Production of site-specific antibody conjugates using metabolic glycoengineering and novel Fc glycovariants.

Molecular conjugation to antibodies has emerged as a growing strategy to combine the mechanistic activities of the attached molecule with the specificity of antibodies. A variety of technologies have been applied for molecular conjugation; however, these approaches face several limitations, including disruption of antibody structure, destabilization of the antibody, and/or heterogeneous conjugation patterns. Collectively, these challenges lead to reduced yield, purity, and function of conjugated antibodies.

Metabolically Glycoengineered Neural Stem Cells Boost Neural Repair After Cardiac Arrest.

Cardiac arrest (CA)-induced cerebral ischemia remains challenging with high mortality and disability. Neural stem cell (NSC) engrafting is an emerging therapeutic strategy with considerable promise that, unfortunately, is severely compromised by limited cell functionality after in vivo transplantation. This groundbreaking report demonstrates that metabolic glycoengineering (MGE) using the "AcManNTProp (TProp)" monosaccharide analog stimulates the Wnt/-catenin pathway, improves cell adhesion, and enhances neuronal differentiation in human NSCs in vitro thereby substantially increasing the therapeutic potential of these cells.

A dual-acting DNASE1/DNASE1L3 biologic prevents autoimmunity and death in genetic and induced lupus models.

A defining feature of systemic lupus erythematosus (SLE) is loss of tolerance to self-DNA, and deficiency of DNASE1L3, the main enzyme responsible for chromatin degradation in blood, is also associated with SLE. This association can be found in an ultrarare population of pediatric patients with DNASE1L3 deficiency who develop SLE, adult patients with loss-of-function variants of DNASE1L3 who are at a higher risk for SLE, and patients with sporadic SLE who have neutralizing autoantibodies against DNASE1L3. To mitigate the pathogenic effects of inherited and acquired DNASE1L3 deficiencies, we engineered a long-acting enzyme biologic with dual DNASE1/DNASE1L3 activity that is resistant to DNASE1 and DNASE1L3 inhibitors.

Protocol Considerations for In Vitro Metabolic Glycoengineering of Non-Natural Glycans.

Metabolic glycoengineering (MGE) refers to a technique where non-natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell-surface-displayed oligosaccharides, where they can modulate a host of biological activities or be exploited as tags for bioorthogonal and chemoselective ligation reactions. Over the past decade, azido-modified monosaccharides have become the go-to analogs for MGE; at the same time, analogs with novel chemical functionalities continue to be developed.

Improving Schwann Cell Differentiation from Human Adipose Stem Cells with Metabolic Glycoengineering.

Schwann cells (SCs) are myelinating cells that promote peripheral nerve regeneration. When nerve lesions form, SCs are destroyed, ultimately hindering nerve repair. The difficulty in treating nerve repair is exacerbated due to SC's limited and slow expansion capacity.

Anticancer Properties of Hexosamine Analogs Designed to Attenuate Metabolic Flux through the Hexosamine Biosynthetic Pathway.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate -acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP.

A versatile design platform for glycoengineering therapeutic antibodies.

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs.

Glycoengineering human neural stem cells (hNSCs) for adhesion improvement using a novel thiol-modified N-acetylmannosamine (ManNAc) analog.

This study sets the stage for the therapeutic use of AcManNTProp, an N-acetylmannosamine (ManNAc) analog that installs thiol-modified sialoglycans onto the surfaces of human neural stem cells (hNSC). First, we compared hNSC adhesion to the extracellular matrix (ECM) proteins laminin, fibronectin, and collagen and found preferential adhesion and concomitant changes to cell morphology and cell spreading for AcManNTProp-treated cells to laminin, compared to fibronectin where there was a modest response, and collagen where there was no observable increase. PCR array transcript analysis identified several classes of cell adhesion molecules that responded to combined AcManNTProp treatment and hNSC adhesion to laminin.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.

Glycoengineering Human Neural and Adipose Stem Cells with Novel Thiol-Modified -Acetylmannosamine (ManNAc) Analogs.

This report describes novel thiol-modified -acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of AcManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that AcManNTGc 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 AcManNTProp and AcManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, -acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols.

Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4--BuManNAc.

Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (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 three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu ManNAc.

Cell Line-, Protein-, and Sialoglycosite-Specific Control of Flux-Based Sialylation in Human Breast Cells: Implications for Cancer Progression.

Sialylation, a post-translational modification that impacts the structure, activity, and longevity of glycoproteins has been thought to be controlled primarily by the expression of sialyltransferases (STs). In this report we explore the complementary impact of metabolic flux on sialylation using a glycoengineering approach. Specifically, we treated three human breast cell lines (MCF10A, T-47D, and MDA-MB-231) with 1,3,4-O-BuManNAc, a "high flux" metabolic precursor for the sialic acid biosynthetic pathway.

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.

Improving Immunotherapy Through Glycodesign.

Immunotherapy is revolutionizing health care, with the majority of high impact "drugs" approved in the past decade falling into this category of therapy. Despite considerable success, glycosylation-a key design parameter that ensures safety, optimizes biological response, and influences the pharmacokinetic properties of an immunotherapeutic-has 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.

Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production.

Sodium butyrate (NaBu) is not only well-known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4-O-Bu ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4-O-Bu ManNAc on EPO glycan quality, while evident in wild-type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β-1,4- and β-1,6-N-acetylglucosaminyltransferases (GnTIV and GnTV) and α-2,6-sialyltransferase (ST6) enzymes responsible for N-glycan antennarity and sialylation.

Integration of genetic and metabolic features related to sialic acid metabolism distinguishes human breast cell subtypes.

In this report we use 'high-flux' tributanoyl-modified N-acetylmannosamine (ManNAc) analogs with natural N-acetyl as well as non-natural azido- and alkyne N-acyl groups (specifically, 1,3,4-O-Bu3ManNAc, 1,3,4-O-Bu3ManNAz, and 1,3,4-O-Bu3ManNAl 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.

Anisotropic biodegradable lipid coated particles for spatially dynamic protein presentation.

Unlabelled: There has been growing interest in the use of particles coated with lipids for applications ranging from drug delivery, gene delivery, and diagnostic imaging to immunoengineering. To date, almost all particles with lipid coatings have been spherical despite emerging evidence that non-spherical shapes can provide important advantages including reduced non-specific elimination and increased target-specific binding. We combine control of core particle geometry with control of particle surface functionality by developing anisotropic, biodegradable ellipsoidal particles with lipid coatings.

Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells.

The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation.

General and Facile Coating of Single Cells via Mild Reduction.

Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups.

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 in silico 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.

Determination of absolute expression profiles using multiplexed miRNA analysis.

Accurate measurement of miRNA expression is critical to understanding their role in gene expression as well as their application as disease biomarkers. Correct identification of changes in miRNA expression rests on reliable normalization to account for biological and technological variance between samples. Ligo-miR is a multiplex assay designed to rapidly measure absolute miRNA copy numbers, thus reducing dependence on biological controls.

A novel sugar analog enhances sialic acid production and biotherapeutic sialylation in CHO cells.

A desirable feature of many therapeutic glycoprotein production processes is to maximize the final sialic acid content. In this study, the effect of applying a novel chemical analog of the sialic acid precursor N-acetylmannosamine (ManNAc) on the sialic acid content of cellular proteins and a model recombinant glycoprotein, erythropoietin (EPO), was investigated in CHO-K1 cells. By introducing the 1,3,4-O-Bu ManNAc analog at 200-300 µM into cell culture media, the intracellular sialic acid content of EPO-expressing cells increased ∼8-fold over untreated controls while the level of cellular sialylated glycoconjugates increased significantly as well.

Glycoengineering of Esterase Activity through Metabolic Flux-Based Modulation of Sialic Acid.

This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity.

Harnessing cancer cell metabolism for theranostic applications using metabolic glycoengineering of sialic acid in breast cancer as a pioneering example.

Abnormal cell surface display of sialic acids - a family of unusual 9-carbon sugars - is widely recognized as distinguishing feature of many types of cancer. Sialoglycans, however, typically cannot be identified with sufficiently high reproducibility and sensitivity to serve as clinically accepted biomarkers and similarly, almost all efforts to exploit cancer-specific differences in sialylation signatures for therapy remain in early stage development. In this report we provide an overview of important facets of glycosylation that contribute to cancer in general with a focus on breast cancer as an example of malignant disease characterized by aberrant sialylation.