VHHs as tools for therapeutic protein delivery to the central nervous system

Abstract Background The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to...

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Main Authors: Yessica Wouters, Tom Jaspers, Laura Rué, Lutgarde Serneels, Bart De Strooper, Maarten Dewilde
Format: Article
Language:English
Published: BMC 2022-10-01
Series:Fluids and Barriers of the CNS
Subjects:
Online Access:https://doi.org/10.1186/s12987-022-00374-4
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author Yessica Wouters
Tom Jaspers
Laura Rué
Lutgarde Serneels
Bart De Strooper
Maarten Dewilde
author_facet Yessica Wouters
Tom Jaspers
Laura Rué
Lutgarde Serneels
Bart De Strooper
Maarten Dewilde
author_sort Yessica Wouters
collection DOAJ
description Abstract Background The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. Methods Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti–β-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aβ1–40. Results Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aβ1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. Conclusion We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients.
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spelling doaj.art-9416f90f5a1549e39005a8a4bb0b1d6c2022-12-22T02:26:24ZengBMCFluids and Barriers of the CNS2045-81182022-10-0119111310.1186/s12987-022-00374-4VHHs as tools for therapeutic protein delivery to the central nervous systemYessica Wouters0Tom Jaspers1Laura Rué2Lutgarde Serneels3Bart De Strooper4Maarten Dewilde5VIB Center for Brain and Disease ResearchVIB Center for Brain and Disease ResearchVIB Center for Brain and Disease ResearchVIB Center for Brain and Disease ResearchVIB Center for Brain and Disease ResearchVIB Center for Brain and Disease ResearchAbstract Background The blood brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders. Previously we found an anti-mouse transferrin receptor (TfR) VHH (Nb62) that was able to deliver a biologically active neuropeptide into the CNS in mice. Here, we aimed to test its potential to shuttle a therapeutic relevant cargo. Since this VHH could not recognize the human TfR and hence its translational potential is limited, we also aimed to find and validate an anti-human transferrin VHH to deliver a therapeutic cargo into the CNS. Methods Alpaca immunizations with human TfR, and subsequent phage selection and screening for human TfR binding VHHs was performed to find a human TfR specific VHH (Nb188). Its ability to cross the BBB was determined by fusing it to neurotensin, a neuropeptide that reduces body temperature when present in the CNS but is not able to cross the BBB on its own. Next, the anti–β-secretase 1 (BACE1) 1A11 Fab and Nb62 or Nb188 were fused to an Fc domain to generate heterodimeric antibodies (1A11AM-Nb62 and 1A11AM-Nb188). These were then administered intravenously in wild-type mice and in mice in which the murine apical domain of the TfR was replaced by the human apical domain (hAPI KI). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed to assess the concentration of the heterodimeric antibodies in the brain over time and the ability to inhibit brain-specific BACE1 by analysing the brain levels of Aβ1–40. Results Selections and screening of a phage library resulted in the discovery of an anti-human TfR VHH (Nb188). Fusion of Nb188 to neurotensin induced hypothermia after intravenous injections in hAPI KI mice. In addition, systemic administration 1A11AM-Nb62 and 1A11AM-Nb188 fusions were able to reduce Aβ1-40 levels in the brain whereas 1A11AM fused to an irrelevant VHH did not. A PK/PD experiment showed that this effect could last for 3 days. Conclusion We have discovered an anti-human TfR specific VHH that is able to reach the CNS when administered systemically. In addition, both the currently discovered anti-human TfR VHH and the previously identified mouse-specific anti-TfR VHH, are both able to shuttle a therapeutically relevant cargo into the CNS. We suggest the mouse-specific VHH as a valuable research tool in mice and the human-specific VHH as a moiety to enhance the delivery efficiency of therapeutics into the CNS in human patients.https://doi.org/10.1186/s12987-022-00374-4NanobodyVHHTransferrin receptorNeurotensinBlood–brain barrierReceptor-mediated transcytosis
spellingShingle Yessica Wouters
Tom Jaspers
Laura Rué
Lutgarde Serneels
Bart De Strooper
Maarten Dewilde
VHHs as tools for therapeutic protein delivery to the central nervous system
Fluids and Barriers of the CNS
Nanobody
VHH
Transferrin receptor
Neurotensin
Blood–brain barrier
Receptor-mediated transcytosis
title VHHs as tools for therapeutic protein delivery to the central nervous system
title_full VHHs as tools for therapeutic protein delivery to the central nervous system
title_fullStr VHHs as tools for therapeutic protein delivery to the central nervous system
title_full_unstemmed VHHs as tools for therapeutic protein delivery to the central nervous system
title_short VHHs as tools for therapeutic protein delivery to the central nervous system
title_sort vhhs as tools for therapeutic protein delivery to the central nervous system
topic Nanobody
VHH
Transferrin receptor
Neurotensin
Blood–brain barrier
Receptor-mediated transcytosis
url https://doi.org/10.1186/s12987-022-00374-4
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