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Article: Exploring the Fate of Antibody-Encoding pDNA after Intramuscular Electroporation in Mice.

Cuypers, Marie-Lynn / Geukens, Nick / Hollevoet, Kevin / Declerck, Paul / Dewilde, Maarten

2023 Volume 15, Issue 4

Abstract: DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the ... ...

Abstract	DNA-based antibody therapy seeks to administer the encoding nucleotide sequence rather than the antibody protein. To further improve the in vivo monoclonal antibody (mAb) expression, a better understanding of what happens after the administration of the encoding plasmid DNA (pDNA) is required. This study reports the quantitative evaluation and localization of the administered pDNA over time and its association with corresponding mRNA levels and systemic protein concentrations. pDNA encoding the murine anti-HER2 4D5 mAb was administered to BALB/c mice via intramuscular injection followed by electroporation. Muscle biopsies and blood samples were taken at different time points (up to 3 months). In muscle, pDNA levels decreased 90% between 24 h and one week post treatment (
Language	English
Publishing date	2023-04-06
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527217-2
ISSN	1999-4923
ISSN	1999-4923
DOI	10.3390/pharmaceutics15041160
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Article ; Online: VHHs as tools for therapeutic protein delivery to the central nervous system.

Wouters, Yessica / Jaspers, Tom / Rué, Laura / Serneels, Lutgarde / De Strooper, Bart / Dewilde, Maarten

Fluids and barriers of the CNS

2022 Volume 19, Issue 1, Page(s) 79

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 ... ...

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 Aβ 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 Aβ 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.
MeSH term(s)	Amyloid Precursor Protein Secretases ; Animals ; Antibodies/metabolism ; Aspartic Acid Endopeptidases/metabolism ; Blood-Brain Barrier/metabolism ; Humans ; Mice ; Neurotensin ; Receptors, Transferrin ; Transferrin/metabolism
Chemical Substances	Antibodies ; Receptors, Transferrin ; Transferrin ; Neurotensin (39379-15-2) ; Amyloid Precursor Protein Secretases (EC 3.4.-) ; Aspartic Acid Endopeptidases (EC 3.4.23.-)
Language	English
Publishing date	2022-10-03
Publishing country	England
Document type	Journal Article
ZDB-ID	2595406-4
ISSN	2045-8118 ; 2045-8118
ISSN (online)	2045-8118
ISSN	2045-8118
DOI	10.1186/s12987-022-00374-4
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Article: Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics.

Rué, Laura / Jaspers, Tom / Degors, Isabelle M S / Noppen, Sam / Schols, Dominique / De Strooper, Bart / Dewilde, Maarten

Pharmaceutics

2023 Volume 15, Issue 6

Abstract: The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. ... ...

Abstract	The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aβ
Language	English
Publishing date	2023-06-16
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527217-2
ISSN	1999-4923
ISSN	1999-4923
DOI	10.3390/pharmaceutics15061748
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Article: Applicability of cerebral open flow microperfusion and microdialysis to quantify a brain-penetrating nanobody in mice

Custers, Marie-Laure / Wouters, Yessica / Jaspers, Tom / De Bundel, Dimitri / Dewilde, Maarten / Van Eeckhaut, Ann / Smolders, Ilse

Analytica chimica acta. 2021 Sept. 15, v. 1178

2021

Abstract: The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier ( ...

Abstract	The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier (BBB) severely hampers biologics from entering the brain. Considerable effort has been put into generating methods to modulate or circumvent the BBB for delivery of therapeutics to the CNS. A promising strategy is receptor-mediated transcytosis (RMT). Recently, Wouters et al. (2020) discovered a mouse anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via RMT. The present study aims to sample a derivative of this brain-penetrating nanobody (Nb105) in the CNS. Therefore, we compared the applicability of cerebral open flow microperfusion (cOFM) and microdialysis as sampling techniques to directly obtain high molecular weight substances from the cerebral interstitial fluid. A custom AlphaScreen™ assay was validated to quantify nanobody concentrations in the samples. In vitro microdialysis probe (AtmosLM™, 1 MDa cut-off) recovery by gain and by loss for Nb105 was 18.3 ± 3.2% and 27.0 ± 2.5% respectively, whereas for cOFM it was 87.2 ± 4.0% and 97.3 ± 1.6%. Although a large difference in in vitro recovery is observed between cOFM and microdialysis, in vivo similar results were obtained. Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher fluorescein isothiocyanate-dextran and immunoglobulin G extravasation around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. This is the first study that samples a bispecific nanobody in the brain's interstitial fluid in function of time, providing a pharmacokinetic profile of nanobodies in the CNS. Furthermore, this is the first time a cOFM study is performed in awake freely moving mice, providing data on inflammation and blood-brain barrier integrity in the mouse brain. Overall, this work demonstrates that, while taking into account the (bio)analytical considerations, both microdialysis and cOFM are suitable in vivo sampling techniques for quantification of nanobodies in the CNS.
Keywords	blood-brain barrier ; brain ; fluorescein ; immunoglobulin G ; immunohistochemistry ; inflammation ; landscapes ; mice ; microdialysis ; molecular weight ; peptides ; pharmacokinetics ; physiological transport ; therapeutics
Language	English
Dates of publication	2021-0915
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	1483436-4
ISSN	1873-4324 ; 0003-2670
ISSN (online)	1873-4324
ISSN	0003-2670
DOI	10.1016/j.aca.2021.338803
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Article ; Online: Selective inhibitors of the PSEN1-gamma-secretase complex.

Serneels, Lutgarde / Narlawar, Rajeshwar / Perez-Benito, Laura / Municoy, Marti / Guallar, Victor / T'Syen, Dries / Dewilde, Maarten / Bischoff, François / Fraiponts, Erwin / Tresadern, Gary / Roevens, Peter W M / Gijsen, Harrie J M / De Strooper, Bart

The Journal of biological chemistry

2023 Volume 299, Issue 6, Page(s) 104794

Abstract: Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III ... ...

Abstract	Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.
MeSH term(s)	Humans ; Alzheimer Disease/drug therapy ; Alzheimer Disease/enzymology ; Alzheimer Disease/metabolism ; Amyloid Precursor Protein Secretases/antagonists & inhibitors ; Amyloid Precursor Protein Secretases/metabolism ; Presenilin-1/antagonists & inhibitors ; Presenilin-1/metabolism ; Multiprotein Complexes/antagonists & inhibitors ; Multiprotein Complexes/metabolism ; Sulfonamides/pharmacology ; Substrate Specificity ; Neoplasms/drug therapy ; Neoplasms/enzymology ; Neoplasms/metabolism
Chemical Substances	Amyloid Precursor Protein Secretases (EC 3.4.-) ; Presenilin-1 ; PSEN1 protein, human ; PSEN2 protein, human ; APH1A protein, human (EC 3.4.-) ; APH1B protein, human (EC 3.4.-) ; Multiprotein Complexes ; Sulfonamides
Language	English
Publishing date	2023-05-09
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2023.104794
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Article ; Online: Applicability of cerebral open flow microperfusion and microdialysis to quantify a brain-penetrating nanobody in mice.

Custers, Marie-Laure / Wouters, Yessica / Jaspers, Tom / De Bundel, Dimitri / Dewilde, Maarten / Van Eeckhaut, Ann / Smolders, Ilse

Analytica chimica acta

2021 Volume 1178, Page(s) 338803

Abstract	The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier (BBB) severely hampers biologics from entering the brain. Considerable effort has been put into generating methods to modulate or circumvent the BBB for delivery of therapeutics to the CNS. A promising strategy is receptor-mediated transcytosis (RMT). Recently, Wouters et al. (2020) discovered a mouse anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via RMT. The present study aims to sample a derivative of this brain-penetrating nanobody (Nb105) in the CNS. Therefore, we compared the applicability of cerebral open flow microperfusion (cOFM) and microdialysis as sampling techniques to directly obtain high molecular weight substances from the cerebral interstitial fluid. A custom AlphaScreen™ assay was validated to quantify nanobody concentrations in the samples. In vitro microdialysis probe (AtmosLM™, 1 MDa cut-off) recovery by gain and by loss for Nb105 was 18.3 ± 3.2% and 27.0 ± 2.5% respectively, whereas for cOFM it was 87.2 ± 4.0% and 97.3 ± 1.6%. Although a large difference in in vitro recovery is observed between cOFM and microdialysis, in vivo similar results were obtained. Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher fluorescein isothiocyanate-dextran and immunoglobulin G extravasation around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. This is the first study that samples a bispecific nanobody in the brain's interstitial fluid in function of time, providing a pharmacokinetic profile of nanobodies in the CNS. Furthermore, this is the first time a cOFM study is performed in awake freely moving mice, providing data on inflammation and blood-brain barrier integrity in the mouse brain. Overall, this work demonstrates that, while taking into account the (bio)analytical considerations, both microdialysis and cOFM are suitable in vivo sampling techniques for quantification of nanobodies in the CNS.
MeSH term(s)	Animals ; Biological Transport ; Blood-Brain Barrier ; Brain ; Extracellular Fluid ; Humans ; Mice ; Microdialysis
Language	English
Publishing date	2021-06-29
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	1483436-4
ISSN	1873-4324 ; 0003-2670
ISSN (online)	1873-4324
ISSN	0003-2670
DOI	10.1016/j.aca.2021.338803
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Article: Clinically relevant dosing and pharmacokinetics of DNA-encoded antibody therapeutics in a sheep model.

Hollevoet, Kevin / Thomas, Debby / Compernolle, Griet / Vermeire, Giles / De Smidt, Elien / De Vleeschauwer, Stéphanie / Smith, Trevor R F / Fisher, Paul D / Dewilde, Maarten / Geukens, Nick / Declerck, Paul

Frontiers in oncology

2022 Volume 12, Page(s) 1017612

Abstract: DNA-encoded delivery ... ...

Abstract	DNA-encoded delivery and
Language	English
Publishing date	2022-10-03
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2649216-7
ISSN	2234-943X
ISSN	2234-943X
DOI	10.3389/fonc.2022.1017612
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Article ; Online: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons.

Libé-Philippot, Baptiste / Lejeune, Amélie / Wierda, Keimpe / Louros, Nikolaos / Erkol, Emir / Vlaeminck, Ine / Beckers, Sofie / Gaspariunaite, Vaiva / Bilheu, Angéline / Konstantoulea, Katerina / Nyitrai, Hajnalka / De Vleeschouwer, Matthias / Vennekens, Kristel M / Vidal, Niels / Bird, Thomas W / Soto, Daniela C / Jaspers, Tom / Dewilde, Maarten / Dennis, Megan Y /

Rousseau, Frederic / Comoletti, Davide / Schymkowitz, Joost / Theys, Tom / de Wit, Joris / Vanderhaeghen, Pierre

Cell

2023 Volume 186, Issue 26, Page(s) 5766–5783.e25

Abstract: The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and ...

Abstract	The enhanced cognitive abilities characterizing the human species result from specialized features of neurons and circuits. Here, we report that the hominid-specific gene LRRC37B encodes a receptor expressed in human cortical pyramidal neurons (CPNs) and selectively localized to the axon initial segment (AIS), the subcellular compartment triggering action potentials. Ectopic expression of LRRC37B in mouse CPNs in vivo leads to reduced intrinsic excitability, a distinctive feature of some classes of human CPNs. Molecularly, LRRC37B binds to the secreted ligand FGF13A and to the voltage-gated sodium channel (Nav) β-subunit SCN1B. LRRC37B concentrates inhibitory effects of FGF13A on Nav channel function, thereby reducing excitability, specifically at the AIS level. Electrophysiological recordings in adult human cortical slices reveal lower neuronal excitability in human CPNs expressing LRRC37B. LRRC37B thus acts as a species-specific modifier of human neuron excitability, linking human genome and cell evolution, with important implications for human brain function and diseases.
MeSH term(s)	Animals ; Humans ; Mice ; Action Potentials/physiology ; Axons/metabolism ; Neurons/metabolism ; Pyramidal Cells ; Voltage-Gated Sodium Channels/genetics ; Voltage-Gated Sodium Channels/metabolism
Chemical Substances	Voltage-Gated Sodium Channels
Language	English
Publishing date	2023-12-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	187009-9
ISSN	1097-4172 ; 0092-8674
ISSN (online)	1097-4172
ISSN	0092-8674
DOI	10.1016/j.cell.2023.11.028
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Article ; Online: AAV-mediated delivery of an anti-BACE1 VHH alleviates pathology in an Alzheimer's disease model.

EMBO molecular medicine

2022 Volume 14, Issue 4, Page(s) e09824

Abstract: Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), ... ...

Abstract	Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood-brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB-crossing adeno-associated virus (AAV)-based vectors to deliver VHH directly into the CNS. As a proof-of-concept, we explored the potential of AAV-delivered VHH to inhibit BACE1, a well-characterized target in Alzheimer's disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH-B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV-VHH-B9 produces positive long-term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the App
MeSH term(s)	Alzheimer Disease/pathology ; Amyloid Precursor Protein Secretases/immunology ; Amyloid Precursor Protein Secretases/metabolism ; Amyloid beta-Peptides/metabolism ; Animals ; Aspartic Acid Endopeptidases/immunology ; Aspartic Acid Endopeptidases/metabolism ; Blood-Brain Barrier ; Dependovirus/genetics ; Disease Models, Animal ; Genetic Vectors/therapeutic use ; Mice ; Mice, Transgenic ; Single-Domain Antibodies
Chemical Substances	Amyloid beta-Peptides ; Single-Domain Antibodies ; Amyloid Precursor Protein Secretases (EC 3.4.-) ; Aspartic Acid Endopeptidases (EC 3.4.23.-) ; Bace1 protein, mouse (EC 3.4.23.46)
Language	English
Publishing date	2022-03-30
Publishing country	England
Document type	Journal Article
ZDB-ID	2467145-9
ISSN	1757-4684 ; 1757-4676
ISSN (online)	1757-4684
ISSN	1757-4676
DOI	10.15252/emmm.201809824
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Article: Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development.

De Vlaeminck, Yannick De / Bonelli, Stefano / Awad, Robin Maximilian / Dewilde, Maarten / Rizzolio, Sabrina / Lecocq, Quentin / Bolli, Evangelia / Santos, Ana Rita / Laoui, Damya / Schoonooghe, Steve / Tamagnone, Luca / Goyvaerts, Cleo / Mazzone, Massimiliano / Breckpot, Karine / Van Ginderachter, Jo A

Cancers

2020 Volume 12, Issue 12

Abstract: Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor- ... ...

Abstract	Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-II
Language	English
Publishing date	2020-11-30
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers12123582
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