Heparan Research Articles

Development of Gold Nanoparticle based Colorimetric Monkeypox Biosensors Suitable for Point of Care (POC) Analysis

AbstractPractical colorimetric biosensors were developed for the detections of A29 and A27L proteins which were defined as Monkeypox virus (MPXV) and smallpox target proteins. For this purpose, gold nanoparticles (AuNPs) were synthesized and used to detect specific interaction between heparan sulphate (HS) receptor and A29 or A27L target proteins. This study is the first colorimetric biosensor that was based on the interaction of HS and A29 or A27L protein. In this work first, HS was labeled with AuNPs. When AuNPs labeled HS was incubated with A29 or A27L protein structure, these proteins specifically bounded to HS. Next, obtained AuNP/HS‐A29 or AuNP/HS‐A27L structures began to aggregate, and then difference in absorbance value of AuNP at 530 nm occurred. Absorbance differences at 530 nm were monitored spectroscopically. After experimental parameters were optimized, analytical characteristics of developed biosensors were investigated. As a result, the limit of detection was calculated as 7.93 ng.mL−1 and 6.09 ng.mL−1for A29 protein and A27L, respectively. The relative standard deviation were calculated as 1.97 % and 5.72 % for 25 ng.mL−1 A29 and 12.5 ng.mL−1 A27, respectively. Sample application studies were also performed with the standard addition of A29 and A27L proteins to real serum samples taken from healthy individuals. As a result, very promising results were obtained.

Systemic immune challenge exacerbates neurodegeneration in a model of neurological lysosomal disease

Mucopolysaccharidosis type IIIA (MPS IIIA) is a rare paediatric lysosomal storage disorder, caused by the progressive accumulation of heparan sulphate, resulting in neurocognitive decline and behavioural abnormalities. Anecdotal reports from paediatricians indicate a more severe neurodegeneration in MPS IIIA patients, following infection, suggesting inflammation as a potential driver of neuropathology. To test this hypothesis, we performed acute studies in which WT and MPS IIIA mice were challenged with the TLR3-dependent viral mimetic poly(I:C). The challenge with an acute high poly(I:C) dose exacerbated systemic and brain cytokine expression, especially IL-1β in the hippocampus. This was accompanied by an increase in caspase-1 activity within the brain of MPS IIIA mice with concomitant loss of hippocampal GFAP and NeuN expression. Similar levels of cell damage, together with exacerbation of gliosis, were also observed in MPS IIIA mice following low chronic poly(I:C) dosing. While further investigation is warranted to fully understand the extent of IL-1β involvement in MPS IIIA exacerbated neurodegeneration, our data robustly reinforces our previous findings, indicating IL-1β as a pivotal catalyst for neuropathological processes in MPS IIIA.

Antiviral activity of temporin-1CEb analogues against gingival infection with herpes simplex virus type 1.

Oral herpes infections caused by herpes simplex virus type 1 (HSV-1) are one of the most common in the human population. Recently, they have been classified as an increasing problem in immunocompromised patients and those suffering from chronic inflammation of the oral mucosa and gums. Treatment mainly involves nucleoside analogues, such as acyclovir and its derivatives, which reduce virus replication and shedding. As drug-resistant strains of herpes emerge rapidly, there is a need for the development of novel anti-herpes agents. The aim of the study was to design an antiviral peptide, based on natural compounds, non-toxic to the host, and efficient against drug-resistant HSV-1. Here, we designed a lysine-rich derivative of amphibian temporin-1CEb conjugated to peptides penetrating the host cell membrane and examined their activity against HSV-1 infection of oral mucosa. We assessed the antiviral efficiency of the tested compound in simple 2D cell models (VeroE6 and TIGKs cells) and a 3D organotypic model of human gingiva (OTG) using titration assay, qPCR, and confocal imaging. To identify the molecular mechanism of antiviral activity, we applied the Azure A metachromatic test, and attachment assays techniques. Toxicity of the conjugates was examined using XTT and LDH assays. Our results showed that temporin-1CEb analogues significantly reduce viral replication in oral mucosa. The mechanism of peptide analogues is based on the interaction with heparan sulfate, leading to the reduce attachment of HSV-1 to the cell membrane. Moreover, temporin-1CEb conjugates effectively penetrate the gingival tissue being effective against acyclovir-resistant strains. Collectively, we showed that temporin-1CEb can be regarded as a novel, naturally derived antiviral compound for HSV-1 treatment.

Laronidase-loaded liposomes reach the brain and other hard-to-treat organs after noninvasive nasal administration

Mucopolysaccharidosis type I (MPS I) is caused by a lack of the lysosomal enzyme α-L-iduronidase (IDUA), responsible for the degradation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate, leading to multisystemic signs and symptoms. Enzyme replacement therapy (ERT) is a treatment that consists of weekly intravenous administrations of laronidase, a recombinant version of IDUA. However, ERT has limited access to certain tissues, such as bone, cartilage, and brain, and laronidase fails to trespass the BBB. In this sense, this study reports the development and characterization of laronidase-loaded liposomes for the treatment of MPS I mice. Liposomal complexes were obtained by the thin film formation method followed by microfluidization. The main characterization results showed mean vesicle size of 103.0 ± 3.3 nm, monodisperse populations of vesicles, zeta potential around + 30.0 ± 2.1 mV, and mucoadhesion strength of 5.69 ± 0.14 mN. Treatment of MPS I mice fibroblasts showed significant increase in enzyme activity. Nasal administration of complexes to MPS I mice resulted in significant increase in laronidase activity in the brain cortex, heart, lungs, kidneys, eyes, and serum. The overall results demonstrate the feasibility of nasal administration of laronidase-loaded liposomes to deliver enzyme in difficult-to-reach tissues, circumventing ERT issues and bringing hope as a potential treatment for MPS I.

The Endothelial Glycocalyx in Pig-to-Baboon Cardiac Xenotransplantation-First Insights.

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface-the endothelial glycocalyx-are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.

Fine-tuning of liposome integrity for differentiated transcytosis and enhanced antitumor efficacy

Transcytosis-inducing nanomedicines have been developed to improve tumor extravasation. However, the fate during transcytosis across multicell layers and the structural integrity of the nanomedicines before reaching tumor cells could impact antitumor therapy. Here, a BAY 87–2243 (a hypoxia-inducible factor-1 inhibitor)-loaded liposomal system (HA-P-LBAY) modified by low molecular weight protamine (LMWP) and crosslinked by hyaluronic acid (HA) was constructed. This system could accomplish differentiate cellular transport in endothelial and tumor cells by fine-tuning its structural integrity, i.e. transcytosis across the endothelial cells while preserving structural integrity, facilitating subsequent retention and drug release within tumor cells via degradation-induced aggregation. In vitro cellular uptake and transwell studies demonstrated that HA-P-LBAY were internalized by endothelial cells (bEnd.3) via an active, caveolin and heparin sulfate proteoglycan (HSPG)-mediated endocytosis, and subsequently achieved transcytosis mainly through the ER/Golgi pathway. Moreover, the fluorescence resonance energy transfer (FRET) study showed that HA-crosslinking maintained higher integrity of HA-P-LBAY after transcytosis, more efficiently than electrostatic coating of HA (HA/P-LBAY). In addition, more HA-P-LBAY was retained in tumor cells (4T1) compared to HA/P-LBAY corresponding to its enhanced in vitro cytotoxicity. This may be attributed to better integrity of HA-P-LBAY post endothelial transcytosis and more degradation of HA in tumor cells, leading to more liposome aggregation and inhibition of their transcytosis, which was inferred by both TEM images and the HAase responsiveness assay proved by FRET. In vivo, HA-P-LBAY exhibited more potency in tumor suppression than the other formulations in both low and high permeability tumor models. This highlighted that fine-tuning of structural integrity of nanocarriers played a key role no matter whether the transcytosis of nanocarriers contributed to cellular transport. Collectively, this study provides a promising strategy for antitumor therapies by fine-tuning liposome integrity to achieve active trans-endothelial transport with structural integrity and selective aggregation for prolonged tumor retention.

Novel insights into the host cell glycan binding profile of human metapneumovirus.

Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.IMPORTANCEAll cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.

Spike N354 glycosylation augments SARS-CoV-2 fitness for human adaptation through structural plasticity.

Selective pressures have given rise to a number of SARS-CoV-2 variants during the prolonged course of the COVID-19 pandemic. Recently evolved variants differ from ancestors in additional glycosylation within the spike protein receptor-binding domain (RBD). Details of how the acquisition of glycosylation impacts viral fitness and human adaptation are not clearly understood. Here, we dissected the role of N354-linked glycosylation, acquired by BA.2.86 sub-lineages, as a RBD conformational control element in attenuating viral infectivity. The reduced infectivity is recovered in the presence of heparin sulfate, which targets the 'N354 pocket' to ease restrictions of conformational transition resulting in a 'RBD-up' state, thereby conferring an adjustable infectivity. Furthermore, N354 glycosylation improved spike cleavage and cell-cell fusion, and in particular escaped one subset of ADCC antibodies. Together with reduced immunogenicity in hybrid immunity background, these indicate a single spike amino acid glycosylation event provides selective advantage in humans through multiple mechanisms.

Heparan sulfate acts as an activator of the NLRP3 inflammasome promoting inflammatory response in the development of acute pancreatitis.

Accumulating evidence has shown that the NOD-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in the inflammatory cascades involved in the development of acute pancreatitis (AP). However, the specific agonist responsible for activating the NLRP3 inflammasome in this process has not yet been identified. The purpose of this study is to clarify whether heparan sulfate (HS) works as an NLRP3 inflammasome activator to evoke inflammatory cascades in the progression of AP. Two experimental mouse models of AP were utilized to investigate the pro-inflammatory activity of HS in the development of AP by measuring the secretion of inflammatory cytokines and the neutrophil infiltration in pancreatic tissue. The ability of HS to activate the NLRP3 inflammasome was evaluated both in vitro and in vivo. The nuclear factor kappa B (NF-κB)-mediated expression of NLRP3 inflammasome components in response to HS treatment was determined to decipher the role of HS in transcriptional priming of NLRP3 inflammasome. Furthermore, HS-triggered deubiquitination of NLRP3 was analyzed to reveal the promoting effect of HS on the NLRP3 inflammasome priming via a non-transcriptional pathway. High plasma level of HS was observed with a positive correlation to that of inflammatory cytokines in AP mice. Administration of HS to mice resulted in an exacerbated inflammatory profile, while reducing HS production by an inhibitor of heparanase significantly attenuated inflammatory response. Pharmacological inhibition or genetic deletion of NLRP3 substantially suppressed the HS-stimulated elevation of IL-1β levels in AP mice. The in vitro data demonstrated that HS primarily serves as a priming signal for the activation of the NLRP3 inflammasome. HS possesses the ability to increase the transcriptional activity of NF-κB and TLR4/NF-κB-driven transcriptional pathway is employed for NLRP3 inflammasome priming. Moreover, HS-induced deubiquitination of NLRP3 is another pathway responsible for non-transcriptional priming of NLRP3 inflammasome. Our current work has unveiled HS as a new activator of the NLRP3 inflammasome responsible for the secondary inflammatory cascades during the development of AP, highlighting the HS-NLRP3 pathway as a potential target for future preventive and therapeutic approaches of AP.

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT).

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of a-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-a-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal a-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in HGSNAT catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

Photobiomodulation in the infrared spectrum reverses the expansion of circulating natural killer cells and brain microglial activation in Sanfilippo mice.

Sanfilippo syndrome results from inherited mutations in genes encoding lysosomal enzymes that catabolise heparan sulfate (HS), leading to early childhood-onset neurodegeneration. This study explores the therapeutic potential of photobiomodulation (PBM), which is neuroprotective and anti-inflammatory in several neurodegenerative diseases; it is also safe and PBM devices are readily available. We investigated the effects of 10-14 days transcranial PBM at 670 nm (2 or 4 J/cm2/day) or 904 nm (4 J/cm2/day) in young (3 weeks) and older (15 weeks) Sanfilippo or mucopolysaccharidosis type IIIA (MPS IIIA) mice. Although we found no PBM-induced changes in HS accumulation, astrocyte activation, CD206 (an anti-inflammatory marker) and BDNF expression in the brains of Sanfilippo mice, there was a near-normalisation of microglial activation in older MPS IIIA mice by 904 nm PBM, with decreased IBA1 expression and a return of their morphology towards a resting state. Immune cell immunophenotyping of peripheral blood with mass cytometry revealed increased pro-inflammatory signalling through pSTAT1 and p-p38 in NK and T cells in young but not older MPS IIIA mice (5 weeks of age), and expansion of NK, B and CD8+ T cells in older affected mice (17 weeks of age), highlighting the importance of innate and adaptive lymphocytes in Sanfilippo syndrome. Notably, 670 and 904 nm PBM both reversed the Sanfilippo-induced increase in pSTAT1 and p-p38 expression in multiple leukocyte populations in young mice, while 904 nm reversed the increase in NK cells in older mice. In conclusion, this is the first study to demonstrate the beneficial effects of PBM in Sanfilippo mice. The distinct reduction in microglial activation and NK cell pro-inflammatory signalling and number suggests PBM may alleviate neuroinflammation and lymphocyte activation, encouraging further investigation of PBM as a standalone, or complementary therapy in Sanfilippo syndrome.

Internalization of PEI-based complexes in transient transfection of HEK293 cells is triggered by coalescence of membrane heparan sulfate proteoglycans like Glypican-4

Polymer-cationic mediated gene delivery is a well-stablished strategy of transient gene expression (TGE) in mammalian cell cultures. Nonetheless, its industrial implementation is hindered by the phenomenon known as cell density effect (CDE) that limits the cell density at which cultures can be efficiently transfected. The rise in personalized medicine and multiple cell and gene therapy approaches based on TGE, make more relevant to understand how to circumvent the CDE. A rational study upon DNA/PEI complex formation, stability and delivery during transfection of HEK293 cell cultures has been conducted, providing insights on the mechanisms for polyplexes uptake at low cell density and disruption at high cell density. DNA/PEI polyplexes were physiochemically characterized by coupling X-ray spectroscopy, confocal microscopy, cryo-transmission electron microscopy (TEM) and nuclear magnetic resonance (NMR). Our results showed that the ionic strength of polyplexes significantly increased upon their addition to exhausted media. This was reverted by depleting extracellular vesicles (EVs) from the media. The increase in ionic strength led to polyplex aggregation and prevented efficient cell transfection which could be counterbalanced by implementing a simple media replacement (MR) step before transfection. Inhibiting and labeling specific cell-surface proteoglycans (PGs) species revealed different roles of PGs in polyplexes uptake. Importantly, the polyplexes uptake process seemed to be triggered by a coalescence phenomenon of HSPG like glypican-4 around polyplex entry points. Ultimately, this study provides new insights into PEI-based cell transfection methodologies, enabling to enhance transient transfection and mitigate the cell density effect (CDE).

The Uptake of Heparanase into Mast Cells Is Regulated by Its Enzymatic Activity to Degrade Heparan Sulfate.

Mast cells take up extracellular latent heparanase and store it in secretory granules. The present study examined whether the enzymatic activity of heparanase regulates its uptake efficiency. Recombinant mouse heparanase mimicking both the latent and mature forms (L-Hpse and M-Hpse, respectively) was internalized into mastocytoma MST cells, peritoneal cell-derived mast cells, and bone marrow-derived mast cells. The internalized amount of L-Hpse was significantly higher than that of M-Hpse. In MST cells, L-Hpse was continuously internalized for up to 8 h, while the uptake of M-Hpse was saturated after 2 h of incubation. L-Hpse and M-Hpse are similarly bound to the MST cell surface. The expression level of cell surface heparan sulfate was reduced in MST cells incubated with M-Hpse. The internalized amount of M-Hpse into mast cells was significantly increased in the presence of heparastatin (SF4), a small molecule heparanase inhibitor that does not affect the binding of heparanase to immobilized heparin. Enzymatically quiescent M-Hpse was prepared with a point mutation at Glu335. The internalized amount of mutated M-Hpse was significantly higher than that of wild-type M-Hpse but similar to that of wild-type and mutated L-Hpse. These results suggest that the enzymatic activity of heparanase negatively regulates the mast cell-mediated uptake of heparanase, possibly via the downregulation of cell surface heparan sulfate expression.

Cut-off Value of HSPG for Early Marker of Plasma Leakage in Adult Dengue Patient

Plasma leakage is a state of increased vascular permeability due to loss of interendothelial junction and focal adhesion. Endothelium glycocalyx plays a major role in the pathogenesis of plasma leakage. Proteoglycans glycocalyx consists of heparan sulfate (HSPG) around 50-90%. Plasma leakage in dengue infection can be fatal and early detection is essential. This study aimed to determine the optimal cut-off value of HSPG levels for early marker of plasma leakage in dengue infection. The study was a retrospective cohort study as a part of the Community Based Dengue Study of the Faculty of Medicine, University of Indonesia, which was conducted between February 2010 and January 2011. Subjects were recruited by consecutive sampling. Dengue infection was confirmed by conventional PCR serotyping. Subjects were categorized into 2 groups, i.e. those with and without plasma leakage. Plasma leakage was confirmed by hemoconcentration or hypoalbuminemia or USG results. The level of HSPG was measured using the ELISA method from the first until the third day of the hospital admission. A total of 40 subjects were involved in this study, consisting of 21 with leakage and 19 without leakage. The optimal cut-off level of HSPG as a marker of early plasma leakage in dengue infection patients on the 1st day, 2nd day, and 3rd day was was 2179.73 pg/mL, 2538.66 pg/mL, and 1294.06 pg/mL, respectively. HSPG could as an early marker of plasma leakage in dengue infection with an optimal cut-off value for each of the first 3 days of the patient's fever. A pediatric study was recommended to obtain the optimal cut-off value for HSPG.

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry–based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

Pathogen‐binding nanoparticles to inhibit host cell infection by heparan sulfate and sialic acid dependent viruses and protozoan parasites (2/2024)

Pathogen‐binding nanoparticles to inhibit host cell infection by heparan sulfate and sialic acid dependent viruses and protozoan parasites (2/2024)

Updated results from first-in-human phase 1 dose-escalation trial of TAK-102, a GPC3-targeted armored CAR T cells, in patients with advanced solid tumors.

2543 Background: Glypican 3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans that are attached to the cell surface by a glycosylphosphatidylinositol anchor. High GPC3 expression rates are reported in numerous cancer types with a high unmet medical need, including hepatocellular carcinoma (HCC). TAK-102 is a GPC3-targeted autologous chimeric antigen receptor T cell (CAR-T) immunotherapy armored with IL-7 and CCL19. The addition of IL-7 and CCL19 to the construct was designed to support the expansion of memory subsets and persistence of CAR-T cells. We hypothesized TAK-102 would help overcome the challenges associated with an immunosuppressive tumor microenvironment that limit the activity of nonarmored CAR-T therapies in solid tumors. Methods: The first-in-human, Phase 1 dose escalation study evaluated TAK-102 in patients (pts) with GPC3-expressing solid tumors who were refractory or intolerant to standard treatments. TAK-102 was administered via a single infusion to 3 dose cohorts after lymphodepleting chemotherapy (LDC; consisting of fludarabine and cyclophosphamide): dose level (DL) 1 (starting cohort), 1x107 CAR+ cells/body; DL2, 1x108 CAR+ cells/body; DL3, 5x108 CAR+ cells/body. Objectives included evaluation of safety, dose-limiting toxicity (DLT), recommended phase 2 dose, cellular kinetic (CK) parameters, tumor markers, cytokine/chemokine and antitumor activity based on RECIST 1.1. Results: Eleven pts were enrolled and infused TAK-102 (DL1: 3 pts, DL2: 3 pts, DL3: 5 pts): 1 gastric neuroendocrine carcinoma, 2 liposarcoma, 8 HCC. Five pts achieved stable disease (SD), HCC (GPC3 H-Score: 36) . In patients achieving SD, the greatest reduction in tumor size was 26.4%. No DLTs or neurotoxicity were observed. Six pts experienced cytokine release syndrome (Grade1: 5 pts, Grade2: 1 pt); all cases were manageable. AFP was measured as a tumor marker for HCC. Among 8 pts with HCC, 4 had SD after treatment with TAK-102 and 3 showed a decrease or stabilization of AFP levels corresponding to their clinical status. CK profiles for pts were assessed by flow cytometry and qPCR-based assays. Overall, there was improvement in TAK-102 exposure (Cmax, AUC) when escalating from DL1 to DL2. There was a slight decrease in Tmax from DL2 to DL3. Homeostatic cytokine (IL-7) spiked post-LDC and showed no further increase after TAK-102 infusion across all the DLs. There was dose-dependency observed in peak CCL19, IFN-γ and IL-6 levels. Conclusions: TAK-102, an armored CAR-T, is well tolerated and has a manageable safety profile with some early signs of anti-tumor activity. For CK, TAK-102 exposure (Cmax, AUC) showed improvement from DL1 to DL2, and slight decrease in Tmax from DL2 to DL3. Dose-dependency was observed in peak CCL19, IFN-γ and IL-6 levels, which may point towards increased signal of activity from DL1 to DL3. Clinical trial information: NCT04405778 .

Galectin-3-Binding Protein Inhibits Extracellular Heparan 6-O-Endosulfatase Sulf-2

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein–protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment.

ATHENA: A phase 1/2 study of AZD5851, a chimeric antigen receptor (CAR) T-cell therapy directed against GPC3 in adult patients with advanced/recurrent hepatocellular carcinoma (HCC).

TPS2675 Background: Checkpoint inhibitor therapy is the standard first-line treatment for advanced/recurrent HCC, but there is a significant unmet therapeutic need for patients (pts) who progress after first-line treatment. Adoptive cellular immunotherapy may provide a novel treatment option. Glypican-3 (GPC3), a heparan sulfate proteoglycan, is overexpressed by tumor cells in HCC and virtually absent in healthy tissues, making it an ideal target for CAR-T therapy. AZD5851 is an autologous CAR-T product that expresses a CAR specific for GPC3 and a dominant negative (dn)TGFβRII as an armoring strategy. Expression of dnTGFβRII can block TGFβ signaling, protecting CAR T-cells from the immunosuppressive effects of this cytokine. Early clinical data with C-CAR031, a CAR-T product with the same GPC3-specific CAR and dnTGFβRII-armoring transgene as AZD5851, showed promising tolerability and antitumor activity in pts with HCC [1]. ATHENA (NCT06084884) is a first-in-human, single-arm, open-label, multicenter phase 1/2 study to evaluate the safety, tolerability, and antitumor activity of AZD5851 in pts with GPC3+ advanced/recurrent HCC, for whom ≥1 prior line of standard therapy failed or was not tolerable. Methods: Eligible pts are aged ≥18 years with histologically confirmed advanced/recurrent or metastatic and/or unresectable HCC, GPC3+ tumor sample as determined by immunohistochemistry, ≥1 prior line of standard systemic therapy, ≥1 measurable lesion per RECIST 1.1, Child-Pugh A, ECOG PS 0/1, and adequate organ and bone marrow function. Approximately 24 pts will be enrolled in Part A (Phase I, dose escalation) and 50 in Part B (Phase II dose expansion). Pts will undergo apheresis to collect peripheral blood mononuclear cells for AZD5851 production, after which they may receive approved bridging therapy for disease control. Upon successful generation of AZD5851 product, pts will undergo lymphodepletion before receiving a single dose of IV AZD5851. Subsequent follow-up is divided into 3 stages: Stage 1, active follow-up of all pts through 6 months post AZD5851 infusion; Stage 2, further evaluation of pts who have not progressed or started a new anticancer regimen during Stage 1; Stage 3, long-term follow-up for all pts who have received AZD5851. The primary endpoints are to assess the safety/tolerability of AZD5851 (both study parts), and to determine the recommended dose(s) of AZD5851 for expansion and phase 2 trials (Part A). Secondary endpoints include efficacy (objective response rate, best overall response, duration of response, disease control rate, durable response rate, time to response, progression-free survival, and overall survival) and pharmacokinetics. Exploratory endpoints include pharmacodynamic biomarkers and immunogenicity. Enrollment began Nov 2023. 1. Zhang et al. AACR 2023. Abstract CT097. Clinical trial information: NCT06084884 .

Beyond amyloid and tau: rethinking Alzheimer's disease through less explored avenues.

Neurodegenerative diseases, particularly Alzheimer's disease (AD), pose a significant challenge in ageing populations. Our current understanding indicates that the onset of toxic amyloid and tau protein pathologies initiates disease progression. However, existing treatments targeting these hallmark symptoms offer symptomatic relief without halting disease advancement. This review offers an alternative perspective on AD, centring on impaired adult hippocampal neurogenesis (AHN) as a potential early aetiological factor. By delving into the intricate molecular events during the initial stages of AD (Braak Stages I-III), a novel hypothesis is presented, interweaving the roles of Notch signalling and heparan sulfate proteoglycans (HSPGs) in compromised AHN. While acknowledging the significance of the amyloid and tau hypotheses, it calls for further exploration beyond these paradigms, suggesting the potential of altered HS sulfation patterns in AD initiation. Future directions propose more detailed investigations into early HS aggregation, aberrant sulfation patterns and examination of their temporal relationship with tau hyperphosphorylation. In challenging the conventional 'triggers' of AD and urging their reconsideration as symptoms, this review advocates an alternative approach to understanding this disease, offering new avenues of investigation into the intricacies of AD pathogenesis.