Extracellular Surface Research Articles

MT1-MMP as a Key Regulator of Metastasis.

Membrane type1-matrix metalloproteinase (MT1-MMP) is a member of metalloproteinases that is tethered to the transmembrane. Its major function in cancer progression is to directly degrade the extracellular matrix components, which are mainly type I-III collagen or indirectly type IV collagen through the activation of MMP-2 with a cooperative function of the tissue inhibitor of metalloproteinase-2 (TIMP-2). MT1-MMP is expressed as an inactive form (zymogen) within the endoplasmic reticulum (ER) and receives truncation processing via furin for its activation. Upon the appropriate trafficking of MT1-MMP from the ER, the Golgi apparatus to the cell surface membrane, MT1-MMP exhibits proteolytic activities to the surrounding molecules such as extracellular matrix components and cell surface molecules. MT1-MMP also retains a non-proteolytic ability to activate hypoxia-inducible factor 1 alpha (HIF-1A) via factors inhibiting the HIF-1 (FIH-1)-Mint3-HIF-1 axis, resulting in the upregulation of glucose metabolism and oxygen-independent ATP production. Through various functions of MT1-MMP, cancer cells gain motility on migration/invasion, thus causing metastasis. Despite the long-time efforts spent on the development of MT1-MMP interventions, none have been accomplished yet due to the side effects caused by off-target effects. Recently, MT1-MMP-specific small molecule inhibitors or an antibody have been reported and these inhibitors could potentially be novel agents for cancer treatment.

Comprehensive Strategy for Identifying Extracellular Vesicle Surface Proteins as Biomarkers for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that has become a global health concern due to its increasing prevalence. There is a need for reliable biomarkers to aid in the diagnosis and prognosis of NAFLD. Extracellular vesicles (EVs) are promising candidates in biomarker discovery, as they carry proteins that reflect the pathophysiological state of the liver. In this review, we developed a list of EV proteins that could be used as diagnostic biomarkers for NAFLD. We employed a multi-step strategy that involved reviewing and comparing various sources of information. Firstly, we reviewed papers that have studied EVs proteins as biomarkers in NAFLD and papers that have studied circulating proteins as biomarkers in NAFLD. To further identify potential candidates, we utilized the EV database Vesiclepedia.org to qualify each protein. Finally, we consulted the Human Protein Atlas to search for candidates' localization, focusing on membrane proteins. By integrating these sources of information, we developed a comprehensive list of potential EVs membrane protein biomarkers that could aid in diagnosing and monitoring NAFLD. In conclusion, our multi-step strategy for identifying EV-based protein biomarkers for NAFLD provides a comprehensive approach that can also be applied to other diseases. The protein candidates identified through this approach could have significant implications for the development of non-invasive diagnostic tests for NAFLD and improve the management and treatment of this prevalent liver disorder.

Cell surface profiling of cultured cells by direct hydrazide capture of oxidized glycoproteins

Glycoproteins are a particularly interesting subset of the cellular proteome as a high proportion of proteins present on the extracellular cell surface are glycosylated. These cell surface proteins are ideal targets for biologic drug therapies or for diagnostics tests. Here, we describe a modification of the well-described Cell Surface Capture (CSC) method for the selective isolation and identification of cell surface glycoproteins that contain N-linked carbohydrates. This modification, which we refer to as Direct Cell Surface Capture (D-CSC), is based on oxidation of cell surface glycans on intact cells, followed by direct conjugation of the oxidized oligosaccharides to a solid support using hydrazide chemistry, with no biotinylation step. As a proof-of-principle, we applied D-CSC to the analysis of cell surface membrane proteins of three adherent cancer cell lines (A549, OVCAR3, and U87MG) and compared our results to those published using the well-established Cell Surface Capture (CSC) method, demonstrating comparable selectivity for cell surface proteins.•A method enabling the identification of cell surface proteins from cells in culture is described.•Application of this method to profile the cell surface on three different cancer cell lines is included.

RNA Crossing Membranes: Systems and Mechanisms Contextualizing Extracellular RNA and Cell Surface GlycoRNAs.

The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs.

Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface to allosterically modulate the receptor. Extracellularly binding allosteric nanobodies have also been investigated, but the structural basis for their activity has not been resolved to date. Here, we report a library of Nbs that bind to the extracellular surface of rhodopsin and allosterically modulate the thermodynamics of its activation process. Crystal structures of Nb2 in complex with native rhodopsin reveal a mechanism of allosteric modulation involving extracellular loop 2 and native glycans. Nb2 binding suppresses Schiff base deprotonation and hydrolysis and prevents intracellular outward movement of helices five and six – a universal activation event for GPCRs. Nb2 also mitigates protein misfolding in a disease-associated mutant rhodopsin. Our data show the power of nanobodies to modulate the photoactivation of rhodopsin and potentially serve as therapeutic agents for disease-associated rhodopsin misfolding.

Carrageenan as a macromolecular crowding agent in human umbilical cord derived mesenchymal stromal cell culture

Cell sheet tissue engineering requires prolonged in vitro culture for the development of implantable devices. Unfortunately, lengthy in vitro culture is associated with cell phenotype loss and substantially higher cost of goods, which collectively hinder clinical translation and commercialisation of tissue engineered medicines. Although macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition, whilst maintaining cellular phenotype, the optimal macromolecular crowding agent still remains elusive. Herein, we evaluated the biophysical properties of seven different carrageenan molecules at five different concentrations and their effect on human umbilical cord-derived mesenchymal stromal cell morphology, viability, metabolic activity, proliferation, extracellular matrix deposition and surface marker expression. All types of carrageenan (CR) assessed demonstrated a hydrodynamic radius increase as a function of increasing concentration; high polydispersity; and negative charge. Two iota CRs were excluded from further analysis due to poor solubility in cell culture. Among the remaining five carrageenans, the lambda medium viscosity type at concentrations of 10 and 50 μg/ml did not affect cell morphology, viability, metabolic activity, proliferation and expression of surface markers and significantly increased the deposition of collagen types I, III and IV, fibronectin and laminin. Our data highlight the potential of lambda medium viscosity carrageenan as a macromolecular crowding agent for the accelerated development of functional tissue engineered medicines.

Development of tandem cation exchange chromatography for high purity extracellular vesicle isolation: The effect of ligand steric availability

Purification of extracellular vesicles for research and therapeutic applications requires updated methodology to address the limitations of traditional ultracentrifugation and other size-based separation techniques. Their downfalls include induced extracellular vesicle aggregation, low yields, poor scalability and one-dimensionality of the separation process, as the size or sedimentation speed of extracellular vesicles is often the only selection criterion. Ion exchange chromatography is a promising alternative or supplementary method candidate, as it offers a different approach for extracellular vesicle separation, which is surface charge. For now, mostly anion exchange chromatography has been evaluated for extracellular vesicle purification, as it successfully relies on the strongly negative surface charge of extracellular vesicles. However, as extracellular vesicles are very complex in their structure, also cation exchange chromatography could be applicable, due to individual cationic domains on the extracellular vesicle surface. Here, we compare anion exchange chromatography to different types of cation exchange chromatography for the purification of platelet extracellular vesicle samples also containing plasma-derived impurities. We found that the choice of resin structure used for cation exchange chromatography is critical for binding platelet extracellular vesicles, as a conventional-type cation exchanger was found to only capture and elute less than 20% of extracellular vesicles. With the tentacle-type resin, it was possible to obtain comparable platelet extracellular vesicle yields (over 90%) with cation exchange chromatography compared to anion exchange chromatography, as well as superior purity, especially when it was combined to conventional cation exchange resin.

Strain and temperature dependent aggregation of Candida auris is attenuated by inhibition of surface amyloid proteins

Candida auris is a multi-drug resistant human fungal pathogen that has become a global threat to human health due to its drug resistant phenotype, persistence in the hospital environment and propensity for patient to patient spread. Isolates display variable aggregation that may affect the relative virulence of strains. Therefore, dissection of this phenotype has gained substantial interest in recent years. We studied eight clinical isolates from four different clades (I-IV); four of which had a strongly aggregating phenotype and four of which did not. Genome analysis identified polymorphisms associated with loss of cell surface proteins were enriched in weakly-aggregating strains. Additionally, we identified down-regulation of chitin synthase genes involved in the synthesis of the chitinous septum. Characterisation of the cells revealed no ultrastructural defects in cytokinesis or cell separation in aggregating isolates. Strongly and weakly aggregating strains did not differ in net surface charge or in cell surface hydrophobicity. The capacity for aggregation and for adhesion to polystyrene microspheres were also not correlated. However, aggregation and extracellular matrix formation were all increased at higher growth temperatures, and treatment with the amyloid protein inhibitor Thioflavin-T markedly attenuated aggregation. Genome analysis further indicated strain specific differences in the genome content of GPI-anchored proteins including those encoding genes with the potential to form amyloid proteins. Collectively our data suggests that aggregation is a complex strain and temperature dependent phenomenon that may be linked in part to the ability to form extracellular matrix and cell surface amyloids.

Two-Dimensional NMR Spectroscopy of the G Protein-Coupled Receptor A2AAR in Lipid Nanodiscs.

Eight hundred and twenty-six human G protein-coupled receptors (GPCRs) mediate the actions of two-thirds of the human hormones and neurotransmitters and over one-third of clinically used drugs. Studying the structure and dynamics of human GPCRs in lipid bilayer environments resembling the native cell membrane milieu is of great interest as a basis for understanding structure-function relationships and thus benefits continued drug development. Here, we incorporate the human A2A adenosine receptor (A2AAR) into lipid nanodiscs, which represent a detergent-free environment for structural studies using nuclear magnetic resonance (NMR) in solution. The [15N,1H]-TROSY correlation spectra confirmed that the complex of [u-15N, ~70% 2H]-A2AAR with an inverse agonist adopts its global fold in lipid nanodiscs in solution at physiological temperature. The global assessment led to two observations of practical interest. First, A2AAR in nanodiscs can be stored for at least one month at 4 °C in an aqueous solvent. Second, LMNG/CHS micelles are a very close mimic of the environment of A2AAR in nanodiscs. The NMR signal of five individually assigned tryptophan indole 15N-1H moieties located in different regions of the receptor structure further enabled a detailed assessment of the impact of nanodiscs and LMNG/CHS micelles on the local structure and dynamics of A2AAR. As expected, the largest effects were observed near the lipid-water interface along the intra- and extracellular surfaces, indicating possible roles of tryptophan side chains in stabilizing GPCRs in lipid bilayer membranes.

Investigation of heavy metal removal from salty wastewater and voltage production using Shewanella oneidensisMR‐1 nanowires in a dual‐chamber microbial fuel cell

AbstractHeavy metal removal and simultaneous energy production were studied using a dual chambered Microbial Fuel Cell inoculated with Shewanella oneidensis MR‐1 in the anode. Synthetic wastewater was prepared with Cu (II), Mg (II), Mn (II), Zn (II), Na, and Phenol based on desalter effluent from refinery processes at different metal concentrations. In this study, a maximum open‐circuit voltage of 517.6 mV was reached at Conc. 5 with wastewater in the anode chamber, and 127.7 mV at Conc. 3 was produced with synthetic wastewater in the cathode chamber. Moreover, μ at Conc. 5 was 0.1133 h−1, demonstrating bacterial growth under metal and phenol concentrations. The highest metal removal in the anode for Cu (II), Mg (II), Mn (II), Zn (II), and Na was 93%, 85%, 93%, 88%, and 36%, respectively. In the cathode chamber the removal of Cu (II), Mg (II), Mn (II), Zn (II), and Na was 98%, 49%, 57%, 59%, and 36%, respectively. During the operation in the anode, SEM images showed that the bacterial nanowires are formed in response to toxic and anaerobic environments which contribute to the bacterial growth. These nanowires increased the metal removal and the voltage production as a consequence of a higher electron rate from the anode to the cathode due to the higher extracellular membrane surface area. S. oneidensis is a bacterium with metal‐reducing characteristics, and it is suitable for metal removal and electron transport from carbon sources, demonstrated in voltage production with microbial fuel cells.

Poria cocos compounds targeting neuropeptide Y1 receptor (Y1R) for weight management: A computational ligand- and structure-based study with molecular dynamics simulations identified beta-amyrin acetate as a putative Y1R inhibitor.

Poria cocos (PC) is a medicinal herb frequently used in weight-loss clinical trials, however the mechanisms by which its compounds target orexigenic receptors including the neuropeptide Y1 receptor (Y1R) remain largely unknown. This study aimed to screen PC compounds for favourable pharmacokinetics profiles and examine their molecular mechanisms targeting Y1R. Forty-three PC compounds were systematically sought from pharmacological databases and docked with Y1R (PDB: 5ZBQ). By comparing the relative binding affinities, pharmacokinetics and toxicity profiles, we hypothesised that compounds designated PC1 3,4-Dihydroxybenzoic acid, PC8 Vanillic acid, PC40 1-(alpha-L-Ribofuranosyl)uracil, could be potential antagonists as they contact major residues Asn283 and Asp287, similar to various potent Y1R antagonists. In addition, PC21 Poricoic acid B, PC22 Poricoic acid G and PC43 16alpha,25-Dihydroxy-24-methylene-3,4-secolanosta-4(28),7,9(11)-triene-3,21-dioic acid, contacting Asn299, Asp104 and Asp200 proximal to the extracellular surface could also interfere with agonist binding by stabilising the extracellular loop (ECL) 2 of Y1R in a closed position. Owing to their selective interaction with Phe302, an important residue in binding of selective Y1R antagonists, PC12 beta-Amyrin acetate, PC26 3-Epidehydrotumulosic acid and PC27 Cerevisterol were proposed as putative antagonists. Following the consensus approach, PC12 beta-Amyrin acetate, PC26 3-Epidehydrotumulosic acid and PC27 Cerevisterol were identified as candidate compounds due to their high affinities (-12.2, -11.0 and -10.8 kcal, respectively), high drug-likeness and low toxicity profiles. Trajectory analyses and energy contributions of PC12-Y1R complex further confirmed their structural stability and favourable binding free energies, highlighting the feasibility and possible development of PC12 beta-Amyrin acetate as a future Y1R inhibitor.

Polymerase-Driven Logic Signal Amplification for the Detection of Small Extracellular Vesicle Surface Proteins and the Identification of Breast Cancer.

Small extracellular vesicles (sEVs) derived from tumors contain a vast amount of cellular information and are regarded as a potential diagnostic biomarker for noninvasive cancer diagnosis. Nevertheless, it remains challenging to accurately measure sEVs from clinical samples due to the low abundance of these vesicles as well as their phenotypic heterogeneity. Herein, a polymerase-driven logic signal amplification system (PLSAS) was developed for the high-sensitivity detection of sEV surface proteins and breast cancer (BC) identification. Aptamers were introduced to serve as sensing modules to specifically recognize target proteins. By changing the input DNA sequences, two polymerase-driven primer exchange reaction systems were rationally designed for DNA logic computing. This allows for autonomous targeting of a limited number of targets using "OR" and "AND" logic, leading to a significant increase in fluorescence signals and enabling the specific and ultrasensitive detection of sEV surface proteins. In this work, we investigated surface proteins of mucin 1 (MUC1) and the epithelial cell adhesion molecule (EpCAM) as model proteins. When MUC1 or EpCAM proteins were used as single signal input in the "OR" DNA logic system, the detection limit of sEVs was 24 or 58 particles/μL, respectively. And MUC1 and EpCAM proteins of sEVs can be simultaneously detected in the AND logic method, which can significantly reduce the effect of phenotypic heterogeneity of sEVs to distinguish the source of sEVs derived from various mammary cell lines, such as MCF-7, MDA MB 231, SKBR3, and MCF-10A. The approach has achieved high discrimination in serologically tested positive BC samples (AUC 98.1%) and holds significant potential in advancing the early diagnosis and prognostic assessments of BC.

Crystal structure of Leptospira LSS_01692 reveals a dimeric structure and induces inflammatory responses through Toll-like receptor 2-dependent NF-κB and MAPK signal transduction pathways.

Leptospirosis is a commonly overlooked zoonotic disease that occurs in tropical and subtropical regions. Recent studies have divided the Leptospira spp. into three groups based on virulence, including pathogenic, intermediate, and saprophytic species. Pathogenic species express a protein family with leucine-rich repeat (LRR) domains, which are less expressed or absent in nonpathogenic species, highlighting the importance of this protein family in leptospirosis. However, the role of LRR domain proteins in the pathogenesis of leptospirosis is still unknown and requires further investigation. In this study, the 3D structure of LSS_01692 (rLRR38) was obtained using X-ray crystallography at a resolution of 3.2 Å. The results showed that rLRR38 forms a typical horseshoe structure with 11 α-helices and 11 β-sheets and an antiparallel dimeric structure. The interactions of rLRR38 with extracellular matrix and cell surface receptors were evaluated using ELISA and single-molecule atomic force microscopy. The results showed that rLRR38 interacted with fibronectin, collagen IV, and Toll-like receptor 2 (TLR2). Incubating HK2 cells with rLRR38 induced two downstream inflammation responses (IL-6 and MCP-1) in the TLR2 signal transduction pathway. The TLR2-TLR1 complex showed the most significant upregulation effects under rLRR38 treatment. Inhibitors also significantly inhibited nuclear factor κB and mitogen-activated protein kinases signals transduction under rLRR38 stimulation. In conclusion, rLRR38 was determined to be a novel LRR domain protein in 3D structure and demonstrated as a TLR2-binding protein that induces inflammatory responses. These structural and functional studies provide a deeper understanding of the pathogenesis of leptospirosis.

#5649 HEPARANASE OVEREXPRESSION MODIFIES PODOCYTE MOTILITY

Abstract Background and Aims Heparanase is an endo-β-d-glucuronidase that degrades heparan sulfate side chains, which are essential components of extracellular matrices and cell surfaces, including in the kidney filtration apparatus, the glomerulus. Upregulation of heparanase in glomerular diseases (GDs) was demonstrated in experimental models of GDs and in humans. Using transgenic mice overexpressing the human heparanase gene, we recently showed that heparanase reduced proteinuria and preserved the glomerular filtration barrier (GFB) after Adriamycin-induced injury. Furthermore, heparanase improved survival of key GFB cells, the podocytes and enhanced their autophagic flux both in vitro and in vivo. Here, we aimed to study the potential role of heparanase on podocyte locomotion and adhesion. Methods We utilized immortalized human podocyte cell line AB8/13 (a kind gift of Prof. Moin Saleem, Bristol, UK) as an experimental platform. Cells were infected with pLenti6/V5-DEST carrying the heparanase gene construct (H) or control empty vector (V) and were allowed to differentiate for 14 days. To induce injury, differentiated podocytes were treated with Adriamycin (0.5 μg/mL), mimicking Adriamycin nephropathy, an established experimental model for progressive proteinuric GDs. For readouts, we used scratch assay, RT-qPCR, Western blot (WB) and immunofluorescence (IF) analyses. Results The in vitro scratch assay showed that differentiated (H) podocytes migrated at a slower rate compared with control (V) podocytes, both at baseline and after a 12-hour treatment with Adriamycin. Hence, we hypothesized that heparanase may alter podocyte adhesion properties. Indeed, IF staining of differentiated (H) podocytes exhibited an increase in focal contacts containing vinculin compared with control (V) cells treated with Adriamycin. Of note, we observed no differences in vinculin abundance by WB, suggesting that heparanase mediates the active form of vinculin. Moreover, among the integrins, we noticed temporal upregulation of ITGB1 transcription, as early as two hours after Adriamycin treatment, in (H) compared with control (V) podocytes (P = 0.032). Conclusions Collectively, our results suggest that in response to Adriamycin injury, constitutive heparanase overexpression could stabilize podocyte focal adhesions and control motility. Although further research is needed, improving the anchoring of podocytes over the basement membrane may provide an additional cellular mechanism by which heparanase conferred protection against Adriamycin toxicity.

Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles

Extracellular vesicles (EVs) are important mediators of intercellular communication in the tumour microenvironment. Many studies suggest that cancer cells release higher amounts of EVs exposing phosphatidylserine (PS) at the surface. There are lots of interconnections between EVs biogenesis and autophagy machinery. Modulation of autophagy can probably affect not only the quantity of EVs but also their content, which can deeply influence the resulting pro-tumourigenic or anticancer effect of autophagy modulators. In this study, we found that autophagy modulators autophinib, CPD18, EACC, bafilomycin A1 (BAFA1), 3-hydroxychloroquine (HCQ), rapamycin, NVP-BEZ235, Torin1, and starvation significantly alter the composition of the protein content of phosphatidylserine-positive EVs (PS-EVs) produced by cancer cells. The greatest impact had HCQ, BAFA1, CPD18, and starvation. The most abundant proteins in PS-EVs were proteins typical for extracellular exosomes, cytosol, cytoplasm, and cell surface involved in cell adhesion and angiogenesis. PS-EVs protein content involved mitochondrial proteins and signalling molecules such as SQSTM1 and TGFβ1 pro-protein. Interestingly, PS-EVs contained no commonly determined cytokines, such as IL-6, IL-8, GRO-α, MCP-1, RANTES, and GM-CSF, which indicates that secretion of these cytokines is not predominantly mediated through PS-EVs. Nevertheless, the altered protein content of PS-EVs can still participate in the modulation of the fibroblast metabolism and phenotype as p21 was accumulated in fibroblasts influenced by EVs derived from CPD18-treated FaDu cells. The altered protein content of PS-EVs (data are available via ProteomeXchange with identifier PXD037164) also provides information about the cellular compartments and processes that are affected by the applied autophagy modulators.AbyDEs_V2PATMNaEJqximCVideo

Selective polypeptide ligand binding to the extracellular surface of the transmembrane domains of the class B GPCRs GLP-1R and GCGR

Crystal and cryo-EM structures of the glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) bound with their peptide ligands have been obtained with full-length constructs, indicating that the extracellular domain (ECD) is indispensable for specific ligand binding. This article complements these data with studies of ligand recognition of the two receptors in solution. Paramagnetic NMR relaxation enhancement measurements using dual labeling with fluorine-19 probes on the receptor and nitroxide spin labels on the peptide ligands provided new insights. The glucagon-like peptide-1 (GLP-1) was found to interact with GLP-1R by selective binding to the extracellular surface. The ligand selectivity toward the extracellular surface of the receptor was preserved in the transmembrane domain (TMD) devoid of the ECD. The dual labeling approach further provided evidence of cross-reactivity of GLP-1R and GCGR with glucagon and GLP-1, respectively, which is of interest in the context of medical treatments usingcombinations of the two polypeptides.

T-Cell Engagers in Solid Cancers—Current Landscape and Future Directions

Monoclonal antibody treatment initially heralded an era of molecularly targeted therapy in oncology and is now widely applied in modulating anti-cancer immunity by targeting programmed cell receptors (PD-1, PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and, more recently, lymphocyte-activation gene 3 (LAG3). Chimeric antigen receptor T-cell therapy (CAR-T) recently proved to be a valid approach to inducing anti-cancer immunity by directly modifying the host’s immune cells. However, such cell-based therapy requires extensive resources such as leukapheresis, ex vivo modification and expansion of cytotoxic T-cells and current Good Manufacturing Practice (cGMP) laboratories and presents significant logistical challenges. Bi-/trispecific antibody technology is a novel pharmaceutical approach to facilitate the engagement of effector immune cells to potentially multiple cancer epitopes, e.g., the recently approved blinatumomab. This opens the opportunity to develop ‘off-the-shelf’ anti-cancer agents that achieve similar and/or complementary anti-cancer effects as those of modified immune cell therapy. The majority of bi-/trispecific antibodies target the tumor-associated antigens (TAA) located on the extracellular surface of cancer cells. The extracellular antigens represent just a small percentage of known TAAs and are often associated with higher toxicities because some of them are expressed on normal cells (off-target toxicity). In contrast, the targeting of intracellular TAAs such as mutant RAS and TP53 may lead to fewer off-target toxicities while still achieving the desired antitumor efficacy (on-target toxicity). Here, we provide a comprehensive review on the emerging field of bi-/tri-specific T-cell engagers and potential therapeutic opportunities.

Exploring Membrane‐tethering Technology for Proteins as a Versatile Tool for Uncovering Novel Disease Targets and Advancing Biotherapeutic Development

AbstractMembrane‐tethering technology for proteins (MTFP) is a promising approach for the development of therapeutic agents that display bioactive proteins, such as antibodies and cytokines, on the cell surface, resulting in the induction of autocrine signalling. In this review article, we provide a comprehensive overview of the MTFP, including its basic principles, selection of agonist antibodies and peptides, and the identification of novel functions of natural cytokines. Furthermore, we discuss the potential of increasing the therapeutic efficacy of existing treatments by engineering active proteins to the cell and extracellular vesicle surfaces. We suggest that the MTFP has the potential to maximize efficiency in drug discovery by identifying proteins with regulatory functions and engineering existing treatments. Our review highlights the importance of MTFP in basic research and translational research, and its potential to apply the development of biotherapeutics.

Neural Stem Cells as Carriers of Nucleoside-Conjugated Nanogels: A New Approach toward Cell-Mediated Delivery

Neural stem cells (NSCs) present attractive natural drug delivery systems (DDSs). Their migratory potential enables crossing of the blood-brain barrier and efficient and selective accumulation near malignant cells. Here, we present the potential of NSCs as DDSs for nucleoside analogue-conjugated nanogels (NGs). Two different approaches were investigated: the intracellular loading and extracellular cell surface decoration with NGs. For both designs, the tumor-specific migratory potentials of NSCs remained unchanged; however, the intracellular loading showed a shorter NG retention. The cell surface decoration protocol yielded a high loading capacity of 100% after 1 h and a prolonged drug retention. A redox-sensitive linker between NGs and the nucleoside analogue 5-ethynyl-2'-deoxycytidine (EdC) allowed a tumor environment-specific drug release and its efficient and preferential incorporation into the DNA of the tumor cells. Interestingly, the tumor-trafficking potentials of NSCs were significantly potentiated by irradiation of tumor cells. In conclusion, this study indicates the potentials of cell surface-decorated NSCs as DDSs for tumor-specific release, cellular uptake, and incorporation of EdC into DNA.

Modification of Extracellular Vesicle Surfaces: An Approach for Targeted Drug Delivery.

Extracellular vesicles (EVs) are a promising drug delivery vehicle candidate because of their natural origin and intrinsic function of transporting various molecules between different cells. Several advantages of the EV delivery platform include enhanced permeability and retention effect, efficient interaction with recipient cells, the ability to traverse biological barriers, high biocompatibility, high biodegradability, and low immunogenicity. Furthermore, EV membranes share approximately similar structures and contents to the cell membrane, which allows surface modification of EVs, an approach to enable specific targeting. Enhanced drug accumulation in intended sites and reduced adverse effects of chemotherapeutic drugs are the most prominent effects of targeted drug delivery. In order to improve the targeting ability of EVs, chemical modification and genetic engineering are the most adopted methods to date. Diverse chemical methods are employed to decorate EV surfaces with various ligands such as aptamers, carbohydrates, peptides, vitamins, and antibodies. In this review, we introduce the biogenesis, content, and cellular pathway of natural EVs and further discuss the genetic modification of EVs, and its challenges. Furthermore, we provide a comprehensive deliberation on the various chemical modification methods for improved drug delivery, which are directly related to increasing the therapeutic index.