| 1. |
- Beretta, Chiara, et al.
(författare)
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Amyloid-β deposits in human astrocytes contain truncated and highly resistant proteoforms
- 2024
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Ingår i: Molecular and Cellular Neuroscience. - : Elsevier. - 1044-7431 .- 1095-9327. ; 128
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer's disease (AD) is a neurodegenerative disorder that develops over decades. Glial cells, including astrocytes are tightly connected to the AD pathogenesis, but their impact on disease progression is still unclear. Our previous data show that astrocytes take up large amounts of aggregated amyloid-beta (Aβ) but are unable to successfully degrade the material, which is instead stored intracellularly. The aim of the present study was to analyze the astrocytic Aβ deposits composition in detail in order to understand their role in AD propagation. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and magnetic beads. Live cell imaging and immunocytochemistry confirmed that the ingested Aβ aggregates and beads were transported to the same lysosomal compartments in the perinuclear region, which allowed us to successfully isolate the Aβ deposits from the astrocytes. Using a battery of experimental techniques, including mass spectrometry, western blot, ELISA and electron microscopy we demonstrate that human astrocytes truncate and pack the Aβ aggregates in a way that makes them highly resistant. Moreover, the astrocytes release specifically truncated forms of Aβ via different routes and thereby expose neighboring cells to pathogenic proteins. Taken together, our study establishes a role for astrocytes in mediating Aβ pathology, which could be of relevance for identifying novel treatment targets for AD.
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| 2. |
- Bonvicini, Gillian, et al.
(författare)
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Comparing in vitro affinity measurements of antibodies to TfR1 : Surface plasmon resonance versus on-cell affinity
- 2024
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Ingår i: Analytical Biochemistry. - : Elsevier. - 0003-2697 .- 1096-0309. ; 686
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Tidskriftsartikel (refereegranskat)abstract
- Despite years of utilizing the transferrin receptor 1 (TfR1) to transport large biomolecules into the brain, there is no consensus on how to optimally measure affinity to it. The aim of this study was to compare different methods for measuring the affinities of anti-TfR1 antibodies.Antibodies 15G11, OX26 and 8D3 are known to successfully carry large biologics across the blood-brain barrier in humans, rats, and mice, respectively. The affinity to their respective species of TfR1 was measured with different surface plasmon resonance setups in Biacore and an on-cell assay.When the antibody was captured and TfR1 was the analyte, the dissociation in Biacore was very slow. The dissociation was faster when the antibody was the analyte and TfR1 was the ligand. The Biacore setup with capture of N-terminal FLAG-tag TfR1 yielded the most similar apparent affinities as the cell assay.In conclusion, it is important to evaluate assay parameters including assay orientation, surface capture method, and antibody format when comparing binding kinetics for TfR1 antibodies. Although it seems possible to determine relative affinities of TfR1 antibodies using the methods described here, both the FLAG-tag TfR1 capture setup and cell assays likely yield apparent affinities that are most translatable in vivo.
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| 3. |
- Pagnon de la Vega, María, et al.
(författare)
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Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion
- 2024
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Ingår i: Acta neuropathologica communications. - : BioMed Central (BMC). - 2051-5960. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Deposition of amyloid beta (Aβ) into plaques is a major hallmark of Alzheimer’s disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aβ. We recently identified the Uppsala APP mutation (APPUpp), which causes Aβ pathology by a triple mechanism: increased β-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aβ conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aβ pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aβ pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased β-secretase cleavage and suppressed α-secretase cleavage, resulting in AβUpp42 dominated diffuse plaque pathology appearing from the age of 5–6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [11C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aβ pathology in all models, whereas the Aβ protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aβ pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AβUpp42 aggregates were found to affect their interaction with anti-Aβ antibodies and profoundly modify the Aβ-mediated glial response, which may be important aspects to consider for further development of AD therapies.
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| 4. |
- Schlein, Eva, et al.
(författare)
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Synthesis and evaluation of fluorine-18 labelled tetrazines as pre-targeting imaging agents for PET
- 2024
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Ingår i: EJNMMI Radiopharmacy and Chemistry. - : Springer Nature. - 2365-421X. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- The brain is a challenging target for antibody-based positron emission tomography (immunoPET) imaging due to the restricted access of antibody-based ligands through the blood-brain barrier (BBB). To overcome this challenge we have previously developed bispecific antibody ligands that pass through the BBB via receptor-mediated transcytosis. These ligands, when radiolabelled, can be used for brain imaging with high affinity and specificity, but their long residence time in the blood and brain can be challenging for their use as PET radioligands. This could be solved by using a two-step approach which involves the administration of a tagged antibody that accumulates at the target site in the brain and then clears from the blood, followed by administration of a radiolabelled molecule, with fast kinetics. This radiolabelled molecule can couple to the tagged antibody and thereby make the antibody localisation visible by PET imaging. The in vivo linkage can be achieved using the inverse electron demand Diels-Alder reaction (IEDDA), with trans-cyclooctene (TCO) and tetrazine groups participating as reactants.In this study, two 18F-labelled tetrazines were synthesized and evaluated for their potential as agents for pre-targeted imaging, i.e. for their ability to rapidly enter the brain and then, if non-bound, be sufficiently cleared with low background retention. The two compounds, a methyl tetrazine [18F]MeTz and an H-tetrazine [18F]HTz were radiolabelled using a two-step procedure via [18F]F-Py-TFP synthesized on solid support followed by amidation with amine-bearing tetrazines, resulting in radiochemical yields of 24% and 22%, respectively, and a radiochemical purity of > 96%. In vivo PET imaging was performed to assess their suitability for in vivo pre-targeting. Time-activity curves from PET-scans revealed that the [18F]MeTz had the most favourable profile for an imaging agent for pre-targeting, due to its fast and homogenous brain distribution and rapid clearance from the brain.
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| 5. |
- Banka, Vinay, et al.
(författare)
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Development of brain-penetrable antibody radioligands for in vivo PET imaging of amyloid-β and tau.
- 2023
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Ingår i: Frontiers in nuclear medicine (Lausanne, Switzerland). - 2673-8880. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Alzheimer's disease (AD) is characterized by the misfolding and aggregation of two major proteins: amyloid-beta (Aβ) and tau. Antibody-based PET radioligands are desirable due to their high specificity and affinity; however, antibody uptake in the brain is limited by the blood-brain barrier (BBB). Previously, we demonstrated that antibody transport across the BBB can be facilitated through interaction with the transferrin receptor (TfR), and the bispecific antibody-based PET ligands were capable of detecting Aβ aggregates via ex vivo imaging. Since tau accumulation in the brain is more closely correlated with neuronal death and cognition, we report here our strategies to prepare four F-18-labeled specifically engineered bispecific antibody probes for the selective detection of tau and Aβ aggregates to evaluate their feasibility and specificity, particularly for in vivo PET imaging.METHODS: We first created and evaluated (via both in vitro and ex vivo studies) four specifically engineered bispecific antibodies, by fusion of single-chain variable fragments (scFv) of a TfR antibody with either a full-size IgG antibody of Aβ or tau or with their respective scFv. Using [18F]SFB as the prosthetic group, all four 18F-labeled bispecific antibody probes were then prepared by conjugation of antibody and [18F]SFB in acetonitrile/0.1 M borate buffer solution (final pH ~ 8.5) with an incubation of 20 min at room temperature, followed by purification on a PD MiniTrap G-25 size exclusion gravity column.RESULTS: Based on both in vitro and ex vivo evaluation, the bispecific antibodies displayed much higher brain concentrations than the unmodified antibody, supporting our subsequent F18-radiolabeling. [18F]SFB was produced in high yields in 60 min (decay-corrected radiochemical yield (RCY) 46.7 ± 5.4) with radiochemical purities of >95%, confirmed by analytical high performance liquid chromatography (HPLC) and radio-TLC. Conjugation of [18F]SFB and bispecific antibodies showed a 65%-83% conversion efficiency with radiochemical purities of 95%-99% by radio-TLC.CONCLUSIONS: We successfully labeled four novel and specifically engineered bispecific antibodies with [18F]SFB under mild conditions with a high RCY and purities. This study provides strategies to create brain-penetrable F-18 radiolabeled antibody probes for the selective detection of tau and Aβ aggregates in the brain of transgenic AD mice via in vivo PET imaging.
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| 6. |
- Bonvicini, Gillian
(författare)
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Harnessing the molecular Trojan horse : Evaluating properties of preclinical Aβ immunoPET radioligands for optimized brain delivery via the transferrin receptor
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- With high specificity and selectivity to targets, antibodies are prime candidates for positron emission tomography (PET) radioligands. They do not passively cross the blood-brain barrier which has hindered their development for imaging intrabrain targets, like amyloid-β (Aβ) in Alzheimer’s disease. The molecular Trojan horse strategy with antibodies that bind to both the transferrin receptor (TfR) and an intrabrain target improves brain delivery of therapeutic antibodies. However, therapeutic antibodies are typically dosed substantially higher than antibody-based PET (immunoPET) radioligands.This thesis evaluated the effects of affinity, valency, and dose on the brain delivery of preclinical Aβ immunoPET radioligands via the TfR.Paper I investigated whether immunoPET with TfR-mediated brain delivery could image Aβ with similar sensitivity in rats as it has in mice. To our knowledge, this was the first time TfR-hijacking to deliver a radioligand to image Aβ was successfully demonstrated in rats; suggesting this strategy could eventually be translated to clinics.Affinity to TfR influences therapeutic delivery to the brain. In Paper II, we compared four Biacore setups and one on-cell assay for determining apparent affinities to the TfR. Absolute affinity determination was challenging since several assay conditions impacted the kinetic parameters. A directional TfR capture in Biacore may be optimal since it determined kinetic parameters while mimicking in vivo receptor conditions. Papers I and III investigated how antibody affinity affects brain delivery at tracer doses and indicated that stronger TfR affinity yielded higher brain delivery. The antibodies in Paper III lacked effector function. The resulting pharmacokinetic profiles in Aβ pathology-presenting mice indicated this may have improved target accumulation of the immunoPET radioligand.In Paper IV, we screened a novel library of monovalent and bivalent affinity variants of the anti-mouse TfR antibody, 8D3. A pair of monovalent and bivalent antibodies with an apparent affinity of 10 nM was identified and evaluated in vivo. Monovalent binding yielded higher brain uptake at a tracer dose but whether bivalent binding steered the antibody towards lysosomal degradation was unclear.In conclusion, monovalency, high affinity binding, and ablated effector function are likely beneficial properties for TfR-mediated brain delivery of an immunoPET radioligand at a tracer dose.
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| 7. |
- 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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| 8. |
- 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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| 9. |
- Gustavsson, Tobias, et al.
(författare)
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Long-term effects of immunotherapy with a brain penetrating Aβ antibody in a mouse model of Alzheimer's disease
- 2023
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Ingår i: Alzheimer's Research & Therapy. - : BioMed Central (BMC). - 1758-9193. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundBrain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer’s disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis.MethodsAppNL−G−F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old AppNL−G−F mice, with evaluation after 3 days. Second, to assess the antibodies’ ability to halt the progression of Aβ pathology, 3 months old AppNL−G−F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4+ T cells. Third, to study the effects of chronic treatment, 7-month-old AppNL−G−F mice were CD4+ T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [125I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining.ResultsNeither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4+ T cell depletion was used for long-term therapy. CD4+ T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [125I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies.ConclusionsBoth RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy.
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| 10. |
- Schlein, Eva
(författare)
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One-click away from higher contrast : Improvements to peripheral clearance for same-day immunoPET in Alzheimer’s disease
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The brain is a challenging target for antibody-based positron emission tomography (immunoPET) to image amyloid-beta (Aβ). Antibodies detect pathology with high sensitivity, but due to their size and biological half-life, they cause a high background radiation, if radiolabelled. Antibodies fused to transferrin-receptor (TfR) binders can penetrate the BBB via receptor-mediated transcytosis. In this thesis, I evaluated several methods to reduce the biological half-life for bispecific antibodies, which bind to Aβ and TfR, to reduce the time between injection and imaging.In paper I, we studied two different clearing approaches – direct clearance and induced clearance – to reduce blood concentrations of a monospecific and a bispecific, brain penetrating antibody, for enhanced contrast. The direct clearing approach was too efficient to show a benefit for brain imaging. The induced clearing strategy, based on the inverse-electron demand Diels−Alder (IEDDA) reaction of a TCO and a tetrazine, proved the concept of induced clearance for the monospecific antibody, but not for the bispecific antibody. For paper II, we changed the antibody design and compared a bispecific antibody with its corresponding monospecific variant, both with and without a mutation that attenuated binding to the neonatal Fc receptor (FcRn), to decrease antibody circulation time in vivo. The mutation reduced the blood half-life and we suggested an imaging time 12 to 24 h after injection. In paper III, we radiolabelled both FcRn mutated antibody constructs with fluorine-18, to compare their pharmacokinetic profiles in WT mice with PET imaging over 9 h. The bispecific antibody, that showed higher brain uptake, was then injected into WT and AD mice (AppNL-G-F). PET scanning 12 h after injection revealed higher antibody retention in AppNL-G-F compared to WT mice. In paper IV, we tested two novel tetrazines for their potential to be used as pre-targeting agents. Pre-targeting describes a two-step approach with the aim to achieve a high contrast PET image. First a TCO-modified antibody is injected and after a while a second substance, a radiolabelled tetrazine is injected. Successful pre-targeting requires a tetrazine which can penetrate the brain and then be efficiently cleared. We could show that both fluorine-18 labelled tetrazines entered the brain, where one of them was more efficient than the other. In conclusion we have shown that it is possible to increase the peripheral clearance of radiolabelled antibodies and get one step closer to same-day immunoPET imaging.
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