Antibody conjugates are the foundation of a wide range of diagnostic and therapeutic applications. Although many antibody-conjugation techniques are robust and efficient, obtaining homogeneous multimeric conjugation products remains challenging. Here we report a modular and versatile technique for the site-directed multivalent conjugation of antibodies via the small-protein ubiquitin. Specifically, multiple ubiquitin fusions with antibodies, antibody fragments, nanobodies, peptides or small molecules such as fluorescent dyes can be conjugated to antibodies and nanobodies within 30 min. The technique, which we named 'ubi-tagging', allowed us to efficiently generate a bispecific T-cell engager as well as nanobodies conjugated to dendritic-cell-targeted antigens that led to potent T-cell responses. Using both recombinant ubi-tagged proteins and synthetic ubiquitin derivatives allows for the iterative, site-directed and multivalent conjugation of antibodies and nanobodies to a plethora of molecular moieties.
© 2025. The Author(s).

Metabolic reprogramming is a hallmark of T-cell activation, and metabolic fitness is fundamental for T-cell-mediated antitumor immunity. Insights into the metabolic plasticity of chimeric antigen receptor (CAR) T cells in patients could help identify approaches to improve their efficacy in treating cancer. Here, we investigated the spatiotemporal immunometabolic adaptation of CD19-targeted CAR T cells using clinical samples from CAR T-cell-treated patients. Context-dependent immunometabolic adaptation of CAR T cells demonstrated the link between their metabolism, activation, differentiation, function, and local microenvironment. Specifically, compared with the peripheral blood, low lipid availability, high IL15, and low TGFβ in the central nervous system microenvironment promoted immunometabolic adaptation of CAR T cells, including upregulation of a lipolytic signature and memory properties. Pharmacologic inhibition of lipolysis in cerebrospinal fluid led to decreased CAR T-cell survival. Furthermore, manufacturing CAR T cells in cerebrospinal fluid enhanced their metabolic fitness and antileukemic activity. Overall, this study elucidates spatiotemporal immunometabolic rewiring of CAR T cells in patients and demonstrates that these adaptations can be exploited to maximize the therapeutic efficacy of CAR T cells.
The spatiotemporal immunometabolic landscape of CD19-targeted CAR T cells from patients reveals metabolic adaptations in specific microenvironments that can be exploited to maximize the therapeutic efficacy of CAR T cells.
©2024 American Association for Cancer Research.

SARS-CoV-2 infects host cells via an ACE2/TMPRSS2 entry mechanism. Monocytes and macrophages, which play a key role during severe COVID-19 express only low or no ACE2, suggesting alternative entry mechanisms in these cells. In silico analyses predicted GRP78, which is constitutively expressed on monocytes and macrophages, to be a potential candidate receptor for SARS-CoV-2 virus entry.
Hospitalized COVID-19 patients were characterized regarding their pro-inflammatory state and cell surface GRP78 (csGRP78) expression in comparison to healthy controls. RNA from CD14+ monocytes of patients and controls were subjected to transcriptome analysis that was specifically complemented by bioinformatic re-analyses of bronchoalveolar lavage fluid (BALF) datasets of COVID-19 patients with a focus on monocyte/macrophage subsets, SARS-CoV-2 infection state as well as GRP78 gene expression. Monocyte and macrophage immunohistocytochemistry on GRP78 was conducted in post-mortem lung tissues. SARS-CoV-2 spike and GRP78 protein interaction was analyzed by surface plasmon resonance, GST Pull-down and Co-Immunoprecipitation. SARS-CoV-2 pseudovirus or single spike protein uptake was quantified in csGRP78high THP-1 cells.
Cytokine patterns, monocyte activation markers and transcriptomic changes indicated typical COVID-19 associated inflammation accompanied by upregulated csGRP78 expression on peripheral blood and lung monocytes/macrophages. Subsequent cell culture experiments confirmed an association between elevated pro-inflammatory cytokine levels and upregulation of csGRP78. Interaction of csGRP78 and SARS-CoV-2 spike protein with a dissociation constant of KD = 55.2 nM was validated in vitro. Infection rate analyses in ACE2low and GRP78high THP-1 cells showed increased uptake of pseudovirus expressing SARS-CoV-2 spike protein.
Our results demonstrate that csGRP78 acts as a receptor for SARS-CoV-2 spike protein to mediate ACE2-independent virus entry into monocytes.
Funded by the Sino-German-Center for Science Promotion (C-0040) and the Germany Ministry BMWi/K [DLR-grant 50WB1931 and RP1920 to AC, DM, TW].
Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

Infection with SARS-CoV-2 can lead to Coronavirus disease-2019 (COVID-19) and result in severe acute respiratory distress syndrome (ARDS). Recent reports indicate an increased rate of fungal coinfections during COVID-19. With incomplete understanding of the pathogenesis and without any causative therapy available, secondary infections may be detrimental to the prognosis. We monitored 11 COVID-19 patients with ARDS for their immune phenotype, plasma cytokines, and clinical parameters on the day of ICU admission and on day 4 and day 7 of their ICU stay. Whole blood stimulation assays with lipopolysaccharide (LPS), heat-killed Listeria monocytogenes (HKLM), Aspergillus fumigatus, and Candida albicans were used to mimic secondary infections, and changes in immune phenotype and cytokine release were assessed. COVID-19 patients displayed an immune phenotype characterized by increased HLA-DR+CD38+ and PD-1+ CD4+ and CD8+ T cells, and elevated CD8+CD244+ lymphocytes, compared to healthy controls. Monocyte activation markers and cytokines IL-6, IL-8, TNF, IL-10, and sIL2Rα were elevated, corresponding to monocyte activation syndrome, while IL-1β levels were low. LPS, HKLM and Aspergillus fumigatus antigen stimulation provoked an immune response that did not differ between COVID-19 patients and healthy controls, while COVID-19 patients showed an attenuated monocyte CD80 upregulation and abrogated release of IL-6, TNF, IL-1α, and IL-1β toward Candida albicans. This study adds further detail to the characterization of the immune response in critically ill COVID-19 patients and hints at an increased susceptibility for Candida albicans infection.
Copyright © 2021 Moser, Biere, Han, Hoerl, Schelling, Choukér and Woehrle.

Chronic exposure to tobacco smoke leads to an increase in the frequency of infections and in the number of CD8(+) and CD4(+) cells as well as the CD4(+) chemoattractant cytokine IL-16 in the airways. Here, we investigated whether tobacco smoke depletes intracellular IL-16 protein and inhibits de novo production of IL-16 in CD8(+) cells from human airways and blood while increasing extracellular IL-16 and whether oxygen free radicals (OFR) are involved. Intracellular IL-16 protein in CD8(+) cells and mRNA in all cells was decreased in bronchoalveolar lavage (BAL) samples from chronic smokers. This was also the case in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro, in which oxidized proteins were markedly increased. Extracellular IL-16 protein was increased in cell-free BAL fluid from chronic smokers and in human blood CD8(+) cells exposed to water-soluble tobacco smoke components in vitro. This was not observed in occasional smokers after short-term exposure to tobacco smoke. A marker of activation (CD69) was slightly increased, whereas other markers of key cellular functions (membrane integrity, apoptosis, and proliferation) in human blood CD8(+) cells in vitro were negatively affected by water-soluble tobacco smoke components. An OFR scavenger prevented these effects, whereas a protein synthesis inhibitor, a β-adrenoceptor, a glucocorticoid receptor agonist, a phosphodiesterase, a calcineurin phosphatase, and a caspase-3 inhibitor did not. In conclusion, tobacco smoke depletes preformed intracellular IL-16 protein, inhibits its de novo synthesis, and distorts key cellular functions in human CD8(+) cells. OFR may play a key role in this context.