| 1. |
- 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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| 2. |
- Faresjö Melander, Rebecca, 1990-
(författare)
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Factors influencing transferrin receptor-mediated brain delivery : Evaluating preclinical antibody-based proteins for PET imaging in Alzheimer’s disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibody-based proteins targeting amyloid-beta (Aβ) could be used as radioligands in positron emission tomography (PET) to study Alzheimer’s disease (AD) pathology in the living brain. The prospective advantages of antibody-based PET are to detect pathology earlier, with higher sensitivity, and to evaluate treatment effects of emerging immunotherapies against Aβ. However, antibodies and other proteins are too large to cross the blood-brain barrier (BBB). This can be circumvented by fusing antibodies with transferrin-receptor (TfR) binders that penetrate the BBB via receptor-mediated transcytosis. In this thesis, I evaluated different bispecific antibody-based proteins that bind both TfR and Aβ. The overall aim was to determine which factors are important for TfR-mediated brain delivery of these proteins and their use as PET radioligands. In paper I, we studied a large, high TfR-avidity antibody compared with a smaller antibody fragment fusion with lower TfR avidity. The small antibody had fast elimination from blood and was cleared from the brain earlier than the large antibody, thus providing better signal-to-noise ratio for brainPET. In paper II, antibody-like proteins (affibodies), even smaller than the previously studied antibody, had enhanced TfR-mediated brain delivery but had an imbalance in binding to TfR and Aβ. This resulted in poor pathology-related retention of 125I-radiolabeled affibodies. In paper III, we observed that aged mice had poorer brain delivery of the bispecific antibody, mAb3D6-scFv8D3, compared with young mice. Age was also related to increased blood cell binding of the bispecific antibody, and a lower dose resulted in higher relative delivery to the brain parenchyma. In paper IV, we evaluated single domain llama-based antibodies, VHHs, which bound both mouse and human TfR, and were characterized by rapid elimination from blood and brain. The VHHs were fused to an Aβ binding antibody fragment, scFv3D6, which enabled increased brain retention of the 125I-radiobeled antibodies in an AD mouse model, and, thus, provided high contrast to healthy controls.In conclusion, antibody format, size, mouse age, dose, and TfR binding were important factors influencing brain delivery and retention.
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| 3. |
- Faresjö, Rebecca, 1990-, et al.
(författare)
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Age, dose, and binding to TfR on blood cells influence brain delivery of a TfR-transported antibody
- 2023
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Ingår i: Fluids and Barriers of the CNS. - : BioMed Central (BMC). - 2045-8118. ; 20:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundTransferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood–brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aβ-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6.MethodsYoung (3–5 months) and aged (17–20 months) WT and tg-ArcSwe mice (AD model) were injected with 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice.ResultsThe aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6.ConclusionAge-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aβ-pathology did not influence the early brain distribution of the bispecific antibody. In summary, age and bispecific antibody dose were important factors determining brain delivery, while genotype was not.
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