation was performed using pGal-ovalbumin (pGal-OVA), which demonstrated suppression of both effector and memory responses using adoptive transfer of OVA-specific CD4⁺ and CD8⁺ T cells. Transcriptional analysis of T cells after treatment revealed co-inhibitory pathways to be the top causal networks induced by pGal-antigen therapy, with PD1 and the co-inhibitory ligand CD276 (B7-H3) driving the tolerogenic response.

Next, the authors applied pGal-antigen-specific tolerance to experimental autoimmune encephalomyelitis (EAE), which is a mouse model of multiple sclerosis (MS), in which autoimmune responses are directed against the myelin insulation around nerves. Several antigen-specific approaches in the EAE model have been reported including targeted delivery of a modified myelin oligodendrocyte glycoprotein (MOG) mRNA in lipid nanoparticles to APCs⁴ and adeno-associated virus (AAV) liver-directed MOG expression.⁵ Both approaches showed efficacy in preventing and treating established disease mediated by induction of MOG-specific Tregs. However, both of these approaches lack the ability to regulate MOG expression in the case of adverse events, i.e., failure to achieve immune tolerance, which is an advantage of the present study. T cells from mice immunized with the MOG autoantigen were used as a source for encephalitogenic T cells to induce chronic EAE in recipient mice, resulting in MS-like paralysis and weight loss. Multiple treatments with a pGal-MOG_18–62 glycoconjugate prevented mice from developing EAE, whereas unconjugated MOG_30–60 treatments did not. Failed recovery of disease-inducing donor T cells and a reduction in inflammatory cytokine production were both attributed to treatment success. More excitingly, the authors were able to show that a glycoconjugate of myelin proteolipid protein (PLP_139–151) immunodominant peptide was able to preclude relapses in mice with a relapsing-remitting (R/R) model of MS. This approach was superior to treatment with FTY720 (fingolimod), a clinically approved immunomodulating therapy for MS. Notably, pGal-PLP treatment was most effective at reducing lymphocyte infiltrates in spinal cords at the study endpoint. In both the chronic and the R/R EAE models, pGal-antigen therapy significantly reduced pathology, even in the presence of systemic inflammation.

Through these findings and in previous studies with celiac disease and type 1 diabetes, the authors have shown that the addition of carbohydrate moieties to peptides and protein antigens is highly flexible.^6,7 Such glycopolymer conjugates are preferentially taken up in the liver, with decreased uptake by the spleen, thereby decreasing the chances of toxicity in other tissues. The major advantage of the synthetic glycosylation system is that it is easily adaptable to any soluble protein antigen (or combination of antigens), and by modifying the glycosylation, one can redirect antigens to different immune compartments, resulting in a desired outcome. Since this approach utilizes the whole antigen, it allows for treatment of a large cohort of patients irrespective of HLA haplotype. Importantly, there would be fewer unwanted side effects associated with long-term systemic IS. Excitingly, the pGal myelin molecule, labeled ANK-700, is now being tested in phase 1 clinical trials for MS (ClinicalTrials.gov: NCT04602390).

A key challenge in MS and other autoimmune diseases is antigen and epitope spread as the disease progresses, leading to a complex autoreactivity profile. This can manifest as a unique specificity of autoreactive T cell clones in individual patients, thus expanding the potential targets needed for antigen-specific tolerization. Although the authors demonstrated that their glycotargeting approach could lead to dampened autoimmunity in a murine model through the activation of antigen-specific Tregs, there are some limitations to their studies that are important to address here. First, the authors did not show bystander tolerance by the induced Tregs. For example, this could have been demonstrated using a more complex EAE model (induction with multiple epitopes or using spinal cord homogenate). This would be critical to show to extend this approach to indications in which the autoantigens are not well defined. Second, the phenotype in the murine EAE model is typically driven by a Th17 response, whereas in humans, it is dependent on both B and T cells. Finally, the authors did not address if their approach could prevent the induction of autoantibodies, which contribute to disease in patients with MS and other autoimmune indications, or the impact of pre-existing antibodies against the antigen, which could potentially redirect the pGal antigen to APCs by FcR and complement receptors.

Nonetheless, the data show potential that warrants further investigations to confirm longevity of suppression. With proven safety and efficacy in the planned clinical study, this “inverse vaccine” platform may have the potential to become a new therapy for autoimmunity, particularly in disease indications with no or limited available therapies.