Structure of the type 38 Streptococcus pneumoniae capsular polysaccharide.

Streptococcus pneumoniae is one of the globally important encapsulated human pathogens and more than 100 different serotypes have been identified. Despite very extensive genetic and immune-serological studies, the capsular polysaccharide repeating unit structure of several serotypes has not been determined yet, including the type 38 (type 38 in Danish nomenclature; type 71 in US nomenclature). Physicochemical data revealed that type 38 polysaccharide is composed of a pentasaccharide repeat unit →3)-[ß-D-Galf(1 â†’ 2)]-ß-D-GalpA6(L-Ser)-(1 â†’ 3)-α-D-GlcpNAc-(1 â†’ 3)-α-D-Sugp-(1 â†’ 4)-α-D-Galp(2OAc)-(1 â†’ . The polysaccharide is O-acetylated at position C2 of the α-Gal residue at approximately (68-87 %) of the repeat units.

Cancer cells and viruses share common glycoepitopes: exciting opportunities toward combined treatments.

Aberrant glycosylation patterns of glycoproteins and glycolipids have long been recognized as one the major hallmarks of cancer cells that has led to numerous glycoconjugate vaccine attempts. These abnormal glycosylation profiles mostly originate from the lack of key glycosyltransferases activities, mutations, over expressions, or modifications of the requisite chaperone for functional folding. Due to their relative structural simplicity, O-linked glycans of the altered mucin family of glycoproteins have been particularly attractive in the design of tumor associated carbohydrate-based vaccines. Several such glycoconjugate vaccine formulations have generated potent monoclonal anti-carbohydrate antibodies useful as diagnostic and immunotherapies in the fight against cancer. Paradoxically, glycoproteins related to enveloped viruses also express analogous N- and O-linked glycosylation patterns. However, due to the fact that viruses are not equipped with the appropriate glycosyl enzyme machinery, they need to hijack that of the infected host cells. Although the resulting N-linked glycans are very similar to those of normal cells, some of their O-linked glycan patterns often share the common structural simplicity to those identified on tumor cells. Consequently, given that both cancer cells and viral glycoproteins share both common N- and O-linked glycoepitopes, glycoconjugate vaccines could be highly attractive to generate potent immune responses to target both conditions.

Chemoenzymatic synthesis and immunological evaluation of sialyl-Thomsen-Friedenreich (sTF) antigen conjugate to CRM197.

sTF (sialyl-Thomsen-Friedenreich) is a type of tumor-associated carbohydrate antigens (TACAs) and is highly expressed in various human malignancies. To validate if sTF could be a valuable molecular target for future cancer vaccine development, in this work the sTF antigen was prepared by adopting a strategy combining chemical and enzymatic methods, and then was covalently conjugated to a carrier protein, CRM197. The preliminary immunological evaluation, performed on BALB/c mice, revealed that the sTF-CRM197 conjugate elicited high titers of specific IgG antibodies. FACS experiments showed that the antisera induced by sTF-CRM197 conjugate could specifically recognize and bind to sTF-positive cancer cells T-47D. Furthermore, the conjugate mediated effective and specific antibody-mediated complement-dependent cytotoxicity (CDC).

Effect of Side-Chain Functional Groups in the Immunogenicity of Bacterial Surface Glycans.

Glycans on the surface of bacteria have diverse and essential biological functions and have widely been employed for treating various bacterial infectious diseases. Furthermore, these glycans comprise various functional groups, such as O-, N-, and carboxyl-modified, which significantly increase the diversity of glycan structures. These functional groups are not only crucial for glycans' structural identity but are also essential for their biological functions. Therefore, a clear understanding of the biological functions of these modified groups in corresponding bacterial glycans is crucial for their medical applications. Thus far, the activities of functional groups in some biomedical active carbohydrates have been elucidated. It has been shown that some functional groups are key constituents of biologically active bacterial glycans, while others are actually not essential and may even mask the functions of the glycans. This paper reviews the structures of naturally occurring side-chain functional groups in glycans located on the bacterial surface and their roles in immunological responses.

A Shiga Toxin B-Subunit-Based Lectibody Boosts T Cell Cytotoxicity towards Gb3-Positive Cancer Cells.

Aberrant glycosylation plays a crucial role in tumour progression and invasiveness. Tumour-associated carbohydrate antigens (TACAs) represent a valuable set of targets for immunotherapeutic approaches. The poor immunogenicity of glycan structures, however, requires a more effective and well-directed way of targeting TACAs on the surface of cancer cells than antibodies. The glycosphingolipid globotriaosylceramide (Gb3) is a well-established TACA present in a multitude of cancer types. Its overexpression has been linked to metastasis, invasiveness, and multidrug resistance. In the present study, we propose to use a dimeric fragment of the Shiga toxin B-subunit (StxB) to selectively target Gb3-positive cancer cells in a StxB-scFv UCHT1 lectibody. The lectibody, comprised of a lectin and the UCHT1 antibody fragment, was produced in E. coli and purified via Ni-NTA affinity chromatography. Specificity of the lectibody towards Gb3-positive cancer cell lines and specificity towards the CD3 receptor on T cells, was assessed using flow cytometry. We evaluated the efficacy of the lectibody in redirecting T cell cytotoxicity towards Gb3-overexpressing cancer cells in luciferase-based cytotoxicity in vitro assays. The StxB-scFv UCHT1 lectibody has proven specific for Gb3 and could induce the killing of up to 80% of Gb3-overexpressing cancer cells in haemorrhagic and solid tumours. The lectibody developed in this study, therefore, highlights the potential that lectibodies and lectins in general have for usage in immunotherapeutic approaches to boost the efficacy of established cancer treatments.

Aberrant Glycosylation as Immune Therapeutic Targets for Solid Tumors.

Glycosylation occurs at all major types of biomolecules, including proteins, lipids, and RNAs to form glycoproteins, glycolipids, and glycoRNAs in mammalian cells, respectively. The carbohydrate moiety, known as glycans on glycoproteins and glycolipids, is diverse in their compositions and structures. Normal cells have their unique array of glycans or glycome which play pivotal roles in many biological processes. The glycan structures in cancer cells, however, are often altered, some having unique structures which are termed as tumor-associated carbohydrate antigens (TACAs). TACAs as tumor biomarkers are glycan epitopes themselves, or glycoconjugates. Some of those TACAs serve as tumor glyco-biomarkers in clinical practice, while others are the immune therapeutic targets for treatment of cancers. A monoclonal antibody (mAb) to GD2, an intermediate of sialic-acid containing glycosphingolipids, is an example of FDA-approved immune therapy for neuroblastoma indication in young adults and many others. Strategies for targeting the aberrant glycans are currently under development, and some have proceeded to clinical trials. In this review, we summarize the currently established and most promising aberrant glycosylation as therapeutic targets for solid tumors.

Smaller size packs a stronger punch - Recent advances in small antibody fragments targeting tumour-associated carbohydrate antigens.

Attached to proteins, lipids, or forming long, complex chains, glycans represent the most versatile post-translational modification in nature and surround all human cells. Unique glycan structures are monitored by the immune system and differentiate self from non-self and healthy from malignant cells. Aberrant glycosylations, termed tumour-associated carbohydrate antigens (TACAs), are a hallmark of cancer and are correlated with all aspects of cancer biology. Therefore, TACAs represent attractive targets for monoclonal antibodies for cancer diagnosis and therapy. However, due to the thick and dense glycocalyx as well as the tumour micro-environment, conventional antibodies often suffer from restricted access and limited effectiveness in vivo. To overcome this issue, many small antibody fragments have come forth, showing similar affinity with better efficiency than their full-length counterparts. Here we review small antibody fragments against specific glycans on tumour cells and highlight their advantages over conventional antibodies.

Cancer Vaccines Based on Fluorine-Modified KH-1 Elicit Robust Immune Response.

KH-1 is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target of antitumor vaccines for cancer immunotherapies. However, most TACAs are thymus-independent antigens (TD-Ag), and they tend to induce immunological tolerance, leading to their low immunogenicity. To overcome these problems, some fluorinated derivatives of the KH-1 antigen were designed, synthesized, and conjugated to the carrier protein CRM197 to form glycoconjugates, which were used for immunological studies with Freund's adjuvant. The results showed that fluorine-modified N-acyl KH-1 conjugates can induce higher titers of antibodies, especially IgG, which can recognize KH-1-positive cancer cells and can eliminate cancer cells through complement-dependent cytotoxicity (CDC). The trifluoro-modified KH-1-TF-CRM197 showed great potential as an anticancer vaccine candidate.

The effect of a methyl group on structure and function: Serine vs. threonine glycosylation and phosphorylation.

A variety of glycan structures cover the surface of all cells and are involved in myriad biological processes, including but not limited to, cell adhesion and communication, protein quality control, signal transduction and metabolism, while also being intimately involved in innate and adaptive immune functions. Immune surveillance and responses to foreign carbohydrate antigens, such as capsular polysaccharides on bacteria and surface protein glycosylation of viruses, are the basis of microbial clearance, and most antimicrobial vaccines target these structures. In addition, aberrant glycans on tumors called Tumor-Associated Carbohydrate Antigens (TACAs) elicit immune responses to cancer, and TACAs have been used in the design of many antitumor vaccine constructs. A majority of mammalian TACAs are derived from what are referred to as mucin-type O-linked glycans on cell-surface proteins and are linked to the protein backbone through the hydroxyl group of either serine or threonine residues. A small group of structural studies that have compared mono- and oligosaccharides attached to each of these residues have shown that there are distinct differences in conformational preferences assumed by glycans attached to either "unmethylated" serine or ß-methylated threonine. This suggests that the linkage point of antigenic glycans will affect their presentation to the immune system as well as to various carbohydrate binding molecules (e.g., lectins). This short review, followed by our hypothesis, will examine this possibility and extend the concept to the presentation of glycans on surfaces and in assay systems where recognition of glycans by proteins and other binding partners can be defined by different attachment points that allow for a range of conformational presentations.

Assessment of Immunologically Potent Carbohydrates.

Carbohydrates have a great potential in generating structural and immunological diversities. Microbial pathogens often decorate their outmost surfaces with specific carbohydrate signatures. Carbohydrate antigens also differ significantly from protein antigens in physiochemical properties, especially in surface display of antigenic determinants in aqueous solutions. Technical optimization or modifications are often needed when we apply standard procedures for protein-based enzyme-linked immunosorbent assay (ELISA) to assess immunologically potent carbohydrates. We present here our laboratory protocols for performing carbohydrate ELISA and discuss several assay platforms that may be applied complementarily to explore the carbohydrate moieties that are critical for host immune recognition and induction of glycan-specific antibody responses.

A bispecific, crosslinking lectibody activates cytotoxic T cells and induces cancer cell death.

BACKGROUND: Aberrant glycosylation patterns play a crucial role in the development of cancer cells as they promote tumor growth and aggressiveness. Lectins recognize carbohydrate antigens attached to proteins and lipids on cell surfaces and represent potential tools for application in cancer diagnostics and therapy. Among the emerging cancer therapies, immunotherapy has become a promising treatment modality for various hematological and solid malignancies. Here we present an approach to redirect the immune system into fighting cancer by targeting altered glycans at the surface of malignant cells. We developed a so-called "lectibody", a bispecific construct composed of a lectin linked to an antibody fragment. This lectibody is inspired by bispecific T cell engager (BiTEs) antibodies that recruit cytotoxic T lymphocytes (CTLs) while simultaneously binding to tumor-associated antigens (TAAs) on cancer cells. The tumor-related glycosphingolipid globotriaosylceramide (Gb3) represents the target of this proof-of-concept study. It is recognized with high selectivity by the B-subunit of the pathogen-derived Shiga toxin, presenting opportunities for clinical development. METHODS: The lectibody was realized by conjugating an anti-CD3 single-chain antibody fragment to the B-subunit of Shiga toxin to target Gb3+ cancer cells. The reactive non-canonical amino acid azidolysine (AzK) was inserted at predefined single positions in both proteins. The azido groups were functionalized by bioorthogonal conjugation with individual linkers that facilitated selective coupling via an alternative bioorthogonal click chemistry reaction. In vitro cell-based assays were conducted to evaluate the antitumoral activity of the lectibody. CTLs, Burkitt´s lymphoma-derived cells and colorectal adenocarcinoma cell lines were screened in flow cytometry and cytotoxicity assays for activation and lysis, respectively. RESULTS: This proof-of-concept study demonstrates that the lectibody activates T cells for their cytotoxic signaling, redirecting CTLs´ cytotoxicity in a highly selective manner and resulting in nearly complete tumor cell lysis-up to 93%-of Gb3+ tumor cells in vitro. CONCLUSIONS: This research highlights the potential of lectins in targeting certain tumors, with an opportunity for new cancer treatments. When considering a combinatorial strategy, lectin-based platforms of this type offer the possibility to target glycan epitopes on tumor cells and boost the efficacy of current therapies, providing an additional strategy for tumor eradication and improving patient outcomes.

Dual-Channel Detection of Breast Cancer Biomarkers CA15-3 and CEA in Human Serum Using Dialysis-Silicon Nanowire Field Effect Transistor.

Background: Breast cancer (BC) is the most common malignant tumors and the leading cause of cancer deaths among women. The early diagnosis and treatment of BC are effective measures that can increase survival rates and reduce mortality. Carbohydrate antigens 15-3 (CA15-3) and carcinoma embryonic antigens (CEA) have been regarded as the most two valuable tumor markers of BC. The combined detection of CA15-3 and CEA could improve the sensitivity and accuracy of early diagnosis for BC. Methods: The multi-channel double-gate silicon nanowire field effect transistor (SiNW-FET) biosensors were fabricated by using the top-down semiconductor manufacturing technology. By surface modification of the different SiNW surfaces with monoclonal CA15-3 and CEA antibodies separately, the prepared SiNW-FET was processed into biosensor for dual-channel detection of CA15-3 and CEA. Results: The prepared SiNW-FET biosensors were proved to have high sensitivity and specificity for the dual-channel detection of CA15-3 and CEA, and the detection limit is as low as 0.1U/mL CA15-3 and 0.01 ng/mL CEA. Moreover, the SiNW-FET biosensors were able to detect CA15-3 and CEA in serum by connecting a miniature hemodialyzer. Conclusion: The present study reported a SiNW-FET biosensor for dual-channel detection of breast cancer biomarkers CA15-3 and CEA in serum, which has potential clinical application value for the early diagnosis and curative effect observation of BC.

Novel lectin-based chimeric antigen receptors target Gb3-positive tumour cells.

The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins  that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.

Specific (sialyl-)Lewis core 2 O-glycans differentiate colorectal cancer from healthy colon epithelium.

Cells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized as promising targets for immunotherapy. These carbohydrates differ from those of the surrounding non-cancerous tissues and contribute to the malignant phenotype of the cancer cells by promoting proliferation, metastasis, and immunosuppression. However, due to tumor tissue heterogeneity and technological limitations, TACAs are insufficiently explored. Methods: A workflow was established to decode the colorectal cancer (CRC)-associated O-linked glycans from approximately 20,000 cell extracts. Extracts were obtained through laser capture microdissection of formalin fixed paraffin embedded tissues of both primary tumors and metastatic sites, and compared to healthy colon mucosa from the same patients. The released O-glycans were analyzed by porous graphitized carbon liquid chromatography-tandem mass spectrometry in negative ion mode. Results: Distinctive O-glycosylation features were found in cancerous, stromal and normal colon mucosal regions. Over 100 O-linked glycans were detected in cancerous regions with absence in normal mucosa. From those, six core 2 O-glycans were exclusively found in more than 33% of the cancers, carrying the terminal (sialyl-)LewisX/A antigen. Moreover, two O-glycans were present in 72% of the analyzed cancers and 94% of the investigated cancers expressed at least one of these two O-glycans. In contrast, normal colon mucosa predominantly expressed core 3 O-glycans, carrying α2-6-linked sialylation, (sulfo-)LewisX/A and Sda antigens. Conclusion: In this study, we present a novel panel of highly specific TACAs, based upon differences in the glycomic profiles between CRC and healthy colon mucosa. These TACAs are promising new targets for development of innovative cancer immune target therapies and lay the foundation for the targeted treatment of CRC.

Glycoconjugate Nanoparticle-Based Systems in Cancer Immunotherapy: Novel Designs and Recent Updates.

For many years, cell-surface glycans (in particular, Tumor-Associated Carbohydrate Antigens, TACAs) have been the target of both passive and active anticancer immunotherapeutic design. Recent advances in immunotherapy as a treatment for a variety of malignancies has revolutionized anti-tumor treatment regimens. Checkpoint inhibitors, Chimeric Antigen Receptor T-cells, Oncolytic virus therapy, monoclonal antibodies and vaccines have been developed and many approvals have led to remarkable outcomes in a subset of patients. However, many of these therapies are very selective for specific patient populations and hence the search for improved therapeutics and refinement of techniques for delivery are ongoing and fervent research areas. Most of these agents are directed at protein/peptide epitopes, but glycans-based targets are gaining in popularity, and a handful of approved immunotherapies owe their activity to oligosaccharide targets. In addition, nanotechnology and nanoparticle-derived systems can help improve the delivery of these agents to specific organs and cell types based on tumor-selective approaches. This review will first outline some of the historical beginnings of this research area and subsequently concentrate on the last 5 years of work. Based on the progress in therapeutic design, predictions can be made as to what the future holds for increasing the percentage of positive patient outcomes for optimized systems.

Advances in the Immunomodulatory Properties of Glycoantigens in Cancer.

Aberrant glycosylation in tumour progression is currently a topic of main interest. Tumour-associated carbohydrate antigens (TACAs) are expressed in a wide variety of epithelial cancers, being both a diagnostic tool and a potential treatment target, as they have impact on patient outcome and disease progression. Glycans affect both tumour-cell biology properties as well as the antitumor immune response. It has been ascertained that TACAs affect cell migration, invasion and metastatic properties both when expressed by cancer cells or by their extracellular vesicles. On the other hand, tumour-associated glycans recognized by C-type lectin receptors in immune cells possess immunomodulatory properties which enable tumour growth and immune response evasion. Yet, much remains unknown, concerning mechanisms involved in deregulation of glycan synthesis and how this affects cell biology on a major level. This review summarises the main findings to date concerning how aberrant glycans influence tumour growth and immunity, their application in cancer treatment and spotlights of unanswered challenges remaining to be solved.

Clinically proven specification setting for a meningococcal serogroup a conjugate vaccine.

GSK is currently working to improve the commercial presentation of the licensed quadrivalent conjugate vaccine (Menveo) for use against meningococcal serogroup A, C, W, Y (MenACWY) infections. Menveo consists of a primary, lyophilized vial, containing the serogroup A antigen that is reconstituted with the content of a second, liquid, vial that contains the serogroup C, W, Y antigens, to give the final liquid MenACWY product. Since the MenA structure is prone to hydrolytic degradation in liquid formulations, we used mathematical models to rationally design a clinical Phase 2 development plan and provide end of shelf-life (EoSL) and release specification setting for the MenACWY liquid product. By using development and clinical stability data, statistical models were built and used to predict both the MenA free saccharide (FS) and O-Acetyl (OAc) content during long-term storage conditions at 5 °C and stressed (accelerated) stability studies at 15 °C, 22.5 °C, 25 °C, 37 °C and 50 °C. This approach allowed us to define an aging plan for the clinical material to reach at least the required levels of MenA FS and OAc levels at product EoSL. The clinical material was then exposed to a temperature of 22.5 ± 2.5 °C for 59 days to generate FS OAc content of about 35% and 40%, respectively, which was then delivered to the patients in the clinical trial. To the best of our knowledge, this work represents the first example in the field of vaccine research where statistical models have been used to rationally design tailored lots, with the goal of setting EoSL and release specification limits based on data collected on artificially aged clinical material, in which the FS and OAc levels tested were intended to support a product shelf-life of at least 24 months.

Glycoconjugate content quantification to assess vaccine potency: A simplified approach.

Several glycoconjugate vaccines have been licensed or are currently in clinical development to prevent bacterial infections. Here we report the development of a single analytical assay to quantify the conjugated saccharide content, as alternative to two separated total and free (unconjugated) saccharide assays used so far, for a quadrivalent conjugate vaccine containing meningococcal serogroup A polysaccharide (α-1,6-linked N-acetylmannosamine phosphate repeating unit partly O-acetylated at position C3 or C4) coupled with CRM197 protein. The results confirm a high linear correlation among the two approaches (conjugated saccharide content vs. difference of total saccharide and free saccharide). Conjugated saccharide content estimation is therefore demonstrated to be a suitable method to monitor the product quality of vaccines containing meningococcal serogroup A conjugate antigen, in the final filled presentation as demonstrated here and potentially on the bulk conjugate before formulation.

Anti-cancer effects of pyrazole-platinum(II) complexes combined with anti-MUC1 monoclonal antibody versus monotherapy in DLD-1 and HT-29 colon cancer cells.

The membrane-bound MUC1 mucin is overexpressed and aberrantly glycosylated in many epithelium origin cancers. One of the promising strategies in cancer therapy is combining monoclonal antibodies against cancer related antigens, like MUC1, with chemotherapeutics. In the study we evaluated the potency of cisplatin (cisPt), two pyrazole-platinum(II) complexes PtPz4, PtPz6, and anti-MUC1 mAb applied as monotherapy, as well as the chemotherapeutics administrated with antibody, towards apoptotic response and cancer-related carbohydrate antigens (TACAs) in DLD-1 and HT-29 colon cancer cells. To assess the impact of the tested compounds on the examined factors flow cytometry, RT-PCR, Western blotting and ELISA were utilized. The combined therapy was more potent than monotherapy towards Bcl-2, Bid, caspases and TACAs of both cell lines. Combined therapy applied in DLD-1 cells induced apoptosis, was more effective than monotherapy in relation to p53, Bcl-xL, Bax, and Bim. In HT-29 cells, anti-MUC1 administrated with the drugs was more potent than monotherapy towards Bad. The proposed anti-MUC1/cisPt and pyrazole-platinum(II) complexes PtPz4, PtPz6 combined therapy may be promising anti-colon cancer therapy.

Anti-Lea monoclonal antibody SPM 522 recognizes an extended Lea epitope.

Insights into the differential binding characteristics of anti-Lea and anti-LeaLex monoclonal antibodies (mAbs) provide information to develop LeaLex-based cancer immunotherapeutics while avoiding anti-Lea autoimmune reactions. We characterized the epitope recognized by anti-Lea mAb SPM 522. We synthesized the Lea 6-aminohexyl glycoside and report experimental evidence of a minor conformation in solution. The Lea and three other 6-aminohexyl glycosides were conjugated to BSA and titration experiments with SPM 522 show that: 1. SPM 522 binds to LeaLex better than to Lea; 2. the non-reducing Lea galactosyl residue is essential to binding. Competitive ELISA experiments using a panel of tri- to pentasaccharide fragments of LeaLex as well as Lea analogues indicate that: 1. the Lea ß-d-galactosyl α hydrophobic patch is crucial to binding; 2. the Lea fucosyl residue contributes to binding; 3. the Lexd-galactosyl residue also contributes to binding. These results indicate that anti-Lea mAb SPM 522 recognizes the Lea[1,3]-ß-d-Gal tetrasaccharide. We propose that a major recognition element is the extended hydrophobic surface defined by the Lea-ß-d-Gal residue extending to the α faces of the ß-d-GlcNAc and ß-d-Gal residues.