ORCID Profile
0000-0002-1050-5948
Current Organisation
Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen
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Publisher: Cold Spring Harbor Laboratory
Date: 09-08-2021
DOI: 10.1101/2021.08.08.455562
Abstract: The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo , human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.
Publisher: Cold Spring Harbor Laboratory
Date: 29-06-2023
DOI: 10.1101/2023.06.27.546763
Abstract: Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don’t eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified three high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis (ADCP). For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography (PET) in novel hSIRPα/hCD47 knock-in (KI) mice, we demonstrated the applicability of 64 Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of in idual responses during cancer immunotherapies.
Publisher: EMBO
Date: 20-12-2021
Publisher: Springer Science and Business Media LLC
Date: 03-05-2022
DOI: 10.1038/S41598-022-10987-2
Abstract: As global vaccination c aigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum s les from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly in idualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.
Publisher: Cold Spring Harbor Laboratory
Date: 04-07-2021
DOI: 10.1101/2021.07.02.450848
Abstract: The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4 + T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processes in vitro and converted them into immune tracers for non-invasive molecular imaging. By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4 + cells in vivo using a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of 64 Cu-radiolabeled CD4-Nb in CD4 + T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring in idual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.
Publisher: American Chemical Society (ACS)
Date: 18-08-2021
Publisher: Elsevier BV
Date: 09-2022
Publisher: Elsevier BV
Date: 11-2021
Publisher: Cold Spring Harbor Laboratory
Date: 10-12-2021
DOI: 10.1101/2021.12.10.472061
Abstract: The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time- lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.
Publisher: American Chemical Society (ACS)
Date: 24-06-2022
DOI: 10.1021/ACS.ANALCHEM.2C01748
Abstract: N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide
Publisher: American Society of Hematology
Date: 09-06-2022
Abstract: Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT–Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT–Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made.
Publisher: Cold Spring Harbor Laboratory
Date: 13-04-2022
DOI: 10.1101/2022.04.13.488165
Abstract: N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry (LC-MS) and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quench conditions (pH 2.5, 0 °C) and in the presence of 0.4 M Tris(2-carboxyethyl)phosphine (TCEP), 4 M urea and 1 M guanidinium chloride (GdmCl). Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).
Publisher: Frontiers Media SA
Date: 09-12-2021
DOI: 10.3389/FIMMU.2021.799910
Abstract: The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4 + cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4 + cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64 Cu-radiolabeled CD4-Nb1 in CD4 + T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring in idual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.
Publisher: Oxford University Press (OUP)
Date: 15-07-2022
DOI: 10.1093/CID/CIAC498
Abstract: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated in iduals showed a substantial reduction. We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated in iduals. While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated s les, antibody binding to Omicron only improved after administration of a third dose. Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.
Publisher: Cold Spring Harbor Laboratory
Date: 22-08-2021
DOI: 10.1101/2021.08.20.21262328
Abstract: As global vaccination c aigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum s les from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly in idualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.
Publisher: Cold Spring Harbor Laboratory
Date: 12-04-2022
DOI: 10.1101/2022.04.12.488002
Abstract: The tumor microenvironment (TME) consists of different cell types that secrete proteins and also control the extracellular concentration of ions and metabolites. Changes in these intra-tumoral analytes and conditions, including K + , glucose, and pH, have been described to alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. However, the mechanisms regulating ion and metabolite levels and their effects on certain characteristics of the TME are still poorly understood. Therefore, accurate determination and visualization of analyte or state changes in real time within the TME is desired. In this study, we genetically combined FRET-based fluorescent biosensors with nanobodies (Nbs) and used them for targeted visualization and monitoring of extracellular changes in K + , pH, and glucose on cell surfaces. We demonstrated that these recombinant biosensors quantitatively visualize extracellular K + alterations on multiple cancer and non-cancer cell lines and primary neurons. By implementing a HER2 specific Nb, we generated K + and pH sensors, which retain their functionality and specifically stained HER2 positive breast cancer cells. Based on the successful technical development of several Nb-biosensor combinations, we anticipate that this approach can be easily extended to design other targeted biosensors. Such versatile probes will open new possibilities for the reliable study of extracellular analytes in advanced 3D cell models or even in vivo systems.
Publisher: MDPI AG
Date: 21-12-2020
DOI: 10.3390/BIOM10121701
Abstract: In biomedical research, there is an ongoing demand for new technologies to elucidate disease mechanisms and develop novel therapeutics. This requires comprehensive understanding of cellular processes and their pathophysiology based on reliable information on abundance, localization, post-translational modifications and dynamic interactions of cellular components. Traceable intracellular binding molecules provide new opportunities for real-time cellular diagnostics. Most prominently, intrabodies derived from antibody fragments of heavy-chain only antibodies of camelids (nanobodies) have emerged as highly versatile and attractive probes to study and manipulate antigens within the context of living cells. In this review, we provide an overview on the selection, delivery and usage of intrabodies to visualize and monitor cellular antigens in living cells and organisms. Additionally, we summarize recent advances in the development of intrabodies as cellular biosensors and their application to manipulate disease-related cellular processes. Finally, we highlight switchable intrabodies, which open entirely new possibilities for real-time cell-based diagnostics including live-cell imaging, target validation and generation of precisely controllable binding reagents for future therapeutic applications.
Publisher: Springer Science and Business Media LLC
Date: 25-05-2021
DOI: 10.1038/S41467-021-23473-6
Abstract: SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva s les from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected in iduals and characterize the antibody response to RBD mutant strains. Vaccinated in iduals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated in iduals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.
Publisher: Frontiers Media SA
Date: 10-03-2022
DOI: 10.3389/FMOLB.2022.835302
Abstract: The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.
Publisher: EMBO
Date: 27-04-2021
Location: United States of America
Location: Germany
No related grants have been discovered for Teresa R. Wagner.