Biology subjects

Menegatti, S.

Publications and source records attributed to Menegatti, S..

15 recordsLinked to original sources

bioRxiv2026

Specific killing of Ewing sarcoma by TCR-T cells targeting public neogene-encoded antigens

EWSR1::FLI1, the oncogenic chimeric transcription factor driving Ewing sarcoma (EwS)induces expression of exquisitely EwS-specific neogenes (Ew_NGs) through neomorphic binding and transcription activation at GGAA microsatellites in genomic regions that are silent in normal tissues. We show that peptides encoded by Ew_NGs are presented on HLA-I complexes on EwS cells. The cytokine secretion of CD8+ T cells specific for Ew_NG-encoded HLA-I-bound peptides is activated by all HLA-I-matched EwS cells but not by non-EwS cells. These T cells kill EwS cells in an HLA-I restricted manner. This cytotoxicity is dependent on the expression of EWSR1::FLI1 and of the corresponding Ew_NG. It can be reproduced by transduction of the TCR into donor T cells (TCR-T) which kill EwS cells in vivo. Moreover, we show that neither off target nor allogeneic activation are observed with TCR-T thus paving the way for cell therapy in relapsed/resistant EwS patients for which therapeutic options are very limited. Statement of significanceThe chimeric transcription factor EWSR1::FLI1 generates tumor-specific neogenes encoding neoantigen presented by the HLA-I molecules of Ewing cells. Neoantigen-specific CD8+ T-cell clones and engineered TCR-T cells can selectively recognize and kill EwS tumor cells in vitro and in vivo.

immunology↗

bioRxiv2025

Adaptive Machine Learning Framework enables Unprecedented Yield and Purity of Adeno-Associated Viral Vectors for Gene Therapy

Adeno-associated viral (AAV) vectors for gene therapy are becoming integral to modern medicine, providing therapeutic options for diseases once deemed incurable. Currently, optimizing viral vector purification is a critical bottleneck in the gene therapy industry, impacting product efficacy and safety as well as accessibility and cost to patients. Traditional optimization methods are resource-intensive and often fail to adjust the purification process parameters to maximize the resulting product yield and quality. To address this challenge, we developed a machine learning framework that leverages Bayesian optimization to systematically refine affinity chromatography parameters (sample load, flow rate, and the formulation of chromatographic media) to improve AAV purification. The efficiency of this closed-loop workflow in iteratively optimizing the vectors yield, purity, and transduction efficiency was demonstrated by purifying clinically-relevant serotypes AAV2, AAV5, and AAV9 from HEK293 cell lysates using the affinity adsorbent AAVidity. We show that three cycles of Bayesian optimization elevated yields from a baseline of 70% to 99%, while reducing host-cell impurities by 230-to-400-fold across all serotypes. The optimized parameters consistently produced vectors with high purity and preserved high transduction activity, essential for therapeutic efficacy and safety, demonstrating serotype versatility - a key challenge in AAV manufacturing. By streamlining parameter optimization and enhancing productivity, our adaptive machine learning framework accelerates process development and reduces costs, advancing the accessibility and clinical translation of AAV-based gene therapies.

molecular biology↗

bioRxiv2025

Integrating affinity chromatography in the platform process for Adenovirus purification

Adenoviral vectors (AdVs) are gaining prominence in cancer therapy and vaccine development, posing the need for a modern AdV manufacturing platform. Current AdV purification by ion-exchange chromatography indeed struggles to achieve the products yield and purity of processes that employ affinity technologies. Addressing these challenges, this study presents the first affinity-based process that delivers high product yield and clearance of host cell proteins and DNA (HCPs and hcDNA) in two chromatography steps. The affinity capture utilizes resins functionalized with peptide ligands that target AdV hexon proteins (AEFFIWNA and TNDGPDYSSPLTGSG), and provide high capacity (>5{middle dot}1010 vp per mL of resin) and yield under mild elution conditions ([~]50% at pH 8.0). Peptide-functionalized adsorbents prepared using different matrices (polymethylmethacrylate vs. agarose) were initially tested to compare the purification performance. AEFFIWNA-SulfoLink resin was selected for its yield of cell- transducing AdVs ([~]50%) and removal of HCPs and hcDNA (144-fold and 56-fold). Similarly, TNDGPDYSSPLTGSG-Toyopearl(R) resin afforded [~]50% yield and >50-fold reduction of con- taminants. Additional gains in product purity were achieved by optimizing the washing step, which removed free hexon proteins and additional HCPs. All peptide-functionalized resins maintained their purification performance for ten cycles upon regeneration at pH [~]2.0. The purification process was assembled to include clarification, affinity capture in bind-and-elute mode using AEFFIWNA-SulfoLink resin, and polishing in flow-through mode using mixed- mode resins. The optimized process provided a yield [~]50% of cell-infecting units (IFU) and a product titer [~]107 IFU/mL, along with residual HCP and hcDNA levels ([~]10 ng/mL and 44 ng per dose, respectively) that meet clinical requirements.

bioengineering↗

bioRxiv2024

The DivisionCounter, a method for counting large ranges of cell divisions in vivo, reveals cell dynamics of leukemic cell killing via CAR-T therapy

Cell division drives multicellular growth and its dysregulation can cause disease. While approximately 44 divisions are needed to produce without death all 1013 cells in the human body, current methods are limited to count 10 cell divisions in vivo across diverse mammalian cell types. Here we introduce the DivisionCounter, a method to count cell division in vivo over large division ranges ([~]70 divisions) using an easy fluorescence readout. We demonstrate that the DivisionCounter accurately measures the average cell division count of cells both in vitro and in vivo. Its use revealed that leukemia tumor division rates are independent of the organs specific microenvironment and CAR-T cell treatment, providing an estimate of tumor death rates in vivo. The DivisionCounter method holds unique potential for quantifying contributions of cell division, death, and migration to the growth of healthy and pathological mammalian tissues.

immunology↗

bioRxiv2024

Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in E. coli

Process Analytical Technologies (PAT) are critical for efficient and automated bioprocessing; moreover, sensor and assay-driven automation will be necessary for the realization of Industry 4.0. Herein, we demonstrate methods for analyzing product yield from a pilot-scale bioreactor (300L) and conduct cross-method comparisons. We used a system of recombinant green fluorescent protein (GFPuv) expressed in Escherichia coli (E. coli), which is a simple, cost-effective model for evaluation at both laboratory and pilot scales. By comparing inline bioreactor measurements, plate reader assays, and a novel image analysis pipeline, we identify optimal harvest timelines and demonstrate the strengths and limitations of each technique. Results indicate peak cell density and GFPuv expression between 12.5h and 18h post-inoculation, with declining viability thereafter. Comparisons across techniques suggest that imaging methods may be more effective in capturing adverse outcomes, such as increased membrane permeability or cell death, which typically occur at later stages of operation. This integrated analysis offers actionable insights for optimizing biomanufacturing workflows and advances the development of scalable PAT applications, bridging the gap between laboratory research and industrial implementation.

biochemistry↗

bioRxiv2023

CD82 expression marks the endothelium to hematopoietic transition at the onset of blood specification in human

SUMMARYDuring embryonic development, all blood progenitors are initially generated from endothelial cells that acquire a hemogenic potential. Blood progenitors emerge through an endothelial-to-hematopoietic transition regulated by the transcription factor RUNX1. To date, we still know very little about the molecular characteristics of hemogenic endothelium and the molecular changes underlying the transition from endothelium to hematopoiesis. Here, we analysed at the single cell level a human embryonic stem cell-derived endothelial population containing hemogenic potential. RUNX1-expressing endothelial cells, which harbour enriched hemogenic potential, show very little molecular differences to their endothelial counterpart suggesting priming toward hemogenic potential rather than commitment. Additionally, we identify CD82 as a marker of the endothelium-to-hematopoietic transition. CD82 expression is rapidly upregulated in newly specified blood progenitors then rapidly downregulated as further differentiation occurs. Together our data suggest that endothelial cells are first primed toward hematopoietic fate, then rapidly undergo the transition from endothelium to blood.

developmental biology↗

bioRxiv2023

Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates

Adeno-associated viruses (AAVs) are the vector of choice for delivering gene therapies that can cure inherited and acquired diseases. Clinical research on various AAV serotypes significantly increased in recent years alongside regulatory approvals of AAV-based therapies. The current AAV purification platform hinges on the capture step, for which several affinity resins are commercially available. These adsorbents rely on protein ligands - typically camelid antibodies - that provide high binding capacity and selectivity, but suffer from low biochemical stability and high cost, and impose harsh elution conditions (pH < 3) that can harm the transduction activity of recovered AAVs. Addressing these challenges, this study introduces peptide ligands that selectively capture AAVs and release them under mild conditions (pH 6.0). The peptide sequences were identified by screening a focused library and modeled in silico against AAV serotypes 2 and 9 (AAV2 and AAV9) to select candidate ligands that target homologous sites at the interface of the VP1-VP2 and VP2-VP3 virion proteins with mild binding strength (KD [~] 10- 5-10-6 M). Selected peptides were conjugated to Toyopearl resin and evaluated via binding studies against AAV2 and AAV9, demonstrating the ability to target both serotypes with values of dynamic binding capacity (DBC10% > 1013 vp per mL of resin) and product yields ([~]50-80%) on par with commercial adsorbents. The peptide-based adsorbents were finally utilized to purify AAV2 from a HEK 293 cell lysate, affording high recovery (50-80%), 80-to-400-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 80%) of the purified viruses.

bioengineering↗

bioRxiv2023

Design of 8-mer Peptides that Block Clostridioides difficile Toxin A in Intestinal Cells

Clostridioides difficile (C. diff.) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff. infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a peptide binding design algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a KD of 56.1 {+/-} 29.8 nM as measured by surface plasmon resonance (SPR). Significance StatementInfections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.

bioengineering↗

bioRxiv2021

In vivo genome-wide CRISPR screens identify SOCS1 as a major intrinsic checkpoint of CD4+ Th1 cell response

The expansion of antigen experienced CD4+ T cells is limited by intrinsic factors. Using in vivo genome-wide CRISPR-Cas9 screens, we identified SOCS1 as a non-redundant checkpoint imposing a brake on CD4+ T-cell proliferation upon rechallenge. We show here that SOCS1 is a critical node integrating both IL-2 and IFN-{gamma} signals and blocking multiple signaling pathways to abrogate CD4+ Th1 cell response. In CD8+ T-cell, SOCS1 does not impact the proliferation but rather reduces survival and effector functions. By targeting SOCS1, both murine and human CD4+ T-cell antitumor adoptive therapies exhibit a restored intra-tumor accumulation, proliferation/survival, persistence and polyfunctionality, promoting long term rejection of established tumors. These findings identify SOCS1 as a major intracellular checkpoint inhibitor of primed CD4+ T cells, opening new possibilities to optimize CAR-T cell therapies composition and efficacy.

immunology↗

bioRxiv2020

Use of target-displaying magnetized yeast in screening mRNA display peptide libraries to identify ligands

This work presents the first use of yeast-displayed protein targets for screening mRNA display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of selected WW-YAP-binding peptides: KD values of 37 M and 4 M were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 M and 3 M for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA and yeast display accelerates the discovery of biospecific peptides.

biochemistry↗

bioRxiv2020

Quantitative yeast-yeast two hybrid for discovery and binding affinity estimation of protein-protein interactions

Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two hybrid (qYY2H) system that not only enables discovery of specific protein-protein interactions, but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semi-empirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of the apparent KD of yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.

biochemistry↗

bioRxiv2020

Screening of yeast display libraries of enzymatically-cyclized peptides to discover macrocyclic peptide ligands

We present the construction and screening of yeast display libraries of cyclic peptides wherein site-selective enzymatic cyclization of linear peptides is achieved using bacterial transglu-taminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve cyclization in the endoplasmic reticulum prior to yeast surface display. The cyclization yield was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-cyclized peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified cyclic peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.67 {micro}M for YAP and 0.84 {micro}M for WW as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of yeast surface display for screening transglutaminase-cyclized peptide libraries, and efficient identification of cyclic peptide ligands.

biochemistry↗

bioRxiv2020

Discovery of membrane-permeating cyclic peptides via mRNA display

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

biochemistry↗

bioRxiv2020

Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display

Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups within a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 {micro}M). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative estimates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human interleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identified a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.

synthetic biology↗

bioRxiv2019

Screening yeast display libraries against magnetized yeast cell targets enables efficient isolation of membrane protein binders.

When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is co-expressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder-target pair with modest binding affinity (KD ~ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched >100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ~ 271-2009 nM) and the extracellular domain of the c-Kit receptor (KD ~ 131 to KD >2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion - in contrast to recombinant soluble expression - makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗