Biology subjects

Seeberger, P. H.

Publications and source records attributed to Seeberger, P. H..

11 recordsLinked to original sources

bioRxiv2025

Cysteine-Engineered CAR-T Cells to Counter Antigen Escape in B Cell Lymphoma

Chimeric Antigen Receptor (CAR-) T cell therapy represents a paradigm shift in immunotherapy of hematological cancers. However, selective pressure on cancer cells often leads to suppression of target antigens, eventually causing cancer relapse1,2. This so-called antigen escape renders CAR-T cells ineffective, posing a significant clinical challenge2-5. Therefore, identifying alternative targets less susceptible to antigen escape is crucial. Here, we describe a novel type of CAR-T cells utilizing cysteine-engineered antibody fragments that target altered redox states on the surface of B cell lymphoma (BCL)6. We demonstrate that cysteine-engineered CAR-T cells exhibit specific cytotoxicity in vitro against various BCL subtypes, including antigen escape models. Additionally, we show that cysteine engineering, achieved through single amino acid substitution in the state-of-the-art anti-CD19-CAR, enables co-targeting of both CD19-positive and -negative BCL. Our findings introduce a novel class of bifunctional CAR-T cells that target conventional antigens and altered redox states simultaneously, potentially reducing the risk of antigen escape. Abnormal redox states occur in several cancers, including breast and leukemia7-12, indicating a broad therapeutic scope.

cancer biology↗

bioRxiv2024

Two-Photon NAD(P)H-FLIM reveals unperturbed energy metabolism of Ascaris suum larvae, in contrast to host macrophages upon artemisinin derivatives exposure

Soil-transmitted helminths (STH) are widespread, with Ascaris lumbricoides infecting millions globally. Malaria and STH co-infections are common in co-endemic regions. Artemisinin derivatives (ARTs)--artesunate, artemether, and dihydroartemisinin--are standard malaria treatments and are also known to influence the energy metabolism of parasites, tumors, and immune cells. Herein, we explore the potential of ARTs to influence ascariasis either by directly affecting larvae or indirectly by modifying macrophage responses. Ascaris suum third-stage larvae and porcine IL-4 polarised (M2-like) macrophages were exposed to ARTs in vitro, and their metabolism was evaluated using two-photon NAD(P)H-FLIM. Both larvae and M2-like macrophages exhibited a steady-state bioenergetic profile of high oxidative phosphorylation and low anaerobic glycolysis. In A. suum larvae, two metabolically distinct regions were identified, with particularly high DUOX activity in the pharynx compared to the midgut. The metabolic profile of both larval regions were, however, unperturbed by ARTs exposure. In contrast, exposure of M2-like macrophages to ARTs induced a metabolic shift towards high anaerobic glycolysis and reduced metabolic activity, suggesting a possible indirect effect of ARTs on the helminth infection. Overall, two-photon NAD(P)H-FLIM proved to be a powerful tool for studying specific metabolic pathways in Ascaris larvae and host macrophages, offering valuable insights into the metabolic mechanisms of drug action on both parasite and host.

biophysics↗

bioRxiv2024

Polyelectrolyte mannan from diatoms reshapes sunlit ocean microbiome

Diatoms are a keystone phylum in Earths ecosystems, specializing in oxygen production and carbohydrate fixation that fuels global food webs. Diatoms host a microbiome, but how they preferentially collect bacteria with complementary traits remains unknown. Here we show that diatoms exude a C6-sulfated -1,3-mannan that serves as a selective carbon source for adapted bacteria. Its structure was resolved by NMR spectroscopy, chromatography, chemical synthesis, and enzymatic dissection. Biochemical, physiological, and structural analyses revealed that specialized Bacteroidota employ a four-enzyme pathway to metabolize this mannan. Metagenomic and transcriptomic data indicate that the mannan globally selects for bacteria carrying these enzymes and associated traits. Because the mannan provides only carbon, oxygen, sulfur, and hydrogen, bacteria must obtain other essential elements from alternative sources, reinforcing metabolic interdependence. We propose that diatoms use sulfated mannans to attract beneficial partners and exclude competitors, thereby engineering a microbiome that enhances their productivity and underpins carbon cycling. Significance statementEukaryotes host microbial partners that shape their health, yet how they selectively assemble beneficial microbes remains unclear. Using diatom microalgae as a model, we show they exude a sulfated mannan that nourishes highly adapted bacteria tracking them across the global ocean. Our findings suggest that single-celled eukaryotes can "domesticate" prokaryotes--analogous to how humans have domesticated animals--albeit on a microscopic scale. Dominating much of Earths aquatic surface, diatoms drive [~]20% of global photosynthesis. We propose that sulfated mannan contributes to this success by helping diatoms shape microbial partnerships that underpin planetary energy balance and atmospheric chemistry.

ecology↗

bioRxiv2022

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/497993v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f47ba9org.highwire.dtl.DTLVardef@1e2bd41org.highwire.dtl.DTLVardef@f7151eorg.highwire.dtl.DTLVardef@18b7681_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

cancer biology↗

bioRxiv2021

Better together - Relative retention time plus spectral matching improves automated glycan characterization using PGC-nLC-IT-ESI-MS/MS

Porous Graphitized Carbon nano-liquid chromatography tandem mass spectrometry (PGC-nLC-MS/MS) is a glycomics technique with the unique capacity to differentiate isobaric glycans. The lack of suitable software tools integrating chromatography and MS-information delivered by PGC-nLC-MS/MS has been limiting fast and robust glycan identification and quantitation. We report a LC-system-independent strategy called GlycoRRT that combines relative retention time (RRT) and negative ion fragment spectra analyses for isobaric structure-specific glycomics of PGC-nLC-MS/MS data. The GlycoRRT toolset is fully customizable and easily adaptable enabling semi-automated high-throughput structural assignments. The current library contains over 200 entries and their individual meta-data (MS instrumentation, experimental conditions, retention times, fragmentation profiles and glycan structural diagnostic ion features) relevant for reliable data analyses. The GlycoRRT workflow was employed to map the N- and O-glycome in blood group matched human plasma and urine as well as decipher Immunoglobulin (IgG) glycosylation features from 13 different animal species. We have also developed visualization tools to enable a consistent, reliable, and reproducible analysis of large sets of multidimensional PGC-nLC-MS/MS glycomics data. This comprehensive glycan resource provides the glycan map of human and animal species, will serve as a reference in dissecting the role of glycans in host pathogen interaction and zoonotic disease transmission.

biochemistry↗

bioRxiv2021

A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae

Streptococcus pneumoniae infections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combining S.pneumoniae proteins, pneumolysin and PspA, with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibit colonization of the nasopharynx, decrease the bacterial load and reduce disease severity in the bacteria challenged model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective ("universal") pneumococcal vaccines.

immunology↗

bioRxiv2021

CLPTM1L is a lipid scramblase involved in glycosylphosphatidylinositol biosynthesis

Glycosylphosphatidylinositols (GPIs) are membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) and the second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the lumenal face for later biosynthetic steps and attachment to proteins. The mechanism of the lumenal translocation of GlcN-PI is unclear. We report that Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L), an ER membrane protein of unknown function, is a lipid scramblase involved in GPI biosynthesis. Purified CLPTM1L scrambles GlcN-PI, PI, and several other phospholipids in vitro. Knockout of CLPTM1L gene in mammalian cultured cells partially decreased GPI-anchored proteins due to impaired usage of GlcN-PI, suggesting a major role of CLPTM1L in lumenal translocation of GlcN-PI. One-Sentence SummaryCLPTM1L translocates glucosaminyl-phosphatidylinositol across the membrane during glycosylphosphatidylinositol biosynthesis.

biochemistry↗

bioRxiv2021

In vitro efficacy of Artemisia extracts against SARS-CoV-2

Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent and fight COVID-19 infections. We sought to study the ability of different A. annua and A. afra extracts and the Covid-Organics drink produced in Madagascar to inhibit SARS-CoV-2 and feline coronavirus (FCoV) replication in vitro. Several extracts as well as Covid-Organics inhibit SARS-CoV-2 and FCoV replication at concentrations that did not affect cell viability. It remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral replication following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment in patients.

pharmacology and toxicology↗

bioRxiv2020

In vitro efficacy of Artemisinin-based treatments against SARS-CoV-2

Effective and affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are needed. We report in vitro efficacy of Artemisia annua extracts as well as artemisinin, artesunate, and artemether against SARS-CoV-2. The latter two are approved active pharmaceutical ingredients of anti-malarial drugs. Proof-of-concept for prophylactic efficacy of the extracts was obtained using a plaque-reduction assay in VeroE6 cells. Subsequent concentration-response studies using a high-throughput antiviral assay, based on immunostaining of SARS-CoV-2 spike glycoprotein, revealed that pretreatment and treatment with extracts, artemisinin, and artesunate inhibited SARS-CoV-2 infection of VeroE6 cells. In treatment assays, artesunate (50% effective concentration (EC50): 7 g/mL) was more potent than the tested plant extracts (128-260 g/mL) or artemisinin (151 g/mL) and artemether (>179 g/mL), while generally EC50 in pretreatment assays were slightly higher. The selectivity index (SI), calculated based on treatment and cell viability assays, was highest for artemisinin (54), and roughly equal for the extracts (5-10), artesunate (6) and artemether (<7). Similar results were obtained in human hepatoma Huh7.5 cells. Peak plasma concentrations of artesunate exceeding EC50 values can be achieved. Clinical studies are required to further evaluate the utility of these compounds as COVID-19 treatment.

pharmacology and toxicology↗

bioRxiv2019

Glycan-dependent two-step cell adhesion mechanism of Tc toxins

Toxin complex (Tc) toxins are virulence factors widespread in insect and human bacterial pathogens. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and prepore-to-pore transition have been well-described, little is known about receptor binding and cellular uptake of Tcs. Here, we identify two classes of glycans, heparins/heparan sulfates and Lewis antigens, that act as receptors for different TcAs from insect- and human pathogenic bacteria. Glycan array screening and electron cryo microscopy (cryo-EM) structures reveal that all tested TcAs bind unexpectedly with their -helical part of the shell domain to negatively charged heparins. In addition, TcdA1 from the insect-pathogen Photorhabdus luminescens binds to Lewis antigens with micromolar affinity. A cryo-EM structure of the TcdA1-Lewis X complex reveals that the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a two-step association mechanism of Tc toxins involving glycans on the surface of host cells.

molecular biology↗

bioRxiv2019

A novel, ultrasensitive approach for quantitative carbohydrate composition and linkage analysis using LC-ESI ion trap tandem mass spectrometry

Glycan identification and characterisation is essential to correlate glycoconjugate structure to biological function. The structural assignment of carbohydrates is often based on MS composition analyses and knowledge on well-studied glycosylation pathways. Nevertheless, many monosaccharide building blocks are indistinguishable by mass alone and detailed linkage information is also not easily obtained by MS/MS analyses, in particular when organisms are studied where the glycosylation pathways are less well defined. Here, we present a novel, simple and sensitive method using Reversed Phase (RP) - Liquid Chromatography Electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) for unambiguous identification and linkage determination of monosaccharides including N-acetylneuraminic acids. Sequential permethylation and reductive amination steps are employed prior and after acid hydrolysis to enable separation and differentiation of the various monosaccharides and their respective linkage positions. The well-established, monosaccharide specific methylation patterns allowed for the identification of the various derivatised monosaccharide alditols based on their retention time and tandem mass spectrometry fingerprint. Absolute quantitation can also be accomplished by including a set of internal standards, thus simultaneously providing qualitative and quantitative information on the monosaccharide residues present.

biochemistry↗