Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 289 records · Page 16Linked to original sources

bioRxiv2023

A robust method for the rapid generation of nanobodies.

Nanobody refers to the variable domain of heavy-chain-only antibodies. The distinctive advantages of nanobodies including small size, feasible expression in Escherichia coli (E. coli), and superior stability make them promising tools for applications in scientific research and therapies. So far, the screening and expression of nanobodies are mainly following similar methods used for conventional antibodies, suffering from amplification-caused losses of the diversity of libraries and requirements of subcloning of interests into the expression vector. Here, based on the unique properties of nanobodies, we developed an integrated method to screen and express nanobodies simultaneously with no bias of the library diversity. The library of nanobodies was cloned and secretively expressed into the culture medium. Target specifical binding nanobodies were isolated through 1-3 rounds of dilution and regrown steps in a way following the Poisson distribution to ensure no positive clones were dismissed, while the population of positive clones increased by more than 10 folds upon each round of dilution. Ultimately, 5 nanobodies against the death domain receptor 5 (DR5) and 5 nanobodies against the Pyrococcus furiosus (Pfu) DNA polymerase were produced directly out of their immunized libraries, respectively. Additionally, our approach allowed nanobody screening even without any specialized instruments/devices, demonstrating general applicability in the routine production of monoclonal nanobodies for diverse biomedical applications.

biochemistry↗

bioRxiv2023

Wingless signaling promotes lipid mobilization through signal-induced transcriptional repression

Conserved Wnt/Wingless signaling plays pivotal roles in regulating normal development and energy metabolism in metazoans, and aberrant activation of Wnt signaling drives the pathogenesis of many diseases including cancer. However, the role of Wnt signaling in regulating cellular lipid homeostasis, particularly lipid mobilization, remains poorly understood. Here we show that canonical Wg signaling inhibits lipid accumulation in Drosophila larval adipocytes by stimulating lipid catabolism while simultaneously inhibiting lipogenesis. Using a combination of RNA-sequencing and CUT&RUN assays, we identified a battery of Wg target genes encoding key factors required for lipogenesis (such as FASN1 and AcCoS), lipolysis (such as lipid droplet-associated proteins Lsd-1 and Lsd-2), and fatty acid {beta}-oxidation in the mitochondria and peroxisome (e.g., CPT1 and CRAT), most of which are directly repressed by active Wg signaling. Furthermore, lipid accumulation defects caused by active Wg signaling are rescued by either ectopically expressing Lsd-1 and Lsd-2 or depleting the transcriptional repressor Aef1, whose binding motif was identified in 52% of Wg signaling-repressed genes. These findings suggest that active Wg signaling reduces intracellular lipid accumulation by inhibiting lipogenesis and fatty acid {beta}-oxidation and by promoting lipolysis and lipid mobilization, and Wg signaling-induced transcriptional repression play a prominent role in these converging mechanisms.

cell biology↗

bioRxiv2023

Loss of genital epithelial barrier function is greater with depot-medroxyprogesterone acetate than intravaginal rings that release etonogestrel and ethinyl estradiol

The injectable progestin depot-medroxyprogesterone acetate (DMPA) is a popular contraceptive choice in sub-Saharan Africa although mouse models indicate it weakens genital epithelial integrity and barrier function and increases susceptibility to genital infection. The intravaginal ring NuvaRing(R) is another contraceptive option that like DMPA suppresses hypothalamic pituitary ovarian (HPO) axis function with local release of progestin (etonogestrel) and estrogen (ethinyl estradiol). As we previously reported that treating mice with DMPA and estrogen averts the loss of genital epithelial integrity and barrier function induced by DMPA alone, in the current investigation we compared genital levels of the cell-cell adhesion molecule desmoglein-1 (DSG1) and genital epithelial permeability in rhesus macaques (RM) treated with DMPA or a NuvaRing(R)re-sized for RM (N-IVR). While these studies demonstrated comparable inhibition of the HPO axis with DMPA or N-IVR, DMPA induced significantly lower genital DSG1 levels and greater tissue permeability to intravaginally administered low molecular mass molecules. By identifying greater compromise of genital epithelial integrity and barrier function in RM administered DMPA vs. N-IVR, our results add to the growing body of evidence that indicate DMPA weakens a fundamental mechanism of anti-pathogen host defense in the female genital tract.

immunology↗

bioRxiv2023

Analysis of Biodeterioration on Ancestral Halls with Different Visitor Flow Rates Using Amplicon Sequencing and Conventional Culturing Methods

AbstractWooden buildings are facing biodeterioration due to natural weathering. Ancestral halls are traditional Chinese wooden architecture with high artistic value. The research analyzed the microbial diversity of nine ancestral halls in Guangdong Province under subtropical monsoon climates. With amplicon sequencing, we found a common core microorganism group, including Bacillus, Pseudomonas, Paenibacillus, Acinetobacter, Toxicocladosporium, Cladosporium, Aspergillus, and Epicoccum. A total of 8 different species of bacteria and 7 different species of fungi were isolated from Chen Clan Ancestral halls, most of which were proven to be capable of degrading cellulose or lignin. The comparison between damaged and undamaged sites (whether the paint coating is flaked off) revealed similar microbial compositions but with differing abundances. Firmicutes and Ascomycota dominated damaged surfaces, while Proteobacteria and Actinobacteria prevailed in undamaged ones. Notably, Cladosporium, widely present in both sites, emerged as the primary cellulose-degrading genus. Given the similar microbial compositions between damaged and undamaged sites, it is suggested that using paint alone as the exclusive means to protect bare wood is insufficient. Apparent correlation between microorganisms on wooden surfaces and humans showed that the increase of visitor flow rates might impact the microbiota composition on cultural relics. We evaluated the effect of four representative biocides and determined the feasibility of low-concentration Isothiazolinone. This research facilitates a basic understanding of the microbial weathering of ancestral halls and add significant reference for preserving wooden structures.

microbiology↗

bioRxiv2023

Arabidopsis TRB proteins function in H3K4me3 demethylation by recruiting JMJ14

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

plant biology↗

bioRxiv2022

Nanofluidic Device for Manipulation and Modification of DNA by Proteins

Single-molecule techniques provide important details supplementing the framework obtained from traditional bulk experiments. Many cellular processes such as DNA replication, DNA repair, and telomere maintenance involve interaction among multiple proteins, their co-factors, and DNA. To investigate such interactions and to differentiate the function of each component necessitate a technique that allows the combinatorial exposure of DNA to multiple proteins and co-factors as well as manipulation of the DNA configuration. We present a nanofluidic device with the capability of active combinatorial exchange of up to three buffers in real-time and dynamic manipulation of DNA under physiologically relevant conditions. We demonstrate its utility in monitoring compaction of DNA by telomeric proteins, DNA modification by an endonuclease, and DNA loop extrusion by cohesin.

biophysics↗

bioRxiv2022

Promoting Axon Regeneration By Inhibiting RNA N6-methyladenosine Demethylase ALKBH5

A key limiting factor of successful axon regeneration is the intrinsic regenerative ability in both the peripheral (PNS) and central nervous system (CNS). Previous studies have identified axon regeneration regulators that act on gene expression in injured neurons. However, it is less known if RNA modifications play a role in this process. Here, we systematically screened the functions of all common m6A modification-related enzymes in axon regeneration and report ALKBH5, an evolutionarily conserved RNA m6A demethylase, as a regulator of axonal regeneration. In PNS, knockdown of ALKBH5 enhanced sensory axonal regeneration, whereas overexpressing ALKBH5 impaired axonal regeneration in an m6A-dependent manner. Mechanistically, ALKBH5 increased the stability of Lpin2 mRNA and thus limited regenerative growth associated lipid metabolism in DRG neurons. Moreover, in CNS, knockdown of ALKBH5 enhanced the survival and axonal regeneration of retinal ganglion cells after optic nerve injury. Together, our results suggest a novel mechanism regulating axon regeneration and point ALKBH5 as a potential target for promoting axon regeneration in both PNS and CNS.

neuroscience↗

bioRxiv2022

Discovery and biological evaluation of a potent small molecule CRM1 inhibitor for its selective ablation of extranodal NK/T cell lymphoma

BackgroundThe overactivation of NF-{kappa}B signaling is a key hallmark for the pathogenesis of extranodal natural killer/T cell lymphoma (ENKTL), a very aggressive subtype of non-Hodgkins lymphoma yet with rather limited control strategies. Previously, we found that the dysregulated exportin-1 (also known as CRM1) is mainly responsible for tumor cells to evade apoptosis and promote tumor-associated pathways such as NF-{kappa}B signaling. MethodsHerein we reported the discovery and biological evaluation of a potent small molecule CRM1 inhibitor, LFS-1107. We validated that CRM1 is a major cellular target of LFS-1107 by biolayer interferometry assay (BLI) and the knockdown of CRM1 conferred tumor cells with resistance to LFS-1107. ResultsWe found that LFS-1107 can strongly suppresses the growth of ENKTL cells at low-range nanomolar concentration yet with minimal effects on human platelets and healthy peripheral blood mononuclear cells. Treatment of ENKTL cells with LFS-1107 resulted in the nuclear retention of IkB and consequent strong suppression of NF-{kappa}B transcriptional activities, NF-{kappa}B target genes downregulation and attenuated tumor cell growth and proliferation. Furthermore, LFS-1107 exhibited potent activities when administered to immunodeficient mice engrafted with human ENKTL cells. ConclusionsTherefore, LFS-1107 holds great promise for the treatment of ENKTL and may warrant translation for use in clinical trials.

pharmacology and toxicology↗

bioRxiv2022

A unified classification system for HIV-1 5' long terminal repeats

The HIV-1 provirus mainly consists of internal coding region flanked by the 2 same long terminal repeats (LTRs) at each terminus. The LTRs play important roles in HIV-1 reverse transcription, integration, and transcription by the association with host factors. However, despite of the significant study advances of the internal coding regions of HIV-1 by using definite reference classification, there are no systematic classifications for HIV-1 5 LTRs, which hinders our elaboration on 5 LTR and a better understanding of the viral origin, spread and therapy. Here, by analyzing all available resources of 5 LTR sequences in public databases following 4 recognized principles for the reference classification, 83 representatives and 14 consensus sequences were identified as representatives of 2 groups, 6 subtypes, 6 sub-subtypes, and 9 CRFs. To test the reliability of our established classification system, the constructed references were applied to identify the 5 LTR assignment of the 22 clinical isolates in China. The results revealed that 16 out of 22 tested strains showed a consistent subtype classification with the previous LTR-independent classification system. However, 6 strains, for which recombination events within 5 LTR were demonstrated, unexpectedly showed a different subtype classification, leading a significant change of binding sites for important transcription factors including SP1, p53, and NF-{kappa}B. The binding change of these transcriptional factors would probably affect the transcriptional activity of 5 LTR. This study established a unified classification system for HIV-1 5 LTRs, which will facilitate HIV-1 characterization and be helpful for both basic and clinical research fields. IMPORTANCEHere, a scientific, reliable, and usable classification system based on the 5 LTR sequences was established, which will allow us to effectively facilitate the precise typing of HIV-1 strains. This classification system was applied to 22 HIV-1 strains circulating in China, we found that 6 out of 22 strains analyzed, belonged to a different subtype when our results were compared to those obtained with the previous LTR-independent classification system. Thus, these data demonstrated that our classification method could greatly improve the HIV-1 subtype classification. We found that 6 5 LTR sequences showed recombination events, leading to a significant exchange of the binding sites of transcriptional factors. Thus, this work established a comprehensive HIV-1 5 LTR classification system, which will help the scientific community to precisely characterize HIV-1 variants, and better understand the origin and spread of HIV-1 strains, and it also may be helpful for pathogenicity and transmissibility evaluation studies.

microbiology↗

bioRxiv2022

Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system as part of autophagy and cell-cycle regulatory network. The highly intrinsic disorder of AMBRA1 has so far precluded its structural determination. To solve this problem, we analyzed the domain organization and dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). High deuterium uptake indicates that AMBRA1 is a dynamic and largely unstructured protein, and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase complex. Here we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3 [A] resolution. The structure shows that parts of N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain, and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. AMBRA1 uses its N-terminal helix-loop-helix and WD40 domain to bind the double-propeller fold of DDB1, whereas different regions target the specific cellular substrates for ubiquitination. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and decreased number of autophagosomes in the cells. Together, these results provide structural insights into AMBRA1-ubiquitin ligase complex and suggests a mechanism by which the AMBRA1 acts as a hub involved in various physiological processes.

biochemistry↗

bioRxiv2022

Fc-gamma receptor-dependent antibody effector functions are required for vaccine protection against infection by antigenic variants of SARS-CoV-2

Emerging SARS-CoV-2 variants with antigenic changes in the spike protein are neutralized less efficiently by serum antibodies elicited by legacy vaccines against the ancestral Wuhan-1 virus. Nonetheless, these vaccines, including mRNA-1273 and BNT162b2, retained their ability to protect against severe disease and death, suggesting that other aspects of immunity control infection in the lung. Although vaccine-elicited antibodies can bind Fc gamma receptors (Fc{gamma}Rs) and mediate effector functions against SARS-CoV-2 variants, and this property correlates with improved clinical COVID-19 outcome, a causal relationship between Fc effector functions and vaccine-mediated protection against infection has not been established. Here, using passive and active immunization approaches in wild-type and Fc-gamma receptor (Fc{gamma}R) KO mice, we determined the requirement for Fc effector functions to protect against SARS-CoV-2 infection. The antiviral activity of passively transferred immune serum was lost against multiple SARS-CoV-2 strains in mice lacking expression of activating Fc{gamma}Rs, especially murine Fc{gamma}R III (CD16), or depleted of alveolar macrophages. After immunization with the preclinical mRNA-1273 vaccine, protection against Omicron BA.5 infection in the respiratory tract also was lost in mice lacking Fc{gamma}R III. Our passive and active immunization studies in mice suggest that Fc-Fc{gamma}R engagement and alveolar macrophages are required for vaccine-induced antibody-mediated protection against infection by antigenically changed SARS-CoV-2 variants, including Omicron strains.

microbiology↗

bioRxiv2022

Alarming antibody evasion properties of rising SARS-CoV-2 BQ and XBB subvariants

The SARS-CoV-2 Omicron variant continues to evolve, with new BQ and XBB subvariants now rapidly expanding in Europe/US and Asia, respectively. As these new subvariants have additional spike mutations, they may possess altered antibody evasion properties. Here, we report that neutralization of BQ.1, BQ.1.1, XBB, and XBB.1 by sera from vaccinees and infected persons was markedly impaired, including sera from individuals who were boosted with a WA1/BA.5 bivalent mRNA vaccine. Compared to the ancestral strain D614G, serum neutralizing titers against BQ and XBB subvariants were lower by 13-81-fold and 66-155-fold, respectively, far beyond what had been observed to date. A panel of monoclonal antibodies capable of neutralizing the original Omicron variant, including those with Emergency Use Authorization, were largely inactive against these new subvariants. The spike mutations that conferred antibody resistance were individually studied and structurally explained. Finally, the ACE2-binding affinities of the spike proteins of these novel subvariants were found to be similar to those of their predecessors. Taken together, our findings indicate that BQ and XBB subvariants present serious threats to the efficacy of current COVID-19 vaccines, render inactive all authorized monoclonal antibodies, and may have gained dominance in the population because of their advantage in evading antibodies.

microbiology↗

bioRxiv2022

The first host-associated anaerobic isolate of Psychrilyobacter provides insights into its potential roles in the abalone gut

Gut bacteria are essential to their hosts, but few studies have focused on marine animals. Psychrilyobacter is frequently related to various marine animals, but the relationship with host remains unknown due to lack of host-associated isolate or genomic information. Here, we combined of high-throughput sequencing, isolating and genomic analyses to uncover its potential role in host abalone. The high-throughput sequencing and literature compiling results indicated that Psychrilyobacter is widely distributed in marine and terrestrial ecosystems with both host-associated and free-living lifestyles. It showed a strong preference for the guts of marine invertebrates, especially abalone, which generally persisted with high relative abundances. By mimicking the gut inner environment for enrichment, the first host-related pure culture of Psychrilyobacter was isolated from the abalone intestine. Phylogenetic, physiological and biochemical characterizations suggested that it represents a novel species named Psychrilyobacter haliotis B1. Carbohydrate utilization experiments and genomic evidence indicated that B1 is good at fermenting diverse host-food-related monosaccharides and disaccharides but not polysaccharides, implying its critical roles in downstream fermentation instead of upstream food degradation in the gut. Furthermore, this strain showed potential to colonize the gut and benefit the host via different strategies, such as short-chain fatty acids (SCFAs) generation by fermenting carbohydrates and amino acids, and the production of diverse vitamins and antibiotics to support the host growth and antipathogenicity. To our knowledge, strain B1 represents the first host-related pure culture of Psychrilyobacter, acting as a potential probiotic gut anaerobe dominating in diverse marine invertebrates. ImportancePsychrilyobacter is a globally distributing bacterial genus and with an inhabiting preference for guts of marine invertebrates. Due to the difficulty of cultivation and the limited genomic information, its role in host remains largely unknown. We isolated the first host-associated Psychrilyobacter species from abalone gut and uncovered its functional potentials to the host through different mechanisms. Our findings provide some insights into the understanding of host-microbe interactions on a core taxon with the marine invertebrates, and the isolate has an application prospect as a probiotic in protection of marine animals.

microbiology↗

bioRxiv2022

Longitudinal analysis of retinal ganglion cell damage at individual axon bundle level in mice using visible-light optical coherence tomography fibergraphy

We developed a new analytic tool based on visible-light optical coherence tomography fibergraphy (vis-OCTF) to longitudinally track individual axon bundle transformation following acute optic nerve crush injury (ONC) in mice. We analyzed four parameters: lateral bundle width, axial bundle height, cross-sectional area, and the shape of individual bundles. We showed that axon bundles became wider and thicker at 3-days post ONC. The bundle swelling at 3-days post-ONC has correlated with about 15% retinal ganglion cell (RGC) soma loss. At 6-days post-ONC, axon bundles showed a significant reduction in lateral width and cross-sectional area, followed by a reduction in bundle height at 9-days post-ONC. Bundle shrinking at 9-days post-ONC has correlated with about 68% RGC soma loss. Both experimental and simulated results suggested that the cross-sectional area of individual RGC axon bundles is more sensitive than the bundle width and height to indicate RGC soma loss. This study is the first to track and quantify individual RGC axon bundles in vivo following ONC injury and establish the correlation between the morphological changes of RGC axon bundles and RGC soma loss.

neuroscience↗

bioRxiv2022

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain, N-terminal domain and S2 region, distal to the ACE2 receptor-interaction site. Our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that peptides and potentially other drug-like molecules can target. Significance statementThis study reports on the discovery of a macrocyclic peptide that is able to inhibit SARS-CoV-2 infection by exploiting a new vulnerable site in the spike glycoprotein. This region is highly conserved across SARS-CoV-2 variants and the subgenus sarbecovirus. Due to the inaccessability and mutational contraint of this site, it is anticipated to be resistant to the development of resistance through antibody selective pressure. In addition to the discovery of a new molecule for development of potential new peptide or biomolecule therapeutics, the discovery of this broadly active conserved site can also stimulate a new direction of drug development, which together may prevent future outbreaks of related viruses.

biochemistry↗

bioRxiv2022

Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1, and XBB.1 by 4 doses of parental mRNA vaccine or a BA.5-bivalent booster

The newly emerged SARS-CoV-2 Omicron BQ.1.1, XBB.1, and other sublineages have accumulated additional spike mutations that may affect vaccine effectiveness. Here we report neutralizing activities of three human serum panels collected from individuals 1-3 months after dose 4 of parental mRNA vaccine (post-dose-4), 1 month after a BA.5-bivalent-booster (BA.5-bivalent-booster), or 1 month after a BA.5-bivalent-booster with previous SARS-CoV-2 infection (BA.5-bivalent-booster-infection). Post-dose-4 sera neutralized USA-WA1/2020, BA.5, BF.7, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 SARS-CoV-2 with geometric mean titers (GMTs) of 1533, 95, 69, 62, 26, 22, and 15, respectively; BA.5-bivalent-booster sera improved the GMTs to 3620, 298, 305, 183, 98, 73, and 35; BA.5-bivalent-booster-infection sera further increased the GMTs to 5776, 1558,1223, 744, 367, 267, and 103. Thus, although BA.5-bivalent-booster elicits better neutralization than parental vaccine, it does not produce robust neutralization against the newly emerged Omicron BA.2.75.2, BQ.1.1, and XBB.1. Previous infection enhances the magnitude and breadth of BA.5-bivalent-booster-elicited neutralization.

microbiology↗

bioRxiv2022

Quartet protein reference materials and datasets for multi-platform assessment of label-free proteomics

Quantitative proteomics is an indispensable tool in life science research. However, there is a lack of reference materials for evaluating the reproducibility of label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements among different instruments and laboratories. We developed the Quartet as a proteome reference material with built-in truths, and distributed the same aliquots to 15 laboratories with nine conventional LC-MS/MS platforms across six cities in China. Relative abundance of over 12,000 proteins on 816 MS files were obtained and compared for reproducibility among the instruments and laboratories to ultimately generate proteomics benchmark datasets. There was a wide dynamic range of proteomes spanning ~7 orders of magnitude (101-108 copies/cell), and the injection order had marked effects on quantitative instead of qualitative. Overall, the Quartet offers valuable standard materials and data resources for improving the quality control of proteomic analyses as well as the reproducibility and reliability of research findings.

molecular biology↗

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↗