Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

Jensen, Rasmus K.; Pedersen, Henrik; Lorentzen, Josefine; Laursen, N.S.; Vorup-Jensen, Thomas; Andersen, Gregers Rom

doi:10.1016/j.jbc.2022.102168

Cited by 7 publications

(8 citation statements)

References 63 publications

(117 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…: L00350, Genscript). Endotoxin levels below 2 EU·mg −1 nanobodies were considered to be endotoxin free [ 26 ]. Human complement component C3b (Cat.No.…”

Section: Methodsmentioning

confidence: 99%

Filtration and tubular handling of EWE‐hC3Nb1, a complement inhibitor nanobody, in wild type mice and a mouse model of proteinuric kidney disease

Fast,

Weyer,

Pedersen

et al. 2024

FEBS Open Bio

Self Cite

View full text Add to dashboard Cite

Tubular activation and deposition of filtered complement proteins have been implicated in the progression of proteinuric kidney disease. The potent C3b‐specific nanobody inhibitor of the alternative pathway, EWE‐hC3Nb1, is likely freely filtered in the glomerulus to allow complement inhibition in the tubular lumen and may provide a novel treatment option to prevent tubulointerstitial injury. However, more information on the pharmacokinetic properties and renal tubular handling of EWE‐hC3Nb1 nanobody is required for its pharmacological application in relation to kidney disease. Here, we examined the pharmacokinetic properties of free EWE‐hC3Nb1 in mouse plasma and urine, following subcutaneous injection in wild‐type control and podocin knock out (KO) mice with severe proteinuria. Tubular handling of filtered EWE‐hC3Nb1 was assessed by immunohistochemistry (IHC) on kidney tissue from control, proteinuric mice, and KO mice deficient in the proximal tubule endocytic receptor megalin. Rapid plasma absorption and elimination of EWE‐hC3Nb1 was observed in both control and proteinuric mice; however, urinary excretion of EWE‐hC3Nb1 was markedly increased in proteinuric mice. Urinary EWE‐hC3Nb1 excretion was amplified in megalin KO mice, and substantial accumulation of EWE‐hC3Nb1 was observed in megalin‐expressing renal proximal tubules by IHC. Moreover, free EWE‐hC3Nb1 was found to be rapidly cleared from plasma. In conclusion, filtered EWE‐hC3Nb1 is reabsorbed by a megalin‐dependent process in the proximal tubules. Increased load of filtered proteins in the tubular fluid may inhibit the megalin‐dependent uptake of EWE‐hC3Nb1 in proteinuric mice. Treatment with EWE‐hC3Nb1 may allow investigation of the effects of complement inhibition in the tubular fluid.

show abstract

“…: L00350, Genscript). Endotoxin levels below 2 EU·mg −1 nanobodies were considered to be endotoxin free [ 26 ]. Human complement component C3b (Cat.No.…”

Section: Methodsmentioning

confidence: 99%

Filtration and tubular handling of EWE‐hC3Nb1, a complement inhibitor nanobody, in wild type mice and a mouse model of proteinuric kidney disease

Fast,

Weyer,

Pedersen

et al. 2024

FEBS Open Bio

Self Cite

View full text Add to dashboard Cite

show abstract

“…RAW264.7 cells were incubated with LPS-buffer, Dulbecco’s modified Eagle’s medium containing 1 µg/mL LPS (L4391) (Merck), in a humidified 5% CO 2 incubator at 37 °C for 4 h. Thereafter, cells were topped up with brefeldin A-buffer, Dulbecco’s modified Eagle’s medium supplemented with 300 ng/mL brefeldin A (Abcam, UK), and placed at 37 °C with a constant supply of 5% CO 2 for 3 h. Stock solutions of Nb1, Nb3, Nb9 and non-targeting Nb (1000 nM) were prepared in Dulbecco’s modified Eagle’s medium and added to the appropriate wells. The anti-inflammatory effects of Panx1-targeting nanobodies were tested by treating RAW264.7 cells with a stock solution of 1000 nM nanobody solution, which is the quantity of nanobody that is commonly applied in in vitro functionality assays [ 19 – 22 ]. After an incubation period of 1 h, RAW264.7 cells were taken out the incubator and topped up with ATP-buffer, Dulbecco’s modified Eagle’s medium containing 5 mM ATP (Thermo Fisher Scientific), for a final incubation step of 30 min at 37 °C with a constant supply of 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury

Van Campenhout,

De Groof,

Kadam

et al. 2023

J Nanobiotechnol

View full text Add to dashboard Cite

Background The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. Results Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. Conclusions This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes. Graphical Abstract

show abstract

“…Native C3 was purified from plasma as described (16). C3 methylamine (C3MA) was generated from freshly purified native C3 by treatment with 100 mM methylamine at pH 8.5, and C3b was generated from native C3 by trypsin cleavage as described (22), whereas iC3b was generated as described in (23). Rat C3 was purified from rat EDTA plasma generously provided by Emma Faddy (Department of Infectious Diseases, Aarhus University).…”

Section: Protein Purificationmentioning

confidence: 99%

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

et al. 2022

Self Cite

View full text Add to dashboard Cite

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

show abstract

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

Cited by 7 publications

References 63 publications

Filtration and tubular handling of EWE‐hC3Nb1, a complement inhibitor nanobody, in wild type mice and a mouse model of proteinuric kidney disease

Filtration and tubular handling of EWE‐hC3Nb1, a complement inhibitor nanobody, in wild type mice and a mouse model of proteinuric kidney disease

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Contact Info

Product

Resources

About