Biology subjects

Lin, X.

Publications and source records attributed to Lin, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Single-Cell Multi-Omics Dissects Transcript Isoform and Immune Repertoire Dynamics in Human Immunosenescence

Immunosenescence, a major hallmark of systemic aging, refers to the progressive functional decline of the immune system. This decline not only compromises host defense and immunological memory but also fuels chronic inflammation and tissue degeneration (collectively known as inflammaging). While single-cell RNA sequencing (scRNA-seq) has revealed transcriptomic alterations in immune aging, analyses restricted to transcript abundance fail to capture deeper regulatory layers, such as transcript isoform diversity and the remodeling of immune receptor repertoires. To address this, we present the human peripheral immune single-cell multi-omics atlas that integrates gene expression, transcript isoforms diversity, and immune receptor repertoires. By combining single-cell full-length transcriptome sequencing (scCycloneSEQ), short-read scRNA-seq, and single-cell immune receptor sequencing (scTCR/BCR-seq), we systematically profiled peripheral blood mononuclear cells (PBMCs) from healthy young and elderly donors. Our analyses uncovered extensive age-related remodeling of immune cell composition, functional states, and TCR/BCR diversity. Notably, we identified in CD4 effector memory T cells exhibited widespread differential isoform usage (DIU), 3'UTR length variation, and a marked reshaping of cytotoxic T lymphocyte (CTL) clonotypes--all closely associated with aging-related inflammation and cellular senescence. This multi-omics atlas delineates key molecular features of immunosenescence and provides a high-resolution resource for deciphering the regulatory architecture underlying immune aging.

immunology↗

bioRxiv2025

mIDEA: An Interpretable Structure-Sequence Model for Methylation-Dependent Protein-DNA Binding Sensitivity

DNA methylation dynamically reshapes protein-DNA interaction landscapes, yet the mechanistic principles governing methylation-dependent recognition remain incompletely understood. Currently, there is no predictive framework that integrates methylation-aware binding specificity with 3D structure-based residue interactions for quantitatively assessing the impact of DNA methylation on protein-DNA interactions. To bridge this gap, we introduce mIDEA (Methylation-informed Interpretable protein-DNA Energy Associative model), a structure-aware, residue-level biophysical framework that predicts and interprets protein-methylated-DNA interactions by optimizing an amino acid-nucleotide energy matrix explicitly accounting for the modulatory effect of 5-methylcytosine. We validate mIDEA across diverse human transcription factors with distinct CpG methylation preferences. Using only structural templates and prior biochemical knowledge, mIDEA accurately recapitulates both "methyl-plus" and "methyl-minus" effects of DNA methylation on protein-DNA binding. Incorporating quantitative methylation-sensitive binding data further enables mIDEA to capture position-specific methylation effects. When integrated with whole-genome methylation profiles, the model enhances in vivo binding-site prediction by reducing false positives while preserving sensitivity compared to models trained on unmethylated sequences. Overall, mIDEA provides a principled framework for elucidating the molecular mechanisms underlying methylation-modulated protein-DNA recognition. The mIDEA source code is available at: https://github.com/LinResearchGroup-NCSU/IDEA_DNA_Methylation.

bioinformatics↗

bioRxiv2025

CONCERT predicts niche-aware perturbation responses in spatial transcriptomics

Spatial perturbation transcriptomics measures how genetic or chemical edits alter gene expression while preserving tissue context. Perturbation outcomes depend on a cells intrinsic state and also on how effects propagate across cellular microenvironments. We present CONCERT, a niche-aware generative model that embeds perturbation context and learns spatial kernels with a Gaussian process variational autoencoder to predict perturbation effects across tissue. We formalize three tasks: patch, border, and niche, predicting responses in nearby unperturbed regions, at tissue interfaces, and as a function of surrounding microenvironments. We evaluate CONCERT on Perturb-map lung datasets. CONCERT outperforms state-of-the-art models (dissociated counterfactuals, spatialized perturbation models, and kNN), reducing E-distance by up to 33.77% (patch), 26.05% (border), and 33.74% (niche) versus the next best, with mean absolute error down by up to 23.28% and Pearson correlation up by up to 9.10%. Two case studies go beyond benchmarking. In dextran sodium sulfate-induced colitis, CONCERT reconstructs spatial gene expression at unmeasured time points, produces longitudinal comparisons across unpaired mice, resolves intermouse heterogeneity, and recovers consistent temporal declines of inflammation-associated genes across regions. In ischemic stroke, CONCERT predicts responses under variable lesion sizes and in a 3D formulation across brain sections, capturing lesion-core and peri-lesion patterns. CONCERT performs niche-aware counterfactual prediction, reconstructs missing spatial data, and models perturbation responses across tissues.

bioinformatics↗

bioRxiv2025

Multimerin1, not Galectin-8, Promotes Gastric Chief Cell Differentiation by Tempering WNT Signaling

Galectins are a family of proteins that bind galactose-containing glycans. One member, galectin-8, preferentially binds galactose that contains a terminal sulfate. Aberrant expression and secretion of sulfated glycosylation epitopes, such as 3-Sulfo-LeA/C, is a feature of high-risk human foregut metaplasias. In addition, recent work has demonstrated that 3-Sulfo-LeC is a marker of mature murine zymogenic chief cells of the stomach and that 3-Sulfo-LeC epitope is secreted via cathartocytosis during the cellular transition to a metaplastic state. Based on those findings, we used Lgals8-/-mice, to determine whether galectin-8 might play a role in chief cell homeostasis. We observed delayed gastric differentiation in the Lgals8-/-mice and discovered that this phenotype was due to an unappreciated deletion of Mmrn1 and Snca in the Lgals8-/- line. We show that multimerin-1 tempers WNT stimulation of the gastric corpus at an early age, as evidenced by nuclear beta-catenin staining and proliferation throughout the gland. Because multimerin-1 is synthesized and secreted from endothelial cells and not from the epithelial compartment, these data uncover a role for mesodermal cells in epithelial developmental and maturation of the mouse stomach. As prior studies have suggested galectin-8 and multimerin-1 have overlapping functions albeit, divergent with respect to bone, future studies using pure knockouts are necessary to refine these phenotypes.

developmental biology↗

bioRxiv2025

Structural Mechanism of Prestin-Membrane Mechanotransduction

Sound frequency discrimination in mammals depends on the conformational transitions of prestin (SLC26A5), the piezoelectric motor in outer hair cells. The mechanism that enables prestins electrically driven interconversion and its dependence on membrane mechanics, remains unresolved. Here, we show that membrane forces represent a strong driver of the same conformational changes generated by transmembrane voltage and stabilized by bound anions. Single particle cryo-EM structures of nanodisc-reconstituted prestin were obtained from varying lipid composition and membrane thickness. These structures show that membrane thinning biases prestin from a compact conformation to a fully expanded conformation, mimicking outer hair cell elongation/contraction during electromotility. In contrast, zebrafish SLC26A5 transporters undergo complete elevator movements with redistribution of areal changes across leaflets. The structures, together with mutagenesis, H/D exchange mass spectrometry data, and NLC measurements, offer a high-resolution understanding of how prestin translates membrane tension into charge and motor movement during sound-evoked vibrations, revealing a process of reciprocal electro-mechanical transduction essential for tuning cochlear amplification.

biophysics↗

bioRxiv2025

Diverse haplotypes at a complex Solanum americanum locus confer resistance to Phytophthora infestans and P. capsici

Plants encounter diverse pathogens and have evolved a two-layered innate immune system to detect pathogen molecules and activate defense mechanisms that restrict infection. Most cloned plant Resistance (R) genes encode NLR immune receptors. NLR genes are often found in clusters of paralogs with sequence and copy number variation; whether these NLR clusters evolve in response to single or multiple pathogens has been unclear. We report here the isolation of a Phytophthora capsici resistance gene, Rpc2, along with a novel P. infestans resistance gene, Rpi-amr5, from two Solanum americanum accessions. These orthologous genes reside in the Rpi-amr1 cluster, which has previously been associated with resistance to P. infestans. By screening RXLR effector libraries of P. infestans and P. capsici, we identified multiple effectors recognized by both NLRs. Our findings highlight the complexity of NLR clusters and evolution driven by interactions with multiple pathogens. This work will underpin efforts to elevate resistance against Phytophthora pathogens and enhances our understanding of NLR evolution.

plant biology↗

bioRxiv2025

B cell transcriptomics reveals lasting dysregulation and rapid decline of protective memory after hepatitis C cure

O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/664545v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@d12426org.highwire.dtl.DTLVardef@9c54daorg.highwire.dtl.DTLVardef@186d559org.highwire.dtl.DTLVardef@120f2f3_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG HighlightsO_LIB cells in CHC patients retained dysregulated transcriptional profiles despite successful DAA treatment C_LIO_LIB cell dysregulation is marked by global B cell hyperactivation and antigen-specific atypical MBC expansion C_LIO_LISustained upregulation of TNF- signaling via NF-{kappa}B (TNF-/NF-{kappa}B) is a central driver of persistent B cell dysregulation and chronic inflammation C_LIO_LIViral clearance leads to restoration of IFN responses and IFN-stimulated gene (ISG) signatures in B cells but not TNF-/NF-{kappa}B C_LIO_LIHCV IGHV1-69 bnAb-producing B cells are enriched within the CD86hi IgG MBC subset before treatment C_LIO_LICD86hi IgG MBC subset contracts rapidly post-cure in parallel with the rapid decline of cross-nAb responses and loss of protective immune memory against HCV reinfection C_LI Chronic hepatitis C (CHC) disrupts host humoral immune response by impairing the timely generation of neutralizing antibody (nAb) and durable immune memory. However, the underlying mechanisms and their reversibility after viral clearance remain poorly defined. Here, through integrated single-cell transcriptomics and antibody repertoire characterization, we show that B cells from CHC patients retain transcriptional dysregulation even after successful antiviral therapy. Sustained TNF- signaling emerged as a central driver of chronic B cell hyperactivation, persistent dysregulation and unresolved inflammation following cure. Furthermore, a CD86hi memory B cell subset, responsible for an IGHV1-69-encoded multi-donor class recall nAb response, declined rapidly following viral clearance, compromising immune memory against reinfection. Together, these findings reveal how CHC imprints lasting B cell dysregulation, impairs nAb memory, and sustains inflammation in the B cell compartment, after viral clearance. The insights underscore the need for strategies aimed at restoring B cell homeostasis to achieve durable immune protection.

immunology↗

bioRxiv2025

The roles of Transposable Elements and Gene Family Dynamics in Shaping Diversity and Evolution in Diatoms

Diatoms are key players in aquatic ecosystems, having evolved through secondary endosymbiosis. Using long-read sequencing, we investigated how transposable elements (TEs) and gene family dynamics have shaped diatom diversification from inter-lineage to intra-species scales. Across diatom lineages, we identified ecological adaptation-linked expansions, including polyamine synthesis genes for silicification and glutathione S-transferases for oxidative stress resistance. Centric diatoms showed lineage-specific expansion of flotation-associated microtubule genes, while pennate diatoms expanded motility-related actin and myosin genes. At the intra-species level, distinct Phaeodactylum tricornutum strains revealed genomic adaptations correlated with their unique features, including strain-specific expansion and contraction in the cruciform strains morphological genes and the Baltic Sea isolates amine metabolism genes. Our estimates of major lineage divergence times in diatoms ([~]202 Ma and [~]173 Ma) were highly consistent with the two deep WGD events ([~]200 Ma and [~]170 Ma). At these evolutionary nodes, gene families showed extensive lineage-specific expansions and contractions, likely linking ancient polyploidy to subsequent gene content evolution. Substantial TE expansions occurred more recently (0.5-5 Ma), with most diatoms showing recent bursts of Long Terminal Repeat Retrotransposons (LTR-RTs) and araphid pennate diatoms displaying more ancient TE insertion peaks. This likely reflects the progressive loss of ancient TE copies, leaving only recent TE insertions detectable. Our findings provide genomic evidence for the adaptive evolution of diatoms, highlighting the crucial roles of TEs and gene family dynamics in shaping their morphological diversity and environmental adaptations, and suggesting a potential connection between WGDs, gene family dynamics, and TE insertions in genome evolution.

genomics↗

bioRxiv2025

A spatially-resolved human brain proteome atlas for understanding function and disease

While the brain performs specialized functions across distinct regions, the spatial organization of the human brain proteome remains largely uncharted. Here we present a comprehensive spatially-resolved proteome atlas of the human brain, analyzing over two thousand MRI- guided locations across four individuals. Proteome analysis integrated with transcriptomics reveals extensive post-transcriptional regulation, with cortical regions showing markedly higher protein diversity than transcript. Unsupervised molecular clustering defines distinct brain territories that transcend anatomical boundaries, instead reflecting metabolic demands and functional specialization patterns. Application to epilepsy brain tissue uncovered disrupted astrocyte metabolism, protein homeostasis and therapeutic targets including the seizure-associated purinergic receptor P2RX7. This resource bridges molecular and systems neuroscience to accelerate neurological drug discovery.

neuroscience↗

bioRxiv2025

A mathematical kinetic model of memory in Bacillus subtilis spore germination

Dormant Bacillus subtilis spores germinate through interaction of germinants with germinant receptors (GRs). Subsequently, the activated GR signal is transduced to SpoVA proteins that constitute transport channels. The opening of the SpoVA channel then leads to calcium dipicolinic acid (CaDPA) release and then completion of spore germination. Spores are known to exhibit memory in germination, as spores given an initial short germinant pulse respond more readily to a second pulse. We have developed a mathematical model to identify a minimal reconstructed molecular network that is crucial for germination kinetics leading to memory of germinant exposure; the model reproduces experimental double germinant pulse germination curves. Analysis of the reconstructed network indicates that a minimal set of inactive and active GRs and a SpoVA channel in three states - closed inactive, closed active and open - is needed to reproduce memory. Spore germination memory is introduced in the network by the activation and deactivation rates of GRs, and by the interplay between activation of closed SpoVA channels and their rate of opening and closing. Author summaryDormant bacterial spores return to life in germination when exposed to germinants which are recognized by germinant receptors (GRs). Spores have memory of germinant exposure, such that more spores germinate after a 2nd germinant pulse than after the 1st one, if intervals between pulses are short. In this work, a mathematical model has been developed that reproduces experimental memory behavior. Computer simulations of the model reveal the crucial factors in spore memory of germinant exposure as: i) active and inactive GR states and ii) a membrane channel for a major spore core molecule, Ca-dipicolinic acid (CaDPA), with the channel existing in closed inactive, closed active and open states, all of which are indispensable for spore memory. The model will be part of an iterative cycle of modelling of network dynamics and generating hypotheses for further experimentation.

microbiology↗

bioRxiv2025

IRIS Integrates Sparse Sequence, Experimental, and AI-Predicted Structures for Protein-RNA Affinity Prediction and Motif Discovery

Protein-RNA interactions are fundamental to numerous cellular processes, yet quantitatively characterizing their binding specificity remains a major challenge. We present IRIS (Integrative RNA-protein interaction prediction Informed by Structure and sequence), a biophysical framework that integrates residue-level sequence and structural features without relying on large-scale affinity data to predict binding affinities and identify binding motifs. Applied across different protein-RNA systems, IRIS predicts relative binding free energies ({Delta}{Delta}G) with consistent correlations and competitive error metrics, and its performance is further improved by incorporating additional high-affinity sequences into the training set. By leveraging predicted structural complexes, IRIS reveals alternative binding modes not observed in experimental structures, extends applicability to systems lacking experimental protein-RNA complexes, and generates a library of favorable RNA-binding motifs at protein-RNA interfaces. Collectively, these results establish IRIS as a versatile framework that leverages increasingly accurate structural predictions to enable quantitative modeling and rational engineering of protein-RNA interactions.

biophysics↗

bioRxiv2025

Therapeutic Salmonella induces long-term protective trained immunity in NK cells against cancer metastasis

Trained immunity, a form of innate immune memory initially recognized in pathogen defense, has recently been reported to aid in combating cancer. Although trained immunity has been well-characterized in myeloid lineage cells, its presence in innate lymphoid cells, particularly natural killer (NK) cells, remains largely unexplored. Here, we report that a single dose of Salmonella YB1, an attenuated candidate anticancer vaccine strain, provides long-lasting protection against cancer metastasis in mice by inducing trained natural killer (NK) cells. Functional assays coupled with multiomics analysis revealed that Salmonella-trained NK (stNK) cells establish an enduring reprogrammed epigenome characterized by enhanced pro-survival signaling and immune effector functions, resulting in more potent IFN-{gamma} release and cytotoxicity upon secondary stimulation. We further revealed that IL-12 and IL-18 are essential but insufficient for this NK cell training effect. Crucially, stNK cells far outperform the common immune checkpoint therapies, including PD-1 and TIGIT blockade, in suppressing metastasis, underscoring the unique immunological mechanisms in combating metastasis. These findings uncover a novel long-term anti-metastatic function of trained immunity in NK cells, highlighting its potential as an effective antitumor strategy.

immunology↗

bioRxiv2025

Targeting the TEA DNA binding Domain of TEAD with a Peptide Inhibitor TEAi Suppresses Oncogenic Transcription and Tumor Growth

The Hippo pathway is a critical regulator of organ size and tumorigenesis, with the TEAD-YAP/TAZ transcriptional complex serving as a key effector frequently hyperactivated in cancers. Targeting this complex has emerged as a promising anticancer strategy. Here, we develop TEAi, a 64-amino acid peptide derived from Drosophila Nerfin-1, which directly binds the TEA DNA-binding domain of TEAD. TEAi potently suppressed TEAD-YAP-driven transcriptional activity, as evidenced by luciferase reporter and qPCR assays targeting canonical downstream genes CTGF and CYR61. Mechanistically, TEAi inhibited the DNA-binding capacity of TEAD2 without direct DNA interaction, thereby abolishing promoter recruitment of the TEAD-YAP complex. unexpectedly, TEAi induced nuclear export and cytoplasmic accumulation of TEAD2, suggesting a non-canonical mechanism distinct from native Nerfin-1 function. Functionally, TEAi significantly suppressed cancer cell proliferation in vitro and tumor growth in vivo. Unlike inhibitors targeting the YAP-binding interface, TEAi directly blocks the DNA-binding activity of TEAD, offering a potential advantage by comprehensively inhibiting all TEAD-dependent oncogenic transcription. Our findings establish TEAi as a promising prototype for therapeutics targeting TEAD, with implications for treating cancers driven by Hippo pathway dysregulation.

cell biology↗

bioRxiv2025

Core gastric microbiota linked to pathogenesis and preserved across age stratified cohorts

Mounting evidences reveal a complex microbial niche in gastric environment. Nevertheless, the phylogenetic stability of gastric microbiota remains undetermined. Here, using full-length 16S rRNA and ITS amplicon sequencing applied to gastric brushes from a pediatric endoscopic cohort (n=243), we identified a core microbiota comprising 11 genera, which clustered into two antagonistic communities (CST1A and CST1B). CST1A was enriched in healthy subjects, while CST1B correlated with gastroduodenal disease. Notably, this bipartite architecture arose through bacterial interactions, with fungal taxa demonstrating minimal integration. Further stratified analyses revealed that H. pylori infection preferentially depleted CST1A taxa. The subclusters exhibited divergent metabolic profiles, with CST1A specializing in motility processes and CST1B in nucleotide/peptidoglycan synthesis. Cross-cohort validation of six datasets (n=1,183) confirmed the persistent relevance of the CST1A-CST1B structure across disease stages. Collectively, our findings define a core gastric microbiota established in early life, linked to gastric disease risk, and persisting into adulthood. HighlightsO_LIIdentification of a gastric bacterial network established in early life. C_LIO_LIDysbiosis in two subclusters within the core microbiota is associated with pediatric peptic ulcers. C_LIO_LIHelicobacter pylori infection reduces the abundance of the core microbiota, with a particularly greater inhibitory effect on CST1A. C_LIO_LIThe core microbiota is preserved across age stratified cohorts and is associated with gastric cancer. C_LI

microbiology↗

bioRxiv2025

Active regulation of the epidermal growth factor receptor by the membrane bilayer

Cell surface receptors transmit information across the plasma membrane to connect the extracellular environment to intracellular function. While the structures and interactions of the receptors have been long established as mediators of signaling, increasing evidence suggests that the membrane itself plays an active role in both suppressing and enhancing signaling. Identifying and investigating this contribution has been challenging owing to the complex composition of the plasma membrane. We used cell-free expression to incorporate the epidermal growth factor receptor (EGFR) into nanodiscs with defined membrane compositions and characterized ligand-induced transmembrane conformational response and interactions with signaling partners using single-molecule and ensemble fluorescence assays. We observed that both the transmembrane conformational response and interactions with signaling partners are strongly lipid dependent, consistent with previous observations of electrostatic interactions between the anionic lipids and conserved basic residues near the membrane adjacent domain. Strikingly, the active conformation of EGFR and high levels of ATP binding were maintained regardless of ligand binding with high anionic lipid content typical of cancer cells, where EGFR signaling is enhanced. In contrast, the conformational response was suppressed in the presence of cholesterol, providing a mechanism for its known inhibitory effect on EGFR signaling. Our findings introduce a model of EGFR signaling in which the lipid environment can override ligand control, providing a biophysical basis for both robust EGFR activity in healthy cells and aberrant activity under pathological conditions. The membrane-adjacent protein sequence, likely responsible for the lipid dependence, is conserved among receptor tyrosine kinases, suggesting that active regulation by the plasma membrane may be a general feature of this important class of proteins.

biophysics↗

bioRxiv2025

Single-cell transcriptome sequencing for opening the blood-brain barrier through specific mode electroacupuncture stimulation

BackgroundThe blood-brain barrier (BBB) interferes with the treatment of central nervous system disorders owing to the complexity of its structure and restrictive function. Thus, it is challenging to develop central nervous system drug delivery strategies. Specific mode electroacupuncture (EA) stimulation can effectively open the BBB in rats. MethodsHere, we used single-cell RNA sequencing (scRNA-seq) to comprehensively map the cell population in the Sprague-Dawley rat cerebral cortex. ResultsWe identified 23 cell subsets and eight types of cells in the brain by cell annotation. scRNA-seq revealed transcriptional changes in the cerebral cortex under EA. ConclusionsOur findings offer valuable insights into the molecular and cellular modifications in the brain resulting from EA intervention and serve as a resource for drug delivery across healthy and diseased states. Innovative approaches to enhance BBB opening will lead to more effective therapeutic plans and enhanced drug delivery.

neuroscience↗

bioRxiv2025

Can large language models reliably extract human disease genes from full-text scientific literature?

Manual extraction of high-fidelity gene-disease-phenotype information from human genetics literature is a labor-intensive task that requires trained human genetics researchers to read through many primary research papers. This presents a major challenge for maintaining up-to-date human disease genetic databases. Recent exploration into large language models (LLMs) opens new directions in automating this manual process. However, most approaches depend on pre-training, finetuning, or specialized generative artificial intelligence (GenAI) tools, but there is a lack of empirical evidence to show whether commercially-available LLMs can be directly used to reliably extract gene-disease-phenotype for human genetic diseases. Herein, we perform a benchmark of the use of three zero-shot prompted LLMs, namely GPT-4, DeepSeek and Claude, without task-specific fine-tuning, to extract human genetic information directly from full text of scientific papers. Using known congenital heart diseases (CHD) genes found in the open access CHDgene database (https://chdgene.victorchang.edu.au/) as the benchmark data set, GPT-4o achieved overall 88.8% extraction accuracy across 23 gene entries containing over 57 references, with 100% accuracy in gene name, 78.3% and 76.7% in disease and phenotype fields respectively. This work introduces a lightweight, easy-to-deploy, and yet robust LLM-based agent named GeneAgent, analyze sources of disagreement, and highlight the feasibility of integrating powerful LLM into genetic evidence synthesis workflows. Highlight- First systematic benchmark of LLMs for extracting human gene-disease-phenotype relationships from full-text biomedical articles - GeneAgent: a lightweight, highly accurate prompt-only LLM agent - New domain task-specific evaluation framework

bioinformatics↗

bioRxiv2025

Functional screening of TCR-like antibodies for targeted cancer immunotherapy

Although hybridomas and display technologies have long been used in antibody discovery, identifying TCR-like antibodies that recognize major histocompatibility (MHC)-presented neoantigens remains an inefficient process. Here, we present a functional screening approach for antibody discovery based on T cell-specific functionality. By constructing Synthetic T cell receptor and Antigen Receptor (STAR)-T cells expressing antibody libraries and co-culturing them with target cells, we observed distinct antigen-specific STAR endocytosis and T cell activation. These two indicators are combined into an E-A functional index, which we demonstrate as an effective screening strategy for identifying antibodies with strict antigen sensitivity and specificity. Using this E-A functional screening method, we successfully discovered antibodies or nanobodies targeting cell-surface tumor antigens and neoantigens presented by human leukocyte antigen (HLA). These antibodies exhibited potent anti-tumor efficacy both in vitro and in vivo. Moreover, converting these antibodies into chimeric antigen receptors (CARs) and bispecific antibodies also induced T cell activation and tumor killing. In summary, our study establishes a robust functional screening strategy for identifying antibodies or TCR-like nanobodies targeting tumor-specific neoantigens, offering significant potential for advancing targeted cancer therapies.

immunology↗