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The Thomsen-Friedenreich (TF) antigen has proven to be a promising target for developing novel therapeutic cancer vaccines. Here, a new strategy that TF antigen covalently coupled with KRN7000 and vizantin was developed. The resulting three-component vaccine KRN7000-TF-vizantin simultaneously triggers invariant natural killer T (iNKT) cells and macrophage-inducible C-type lectin (Mincle) signaling pathways, eliciting much stronger TF-specific immune responses than glycoprotein vaccine TF-KLH/alum and the corresponding two-component conjugate vaccines TF-KRN7000 and TF-vizantin. The analysis of IgG isotypes and the secretion of cytokines revealed that KRN7000-TF-vizantin induced Th1/Th2 mixed immune responses, where Th1 was dominant. In vivo experiments demonstrated that KRN7000-TF-vizantin increased the survival rate and survival time of tumor-challenged mice, and surviving mice rejected further tumor attacks without any additional treatment. This work demonstrates that covalently coupled KRN7000 and vizantin could serve as a promising TF-based vaccine carrier for antitumor immune therapy, and KRN7000-TF-vizantin features great potential to be a vaccine candidate.

Interactions between the tumor-associated carbohydrate antigens of Mucin 1 (MUC1) and the carbohydrate-binding proteins, lectins, often lead to the creation of a pro-tumor microenvironment favoring tumor initiation, progression, metastasis, and immune evasion. Macrophage galactose binding lectin (MGL) is a C-type lectin receptor found on antigen-presenting cells that facilitates the uptake of carbohydrate antigens for antigen presentation, modulating the immune response homeostasis, autoimmunity, and cancer. Considering the crucial role of tumor-associated forms of MUC1 and MGL in tumor immunology, a thorough understanding of their binding interaction is essential for it to be exploited for cancer vaccine strategies. The synthesis of MUC1 glycopeptide models carrying a single or multiple Tn and/or sialyl-Tn antigen(s) is described. A novel approach for the sialyl-Tn threonine building block suitable for the solid phase peptide synthesis was developed. The thermodynamic profile of the binding interaction between the human MGL and MUC1 glycopeptide models was analyzed using isothermal titration calorimetry. The measured dissociation constants for the sialyl-Tn-bearing peptide epitopes were consistently lower compared to the Tn antigen and ranged from 10 μM for mono- to 1 μM for triglycosylated MUC1 peptide, respectively. All studied interactions, regardless of the glycan's site of attachment or density, exhibited enthalpy-driven thermodynamics., (© 2024 Wiley-VCH GmbH.)

Purpose: OBI-888 is a humanized, monoclonal IgG1 antibody specific to the tumor-associated carbohydrate antigen Globo H. We conducted a phase I-II study of OBI-888 in patients with advanced cancer., Methods: Patients were treated with OBI-888 5, 10, or 20 mg/kg IV weekly in Part A ("3 + 3" design) and 20 mg/kg IV weekly in Part B (Simon's 2-stage design) (1 cycle = 28 days)., Results: Overall, 54 patients were treated (Part A, n = 14; Part B, n = 40). OBI-888 was safe and well tolerated across the doses studied, with a low incidence of OBI-888-related treatment emergent adverse events. The maximum tolerated dose of OBI-888 was not reached. No dose-limiting toxicities were noted up to the 20 mg/kg dose level (recommended phase 2 dose). Stable disease (SD) was noted in 28.6% and 20% of Parts A and B, respectively, including three patients with SD for 6+, 7+, and 9 months. Antibody-dependent cellular cytotoxicity (ADCC) was induced after each OBI-888 treatment (average increase, 3.8-fold and 4.7-fold in Parts A and B, respectively), suggesting that ADCC induction is a potential mechanism of action of OBI-888., Conclusions: OBI-888 was well tolerated. Prolonged SD was noted in three patients. ADCC was induced after each OBI-888 treatment., Competing Interests: Declarations Competing interests Apostolia Maria Tsimberidou: Clinical Trial Research Funding (received through the institution): OBI Pharma, Agenus, Vividion, Macrogenics, AbbVie, IMMATICS, Novocure, Tachyon, Parker Institute for Cancer Immunotherapy, Tempus, and Tvardi; fees for consulting or advisory roles for Avstera Therapeutics, Bioeclipse, BrYet, Diaccurate, Macrogenics, NEX-I, and VinceRx. Dong Xu and M. Wayne Saville are employees of OBI Pharma USA, Inc. The remaining authors declare no relevant conflict of interest., (© 2024. The Author(s).)

Tn antigen is a tumor-associated carbohydrate antigen, which is present prominently on the tumor cell surfaces and attracts an interest in vaccine development. This work demonstrates that a synthetic Tn antigen carrying glycoconjugate forms a complex with circulating albumin, delivers the antigen to lymph nodes (LNs), and leads to the efficient production of antibodies against the antigen. Synthetic Tn antigen glycoconjugate, possessing DSPE-PEG2000 linker and lipophilic moieties, undergoes micellization in PBS buffer. In the presence of bovine serum albumin (BSA), demicellization of the glycolipid occurs, with a rate constant of 0.18 min -1 . In vitro studies show that the glycoconjugate binds preferentially to BSA in the presence of cells. Immunological assessments in mice models reveal the albumin-enabled delivery of the Tn glycoconjugate to antigen-presenting cells in the LNs, specifically leading to a robust humoral immune response. ELISA titers show superior binding, with a saturation dilution of 1:51 200 for Tn glycoconjugate, in comparison to that mediated by the Tn-BSA covalent conjugate with a saturation dilution of 1:6400. Immunohistochemical staining shows delivery of Tn glycoconjugate at the LNs, specifically at the subcapsular sinus and interfollicular areas. The work highlights the potential of albumin-mediated target delivery strategy for cancer immunotherapies., (© 2024 Wiley‐VCH GmbH.)

Glycosylation of cancerous epithelial MUC1 protein is specifically altered in comparison to that which is presented by healthy cells. One of such changes is appearing tumor-associated carbohydrate antigens (TACAs) which are rare in normal tissues and are highly correlated with poor clinical outcomes and cancer progression. This review summarizes and describes the role of Tn, T antigens, their sialylated forms as well as fucosylated Lewis epitopes in different aspects of tumor development, progression, and metastasis. Finally, applications of MUC1 glycan epitopes as potential targets for therapeutic strategy of cancers are notified. One of the novelties of this review is presentation of TACAs as inherently connected with MUC1 mucin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Glycans, chains of sugar molecules found conjugated to cell proteins and lipids, contribute to their growth, movement and differentiation. Aberrant glycosylation is a hallmark of several medical conditions including tumorigenesis. Glycosphingolipids (GSLs), consisting of glycans conjugated to a lipid (ceramide) core, are found in the lipid bilayer of eukaryotic cell membranes. GSLs, play an active role in cell processes. Several GSLs are expressed by human embryonic stem cells and have been found to be overexpressed in several types of cancer. In this review, we discuss the data, hypotheses and perspectives related to the GSLs Globo H and SSEA-4.

Recent studies support the development of cancer therapeutics to target Globo H-ceramide, the most prevalent tumor-associated carbohydrate antigen in epithelial cancers. Herein, we evaluated the expression of Globo H and its prognostic significance in intrahepatic cholangiocarcinoma (ICC) and conducted preclinical studies to assess the antitumor activity of Globo H-specific antibody in thioacetamide (TAA)-induced ICC in rats. Globo H-ceramide in tumor specimens was detected by immunohistochemistry (IHC) and mass spectrometry. Antitumor efficacy of anti-Globo H mAbVK9 was evaluated in TAA-induced ICC in rat. Natural killer (NK) cells and their related genes were analyzed by IHC and quantitative real-time polymerase chain reaction. Data mining revealed that B3GALT5 and FUT2, the key enzymes for Globo H biosynthesis, were significantly up-regulated in human ICC. In addition, Globo H expression was detected in 41% (63 of 155) of ICC tumor specimens by IHC staining, and validated by mass spectrometric analysis of two IHC-positive tumors. Patients with Globo H positive tumors had significantly shorter relapse-free survival (RFS) and overall survival (P = 0.0003 and P = 0.002, respectively). Multivariable Cox regression analysis identified Globo H expression as an independent unfavorable predictor for RFS (hazard ratio: 1.66, 95% confidence interval: 1.08-2.36, P = 0.02) in ICC. Furthermore, gradual emergence of Globo H in liver tissues over 6 months in TAA-treated rats recapitulated the multistage progression of ICC in vivo. Importantly, administration of anti-Globo H mAbVK9 in rats bearing TAA-induced ICC significantly suppressed tumor growth with increased NK cells in the tumor microenvironment. Conclusion: Globo H is a theranostic marker in ICC., (© 2021 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

The Tn antigen, an N-acetylgalactosamine structure linked to serine or threonine, has been shown to induce high-specificity, high-affinity anti-Tn antibodies in mice. Maternal immunization with the Tn vaccine increases serum anti-Tn antibody titers and attenuates hyperoxia-induced kidney injury in neonatal rats. However, immunizing mothers to treat neonatal kidney disease is clinically impractical. This study is aimed at determining whether anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury in neonatal mice. Newborn BALB/c mice were exposed to room air (RA) or normobaric hyperoxia (85% O 2 ) for 1 week. On postnatal days 2, 4, and 6, the mice were injected intraperitoneally with PBS alone or with anti-Tn monoclonal antibodies at 25  μ g/g body weight in 50  μ L phosphate-buffered saline (PBS). The mice were divided into four study groups: RA + PBS, RA + anti-Tn monoclonal antibody, O 2 + PBS, and O 2 + anti-Tn monoclonal antibody. The kidneys were excised for histology, oxidative stress, cytokine, and Western blot analyses on postnatal day 7. The O 2 + PBS mice exhibited significantly higher kidney injury scores, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear factor- κ B (NF- κ B) expression, and cytokine levels than did the RA + PBS mice or RA + anti-Tn mice. Anti-Tn monoclonal antibody treatment reduced kidney injury and cytokine levels to normoxic levels. The attenuation of kidney injury was accompanied by a reduction of oxidative stress and NF- κ B expression. Therefore, we propose that anti-Tn monoclonal antibody treatment ameliorates hyperoxia-induced kidney injury by suppressing oxidative stress and inflammation in neonatal mice., Competing Interests: The authors have indicated no financial conflicts of interest., (Copyright © 2021 Julie Chi Chow et al.)

Tn is a tumor-associated carbohydrate antigen that constitutes both a diagnostic tool and an immunotherapeutic target. It originates from interruption of the mucin O-glycosylation pathway through defects involving, at least in part, alterations in core-1 synthase activity, which is highly dependent on Cosmc, a folding chaperone. Tn antigen is recognized by the Macrophage Galactose-type Lectin (MGL), a C-type lectin receptor present on dendritic cells and macrophages. Specific interactions between Tn and MGL shape anti-tumoral immune responses by regulating several innate and adaptive immune cell programs. In this work, we generated and characterized a variant of the lung cancer murine cell line LL/2 that expresses Tn by mutation of the Cosmc chaperone gene (Tn + LL/2). We confirmed Tn expression by lectin glycophenotyping and specific anti-Tn antibodies, verified abrogation of T-synthase activity in these cells, and confirmed its recognition by the murine MGL2 receptor. Interestingly, Tn + LL/2 cells were more aggressive in vivo, resulting in larger and highly vascularized tumors than those generated from wild type Tn - LL/2 cells. In addition, Tn + tumors exhibited an increase in CD11c + F4/80 + cells with high expression of MGL2, together with an augmented expression of IL-10 in infiltrating CD4 + and CD8 + T cells. Importantly, this immunosuppressive microenvironment was dependent on the presence of MGL2 + cells, since depletion of these cells abrogated tumor growth, vascularization and recruitment of IL-10 + T cells. Altogether, our results suggest that expression of Tn in tumor cells and its interaction with MGL2-expressing CD11c + F4/80 + cells promote immunosuppression and angiogenesis, thus favoring tumor progression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

Cancer-associated antigens are not only a good marker for monitoring cancer progression but are also useful for molecular target therapy. In this study, we aimed to generate a monoclonal antibody that preferentially reacts with colorectal cancer cells relative to noncancerous gland cells. We prepared antigens composed of HT-29 colorectal cancer cell lysates that were adsorbed by antibodies to sodium butyrate-induced enterocytically differentiated HT-29 cells. Subsequently, we generated a monoclonal antibody, designated 12G5A, which reacted with HT-29 colon cancer cells, but not with sodium butyrate-induced differentiated HT-29 cells. Immunohistochemical staining revealed 12G5A immunoreactivity in all 73 colon cancer tissue specimens examined at various degrees, but little or no immunoreactivity in noncancerous gland cells. Notably, high 12G5A immunoreactivity, which was determined as more than 50% of colon cancer cells intensively stained with 12G5A antibody, exhibited significantly higher association with a poor overall survival rate of patients with colorectal cancer (P = 0.0196) and unfavorable progression-free survival rate of patients with colorectal cancer (P = 0.0418). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, si-RNA silencing analysis, enzymatic deglycosylation, and tunicamycin treatment revealed that 12G5A recognized the glycosylated epitope on annexin A2 protein. Our findings indicate that 12G5A identified a cancer-associated glycosylation epitope on annexin A2, whose expression was related to unfavorable colorectal cancer behavior. KEY MESSAGE: • 12G5A monoclonal antibody recognized a colorectal cancer-associated epitope. • 12G5A antibody recognized the N-linked glycosylation epitope on annexin A2. • 12G5A immunoreactivity was related to unfavorable colorectal cancer behavior., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The aim of this work was to evaluate the physicochemical and biological properties of docetaxel (DCX) loaded chitosan nanocapsules (CS Nc) functionalized with the monoclonal antibody Chi-Tn (CS-PEG-ChiTn mAb Nc) as a potential improvement treatment for cancer therapy. The Tn antigen is highly specific for carcinomas, and this is the first time that such structure is targeted for drug delivery. The nanocapsules (Ncs), formed as a polymeric shell around an oily core, allowed a 99.9% encapsulation efficiency of DCX with a monodispersity particle size in the range of 200 nm and a high positive surface charge that provide substantial stability to the nanosystems. Release profile of DCX from Ncs showed a sustained and pH dependent behavior with a faster release at acidic pH, which could be favorable in the intracellular drug delivery. We have designed PEGylated CS Nc modified with a monoclonal antibody which recognize Tn antigen, one of the most specific tumor associated antigen. A biotin-avidin approach achieved the successful attachment of the antibody to the nanocapsules. Uptake studies and viability assay conducted in A549 human lung cancer cell line in vitro demonstrate that ChiTn mAb enhance nanoparticles internalization and cell viability reduction. Consequently, these ChiTn functionalized nanocapsules are promising carriers for the active targeting of DCX to Tn expressing carcinomas., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Tumor cells are characterized by the expression of tumor-specific carbohydrate structures that differ from their normal counterparts. Carbohydrates on tumor cells have phenotypical as well as functional implications, impacting the tumor progression process, from malignant transformation to metastasis formation. Importantly, carbohydrates are structures that play a role in receptor-ligand interaction and elicit the activity of growth factor receptors, integrins, lectins, and other type 1 transmembrane proteins. They have been recognized as biomarkers for cancer diagnosis, and evidence demonstrating their relevance as targets for anticancer therapeutic strategies, including immunotherapy, continues to accumulate. Different approaches targeting carbohydrates include monoclonal antibodies (mAbs), antibody (Ab)-drug conjugates, vaccines, and adhesion antagonists. Development of bispecific antibodies and chimeric antigen receptor (CAR)-modified T cells against tumor-associated carbohydrate antigens (TACAs) as promising cancer immunotherapeutic agents is rapidly evolving. As reviewed here, there are several cancer-associated glycan features that can be leveraged to design rational drug or immune system targets, applying multiple TACA structural and functional features to be targeted as the standard treatment paradigm. Many of the underlying targets were defined by researchers at the Wistar Institute in Philadelphia, Pennsylvania, which provide basis for different immunotherapy approaches.

Background: Hepatocellular carcinoma-associated antigen 59 (HCA59) from excretory/secretory products of Haemonchus contortus is known to have the ability to modulate the functions of host cells. However, its immunogenicities using different nanoparticles adjuvants remain poorly understood., Purpose: The study aimed to select an efficient nanoparticle antigen delivery system, which could enhance the immune responses of Haemonchus contortus HCA59 in mice., Methods: Here, the immune responses induced by the recombinant protein of HCA59 (rHCA59) with poly-D,L-lactide-co-glycolide (PLGA) nanoparticles, Chitosan nanoparticles, mixture of PLGA and Chitosan nanoparticles (rHCA59-Chitosan-PLGA), and Freund's complete adjuvant were observed, respectively, in mice. Cytokine and antibody levels induced by different groups were detected by ELISA assay. The effects of lymphocyte proliferations on different groups were examined using CCK-8 kit. Phenotypes of T cells and dendritic cells were analyzed by flow cytometry., Results: On day 14 post vaccination, levels of IgM, IgG1, IgG2a, IFN-γ, IL-4, and IL-17 were significantly increased in the groups immunized with rHCA59 encapsulated with nanoparticles. After mice were vaccinated with rHCA59 loaded with Chitosan/PLGA nanoparticles, lymphocytes proliferated significantly. Additionally, the percentages of CD4 + T cells (CD3 + CD4 + ), CD8 + T cells (CD3 + CD8 + ), and dendritic cells (CD11c + CD83 + , CD11c + CD86 + ) were obviously up-regulated in the mice immunized with nanoparticles, especially in the rHCA59-Chitosan-PLGA antigen delivery system group., Conclusion: The findings of this research demonstrated that rHCA59-Chitosan-PLGA antigen delivery system could induce higher immune responses in mice model and indicated that rHCA59 might be a good candidate molecule to develop nanovaccines against Haemonchus contortus in future study., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Wang et al.)

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N- acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O- glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N- acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize Lewis X and GalNAc, respectively.

Rationale: Advanced signet ring cell (SRC) carcinoma has a worse prognosis. Therefore, early diagnosis and prevention is particularly important; SRC tumors have lower R0 resection rate and are thought to be less chemosensitive than non-SRCC. Consequently, a novel postoperative adjuvant treatment is urgently needed to improve clinical outcomes., Patient Concerns: A 41-year-old female with advanced gastric SRC carcinoma was treated with radical gastrectomy and oxaliplatin-based regimen for 6 cycles after surgery. She was suspected of recurrence with the high level of carbohydrate antigen (CA) 72-4., Diagnoses: The gastroscopy revealed SRC carcinoma of gastric antrum and poorly differentiated adenocarcinoma in some areas. The diagnosis of postoperative pathology report was gastric cancer with stage III C (T4a, N3a, M0)., Interventions: The level of CA72-4 rapidly increased during the 2 follow-up after the completion of conventional treatment, ex vivo-cultured allogeneic natural killer (NK) cell infusion was offered to prevent recurrence., Outcomes: Intravenous injections of NK cells combination with surgical treatment and chemotherapy showed therapeutic effects in this patient with possible relapse. The patient remained disease-free 46 months after the infusion of NK cells until the latest follow-up., Lessons: CA72-4 appeared to be the most sensitive and specific marker in the gastric cancer patient, and the high level of CA72-4 may indicate the risk of recurrence. This case report provide rationale for NK cell infusion following the rapid increase of CA72-4 to prevent recurrence., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Neonatal hyperoxia increases oxidative stress and adversely disturbs glomerular and tubular maturity. Maternal Tn immunization induces anti-Tn antibody titer and attenuates hyperoxia-induced lung injury in neonatal rats., Methods: We intraperitoneally immunized female Sprague-Dawley rats (6 weeks old) with Tn immunogen (50 μg/dose) or carrier protein five times at biweekly intervals on 8, 6, 4, 2, and 0 weeks before the delivery day. The pups were reared for 2 weeks in either room air (RA) or in 85% oxygen-enriched atmosphere (O 2 ), thus generating four study groups, namely carrier protein + RA, Tn vaccine + RA, carrier protein + O 2 , and Tn vaccine + O 2 . On postnatal day 14, the kidneys were harvested for the oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), nuclear factor-κB (NF-κB), and collagen expression and histological analyses., Results: Hyperoxia reduced body weight, induced tubular and glomerular injuries, and increased 8-OHdG and NF-κB expression and collagen deposition in the kidneys. By contrast, maternal Tn immunization reduced kidney injury and collagen deposition in neonatal rats. Furthermore, kidney injury attenuation was accompanied by a reduction in 8-OHdG and NF-κB expression., Conclusion: Maternal Tn immunization protects against hyperoxia-induced kidney injury in neonatal rats by attenuating oxidative stress and NF-κB activity., Impact: Hyperoxia increased nuclear factor-κB (NF-κB) activity and collagen deposition in neonatal rat kidney. Maternal Tn immunization reduced kidney injury as well as collagen deposition in neonatal rats. Maternal Tn immunization reduced kidney injury and was associated with a reduction in 8-hydroxy-2'-deoxyguanosine and NF-κB activity. Tn vaccine can be a promising treatment modality against hyperoxia-induced kidney injury in neonates.

Purpose: A subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that a glycan biomarker for PDACs called sialylated tumor-related antigen (sTRA) could be used for this purpose., Experimental Design: We tested for differences between PDACs classified by glycan expression in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from human subjects., Results: The sTRA-expressing models tended to have stem-like gene expression and the capacity for mesenchymal differentiation, in contrast to the nonexpressing models. The sTRA cell lines also had significantly increased resistance to seven different chemotherapeutics commonly used against pancreatic cancer. Patients with primary tumors that were positive for a gene expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, based on direct measurements of sTRA in tissue microarrays, the patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Furthermore, a blood plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy., Conclusions: This research demonstrates that a glycan biomarker could have value to detect chemotherapy-resistant PDAC in clinical settings. This capability could aid in the development of stratified treatment plans and facilitate biomarker-guided trials targeting resistant PDAC., (©2020 American Association for Cancer Research.)

Malignant transformation is often associated with abnormal protein glycosylation expressed, amongst others, by the accumulation of simple mucin-type carbohydrates namely Tn and Sialyl-Tn (STn) antigens. These are usually limited in normal tissues and their increased expression has been associated with cancer progression and poor prognosis. This study aims to evaluate the role of Tn and STn antigens in the neoplastic transformation of the canine gastric mucosa and to correlate their putative immunoexpression alterations with some pathological features. Tn and STn antigens expression were immunohistochemically evaluated in canine normal gastric mucosa (n = 3), gastric polyps (n = 9) and gastric carcinomas (n = 25), neoplastic emboli (n = 12) and metastases (n = 8). In normal gastric mucosa, Tn antigen was detected in the gastric epithelial cells, while STn antigen was absent. Similarly, all gastric polyps expressed Tn antigen, but none displayed STn antigen immunostaining. In carcinomas, Tn antigen was expressed in 96% of the cases and STn antigen in 68% of the neoplasms. STn antigen was significantly higher in carcinomas compared with normal mucosa (P < .05). No correlation was found between each antigen and the different subtypes of tumours according to WHO classification, tumour differentiation, lymph vascular invasion or metastasis. All neoplastic emboli expressed both antigens, and the expression score was similar or higher than that displayed by the neoplastic cells of the primary tumour. The high prevalence of STn antigen in gastric carcinomas compared with normal mucosa highlights the cancer-associated nature of this antigen. Our results link STn antigen expression to neoplastic transformation and suggest that it may be a useful marker of gastric cancer progression in dogs., (© 2020 John Wiley & Sons Ltd.)

Objective: To estimate the diagnostic value of tumor and immune related proteins in the discrimination between benign and malignant adnexal masses, and between different subgroups of tumors., Methods: In this exploratory diagnostic study, 254 patients with an adnexal mass scheduled for surgery were consecutively enrolled at the University Hospitals Leuven (128 benign, 42 borderline, 22 stage I, 55 stage II-IV, and 7 secondary metastatic tumors). The quantification of 33 serum proteins was done preoperatively, using multiplex high throughput immunoassays (Luminex) and electrochemiluminescence immuno-assay (ECLIA). We calculated univariable areas under the Receiver Operating Characteristic Curves (AUCs). To discriminate malignant from benign tumors, multivariable ridge logistic regression with backward elimination was performed, using bootstrapping to validate the resulting AUCs., Results: CA125 had the highest univariable AUC to discriminate malignant from benign tumors (0.85, 95% confidence interval 0.79-0.89). Combining CA125 with CA72.4 and HE4 increased the AUC to 0.87. For benign vs borderline tumors, CA125 had the highest univariable AUC (0.74). For borderline vs stage I malignancy, no proteins were promising. For stage I vs II-IV malignancy, CA125, HE4, CA72.4, CA15.3 and LAP had univariable AUCs ≥0.80., Conclusions: The results confirm the dominant role of CA125 for identifying malignancy, and suggest that other markers (HE4, CA72.4, CA15.3 and LAP) may help to distinguish between stage I and stage II-IV malignancies. However, further research is needed, also to investigate the added value over clinical and ultrasound predictors of malignancy, focusing on the differentiation between subtypes of malignancy., Competing Interests: Declaration of Competing Interest TB reports conflicts of interests with Roche Diagnostics, outside the submitted work. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aberrant Mucin-1 (MUC1) glycosylation with the Thomsen-Friedenreich (TF) tumor-associated antigen (CD176) is a hallmark of epithelial carcinoma progression and poor patient prognosis. Recognition of TF by glycan-binding proteins, such as galectins, enables the pathological repercussions of this glycan presentation, yet the underlying binding specificities of different members of the galectin family is a matter of continual investigation. While Galectin-3 (Gal-3) recognition of TF has been well-documented at both the cellular and molecular level, Galectin-1 (Gal-1) recognition of TF has only truly been alluded to in cell-based platforms. Immunohistochemical analyses have purported Gal-1 binding to TF on MUC1 at the cell surface, however binding at the molecular level was inconclusive. We hypothesize that glycan scaffold (MUC1's tandem repeat peptide sequence) and/or multivalency play a role in the binding recognition of TF antigen by Gal-1. In this study we have developed a method for large-scale expression of Gal-1 and its histidine-tagged analog for use in binding studies by isothermal titration calorimetry (ITC) and development of an analytical method based on AlphaScreen technology to screen for Gal-1 inhibitors. Surprisingly, neither glycan scaffold or multivalent presentation of TF antigen on the scaffold was able to entice Gal-1 recognition to the level of affinity expected for functional significance. Future evaluations of the Gal-1/TF binding interaction in order to draw connections between immunohistochemical data and analytical measurements are warranted.

Mucin-type O-glycans are involved in cancer initiation and progression, although details of their biological and clinicopathological roles remain unclear. The aim of this study was to investigate the clinicopathological significance of β1,3-N-acetylglucosaminyltransferase 6 (β3Gn-T6), an essential enzyme for the synthesis of core 3 O-glycan and several other O-glycans in pancreatic ductal adenocarcinoma (PDAC). We performed immunohistochemical and lectin-histochemical analyses to detect the expression of β3Gn-T6 and several O-glycans in 156 cases of PDAC with pancreatic intraepithelial neoplasias (PanINs) and corresponding normal tissue samples. The T antigen, Tn antigen, sialyl Lewis X (sLeX) antigen, and sLeX on core 2 O-glycan were more highly expressed in PDAC cells than in normal pancreatic duct epithelial cells (NPDEs). Conversely, the expression of 6-sulfo N-acetyllactosamine on extended core 1 O-glycan was found in NPDEs and was low in PDAC cells. These glycan expression levels were not associated with patient outcomes. β3Gn-T6 was expressed in ~20% of PDAC cases and 30-40% of PanINs but not in NPDEs. Higher expression of β3Gn-T6 was found in PDAC cells in more differentiated adenocarcinoma cases showing significantly longer disease-free survival in both univariate and multivariate analyses. In addition, the expression of β3Gn-T6 in PDAC cells and PanINs significantly correlated with the expression of MUC5AC in these cells, suggesting that β3Gn-T6 expression is related to cellular differentiation status of the gastric foveolar phenotype. Thus, it is likely that β3Gn-T6 expression in PDAC cells is a favorable prognostic factor in PDAC patients, and that the expression of β3Gn-T6 correlates with the gastric foveolar phenotype in pancreatic carcinogenesis., Competing Interests: The authors have declared that no competing interests exist.

Rhamnose and sTn antigen were co-conjugated to bovine serum albumin (BSA), a weakly immunogenic carrier protein, for cancer vaccine development. The immune responses against sTn have been significantly augmented with the involvement of Rha-specific antibodies to enhance antigen uptake.

The monosaccharide Tn and the disaccharide STn are tumor antigens with similar structures and common biosynthetic pathways. Both are always over-expressed simultaneously on tumor cell surfaces. We report herein the efficient synthesis of the STnThr antigen analogue 2, featuring the immunogenic TnThr mimetic 1 aglycon. Analogously to the native STn, 2 is recognized by the influenza N1 neuraminidase. A model of the N1·2 complex showed the sialyl moiety of 2 well nested in the active site pocket, with docking unaffected by the rigid aglycon. The analogue 2 is, therefore, in association with mimetic 1, a good determinant for the design of new multiantigen cancer vaccines.

Tn antigen is a tumor-associated antigen that appears on cancer cells as a result of aberrant O-glycosylation. The most studied form of Tn antigen is found in mucins, in particular, in mucin 1 (MUC1). Antibodies against this form of Tn antigen are used to diagnose tumors, as well as to generate T-killers with a chimeric receptor. Some carcinomas do not carry MUC1 and antibodies of a different specificity are required to detect Tn antigen on these cells. In our work, we searched for anti-Tn antibodies without preliminary assumptions about the proteins that may be carriers of the Tn antigen. For this purpose, we obtained several pairs of isogenic cell lines with the wild type and knockout of the Cosmc gene, which is essential for correct protein O-glycosylation. Using the created lines as immunogens, we generated a monoclonal antibody AKC3, which reacted with the Cosmc-deficient A549 lung adenocarcinoma cells and did not bind to the wild-type cells. Using mass spectrometry, as well as co-immunoprecipitation, it was shown that the AKC3 antibody recognized the Tn antigen in the context of CD44 protein - a protein important for tumor growth. The AKC3 antibody can be used for tumor diagnosis, and to generate T cells with a chimeric receptor for treatment of tumors that do not express mucins.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris . In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn + cell lines was higher than that of granulysin alone.

In many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α- O -GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity. Two different glycopeptide libraries presenting the natural Tn antigen or the sp 2 -iminosugar analogue were synthesized and evaluated with anti-MUC1 monoclonal antibodies in a microarray platform. The most promising candidates were tested with healthy and breast cancer sera aiming for potential autoantibody-based biomarkers. The suitability of sp 2 -iminosugar glycopeptides to detect anti-MUC1 antibodies was demonstrated, and serological experiments showed stage I breast cancer autoantibodies binding with a specific unnatural glycopeptide with almost no healthy serum interaction. These results will promote further studies on their capabilities as early cancer biomarkers.

Electrochemical immunosensors have been developed to determine the carbohydrate antigen 19-9 (CA19-9). They are based on screen-printed carbon electrodes (SPCEs) coated with layer-by-layer (LbL) films of carbon black (CB) and polyelectrolytes. Owing to a suitable choice of LbL film architecture, the procedures for immobilization of anti-CA19-9 antibodies on the electrode surfaces were straightforward. Mechanically flexible immunosensors were capable of detecting CA19-9 within a dynamic range of 0.01 to 40 U mL -1 and a limit of detection of 0.07 U mL -1 using differential pulse voltammetry. In addition to detecting CA19-9 at clinically relevant concentrations for pancreatic cancer in standard solutions, the immunosensors provide the determination of CA19-9 on cell lysate and human serum samples. Using LbL films led to immunosensors with superior performance compared to similar systems obtained by drop casting. The fabrication of this relatively simple, inexpensive platform is a demonstration that SPCEs modified with cost-effective materials are able to detect cancer biomarkers and may be adapted to other disposable immunosensors. Graphical abstract Schematic representation of assembly and characterization of electrochemical immunosensors for the determination of carbohydrate antigen 19-9 based on printed electrodes modified with composites of carbon black and polyelectrolyte films.

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N -acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors., Competing Interests: Competing interest statement: P.S., Y.H., K.S., H.S., and D.M.K. are listed as inventors on a US patent application involving this study. Q.C. is employed by Gilead Sciences, Inc. D.M.K. is a consultant for AbbVie. H.C. is a co-inventor on patents related to antibodies to Tn-glycopeptides, including Tn-MUC1 held by the University of Copenhagen. H.C. is co-founder of GlycoDisplay Aps and GO Therapeutics Inc., formerly GlycoZym Inc.

Tumor-associated carbohydrate antigens Lewis X (Le x ), Lewis Y (Le y ), and KH-1 are useful targets for cancer immunotherapy. In this regard, an insight into the structure-immunogenicity relationships of these antigens is important but this has not been systematically investigated yet. In the current study, Le x , Le y , and KH-1 antigens with a lactose unit at the reducing end as a spacer were synthesized and coupled with keyhole limpet hemocyanin (KLH) protein. Immunological evaluations of the resultant conjugates revealed that they all could elicit robust immune responses whilst the Le y conjugate could provoke the highest titers of total and IgG antibodies. The binding assays of their antisera to each antigen and to cancer cells showed that each antiserum had extensive cross-reaction with all three antigens as protein conjugates and strong but somewhat antigen-selective binding towards MCF-7 cancer cell. Moreover, none of these antisera had obvious binding to SKMEL-28 cancer cell that does not express Le x , Le y and KH-1. The results of assays of these antisera to mediate complement-dependent cytotoxicity (CDC) to MCF-7 and SKMEL-28 cancer cells were very similar to the results of binding assays. Thus, it was concluded that all three antigens could form effective conjugate vaccines whereas the Le y conjugate induced the most robust immune responses and the antiserum of Le x had the highest binding and cytotoxicity to target cancer cells. In addition, as the antibodies induced by each antigen had extensive cross-reaction with other two antigens, either Le x or Le y or the two combined can be utilized to formulate effective conjugate vaccines for cancer immunotherapy. Another paradigm-shifting discovery of this study is that the presentation of Le x , Le y , and KH-1 antigens on cancer cell can be different from that in synthetic conjugates, which should be taken into consideration during the design and optimization of related cancer vaccines or immunotherapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn)., Methods: The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM., Results: The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM., Conclusion: In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT 830-844 CD4 + T-cell epitope. This promiscuous CD4 + T-cell epitope can bind to a wide range of HLA-DRB molecules and is thus expected to activate CD4 + T-cell responses in a large part of the human population. The MAG-Tn3 vaccine was formulated with the GSK-proprietary immunostimulant AS15, composed of CpG7909, MPL, and QS21, which has been shown to stimulate both innate and humoral responses, in addition to being well tolerated. Here, seven patients with localized breast cancer with a high-risk of relapse were immunized with the MAG-Tn3 vaccine formulated with AS15. The first results of phase I clinical trial demonstrated that all vaccinated patients developed high levels of Tn-specific antibodies. Moreover, these antibodies specifically recognized Tn-expressing human tumor cells and killed them through a complement-dependent cytotoxicity mechanism. Overall, this study establishes, for the first time, the capacity of a fully synthetic glycopeptide cancer vaccine to induce specific immune responses in humans.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galβ1-3(4)GlcNAcβ. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs., Competing Interests: Declaration of Competing Interest Authors declare there is no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The Thomsen-Friedenreich (TF) antigen is expressed in a majority of human tumors due to aberrant glycosylation in cancer cells. There is strong evidence that humoral immune response to TF represents an effective mechanism for the elimination of cancer cells that express TF-positive glycoconjugates. The presence of naturally occurring antibodies to tumor-associated TF and cancer-specific changes in their levels, isotype distribution and interrelation, avidity, and glycosylation profile make these Abs a convenient and ubiquitous marker for cancer diagnostics and prognostics. In this review, we attempt to summarize the latest data on the potential of TF-specific Abs for cancer diagnostics and prognostics., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Oleg Kurtenkov.)

The Thomsen-Friedenreich (TF) antigen is a key target for the development of anticancer vaccines, and this ongoing challenge remains relevant due to the poor immunogenicity of the TF antigen. To overcome this challenge, we adopted a bivalent conjugate design which introduced both the TF antigen and the Thomsen-nouveau (Tn) antigen onto the immunologically relevant polysaccharide A1 (PS A1). The immunological results in C57BL/6 mice revealed that the bivalent, Tn-TF-PS A1 conjugate increased the immune response towards the TF antigen as compared to the monovalent TF-PS A1. This phenomenon was first observed with enzyme-linked immunosorbent assay (ELISA) where the bivalent conjugate generated high titers of IgG antibodies where the monovalent conjugate generated an exclusive IgM response. Fluorescence-activated cell sorting (FACS) analysis also revealed increased binding events to the tumor cell lines MCF-7 and OVCAR-5, which are consistent with the enhanced tumor cell lysis observed in a complement dependent cytotoxicity (CDC) assay. The cytokine profile generated by the bivalent construct revealed increased pro-inflammatory cytokines IL-17 and IFN-γ. This increase in cytokine concentration was matched with an increase in cytokine producing cells as observed by ELISpot. We hypothesized the mechanisms for this phenomenon to involve the macrophage galactose N -acetylgalactosamine specific lectin 2 (MGL2). This hypothesis was supported by using biotinylated probes and recombinant MGL2 to measure carbohydrate-protein interactions.

Structuring of noble metal nanoparticles on transition metal dichalcogenide nanosheets induces significantly enhanced electronic, optical, and catalytic functions. However, the synthesis of multifunctional hybrids is always time-consuming and involves multiple steps. Herein, a ternary Au nanoparticle-ferrocene-WS 2 nanosheet (AFW) composite has been prepared by a facile one-step sonochemical approach. Stripped WS 2 nanosheets were functionalized with ferrocene monocarboxylic acid (FMC) and gold nanoparticles (AuNPs) by making use of the strong coordinative interaction of carboxyl groups with tungsten atoms. The AuNPs decorating the WS 2 nanosheet not only increase the water solubility of WS 2 nanosheet and surface area of the modified electrode, but also act as electron transport bridges to aid the tunneling of electrons from the small redox molecule, FMC, through the space to the electrode on which they are mounted. Furthermore, the ternary AFW nanocomposite could effectively avoid the leaching of FMC from the nanocomposite matrix and provided a suitable environment for the immobilized biomolecules. Combining the immune magnetic beads technology and the AFW nanocomposite with aforementioned advantages, a high-performance electrochemical immunosensor was successfully developed using carbohydrate antigen 72-4 (CA72-4) as a model analyte. A linear relationship in the range of 2-50 U/L for the detection of CA72-4 was found with a low detection limit of 0.6 U/L. In addition, the biosensor showed excellent performance in selectivity, stability, and reproducibility. Thus, this work not only proposes a facile avenue for preparing a 2D WS 2 nanocomposite with multifunctional properties but also opens up a new method to extend the application of WS 2 -based materials in biological sensing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: The TF (Thomsen-Friedenreich, CD176, Galβ1-3GalNAc) carbohydrate moiety is known as a specific oncofetal carbohydrate epitope present in fetal and neoplastic tissue as well as in stem cells. TF was demonstrated to mediate tumor-promoting features and to be highly immunogenic. The current study aimed to evaluate whether presence of the TF antigen is associated with clinico-pathological parameters and prognosis of early breast cancer (BC)., Methods: Primary BC tissue (n = 226) was stained for TF using two monoclonal anti-TF antibodies (Nemod-TF1, Nemod-TF2). Staining results were correlated to clinical data including survival., Results: Nemod-TF1 staining was positively correlated to lymph node metastasis (p = 0.03) and the presence of tumor-associated MUC1 (TA-MUC1; p = 0.003). Further, the presence of the Nemod-TF1 epitope predicted worse prognosis in TA-MUC1 positive (overall survival: p = 0.026) as well as in triple negative (overall survival: p = 0.002; distant metastasis-free survival: p = 0.012) BC., Conclusions: The data presented here further support a role of TF in BC tumor biology. Whether anti-TF directed treatment approaches may gain clinical relevance in those cases determined as triple negative or TA-MUC1 positive remains to be determined.

Tumor associated carbohydrate antigens (TACAs) are a class of attractive antigens for the development of anti-cancer immunotherapy. Besides monoclonal antibodies and vaccines, chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs) targeting TACA are exciting directions to harness the power of the immune system to fight cancer. In this review, we focus on two TACAs, i.e., the GD2 ganglioside and the mucin-1 (MUC1) protein. The latest advances in CAR T cells and bispecific antibodies targeting these two antigens are presented. The roles of co-stimulatory molecules, structures of the sequences for antigen binding, methods for CAR and antibody construction, as well as strategies to enhance solid tumor penetration and reduce T cell exhaustion and death are discussed. Furthermore, approaches to reduce "on target, off tumor" side effects are introduced. With further development, CAR T cells and BsAbs targeting GD2 and MUC1 can become powerful agents to effectively treat solid tumor., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. It has frequently been reported that MUC1, the heavily glycosylated cell-surface mucin, is altered in both, expression and glycosylation pattern, in human carcinomas of the epithelium. The presence of incomplete or truncated glycan structures, often capped by sialic acid, commonly known as tumor-associated carbohydrate antigens (TACAs), play a key role in tumor initiation, progression, and metastasis. Accumulating evidence suggests that expression of TACAs is associated with tumor escape from immune defenses. In this report, we will give an overview of the oncogenic functions of MUC1 that are exerted through TACA interactions with endogenous carbohydrate-binding proteins (lectins). These interactions often lead to creation of a pro-tumor microenvironment, favoring tumor progression and metastasis, and tumor evasion. In addition, we will describe current efforts in the design of cancer vaccines with special emphasis on synthetic MUC1 glycopeptide vaccines. Analysis of the key factors that govern structure-based design of immunogenic MUC1 glycopeptide epitopes are described. The role of TACA type, position, and density on observed humoral and cellular immune responses is evaluated., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

MUC1 is a membrane glycoprotein, which in adenocarninomas is overexpressed and exhibits truncated O-glycosylation. Overexpression and altered glycosylation make MUC1 into a candidate for immunotherapy. Monoclonal antibodies directed against MUC1 frequently bind an immunodominant epitope that contains a single site for O-glycosylation. Glycosylation with tumor carbohydrate antigens such as the Tn-antigen (GalNAc-O-Ser/Thr) results in antibodies binding with higher affinity. One proposed model to explain the enhanced affinity of antibodies for the glycosylated antigen is that the addition of a carbohydrate alters the conformational properties, favoring a binding-competent state. The conformational effects associated with Tn glycosylation of the MUC1 antigen was investigated using solution-state NMR and molecular dynamics. NMR experiments revealed distinct substructures of the glycosylated MUC1 peptides compared with the unglycosylated peptide. Molecular dynamics simulations of the MUC1 glycopeptide and peptide revealed distinguishing differences in their conformational preferences. Furthermore, the glycopeptide displayed a smaller conformational sampling compared with the peptide, suggesting that the glycopeptide sampled a narrower conformational space and is less dynamic. A comparison of the computed ensemble of conformations assuming random distribution, NMR models, and molecular dynamics simulations indicated that the MUC1 glycopeptide and aglycosylated peptide sampled structurally distinctly ensembles and that these ensembles were different from that of the random coil. Together, these data support the hypothesis that that conformational pre-selection could be an essential feature of these peptides that dictates the binding affinities to MUC1 specific antibodies., (© 2019 European Peptide Society and John Wiley & Sons, Ltd.)

The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc- O -Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V vs. Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L -1 . Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.

Background: The Sialyl-Thomsen-nouveau antigen (STn) is abundantly produced on many types of human epithelial cancers including epithelial ovarian cancer (EOC). We previously developed an EOC-specific lectin sandwich immunoassay (CA125 MGL ) using a human macrophage galactose-binding lectin coated on fluorescent europium nanoparticles (Eu +3 -NPs) as a tracer and an anti-CA125-specific mAb for capture. Here we have identified a novel STn-mAb that efficiently recognizes the EOC-associated STn antigen on CA125 when coated on Eu +3 -NPs., Method: CA125 from the ovarian cancer cell line OVCAR-3, placental homogenate, and ascites fluid from patients with liver cirrhosis was captured by anti-CA125 antibody immobilized on microtitration wells and traced with anti-STn-mAb-Eu +3 -NPs. Samples from EOC or patients with endometriosis with marginally increased conventional CA125 immunoassay (CA125 IA ; 35-200 U/mL) and healthy controls were analyzed., Results: An analytically sensitive CA125 STn assay that specifically recognized the CA125 isoform produced by OVCAR-3 was achieved. Serum CA125 STn concentration was significantly higher in EOC patients than in those with endometriosis ( P < 0.001). Furthermore, the sensitivity for detection of EOC with CA125 STn assay was 73.3% when 95% of endometriosis cases were undetectable., Conclusion: Our findings suggest that Eu +3 -NPs-based CA125 STn assay could help reduce the false-positive rates of CA125 IA to improve differential diagnosis. The results encourage studying further the potential use of CA125 STn to detect EOC at earlier clinical stages. This approach warrants further investigation in other cancers as well., (© 2019 American Association for Clinical Chemistry.)

Human cancer cells were eradicated by adoptive transfer of T cells transduced with a chimeric antigen receptor (CAR) made from an antibody (237Ab) that is highly specific for the murine Tn-glycosylated podoplanin (Tn-PDPN). The objectives were to determine the specificity of these CAR-transduced T (CART) cells and the mechanism for the absence of relapse. We show that although the 237Ab bound only to cell lines expressing murine Tn-PDPN, the 237Ab-derived 237CART cells lysed multiple different human and murine cancers not predicted by the 237Ab binding. Nevertheless, the 237CART cell reactivities remained cancer specific because all recognitions were dependent on the Tn glycosylation that resulted from COSMC mutations that were not present in normal tissues. While Tn was required for the recognition by 237CART, Tn alone was not sufficient for 237CART cell activation. Activation of 237CART cells required peptide backbone recognition but tolerated substitutions of up to 5 of the 7 amino acid residues in the motif recognized by 237Ab. Together, these findings demonstrate what we believe is a new principle whereby simultaneous recognition of multiple independent Tn-glycopeptide antigens on a cancer cell makes tumor escape due to antigen loss unlikely.

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen-Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro . Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.

Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.

Protein glycosylation is a diverse form of post-translational modification. Two to three consecutive O-linked N-acetylgalactosamines (Tn-antigens) are recognized by antibodies such as MLS128. MLS128 mAb inhibited cell growth and bound to a 110 kDa glycoprotein (GP) in LS180 and HT29 colon cancer cells. However, purification and identification of the 110 kDa GP was unsuccessful due to its low abundance. The present study used a highly sophisticated and sensitive mass spectrometry method to identify proteins immunoprecipitated with MLS128 and separated by two-dimensional gel electrophoresis. Three desmosome components were identified. Of these, desmocollin and desmoglein shared many similar characteristics, including molecular mass, pI, and potential Tn-antigen sites. Western blotting analyses of LS180 cell lysates revealed a common 110 kDa band recognized by MLS128 and anti-desmocollin, but not by anti-desmoglein. Immunofluorescence microscopy of LS180 cells revealed that desmocollin is membrane-bound, while desmoglein is primarily localized in the cytosol. Confocal microscopy demonstrated colocalization of the desmocollin-specific antibody with the MLS128 antibody on the cell membrane, suggesting that desmocollin may contain Tn-antigens recognized by MLS128. Treatment of LS180 cells with siRNA to knock down desmocollin expression or a desmocollin-specific antibody decreased cell viability, suggesting a critical role for this protein in cell growth and survival. N-glycosidase F digestion of the 110 kDa GP and desmocollin suggested that although both proteins contain N-glycosylation sites, they are not identical. These findings suggest that desmocollin colocalizes with the 110 kDa GP and that growth inhibition induced by the MLS128 antibody may be mediated through a mechanism that involves desmocollin.

In the last two decades, the paramount importance of Tumor Associated Carbohydrate Antigens (TACAs) as targets for anticancer vaccine development has been firmly assessed. The Tn antigen is an ideal target for immunotherapy, in that it is masked on normal cells, but exposed on cancer cells. However, it is difficult to elicit an effective and long-lasting response against Tn antigen and other TACAs. Here we report on the Tn antigen analogues developed to boost the latent Tn immune response. Hopefully, the results reported herein will be of help for the rational design of effective TACA-based immunostimulants.

Antibodies to carbohydrate epitopes are often of the IgM isotype and require multiple binding for sufficient avidity. Therefore clusters of epitopes are preferred antigenic sites in these cases. We have examined the type of clusters recognized by two anti-Thomsen-Friedenreich (TF, core-1, CD176) IgM antibodies, NM-TF1 and NM-TF2, using several different sets of TF-carrying synthetic glycoconjugates in ELISA experiments. To our surprise, the single most important factor determining binding strength was a close vicinity of several TF glycans at distances of ≤1 nm. Considering the known dimensions of IgM antibodies, our data strongly suggest that a cluster of up to four TF moieties, presenting as a "multiple epitope", is required to attach to a single combining site in order to result in adequate binding strength. This effect can also be achieved by "surrogate-multiple epitopes" consisting of separate TF-carrying molecules in close vicinity. In addition, it was found that serine-linked TFs are stronger bound than threonine-linked TFs by both antibodies. This peculiar type of cluster recognition may contribute to improved avidity and explicit tumor specificity., (Copyright © 2019. Published by Elsevier B.V.)