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.

Safety and tolerability of KAN-101, a liver-targeted immune tolerance therapy, in patients with coeliac disease (ACeD): a phase 1 trial.

BACKGROUND: Coeliac disease management is limited to strict adherence to a gluten-free diet with no approved therapies. This first-in-human phase 1 study evaluated the safety and tolerability of KAN-101, a liver-targeting glycosylation signature conjugated to a deaminated gliadin peptide designed to induce immune tolerance to gliadin. METHODS: Adults (aged 18-70 years) with biopsy-confirmed, HLA-DQ2.5 genotype coeliac disease were enrolled from clinical research units and hospitals in the USA. Part A of the trial was an open-label, single ascending dose study of intravenous KAN-101 using sentinel dosing in evaluation of the following cohorts: 0·15 mg/kg, 0·3 mg/kg, 0·6 mg/kg, 1·2 mg/kg, and 1·5 mg/kg. Following safety monitoring committee review of the 0·3 mg/kg dose level in part A, part B was initiated as a randomised, placebo-controlled, multiple ascending dose study. In part B, interactive response technology was used to randomly assign (5:1) patients to receive intravenous KAN-101 (0·15 mg/kg, 0·3 mg/kg, or 0·6 mg/kg) or placebo following a 1:1 assignment of the first two eligible patients in each cohort for sentinel dosing. Patients in part B received three administrations of KAN-101 or placebo followed by a 3-day oral gluten challenge (9 g per day) 1 week after completing dosing. Study personnel and patients were masked to treatment assignments in part B, and not in part A. The primary endpoint was the incidence and severity of adverse events with escalating doses of KAN-101, assessed in all patients who received any amount of study drug based on dose level received. The secondary endpoint was assessment of plasma concentrations and pharmacokinetic parameters of KAN-101 following single and multiple doses, assessed in all patients who received at least one dose and had one or more values for drug concentration. This study is registered with ClinicalTrials.gov, NCT04248855, and is completed. FINDINGS: Between Feb 7, 2020, and Oct 8, 2021, 41 patients were enrolled at ten US sites. 14 patients were assigned to part A (four 0·15 mg/kg, three 0·3 mg/kg, three 0·6 mg/kg, three 1·2 mg/kg, one 1·5 mg/kg) and 27 patients to part B (six 0·15 mg/kg with two placebo, seven 0·3 mg/kg with two placebo, and eight 0·6 mg/kg with two placebo). Treatment-related adverse events were reported in 11 (79%) of 14 patients in part A and 18 (67%) of 27 in part B (placebo two [33%] of six patients; KAN-101 16 [76%] of 21 patients), were grade 2 or lower, and were mild to moderate in severity. The most commonly observed adverse events were nausea, diarrhoea, abdominal pain, and vomiting, consistent with symptoms had by patients with coeliac disease on gluten ingestion. No grade 3-4 adverse events, serious adverse events, dose-limiting toxicities, or deaths occurred. Pharmacokinetic analyses showed KAN-101 was cleared from systemic circulation within roughly 6 h with a geometric mean half-life of 3·72 min (CV% 6·5%) to 31·72 min (83·7%), and no accumulation with repeated dosing. INTERPRETATION: KAN-101 has an acceptable safety profile in patients with coeliac disease with no dose-limiting toxicities and no maximum tolerated dose was observed. Rapid systemic clearance of KAN-101 was observed and no accumulation on repeated dosing. A future study will evaluate the safety and efficacy, including biomarker responses with a gluten challenge, of KAN-101 at doses 0·6 mg/kg and greater in patients with coeliac disease. FUNDING: Kanyos Bio.

Persistent antigen exposure via the eryptotic pathway drives terminal T cell dysfunction.

Although most current treatments for autoimmunity involve broad immunosuppression, recent efforts have aimed to suppress T cells in an antigen-specific manner to minimize risk of infection. One such effort is through targeting antigen to the apoptotic pathway to increase presentation of the antigen of interest in a tolerogenic context. Erythrocytes present a rational candidate to target because of their high rate of eryptosis, which facilitates continual uptake by antigen-presenting cells in the spleen. Here, we develop an approach that binds antigens to erythrocytes to induce sustained T cell dysfunction. Transcriptomic and phenotypic analyses revealed signatures of self-tolerance and exhaustion, including up-regulation of PD-1, CTLA4, Lag3, and TOX. Antigen-specific T cells were incapable of responding to an adjuvanted antigenic challenge even months after antigen clearance. With this strategy, we prevented pathology in a mouse experimental autoimmune encephalomyelitis model. CD8+ T cell education occurred in the spleen and was dependent on cross-presenting Batf3+ dendritic cells. These results demonstrate that antigens associated with eryptotic erythrocytes induce lasting T cell dysfunction that could be protective in deactivating pathogenic T cells.

Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase.

Antigen-specific immune responses to protein drugs can hinder efficacy and compromise safety because of drug neutralization and secondary clinical complications. We report a tolerance induction strategy to prevent antigen-specific humoral immune responses to therapeutic proteins. Our modular, biomolecular approach involves engineering tolerizing variants of proteins such that they bind erythrocytes in vivo upon injection, on the basis of the premise that aged erythrocytes and the payloads they carry are cleared tolerogenically, driving the deletion of antigen-specific T cells. We demonstrate that binding the clinical therapeutic enzyme Escherichia coli l-asparaginase to erythrocytes in situ antigen-specifically abrogates development of antibody titers by >1000-fold and extends the pharmacodynamic effect of the drug 10-fold in mice. Additionally, a single pretreatment dose of erythrocyte-binding asparaginase tolerized mice to multiple subsequent doses of the wild-type enzyme. This strategy for reducing antigen-specific humoral responses may enable more effective and safer treatment with therapeutic proteins and drug candidates that are hampered by immunogenicity.

Memory of tolerance and induction of regulatory T cells by erythrocyte-targeted antigens.

New approaches based on induction of antigen-specific immunological tolerance are being explored for treatment of autoimmunity and prevention of immunity to protein drugs. Antigens associated with apoptotic debris are known to be processed tolerogenically in vivo. Our group is exploring an approach toward antigen-specific tolerization using erythrocyte-binding antigens, based on the premise that as the erythrocytes circulate, age and are cleared, the erythrocyte surface-bound antigen payload will be cleared tolerogenically along with the eryptotic debris. Here, we characterized the phenotypic signatures of CD8+ T cells undergoing tolerance in response to soluble and erythrocyte-targeted antigen. Signaling through programmed death-1/programmed death ligand-1 (PD-1/PD-L1), but not through cytotoxic T lymphocyte antigen 4 (CTLA4), was shown to be required for antigen-specific T cell deletion, anergy and expression of regulatory markers. Generation of CD25+FOXP3+ regulatory T cells in response to erythrocyte-targeted antigens but not soluble antigen at an equimolar dose was observed, and these cells were required for long-term maintenance of immune tolerance in both the CD4+ and CD8+ T cell compartments. Evidence of infectious tolerance was observed, in that tolerance to a one antigenic epitope was able to regulate responses to other epitopes in the same protein antigen.

Engineering antigen-specific immunological tolerance.

Unwanted immunity develops in response to many protein drugs, in autoimmunity, in allergy, and in transplantation. Approaches to induce immunological tolerance aim to either prevent these responses or reverse them after they have already taken place. We present here recent developments in approaches, based on engineered peptides, proteins and biomaterials, that harness mechanisms of peripheral tolerance both prophylactically and therapeutically to induce antigen-specific immunological tolerance. These mechanisms are based on responses of B and T lymphocytes to other cells in their immune environment that result in cellular deletion or ignorance to particular antigens, or in development of active immune regulatory responses. Several of these approaches are moving toward clinical development, and some are already in early stages of clinical testing.

Bicyclic peptides conjugated to an albumin-binding tag diffuse efficiently into solid tumors.

Monoclonal antibodies have long in vivo half-lives and reach high concentrations in tumors but cannot access all regions in the tissue, whereas smaller ligands such as peptides distribute better but are limited by low concentrations due to fast renal clearance. A potential solution to this problem might be offered by peptide-based ligands that are conjugated to an albumin-binding tag, and thus have a long plasma half-life. Herein, we tested if a small ligand based on a bicyclic peptide (1.9 kDa) conjugated to an albumin-binding peptide (2.3 kDa) can diffuse into tissues. Although the peptide conjugate (4.6 kDa) was most of the time bound to the large protein serum albumin (66.5 kDa), it diffused deeply into tissues and reached high nanomolar concentrations in wide areas of solid tumors. Most of the peptide conjugate isolated from tumor tissue was found to be fully intact 24 hours after administration. Because of its noncovalent interaction with albumin, the bicyclic peptide might dissociate to diffuse to tumor regions that are not accessible to larger ligands. Bicyclic peptides having high binding affinity for targets of interest and being proteolytically stable can be evolved by phage display; in conjunction with albumin-binding tags, they offer a promising format to access targets in solid tumors.

Peripherally administered nanoparticles target monocytic myeloid cells, secondary lymphoid organs and tumors in mice.

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ∼50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet ß cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Long-term maintenance of mouse embryonic stem cell pluripotency by manipulating integrin signaling within 3D scaffolds without active Stat3.

We engineered an acellular biomimetic microenvironment to regulate stem cell fate and applied it to maintain mouse embryonic stem (ES) cell self-renewal. In the 3D environment formed using hydrogel scaffolds in which specific integrin ligation was provided, Stat3 activation by exogenous leukemia inhibitory factor (LIF) no longer acted as a limiting factor for stem cell self-renewal. Instead, simultaneous stimulation of integrins α(5)ß(1), α(v)ß(5), α(6)ß(1) and α(9)ß(1) within the 3D scaffold greatly increased Akt1 and Smad 1/5/8 activation, which resulted in prolonged self-renewal of the ES cells. The ES cells exposed to the combined stimulation of the integrins for 4 wk in LIF-free 3D scaffolds maintained the spherical morphology of cell colonies without losing any activity of pluripotency. In conclusion, cell niche-specific integrin signaling within the 3D environment supported mouse ES cell self-renewal, and the resulting integrin signaling replaced Stat3 with Akt1 and Smad 1/5/8 as critical signals for mouse ES cell self-renewal.

Drug development: longer-lived proteins.

Protein therapeutics represent a powerful class of clinically approved drugs for the prevention and treatment of various diseases. Once administered, the biological fate of protein therapeutics is governed by the body's various complex biochemical and biophysical clearance mechanisms, several of which may decrease the drug's circulation time and efficiency. In this tutorial review, we introduce the concepts of physiological protein clearance from the body, and describe several chemical modification and protein engineering approaches used to improve the life span of administered protein therapeutics.

Engineered aprotinin for improved stability of fibrin biomaterials.

Fibrin has been long used clinically for hemostasis and sealing, yet extension of use in other applications has been limited due to its relatively rapid resorption in vivo, even with addition of aprotinin or other protease inhibitors. We report an engineered aprotinin variant that can be immobilized within fibrin and thus provide extended longevity. When recombinantly fused to a transglutaminase substrate domain from α(2)-plasmin inhibitor (α(2)PI(1-8)), the resulting variant, aprotinin-α(2)PI(1-8), was covalently crosslinked into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Challenge with physiological plasmin concentrations revealed that aprotinin-α(2)PI(1-8)-containing matrices retained 78% of their mass after 3 wk, whereas matrices containing wild type (WT) aprotinin degraded completely within 1 wk. Plasmin challenge of commercial sealants Omrixil and Tisseel, supplemented with aprotinin-α(2)PI(1-8) or WT aprotinin, showed extended longevity as well. When seeded with human dermal fibroblasts, aprotinin-α(2)PI(1-8)-supplemented matrices supported cell growth for at least 33% longer than those containing WT aprotinin. Subcutaneously implanted matrices containing aprotinin-α(2)PI(1-8) were detectable in mice for more than twice as long as those containing WT aprotinin. We conclude that our engineered recombinant aprotinin variant can confer extended longevity to fibrin matrices more effectively than WT aprotinin in vitro and in vivo.

Improving protein pharmacokinetics by engineering erythrocyte affinity.

Poor pharmacokinetic profiles are often the underlying reason for the failure of novel protein drugs to reach clinical translation. Current passive half-life improvement methods focus on increasing the apparent hydrodynamic radius of the drug. We sought to develop an active method to increase the circulation half-life of proteins by binding to erythrocytes in blood. Screening a naive phage-displayed peptide library against whole mouse erythrocytes yielded a 12 amino acid peptide (ERY1) that binds the erythrocyte surface with high specificity. ERY1-displaying phage bind mouse and rat erythrocytes 95-fold higher than wild-type phage and exhibit negligible binding to mouse leukocytes, as determined by flow cytometry. Affinity experiments with soluble peptide revealed the extracellular domain of glycophorin-A as the membrane protein ligand. When expressed as an N-terminal fusion to maltose-binding protein and administered intravenously, the erythrocyte-binding variant exhibits a 3.2- to 6.3-fold increase in circulation half-life, 2.15-fold decrease in clearance, and 1.67-fold increase in bioavailability as compared to the wild-type protein. The peptide fails to induce ERY1-reactive immunoglobulin production, furthering the potential of the concept in therapeutic design, although this sequence does not bind human erythrocytes. We conclude that engineering erythrocyte affinity into proteins effectively increases their circulation half-life, thereby offering a solution to improve pharmacokinetic profiles of the numerous therapeutic protein drugs in clinical development.

Engineering integrin signaling for promoting embryonic stem cell self-renewal in a precisely defined niche.

We present development and use of a 3D synthetic extracellular matrix (ECM) analog with integrin-specific adhesion ligands to characterize the microenvironmental influences in embryonic stem cell (ESC) self-renewal. Transcriptional analysis of 24 integrin subunits followed by confirmation at the translational and functional levels suggested that integrins alpha(5)beta(1), alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1) play important roles in maintenance of stemness in undifferentiated mouse ESCs. Using the well-defined matrix as a tool to activate integrins alpha(5)beta(1) plus alpha(v)beta(5), alpha(6)beta(1) and alpha(9)beta(1), individually and in combination, differential integrin activation was demonstrated to exert exquisite control over ESC fate decisions. Simultaneous ligation of these four integrin heterodimers promoted self-renewal, as evidence by prolonged SSEA-1, Oct4 and Nanog expression, and induced Akt1 kinase signaling along with translational regulation of other stemness-related genes. The biofunctional network we have designed based on this knowledge may be useful as a defined niche for regulating ESC pluripotency through selective cell-matrix interactions, and the method we present may be more generally useful for probing matrix interactions in stem cell self-renewal and differentiation.