21 research outputs found
Improving Immunotherapy Through Glycodesign
Immunotherapy is revolutionizing health care, with the majority of high impact âdrugsâ approved in the past decade falling into this category of therapy. Despite considerable success, glycosylationâa key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeuticâhas slowed the development of this class of drugs in the past and remains challenging at present. This article describes how optimizing glycosylation through a variety of glycoengineering strategies provides enticing opportunities to not only avoid past pitfalls, but also to substantially improve immunotherapies including antibodies and recombinant proteins, and cell-based therapies. We cover design principles important for early stage pre-clinical development and also discuss how various glycoengineering strategies can augment the biomanufacturing process to ensure the overall effectiveness of immunotherapeutics
Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs
This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac5ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac5ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac5ManNTProp and Ac5ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac5ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac5ManNTProp and Ac5ManNTBut suppressed adipogenic differentiation, with Ac5ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac5ManNTProp and Ac5ManNTBut) for manipulating human stem cells
Cell Surface and Membrane Engineering: Emerging Technologies and Applications
Membranes constitute the interface between the basic unit of lifeâa single cellâand the outside environment and thus in many ways comprise the ultimate âfunctional biomaterialâ. To perform the many and often conflicting functions required in this role, for example to partition intracellular contents from the outside environment while maintaining rapid intake of nutrients and efflux of waste products, biological membranes have evolved tremendous complexity and versatility. This article describes how membranes, mainly in the context of living cells, are increasingly being manipulated for practical purposes with drug discovery, biofuels, and biosensors providing specific, illustrative examples. Attention is also given to biology-inspired, but completely synthetic, membrane-based technologies that are being enabled by emerging methods such as bio-3D printers. The diverse set of applications covered in this article are intended to illustrate how these versatile technologiesâas they rapidly matureâhold tremendous promise to benefit human health in numerous ways ranging from the development of new medicines to sensitive and cost-effective environmental monitoring for pathogens and pollutants to replacing hydrocarbon-based fossil fuels
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Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified N-Acetylmannosamine (ManNAc) Analogs
This report describes novel thiol-modified N-acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac(5)ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac(5)ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac(5)ManNTProp and Ac(5)ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N-acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac(5)ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac(5)ManNTProp and Ac(5)ManNTBut suppressed adipogenic differentiation, with Ac(5)ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac(5)ManNTProp and Ac(5)ManNTBut) for manipulating human stem cells
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Exploiting metabolic glycoengineering to advance healthcare
Metabolic glycoengineering (MGE) is a technique for manipulating cellular metabolism to modulate glycosylation. MGE is used to increase the levels of natural glycans and, more importantly, to install non-natural monosaccharides into glycoconjugates. In this Review, we summarize the chemistry underlying MGE that has been developed over the past three decades and highlight several recent advances that have set the stage for clinical translation. In anticipation of near-term application to human healthcare, we describe emerging efforts to deploy MGE in diverse applications, ranging from the glycoengineering of biotherapeutic proteins and the diagnosis and treatment of complex diseases such as cancer to the development of new immunotherapies
Pharmacological, Physiochemical, and Drug-Relevant Biological Properties of Short Chain Fatty Acid Hexosamine Analogues Used in Metabolic Glycoengineering
In this study, we
catalog structure activity relationships (SAR)
of several short chain fatty acid (SCFA)-modified hexosamine analogues
used in metabolic glycoengineering (MGE) by comparing <i>in silico</i> and experimental measurements of physiochemical properties important
in drug design. We then describe the impact of these compounds on
selected biological parameters that influence the pharmacological
properties and safety of drug candidates by monitoring P-glycoprotein
(Pgp) efflux, inhibition of cytochrome P450 3A4 (CYP3A4), hERG channel
inhibition, and cardiomyocyte cytotoxicity. These parameters are influenced
by length of the SCFAs (e.g., acetate vs n-butyrate), which are added
to MGE analogues to increase the efficiency of cellular uptake, the
regioisomeric arrangement of the SCFAs on the core sugar, the structure
of the core sugar itself, and by the type of <i>N</i>-acyl
modification (e.g., <i>N</i>-acetyl vs <i>N</i>-azido). By cataloging the influence of these SAR on pharmacological
properties of MGE analogues, this study outlines design considerations
for tuning the pharmacological, physiochemical, and the toxicological
parameters of this emerging class of small molecule drug candidates
Improving the Pharmacodynamics and In Vivo Activity of ENPP1âFc Through Protein and Glycosylation Engineering
Enzyme replacement with ectonucleotide pyrophosphatase phospodiesteraseâ1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a threeâprong strategy. First, we added new Nâglycans to ENPP1; second, we optimized pHâdependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a twoâstep process to improve sialylation by first producing ENPP1âFc in cells stably transfected with human αâ2,6âsialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4â
O
âBu
3
ManNAc. These steps sequentially increased the halfâlife of the parent compound in rodents from 37Â hours to ~Â 67Â hours with an added Nâglycan, to ~Â 96Â hours with optimized pHâdependent Fc recycling, to ~Â 204Â hours when the therapeutic was produced in ST6âoverexpressing cells with 1,3,4â
O
âBu
3
ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1âdeficient mice when the optimized biologic was administered at a 10âfold lower mass dose less frequently than the parent compoundâonce every 10Â days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics
Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes
<div><p>In this report we use âhigh-fluxâ tributanoyl-modified <i>N</i>-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu<sub>3</sub>ManNAc, 1,3,4-O-Bu<sub>3</sub>ManNAz, and 1,3,4-O-Bu<sub>3</sub>ManNAl respectively) to probe intracellular sialic acid metabolism in the near-normal MCF10A human breast cell line in comparison with earlier stage T-47D and more advanced stage MDA-MB-231 breast cancer lines. An integrated view of sialic acid metabolism was gained by measuring intracellular sialic acid production in tandem with transcriptional profiling of genes linked to sialic acid metabolism. The transcriptional profiling showed several differences between the three lines in the absence of ManNAc analog supplementation that helps explain the different sialoglycan profiles naturally associated with cancer. Only minor changes in mRNA transcript levels occurred upon exposure to the compounds confirming that metabolic flux alone can be a key determinant of sialoglycoconjugate display in breast cancer cells; this result complements the well-established role of genetic control (e.g., the transcription of STs) of sialylation abnormalities ubiquitously associated with cancer. A notable result was that the different cell lines produced significantly different levels of sialic acid upon exogenous ManNAc supplementation, indicating that feedback inhibition of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE)âgenerally regarded as the âgatekeeperâ enzyme for titering flux into sialic acid biosynthesisâis not the only regulatory mechanism that limits production of this sugar. A notable aspect of our metabolic glycoengineering approach is its ability to discriminate cell subtype based on intracellular metabolism by illuminating otherwise hidden cell type-specific features. We believe that this strategy combined with multi-dimensional analysis of sialic acid metabolism will ultimately provide novel insights into breast cancer subtypes and provide a foundation for new methods of diagnosis.</p></div
Metabolic profiles of Compartment 1 in analog-supplemented cells.
<p>(<b>A</b>) 3D surface plots were generated for each cell line in response to treatments with analogs; sialic acid production is shown as a function of both dose and time (AZ stands for azimuth values, El stands for elevation values, and the three plots provided for each data set are rotated to depict different vantage points). (<b>B</b>) The Reserve Capacity for each cell line was calculated based on the highest observed increase in Compartment 1 sialic acid levels in analog-supplemented cells compared to untreated controls.</p
âCompartment Ratiosâ of ManNAc analog-treated cells.
<p>The ratio of sialic acid in Compartment 1 (i.e., unconjugated sialometabolites) compared to Compartment 2 (i.e., predominantly glycoconjugate-bound sialic acids) was calculated for cells after 24 h of treatment with each analog at the indicated concentrations.</p