Development and Validation of Novel VHHs Targeting Astrocyte-Enriched Glutamate Transporters GLT-1 and GLAST-1.
4 affiliations
- 1Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, 3000 Leuven, Belgium.
- 2VIB-KU Leuven Center for Neuroscience, 3000 Leuven, Belgium.
- 3Department of Neurosciences, KU Leuven, 3000 Leuven, Belgium.
- 4PharmAbs-The KU Leuven Antibody Center, KU Leuven, 3000 Leuven, Belgium.
Abstract
Background/objectives
VHHs (Nanobodies®) are camelid single-domain antibody fragments with broad applicability in central nervous system (CNS) research, including blood-brain barrier transport, modulation of pathological proteins and molecular detection. However, few VHHs have been developed for cell-type-specific targeting within the brain. Astrocytes play essential roles in CNS homeostasis and contribute to many neurological disorders, making them an appealing target for diagnostic and therapeutic approaches. Here, we report the development and validation of the first VHHs directed against the astrocyte-enriched glutamate transporters GLT-1 and GLAST-1.Methods
VHHs were generated from a camelid immune library using phage display. Target specificity and affinity were evaluated in cells overexpressing the mouse and human transporters, as well as in iPSC-derived astrocytes. Target engagement and astrocyte specificity was further assessed ex vivo on brain sections using immunohistochemistry and in dissociated mouse brain cell suspensions by flow cytometry. VHH in vivo binding was evaluated by stereotactic injections within the brain striatum.Results
One anti-GLT-1 VHH and four anti-GLAST-1 VHHs demonstrated high specificity and apparent nanomolar affinity when binding tocells overexpressing the mouse transporters. The anti-GLT-1 VHH was additionally cross-reactive with the human ortholog, as demonstrated in overexpressing cells and iPSC-derived astrocytes. All VHHs showed target engagement on mouse brain sections and astrocyte specificity in dissociated mouse brain cell suspensions. VHH direct brain delivery resulted in localized transporter-specific signal around the injection site, whereas the non-binding isotype control VHH produced no detectable signal.Conclusions
Here we present the first set of VHHs capable of selectively recognizing astrocyte-enriched glutamate transporters GLT-1 and GLAST-1 in vitro, ex vivo, and in vivo. These VHHs constitute promising tools for astrocyte detection in research applications and the cross-reactive anti-GLT-1 lead might hold potential for future diagnostic or therapeutic strategies, including cell-targeted payload delivery.
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BibTeX
@article{gesteiraperez2026development,
title = {{Development and Validation of Novel VHHs Targeting Astrocyte-Enriched Glutamate Transporters GLT-1 and GLAST-1.}},
author = {Noelia Gesteira-Pérez and Pieterjan Van Maele and Benedetta Frizzi and Ludo Van Den Bosch and Laura Rué and Maarten Dewilde},
journal = {Antibodies (Basel, Switzerland)},
year = {2026},
month = sep,
doi = {10.3390/antib15050089},
url = {https://europepmc.org/article/MED/42930031},
note = {CC BY},
}
RIS
TY - JOUR
TI - Development and Validation of Novel VHHs Targeting Astrocyte-Enriched Glutamate Transporters GLT-1 and GLAST-1.
AU - Noelia Gesteira-Pérez
AU - Pieterjan Van Maele
AU - Benedetta Frizzi
AU - Ludo Van Den Bosch
AU - Laura Rué
AU - Maarten Dewilde
PY - 2026
DA - 2026/09/29
JO - Antibodies (Basel, Switzerland)
DO - 10.3390/antib15050089
UR - https://europepmc.org/article/MED/42930031
AB - Background/objectives VHHs (Nanobodies®) are camelid single-domain antibody fragments with broad applicability in central nervous system (CNS) research, including blood-brain barrier transport, modulation of pathological proteins and molecular detection. However, few VHHs have been developed for cell-type-specific targeting within the brain. Astrocytes play essential roles in CNS homeostasis and contribute to many neurological disorders, making them an appealing target for diagnostic and therapeutic approaches. Here, we report the development and validation of the first VHHs directed against the astrocyte-enriched glutamate transporters GLT-1 and GLAST-1.Methods VHHs were generated from a camelid immune library using phage display. Target specificity and affinity were evaluated in cells overexpressing the mouse and human transporters, as well as in iPSC-derived astrocytes. Target engagement and astrocyte specificity was further assessed ex vivo on brain sections using immunohistochemistry and in dissociated mouse brain cell suspensions by flow cytometry. VHH in vivo binding was evaluated by stereotactic injections within the brain striatum.Results One anti-GLT-1 VHH and four anti-GLAST-1 VHHs demonstrated high specificity and apparent nanomolar affinity when binding tocells overexpressing the mouse transporters. The anti-GLT-1 VHH was additionally cross-reactive with the human ortholog, as demonstrated in overexpressing cells and iPSC-derived astrocytes. All VHHs showed target engagement on mouse brain sections and astrocyte specificity in dissociated mouse brain cell suspensions. VHH direct brain delivery resulted in localized transporter-specific signal around the injection site, whereas the non-binding isotype control VHH produced no detectable signal.Conclusions Here we present the first set of VHHs capable of selectively recognizing astrocyte-enriched glutamate transporters GLT-1 and GLAST-1 in vitro, ex vivo, and in vivo. These VHHs constitute promising tools for astrocyte detection in research applications and the cross-reactive anti-GLT-1 lead might hold potential for future diagnostic or therapeutic strategies, including cell-targeted payload delivery.
SN - 2073-4468
ER -