Biology subjects

Lee, M. S.

Publications and source records attributed to Lee, M. S..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural Basis of Serine Protease Inhibition by Antibodies from Biased Fab Phage-Display Libraries

Biased Fab phage-display libraries were designed to determine whether inhibitory CDR H3 motifs from potent anti-matriptase antibodies could be transferred to target homologous serine proteases. Using reverse-binding and substrate-like H3 motifs from parental clones A11 and E2 as templates, six synthetic libraries with 1010 diversity were constructed. Selection against matriptase identified sixteen inhibitors with sub-100 nM potency, representing 100,000-fold improvement over circularized H3 loops alone. Selection against TMPRSS2, a serine protease implicated in viral entry and prostate cancer with 43% sequence identity to matriptase, yielded binders with micromolar inhibitory potency. Selection against urokinase plasminogen activator (uPA, 35% identity) identified binders that adopted a substrate-like CDR H3 binding mode in our structural models. Across all reference structures, including the separately identified uPA inhibitor AB2 (PDB: 9PYF, deposited with this work), benchmarking of five co-folding methods and rigid-body docking showed that co-folding consistently achieved acceptable to high quality DockQ scores, outperforming traditional docking and capturing the recognition of key active site determinants. Ensemble predictions of mutational binding energy changes ({Delta}{Delta}G) using these models identified key paratope-epitope interactions, with predictions validated through mutagenesis. This work establishes a framework integrating biased antibody libraries with computational structure prediction and analysis for targeting conserved protease epitopes.

molecular biology↗

bioRxiv2026

Focused Ultrasound Thermal Ablation and CD40 Agonism Reprograms Breast Tumor Immunity to Drive Regression and Memory

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we demonstrate that subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across four immunologically and hormonally distinct murine BC models. Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4+ and CD8+ T cells. When layered onto this immunogenic milieu, CD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 33% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Efficacy required both CD4+ and CD8+ T cells, and complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T cell activation. Together, these findings establish T-FUS as an immune-potentiating partner for CD40 agonism, capable of driving durable, robust BC regression and immunological memory. This work positions T-FUS+CD40 agonism as a clinically scalable, in situ vaccination-like strategy with potential to benefit breast cancers, including luminal subtypes, that remain largely refractory to immune checkpoint blockade.

cancer biology↗

bioRxiv2025

In situ cell-only bioprinting of patterned prevascular tissue into bioprinted high-density stem cell-laden microgel bioinks for vascularized bone tissue regeneration

Recently, microgels have been widely used in three-dimensional (3D) bioprinting as both supporting baths and bioinks. As a bioink, microgels have several unique properties, such as shear-thinning and self-healing behaviors with tunable mechanics, making them useful in 3D bioprinting. While cell encapsulated microgels offer many advantages in 3D bioprinting, they also have some limitations. It is still challenging to produce large quantities of cell encapsulated microgels with consistent quality and properties due to processes that are often complex and time-consuming. In this study, stem cell encapsulated, photocrosslinkable, shear-thinning and self-healing alginate microgel (SSAM) bioinks have been successfully fabricated via simple mixing of an oxidized and methacrylated alginate solution with suspended stem cells and a supersaturated calcium sulfate slurry solution through a custom-made spiral mixing unit. The SSAM bioinks can be bioprinted into complex 3D structures with both high resolution and shape fidelity due to their shear-thinning and self-healing properties. The 3D bioprinted SSAM bioinks can then serve as a supporting bath for the creation of prevascular network patterns using an individual cell-only prevasculogenic bioink within the 3D printed constructs. The prevascular network patterned 3D bioprinted constructs can be further stabilized by secondary photocrosslinking of the SSAMs, which enables long-term culture of the printed constructs for functional vascularized osteogenic tissue formation by differentiation of the bioprinted cells. The SSAM bioinks and individual cell-only printing technique enable in situ bioprinting of prevascularized tissue constructs in a mouse calvarial bone defect, achieving mechanical stability and ensuring the in situ bioprinted constructs remain within the defect.

bioengineering↗

bioRxiv2025

Precise, Specific, and Sensitive De Novo Antibody Design Across Multiple Cases

The precision design of antibodies, which naturally recognize diverse molecules through six variable loops, remains a critical challenge in therapeutic molecule discovery. In this study, we demonstrate that precise, sensitive, and specific antibody design can be achieved without prior antibody information across eight distinct target proteins. For each target, binders were identified from a yeast display scFv library of approximately 106 sequences, constructed by combining 102 designed light chain sequences with 104 designed heavy chain sequences. Binders with varying binding strengths were identified for all eight targets, including a case where no experimentally resolved target protein structure was available, demonstrating the highest level of precision compared to previous de novo antibody design reports. To further validate the designed antibodies, they were characterized in the IgG format for five distinct targets. The antibodies exhibited favorable developability properties, positioning them as promising hit or lead candidates. Notably, for one target in particular, the IgG-formatted antibodies exhibited affinity, activity, and developability, comparable to a commercial antibody, highlighting the sensitivity of the design method. Furthermore, binders capable of distinguishing closely related protein subtypes or mutants were identified, demonstrating that the method can achieve high molecular specificity. Cryo-EM analysis experimentally validated the designs accuracy by confirming that the key binding interactions were precisely as designed. These findings underscore the effectiveness of precision molecular design based on atomic-accuracy structure prediction. This study establishes computational antibody design as a viable approach for generating therapeutic molecules with tailored properties, with promising potential for achieving the efficacy and safety required for successful therapeutics.

bioengineering↗

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2024

Accurate antibody loop structure prediction enables zero-shot design of target-specific antibodies

Protein loops, characterized by their versatile structures with varying sizes and shapes, can recognize a wide range of targets with high specificity and affinity. The variable loops of the antibody complementarity-determining region (CDR) are particularly crucial for immune responses and therapeutic applications due to their effective target recognition capabilities. Accurate structure prediction of these antibody loops is essential for the efficient in silico design of target-binding antibodies for therapeutic or industrial use. However, predicting antibody loop structures is challenging due to the lack of evolutionary information from related proteins. Thus, a successful ab initio structure prediction method, which operates without structural templates or related sequences, is crucial for the effective design of antibody loop-mediated interactions. This study demonstrates that highly accurate antibody loop structure prediction enables the effective zero-shot design of target-binding antibody loops. The performance of loop design has been shown to depend on the accuracy of ab initio loop structure prediction, as tested with two versions of our design model. The high affinity, diversity, novelty, and specificity of the antibody loops designed with these new methods were validated experimentally on four target proteins.

bioengineering↗

bioRxiv2024

T cell cholesterol transport is a metabolic checkpoint that links intestinal immune responses to dietary lipid absorption

The intrinsic pathways that control membrane organization in immune cells and the impact of such pathways on cellular function are not well defined. Here we report that the non-vesicular cholesterol transporter Aster-A links plasma membrane (PM) cholesterol availability in T cells to immune signaling and systemic metabolism. Aster-A is recruited to the PM during T-cell receptor (TCR) activation, where it facilitates the removal of newly generated "accessible" membrane cholesterol. Loss of Aster-A leads to excess PM cholesterol accumulation, resulting in enhanced TCR nano-clustering and signaling, and Th17 cytokine production. Finally, we show that the mucosal Th17 response is restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells leads to enhanced IL-22 production, reduced intestinal fatty acid absorption, and resistance to diet-induced obesity. These findings delineate a multi-tiered regulatory scheme linking immune cell lipid flux to nutrient absorption and systemic physiology.

physiology↗

bioRxiv2024

Discovery of Highly Active Kynureninases for Cancer Immunotherapy through Protein Language Model

Overcoming the immunosuppressive tumor microenvironment is a promising strategy in anticancer therapy. L-kynurenine, a strong immunosuppressive metabolite can be degraded through kynureninases. Through homology searches and protein language models, we identified and then experimentally determined the efficacy of four top-ranked kynureninases. The catalytically most active one nearly doubles turnover number over the prior best, reducing tumor weight by 3.42 times in mouse model comparisons, and thus, presenting substantial therapeutic potential.

cancer biology↗

bioRxiv2023

Cell-Free Optimized Production of Protoporphyrin IX

BackgroundThe asymmetric and aromatic structures of porphyrins enable semiconducting properties allowing them to absorb light to initiate complex photocatalytic activity. These properties coupled with biological origins have garnered these molecules and their derivatives wide interest as a biotechnological platform. However, porphyrin production in cells is challenging due to the limited titer, long production timescales, and difficult purification. ResultsA cell-free metabolic engineering platform was constructed to produce protoporphyrin IX (PPIX) from E. coli crude extracts. Using acoustic liquid-handling, design of experiments for high-throughput buffer optimization and co-culturing techniques for extract production, our cell-free reactions effectively produced 0.109 mg/mL quantities of porphyrins. ConclusionsThe use of cell-free metabolic engineering as a bioproduction platform could improve the production of toxic or inefficient biomolecules such as PPIX. The engineering strategies including DOE and co-culturing applied in this study provide a roadmap towards increasing the scale of cell-free metabolic engineering by elucidating batch to batch variability, as well as the need for batch specific optimization of reaction conditions.

bioengineering↗

bioRxiv2023

Virtual biomarkers: predicting immune status using label-free holotomography of individual human monocytes and machine learning analysis

Precise evaluation of immune status is critical for managing diseases such as sepsis, in which the immune system transitions between hyper-inflammatory and immune-suppressed states. However, current biomarkers are limited by low specificity and time-consuming protocols. Here, we present a label-free, imaging-based framework for single-cell immune profiling of human monocytes using three-dimensional holotomography (HT) and deep learning. HT captures subcellular refractive index (RI) distributions of live, unlabeled cells, enabling quantitative extraction of morphological and biophysical features. Using an in vitro LPS-based model, we classified three functional immune states--control, hyper-inflammation, and immune suppression--based on 4,059 holotomograms from 11 donors. Immune state transitions were associated with significant changes in cell volume, surface area, RI variability, and the abundance of lipid droplets. A 3D convolutional neural network trained on HT images achieved 83.7% accuracy for single-cell predictions, increasing to 99.9% with ensemble averaging. This study establishes HT as a scalable, label-free platform for real-time immune monitoring and introduces subcellular RI features as robust correlates of immune dysregulation.

immunology↗

bioRxiv2023

The CD4 transmembrane GGXXG and juxtamembrane (C/F)CV+C motifs mediate pMHCII-specific signaling independently of CD4-Lck interactions

CD4+ T cell activation is driven by 5-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3{gamma}{varepsilon}, CD3{delta}{varepsilon}, and CD3{zeta}{zeta}) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee, et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3{zeta}, ZAP70, and PLC{gamma}1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

immunology↗

bioRxiv2021

Targeting mitochondrial double-stranded RNAs ameliorates autoimmune characteristics of Sjogrens syndrome

ObjectiveSjogrens syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. This study investigates the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFN response in SS. MethodsSaliva and tear from SS patients and controls (n=73 for saliva and n=16 for tear), the salivary glands of the SS-prone-non-obese-diabetic mouse, and primary human salivary glandular cells were screened for mt-dsRNAs by RT-qPCR. The human salivary cell line (NS-SV-AC) grown as three-dimensional spheroids were subject to dsRNA stress to measure mt-dsRNA induction and recapitulation of SS glandular inflammatory features. Acetylcholine, SS-IgG, upadacitinib (JAK1 inhibitor), or 2-C'-methyladenosine (mitochondrial transcription inhibitor) were applied to characterize the roles of mt-dsRNAs. To identify endogenous dsRNA-sensor and confirm the mitochondrial origin of cytoplasmic dsRNAs, the immunoprecipitation of dsRNAs was performed. Resultsmt-dsRNAs were elevated in the SS specimens with salivary ND5 and tear CYTB1 being statistically associated with secretory dysfunction/inflammation and corneal/conjunctival damage, respectively. Stimulation of the spheroids with dsRNA stress of poly I:C induced mt-dsRNAs, p-PKR, and I-IFNS via the JAK1/STAT pathway whereas the inhibition of mt-RNA synthesis or JAK1 attenuated the glandular signature. The inhibitory effect of acetylcholine on mt-dsRNAs and I-IFNS induction was reversed by SS-IgG. Conclusionmt-dsRNAs amplify the impact of dsRNA stress on SS glandular signatures in vitro, potentially propagating a pseudo-viral signal in the SS target tissue. SummaryMitochondrial double-stranded RNA levels were elevated in the tear and saliva of SS patients, which was associated with secretory dysfunction and tissue inflammation. These RNAs amplified type I interferon signature as well as glandular phenotypes reported in SS. Inhibitors of mitochondrial RNA transcription or JAK1 in salivary gland acinar cell spheroids attenuated the mitochondrial RNA-mediated changes.

molecular biology↗

bioRxiv2021

Enhancing and inhibitory motifs have coevolved to regulate CD4 activity

CD4+ T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through [~]435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that both enhance pMHCII responses and are coevolving with residues in an intracellular motif that inhibits pMHCII responses. Importantly, while CD4 interactions with the Src kinase, Lck, are classically viewed as the key determinant of CD4s contribution to pMHCII responses, we found that without the inhibitory motif CD4-Lck interactions are not necessary for robust responses to pMHCII. In summary, motifs that mediate events on the outside and inside of CD4+ T cells coevolved to finetune the relay of pMHCII-specific information across the membrane. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.

immunology↗

bioRxiv2021

Analysis of the blood microbiome in a porcine model of fecal-induced peritonitis

BackgroundRecent studies have proposed the existence of a blood microbiome, even in the healthy host. However, we do not know how the blood microbiome changes when a bloodstream infection (BSI) occurs. Here, we analyzed the dynamics of the blood microbiome in a porcine model of polymicrobial bacteremia induced by fecal peritonitis. Serial blood samples were taken over 12 hours post-induction of fecal peritonitis, and BSI was validated by conventional blood culture and assessment of clinical symptoms. ResultsThe bacterial populations in the blood microbiome were retained throughout the experimental period. However, there were significant taxonomic differences between the profile in the fecal and blood microbiomes, reflecting tropism for the blood environment. We also confirmed that the microbiota we detected was not contaminated by low mass bacteria in the bloodstream. However, at the same time, we noted a slight increase in Bacteroidetes, which is a major component of the gut microbiome, as sepsis developed. Comparison of the functional pathways in the blood and fecal microbiomes revealed upregulation of pathways involved in environmental interactions, and downregulation of those related to cell proliferation, in the former. Based on the enriched biological pathways, we concluded that communication and stress management pathways are essential for the survival of the blood microbiome under harsh conditions. ConclusionThis study suggests that the microbiota can be stably retained in the bloodstream over time. Although further investigation in humans is required, we suggest that the blood microbiome may be another factor to be considered in the context of BSI and subsequent sepsis.

microbiology↗

bioRxiv2020

Fab-dimerized glycan-reactive antibodies neutralize HIV and are prevalent in humans and rhesus macaques

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Lyophilized cell-free systems display tolerance to organic solvent exposure

Cell-free systems offer a powerful way to deliver biochemical activity to the field without cold chain storage. These systems are capable of sensing as well as biosynthesis of useful molecules at the point of need. So far, cell-free protein synthesis (CFPS) reactions have been studied as aqueous solutions in test tubes or absorbed into paper or cloth. Embedding biological functionality into broadly-used materials, such as plastic polymers, represents an attractive goal. Unfortunately, this goal has for the most part remained out of reach, presumably due to the fragility of biological systems outside of aqueous environments. Here, we describe a surprising and useful feature of lyophilized cell-free lysate systems: tolerance to a variety of organic solvents. Screens of individual CFPS reagents and different CFPS methods reveal that solvent tolerance varies by CFPS reagent composition. Tolerance to suspension in organic solvents may facilitate the use of polymers to deliver dry cell-free reactions in the form of coatings or fibers, or allow dosing of analytes or substrates dissolved in non-aqueous solvents, among other processing possibilities.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsCFPScell-free protein synthesisGFPgreen fluorescent proteinDCMdichloromethaneDMFdimethylformamideDMSOdimethylsulfoxideTHFtetrahydrofuranView Full Text

synthetic biology↗

bioRxiv2020

Silk Fibroin as an Additive for Cell-Free Protein Synthesis

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions. Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing, point-of-need manufacturing, and responsive materials. Meanwhile, silk fibroin from the silk worm, Bombyx mori, has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes. In this work, we explore the effects of silk fibroin as an additive in cell-free protein synthesis (CFPS) reactions. Impacts of silk fibroin on CFPS activity and stability after drying, as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed. We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%, which we attribute to macromolecular crowding effects. However, we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes. Further, the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture. Crosslinking attempts did not impact CFPS activity, but did yield localized protein aggregates rather than a hydrogel. We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsCFPSCell-free Protein SynthesisSFSilk FibroinHRPhorse radish peroxidasesfGFPsuperfolder green fluorescent proteinSEMScanning Electron MicroscopyView Full Text

synthetic biology↗

bioRxiv2020

Peptide recognition and dephosphorylation by the vaccinia VH1 phosphatase

The VH1 protein encoded by the highly conserved H1 locus of orthopoxviruses is a dual-specificity phosphatase (DUSPs) that hydrolyzes phosphate groups from phosphorylated tyrosine, serine, and threonine residues of viral and host cell proteins. Because the DUSP activities are required for virus replication, VH1 is a prime target for the development of therapeutic inhibitors. However, the presentation of a shallow catalytic site has thwarted all drug development efforts. As an alternative to direct targeting of catalytic pockets, we describe surface contacts between VH1 and substrates that are essential for full activity and provide a new pathway for developing inhibitors of protein-protein interactions. Critical amino acid residues were manipulated by site-directed mutagenesis of VH1, and perturbation of peptide substrate interactions based on these mutations were assessed by high-throughput assays that employed surface plasmon resonance and phosphatase activities. Two positively-charged residues (Lys-20 and Lys-22) and the hydrophobic side chain of Met-60 appear to orient the polarity of the pTyr peptide on the VH1 surface, while additional amino acid residues that flank the catalytic site contribute to substrate recognition and productive dephosphorylation. We propose that the enzyme-substrate contact residues described here may serve as molecular targets for the development of inhibitors that specifically block VH1 catalytic activity and thus poxvirus replication.

biochemistry↗