Abstract Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive B cell lymphoma, with poor outcomes for patients who relapse or are refractory to treatment. Chimeric antigen receptor (CAR)-T cell therapy offers a promising therapeutic option, yet limited persistence and functional impairment remain major barriers to durable responses. Galectin (Gal)-3, a carbohydrate-binding protein highly expressed in the DLBCL microenvironment, has been implicated in T cell dysfunction, but its role in CAR-T cell impairment has not been fully defined. We hypothesized that Gal-3 binds to CAR-T cell surface glycans, promoting apoptosis and loss of effector function. ELISA revealed significantly elevated Gal-3 levels in serum from DLBCL patients versus healthy controls (p<0.001). Flow cytometry confirmed strong Gal-3 binding to CAR-T cells, which correlated with increased apoptosis (p<0.01). To overcome this, we engineered CAR-T cells to overexpress the α2,6 sialyltransferase ST6GAL1 (ST6OE)CAR-T cells, which adds α2,6-sialic acids known to mask Gal-3-binding glycans. ST6OECAR T cells displayed markedly reduced Gal-3 binding, lower apoptosis (p<0.001), and improved viability and cytotoxicity in Gal-3high DLBCL co-cultures. Cytokine profiling further revealed that control CAR-T cells secreted elevated levels of interleukin-5 (IL-5),a Th2-associated cytokine linked to reduced antitumor activity. In contrast, ST6OE CAR-T cells exhibited significantly lower IL-5 production (p<0.01), indicating a shift toward amore cytotoxic, Th1-like functional profile. In a DLBCL xenograft model, ST6OE CAR-T treatment significantly reduced tumor burden and improved survival compared to control CAR-T cells (p<0.001). These findings demonstrate that ST6GAL1 overexpression mitigates apoptosis and IL-5-associated dysfunction, enhancing CAR-T cell persistence and antitumor efficacy. Glycoengineering CAR-T cells via ST6GAL1 provides a novel strategy to improve therapeutic outcomes for patients with DLBCL. Citation Format: Lee Seng Mari Lau, Maria Suarez, Brandon Fernandez, Aiza Berdalinova, Joseph Souchack, Aristotelis Antonopoulos, Anne Dell, Stuart Haslam, Avery D. Posey, Charles J. Dimitroff. ST6GAL1 enhanced CAR-T cells improve persistence and antitumor efficacy in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1529.

Protein glycosylation has been considered as a fundamental phenomenon shared by all domains of life. In Helicobacter pylori, glycosylation of flagellins A and B with pseudaminic acid have been rigorously confirmed and shown to be essential for flagella assembly and bacterial colonization. In addition to flagellins, several other proteins including RecA, AlpA/B, and BabA/B in H. pylori have also been reported to be glycosylated and to be dependent on the lipopolysaccharide (LPS) biosynthetic pathway. However, these proteins have not been purified for sugar-specific staining or structural analysis to confirm the existence of carbohydrate motifs. Here, using a combined approach of genetics, protein purification, and sugar-specific staining, we demonstrate that RecA is not a glycoprotein. Moreover, using LPS-protein reconstitution experiments, we demonstrate that the presence of O-antigen containing full-length LPS interferes with the electrophoretic mobility of H. pylori RecA and many other proteins including AlpA/B on SDS-PAGE. Finally, we demonstrate that full-length LPS extracted from E. coli affects electrophoretic migration of H. pylori proteins, while full-length LPS extracted from H. pylori similarly influences the electrophoretic migration of E. coli proteins. The impact is more subtle with E. coli LPS compared to H. pylori LPS, indicating that the magnitude of effect of LPS effects on protein mobility is dependent on bacterial source of the LPS. These findings suggest that the effects of full-length LPS on protein electrophoresis may represent a more general phenomenon. As LPS is a unique component of virtually all Gram-negative bacteria, our data suggest that when observing protein electrophoretic mobility shifts between wild-type and LPS mutant strains or between subcellular fractionation samples, the influence of LPS on protein electrophoretic migration should be considered first, rather than interpreting it as potential protein glycosylation that is dependent upon LPS biosynthetic pathway.

While Chinese hamster ovary (CHO) cells continue to be the workhorse of recombinant therapeutic protein production, decades of genetic divergence in the industrially relevant CHO-K1 and CHO-S cell lines, are likely to have resulted in differing glycosylation capabilities. Glycosylation can influence the efficacy, serum half-life, and safety of biologics, and as a critical quality attribute of glycoprotein biopharmaceuticals, it is essential to better understand how major CHO cell manufacturing platforms diverge. We used matrix-assisted laser desorption ionization-time of flight mass spectrometry to perform N-glycomic analyses comparing CHO-K1 cells, CHO-S cells and antibody-producing daughter cell lines. The results reveal that genetic divergence in these industrially relevant cell lines, as well as the burden of antibody production, lead to significant differences in antennal branching and terminal elaboration in the cellular N-glycome. More specifically, CHO-K1 cells produce larger and more complex N-glycans with higher levels of sialylation than CHO-S cells, and antibody production was associated with increased antennal branching. Additionally, these findings were also reflected in the N-glycomic profiles of IgG1-Fc constructs produced in either CHO-K1 and CHO-S cells.

The mechanisms by which vaginal microbiota shape spontaneous preterm birth (sPTB) risk remain poorly defined. Using electronic clinical records data from 74,913 maternities in conjunction with metaxanomic (n = 596) and immune profiling (n = 314) data, we show that the B blood group phenotype associates with increased risk of sPTB and adverse vaginal microbiota composition. The O blood group associates with sPTB in women who have a combination of a previous history of sPTB, an adverse vaginal microbial composition and pro-inflammatory cervicovaginal milieu. In contrast, women of blood group A have a higher prevalence of vaginal Lactobacillus crispatus, a lower risk of sPTB, with sPTB cases showing no association with vaginal microbiota composition or inflammation. We found that cervicovaginal fluid contains ABH(O) glycans and shows variable binding to key vaginal bacteria. This indicates that cervicovaginal ABH(O) glycans influence microbiota-host interactions implicated in sPTB risk, suggesting a novel target for sPTB prediction and prevention.

Mutations in the FUT2 gene that result in a lack of expression of histo-blood group antigens on secreted glycoproteins may shape the vaginal microbiota with consequences for birth outcome. To test this, we analysed the relationship between secretor status, vaginal microbiota and gestational length in an ethnically diverse cohort of 302 pregnant women, including 82 who delivered preterm. Lactobacillus gasseri and L. jensenii were found to have distinct co-occurrence patterns with other microbial taxa in non-secretors. Moreover, non-secretors with Lactobacillus spp. depleted high diversity vaginal microbiota in early pregnancy had significantly shorter gestational length than Lactobacillus spp. dominated non-secretors (mean of 241.54 days (sd=47.14) versus 266.21 (23.61); P-value=0.0251). Similar gestational length differences were observed between non-secretors with high vaginal diversity and secretors with Lactobacillus spp. dominance (mean of 262.52 days (SD=27.73); p-value=0.0439) or depletion (mean of 266.05 days (SD=20.81); p-value=0.0312). Our data highlight secretor status and blood-group antigen expression as being important mediators of vaginal microbiota-host interactions in the context of preterm birth risk.

Glycosylation is a critical post-translational modification of proteins, improving properties such as folding, half-life and functionality. However, glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. Here we describe a platform for s eq u ential g lycosyl a tion r eactions for ta ilo r ed su g ar s t ructures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro . This novel proof-of-concept system is enabled by immobilised enzymes produced with a “one-step immobilisation/purification” method to express, biotinylate in vivo and immobilise glycosyltransferases. The immobilised enzymes are used in a reaction cascade mimicking a human-like N-linked glycosylation pathway where promiscuity naturally exists. The enzyme cascade is applied to free glycans, and a monomeric Fc domain expressed in glycoengineered Pichia pastoris , yielding near homogeneous glycoforms (>95% conversion). Finally, immobilised β-1,4 galactosyltransferase is used to enhance the galactosylation profile of three different IgGs yielding 80.2 – 96.3 % terminal galactosylation. Enzyme recycling was further demonstrated for 7 cycles, with a combined reaction time greater than 140 hours. The novel SUGAR-TARGET platform is easy to implement, modular and reusable, and therefore can lead to the development of homogeneous glycan structures for functional and clinical evaluation. The use of immobilised enzymes enables the economical modification of cell-based material supporting applications at a large industrial scale.

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses. However, the specific glycan epitopes involved in these processes are not well understood. To address this, we conducted glycomic analyses of cervicovaginal fluid (CVF) from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Our analysis of N- and O-glycans revealed a rich CVF glycome. While O-glycans were shown to be the main carriers of ABO blood group epitopes, the main features of N-glycans were the presence of abundant paucimannose and high mannose glycans, and a remarkable diversity of complex bi-, tri-, and tetra-antennary glycans decorated with fucose and sialic acid. We identified immuno-regulatory epitopes, such as Lewis antigens, and found that fucosylation was negatively correlated to pro-inflammatory factors, such as IL-1β, MMP-8, C3a and C5a, while glycans with only sialylated antennae were mainly positively correlated to those. Similarly, paucimannose glycans showed a positive correlation to pro-inflammatory factors. We revealed a high abundance of glycans which have previously been identified as hallmarks of cancer and viral glycosylation, such as Man8 and Man9 high mannose glycans. Although each pregnant woman had a unique glycomic profile, longitudinal studies showed that the main glycosylation features were consistent throughout pregnancy in women who delivered at term, whereas women who experienced extreme preterm birth exhibited sharp changes in the CVF glycome shortly before delivery. These findings shed light on the processes underlying the role of glycosylation in maintaining a healthy vaginal microbiome and associated host immune responses. In addition, these discoveries facilitate our understanding of the lower female reproductive tract which has broad implications for women’s health.

PDF file 299K, Additional characterization of human breast cancer cell lines; immunofluorescence analysis and lung retention assays

PDF file 325K, Additional validation of ST6GalNAc2 as a metastasis suppressor including in a spontaneous metastasis assay using the parental 4T1 cells

Chinese hamster ovary (CHO) cells are extensively used for the production of glycoprotein therapeutics proteins, for which N-linked glycans are a critical quality attribute due to their influence on activity and immunogenicity. Manipulation of protein glycosylation is commonly achieved through cell or process engineering, which are often guided by mathematical models. However, each study considers a unique glycosylation reaction network that is tailored around the cell line and product at hand. Herein, we use 200 glycan datasets for both recombinantly produced and native proteins from different CHO cell lines to reconstruct a comprehensive reaction network, CHOGlycoNET, based on the individual minimal reaction networks describing each dataset. CHOGlycoNET is used to investigate the distribution of mannosidase and glycosyltransferase enzymes in the Golgi apparatus and identify key network reactions using machine learning and dimensionality reduction techniques. CHOGlycoNET can be used for accelerating glycomodel development and predicting the effect of glycoengineering strategies. Finally, CHOGlycoNET is wrapped in a SBML file to be used as a standalone model or in combination with CHO cell genome scale models.

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galβ1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing β3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines.

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with “protein glycosylation” and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders.

Host-vaginal-microbial interactions have been shown to influence spontaneous preterm birth (sPTB) risk(1,2). In other body niches, histo-blood group antigens are associated with microbiota composition and disease risk(3,4). To investigate whether ABO blood group influences sPTB risk, and if this is associated with changes in vaginal microbial composition and host immune response. Prospective study of women defined as at-risk of sPTB (n=1935), where cervicovaginal fluid (CVF) was collected in pregnancy (20-24 weeks). Bacterial DNA was extracted, and the composition assessed using 16 S rRNA gene sequencing surveying the V1-V2 region (n=238). Cytokine immunoassays were performed on matched CVF supernatant (n=103). Results were analysed according to ABO blood group status and risk factor for sPTB. In women at risk of sPTB with previous cervical treatment blood group B was associated with a higher risk of sPTB<34weeks than A (RR 2.94(1.22-7.64), p=0.01), yet no correlation was seen with CVF cytokine concentration. In women with previous mid-trimester loss (MTL)/PTB those with blood group O were at increased risk of sPTB <34 weeks compared to those of blood group A (RR 1.42(0.94-2.17), p=0.04). IL-8 levels were higher in women of blood group O with a previous MTL/PTB than those of blood group A or B. IL-8 was also correlated with L. iners and bacterial vaginosis-associated taxa in women of blood group O with sPTB. ABO blood groups influence vaginal microbial composition, local inflammation and risk of sPTB, and provide mechanistic insight on the different aetiologies of PTB.

PDF file 149K, In vivo validation of 3 hits identified in the screen (Mre11a, Fen1, Wwc1)

PDF file 174K, Additional characterization of the 4T1-Luc cells in lung retention assays

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, (beta 1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM gly-come-associated genes and found that (beta 1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low (beta 1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed (beta 1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell-intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Abstract Placental abnormalities cause impaired fetal growth and poor pregnancy outcome (e.g. preeclampsia [PE]) with long-lasting consequences for the mother and offspring. The molecular dialogue between the maternal niche and the developing placenta is critical for the function of this organ. Galectin-1 (gal-1), a highly expressed glycan-binding protein at the maternal–fetal interface, orchestrates the maternal adaptation to pregnancy and placenta development. Down-regulation or deficiency of gal-1 during pregnancy is associated with the development of PE; however, the maternal- and placental-derived gal-1 contributions to the disease onset are largely unknown. We demonstrate that lack of gal-1 imposes a risk for PE development in a niche-specific manner, and this is accompanied by a placental dysfunction highly influenced by the absence of maternal-derived gal-1. Notably, differential placental glycosylation through the Sda-capped N-glycans dominates the invasive trophoblast capacity triggered by maternal-derived gal-1. Our findings show that gal-1 derived from the maternal niche is essential for healthy placenta development and indicate that impairment of the gal-1 signaling pathway within the maternal niche could be a molecular cause for maternal cardiovascular maladaptation during pregnancy.

Neutrophils have been implicated in the process of labour. Preterm birth (PTB) is associated with adverse vaginal microbial compositions (Community State Type, CST IV, Lactobacillus spp. depletion; or L. iners, CST III, dominance), whereas L. crispatus (CST I) dominance is associated with a lower risk. However, it is unclear how microbial composition influences the local immune milieu and risk of PTB. Women at high-risk of PTB (n=98) were recruited from Queen Charlotte's and St Mary's Hospitals, providing matched cervicovaginal swabs, cytobrush and peripheral blood samples. Microbial composition was assessed surveying the V1-V2 region of the 16 S rRNA gene on Illumina MiSeq. Peripheral blood and cytobrush were assessed with Aurora CytekTM. On a subset of women, N-glycan profiling of cervicovaginal samples was done using MALDI-TOF/TOF. Cervical neutrophils exhibited a more activated immunophenotype compared to peripheral neutrophils, including reduced median fluorescence intensity (MFI) of migration markers CD11b and CD62L (p<0.0001), increased expression of CD63, CD66b (degranulation, phagocytosis, p<0.0001), CD88 (C5a receptor, p<0.0001) and CD55 (complement regulator, p=0.0007). Cervical neutrophils from women with CST IV vaginal dominance exhibited higher CD63 (p=0.0441) and CD66b (p=0.0049) MFI compared to cervical neutrophils from women with CST I. Cervicovaginal glycomic studies have identified neutrophil specific paucimannose N-glycan structures that are positively correlated with proinflammatory cytokines IL-1β and IL-8, and increased in women who delivered PTB. Cervical neutrophils are more activated than peripheral neutrophils and their activation status is associated with high-risk microbial profiles. These findings support a role for neutrophils in microbial-driven PTB.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan–GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan–GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.