Biology subjects

Song, X.

Publications and source records attributed to Song, X..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Lab on a Capillary: Instrument-Free Compartmentalization Using Photopatterned Hydrogel Rings Embedded inside a Glass Capillary for Amplified Bioassays

Sensitive and low-cost protein biomarker detection is critical for disease diagnosis. Advanced microfluidic systems can generate miniature reaction compartments for a high-sensitivity assay. However, these platforms often require external instruments, skilled operators, and complex setups. Here, we develop a Lab on a Capillary (LabCap) platform that integrates photopatterned hydrogel rings within a glass capillary using a reconfigurable stop-flow lithography system. During sample loading and unloading steps, nanoliter-scale aqueous droplets (torodrops) are spontaneously formed around the hydrogel rings, creating isolated reaction compartments without the need for external instruments or an immiscible oil phase. The LabCap platform enables quantitative detection of clinically relevant biomarkers, including C-reactive protein (CRP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP). By adjusting the incubation protocol, assay speed and sensitivity can be tuned to meet different analytical requirements. A periodic medium exchange protocol enables biomarker detection at concentrations as low as 1 ng/mL, whereas prolonged static incubation extends detection to 0.1 ng/mL. In addition, LabCap offers practical advantages, including low fabrication cost (<{euro}1 per device), low reagent consumption (<100 L per assay step), and minimal wash-buffer usage (1 mL). These results demonstrate that LabCap is a simple, cost-effective, and versatile platform for biomarker detection.

bioengineering↗

bioRxiv2026

Engineered probiotic Escherichia coli-mediated intestinal nicotine clearance alleviates nonalcoholic steatohepatitis in mice

Nicotine accumulates in the gut and drives non-alcoholic steatohepatitis (NASH) via the gut-liver axis, yet no effective clinical intervention is currently available. To address this challenge, the probiotic Escherichia coli Nissle 1917 (EcN) was engineered for in situ nicotine clearance in the gut. Mutational screening of nicotine oxidoreductase 2 (PpNicA2) identified a highly active variant, PpNicA2A107R. Its incorporation into EcN together with an electron transfer protein (CycN) and a newly identified transporter (T3/T7) yielded 80% nicotine-degrading activity. Chromosomal integration of this module generated a stable strain, EcN-N12, which in NASH mouse models depleted intestinal nicotine, rescued hepatic lipid metabolism, alleviated tissue damage, and intercepted the nicotine-mediated gut-liver axis pathological progression. This work thus offers an effective and clinically translatable approach for nicotine-associated diseases.

synthetic biology↗

bioRxiv2026

A High-Affinity Nanobody Recognizing mNeonGreen Enables Versatile Biochemical, Cellular, and in vivo Applications.

mNeonGreen (mNG) is among the brightest and most photostable monomeric green fluorescent proteins and is widely used for protein tagging. Here, we present sdAb(mNG), a high-affinity single-domain antibody (sdAb) that enables biochemical capture, imaging, and manipulation of mNG-tagged proteins. A 1.26 [A] crystal structure reveals an extensive interaction surface between mNG and sdAb(mNG), accounting for its high affinity (KD = 0.39 nM) and robust target recognition across diverse experimental conditions. This allows a single sdAb to support applications that typically require multiple specialized tools. We demonstrate the utility of sdAb(mNG) in several example applications including highly specific immunoprecipitation, direct immunofluorescence, and super-resolution imaging. Importantly, sdAb(mNG) retains high-performance target recognition even in intracellular environments. When expressed as an intrabody in living mammalian cells, sdAb(mNG) enables relocalization of mNG-tagged proteins to defined compartments or visualization of synaptic vesicle transport in primary neurons. In zebrafish, fusion of sdAb(mNG) to an F-box degradation domain induces cell-autonomous depletion of an endogenous mNG-tagged transcription factor and produces a clear developmental phenotype. These findings establish sdAb(mNG) as a versatile and robust affinity reagent that converts mNG from a passive fluorescent reporter into a multifunctional handle for imaging, proteomics, and programmable manipulation of endogenous and engineered proteins.

biochemistry↗

bioRxiv2026

EZH1/2 inhibition improves immunotherapy response through MHC Class II de-repression and neutrophil reprogramming

Squamous cell carcinoma of the lung is a difficult-to-treat cancer with high prevalence in the US, and particularly in Kentucky. Here, we sought to test if the EZH1/2 inhibitor valemetostat improves anti-PD1 responses in squamous cell lung cancer models, and to develop ex vivo models to test immunotherapy drug combinations. We found that valemetostat augmented anti-tumor responses to anti-PD1 therapy, led to up-regulation of tumor cell specific Major Histocompatibility Complex Class II (MHC Class II), and drove systemic neutrophil maturation. Likewise, Ezh2 knock-out mice produced neutrophils that were more apoptotic, less migratory, and less able to produce extracellular nets, but had similar ability to kill bacteria as Ezh2-WT neutrophils. To test tumor responses to differing neutrophil populations, we engineered three-dimensional air-liquid interface cultures with tumoroids, lung mesenchymal cells, and T cells, with and without bone marrow containing neutrophils and myeloid progenitors from distinct donors. Bone marrow from tumor-naive or mice with actively growing untreated tumors boosted tumoroid growth, while bone marrow from spontaneously tumor-rejected or mice with tumors treated with valemetostat was anti-tumor. MHC Class II blockade lowered the ability of bone marrow to boost tumor growth, and reduced the ability of valemetostat with anti-PD1 to reduce tumoroid growth. Patient samples revealed a strong negative correlation between EZH2 and MHC Class II, suggesting that targeting EZH2 activity could lead to marked increase in MHC Class II and improve treatment responses in lung squamous cell carcinomas.

cancer biology↗

bioRxiv2026

AAV2 Crosslinks Actin Filaments: Implications for AAV Gene Therapy Vector Design

Adeno-associated virus (AAV) capsids are important gene therapy vectors, allowing for the one-time treatment of monogenetic disorders, with durable gene expression lasting for years. However, despite the clinical success of AAV usage, low transduction efficiencies require high dosage to achieve therapeutic efficacy, resulting in prohibitive costs and rare, but life-threatening immune responses. One key knowledge gap is how the capsids traffic to the nucleus following endocytosis. Here, we identify a direct interaction between AAV2 and actin filaments. Our results show that AAV2 bundles multiple actin filaments in a highly periodic hexagonal lattice. Therefore, we propose that AAV2 is the founding member of a new class of actin binding proteins, the actin bundling viruses (ABVs). Using cryogenic electron microscopy, we determined the structure of AAV2-actin filaments, identified the interaction interfaces, and engineered capsid variants that no longer bundle actin. Together, our results suggest that the AAV2-actin interaction may be responsible for trapping capsids and mediating peri-nuclear accumulation. Overall, this interaction expands the current understanding of AAV2 biology and offers new directions for capsid engineering.

biochemistry↗

bioRxiv2026

Myeloma and therapy reshape the bone marrow niche to durably constrain immune reconstitution and vaccine responsiveness

Infections are the most common cause of non-relapse mortality in multiple myeloma (MM), but the basis of persistent immune dysfunction is obscured by patient heterogeneity and complex treatment regimens, including autologous stem cell transplant (ASCT). We performed longitudinal multi-omic profiling of matched bone marrow and peripheral blood from MM patients across diagnosis, induction, ASCT, and recovery. We found the tumor imposes a compartment-specific immune program where the marrow exhibits metabolic and inflammatory changes that bias hematopoiesis and alter cytotoxic effector programs not mirrored in blood. Adaptive immune reconstitution is impaired up to two years post-ASCT. Half of patients fail to mount IgG responses to high-dose non-adjuvanted influenza vaccine, a defect overcome by the lipid nanoparticle (LNP) adjuvanted COVID mRNA vaccine, which elicited responses in all patients, supporting adjuvanted influenza vaccine strategies in MM. Together these findings define how myeloma and its treatment durably reshape immunity from the marrow outward. HighlightsO_LIMultiple Myeloma marrow and blood show opposing metabolic and inflammatory states C_LIO_LIInduction therapy selects durable myeloma plasma-cell transcriptional states C_LIO_LIB cell and follicular helper T deficits blunt antigen responses after transplant C_LIO_LICOVID-19 vaccination builds immune memory with variable responses to flu vaccination C_LI eTOCMultiple myeloma and its treatment leave a lasting imprint on the bone marrow niche. By profiling bone marrow and blood longitudinally at diagnosis, through induction, autologous transplant, and recovery, we show that marrow-local metabolic and inflammatory constraints persist and help explain why influenza vaccination often fails while mRNA vaccination succeeds.

cancer biology↗

bioRxiv2026

Structural and biochemical characterization of a novel inhibitor of NMNAT1, the gatekeeper of nuclear NAD+ biosynthesis

Nicotinamide adenine dinucleotide (NAD+) is crucial for cellular functions including DNA repair and metabolism. Nicotinamide mononucleotide adenylyltransferase (NMNAT) enzymes catalyze the final step of NAD+ synthesis from NMN and ATP. There are three NMNAT isoforms: NMNAT1, NMNAT2, and NMNAT3, located in the nucleus, cytoplasm, and mitochondria, respectively. Nuclear NAD+ promotes disease progression in NAD+-dependent cancers, and it is hypothesized that targeting NMNAT1 with small-molecule inhibitors could be an effective therapeutic strategy. Here, we identify an NMNAT1 inhibitor from a bioactive compound screen and report its effects on NAD+ levels and the viability of NMNAT1-dependent cancer cell lines. The compound AMI-1 is a known inhibitor of Protein Arginine N-Methyltransferase 1, and we find that it also inhibits NMNAT1 with similar potency. Additionally, we determined a cryo-EM structure of NMNAT1 bound to AMI-1 and revealed its mechanism of inhibition. This provides proof of principle for inhibiting NMNAT1 to target NAD+ metabolism in dependent cancers, while also highlighting that caution is warranted when interpreting studies using AMI-1 as a PRMT1 inhibitor, given its effect on NAD+ through NMNAT1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/716846v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@139e792org.highwire.dtl.DTLVardef@923869org.highwire.dtl.DTLVardef@1b5aa1borg.highwire.dtl.DTLVardef@1b2d797_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Multi-component functionalized Bifidobacterium longum hydrogel for multi-target integrated therapy of colitis-associated anxiety and depression

Inflammatory bowel diseases (IBDs) are frequently accompanied by anxiety and depression, largely driven by perturbed gut-brain axis signaling. However, current oral therapies remain constrained by the spatial and functional separation between intestinal inflammation and central nervous system dysfunction. Here, we present a comprehensive gut-brain dual region integrated therapeutic strategy based on functionalized Bifidobacterium longum hydrogel (INPs@BL@Gel), in which baicalin and tyrosine are coordinated with Fe(III) to form infinite coordination polymers (ICPs), coated with inulin, assembled onto Bifidobacterium longum (BL), and subsequently encapsulated within a pH- and matrix metalloproteinase-responsive silk fibroin-gelatin hydrogel. INPs@BL@Gel exhibits high drug-loading, effective gastric protection, inflammation-triggered release, and long-term intestinal colonization. Within the inflamed intestine, BL and components synergistically suppress inflammatory responses, restore gut microbiota homeostasis, and promote intestinal barrier repair through multi-target integrated therapy. Importantly, BL combined with components markedly enhances the production of beneficial neuroactive metabolites such as homovanillic acid and short-chain fatty acids, which integrated regulate neuroinflammation, preserve synaptic function, and facilitate blood-brain barrier repair via the gut-brain axis. In vivo studies demonstrate that INPs@BL@Gel not only exert potent therapeutic efficacy against colitis and effectively alleviate associated depression, but also reshape the gut microbiota and restore barrier integrity, achieving an remarkable comprehensive therapeutic effect. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/710940v1_fig1a.gif" ALT="Figure 11"> View larger version (59K): org.highwire.dtl.DTLVardef@1a831f3org.highwire.dtl.DTLVardef@1676617org.highwire.dtl.DTLVardef@15f99c1org.highwire.dtl.DTLVardef@1677cc4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOScheme 1.C_FLOATNO (a) Schematic diagram of the design and preparation of functionalized Bifidobacterium longum hydrogel. (b) Exploration of the mechanism of INPs@BL@Gel in treating colitis-associated anxiety and depression through a dual-site multi-target synergistic strategy. C_FIG

pathology↗

bioRxiv2026

PD1-induced Shp2 condensation organizes inhibitory signalosomes through selective substrate partitioning

The formation of microclusters is a hallmark of PD1 engagement with its ligands, yet the physical basis and functional significance of this phenomenon remain unclear. Here we show that ligand-bound PD1 licenses Shp2 self-association and liquid-liquid phase separation (LLPS), producing dynamic PD1:Shp2 condensates whose liquidity depends on Shp2 catalytic activity. Mutations that selectively disrupt Shp2 self-association weaken PD1 microcluster formation and impair PD1 inhibitory function. Mechanistically, PD1-induced Shp2 LLPS promotes the co-compartmentalization of signaling substrates such as CD3{zeta} and CD28, thereby facilitating its dephosphorylation. These findings identify Shp2 LLPS as an intrinsic organizing principle of the PD1 inhibitory pathway that links enzymatic activation, substrate selectivity and mesoscale assembly to suppress T cell activation.

biochemistry↗

bioRxiv2026

Super-resolution single-cell spatial atlas of plant de novo regeneration

Plant de novo regeneration, a capacity absent in animals, relies on stem-cell-niche formation rather than pre-existing reservoirs. Growth hormones such as auxin and cytokinin, together with numerous genetic regulators, participate in regeneration, but the architectural principles coordinating extensive cellular reprogramming at the individual-cell scale remain elusive. Using super-resolution multimodal spatial transcriptomics to 1.16 million cells, we tracked tomato regeneration across temporal intervals from wounding to organogenesis. Datasets are available at http://www.single-cell-spatial.com.

cell biology↗

bioRxiv2026

Protein aggregation inhibitors induce divergent transcriptional responses in a cellular model of a-synuclein seeded aggregation

Parkinsons disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) are progressive neurodegenerative disorders marked by the pathological aggregation of alpha-synuclein ([a]Syn). Despite significant research efforts, effective therapeutic interventions remain elusive due to limited understanding of the cellular effects of [a]Syn aggregation and propagation. This study presents the development of a scalable cellular seeding assay for screening small molecules targeting cellular [a]Syn seeded aggregation. By leveraging a fluorescent reporter of [a]Syn and phenotypic screening, the assay enables high-throughput evaluation of potential inhibitors in a cellular environment mimicking disease pathology. We evaluated three different Syn aggregation inhibitors tested in clinical trials for PD: Minzasolmin, Emrusolmin and EGCG and profiled gene expression using multiplexed single cell RNA sequencing in order to examine their distinct effects on cellular pathways associated with [a]Syn overexpression or seeded aggregation. Two cellular activities were prominently affected: lipid metabolism and rRNA processing. Notably, while EGCG effects were confined to cells with aggregated Syn, Minzasolmin and Emrusolmin also produced transcriptional changes in cells without aggregated Syn. Each of the compounds tested induced a partial reversal of transcriptional effects resulting from Syn seeded aggregation. We identified 391 genes that were no longer significantly differentially expressed upon addition of compound, relative to cells with seeded aggregation. This platform bridges phenotypic screening and molecular pathway analysis, providing insights into druggable pathways for synucleinopathies. The molecular signatures identified here can assist in testing and benchmarking future drug discovery leads.

neuroscience↗

bioRxiv2026

Inhalable Polymeric Nanoparticle Vaccine for Lysosome-targeting Co-delivery of Antigen and Adjuvant with Enhanced Immunoprotection

Conventional subunit vaccines suffer from premature clearance and poor synchronization of antigen and adjuvant, limiting coordinated immune activation. Here, we develop an amphipathic polymer (YAXA) with acid-labile imine bonds for acid-sensitive degradation and terminal NHS-activated esters for antigen conjugation. YAXA is co-assembled with hydrophobic TLR7 agonist 3M-052 and conjugated to protein antigen, forming nanoparticle vaccine YM3.7. Following aerosolized intratracheal inoculation into the lung, YM3.7 is efficiently internalized by antigen-presenting cell and trafficked into lysosome, where acidic conditions trigger its dissociation and co-release of antigen and adjuvant. This spatiotemporally synchronized delivery promotes robust immune activation, including cell maturation, germinal center formation, systemic and lung-resident B/T cell response, and IgG/sIgA production. In lethal pneumonia models induced by either Pseudomonas aeruginosa or Staphylococcus aureus, YM3.7 markedly improves survival over conventional antigen and adjuvant mixture. This study presents an inhalable nanoparticle platform that coordinately activates innate, humoral, mucosal, and cellular immunity for enhanced protection.

immunology↗

bioRxiv2026

Integrated Multi-omics Analysis Identifies Molecular Signatures of Regular Physical Activity in Human Peripheral Blood

Regular physical activity is well established to protect against metabolic disorders and bolster immunity; yet, the underlying molecular and cellular mechanisms remain incompletely understood. We integrated plasma metabolomics and lipidomics with single-cell transcriptomic and chromatin accessibility profiles to decode the systemic impact of physical activity on human immunity and metabolism. Our data reveal that regular physical activity is linked to a coordinated metabolic signature marked by enhanced fatty acid oxidation and antioxidant defense. In circulating immune cells, regularly active individuals exhibited synchronous enhancement at both the chromatin accessibility and transcriptional levels for antigen presentation-related genes in antigen-presenting cells (APCs), particularly in classical monocytes, naive B cells, and switched memory B cells. Meanwhile, cytotoxic programs in CD8+ cytotoxic T and mature NK cells showed epigenetic pre-activation of effector function regulators. Intercellular communication analysis further revealed that regular exercise enhanced MHC-I/II signaling between APCs and T cells and suppressed inflammatory signaling networks. Together, these findings elucidate molecular mechanisms underlying the health benefits of regular exercise and offer a theoretical basis for enhancing public health and preventing chronic diseases.

bioinformatics↗

bioRxiv2026

Odor Sampling Bags Enable Reliable Delivery of Controlled Odor Concentrations

Precise control of odorant concentration is essential for reliable olfactory research, yet existing odorant delivery methods often suffer from solvent interactions and dilution from ambient air, limiting stimulus consistency in olfactory research. We developed an odor sampling bag system using Nalophan plastic to create a closed headspace with air as the carrier medium, eliminating solvent-related variability and ambient air dilution. In two independent experiments, 15 trained panelists each rated the perceived intensity of seven concentrations of benzaldehyde and 2-heptanone using both gas-sampling bags and glass jars. Bags produced higher maximum perceptual intensities (p < 0.001) and greater test-retest reliability than jars (Experiment 1: r = 0.89 vs. 0.81, p < 0.001; Experiment 2: r = 0.86 vs. 0.72, p < 0.001). Notably, the two tested odorants showed different maximum intensities in bags (p < 0.001) but not jars (p = 0.85), suggesting bags better preserve odorant-specific concentration differences. Photoionization detector measurements confirmed stable headspace concentrations over time, comparable to industry-standard Tedlar bags. This cost-effective approach offers improved stimulus control for olfactory psychophysics research.

neuroscience↗

bioRxiv2026

Development of Integrin α5β1-targeted PET/NIR imaging probes for glioblastoma intraoperative navigation and intracavity targeted radionuclide therapy

Incomplete extent of tumor resection is the major factor of glioblastoma recurrence, leading to its poor prognosis. Developing therapies that enable the minimal tumor cell remnant during surgical resection is a major challenge to combat this aggressive cancer. This study develops a glioblastoma integrin 5{beta}1-selective peptide binder through series strategy screening and labeling it with 68Ga, indocyanine green (ICG), or 177Lu for efficacious multimodal treatment. This strategy combines positron emission tomography (PET) imaging for preoperative diagnosis, intraoperative NIR-II fluorescence-guided surgery for maximal tumor resection, and followed by intracavity targeted radionuclide therapy for elimination of residual tumor cells. We identified GR, with five arginine mutation, exhibiting superior integrin 5{beta}1 binding affinity and brain penetration. [68Ga]GR demonstrated substantial tumor uptake and prolonged retention time in both mouse models and tissues from glioblastoma patients. The guidance of ICG-GR achieved accurately intraoperative tumor imaging and maximal tumor resection in orthotopic mouse glioblastoma models. Notably, combining intracavity administration of [177Lu]GR following ICG-GR guiding resection markedly inhibited tumor growth and reduced possibility of tumor recurrence compared with surgery alone (unguided or ICG-GR-guided) or unguided surgery followed by [177Lu]GR. Therefore, we have reported the integrin 5{beta}1-targeted radionuclide and optical agents for maximal elimination of tumor cells during surgical resection in glioblastoma patients. HIGHLIGHTSO_LIGR is a glioblastoma-specific peptide with both favorable binding affinity superior brain penetration. C_LIO_LIICG-GR is a glioblastoma-targeted NIR probe enabling maximal surgical resection. C_LIO_LIICG-GR-guided surgery with [177Lu]GR-based adjuvant therapy allows robust elimination of tumor cells. C_LI

cancer biology↗

bioRxiv2025

Collagen remodeling promotes a GPCR-mediated mechanosensory immune checkpoint in ADGRG1+ CD8+ T cells and serves as a spatial biomarker of response to immunotherapy

Accessibility of immune cells to the tumor microenvironment (TME) in many solid tumors can be influenced by extracellular matrix (ECM) deposition, organization, and remodeling within tumor stroma. Specifically, lysyl hydroxylase-2 (LH2)-catalyzed lysine hydroxylation in type-I collagen telopeptides leads to formation of intermolecular collagen cross-links creating a stiffened and proteolytically resistant, stable ECM. Advanced head and neck squamous cell carcinomas (HNSCC) frequently exhibit desmoplasia with elevated LH2 expression, and Immune-checkpoint (ICI) therapy is effective in only <20% patients, suggesting that ECM remodeling mechanistically governs the composition and function of TME infiltrates. We show that elevated LH2 and collagen alignment promotes stromal accumulation of CD8 T-cells, and poor response to ICI in HPV-HNSCCs. Integration of clinical biopsies, transcriptomic datasets, and an immunocompetent syngeneic mouse model revealed that aligned collagen spatially restricts adhesion G protein-coupled receptor positive (ADGRG1) CD8 T-cells to activate a non-canonical GPCR-mediated mechanosensory program that drives dysfunction and exhaustion. Statement of significanceDesmoplasia is common in solid tumors, and immune checkpoint inhibitors benefit only some patients. Current biomarkers like PD-L1 and TMB have limited value. Our findings reveal a previously unrecognized collagen-ADGRG1 mechanosensory immune checkpoint, offering a clinically tractable, spatially resolved biomarker to better stratify patients for immunotherapy.

cancer biology↗

bioRxiv2025

High-Fidelity Tuning of Olfactory Mixture Distances in the Perceptual Space of Smell Through a Community Effort

A central goal in sensory science is to establish quantitative mappings between physical stimuli and perceptual responses. While such mappings are well characterized in vision and audition, they remain poorly defined in olfaction, limiting progress toward understanding the representations of smell. Predicting perceptual similarity between odor mixtures offers a promising route to formalize these relationships. To advance this effort, the DREAM (Dialogue for Reverse Engineering Assessment and Methods) Olfactory Mixtures Prediction Challenge assembled a curated, cross-study dataset describing the similarity of 507 mixture pairs and an unpublished test set of 46 mixture pairs. Teams competed to predict the perceptual similarity of mixture pairs, and then collaborated post-challenge to create an ensemble combining top-performing models that notably improves predictions over the existing state-of-the-art models. Moreover, ensemble model maintains high predictive accuracy in novel validation set. Our model provides a reproducible framework for neuroscientists, chemists, and engineers to compare odor mixtures and provides a foundation for future efforts towards better understanding the olfactory properties of mixtures.

neuroscience↗

bioRxiv2025

Precise Control of Switchable Chimeric Antigen Receptor T Cells Allows Enhanced Safety and Less T Cell Exhaustion

BackgroundChimeric antigen receptor (CAR)-T cell therapies have achieved remarkable success in hematologic malignancies, yet their clinical utility remains limited by safety concerns, limited persistence, and T-cell exhaustion driven by continuous receptor signaling. While switchable CAR designs provide external control, many reported systems are irreversible, strictly binary, or compromise CAR-T potency. MethodsWe engineered an optimized chemically switchable CAR platform (CSN CAR) that pharmacologically regulates antigen engagement by controlling the surface expression of the full-length CAR. An NS3 protease module was embedded within the CAR construct to enable drug-dependent stabilization of intact CAR on T cells. Using engineered CAR-T cells, we quantified drug-controlled activation, cytotoxicity, and cytokine release against CD19+ tumor cells by flow cytometry and ELISA in vitro. We further screened clinically approved NS3/4A inhibitors in CAR-HEK and CAR-T cells to identify optimal small-molecule controllers. A chronic stimulation model was established to assess CAR-T persistence and exhaustion-associated phenotypes in vitro. ResultsCSN CAR-T cells enabled precise, dose-dependent regulation of CAR surface density, cytokine production, and cytotoxicity. In the OFF state, switchable CAR-T cells showed minimal basal activity, consistent with reduced antigen-driven activation and cytokine release in the absence of drug in the experimental conditions. Upon drug addition, intact surface CAR was detectable within 1 hour, reaching [~]80% of peak observed CAR expression by 4 hours. Reversible suppression of CAR expression enabled attenuation of cytotoxicity toward normal CD19+ B cells in vitro after target-cell reduction, supporting a potential strategy to mitigate prolonged on-target/off-tumor activity. Under chronic stimulation, switchable CAR-T cells exhibited reduced exhaustion-associated markers, more stable CAR expression, and preferential differentiation toward a central memory phenotype. ConclusionTogether, these findings establish CSN CAR as a reversible and tunable switchable CAR-T platform enabled by clinically approved NS3/4A inhibitors, supporting controllable modulation of CAR activity with potential applications for improving the precision and safety of CAR-T cell therapy.

cancer biology↗