Biology subjects

Wagner, T. R.

Publications and source records attributed to Wagner, T. R..

8 recordsLinked to original sources

bioRxiv2025

Conserved programs and specificities of T cells targeting hematological malignancies

T cell-mediated immune surveillance is critical for cancer control, yet its endogenous effectiveness in hematological malignancies remains limited and poorly understood. Here, we integrate single-cell T cell receptor (TCR) profiling, HLA immunopeptidomics and functional antigen mapping to dissect the specificity landscape of bone marrow lymphocytes (BMLs) in multiple myeloma (MM) and acute myeloid leukemia (AML). We identify a rare subset of tumor-reactive T cells that exhibit a stereotyped transcriptional state distinct from bystander and virus-specific populations. Across both malignancies, immunopeptidomic profiling uncovers a partially conserved antigen repertoire enriched for noncanonical peptides, including products of novel or unannotated open reading frames (nuORFs), pseudogenes, and clonotypic immunoglobulin sequences. Several of these epitopes are recurrently presented and associated with convergent TCR responses across individuals. Based on this immune architecture, we develop a TCR-intrinsic fitness model that infers BML tumor specificity from transcriptional cues and stratifies immunotherapy response across three independent patient cohorts. Together, these findings map the latent potential of endogenous anti-tumor immunity in two biologically distinct diseases and provide a framework for decoding and restoring productive immune surveillance of hematological malignancies. HighlightsO_LISingle-cell resolved TCR profiling maps rare tumor-reactive T cells in the bone marrow of multiple myeloma (MM) and acute myeloid leukemia (AML) reveals conserved transcriptional programs C_LIO_LIA shared immunopeptidome across MM and AML includes noncanonical epitopes from nuORFs and idiotype sequences C_LIO_LIConserved tumor antigens elicit convergent T cell responses across patients C_LI O_LIA TCR fitness model predicts tumor specificity in bone marrow lymphocytes and stratifies immunotherapy response in both hematological malignancies C_LI

immunology↗

bioRxiv2024

Making the effect visible - OX40 targeting nanobodies for in vivo imaging of activated T cells

PurposeHuman OX40 (hOX40/CD134), a member of the TNF receptor superfamily, is mainly expressed on activated T lymphocytes. Triggered by its ligand OX40L (CD252), it provides costimulatory signals that support the differentiation, proliferation and long-term survival of T cells. Besides being a relevant therapeutic target, hOX40 is also an important biomarker for monitoring the presence or infiltration of activated T cells within the tumor microenvironment (TME), the inflammatory microenvironment (IME) in immune-mediated diseases (IMIDs) and the lymphatic organs. Here, we developed novel single domain antibodies (nanobodies, Nbs) targeting hOX40 to monitor the activation status of T cells by in vivo molecular imaging. MethodsNbs against hOX40 (hOX40-Nbs) were selected from an immunized Nb-library by phage display. The identified hOX40-Nbs were characterized in vitro, including determination of their specificity, affinity, stability, epitope recognition and their impact on OX40 signaling and T cell function. A lead candidate was site-specifically conjugated with a fluorophore via sortagging and applied for noninvasive in vivo optical imaging (OI) of hOX40-expressing cells in a xenograft mouse model. ResultsOur selection campaign revealed four unique Nbs that exhibit strong binding affinities and high stabilities under physiological conditions. Epitope binning and domain mapping indicated the targeting of at least two different epitopes on hOX40. When analyzing their impact on OX40 signaling, an agonistic effect was excluded for all validated Nbs. Incubation of activated T cells with hOX40-Nbs did not affect cell viability or proliferation patterns, whereas differences in cytokine release were observed. In vivo OI with a fluorophore-conjugated lead candidate in experimental mice with hOX40-expressing xenografts demonstrated its specificity and functionality as an imaging probe. ConclusionConsidering the need for advanced probes for noninvasive in vivo monitoring of T cell activation dynamics, we propose, that our hOX40-Nbs have a great potential as imaging probes for noninvasive and longitudinal in vivo diagnostics. Quantification of OX40+ T cells in TME or IME will provide crucial insights into the activation state of infiltrating T cells, offering a valuable biomarker for assessing immune responses, predicting treatment efficacy, and guiding personalized immunotherapy strategies in patients with cancer or IMIDs.

immunology↗

bioRxiv2023

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

Signal-regulatory protein (SIRP) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRP and the "dont 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 SIRP (hSIRP)-specific nanobodies (Nbs). We identified three high-affinity Nbs targeting the hSIRP/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 hSIRP/hCD47 knock-in (KI) mice, we demonstrated the applicability of 64Cu-hSIRP-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRP-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 individual responses during cancer immunotherapies.

immunology↗

bioRxiv2022

A novel PNGase Rc for improved protein N-deglycosylation in bioanalytics and HDX-MS epitope mapping under challenging conditions

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 {degrees}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 (SIRP).

biochemistry↗

bioRxiv2022

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

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.

biochemistry↗

bioRxiv2021

A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1

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 Parkinsons 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.

cell biology↗

bioRxiv2021

A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern

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.

biochemistry↗

bioRxiv2021

Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

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 64Cu-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 individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

immunology↗