Glycan Antibodies Research Articles

Structure, Dynamics, Receptor Binding, and Antibody Binding of the Fully Glycosylated Full-Length SARS-CoV-2 Spike Protein in a Viral Membrane.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2) and is considered the major target for drug and vaccine development. We previously built fully glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of the receptor-binding domain (RBD) and different templates for the stalk region. In this work, multiple μs-long all-atom molecular dynamics simulations were performed to provide deeper insights into the structure and dynamics of S protein and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of the RBD and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody–glycan clash analysis instead of commonly used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibodies. Overall, our observations offer structural and dynamic insights into the SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.

Mass Spectrometry-Based Methods for Immunoglobulin G N-Glycosylation Analysis.

Mass spectrometry and its hyphenated techniques enabled by the improvements in liquid chromatography, capillary electrophoresis, novel ionization, and fragmentation modes are truly a cornerstone of robust and reliable protein glycosylation analysis. Boost in immunoglobulin G (IgG) glycan and glycopeptide profiling demands for both applied biomedical and research applications has brought many new advances in the field in terms of technical innovations, sample preparation, improved throughput, and confidence in glycan structural characterization. This chapter summarizes mass spectrometry basics, focusing on IgG and monoclonal antibody N-glycosylation analysis on several complexity levels. Different approaches, including antibody enrichment, glycan release, labeling, and glycopeptide preparation and purification, are covered and illustrated with recent breakthroughs and examples from the literature omitting excessive theoretical frameworks. Finally, selected highly popular methodologies in IgG glycoanalytics such as liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization are discussed more thoroughly yet in simple terms making this text a practical starting point either for the beginner in the field or an experienced clinician trying to make sense out of the IgG glycomic or glycoproteomic dataset.

新しい糖鎖マーカー血中 10-7G 値は、膵がんの肝転移予測マーカーになる

新しい糖鎖マーカー血中 10-7G 値は、膵がんの肝転移予測マーカーになる

Glycan Positioning Impacts HIV-1 Env Glycan-Shield Density, Function, and Recognition by Antibodies.

SummaryHIV-1 envelope (Env) N-glycosylation impact virus-cell entry and immune evasion. How each glycan interacts to shape the Env-protein-sugar complex and affects Env function is not well understood. Here, analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation sites affected the Env structure at distant locations and had a ripple effect on Env-wide glycan processing, virus infectivity, antibody recognition, and virus neutralization. Specifically, the N262 glycan, although not in the CD4-binding site, modulated Env binding to the CD4 receptor, affected Env recognition by several glycan-dependent neutralizing antibodies, and altered site-specific glycosylation heterogeneity, with, for example, N448 displaying limited glycan processing. Molecular-dynamic simulations visualized differences in glycan density and how specific oligosaccharide positions can move to compensate for a glycan loss. This study demonstrates how changes in individual glycans can alter molecular dynamics, processing, and function of the Env-glycan shield.

A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology

N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.

Abstract 4524: Non-genetic generation of IgG-like immune cell-redirecting antibodies lacking effector function

Abstract Since the early approval of catumaxomab and blinatumomab, T cell-redirecting antibodies have emerged as a promising biologics in cancer treatment. New technologies are overcoming the limitations of the 1st generation T-cell redirecting bispecifics, especially extending half-life to allow intermittent dosing, reducing immunogenicity and improving the safety profile. Today, T cell-engagers are generated as a wide variety of molecular architectures, typically IgG-type. However, nearly without exception, these bispecifics are generated by genetic fusion of a cancer-binding and an anti-CD3 antibody, which inevitably requires genetic reengineering of an existing targeting antibody and/or extensive optimization of antibody format. We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for controlled attachment of small-molecule cytotoxic drugs.1 The resulting antibody-drug conjugates (ADCs) were found to display significantly improved therapeutic index versus conventional technologies, while improving manufacturability. This proprietary technology, known as GlycoConnect™, has been adapted by various ADC developers, and entered clinical phase early 2019. Here we present the adaption of GlycoConnect™ technology for the generation of immune cell-redirecting antibodies, without requiring prior protein reengineering. Instead, a two-stage chemoenzymatic process is applied that allows the conversion of any IgG isotype into a T cell-engager by selective attachment of anti-CD3 scFv to the antibody glycan. Studies are presented to precisely control conjugation stoichiometry, providing molecular architectures with defined 2:1 or 2:2 ratio (target-binding:CD3-binding), as well as functional studies to corroborate the biological activity of these GlycoConnect™ bispecifics. Finally, early results will be presented regarding extension of the concept to bispecifics designed to redirect natural killer (NK) cells, or to engage T-cells by activation of costimulatory molecules, such as CD137 (4-1BB). 1 van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved NGlycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. Citation Format: Floris L. van Delft, Remon van Geel. Non-genetic generation of IgG-like immune cell-redirecting antibodies lacking effector function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4524.

Glycomic analysis of antibody indicates distinctive glycosylation profile in patients with autoimmune cholangitis

Glycosylation of antibodies, particularly in the Fc domain, critically modulate the ability of antibodies to bind to FcRs, maintaining immune quiescence to achieve a finely orchestrated immune response. The removal of sialic acid and galactose residues dramatically alters the physiological function of IgGs, and alterations of Ig glycosylation have been associated with several autoimmune disorders. However, Ig glycosylation has not been extensively studied in autoimmune cholangitis. We applied triple quadruple mass spectroscopy with subsequent multiple reaction monitoring to elucidate the profile, composition and linkage of sugar residues of antibody glycans in patients with primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and healthy controls (HC). Agalactosylated, HexNAc terminated IgG1 glycoforms were enriched in both PBC and PSC. Levels of IgM glycans at site N439 and fucosylated glycans in J chain, were significantly decreased in PBC compared to PSC and HC. PSC patients had decreased bisecting glycoforms and increased biantennary glycoforms on IgA compared to PBC. Importantly, our data demonstrate the association of distinct branching and composition patterns of Ig glycoforms with disease severity and liver cirrhosis, which highlight the importance of glycan biology as a potential mechanism and/or a disease specific signal of inflammation.

Impact of Glycan Positioning on HIV-1 Env Glycan Shield Density, Function, and Antibody Recognition

N-glycans, which represent >50% mass of the HIV-1 envelope (Env) trimer, play important roles for virus-cell entry and immune evasion. How each glycan unit interacts to shape the Env protein-sugar complex and affects Env function is not well understood. Here, high-resolution glycomics analysis of two Env variants from the same donor, with differing functional characteristics and N-glycosylation-site composition, revealed that changes to key N-glycosylation-site not only affected the Env structure at distant locations, but also had a ripple effect on Env-wide glycan processing, virus infectivity, and antibody recognition and virus neutralization. Specifically, the N262 glycan, although not located in the CD4-binding site, controlled Env binding to the CD4 receptor, affected the recognition of Env by several glycan-dependent broadly neutralizing antibodies, and altered heterogeneity of glycosylation at several sites, with N156, N160, and N448 displaying limited glycan processing. Molecular dynamic simulations visualized how specific oligosaccharide positions can move to compensate for loss of a glycan. This study demonstrates how changes in individual glycan units can alter molecular dynamics and processing of the Env-glycan shield and, consequently, Env function.

Identification of the epitope of 10-7G glycan antibody to recognize cancer-associated haptoglobin

We previously identified fucosylated haptoglobin (Fuc-Hpt) as a clinical serum biomarker of pancreatic cancer and established the novel glycan monoclonal antibody (mAb) 10-7G. This antibody recognizes cancer-associated haptoglobin including Fuc-Hpt and the precursor of haptoglobin. Interestingly, Western blot analysis showed that the 10-7G mAb reacts with the haptoglobin α chain, which has no N-glycan potential sites; haptoglobin β chain has four N-glycan sites. In this study, we identified the epitope for the 10-7G mAb using haptoglobin deletion mutants, as well as inhibition ELISA with recombinant peptides. We illustrated molecular graphics to show a relationship between the epitope and the β chain. Furthermore, we hypothesized that the 10-7G mAb minimally recognizes normal haptoglobin, but aberrant glycosylation on the β chain causes conformational changes, enabling the 10-7G mAb to easily access the epitope within the α chain. Because 10-7G values, an enzyme-linked immunosorbent assay–immobilized 10-7G mAb, in patients with pancreatic cancer varied by haptoglobin phenotype, the amount of aberrant glycosylation needed to affect haptoglobin conformation probably depends on haptoglobin phenotype. Taken together, the 10-7G mAb recognized characteristic peptides on the haptoglobin α chain as a result of conformational changes and is a promising tool for diagnosing pancreatic cancer by haptoglobin phenotype.

Comparison of two glycoengineering strategies to control the fucosylation of a monoclonal antibody

Core fucosylation of an Fc N-linked glycan affects antibody effector functions, as the absence of fucose increases the antibody dependent cell cytotoxicity (ADCC) response with increased binding to the Fcγ receptors. The work presented here compares two different approaches to incrementally reduce core fucosylation of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- fluoro peracetylated fucose (2FF), which was added to cell cultures expressing the EG2-hFc antibody in increasing concentrations up to 50μM. At this concentration there was no observed effect on cell growth. Glycan analysis was performed on antibodies collected from culture samples using HILIC-HPLC. The inhibitor reduced total fucosylation from 80% to 17.5% at 20μM 2FF.The second method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de novo pathway into producing rhamnose which is not incorporated into a glycan. Stable clones from transfected pools were isolated following flow cytometry using the green fluorescent protein (GFP) marker which was co-expressed with the RMD gene. High expressing RMD clones reduced the fucosylation of the antibody glycan to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the fucosylation level even further to 3% of the antibody glycan. An incremental increase in fucosylation was obtained by step-wise addition of fucose (up to 1 mM) to the RMD cells, in which the fucosylation level increased to a maximum of 87%.We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with addition of increasing concentrations of 2FF. This showed that 2FF inhibits the addition of fucose in a concentration- dependent and specific manner with the inhibition of fucose occurring one fucose at a time. Control cultures showed the presence of a predominant peak indicating two fucose moieties per antibody. As the 2FF inhibitor concentration was increased peaks corresponding to one fucose per antibody and non-fucosylated antibody predominated with a gradual decrease of the 2 fucose peak to insignificance at 15 μM 2FF.

Cooperation between somatic mutation and germline-encoded residues enables antibody recognition of HIV-1 envelope glycans.

Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.

Establishment and characterization of a fucosylated \u03b1-fetoprotein-specific monoclonal antibody: a potential application for clinical research

The Lens culinaris agglutinin (LCA)-reactive fraction of α-fetoprotein (AFP-L3) is a well-known cancer biomarker for hepatocellular carcinoma (HCC) with very high specificity. Because LCA recognizes only bi-antennary N-glycans with a core fucose, some of fucosylated AFP in HCC patients may not be detected. Then glycan antibodies, which recognize both specific glycan and protein, are desired for glycobiology. Here, we successfully established a novel glycan antibody for fucosylated AFP and demonstrated its potential clinical application. After immunization with a fucosylated AFP peptide, positive screening was performed for fucosylated AFP peptides using solid-phase enzyme-linked immunosorbent assay (ELISA). The newly developed antibody was designated: fucosylated AFP-specific mAb (FasMab). Western blot analysis showed that FasMab reacted with AFP produced by HepG2 cells, but not with AFP produced by α-1,6-fucosyltransferase deficient HepG2 cells. The specific binding of FasMab to fucosylated AFP was confirmed with ELISA as well as western blot analysis. A preliminary high sensitivity chemiluminescence enzyme immunoassay kit showed increased levels of fucosylated AFP in the sera of patients with HCC, but not in the sera of normal patients, or patients with chronic liver diseases. Thus, the novel glycan antibody, FasMab, is a promising tool to study fucosylated AFP with clinical and basic research applications.

Constrained global sensitivity analysis for bioprocess design space identification

The manufacture of protein-based therapeutics presents unique challenges due to limited control over the biotic phase. This typically gives rise to a wide range of protein structures of varying safety and in vivo efficacy. Herein we propose a computational methodology, enabled by the application of constrained Global Sensitivity Analysis, for efficiently exploring the operating range of process inputs in silico and identifying a design space that meets output constraints. The methodology was applied to an antibody-producing Chinese hamster ovary (CHO) cell culture system: we explored >8000 feeding strategies to identify a subset of manufacturing conditions that meet constraints on antibody titre and glycan distribution as an attribute of product quality. Our computational findings were then verified experimentally, confirming the applicability of this approach to a challenging production system. We envisage that this methodology can significantly expedite bioprocess development and increase operational flexibility.

Antibody responses to the HIV-1 envelope high mannose patch.

Neutralizing antibodies against human immunodeficiency virus subtype 1 (HIV-1) bind to its envelope glycoprotein (Env). Half of the molecular mass of Env is carbohydrate making it one of the most heavily glycosylated proteins known in nature. HIV-1 Env glycans are derived from the host and present a formidable challenge for host anti-glycan antibody induction. Anti-glycan antibody induction is challenging because anti-HIV-1 glycan antibodies should recognize Env antigen while not acquiring autoreactivity. Thus, the glycan network on HIV-1 Env is referred to as the glycan shield. Despite the challenges presented by immune recognition of host-derived glycans, neutralizing antibodies capable of binding the glycans on HIV-1 Env can be generated by the host immune system in the setting of HIV-1 infection. In particular, a cluster of high mannose glycans, including an N-linked glycan at position 332, form the high mannose patch and are targeted by a variety of broadly neutralizing antibodies. These high mannose patch-directed HIV-1 antibodies can be categorized into distinct categories based on their antibody paratope structure, neutralization activity, and glycan and peptide reactivity. Below we will compare and contrast each of these classes of HIV-1 glycan-dependent antibodies and describe vaccine design efforts to elicit each of these antibody types.

Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice.

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

Development, Optimization, and Structural Characterization of an Efficient Peptide-Based Photoaffinity Cross-Linking Reaction for Generation of Homogeneous Conjugates from Wild-Type Antibodies.

Site-specific conjugation of small molecules to antibodies represents an attractive goal for the development of more homogeneous targeted therapies and diagnostics. Most site-specific conjugation strategies require modification or removal of antibody glycans or interchain disulfide bonds or engineering of an antibody mutant that bears a reactive handle. While such methods are effective, they complicate the process of preparing antibody conjugates and can negatively impact biological activity. Herein we report the development and detailed characterization of a robust photoaffinity cross-linking method for site-specific conjugation to fully glycosylated wild-type antibodies. The method employs a benzoylphenylalanine (Bpa) mutant of a previously described 13-residue peptide derived from phage display to bind tightly to the Fc domain; upon UV irradiation, the Bpa residue forms a diradical that reacts with the bound antibody. After the initial discovery of an effective Bpa mutant peptide and optimization of the reaction conditions to enable efficient conjugation without concomitant UV-induced photodamage of the antibody, we assessed the scope of the photoconjugation reaction across different human and nonhuman antibodies and antibody mutants. Next, the specific site of conjugation on a human antibody was characterized in detail by mass spectrometry experiments and at atomic resolution by X-ray crystallography. Finally, we adapted the photoconjugation method to attach a cytotoxic payload site-specifically to a wild-type antibody and showed that the resulting conjugate is both stable in plasma and as potent as a conventional antibody-drug conjugate in cells, portending well for future biological applications.

Micro Array-Assisted Analysis of Anti-Schistosome Glycan Antibodies Elicited by Protective Vaccination With Irradiated Cercariae.

Baboons vaccinated with radiation-attenuated cercariae develop high levels of protection against schistosome infection, correlating to high antibody titres towards schistosome antigens with unknown molecular identity. Using a microarray consisting of glycans isolated from different life-stages of schistosomes, we studied the anti-glycan immunoglobulin (Ig) G and IgM responses in vaccinated and challenged baboons over a time course of 25 weeks. Anti-glycan IgM responses developed early after vaccination, but did not rise in response to later vaccinations. In contrast, anti-glycan IgG developed more slowly, but was boosted by all five subsequent vaccinations. High IgM and IgG levels against O-glycans and glycosphingolipid glycans of cercariae were observed. At the time of challenge, while most antibody levels decreased in the absence of vaccination, IgG towards a subset of glycans containing multiple-fucosylated motifs remained high until 6 weeks post-challenge during challenge parasite elimination, suggesting a possible role of this IgG in protection.

Haptoglobin phenotype is a critical factor in the use of fucosylated haptoglobin for pancreatic cancer diagnosis

Fucosylation is one of the most important glycosylations involved in cancer and inflammation. Many studies have reported significant increases in serum fucosylated haptoglobin (Fuc-Hpt) in a variety of cancer patients. In this study, we measured Fuc-Hpt using a lectin-antibody enzyme-linked immunosorbent assay (ELISA) or a novel ELISA system that used a glycan antibody for Fuc-Hpt. Hpt is known to be divided into three phenotypes (Hpt1–1, Hpt2–1, and Hpt2–2), depending on its genetic background. Normal levels of serum Hpt are different in each Hpt phenotype and these phenotypes are associated with the incidence of several human diseases. Here, we investigated how Hpt phenotype affected measurements of Fuc-Hpt, using two kinds of ELISA. Interestingly, we found that serum Fuc-Hpt levels were dramatically lower in the Hpt1–1 phenotype for both types of ELISA. For Hpt2–1 and Hpt2–2, we observed significantly increased serum Fuc-Hpt levels in patients with pancreatic cancer. When cases of the Hpt1–1 phenotype were depleted, our receiver operating characteristic (ROC) curve analyses showed that the area under the curve (AUC) value for pancreatic cancer diagnosis increased in each ELISA. Taken together, our results indicate that Hpt phenotype is a critical for the clinical application of Fuc-Hpt as a cancer biomarker.

Through the barricades: overcoming the barriers to effective antibody-based cancer therapeutics.

Since the turn of the century, cancer therapy has undergone a transformation in terms of new treatment modalities and renewed optimism in achieving long-lived tumor control and even cure. This is, in large part, thanks to the widespread incorporation of monoclonal antibodies (mAbs) into standard treatment regimens. These new therapies have, across many settings, significantly contributed to improved clinical responses, patient quality of life and survival. Moreover, the flexibility of the antibody platform has led to the development of a wide range of innovative and combinatorial therapies that continue to augment the clinician's armory. Despite these successes, there is a growing awareness that in many cases mAb therapy remains suboptimal, primarily due to inherent limitations imposed by the immune system's own homeostatic controls and the immunosuppressive tumor microenvironment. Here, we discuss the principal barriers that act to constrain the tumor-killing activity of antibody-based therapeutics, particularly those involving antibody glycans, using illustrative examples from both pre-clinical and market approved mAbs. We also discuss strategies that have been, or are in development to overcome these obstacles. Finally, we outline how the growing understanding of the biological terrain in which mAbs function is shaping innovation and regulation in cancer therapeutics.

Synthesis, Liposomal Formulation, and Immunological Evaluation of a Minimalistic Carbohydrate-α-GalCer Vaccine Candidate.

Fully synthetic glycan-based vaccines hold great potential as preventive and therapeutic vaccines against infectious diseases as well as cancer. Here, we present a two-component platform based on the facile conjugation of carbohydrate antigens to α-galactosylceramide (α-GalCer) to yield fully synthetic vaccine candidates. Formulation of the cancer-associated Tn antigen glycolipid model vaccine candidate into liposomes of different sizes and subsequent immunization of mice generated specific, high-affinity antibodies against the carbohydrate antigen with characteristics of T cell-dependent immunity. Liposome formulation elicited more reproducible glycan immunity than a conventional glycoconjugate vaccine bearing the same glycan antigen did. Further evaluation of the immune response revealed that the size of the liposomes influenced the glycan antibody responses toward either a cellular (Th1) or a humoral (Th2) immune phenotype. The glycolipid vaccine platform affords strong and robust antiglycan antibody responses in vivo without the need for an external adjuvant.