RESUMO
Pancreatic islet-reactive B lymphocytes promote Type 1 diabetes (T1D) by presenting an antigen to islet-destructive T cells. Teplizumab, an anti-CD3 monoclonal, delays T1D onset in patients at risk, but additional therapies are needed to prevent the disease entirely. Therefore, bifunctional molecules were designed to selectively inhibit T1D-promoting anti-insulin B cells by conjugating a ligand for the B cell inhibitory receptor CD22 (i.e., CD22L) to insulin, which permit these molecules to concomitantly bind to anti-insulin B cell receptors (BCRs) and CD22. Two prototypes were synthesized: 2:2 insulin-CD22L conjugate on a 4-arm PEG backbone, and 1:1 insulin-CD22L direct conjugate. Transgenic mice (125TgSD) expressing anti-insulin BCRs provided cells for in vitro testing. Cells were cultured with constructs for 3 days, then assessed by flow cytometry. Duplicate wells with anti-CD40 simulated T cell help. A 2-insulin 4-arm PEG control caused robust proliferation and activation-induced CD86 upregulation. Anti-CD40 further boosted these effects. This may indicate that BCR-cross-linking occurs when antigens are tethered by the PEG backbone as soluble insulin alone has no effect. Addition of CD22L via the 2:2 insulin-CD22L conjugate restored B cell properties to that of controls without an additional beneficial effect. In contrast, the 1:1 insulin-CD22L direct conjugate significantly reduced anti-insulin B cell proliferation in the presence of anti-CD40. CD22L alone had no effect, and the constructs did not affect the WT B cells. Thus, multivalent antigen constructs tend to activate anti-insulin B cells, while monomeric antigen-CD22L conjugates reduce B cell activation in response to simulated T cell help and reduce pathogenic B cell numbers without harming normal cells. Therefore, monomeric antigen-CD22L conjugates warrant futher study and may be promising candidates for preclinical trials to prevent T1D without inducing immunodeficiency.
Assuntos
Diabetes Mellitus Tipo 1 , Insulina , Camundongos , Animais , Humanos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Linfócitos B , Ativação Linfocitária , Linfócitos T , Camundongos Transgênicos , AntígenosRESUMO
Autoimmune diseases are characterized by aberrant immune responses toward self-antigens. Current treatments lack specificity, promoting adverse effects by broadly suppressing the immune system. Therapies that specifically target the immune cells responsible for disease are a compelling strategy to mitigate adverse effects. Multivalent formats that display numerous binding epitopes off a single scaffold may enable selective immunomodulation by eliciting signals through pathways unique to the targeted immune cells. However, the architecture of multivalent immunotherapies can vary widely, and there is limited clinical data with which to evaluate their efficacy. Here, we set forth to review the architectural properties and functional mechanisms afforded by multivalent ligands and evaluate four multivalent scaffolds that address autoimmunity by altering B cell signaling pathways. First, we address both synthetic and natural polymer backbones functionalized with a variety of small molecule, peptide, and protein ligands for probing the effects of valency and costimulation. Then, we review nanoparticles composed entirely from immune signals which have been shown to be efficacious. Lastly, we outline multivalent liposomal nanoparticles capable of displaying high numbers of protein antigens. Taken together, these examples highlight the versatility and desirability of multivalent ligands for immunomodulation and illuminate strengths and weaknesses of multivalent scaffolds for treating autoimmunity.
Assuntos
Doenças Autoimunes , Linfócitos B , Humanos , Ligantes , Tolerância Imunológica , Autoantígenos , ImunoterapiaRESUMO
Cancer-associated alterations to glycosylation have been shown to aid cancer development and progression. An increased abundance of high mannose N-glycans has been observed in several cancers. Here, we describe the preparation of lectin drug conjugates (LDCs) that permit toxin delivery to cancer cells presenting high mannose N-glycans. Additionally, we demonstrate that cancer cells presenting low levels of high mannose N-glycans can be rendered sensitive to the LDCs by co-treatment with a type I mannosidase inhibitor. Our findings establish that an increased abundance of high mannose N-glycans in the glycocalyx of cancer cells can be leveraged to enable toxin delivery.
Assuntos
Lectinas , Manose , Manosidases , Preparações Farmacêuticas , PolissacarídeosRESUMO
ß-glucans are polymers of glucose that have been isolated from a variety of organisms. Isolated ß-glucans have been used for medical purposes for centuries; however, efforts to define the biological activities of ß-glucans experimentally were initiated in the 1940's. The diversity of structure associated with isolated ß-glucans has impeded said investigations, and efforts to leverage the biological activity of ß-glucans for clinical applications. In recognition of the need for defined ß-glucans that retain the biological activity of isolated ß-glucans, considerable investment has been made to facilitate the synthesis of structurally defined ß-glucans. Here, we review the different approaches that have been applied to prepare ß-glucans. In addition, we summarize the approaches that have been utilized to conjugate ß-glucans to proteins.
Assuntos
beta-Glucanas , PolímerosRESUMO
An engineered cyanovirin-N homologue that exhibits specificity for high mannose N-glycans has been constructed to aid typeâ I α 1,2-mannosidase inhibitor discovery and development. Engineering the lectins C-terminus permitted facile functionalization with fluorophores via a sortase and click strategy. The resulting lectin constructs exhibit specificity for cells presenting high mannose N-glycans. Importantly, these lectin constructs can also be applied to specifically assess changes in cell surface glycosylation induced by typeâ I mannosidase inhibitors. Testing the utility of these lectin constructs led to the discovery of typeâ I mannosidase inhibitors with nanomolar potency. Cumulatively, these findings reveal the specificity and utility of the functionalized cyanovirin-N homologue constructs, and highlight their potential in analytical contexts that require high mannose-specific lectins.
Assuntos
Lectinas/química , Manosidases/antagonistas & inibidores , Alcaloides/química , Alcaloides/metabolismo , Motivos de Aminoácidos , Aminoaciltransferases/química , Proteínas de Bactérias/química , Linhagem Celular , Cisteína Endopeptidases/química , Desenho de Fármacos , Corantes Fluorescentes/química , Glicosilação , Humanos , Lectinas/metabolismo , Manose/química , Manose/metabolismo , Manosidases/metabolismo , Microscopia de FluorescênciaRESUMO
The abundance of sialic acid-containing glycans in the glycocalyx of malignant cells enables immune evasion. Here, we leverage the biosynthetic pathways that permit pervasive sialylation to incorporate a chimeric antigen receptor (CAR) ligand into malignant cell glycans, and demonstrate that this increases the susceptibility of malignant cells to the cytolytic activity of CAR-expressing natural killer (NK) cells. Specifically, we applied a C-9-functionalized nonnatural sialic acid [i.e., fluorescein sialic acid (FL-SA)] to modify malignant cell glycans. We confirm the metabolic incorporation of FL-SA into plasma membrane-associated glycans. The preparation of anti-fluorescein CAR NK cells permitted studies demonstrating that treating malignant cells with FL-SA increased susceptibility to CAR NK cell-mediated cytolysis. Furthermore, we observed that the specificity of the anti-fluorescein CAR NK cells is enhanced for fluorescein-labeled cells, and an increased release of cytokines from the CAR NK cells upon incubation with FL-SA-treated cells. The results arising from this study demonstrate that CAR ligands can be metabolically incorporated into malignant cells, and we reason that such strategies could be leveraged to tackle the issue of antigen heterogeneity that limits the clinical efficacy of CAR T/NK cell therapies.
Assuntos
Receptores de Antígenos Quiméricos , Linhagem Celular Tumoral , Fluoresceínas/metabolismo , Células Matadoras Naturais , Ligantes , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismoRESUMO
Kifunensine is a known inhibitor of type I α-mannosidase enzymes and has been shown to have therapeutic potential for a variety of diseases and application in the expression of high-mannose N-glycan bearing glycoproteins; however, the compound's hydrophilic nature limits its efficacy. We previously synthesized two hydrophobic acylated derivatives of kifunensine, namely, JDW-II-004 and JDW-II-010, and found that these compounds were over 75-fold more potent than kifunensine. Here we explored the effects of these compounds on different mice and human B cells, and we demonstrate that they affected the cells in a similar fashion to kifunensine, further demonstrating their functional equivalence to kifunensine in assays utilizing primary cells. Specifically, a dose-dependent increase in the formation of high-mannose N-glycans decorated glycoproteins were observed upon treatment with kifunensine, JDW-II-004, and JDW-II-010, but greater potency was observed with the acylated derivatives. Treatment with kifunensine or the acylated derivatives also resulted in impaired B-cell receptor (BCR) signaling of the primary mouse B cells; however, primary human B cells treated with kifunensine or JDW-II-004 did not affect BCR signaling, while a modest increase in BCR signaling was observed upon treatment with JDW-010. Nevertheless, these findings demonstrate that the hydrophobic acylated derivatives of kifunensine can help overcome the mass-transfer limitations of the parent compound, and they may have applications for the treatment of ERAD-related diseases or prove to be more cost-effective alternatives for the generation and production of high-mannose N-glycan bearing glycoproteins.
RESUMO
Microtiter plate colorimetric assays are widely used for analysis of carbohydrates and glycoconjugates. However, mucins are often not easily detected, as they have low neutral sugar content. We have adapted and optimised the periodic acid-Schiff's reagent (PAS) staining for microtiter plate assay by examining five factors: concentration and volume of periodic acid, oxidation time, volume of Schiff's reagent, and color development time. This assay requires just 25 µl of sample, utilises standardised Schiff's reagent, and has decreased assay time (140 min to completion). Seventeen monosaccharides (acidic, neutral, basic, phosphorylated, and deoxy) and four disaccharides were assessed. PAS-positive carbohydrates (amino, N-acetylamino, deoxy, and certain neutral monosaccharides, and sialic acids) responded linearly within a 10-100 nmol range approximately, which varied for each carbohydrate. The assay response for fetuin and porcine gastric mucin (PGM) was linear up to 150 µg (highest concentration tested), with no response from nonglycosylated protein. A lower response for asialofetuin was observed, but desialylated PGM preparations were similar or higher in response than their sialylated counterparts. The simplicity and low sample consumption of this method make it an excellent choice for screening or quantitation of chromatographic fractions containing carbohydrates and glycoconjugates, especially in the case of mucins.
Assuntos
Carboidratos/análise , Colorimetria/instrumentação , Colorimetria/métodos , Ácido Periódico/química , Configuração de Carboidratos , Carboidratos/química , Concentração de Íons de Hidrogênio , Oxirredução , Corantes de Rosanilina/química , EstereoisomerismoRESUMO
Endo-ß-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man5GlcNAc and Man7GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.