Chimeric antigen receptor T cells targeting the GM3(Neu5Gc) ganglioside.

Chimeric antigen receptor (CAR) T cell technology has ushered in a new era of immunotherapy, enabling the targeting of a broad range of surface antigens, surpassing the limitations of traditional T cell epitopes. Despite the wide range of non-protein tumor-associated antigens, the advancement in crafting CAR T cells for these targets has been limited. Owing to an evolutionary defect in the CMP-Neu5Ac hydroxylase (CMAH) that abolishes the synthesis of CMP-Neu5Gc from CMP-Neu5Ac, Neu5Gc is generally absent in human tissues. Despite this, Neu5Gc-containing antigens, including the ganglioside GM3(Neu5Gc) have consistently been observed on tumor cells across a variety of human malignancies. This restricted expression makes GM3(Neu5Gc) an appealing and highly specific target for immunotherapy. In this study, we designed and evaluated 14F7-28z CAR T cells, with a targeting unit derived from the GM3(Neu5Gc)-specific murine antibody 14F7. These cells exhibited exceptional specificity, proficiently targeting GM3(Neu5Gc)-expressing murine tumor cells in syngeneic mouse models, ranging from B cell malignancies to epithelial tumors, without compromising safety. Notably, human tumor cells enhanced with murine Cmah were effectively targeted and eliminated by the 14F7 CAR T cells. Nonetheless, despite the detectable presence of GM3(Neu5Gc) in unmodified human tumor xenografts, the levels were insufficient to trigger a tumoricidal T-cell response with the current CAR T cell configuration. Overall, our findings highlight the potential of targeting the GM3(Neu5Gc) ganglioside using CAR T cells across a variety of cancers and set the stage for the optimization of 14F7-based therapies for future human clinical application.

Synthesis, biological activity evaluation and mechanism analysis of new ganglioside GM3 derivatives as potential agents for nervous functional recovery.

Neuronal regenerative ability is vital for the treatment of neurodegenerative diseases and neuronal injuries. Recent studies have revealed that Ganglioside GM3 and its derivatives may possess potential neuroprotective and neurite growth-promoting activities. Herein, six GM3 derivatives were synthesized and evaluated their potential neuroprotective effects and neurite outgrowth-promoting activities on a cellular model of Parkinson's disease and primary nerve cells. Amongst these derivatives, derivatives N-14 and 2C-12 demonstrated neuroprotective effects in the MPP + model in SH-SY5Y cells. 2C-12 combined with NGF (nerve growth factor) induced effecially neurite growth in primary nerve cells. Further action mechanism revealed that derivative 2C-12 exerts neuroprotective effects by regulating the Wnt signaling pathway, specifically involving the Wnt7b gene. Overall, this study establishes a foundation for further exploration and development of GM3 derivatives with neurotherapeutic potential.

Detection of N-glycolyl-neuraminic acid-containing glycolipids in human skin.

Humans lack the enzyme that produces the sialic acid N-glycolyl neuraminic acid (Neu5Gc), but several lines of evidence have shown that Neu5Gc can be taken up by mammalian food sources and replace the common human sialic acid N-acetyl neuraminic acid (Neu5Ac) in glycans. Cancer tissue has been shown to have increased the presence of Neu5Gc and Neu5Gc-containing glycolipids such as the ganglioside GM3, which have been proposed as tumor-specific antigens for antibody treatment. Here, we show that a previously described antibody against Neu5Gc-GM3 is binding to Neu5GC-containing gangliosides and is strongly staining different cancer tissues. However, we also found a strong intracellular staining of keratinocytes of healthy skin. We confirmed this staining on freshly isolated keratinocytes by flow cytometry and detected Neu5Gc by mass spectrometry. This finding implicates that non-human Neu5Gc can be incorporated into gangliosides in human skin, and this should be taken into consideration when targeting Neu5Gc-containing gangliosides for cancer immunotherapy.

Characterization of GM3 Gangliosides in Human Milk throughout Lactation: Insights from the Analysis with the Use of Reversed-Phase Liquid Chromatography Coupled to Quadrupole Time-Of-Flight Mass Spectrometry.

Gangliosides are complex lipids found in human milk that play important structural and biological functions. In this study, we utilized reversed-phase liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to evaluate the molecular distribution of GM3 in human milk samples collected at distinct lactation stages, ranging from colostrum to advanced lactation samples. Throughout lactation, GM3 d40:1 emerged as the most abundant GM3 species, except in colostrum, where GM3 d42:2 prevailed. The relative content of GM3 species containing very long N-fatty acyl (N-FA) substituents with >22 carbon atoms decreased, while the content of GM3 species containing 14:0, 18:0, 18:1, and 20:0 N-FA substituents increased in the later months of lactation. These findings highlight the divergence of GM3 profiles across the lactation period. Moreover, considerable interindividual variance was observed among the analyzed samples. The assessment of the GM3 profiles contributes to our understanding of the dynamic composition of human milk.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

Comparison and Possible Binding Orientations of SARS-CoV-2 Spike N-Terminal Domain for Gangliosides GM3 and GM1.

SARS-CoV-2 spike glycoprotein is anchored by gangliosides. The sialic acid in the ganglioside headgroup is responsible for virus attachment and entry into host cells. We used coarse-grained (CG) molecular dynamics simulations to expand on our previous study of GM1 interaction with two different orientations of the SARS-CoV-2 S1 subunit N-terminal domain (NTD) and to confirm the role of sialic acid receptors in driving the viral receptor; GM3 was used as another ganglioside on the membrane. Because of the smaller headgroup, sialic acid is crucial in GM3 interactions, whereas GM1 interacts with NTD via both the sialic acid and external galactose. In line with our previous findings for NTD orientations in GM1 binding, we identified two orientations, "compact" and "distributed", comprising sugar receptor-interacting residues in GM3-embedded lipid bilayers. Gangliosides in closer proximity to the compact NTD orientation might cause relatively greater restrictions to penetrate the bilayer. However, the attachment of a distributed NTD orientation with more negative interaction energies appears to facilitate GM1/GM3 to move quickly across the membrane. Our findings likely shed some light on the orientations that the NTD receptor acquires during the early phases of interaction with GM1 and GM3 in a membrane environment.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Ganglioside GM3 deficiency enhances mast cell sensitivity.

Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction.

Synthesis and biological activity of ganglioside GM3 analogues with a (S)-CHF-Sialoside linkage and an alkyne tag.

The alkyne tag, consisting of only two carbons, is widely used as a bioorthogonal functional group due to its compactness and nonpolar structure, and various probes consisting of lipids bearing an alkyne tag have been developed. Here, we designed and synthesized analogues of ganglioside GM3 bearing an alkyne tag in the fatty acid moiety and evaluated the effect of the alkyne tag on the biological activity. To eliminate the influence of other factors such as degradation of the glycan chain when evaluating biological activity in a cellular environment, we introduced the tag into sialidase-resistant (S)-CHF-linked GM3 analogues developed by our group. The designed analogues were efficiently synthesized by tuning the protecting group of the glucosylsphingosine acceptor. The growth-promoting effect of these analogues on Had-1 cells was dramatically altered depending upon the position of the alkyne tag.

Ganglioside GM3 prevents high fat diet-induced hepatosteatosis via attenuated insulin signaling pathway.

Gangliosides, sialic acid-containing glycosphingolipids, are widely involved in regulations of signal transductions to control cellular functions. It has been suggested that GM3, the simplest structure among gangliosides, is involved in insulin resistance, whereas it remains unclear whether insulin signaling diminished by GM3 actually aggravates the pathological conditions in metabolic disorders. Moreover, the functional roles of gangliosides in the regulation of insulin signaling have not yet been fully elucidated in liver or hepatocytes despite that it is one of the major insulin-sensitive organs. To understand physiological roles of GM3 in metabolic homeostasis in liver, we conducted a high fat diet (HFD) loading experiment using double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase, which lack all gangliosides except GM3, as well as wild-type (WT) mice. DKO mice were strikingly resistant to HFD-induced hepatosteatosis, and hepatic lipogenesis-related molecules including insulin signaling components were down-regulated in HFD-fed DKO. Furthermore, we established primary hepatocyte cultures from DKO and WT mice, and examined their responses to insulin in vitro. Following insulin stimulation, DKO hepatocytes expressing GM3 showed attenuated expression and/or activations in the downstream components compared with WT hepatocytes expressing GM2. While insulin stimulation induced lipogenic proteins in hepatocytes from both genotypes, their expression levels were lower in DKO than in WT hepatocytes after insulin treatment. All our findings suggest that the modified gangliosides, i.e., a shift to GM3 from GM2, might exert a suppressive effect on lipogenesis by attenuating insulin signaling at least in mouse hepatocytes, which might result in protection of HFD-induced hepatosteatosis.

Sialidase-Resistant Ganglioside GM3 Analogues: Evaluation of Biological Activity.

Glycolipids play important biological roles mainly in biological membranes, but their functions at the molecular level remain to be fully established. A chemical biology approach using exogenously added glycolipid probes would be promising, but the possibility of cleavage by cellular glycohydrolases complicates the interpretation of results. Thus, there is a need for non-hydolyzable analogues. In the present study, we designed and synthesized GM3 analogues resistant to GM3-degrading sialidase by replacing the O-sialoside linkage with a C-sialoside linkage. The bioactivity of the analogues was also investigated.

Sialyltransferase Activity Assay for Ganglioside GM3 Synthase.

GM3 synthase (GM3S) is a sialyltransferase that transfers sialic acid from CMP-sialic acid to lactosylceramide. This reaction results in formation of ganglioside GM3 and is essential for biosynthesis of its downstream derivatives, which include a- and b-series gangliosides. Here, we describe a method for GM3S enzymatic assay using fluorescence-labeled alkyl lactoside as acceptor substrate, followed by HPLC for separation of enzymatic product. The method allows quantitative assay of GM3S sialyltransferase activity in cultured cells and mouse brain tissues.

Ganglioside GM3 stimulates lipid-protein co-assembly in α-synuclein amyloid formation.

Parkinson's disease is characterized by the aggregation of the presynaptic protein α-synuclein (αSyn), and its co-assembly with lipids and other cellular matter in the brain. Here we investigated lipid-protein co-assembly in a system composed of αSyn and model membranes containing the glycolipid ganglioside GM3. We quantified the uptake of lipids into the co-assembled aggregates and investigated how lipid molecular dynamics is altered by being present in the co-assemblies using solution 1H- and solid-state 13C NMR spectroscopy. Aggregate morphology was studied using cryo-TEM. The overall lipid uptake in the co-assembled aggregates was found to increase with the molar ratio of GM3 in the vesicles. The lipids present in the co-assembled aggregates have reduced acyl chain and headgroup dynamics compared to the protein-free bilayer system. These findings may improve our understanding of how different types of lipids can influence the composition of αSyn aggregates, which may have consequences for amyloid formation in vivo.

GM3 synthase deficiency increases brain glucose metabolism in mice.

GM3 synthase (GM3S) deficiency is a rare neurodevelopmental disorder caused by an inability to synthesize gangliosides, for which there is currently no treatment. Gangliosides are brain-enriched, plasma membrane glycosphingolipids with poorly understood biological functions related to cell adhesion, growth, and receptor-mediated signal transduction. Here, we investigated the effects of GM3S deficiency on metabolism and mitochondrial function in a mouse model. By indirect calorimetry, GM3S knockout mice exhibited increased whole-body respiration and an increased reliance upon carbohydrate as an energy source. 18F-FDG PET confirmed higher brain glucose uptake in knockout mice, and GM3S deficient N41 neuronal cells showed higher glucose utilization in vitro. Brain mitochondria from knockout mice respired at a higher rate on Complex I substrates including pyruvate. This appeared to be due to higher expression of pyruvate dehydrogenase (PDH) and lower phosphorylation of PDH, which would favor pyruvate entry into the mitochondrial TCA cycle. Finally, it was observed that blocking glucose metabolism with the glycolysis inhibitor 2-deoxyglucose reduced seizure intensity in GM3S knockout mice following administration of kainate. In conclusion, GM3S deficiency may be associated with a hypermetabolic phenotype that could promote seizure activity.

Regulation of signal transduction by gangliosides in lipid rafts: focus on GM3-IR and GM1-TrkA interactions.

The interactions between gangliosides and proteins belonging to the same or different lipid domains and their influence on physiological and pathological states have been analysed in detail. A well-known factor impacting on lipid-protein interactions and their biological outcomes is the dynamic composition of plasma membrane. This review focuses on GM1 and GM3 gangliosides because they are an integral part of protein-receptor complexes and dysregulation of their concentration shows a direct correlation with the onset of pathological conditions. We first discuss the interaction between GM3 and insulin receptor in relation to insulin responses, with an increase in GM3 correlating with the onset of metabolic dysfunction. Next, we describe the case of the GM1-TrkA interaction, relevant to nerve-cell differentiation and homeostasis as deficiency in plasma-membrane GM1 is known to promote neurodegeneration. These two examples highlight the fact that interactions between gangliosides and receptor proteins within the plasma membrane are crucial in controlling cell signalling and pathophysiological cellular states.

Ganglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse.

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be an effective strategy against hematological malignancies but persistence and activity against solid tumors must be further improved. One emerging strategy for enhancing efficacy is based on directing CAR T cells to antigen presenting cells (APCs). Activation of CAR T cells at the immunological synapse (IS) formed between APC and T cell is thought to promote strong, persistent antigen-specific T cell-mediated immune responses but requires integration of CAR ligands into the APC/T-cell interface. Here, we demonstrate that CAR ligand functionalized, lipid-coated, biodegradable polymer nanoparticles (NPs) that contain the ganglioside GM3 (GM3-NPs) bind to CD169 (Siglec-1)-expressing APCs and localize to the cell contact site between APCs and CAR T cells upon initiation of cell conjugates. The CD169+ APC/CAR T-cell interface is characterized by a strong optical colocalization of GM3-NPs and CARs, enrichment of F-actin, and recruitment of ZAP-70, indicative of integration of GM3-NPs into a functional IS. Ligands associated with GM3-NPs localized to the APC/T-cell contact site remain accessible to CARs and result in robust T-cell activation. Overall, this work identifies GM3-NPs as a potential antigen delivery platform for active targeting of CD169 expressing APCs and enhancement of CAR T-cell activation at the NP-containing IS.

CAR T cells targeting the ganglioside NGcGM3 control ovarian tumors in the absence of toxicity against healthy tissues.

Chimeric antigen receptor (CAR) T cells have emerged as a powerful immunotherapeutic tool against certain hematological malignancies but a significant proportion of patients either do not respond or they relapse, sometimes as a result of target antigen loss. Moreover, limited clinical benefit has been reported for CAR therapy against epithelial derived solid tumors. A major reason for this is the paucity of solid tumor antigens identified to date that are broadly, homogeneously and stably expressed but not found on healthy tissues. To address this, here we describe the development and evaluation of CAR T cells directed against N-glycoslylated ganglioside monosialic 3 (NGcGM3). NGcGM3 derives from the enzymatic hydroxylation of N-acetylneuraminic acid (NAc) GM3 (NAcGM3) and it is present on the surface of a range of cancers including ovarian, breast, melanoma and lymphoma. However, while NAcGM3 is found on healthy human cells, NGcGM3 is not due to the 7deletion of an exon in the gene encoding for the enzyme cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH). Indeed, unlike for most mammals, in humans NGcGM3 is considered a neoantigen as its presence on tumors is the result of metabolic incorporation from dietary sources. Here, we have generated 3 CARs comprising different single chain variable fragments (scFvs) originating from the well-characterized monoclonal antibody (mAb) 14F7. We show reactivity of the CAR T cells against a range of patient tumor fragments and we demonstrate control of NGcGM3+ SKOV3 ovarian tumors in the absence of toxicity despite the expression of CMAH and presence of NGcGM3+ on healthy tissues in NSG mice. Taken together, our data indicate clinical potential for 14F7-based CAR T cells against a range of cancers, both in terms of efficacy and of patient safety.

GM2/GM3 controls the organizational status of CD82/Met microdomains: further studies in GM2/GM3 complexation.

At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.