Interrogating the Theranostic Capacity of a MUC16-Targeted Antibody for Ovarian Cancer.

Aberrantly expressed glycans on mucins such as mucin-16 (MUC16) are implicated in the biology that promotes ovarian cancer (OC) malignancy. Here, we investigated the theranostic potential of a humanized antibody, huAR9.6, targeting fully glycosylated and hypoglycosylated MUC16 isoforms. Methods: In vitro and in vivo targeting of the diagnostic radiotracer [89Zr]Zr-DFO-huAR9.6 was investigated via binding experiments, immuno-PET imaging, and biodistribution studies on OC mouse models. Ovarian xenografts were used to determine the safety and efficacy of the therapeutic version, [177Lu]Lu-CHX-A″-DTPA-huAR9.6. Results: In vivo uptake of [89Zr]Zr-DFO-huAR9.6 supported in vitro-determined expression levels: high uptake in OVCAR3 and OVCAR4 tumors, low uptake in OVCAR5 tumors, and no uptake in OVCAR8 tumors. Accordingly, [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in the OVCAR3 model and improved overall survival in the OVCAR3 and OVCAR5 models in comparison to the saline control. Hematologic toxicity was transient in both models. Conclusion: PET imaging of OC xenografts showed that [89Zr]Zr-DFO-huAR9.6 delineated MUC16 expression levels, which correlated with in vitro results. Additionally, we showed that [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in highly MUC16-expressing tumors. These findings demonstrate great potential for 89Zr- and 177Lu-labeled huAR9.6 as theranostic tools for the diagnosis and treatment of OC.

Combined Immune Checkpoint Blockade Enhances Antiviral Immunity against Bovine Leukemia Virus.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

An aqueous extract from Artemisia capillaris inhibits acute gastric injury through mucosal stabilization.

BACKGROUND: Artemisia capillaris is among the most abundantly used traditional medicines, utilized in East Asia to treat diverse illnesses, including gastrointestinal tract diseases. We previously reported that an aqueous extract of A. capillaris (AEAC) inhibited gastric inflammation induced by HCl/ethanol via reactive oxygen species scavenging and NF-κB downregulation. To date, the pharmacological potential of AEAC for promoting mucosal integrity has not been studied. RESULTS: Here, we report that a single treatment with AEAC increased mucus production, and repeated administration of AEAC abolished HCl/ethanol-induced mucosal injury in vivo. Single- and multiple-dose AEAC treatments measurably increased the expression of mucosal stabilizing factors in vivo, including mucin (MUC) 5 AC, MUC6, and trefoil factor (TFF) 1 and TFF2 (but not TFF3). AEAC also induced mucosal stabilizing factors in both SNU-601 cells and RGM cells through phosphorylation of extracellular signal-regulated kinases. CONCLUSION: Taken together, our results suggest that AEAC protects against HCl/ethanol-induced gastritis by upregulating MUCs and TFFs and stabilizing the mucosal epithelium. © 2021 Society of Chemical Industry.

The Development of Vaccines from Synthetic Tumor-Associated Mucin Glycopeptides and their Glycosylation-Dependent Immune Response.

Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood. The current state of the art suggests that the identity, abundancy and location of the glycans on the MUC backbone are all key parameters in the cellular and humoral response. This review shares lessons learned by us in over two decades of research in glycopeptide vaccines. By bridging synthetic chemistry and immunology, we discuss efforts in designing synthetic MUC1/4/16 vaccines and focus on the role of glycosylation patterns. We provide a brief introduction into the mechanisms of the immune system and aim to promote the development of cancer subunit vaccines.

Autoimmune Epithelitis and Chronic Inflammation in Sjögren's Syndrome-Related Dry Eye Disease.

Autoimmune epithelitis and chronic inflammation are one of the characteristic features of the immune pathogenesis of Sjögren's syndrome (SS)-related dry eye disease. Autoimmune epithelitis can cause the dysfunction of the excretion of tear fluid and mucin from the lacrimal glands and conjunctival epithelia and meibum from the meibomian glands. The lacrimal gland and conjunctival epithelia express major histocompatibility complex class II or human leukocyte antigen-DR and costimulatory molecules, acting as nonprofessional antigen-presenting cells for T cell and B cell activation in SS. Ocular surface epithelium dysfunction can lead to dry eye disease in SS. Considering the mechanisms underlying SS-related dry eye disease, this review highlights autoimmune epithelitis of the ocular surface, chronic inflammation, and several other molecules in the tear film, cornea, conjunctiva, lacrimal glands, and meibomian glands that represent potential targets in the treatment of SS-related dry eye disease.

Preclinical Assessment of a MUC12-Targeted BiTE (Bispecific T-cell Engager) Molecule.

MUC12 is a transmembrane mucin that is highly expressed in >50% of primary and metastatic colorectal tumors. MUC12 is also expressed by normal epithelial cells of the colon and small intestine. Although MUC12 localization in normal epithelial cells is restricted to the apical membrane, expression in tumors is depolarized and shows broad membrane localization. The differential localization of MUC12 in tumor cells as compared with normal cells makes it a potential therapeutic target. Here, we evaluated targeting of MUC12 with a BiTE (bispecific T-cell engager) molecule. We generated a panel of proof-of-concept half-life extended (HLE) BiTE molecules that bind MUC12 on tumor cells and CD3 on T cells. We prioritized one molecule based on in vitro activity for further characterization in vivo In vitro, the MUC12 HLE BiTE molecule mediated T-cell-redirected lysis of MUC12-expressing cells with half-maximal lysis of 4.4 ± 0.9 to 117 ± 78 pmol/L. In an exploratory cynomolgus monkey toxicology study, the MUC12 HLE BiTE molecule administered at 200 µg/kg with a step dose to 1,000 µg/kg was tolerated with minimal clinical observations. However, higher doses were not tolerated, and there was evidence of damage in the gastrointestinal tract, suggesting dose levels projected to be required for antitumor activity may be associated with on-target toxicity. Together, these data demonstrate that the apically restricted expression of MUC12 in normal tissues is accessible to BiTE molecule target engagement and highlight the difficult challenge of identifying tumor-selective antigens for solid tumor T-cell engagers.

Experimental and molecular predictions of the adjuvanticity of snail mucin on hepatitis B vaccine in albino mice.

Although aluminum-containing adjuvants are widely used in human vaccination due to their excellent safety profile, they exhibit low effectiveness with many recombinant antigens. This study investigated the adjuvanticity of snail mucin with recombinant Hepatitis B Vaccine (rHBsAg). Twenty-five (25) female mice distributed unbiasedly into 5 groups were used in the study and were administered different rHBsAg/Mucin formulation at 7 days intervals. Blood samples were collected a day following the administration for analysis. The results of liver function and body weight analysis were indications that snail mucin had no adverse effect on the mice. The treatment group (administer mucin and rHBsAg) showed significantly (P<0.05) higher mean titres of anti-HBsAg antibodies when compared with the negative controls and the positive control administered with two doses of rHBsAg after the boost doses (day 28). Furthermore, a comparable immune response to positive control administered with three doses rHBaAG was recorded. In silico prediction, studies of the protein-protein interaction of a homology modelled snail mucus protein and HBsAg gave an indication of enhanced HBV antigen-antibody interaction. Therefore, this study has shown that snail mucin possesses some adjuvant properties and enhances immune response towards rHBsAg vaccine. However, there is a need for further molecular dynamics studies to understand its mechanism of action.

The Role of Glycosyltransferases in Colorectal Cancer.

Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), ß1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-ß) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell-cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.

Clinical Features, Molecular Alterations and Prognosis of Colorectal Adenocarcinoma With Mucinous Component in Chinese Patients.

Mucinous adenocarcinoma (MAC) is conventionally diagnosed by WHO definition when the extracellular mucin is >50% of the tumor area, while tumors with <50% mucin are designated as having a mucinous component. The study is aimed at analyzing the clinicopathologic characteristics, mutation spectrum, and prognosis of colorectal adenocarcinoma with mucinous component (CAWMC). Mutation analyses for exon 2 to 4 of KRAS gene and exon 15 of BRAF gene were performed by Sanger sequencing. Expression of DNA mismatch repairs and P53 proteins were evaluated by immunohistochemistry. Density of tumor-infiltrating lymphocyte (TIL) status was scored. We also evaluated the percentage of glands producing mucin and the morphology of the different tumor cell types in mucin pools. We retrospectively analyzed the prognosis of 43 patients with stage II/III. The overall frequencies of KRAS and BRAF mutations were 36% and 8%, respectively. Patients with MAC exhibiting high levels of mucin were related to the increase of tumor diameter (P=0.038) but were not associated with any of the other clinicopathologic parameters. The proportion or variable morphology of mucinous component did not stratify progression-free survival in stage II/III cases. TIL was the most significant predictor of progression-free survival among stage II/III CAWMC. It is interesting to note that signet ring cell carcinoma does not portend a worse prognosis for patients with high TIL levels. Combining use the grade of TIL status with the WHO grade of the entire tumor can help identify patients with a high risk of recurrence more accurately.

Mucins as anti-cancer targets: perspectives of the glycobiologist.

Mucins are highly O-glycosylated glycoproteins that carry a heterogenous variety of O-glycan structures. Tumor cells tend to overexpress specific mucins, such as the cell surface mucins MUC1 and MUC4 that are engaged in signaling and cell growth, and exhibit abnormal glycosylation. In particular, the Tn and T antigens and their sialylated forms are common in cancer mucins. We review herein methods chosen to use cancer-associated glycans and mucins as targets for the design of anti-cancer immunotherapies. Mucin peptides from the glycosylated and transmembrane domains have been combined with immune-stimulating adjuvants in a wide variety of approaches to produce anti-tumor antibodies and vaccines. These mucin conjugates have been tested on cancer cells in vitro and in mice with significant successes in stimulating anti-tumor responses. The clinical trials in humans, however, have shown limited success in extending survival. It seems critical that the individual-specific epitope expression of cancer mucins is considered in future therapies to result in lasting anti-tumor responses.

Molecular endoscopic imaging for the detection of Barrett's metaplasia using biodegradable inorganic nanoparticles: An ex-vivo pilot study on human tissue.

BACKGROUND AND STUDY AIMS: Despite major technical advancements, endoscopic surveillance for detecting premalignant lesions in Barrett's esophagus is challenging because of their flat appearance with only subtle morphological changes. Molecular endoscopic imaging (MEI) using nanoparticles (NPs), coupled with fluorescently labeled antibody permits visualization of disease-specific molecular alterations. The aim of this ex vivo study was to assess the diagnostic applicability of MEI with NPs to detect Barrett's metaplasia. PATIENTS AND METHODS: Seven patients undergoing endoscopic surveillance of known Barrett's esophagus were recruited. Freshly resected biopsy specimens were incubated with NPs coupled with FITC labeled Muc-2 antibodies and examined with MEI. Fluorescence intensity from Barrett's mucosa and control specimens were compared, followed by histological confirmation. RESULTS: Fluorescence signals, indicating the presence of goblet cells, were noted for traditional MEI using Muc-2 antibodies in Barrett's intestinal metaplasia. Significantly stronger fluorescence signals were achieved with NPs coupled with FITC-conjugated Muc-2 antibodies. The results of MEI with NPs for the prediction of Barrett's metaplasia correlated with the final histopathological examination in all the cases. CONCLUSIONS: Highly-specific NPs detected Barrett's metaplasia more efficiently than conventional MEI in this first feasibility study. MEI was as effective as standard histopathology for identifying Muc-2 containing goblet cells for diagnosis of Barrett's metaplasia. (DRKS-ID: DRKS00017747).

The Glycan Structure of T. cruzi mucins Depends on the Host. Insights on the Chameleonic Galactose.

Trypanosoma cruzi, the protozoa that causes Chagas disease in humans, is transmitted by insects from the Reduviidae family. The parasite has developed the ability to change the structure of the surface molecules, depending on the host. Among them, the mucins are the most abundant glycoproteins. Structural studies have focused on the epimastigotes and metacyclic trypomastigotes that colonize the insect, and on the mammal trypomastigotes. The carbohydrate in the mucins fulfills crucial functions, the most important of which being the accepting of sialic acid from the host, a process catalyzed by the unique parasite trans-sialidase. The sialylation of the parasite influences the immune response on infection. The O-linked sugars have characteristics that differentiate them from human mucins. One of them is the linkage to the polypeptide chain by the hexosamine, GlcNAc, instead of GalNAc. The main monosaccharide in the mucins oligosaccharides is galactose, and this may be present in three configurations. Whereas ß-d-galactopyranose (ß-Galp) was found in the insect and the human stages of Trypanosoma cruzi, ß-d-galactofuranose (ß-Galf) is present only in the mucins of some strains of epimastigotes and α-d-galactopyranose (α-Galp) characterizes the mucins of the bloodstream trypomastigotes. The two last configurations confer high antigenic properties. In this review we discuss the different structures found and we pose the questions that still need investigation on the exchange of the configurations of galactose.

Interactions of microorganisms with host mucins: a focus on Candida albicans.

Mucus is an important host innate defense factor that lines most epithelial cell layers of the body and provides crucial physical and biological protection against pathogenic microorganisms. Mucins are the main glycoproteins of mucus that are responsible for interacting with microorganisms and are critical for the antimicrobial properties of mucus. The mechanisms by which microorganisms interact with mucins are poorly understood, especially in terms of fungi, and these interactions are continually evolving. Work in bacterial pathogens has shown that mucins inhibit bacterial virulence traits, including quorum sensing, toxin secretion and biofilm formation. Among the fungal clade, the common opportunistic human fungal pathogen and commensal Candida albicans engages in constant battle with the host innate immune system. This battle creates strong selective pressures for C. albicans to evolve in response to the host. Recent work in C. albicans found that mucins inhibit specific virulence traits, such as surface adherence, filamentation, biofilm formation and the production of secreted proteases. Here we review the current knowledge of microbial interactions with mucins, with a special emphasis on the interactions between C. albicans and mucins.

Unraveling mucin domains in cancer and metastasis: when protectors become predators.

A dynamic mucosal layer shields the epithelial cells lining the body cavities and is made up of high molecular weight, heavily glycosylated, multidomain proteins called mucins. Mucins, broadly grouped into transmembrane and secreted mucins, are the first responders to any mechanical or chemical insult to the epithelia and help maintain tissue homeostasis. However, their intrinsic properties to protect and repair the epithelia are exploited during oncogenic processes, where mucins are metamorphosed to aid the tumor cells in their malignant journey. Diverse domains, like the variable number tandem repeats (VNTR), sea urchin sperm protein enterokinase and agrin (SEA), adhesion-associated domain (AMOP), nidogen-like domain (NIDO), epidermal growth factor-like domain (EGF), and von Willebrand factor type D domain (vWD) on mucins, including MUC1, MUC4, MUC5AC, MUC5B, and MUC16, have been shown to facilitate cell-to-cell and cell-to-matrix interactions, and cell-autonomous signaling to promote tumorigenesis and distant dissemination of tumor cells. Several obstacles have limited the study of mucins, including technical difficulties in working with these huge glycoproteins, the dearth of scientific tools, and lack of animal models; thus, the tissue-dependent and domain-specific roles of mucins during mucosal protection, chronic inflammation, tumorigenesis, and hematological dissemination of malignant cells are still unclear. Future studies should try to integrate information on the rheological, molecular, and biological characteristics of mucins to comprehensively delineate their pathophysiological role and evaluate their suitability as targets in future diagnostic and therapeutic strategies.

Targeting MUC15 Protein in Cancer: Molecular Mechanisms and Therapeutic Perspectives.

MUC15, a member of the mucin family, is a heavily glycosylated transmembrane protein with the primary functions of lubricating surfaces, establishing a selective molecular barrier at the epithelium and mediating signal transduction. Aberrant expression of MUC15 plays a crucial role in the progression of multiple diseases, including malignant tumors. MUC15 has been identified as a tumor suppressor, but current evidence indicate its function as an oncogene in different types of cancers. MUC15 has been shown to be involved in the development of cancer and influence cellular growth, adhesion, invasion, metastasis and immune immunomodulation. However, the precise role of MUC15 in tumour development has not been thoroughly clarified. Here, we systematically summarize the structure and function of MUC15 in cancer, and discuss its potential role in cancer treatment.

Mucin-Like Domain of Ebola Virus Glycoprotein Enhances Selective Oncolytic Actions against Brain Tumors.

Given that the Ebola virus (EBOV) infects a wide array of organs and cells yet displays a relative lack of neurotropism, we asked whether a chimeric vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) might selectively target brain tumors. The mucin-like domain (MLD) of the EBOV GP may enhance virus immune system evasion. Here, we compared chimeric VSVs in which EBOV GP replaces the VSV glycoprotein, thereby reducing the neurotoxicity associated with wild-type VSV. A chimeric VSV expressing the full-length EBOV GP (VSV-EBOV) containing the MLD was substantially more effective and safer than a parallel construct with an EBOV GP lacking the MLD (VSV-EBOVΔMLD). One-step growth, reverse transcription-quantitative PCR, and Western blotting assessments showed that VSV-EBOVΔMLD produced substantially more progeny faster than VSV-EBOV. Using immunodeficient SCID mice, we focused on targeting human brain tumors with these VSV-EBOVs. Similar to the findings of our previous study in which we used an attenuated VSV-EBOV with no MLD that expressed green fluorescent protein (GFP) (VSV-EBOVΔMLD-GFP), VSV-EBOVΔMLD without GFP targeted glioma but yielded only a modest extension of survival. In contrast, VSV-EBOV containing the MLD showed substantially better targeting and elimination of brain tumors after intravenous delivery and increased the survival of brain tumor-bearing mice. Despite the apparent destruction of most tumor cells by VSV-EBOVΔMLD, the virus remained active within the SCID mouse brain and showed widespread infection of normal brain cells. In contrast, VSV-EBOV eliminated the tumors and showed relatively little infection of normal brain cells. Parallel experiments with direct intracranial virus infection generated similar results. Neither VSV-EBOV nor VSV-EBOVΔMLD showed substantive infection of the brains of normal immunocompetent mice.IMPORTANCE The Ebola virus glycoprotein contains a mucin-like domain which may play a role in immune evasion. Chimeric vesicular stomatitis viruses with the EBOV glycoprotein substituted for the VSV glycoprotein show greater safety and efficacy in targeting brain tumors in immunodeficient mice when the MLD was expressed within the EBOV glycoprotein than when EBOV lacked the mucin-like domain.

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic.

Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.

A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies.

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), Galß1-3GalNAc (T-antigen), NeuAcα2-3Galß1-3GalNAc (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.

Differential requirements for FcγR engagement by protective antibodies against Ebola virus.

Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.