Biology subjects

Zheng, H.

Publications and source records attributed to Zheng, H..

At least 145 records · Page 8Linked to original sources

bioRxiv2023

Elevated ENO2 Disrupts VSMCs Homeostasis Facilitating the Development of Aortic Dissection

OBJECTIVEAortic dissection (AD) is a dangerous cardiovascular disease. However, its regulatory mechanism remains poorly understood. Autophagy is an important pathway for maintaining cellular homeostasis. Recent studies have shown that glycolysis and autophagy are essential for the development of AD. Elevated glycolysis can promote cells autophagy, but the relationship between the two is unclear. Enolase 2 (ENO2) is a glycolytic enzyme. Here, we investigated whether ENO2 can regulate autophagy in vascular smooth muscle cells (VSMCs) involved in the progression of AD. APPROACH AND RESULTSThe levels of ENO2 and autophagy-related proteins in aortic tissues from AD patients were identified by qRT-PCR, Western blot and IHC. We found that autophagosome clearance was impaired in aortic tissues and positively correlated with elevated ENO2 expression. Furthermore, aortic application of smooth muscle-specific adeno-associated virus (AAV) inhibiting ENO2 expression attenuated the development of AD in mice. in vitro experiments, downregulation of ENO2 partially restored PDGF-BB-induced impairment of autophagic flux, as evidenced by reduced expression of Beclin-1, SQSTM1, LC3BII/I and increased levels of LAMP2 in human aortic vascular smooth muscle cells (HAVSMCs). Similarly, increased levels of ENO2 exacerbated the autophagic dysfunction in HAVSMCs. Mechanistically, ENO2 may block autophagic flux through the ENO2-GAP43-ATF4 pathway and disrupt cellular homeostasis. CONCLUSIONSOur data reveal a perturbing role of ENO2 in the homeostasis of VSMCs, suggesting ENO2 as a potential target for intervention in AD.

molecular biology↗

bioRxiv2023

Brain structure-function relationships across the human lifespan based on network eigenmodes

While brain function is considered to be tightly supported by the underlying structure, the connectome-based link estimated by current models is relatively moderate, leaving the structure-function relationship an ongoing challenge in neuroscience. Here, by proposing a novel mapping method based on network eigendecomposition, we present a concise and strong correspondence between structure and function. We show that the explanation of functional connectivity can be significantly improved by incorporating interactions between different structural eigenmodes, highlighting the potential importance of collective, higher-order coupling patterns between structure and function. We also demonstrate the pronounced advantage of the present mapping in capturing individual-specific information, and apply it to assess individual differences of structure-function coupling across the lifespan. We find that structure-function liberality weakens with age, which is driven by the decreases in functional components that are less constrained by anatomy, while the magnitude of structure-aligned components is preserved. Our results contribute to a more refined understanding of structure-function coupling and how it evolves with age.

neuroscience↗

bioRxiv2023

Distinct disease mutations in DNMT3A result in a spectrum of behavioral, epigenetic, and transcriptional deficits

Phenotypic heterogeneity is a common feature of monogenic neurodevelopmental disorders that can arise from differential severity of missense variants underlying disease, but how distinct alleles impact molecular mechanisms to drive variable disease presentation is not well understood. Here, we investigate missense mutations in the DNA methyltransferase DNMT3A associated with variable overgrowth, intellectual disability, and autism, to uncover molecular correlates of phenotypic heterogeneity in neurodevelopmental disease. We generate a DNMT3A P900L/+ mouse model mimicking a disease mutation with mild-to-moderate severity and compare phenotypic and epigenomic effects with a severe R878H mutation. We show that the P900L mutation leads to disease-relevant overgrowth, obesity, and social deficits shared across DNMT3A disorder models, while the R878H mutation causes more extensive epigenomic disruption leading to differential dysregulation of enhancers elements. We identify distinct gene sets disrupted in each mutant which may contribute to mild or severe disease, and detect shared transcriptomic disruption that likely drives common phenotypes across affected individuals. Finally, we demonstrate that core gene dysregulation detected in DNMT3A mutant mice overlaps effects in other developmental disorder models, highlighting the importance of DNMT3A-deposited methylation in neurodevelopment. Together, these findings define central drivers of DNMT3A disorders and illustrate how variable disruption of transcriptional mechanisms can drive the spectrum of phenotypes in neurodevelopmental disease.

neuroscience↗

bioRxiv2023

Histone demethylase KDM2A is a selective vulnerability of cancers relying on alternative telomere maintenance

Telomere length maintenance is essential for cellular immortalization and tumorigenesis. 5% - 10% of human cancers rely on a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to sustain their replicative immortality, yet there are currently no targeted therapies. Through CRISPR/Cas9-based genetic screens in an ALT-immortalized isogenic cellular model, here we identify histone lysine demethylase KDM2A as a molecular vulnerability selectively for cells contingent on ALT-dependent telomere maintenance. Mechanistically, we demonstrate that KDM2A is required for dissolution of the ALT-specific telomere clusters following homology-directed telomere DNA synthesis. We show that KDM2A promotes de-clustering of ALT multitelomeres through facilitating isopeptidase SENP6-mediated SUMO deconjugation at telomeres. Inactivation of KDM2A or SENP6 impairs post-recombination telomere de-SUMOylation and thus dissolution of ALT telomere clusters, leading to gross chromosome missegregation and mitotic cell death. These findings together establish KDM2A as a selective molecular vulnerability and a promising drug target for ALT-dependent cancers.

cancer biology↗

bioRxiv2023

Single-cell transcriptomic analyses reveal the cellular and genetic basis of aquatic locomotion in scyphozoan jellyfish

The authors have withdrawn their manuscript because the authors need to re-organize the data and writing. More experimental evidence from whole-mount ISH assay and RNAi assays will be added to support the viewpoints. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

ecology↗

bioRxiv2023

TFEB-vacuolar ATPase signaling regulates lysosomal function and microglial activation in tauopathy

Transcription factor EB (TFEB) mediates gene expression through binding to the Coordinated Lysosome Expression And Regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase) essential for lysosome acidification. Single nucleus RNA-sequencing (snRNA-seq) of wild-type and PS19 (Tau) transgenic mice identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. We show that the CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and was locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homoeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimers disease.

neuroscience↗

bioRxiv2023

CALCIUM-DEPENDENT PROTEIN KINASE32 regulates cellulose biosynthesis through post-translational modification of cellulose synthase

Cellulose is an economically important source of food, paper, textiles, and biofuel. As an essential component of plant cell walls, cellulose is critical for plant cell growth. Despite its economic and biological significance, the regulation of cellulose biosynthesis is poorly understood. Phosphorylation and dephosphorylation of cellulose synthases (CESAs) were shown to impact the direction and velocity of cellulose synthase complexes (CSCs). Despite a high prevalence of phosphorylation sites in CESAs, the protein kinases that phosphorylate CESAs are largely unknown. Here, we demonstrate that CALCIUM-DEPENDENT PROTEIN KINASE32 (CPK32) regulates cellulose biosynthesis via phosphorylation of CESA3. Phosphorylation of CESA3 is important for the motility and stability of CSCs. Hence, we uncovered a new function of CPKs that regulates cellulose biosynthesis and a novel mechanism by which phosphorylation regulates the stability of CSCs.

plant biology↗

bioRxiv2023

Profiling extracellular vesicles in circulation enables the early detection of ovarian cancer

Ovarian cancer is a heterogeneous group of tumors in both cell type and natural history. While outcomes are generally favorable when detected early, the most common subtype, high-grade serous carcinoma (HGSOC), typically presents at an advanced stage and portends less favorable prognoses. Its aggressive nature has thwarted early detection efforts through conventional detection methods such as serum CA125 and ultrasound screening and thus inspired the investigation of novel biomarkers. Here, we report the systematic development of an extracellular-vesicle (EV)-based test to detect early-stage HGSOC. Our study is based on emerging insights into HGSOC biology, notably that it arises from precursor lesions within the fallopian tube before traveling to ovarian and/or peritoneal surfaces. To identify HGSOC marker candidates, we established murine fallopian tube (mFT) cells with oncogenic mutations in Brca1/2, Tp53, and Pten genes, and performed proteomic analyses on mFT EVs. The identified markers were then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood samples of tumor-bearing mice, mFT-EV markers increased with tumor initiation, supporting their potential use in early cancer detection. A pilot human clinical study (n = 51) further narrowed EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. Combined expression of these markers achieved high OvCa diagnostic accuracy (cancer vs. non-cancer) with a sensitivity of 0.89 and specificity of 0.93. The same five markers were also effective in a three-group classification: non-cancer, early-stage (I & II) HGSOC, and late-stage (III & IV) HGSOC. In particular, they differentiated early-stage HGSOC from the rest with a specificity of 0.91. Minimally invasive and repeatable, this EV-based testing could be a versatile and serial tool for informing patient care and monitoring women at high risk for ovarian cancer.

cancer biology↗

bioRxiv2023

Engineered symbiotic bacteria interfering Nosema redox system inhibit microsporidia parasitism in honeybees

Nosema ceranae is an emergent microsporidia parasite of the European honey bee (Apis mellifera), which causes serious nosemosis implicated in honeybee colony losses worldwide. N. ceranae is an obligate intracellular eukaryotic parasite that mainly invades the midgut of honeybees. Recent studies find that bee gut microbiota is potentially involved in protecting against parasitism. Here, using laboratory-generated bees mono-associated with gut members, we find that Snodgrassella alvi inhibited microsporidia proliferation, potentially via the stimulation of host oxidant-mediated immune response. Accordingly, N. ceranae employs the thioredoxin and glutathione systems to defend against oxidative stress and maintain a balanced redox equilibrium, which is essential for the infection process. We knocked down the gene expression using nanoparticle-mediated RNA interference, which targets the {gamma}-glutamyl-cysteine synthetase and thioredoxin reductase genes of microsporidia. It significantly reduces the spore load, confirming the importance of the antioxidant mechanism for the intracellular invasion of the N. ceranae parasite. Finally, we genetically modified the symbiotic S. alvi to deliver dsRNA corresponding to the genes involved in the redox system of the microsporidia. The engineered S. alvi induces RNA interference and represses parasite gene expression, thereby inhibits the parasitism by up to 99.8%. Specifically, N. ceranae was most suppressed by the recombinant strain corresponding to the glutathione synthetase or by a mixture of bacteria expressing variable dsRNA. Our findings extend our previous understanding of the protection of gut symbionts against N. ceranae and provide a symbiont-mediated RNAi system for inhibiting microsporidia infection in honeybees.

microbiology↗

bioRxiv2023

Noise improves the association between effects of local stimulation and structural degree of brain networks

Stimulation to local areas remarkably affects brain activity patterns, which can be exploited to investigate neural bases of cognitive function and modify pathological brain statuses. There has been growing interest in exploring the fundamental action mechanisms of local stimulation. Nevertheless, how noise amplitude, an essential element in neural dynamics, influences stimulation-induced brain states remains unknown. Here, we systematically examine the effects of local stimulation by using a large-scale biophysical model under different combinations of noise amplitudes and stimulation sites. We demonstrate that noise amplitude nonlinearly and heterogeneously tunes the stimulation effects from both regional and network perspectives. Furthermore, by incorporating the role of the anatomical network, we show that the peak frequencies of unstimulated areas at different stimulation sites averaged across noise amplitudes are highly positively related to structural connectivity. Crucially, the association between the overall changes in functional connectivity as well as the alterations in the constraints imposed by structural connectivity with the structural degree of stimulation sites is nonmonotonically influenced by the noise amplitude, with the association increasing in specific noise amplitude ranges. Moreover, the impacts of local stimulation of cognitive systems depend on the complex interplay between the noise amplitude and average structural degree. Overall, this work provides theoretical insights into how noise amplitude and network structure jointly modulate brain dynamics during stimulation and introduces possibilities for better predicting and controlling stimulation outcomes. Author summaryDespite the extensive application of local stimulation in cognition research and disease treatments, how regional perturbations alter brain-wide dynamics has not yet been fully understood. Given that noninvasive stimulation is associated with changes in the signal-noise relationship, we assume that noise amplitude is one of the plausible factors modulating the stimulation effects. Using a whole-brain biophysical model under different stimulation sites and noise amplitudes, we explore the influence of noise amplitude on stimulation effects and, more importantly, the interplay between noise amplitude and network structure. From a regional perspective, noise amplitude reduces the peak frequencies in unstimulated areas during stimulation. Moreover, we find a high similarity between the noise-averaged peak frequency matrix and the structural network. From a network perspective, we show that the changes in functional connectivity are decreased by noise amplitude, while the alterations in structural constraints display nonmonotonic trends. Intriguingly, increasing the noise amplitude in specific ranges can improve the association between network-level effects and structural degree, promoting better predicting and controlling therapeutic performance. Finally, the behaviors of cognitive systems quantified by network-level effects are jointly modulated by the noise amplitude and average structural degree.

neuroscience↗

bioRxiv2023

A pilot study of stable isotope fractionation in Bombyx mori rearing

Hydrogen, oxygen, carbon, and nitrogen isotopes derived from three different strains of silkworms at different life stages involved in silkworm rearing, were measured to understand the fractionation characteristics of stable isotopes at different stages of silkworm development, and to trace the movement of these isotopes from food to larva to excrement and finally to silk. We found that silkworm strain had little effect on {delta}2H, {delta}18O and {delta}13C values. However, a large difference was found in the {delta}15N levels of newly-hatched silkworms between Jingsong Haoyue and Hua Kang No.3 orthogonal strains, suggesting that the mating and egg laying differences may result in an inconsistent kinetic nitrogen isotope fractionation. The {delta}13C values of silkworm pupae and silkworm cocoon also displayed significant differences, suggesting that heavy carbon isotopes are greatly fractionated from the larva to the silk during cocoon formation. Overall, these results may be used to clarify the relationship between isotope fractionation and the ecological process of the Bombyx mori and expand our ability to resolve stable isotope anomalies at a small regional-scale level.

biochemistry↗

bioRxiv2023

Loss of PTPMT1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here we report that the deletion of PTPMT1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection - the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. PTPMT1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, PTPMT1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that PTPMT1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

cell biology↗

bioRxiv2023

Loss of secondary motor cortex neurons in chronic neuropathic pain

Chronic neuropathic pain is associated with structural plasticity of the brain on different spatial scales, yet, little is known on the mesoscopic scale of tissue composition. Here, we determined the cellular composition of cortical areas and structural variability of entire neurons during the development of chronic neuropathic pain using longitudinal in vivo two-photon microscopy and behavioral assessment. When monitoring cell type composition in response to spared-nerve injury in 84 cortical volumes containing {bsim}25000 cells each, we found neuronal loss in the secondary motor cortex region M2 immediately adjacent to the cingulate cortex already one week after surgery. Loss of mostly interneurons was also evident when monitoring individual M2 neurons over time. This neuronal loss was preceded by decreased spine density and loss of distal dendritic branches. In conclusion, our work delineates M2 as a novel site and neuronal loss as a so far underappreciated mechanism underlying chronic neuropathic pain states.

neuroscience↗

bioRxiv2022

Large-scale analysis of the N-terminal regulatory elements of the kinase domain in plant receptor-like kinase family

The N-terminal regulatory element of the kinase domain (NRE), which includes juxtamembrane segment (JM) of Receptor-like kinases (RLKs) and the N-terminal extension segment of the kinase domain in RLCKs, is the key component that regulates the activities of RLKs and RLCKs. However, their feature and functions remain largely unexplored. Herein, we perform a systematic analysis of 510,233 NRE sequences in RLKs and RLCKs from 528 plants by integrating information theory and genome-wide analysis to unravel their common characteristics and diversity. Recombinant RLKs are used to explore the structural-functional relationship of the newly-discovered motifs in vitro. Most NRE segments are around 40-80 amino acids, featuring a serine-rich region and a 14-amino-acid motif FSYEELEKATBNFS which harbors a characteristic -helix connecting to the core kinase domain. This -helix suppresses FERONIAs kinase activity. Motif discovery algorithm has identified 29 motifs with known phosphorylation sites highly conserved in RLK and RLCK classes, especially the motif VGPWKpTGLpSGQLQKAFVTGVP in LRR-VI-2 class. The NRE phosphorylation of an LRR-VI-2 member MDIS1 modulates the auto-phosphorylation of its co-receptor MIK1, indicating NREs potential role as a "kinase switch" in RLK activation. Consequently, the characterization of phosphorylatable NRE motifs improves the accuracy in predicting phosphorylatable serines. Altogether, our study provides an enriched and comprehensive dataset to investigate NRE segments from individual RLKs and helps understand the underlying mechanism of action of the RLK signal transduction and kinase activation processes in plant adaptation.

plant biology↗

bioRxiv2022

Stat3 has a different role in axon growth during development than it does in axon regeneration after injury

Signal transducer and activator of transcription 3 (STAT3) is essential for neural development and regeneration as a key transcription factor and mitochondrial activator. In this study, using zebrafish posterior lateral line (PLL) axons, we demonstrate that Stat3 plays distinct roles in PLL axon embryonic growth and regeneration. In zebrafish stat3 mutants the PLL axons were truncated during embryonic growth. Expressing stat3 in neurons of the PLL ganglion rescued their axon growth suggesting stat3 promoted PLL axon growth in a cell-autonomous manner. Using Jak/Stat signaling inhibitors or modified Stat3, we found Jak/Stat signaling was dispensable for PLL axon growth. We showed that Stat3 was co-localized with mitochondria in PLL axon and lower ATPase activity in stat3 mutant embryos than wild-type embryos. The PLL axon growth was affected by mitochondrial modulators indicating that Stat3 regulated PLL axon growth through mitochondrial Stat3 function. Mutation of stat3 or treating with Jak/Stat signaling inhibitors retarded PLL axon regeneration and Schwann cell migration. Taken together, Stat3 is required for embryonic PLL axon growth by regulating the ATP synthesis efficiency of mitochondria, whereas Stat3 stimulates PLL axon regeneration through regulating Schwann cell migration via Jak/Stat signaling. Our findings show a new mechanism of Stat3 in axon growth and regeneration. Author SummaryNeurons are one of the most specialized cell type in our body. A slender axon used for transmitting information between neuron and target cell extends from the cell body. Axon extension during both development and regeneration is regulated by intrinsic and extrinsic factors. In this study, we used the zebrafish posterior lateral line (PLL) axons which extends a long distance to study the regulation of axon extension. Signal transducer and activator of transcription 3 (STAT3) is a key transcription factor in Jak/Stat signaling and an activator for mitochondria functions. Our results demonstrate that Stat3 plays distinct roles in PLL axons development and regeneration. During embryonic development, Jak/Stat signaling is dispensable for PLL axons extension, whereas the mitochondrial Stat3 is required for embryonic axon growth through promoting ATP synthesis. During PLL axons regeneration, Jak/Stat is essential for Schwann cells migration which mediates axon regeneration.

developmental biology↗

bioRxiv2022

Measuring cell-to-cell expression variability in single-cell RNA-sequencing data: a comparative analysis and applications to B cell ageing

BackgroundSingle-cell RNA-sequencing (scRNA-seq) technologies enable the capture of gene expression heterogeneity and consequently cell-to-cell variability at the cell type level. Although different methods have been proposed to quantify cell-to-cell variability, it is unclear what the optimal statistical approach is, especially in light of challenging data structures that are unique to scRNA-seq data like zero inflation. ResultsIn this study, we conducted a systematic evaluation of cell-to-cell gene expression variability using 14 different variability metrics that are commonly applied to transcriptomic data. Performance was evaluated with respect to data-specific features like sparsity and sequencing platform, biological properties like gene length, and the ability to recapitulate true levels of variability based on simulation and known biological gene sets like ribosomal genes and stably expressed genes. scran had the strongest all-round performance, and this metric was then applied to investigate the changes in cell-to-cell variability that occur during ageing. Studying ageing showcases the value of cell-to-cell variability as it is a genetically-regulated program that is influenced by stochastic processes.scRNA-seq datasets from hematopoietic stem cells (HSCs) and B lymphocytes and other cell types from this differentiation lineage were used with scran to identify the genes with consistent patterns of variable and stable expression profiles during differentiation. Furthermore, to understand the regulatory relationship for genes that were differentially-variable in their expression between young and old mice, we constructed networks using transcription factors and their known targets for HSC and B lymphocyte cells. Comparisons of these networks identified a shared TF Sfpi1 that although was seen to increase in gene expression variability in old mice versus young in both cell types, the corresponding targets were distinct and their gene expression variability had different directions between cell types. ConclusionsThrough these analyses, we highlight the importance of capturing cell-to-cell gene expression variability in a complex biological process like differentiation and ageing, and emphasise the value and specificity of interpreting these findings at the level of individual cell types.

bioinformatics↗

bioRxiv2022

Taming the data deluge: a novel end-to-end deep learning framework for classifying marine biological and environmental images

Marine underwater imaging facilitates non-destructive sampling of species at frequencies, durations, and accuracies that are unattainable by conventional sampling methods. These systems necessitate complex automated processes to identify organisms efficiently, however, current frameworks struggle to disentangle ecological foreground components from their dispensable background content. Underwater image processing relies on common architecture: namely image binarization for segmenting potential targets, prior to information extraction and classification by deep learning models. While intuitive, this infrastructure underperforms as it has difficulty in handling: high concentrations of biotic and abiotic particles, rapid changes in dominant taxa, and target sizes that vary by several orders of magnitude. To overcome these issues, a new framework is presented that begins with a scene classifier to capture large within-image variation, such as disparities in particle concentration and dominant taxa. Following scene classification, scene-specific regional convolutional neural network (Mask R-CNN) models were trained to separate target objects into different taxonomic groups. The procedure allows information to be extracted from different image types, while minimizing potential bias for commonly occurring features. Using in situ coastal PlanktonScope images, we compared the scene-specific models to the Mask R-CNN model including all scene categories without scene classification, defined as the full model, and found that the scene-specific approach outperformed the full model with >20% accuracy in noisy images. The full model missed up to 78% of the dominant taxonomic groups, such as Lyngbya, Noctiluca, and Phaeocystis colonies. This performance improvement is due to the scene classifier, which reduces the variation among images and allows an improved match between the observed taxonomic groups and the taxonomic groups in pre-trained models. We further tested the framework on images from a benthic video camera and an imaging sonar system. Results demonstrate that the procedure is applicable to different types of underwater images and achieves significantly more accurate results than the full model. Given that the unified framework is neither instrument nor ecosystem-specific, the proposed model facilitates deployment throughout the marine biome.

ecology↗

bioRxiv2022

Development of an affinity-enhanced clinical candidate TCR targeting NY-ESO-1 with optimal potency and high specificity

The clinical success of T-cell receptor (TCR)-based immunotherapy depends on the efficacy and specificity of TCRs. Naturally occurring TCRs have limited anti-tumor potency due to their low affinity for tumor antigens. Affinity enhancement is a promising strategy to generate highly potent TCRs. However, it is concerned that affinity-enhanced TCRs are prone to lose specificity. We isolated low affinity TCRs specific for NY-ESO-1157-165/HLA-A*02:01 from peripheral blood mononuclear cells of healthy donors. An affinity-enhanced TCR candidate with optimal affinity and specificity was generated using phage display and an extensive set of in vitro and in vivo assays. Alanine scanning mutagenesis showed that the TCR candidate retained specificity by making extensive contacts to the side chains of NY-ESO-1157-165 peptide. Adoptive transfer of T cells engineered with this candidate (termed TAEST16001) significantly inhibited tumor growth in subcutaneous, metastatic, and patient-derived xenograft (PDX) mouse tumor models. This study demonstrates that sophisticated engineering and screening techniques can be utilized to generate a clinical candidate TCR with potent anti-tumor activity without losing specificity. TAEST16001 was approved by the Center for Drug Evaluation (CDE) as the first TCR-based immunotherapy clinical trial in China (ClinicalTrials.gov Identifier: NCT03159585).

immunology↗