A Robust α-l-Fucosidase from Prevotella nigrescens for Glycoengineering Therapeutic Antibodies.

Eliminating the core fucose from the N-glycans of the Fc antibody segment by pathway engineering or enzymatic methods has been shown to enhance the potency of therapeutic antibodies, especially in the context of antibody-dependent cytotoxicity (ADCC). However, there is a significant challenge due to the limited defucosylation efficiency of commercially available α-l-fucosidases. In this study, we report a unique α-l-fucosidase (PnfucA) from the bacterium Prevotella nigrescens that has a low sequence identity compared with all other known α-l-fucosidases and is highly reactive toward a core disaccharide substrate with fucose α(1,3)-, α (1,4)-and α(1,6)-linked to GlcNAc, and is less reactive toward the Fuc-α(1,2)-Gal on the terminal trisaccharide of the oligosaccharide Globo H (Bb3). The kinetic properties of the enzyme, such as its Km and kcat, were determined and the optimized expression of PnfucA gave a yield exceeding 30 mg/L. The recombinant enzyme retained its full activity even after being incubated for 6 h at 37 °C. Moreover, it retained 92 and 87% of its activity after freezing and freeze-drying treatments, respectively, for over 28 days. In a representative glycoengineering of adalimumab (Humira), PnfucA showed remarkable hydrolytic efficiency in cleaving the α(1,6)-linked core fucose from FucGlcNAc on the antibody with a quantitative yield. This enabled the seamless incorporation of biantennary sialylglycans by Endo-S2 D184 M in a one-pot fashion to yield adalimumab in a homogeneous afucosylated glycoform with an improved binding affinity toward Fcγ receptor IIIa.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

Increased expression of SSEA-4 on TKI-resistant non-small cell lung cancer with EGFR-T790M mutation.

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme ß3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Single Site N-Glycosylation of B Cell Maturation Antigen (BCMA) Inhibits γ-Secretase-Mediated Shedding and Improves Surface Retention and Cell Survival.

B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor (TNFR) family, on the cell surface plays a key role in maintaining the survival of plasma cells and malignant as well as inflammatory accessory cells. Therefore, targeting BCMA or disrupting its interaction with ligands has been a potential approach to cancer therapy. BCMA contains a single N-glycosylation site, but the function of N-glycan on BCMA is not understood. Here, we found that the N-glycosylation of BCMA promoted its cell-surface retention while removing the N-glycan increased BCMA secretion through γ-secretase-mediated shedding. Addition of γ-secretase inhibitor prevented nonglycosylated BCMA from shedding and protected cells from dexamethasone and TRAIL-induced apoptosis.

Low-sugar universal mRNA vaccine against coronavirus variants with deletion of glycosites in the S2 or stem of SARS-CoV-2 spike messenger RNA (mRNA).

Since the outbreak of Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) in 2019, more than 15 million spike protein sequences have been identified, raising a new challenge for the development of a broadly protective vaccine against the various emerging variants. We found that the virus, like most other human viruses, depends on host-made glycans to shield the conserved epitopes on spike protein from immune response and demonstrated that deletion of the glycan shields exposed highly conserved epitopes and elicited broadly protective immune responses. In this study, we identified 17 conserved epitopes from 14 million spike protein sequences and 11 of the conserved epitopes are in the S2 domain, including the six most conserved epitopes in the stem region. We also demonstrated that deletion of the glycosites in the spike messenger RNA (mRNA) S2 domain or the stem region exposed the highly conserved epitopes and elicited broadly protective immune responses, particularly CD-8+ T cell response against various SARS-CoV-2 variants, and other human coronaviruses including MERS, SARS viruses, and those causing common cold.

Quantitative mass spectrometric analysis of hepatocellular carcinoma biomarker alpha-fetoprotein.

Serum alpha-fetoprotein (AFP) has been used as a marker for the diagnosis of hepatocellular carcinoma (HCC) and its core fucosylation is associated with the early stage of HCC. However, current methods for the detection of AFP with core fucose are not highly accurate for early diagnosis. In this study, we established an enzyme-assisted mass spectrometric method for the quantitative analysis of AFP/core fucose with high specificity and sensitivity. We employed endoglycosidase treatment of AFP to improve the biomarker analysis. The accuracy and precision are within the US FDA-suggested value, and a good linearity (r2 = 0.9930) and a detection limit of 15.6 ng mL-1 can be achieved.

Isolation of Anti-SARS-CoV-2 Natural Products Extracted from Mentha canadensis and the Semi-synthesis of Antiviral Derivatives.

Traditional herbal medicine offers opportunities to discover novel therapeutics against SARS-CoV-2 mutation. The dried aerial part of mint (Mentha canadensis L.) was chosen for bioactivity-guided extraction. Seven constituents were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Syringic acid and methyl rosmarinate were evaluated in drug combination treatment. Ten amide derivatives of methyl rosmarinate were synthesized, and the dodecyl (13) and 3-ethylphenyl (19) derivatives demonstrated significant improvement in the anti-SARS-CoV-2 plaque reduction assay, achieving IC50 of 0.77 and 2.70 µM, respectively, against Omicron BA.1 as compared to methyl rosmarinate's IC50 of 57.0 µM. Spike protein binding and 3CLpro inhibition assays were performed to explore the viral inhibition mechanism. Molecular docking of compounds 13 and 19 to 3CLpro was performed to reveal potential interaction. In summary, natural products with anti-Omicron BA.1 activity were isolated from Mentha canadensis and derivatives of methyl rosmarinate were synthesized, showing 21- to 74-fold improvement in antiviral activity against Omicron BA.1.

Effective Organotin-Mediated Regioselective Functionalization of Unprotected Carbohydrates.

Regioselective functionalization of unprotected carbohydrates at a secondary OH group in the presence of primary OH groups based on the commonly used organotin-mediated reaction has been improved. We found that the preactivation of the dibutylstannylene acetal intermediate with tetrabutylammonium bromide in toluene is a key to the improved condition for the efficient, high-yielding, and regioselective tosylation, benzoylation, or benzylation of unprotected carbohydrates. The counteranion of tetrabutylammonium ion with a weak coordination ability plays a crucial role in the improved regioselective reactions. A convenient access to the intermediates of synthetic value is also demonstrated in the organotin-mediated regioselective tosylation of unprotected carbohydrates, followed by the nucleophilic inversion reaction to give sulfur-containing and azide-modified carbohydrates.

Chemical Synthesis of a Keto Sugar Nucleotide.

Keto sugar nucleotides (KSNs) are common and versatile precursors to various deoxy sugar nucleotides, which are substrates for the corresponding glycosyltransferases involved in the biosynthesis of glycoproteins, glycolipids, and natural products. However, there has been no KSN synthesized chemically due to the inherent instability. Herein, the first chemical synthesis of the archetypal KSN TDP-4-keto-6-deoxy-d-glucose (1) is achieved by an efficient and optimized route, providing feasible access to other KSNs and analogues, thereby opening a new avenue for new applications.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Study on antibody Fc-glycosylation for optimal effector functions.

A comprehensive structure-activity relationship study on antibody Fc-glycosylation has been performed using the chimeric anti-SSEA4 antibody chMC813-70 as a model. The α-2,6 sialylated biantennary complex type glycan was identified as the optimal Fc-glycan with significant enhancement in antibody effector functions, including binding to different Fc receptors and ADCC.

Cell-based production of Fc-GlcNAc and Fc-alpha-2,6 sialyl glycan enriched antibody with improved effector functions through glycosylation pathway engineering.

Glycosylation of antibody plays an important role in Fc-mediated killing of tumor cells and virus-infected cells through effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP) and vaccinal effect. Previous studies showed that therapeutical humanized antibodies with α2-6 sialyl complex type (SCT) glycan attached to Fc-Asn297 exhibited optimal binding to the Fc receptors on effector cells associated with ADCC, ADCP and vaccinal effect. However, the production of antibodies with homogeneous Fc-SCT needs multiple in vitro enzymatic and purification steps. In this study, we report two different approaches to shorten the processes to produce SCT-enriched antibodies. First, we expressed a bacterial endoglycosidase in GNT1-KO EXPI293 cells to trim all N -glycans to mono-GlcNAc glycoforms for in vitro transglycosylation to generate homogeneous SCT antibody. Second, we engineered the glycosylation pathway of HEK293 cells through knockout of the undesired glycosyltransferases and expression of the desired glycosyltransferases to produce SCT enriched antibodies with similar binding affinity to Fc receptors and ADCC activity to homogenous SCT antibody.

Probing the Internalization and Efficacy of Antibody-Drug Conjugate via Site-Specific Fc-Glycan Labelling of a Homogeneous Antibody Targeting SSEA-4 Bearing Tumors.

Antibody drug conjugates (ADC) are an emerging class of pharmaceuticals consisting of cytotoxic agents covalently attached to an antibody designed to target a specific cancer cell surface molecule followed by internalization and intracellular release of payload to exhibit its anticancer activity. Targeted delivery of cytotoxic payload to a variety of specific cells has been demonstrated to have significant enhancement in clinical efficacy and dramatic reduction in off-target toxicity. Site-specific conjugation of payload to the antibody is highly desirable for development of ADC with well-defined antibody-to-drug ratio, enhanced internalization, reduced toxicity, improved stability, desired pharmacological profile and optimal therapeutic index. Here, we reported a site-specific conjugation strategy for evaluation of antibody internalization and efficacy of ADC designed to target SSEA4 on solid tumors. This strategy stems from the azido-fucose tag of a homogeneous antibody Fc-glycan generated via in vitro glycoengineering approach for site-specific conjugation and optimization of antibody-drug ratio to exhibit optimal efficacy. The ADC consisting of a chimeric anti-SSEA4 antibody chMC813-70, conjugated to the antineo-plastic agent monomethyl auristatin E via both cleavable and non-cleavable linkers showed excellent cytotoxicity profile towards SSEA4-bearing cancer cells. A clear distinction in cytotoxicity was observed among cancer cells with different SSEA4 expression levels.

Combating multidrug-resistant Helicobacter pylori with moenomycin A in combination with clarithromycin or metronidazole.

Current treatment of Helicobacter pylori involves a triple therapy comprising one proton pump inhibitor and two other antibiotics; however, the outcomes are limited due to the existence of antibiotic resistant strains. We previously reported that moenomycin A, a cell-wall transglycosylase inhibitor, is highly active against multidrug-resistant Helicobacter pylori. Herein we show that combination of moenomycin A with the protein synthesis inhibitor clarithromycin or metronidazole can synergistically achieve almost 95% eradication of multidrug-resistant Helicobacter pylori. We also found that the moenomycin A-non-susceptible strains of Helicobacter pylori with deletion of transglycosylase exhibit moenomycin A hyposensitivity, faster growth and impaired biofilm formation compared to the parental strain. Overall, the combination of moenomycin A and clarithromycin or metronidazole to achieve a synergistic effect on different targets is a promising treatment for multidrug-resistant Helicobacter pylori.

Glycoconjugates: Synthesis, Functional Studies, and Therapeutic Developments.

Glycoconjugates are major constituents of mammalian cells that are formed via covalent conjugation of carbohydrates to other biomolecules like proteins and lipids and often expressed on the cell surfaces. Among the three major classes of glycoconjugates, proteoglycans and glycoproteins contain glycans linked to the protein backbone via amino acid residues such as Asn for N-linked glycans and Ser/Thr for O-linked glycans. In glycolipids, glycans are linked to a lipid component such as glycerol, polyisoprenyl pyrophosphate, fatty acid ester, or sphingolipid. Recently, glycoconjugates have become better structurally defined and biosynthetically understood, especially those associated with human diseases, and are accessible to new drug, diagnostic, and therapeutic developments. This review describes the status and new advances in the biological study and therapeutic applications of natural and synthetic glycoconjugates, including proteoglycans, glycoproteins, and glycolipids. The scope, limitations, and novel methodologies in the synthesis and clinical development of glycoconjugates including vaccines, glyco-remodeled antibodies, glycan-based adjuvants, glycan-specific receptor-mediated drug delivery platforms, etc., and their future prospectus are discussed.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Vaccination with SARS-CoV-2 spike protein lacking glycan shields elicits enhanced protective responses in animal models.

A major challenge to end the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to develop a broadly protective vaccine that elicits long-term immunity. As the key immunogen, the viral surface spike (S) protein is frequently mutated, and conserved epitopes are shielded by glycans. Here, we revealed that S protein glycosylation has site-differential effects on viral infectivity. We found that S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared to the fully glycosylated S protein, immunization of S protein with N-glycans trimmed to the mono-GlcNAc-decorated state (SMG) elicited stronger immune responses and better protection for human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). In addition, a broadly neutralizing monoclonal antibody was identified from SMG-immunized mice that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. Together, these results demonstrate that removal of glycan shields to better expose the conserved sequences has the potential to be an effective and simple approach for developing a broadly protective SARS-CoV-2 vaccine.

Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine.

Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8+ T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8+ T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.

Synthesis of Azido-Globo H Analogs for Immunogenicity Evaluation.

Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.