| 1. |
- Bonvicini, Gillian, et al.
(författare)
-
Stronger affinity to Transferrin receptor enhances detection of amyloid-β pathology with bispecific antibody radioligands at a tracer dose
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A popular method for delivering biologic therapeutics and diagnostics to the brain is by hijacking transferrin receptor (TfR)-mediated transcytosis. Moderate affinity towards TfR is beneficial for TfR-mediated brain delivery at therapeutic doses while a few studies have indicated that high TfR affinity may be more beneficial at tracer doses. With the development of antibody-based PET radioligands for neurodegenerative diseases, such as Alzheimer’s disease, a better understanding of the pharmacokinetics of TfR-binders at tracer dose is essential. Thus the aim of this study was to evaluate the effect of TfR affinity on brain uptake at a tracer dose in both wild-type (WT) mice and in a mouse model of Aβ pathology.Three different affinity variants of 8D3, produced by alanine point mutations, were selected. Bispecific antibodies were designed with knob-into-hole technology where one arm was the anti-mouse TfR antibody, 8D3, and the other arm was the anti-human Aβ antibody, bapineuzumab (Bapi). Antibody affinities were measured in an in vitro cell assay. In vivo pharmacokinetic analyses of radioiodinated bispecific antibodies and Bapi in brain, blood and peripheral organs were performed over 7 days post-injection in WT and Aβ mice.The affinities of the three bispecific antibodies to TfR were 10 nM, 20 nM and 240 nM. Independent of genotype, stronger TfR-affinity resulted in higher brain uptake. The two bispecific antibodies with stronger affinity behaved similarly and differentiated between WT and Aβ model mice at an earlier time point than the low affinity variant.This study supports the hypothesis that stronger TfR affinity yields better brain uptake at a tracer dose. With the better detection of Aβ pathology, stronger affinity to TfR is a critical feature for the design of future bispecific immunoPET radioligands for intrabrain targets using TfR-mediated transcytosis.
|
|
| 2. |
- Rokka, Johanna, et al.
(författare)
-
Improved synthesis of SV2A targeting radiotracer [11C]UCB-J
- 2019
-
Ingår i: EJNMMI Radiopharmacy and Chemistry. - : Springer Nature. - 2365-421X. ; 4:1
-
Tidskriftsartikel (refereegranskat)abstract
- [11C]UCB-J is a tracer developed for PET (positron emission tomography) that has high affinity towards synaptic vesicle glycoprotein 2A (SV2A), a protein believed to participate in the regulation of neurotransmitter release in neurons and endocrine cells. The localisation of SV2A in the synaptic terminals makes it a viable target for in vivo imaging of synaptic density in the brain. Several SV2A targeting compounds have been evaluated as PET tracers, including [11C]UCB-J, with the aim to facilitate studies of synaptic density in neurological diseases.The original two-step synthesis method failed in our hands to produce sufficient amounts of [11C]UCB-J, but served as an excellent starting point for further optimizations towards a high yielding and simplified one-step method. [11C]Methyl iodide was trapped in a clear THF-water solution containing the trifluoroborate substituted precursor, potassium carbonate and palladium complex. The resulting reaction mixture was heated at 70 °C for 4 min to produce [11C]UCB-J.After semi-preparative HPLC purification and reformulation in 10% ethanol/phosphate buffered saline, the product was obtained in 39 ± 5% radiochemical yield based on [11C]methyl iodide, corresponding to 1.8 ± 0.5 GBq at EOS. The radiochemical purity was > 99% and the molar activity was 390 ± 180 GBq/μmol at EOS. The product solution contained < 2 ppb palladium.A robust and high yielding production method has been developed for [11C]UCB-J, suitable for both preclinical and clinical PET applications.
|
|
| 3. |
|
|
| 4. |
- Schlein, Eva, et al.
(författare)
-
Aβ targeting ImmunoPET : Brain pharmacokinetics comparison between a brain penetrating and a regular antibody
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bispecific antibodies utilizing the transferrin receptor (TfR) for transport into the brain are being developed for both therapeutic and diagnostic applications. Compared with regular monospecific antibodies, the brain uptake of TfR-binding bispecific antibodies is rapid and efficient. However, due to differences in pharmacokinetic properties, it has been challenging to directly compare their brain uptake in vivo. In this study, we have studied the amyloid-β (Aβ) antibody Bapineuzumab (Bapi) and its bispecific variant Bapi-Fab8D3, which contains a fragment of the TfR-binding antibody 8D3. Both antibodies were recombinantly engineered to harbour a mutation that reduces binding to the neonatal Fc receptor (FcRn) and thus contributes to an increased clearance rate from blood.The antibodies were labelled with fluorine-18 (18F) and administered to wildtype (WT) mice, which were PET scanned for 2 h in an alternating manner to cover a period of 9 h, followed by ex vivo analyses. Next, the bispecific antibody [18F]Bapi-Fab8D3 was used for PET imaging if Aβ pathology in the AD mouse model AppNL-G-F compared with WT mice at 12 h after antibody administration. [18F]Bapi and [18F]Bapi-Fab8D3 had identical blood elimination curves in WT mice and PET data quantification demonstrated that [18F]Bapi brain concentration declined from the start and throughout the 9 h time period, while [18F]Bapi-Fab8D3 displayed a higher brain concentration, indicative of its active transport into the brain.[18F]Bapi-Fab PET imaging discriminated AppNL-G-F from WT mice already at 12 h after administration, suggesting that this novel antibody-based ligand could be used for same-day PET imaging.
|
|
| 5. |
- Schlein, Eva, et al.
(författare)
-
Functionalization of Radiolabeled Antibodies to Enhance Peripheral Clearance for High Contrast Brain Imaging
- 2022
-
Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 19:11, s. 4111-4122
-
Tidskriftsartikel (refereegranskat)abstract
- Small molecule imaging agents such as [11C]PiB, which bind to the core of insoluble amyloid-β (Aβ) fibrils, are useful tools in Alzheimer’s disease (AD) research, diagnostics, and drug development. However, the [11C]PiB PET signal saturates early in the disease progression and does not detect soluble or diffuse Aβ pathology which are believed to play important roles in the disease progression. Antibodies, modified into a bispecific format to enter the brain via receptor-mediated transcytosis, could be a suitable alternative because of their diversity and high specificity for their target. However, the circulation time of these antibodies is long, resulting in an extended exposure to radiation and low imaging contrast. Here, we explore two alternative strategies to enhance imaging contrast by increasing clearance of the antibody ligand from blood. The bispecific Aβ targeting antibody RmAb158-scFv8D3 and the monospecific RmAb158 were radiolabeled and functionalized with either α-d-mannopyranosylphenyl isothiocyanate (mannose) or with trans-cyclooctene (TCO). While mannose can directly mediate antibody clearance via the liver, TCO-modified antibody clearance was induced by injection of a tetrazine-functionalized, liver-targeting clearing agent (CA). In vivo experiments in wild type and AD transgenic mice demonstrated the ability of both strategies to drastically shorten the circulation time of RmAb158, while they had limited effect on the bispecific variant RmAb158-8D3. Furthermore, single photon emission computed tomography imaging with TCO-[125I]I-RmAb158 in AD mice showed higher contrast 1 day after injection of the tetrazine-functionalized CA. In conclusion, strategies to enhance the clearance of antibody-based imaging ligands could allow imaging at earlier time points and thereby open the possibility to combine antibodies with short-lived radionuclides such as fluorine-18.
|
|
| 6. |
- Schlein, Eva
(författare)
-
One-click away from higher contrast : Improvements to peripheral clearance for same-day immunoPET in Alzheimer’s disease
- 2023
-
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.
|
|
| 7. |
- Schlein, Eva, et al.
(författare)
-
Reduced neonatal Fc receptor binding increases clearance and brain-to-blood ratio of a brain penetrating amyloid-β antibody
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Recent advances in the development of amyloid-β (Aβ) targeted immunotherapies for Alzheimer’s disease (AD) has highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed towards the same target as the therapeutic antibody. Bispecific brain penetrating antibodies are interesting for this purpose, but the slow clearance of antibodies remains a challenge for their use PET radioligands since antibody in the circulation as well as unbound antibody in the brain contribute to a non-specific background signal. Thus, a substantial time between the injection and the time of PET scanning is required to achieve an acceptable specific-to-nonspecific signal in PET. In this study, two antibody pairs were designed based on the Aβ antibody Bapineuzumab (Bapi), one regular IgG (Bapi) and one bispecific antibody with a Fab fragment of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3), for active TfR-mediated transport into the brain. One of the pairs was engineered to harbor a mutation in the Fc domain which reduced its binding to the neonatal Fc receptor (FcRn) and thereby increased the clearance of the antibody. Blood and brain pharmacokinetics of the antibody pairs were studied in WT mice and in the AD mouse model AppNL-G-F. The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. With the high brain uptake of Bapi-Fab8D3, the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F than WT mice already 12 h after injection and this difference increased further up to 24 h after antibody injection. Ex vivo autoradiography, used to visualize antibody distribution in the brain, showed specific antibody retention in areas with high amounts of Aβ pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody could drastically reduce the time from injection to imaging.
|
|
| 8. |
- Schlein, Eva, et al.
(författare)
-
Reducing neonatal Fc receptor binding enhances clearance and brain-to-blood ratio of TfR-delivered bispecific amyloid-β antibody
- 2024
-
Ingår i: mAbs. - : Taylor & Francis. - 1942-0862 .- 1942-0870. ; 16:1
-
Tidskriftsartikel (refereegranskat)abstract
- Recent development of amyloid-β (Aβ)-targeted immunotherapies for Alzheimer’s disease (AD) have highlighted the need for accurate diagnostic methods. Antibody-based positron emission tomography (PET) ligands are well suited for this purpose as they can be directed toward the same target as the therapeutic antibody. Bispecific, brain-penetrating antibodies can achieve sufficient brain concentrations, but their slow blood clearance remains a challenge, since it prolongs the time required to achieve a target-specific PET signal. Here, two antibodies were designed based on the Aβ antibody bapineuzumab (Bapi) – one monospecific IgG (Bapi) and one bispecific antibody with an antigen binding fragment (Fab) of the transferrin receptor (TfR) antibody 8D3 fused to one of the heavy chains (Bapi-Fab8D3) for active, TfR-mediated transport into the brain. A variant of each antibody was designed to harbor a mutation to the neonatal Fc receptor (FcRn) binding domain, to increase clearance. Blood and brain pharmacokinetics of radiolabeled antibodies were studied in wildtype (WT) and AD mice (AppNL-G-F). The FcRn mutation substantially reduced blood half-life of both Bapi and Bapi-Fab8D3. Bapi-Fab8D3 showed high brain uptake and the brain-to-blood ratio of its FcRn mutated form was significantly higher in AppNL-G-F mice than in WT mice 12 h after injection and increased further up to 168 h. Ex vivo autoradiography showed specific antibody retention in areas with abundant Aβ pathology. Taken together, these results suggest that reducing FcRn binding of a full-sized bispecific antibody increases the systemic elimination and could thereby drastically reduce the time from injection to in vivo imaging.
|
|
| 9. |
- Schlein, Eva, et al.
(författare)
-
Synthesis and evaluation of fluorine-18 labelled tetrazines as pre-targeting imaging agents for PET
- 2024
-
Ingår i: EJNMMI Radiopharmacy and Chemistry. - : Springer Nature. - 2365-421X. ; 9:1
-
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.
|
|
| 10. |
- Syvänen, Stina, et al.
(författare)
-
PET Imaging in Preclinical Anti-Aβ Drug Development
- 2022
-
Ingår i: Pharmaceutical research. - : Springer Nature. - 0724-8741 .- 1573-904X. ; 39:7, s. 1481-1496
-
Forskningsöversikt (refereegranskat)abstract
- Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer’s disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab, an antibody directed towards amyloid-beta (Aβ) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aβ pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aβ drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aβ, and its present and potential future role in the development of drugs aimed at reducing brain Aβ levels as a therapeutic strategy to halt disease progression in AD.
|
|
