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- de la Rosa, Andres, et al.
(författare)
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Introducing or removing heparan sulfate binding sites does not alter brain uptake of the blood-brain barrier shuttle scFv8D3
- 2022
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- The blood-brain barrier (BBB) greatly limits the delivery of protein-based drugs into the brain and is a major obstacle for the treatment of brain disorders. Targeting the transferrin receptor (TfR) is a strategy for transporting protein-based drugs into the brain, which can be utilized by using TfR-binding BBB transporters, such as the TfR-binding antibody 8D3. In this current study, we investigated if binding to heparan sulfate (HS) contributes to the brain uptake of a single chain fragment variable of 8D3 (scFv8D3). We designed and produced a scFv8D3 mutant, engineered with additional HS binding sites, HS(+)scFv8D3, to assess whether increased HS binding would improve brain uptake. Additionally, a mutant with a reduced number of HS binding sites, HS(-)scFv8D3, was also engineered to see if reducing the HS binding sites could also affect brain uptake. Heparin column chromatography showed that only the HS(+)scFv8D3 mutant bound HS in the experimental conditions. Ex vivo results showed that the brain uptake was unaffected by the introduction or removal of HS binding sites, which indicates that scFv8D3 is not dependent on the HS binding sites for brain uptake. Conversely, introducing HS binding sites to scFv8D3 decreased its renal excretion while removing them had the opposite effect.
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- Morrison, Jamie I., et al.
(författare)
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A single-chain fragment constant design enables easy production of a monovalent blood-brain barrier transporter and provides an improved brain uptake at elevated doses
- 2023
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Ingår i: Journal of Neurochemistry. - : John Wiley & Sons. - 0022-3042 .- 1471-4159. ; 165:3, s. 413-425
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Tidskriftsartikel (refereegranskat)abstract
- The interest for developing antibody-driven therapeutic interventions has exponentially grown over the last few decades. Even though there have been promising leaps in the development of efficacious antibody therapies, problems revolving around production and site-directed delivery of these large macromolecules persist. This is especially pertinent when it comes to designing and producing antibodies to penetrate the blood-brain barrier (BBB) to tackle neurodegenerative diseases. One of the most effective approaches to alleviating this problem is to employ a "Trojan Horse " approach, using receptor-mediated transcytosis, such as those governed by the transferrin receptor (TfR)-mediated pathways, to deliver large protein payloads into the brain. Even though this method is effective, ideal limiting factors, related to how the antibody binds to the TfR, need to be elucidated to improve BBB penetrance. With this said, we have designed and produced a single-chain Fc antibody, conjugated to an scFv8D3 TfR binding motif, creating a single-chain monovalent BBB transporter (scFc-scFv8D3). This recombinant protein is easy to produce and purify, demonstrates monovalent binding to the TfR and is structurally stable at physiologically relevant temperatures. Using an in vitro BBB model system, we show a positive correlation between the concentration of administered antibody and transcytosis efficacy, with scFc-scFv8D3 demonstrating significantly higher transcytosis levels compared with scFv8D3-conjugated bivalent antibodies at elevated administered concentrations. Furthermore, in vivo studies recapitulate the in vitro results, with the scFc-scFv8D3 demonstrating an elevated brain uptake at higher therapeutic doses in wild-type mice, comparable with that of the scFv8D3-conjugated bivalent antibody control. In addition, the half-life of the single-chain monovalent BBB transporter is comparable with that of standard IgG antibodies, indicating that the scFc format does not exacerbate physiological degradation. Our results lead us to the conclusion that valency and affinity are important variables to consider when discerning optimal transport across the BBB using TfR-mediated transcytosis pathways. In addition, we believe the single-chain Fc antibody we have described, which can easily be manipulated to accommodate a bispecific target tactic, provides a simple and efficacious approach for delivering therapeutic payloads to the brain milieu.
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- Morrison, Jamie, et al.
(författare)
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Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways
- 2023
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 20:3, s. 1564-1576
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Tidskriftsartikel (refereegranskat)abstract
- The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.
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- Perry, Sharn, et al.
(författare)
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Characterization of Dmrt3-Derived Neurons Suggest a Role within Locomotor Circuits
- 2019
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Ingår i: Journal of Neuroscience. - : SOC NEUROSCIENCE. - 0270-6474 .- 1529-2401. ; 39:10, s. 1771-1782
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the Dmrt3 gene has major effects on gaiting ability, whereas mice lacking the Dmrt3 gene display impaired locomotor activity. Although the Dmrt3 gene is necessary for normal spinal network formation and function in mice, a direct role for Dmrt3-derived neurons in locomotor-related activities has not been demonstrated. Here we present the characteristics of the Dmrt3-derived spinal cord interneurons. Using transgenic mice of both sexes, we characterized interneurons labeled by their expression of Cre driven by the endogenous Dmrt3 promoter. We used molecular, retrograde tracing and electrophysiological techniques to examine the anatomical, morphological, and electrical properties of the Dmrt3-Cre neurons. We demonstrate that inhibitory Dmrt3-Cre neurons receive extensive synaptic inputs, innervate surrounding CPG neurons, intrinsically regulate CPG neuron's electrical activity, and are rhythmically active during fictive locomotion, bursting at frequencies independent to the ventral root output. The present study provides novel insights on the character of spinal Dmrt3-derived neurons, data demonstrating that these neurons participate in locomotor coordination.
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- Petersen, Inga, et al.
(författare)
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Multivalent design of the monoclonal SynO2 antibody improves binding strength to soluble α-Synuclein aggregates
- 2023
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Ingår i: mAbs. - : Taylor & Francis. - 1942-0862 .- 1942-0870. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Soluble aggregates are reported to be the most neurotoxic species of alpha-Synuclein (alpha Syn) in Parkinson's disease (PD) and hence are a promising target for diagnosis and treatment of PD. However, the predominantly intracellular location of alpha Syn limits its accessibility, especially for antibody-based molecules and prompts the need for exceptionally strong soluble alpha Syn aggregate binders to enhance their sensitivity and efficacy for targeting the extracellular alpha Syn pool. In this study, we have created the multivalent antibodies TetraSynO2 and HexaSynO2, derived from the alpha Syn oligomer-specific antibody SynO2, to increase avidity binding to soluble alpha Syn aggregate species through more binding sites in close proximity. The multivalency was achieved through recombinant fusion of single-chain variable fragments of SynO2 to the antibodies' original N-termini. Our ELISA results indicated a 20-fold increased binding strength of the multivalent formats to alpha Syn aggregates, while binding to alpha Syn monomers and unspecific binding to amyloid beta protofibrils remained low. Kinetic analysis using LigandTracer revealed that only 80% of SynO2 bound bivalently to soluble aSyn aggregates, whereas the proportion of TetraSynO2 and HexaSynO2 binding bi- or multivalently to soluble alpha Syn aggregates was increased to similar to 95% and 100%, respectively. The overall improved binding strength of TetraSynO2 and HexaSynO2 implies great potential for immunotherapeutic and diagnostic applications with targets of limited accessibility, like extra-cellular alpha Syn aggregates. The ability of the multivalent antibodies to bind a wider range of alpha Syn aggregate species, which are not targetable by conventional bivalent antibodies, thus could allow for an earlier and more effective intervention in the progression of PD.
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- Rofo, Fadi, et al.
(författare)
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A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer's Disease Mice
- 2022
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Ingår i: NEUROTHERAPEUTICS. - : Springer Nature. - 1933-7213 .- 1878-7479. ; 19:5, s. 1588-1602
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-beta (A beta) oligomers and protofibrils are suggested to be the most neurotoxic A beta species in Alzheimer's disease (AD). Hence, antibodies with strong and selective binding to these soluble A beta aggregates are of therapeutic potential. We have recently introduced HexaRmAb158, a multivalent antibody with additional A beta-binding sites in the form of single-chain fragment variables (scFv) on the N-terminal ends of A beta protofibril selective antibody (RmAb158). Due to the additional binding sites and the short distance between them, HexaRmAb158 displayed a slow dissociation from protofibrils and strong binding to oligomers in vitro. In the current study, we aimed at investigating the therapeutic potential of this antibody format in vivo using mouse models of AD. To enhance BBB delivery, the transferrin receptor (TfR) binding moiety (scFv8D3) was added, forming the Bispecific-multivalent antibody (HexaRmAb158-scFv8D3). The new antibody displayed a weaker TfR binding compared to the previously developed RmAb158-scFv8D3 and was less efficiently transcytosed in a cell-based BBB model. HexaRmAb158 detected soluble A beta aggregates derived from brains of tg-ArcSwe and App(NL-G-F) mice more efficiently compared to RmAb158. When intravenously injected, HexaRmAb158-scFv8D3 was actively transported over the BBB into the brain in vivo. Brain uptake was marginally lower than that of RmAb158-scFv8D3, but significantly higher than observed for conventional IgG antibodies. Both antibody formats displayed similar brain retention (72 h post injection) and equal capacity in clearing soluble A beta aggregates in tg-ArcSwe mice. In conclusion, we demonstrate a Bispecific-multivalent antibody format capable of passing the BBB and targeting a wide-range of sizes of soluble A beta aggregates.
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| 10. |
- Rofo, Fadi, et al.
(författare)
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Blood-brain barrier penetrating neprilysin degrades monomeric amyloid-beta in a mouse model of Alzheimer’s disease
- 2022
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Ingår i: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundAggregation of the amyloid-β (Aβ) peptide in the brain is one of the key pathological events in Alzheimer’s disease (AD). Reducing Aβ levels in the brain by enhancing its degradation is one possible strategy to develop new therapies for AD. Neprilysin (NEP) is a membrane-bound metallopeptidase and one of the major Aβ-degrading enzymes. The secreted soluble form of NEP (sNEP) has been previously suggested as a potential protein-therapy degrading Aβ in AD. However, similar to other large molecules, peripherally administered sNEP is unable to reach the brain due to the presence of the blood–brain barrier (BBB).MethodsTo provide transcytosis across the BBB, we recombinantly fused the TfR binding moiety (scFv8D3) to either sNEP or a previously described variant of NEP (muNEP) suggested to have higher degradation efficiency of Aβ compared to other NEP substrates, but not per se to degrade Aβ more efficiently. To provide long blood half-life, an Fc-based antibody fragment (scFc) was added to the designs, forming sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3. The ability of the mentioned recombinant proteins to degrade Aβ was first evaluated in vitro using synthetic Aβ peptides followed by sandwich ELISA. For the in vivo studies, a single injection of 125-iodine-labelled sNEP-scFc-scFv8D3 and muNEP-scFc-scFv8D3 was intravenously administered to a tg-ArcSwe mouse model of AD, using scFc-scFv8D3 protein that lacks NEP as a negative control. Different ELISA setups were applied to quantify Aβ concentration of different conformations, both in brain tissues and blood samples.ResultsWhen tested in vitro, sNEP-scFc-scFv8D3 retained sNEP enzymatic activity in degrading Aβ and both constructs efficiently degraded arctic Aβ. When intravenously injected, sNEP-scFc-scFv8D3 demonstrated 20 times higher brain uptake compared to sNEP. Both scFv8D3-fused NEP proteins significantly reduced aggregated Aβ levels in the blood of tg-ArcSwe mice, a transgenic mouse model of AD, following a single intravenous injection. In the brain, monomeric and oligomeric Aβ were significantly reduced. Both scFv8D3-fused NEP proteins displayed a fast clearance from the brain.ConclusionA one-time injection of a BBB-penetrating NEP shows the potential to reduce, the likely most toxic, Aβ oligomers in the brain in addition to monomers. Also, Aβ aggregates in the blood were reduced.
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