RESUMO
The elimination of amyloid-beta (Aß) plaques in Alzheimer's disease patients after treatment with anti-Aß antibodies such as lecanemab and aducanumab is supported by a substantially decreased signal in amyloid positron emission tomography (PET) imaging. However, this decreased PET signal has not been matched by a similar substantial effect on cognitive function. There may be several reasons for this, including short treatment duration and advanced disease stages among the patients. However, one aspect that has not been investigated, and the subject of this study, is whether antibody engagement with amyloid plaques inhibits the binding of amyloid-PET ligands, leading to a false impression of Aß removal from the brain. In the present study, tg-ArcSwe mice received three injections of RmAb158, the murine version of lecanemab or phosphate-buffered saline (PBS) before the administration of the amyloid-PET radioligand [11C]PiB, followed by isolation of brain tissue. Autoradiography showed that RmAb158- and PBS-treated mice displayed similar [11C]PiB binding. Moreover, the total Aß1-40 levels, representing the major Aß species of plaques in the tg-ArcSwe model, as well as soluble triggering receptor on myeloid cells 2 (sTREM2) levels, were similar in both groups. Interestingly, the concentration of soluble Aß aggregates was decreased in the RmAb158-treated group, along with a small but significant decrease in the total Aß1-42 levels. In conclusion, this study indicates that the binding of [11C]PiB to Aß accurately mirrors the load of Aß plaques in the brain, aligning with how amyloid-PET is interpreted in clinical studies of anti-Aß antibodies. However, early treatment effects on soluble Aß aggregates and Aß1-42 levels were not detected.
RESUMO
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.
Assuntos
Anticorpos , Ressonância de Plasmônio de Superfície , Ratos , Camundongos , Humanos , Animais , Ressonância de Plasmônio de Superfície/métodos , Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Receptores da Transferrina/metabolismoRESUMO
Synaptic alterations in certain brain structures are related to cognitive decline in neurodegeneration and in aging. Synaptic loss in many neurodegenerative diseases can be visualized by positron emission tomography (PET) imaging of synaptic vesicle glycoprotein 2A (SV2A). However, the use of SV2A PET for studying synaptic changes during aging is not particularly explored. Thus, in the present study, PET ligand [18F]SynVesT-1, which binds to SV2A, was used to investigate synaptic density at different ages in healthy mice. Wild type C57BL/6 mice divided into three age groups (4-5 months (n = 7), 12-14 months (n = 11), 17-19 months (n = 7)) were PET scanned with [18F]SynVesT-1. Brain retention of [18F]SynVesT-1 expressed as the volume of distribution (VIDIF) was calculated using an image-derived input function. Estimates of VIDIF were derived using either a one-tissue compartment model (1TCM), a two-tissue compartment model (2TCM), or the Logan plot with blood input to find the best-fit model for [18F]SynVesT-1. After the PET scans, tissue sections were immunostained for the detection of SV2A and neuronal markers. We found that [18F]SynVesT-1 data acquired 60 min post intravenously injection and analyzed with 1TCM described the brain pharmacokinetics of the radioligand in mice well. [18F]SynVesT-1 brain retention was lower in the oldest group of mice, indicating a decrease in synaptic density in this age group. However, no gradual age-dependent decrease in synaptic density at a region-specific level was observed. Immunostaining indicated that SV2A expression and neuron numbers were similar across all three age groups. In general, these data obtained in healthy aging mice are consistent with previous findings in humans where synaptic density appeared stable during aging up to a certain age, after which a small decrease is observed.
Assuntos
Tomografia por Emissão de Pósitrons , Pirrolidinas , Humanos , Camundongos , Animais , Lactente , Camundongos Endogâmicos C57BL , Tomografia por Emissão de Pósitrons/métodos , Pirrolidinas/farmacocinética , Piridinas/farmacocinética , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismoRESUMO
Tetrazine (Tz)-trans-cyclooctene (TCO) ligation is an ultra-fast and highly selective reaction and it is particularly suited to label biomolecules under physiological conditions. As such, a 3 H-Tz based synthon would have wide applications for inâ vitro/ex vivo assays. In this study, we developed a 3 H-labeled Tz and characterized its potential for application to pretargeted autoradiography. Several strategies were explored to synthesize such a Tz. However, classical approaches such as reductive halogenation failed. For this reason, we designed a Tz containing an aldehyde and explored the possibility of reducing this group with NaBT4 . This approach was successful and resulted in [3 H]-(4-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)phenyl)methan-t-ol with a radiochemical yield of 22 %, a radiochemical purity of 96 % and a molar activity of 0.437â GBq/µmol (11.8â Ci/mmol). The compound was successfully applied to pretargeted autoradiography. Thus, we report the synthesis of the first 3 H-labeled Tz and its successful application as a labeling building block.
Assuntos
Compostos Heterocíclicos , Compostos Radiofarmacêuticos , Linhagem Celular Tumoral , Compostos Radiofarmacêuticos/química , Ciclo-Octanos/químicaRESUMO
Small molecule imaging agents such as [11C]PiB, which bind to the core of insoluble amyloid-ß (Aß) 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 Aß 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 Aß 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.
Assuntos
Doença de Alzheimer , Imunoconjugados , Animais , Camundongos , Manose , Imunoconjugados/farmacologia , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Camundongos Transgênicos , Amiloide/metabolismo , Anticorpos/metabolismo , Progressão da Doença , Neuroimagem , Tomografia por Emissão de Pósitrons/métodosRESUMO
Affibodies targeting amyloid-beta (Aß) could potentially be used as therapeutic and diagnostic agents in Alzheimer's disease (AD). Affibodies display suitable characteristics for imaging applications such as high stability and a short biological half-life. The aim of this study was to explore brain delivery and retention of Aß protofibril-targeted affibodies in wild-type (WT) and AD transgenic mice and to evaluate their potential as imaging agents. Two affibodies, Z5 and Z1, were fused with the blood-brain barrier (BBB) shuttle single-chain variable fragment scFv8D3. In vitro binding of 125I-labeled affibodies with and without scFv8D3 was evaluated by ELISA and autoradiography. Brain uptake and retention of the affibodies at 2 h and 24 h post injection was studied ex vivo in WT and transgenic (tg-Swe and tg-ArcSwe) mice. At 2 h post injection, [125I]I-Z5 and [125I]I-Z1 displayed brain concentrations of 0.37 ± 0.09% and 0.46 ± 0.08% ID/g brain, respectively. [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 showed increased brain concentrations of 0.53 ± 0.16% and 1.20 ± 0.35%ID/g brain. At 24 h post injection, brain retention of [125I]I-Z1 and [125I]I-Z5 was low, while [125I]I-scFv8D3-Z1 and [125I]I-scFv8D3-Z5 showed moderate brain retention, with a tendency towards higher retention of [125I]I-scFv8D3-Z5 in AD transgenic mice. Nuclear track emulsion autoradiography showed greater parenchymal distribution of [125I]I-scFv8D3-Z5 and [125I]I-scFv8D3-Z1 compared with the affibodies without scFv8D3, but could not confirm specific affibody accumulation around Aß deposits. Affibody-scFv8D3 fusions displayed increased brain and parenchymal delivery compared with the non-fused affibodies. However, fast brain washout and a suboptimal balance between Aß and mTfR1 affinity resulted in low intrabrain retention around Aß deposits.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Tomografia por Emissão de Pósitrons/métodos , Receptores da Transferrina/metabolismoRESUMO
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 (Aß) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aß pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aß 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 Aß, and its present and potential future role in the development of drugs aimed at reducing brain Aß levels as a therapeutic strategy to halt disease progression in AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Desenvolvimento de Medicamentos , Tomografia por Emissão de Pósitrons/métodosRESUMO
It is crucial to understand the basic principles of drug transport, from the site of delivery to the site of action within the CNS, in order to evaluate the possible utility of a new drug candidate for CNS action, or possible CNS side effects of non-CNS targeting drugs. This includes pharmacokinetic aspects of drug concentration-time profiles in plasma and brain, blood-brain barrier transport and drug distribution within the brain parenchyma as well as elimination processes from the brain. Knowledge of anatomical and physiological aspects connected with drug delivery is crucial in this context. The chapter is intended for professionals working in the field of CNS drug development and summarizes key pharmacokinetic principles and state-of-the-art experimental methodologies to assess brain drug disposition. Key parameters, describing the extent of unbound (free) drug across brain barriers, in particular blood-brain and blood-cerebrospinal fluid barriers, are presented along with their application in drug development. Special emphasis is given to brain intracellular pharmacokinetics and its role in evaluating target engagement. Fundamental neuropharmacokinetic differences between small molecular drugs and biologicals are discussed and critical knowledge gaps are outlined.
Assuntos
Barreira Hematoencefálica , Encéfalo , Transporte Biológico/fisiologia , Fármacos do Sistema Nervoso Central/farmacocinética , Humanos , Preparações FarmacêuticasRESUMO
Drug delivery to the brain is challenging to study due to the complexity of the barriers of the central nervous system (CNS). The present chapter describes and compares experimental methods such as microdialysis, two-photon laser scanning fluorescence microscopy and positron emission tomography (PET) that can be used for in vivo studies of drug transport across the blood-brain barrier (BBB). The selection of appropriate method is based on the research question, and the different methods will in most cases provide complementary information. Attention is also given to the fact that the BBB might undergo changes in integrity, protein expression and other morphological alterations as a result of disease. The use of animal models of human disease is therefore also discussed. Special emphasis is given to translational aspects of the different methods and readouts.
Assuntos
Barreira Hematoencefálica , Encéfalo , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Neuroimagem , Tomografia por Emissão de PósitronsRESUMO
The positron emission tomography (PET) radioligand [11C]UCB-J binds to synaptic vesicle protein 2A (SV2A) and is used to investigate synaptic density in the living brain. Clinical studies have indicated reduced [11C]UCB-J binding in Alzheimer's disease (AD) and Parkinson's disease (PD) brains compared to healthy controls. Still, it is unknown whether [11C]UCB-J PET can visualise synaptic loss in mouse models of these disorders. Such models are essential for understanding disease pathology and for evaluating the effects of novel disease-modifying drug candidates. In the present study, synaptic density in transgenic models of AD (ArcSwe) and PD (L61) was studied using [11C]UCB-J PET. Data were acquired during 60 min after injection, and time-activity curves (TACs) in different brain regions and the left ventricle of the heart were generated based on the dynamic PET images. The [11C]UCB-J brain concentrations were expressed as standardised uptake value (SUV) over time. The area under the SUV curve (AUC), the ratio of AUC in the brain to that in the heart (AUCbrain/blood), and the volume of distribution (VT) obtained by kinetic modelling using the heart TAC as input were compared between transgenic and age-matched wild type (WT) mice. The L61 mice displayed 11-13% lower AUCbrain/blood ratio and brain VT generated by kinetic modeling compared to the control WT mice. In general, also transgenic ArcSwe mice tended to show lower [11C]UCB-J brain exposure than age-matched WT controls, but variation within the different animal groups was high. Older WT mice (18-20 months) showed lower [11C]UCB-J brain exposure than younger WT mice (8-9 months). Together, these data imply that [11C]UCB-J PET reflects synaptic density in mouse models of neurodegeneration and that inter-subject variation is large. In addition, the study suggested that model-independent AUCbrain/blood ratio can be used to evaluate [11C]UCB-J binding as an alternative to full pharmacokinetic modelling.
Assuntos
Peptídeos beta-Amiloides/análise , Encéfalo/diagnóstico por imagem , Radioisótopos de Carbono/farmacocinética , Modelos Animais de Doenças , Glicoproteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Fragmentos de Peptídeos/análise , Tomografia por Emissão de Pósitrons/métodos , Piridinas/farmacocinética , Pirrolidinonas/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Vesículas Sinápticas/ultraestrutura , Sinucleinopatias/diagnóstico por imagem , Envelhecimento , Doença de Alzheimer , Peptídeos beta-Amiloides/genética , Animais , Área Sob a Curva , Encéfalo/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Doença de Parkinson , Fragmentos de Peptídeos/genéticaRESUMO
Transferrin receptor 1 (TfR1) mediated transcytosis is an attractive strategy to enhance brain uptake of protein drugs, but translation remains a challenge. Here, a single domain shark antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was used to shuttle protein cargo into the brain. TXB2 was fused to a human IgG1 Fc domain (hFc) or to the amyloid-ß (Aß) antibody bapineuzumab (Bapi). TXB2-hFc displayed 20-fold higher brain concentrations compared with a control VNAR-hFc at 18 hours post-injection in wt mice. At the same time point, brain concentrations of Bapi-TXB2 was threefold higher than Bapi. In transgenic mice overexpressing human Aß, the brain-to-blood concentration ratio increased with time due to interaction with intracerebral Aß deposits. The relatively stable threefold difference between Bapi-TXB2 and Bapi was observed for up to 6 days after injection. PET imaging and ex vivo autoradiography revealed more parenchymal distribution of Bapi-TXB2 compared with Bapi. In conclusion, the TXB2 VNAR shuttle markedly increased brain uptake of protein cargo and increased brain concentrations of the Aß binding antibody Bapi.
Assuntos
Antígenos CD/metabolismo , Produtos Biológicos/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Tromboxano B2/metabolismo , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/genética , Produtos Biológicos/farmacocinética , Barreira Hematoencefálica/diagnóstico por imagem , Sistemas de Liberação de Medicamentos , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/genética , Tromboxano B2/genética , TranscitoseRESUMO
Amyloid-ß (Aß) pathology in Alzheimer's disease (AD) is characterized by the formation of polymorphic deposits comprising diffuse and cored plaques. Because diffuse plaques are predominantly observed in cognitively unaffected, amyloid-positive (CU-AP) individuals, pathogenic conversion into cored plaques appears to be critical to AD pathogenesis. Herein, we identified the distinct Aß species associated with amyloid polymorphism in brain tissue from individuals with sporadic AD (s-AD) and CU-AP. To this end, we interrogated Aß polymorphism with amyloid conformation-sensitive dyes and a novel in situ MS paradigm for chemical characterization of hyperspectrally delineated plaque morphotypes. We found that maturation of diffuse into cored plaques correlated with increased Aß1-40 deposition. Using spatial in situ delineation with imaging MS (IMS), we show that Aß1-40 aggregates at the core structure of mature plaques, whereas Aß1-42 localizes to diffuse amyloid aggregates. Moreover, we observed that diffuse plaques have increased pyroglutamated Aßx-42 levels in s-AD but not CU-AP, suggesting an AD pathology-related, hydrophobic functionalization of diffuse plaques facilitating Aß1-40 deposition. Experiments in tgAPPSwe mice verified that, similar to what has been observed in human brain pathology, diffuse deposits display higher levels of Aß1-42 and that Aß plaque maturation over time is associated with increases in Aß1-40. Finally, we found that Aß1-40 deposition is characteristic for cerebral amyloid angiopathy deposition and maturation in both humans and mice. These results indicate that N-terminal Aßx-42 pyroglutamation and Aß1-40 deposition are critical events in priming and maturation of pathogenic Aß from diffuse into cored plaques, underlying neurotoxic plaque development in AD.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Progressão da Doença , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
One of the major hallmarks of Alzheimer's disease (AD) pathology is the formation of extracellular amyloid ß (Aß) plaques. While Aß has been suggested to be critical in inducing and, potentially, driving the disease, the molecular basis of AD pathogenesis is still under debate. Extracellular Aß plaque pathology manifests itself upon aggregation of distinct Aß peptides, resulting in morphologically different plaque morphotypes, including mainly diffuse and cored senile plaques. As plaque pathology precipitates long before any clinical symptoms occur, targeting the Aß aggregation processes provides a promising target for early interventions. However, the chain of events of when, where and what Aß species aggregate and form plaques remains unclear. The aim of this study was to investigate the potential of matrix-assisted laser desorption/ionization imaging mass spectrometry as a tool to study the evolving pathology in transgenic mouse models for AD. To that end, we used an emerging, chemical imaging modality - matrix-assisted laser desorption/ionization imaging mass spectrometry - that allows for delineating Aß aggregation with specificity at the single plaque level. We identified that plaque formation occurs first in cortical regions and that these younger plaques contain higher levels of 42 amino acid-long Aß (Aß1-42). Plaque maturation was found to be characterized by a relative increase in deposition of Aß1-40, which was associated with the appearance of a cored morphology for those plaques. Finally, other C-terminally truncated Aß species (Aß1-38 and Aß1-39) exhibited a similar aggregation pattern as Aß1-40, suggesting that these species have similar aggregation characteristics. These results suggest that initial plaque formation is seeded by Aß1-42; a process that is followed by plaque maturation upon deposition of Aß1-40 as well as deposition of other C-terminally modified Aß species.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Encéfalo/patologia , Placa Amiloide/patologia , Agregação Patológica de Proteínas/patologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
PET imaging of amyloid-beta (Aß) deposits in brain has become an important aid in Alzheimer's disease diagnosis, and an inclusion criterion for patient enrolment into clinical trials of new anti-Aß treatments. Available PET radioligands visualizing Aß bind to insoluble fibrils, i.e. Aß plaques. Levels of prefibrillar Aß forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of Aß leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary Aß, but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the Aß N-terminus and the transferrin receptor (TfR). Natively expressed at the blood-brain barrier (BBB), TfR could thus be used as a brain-blood shuttle. Di-scFv 3D6-8D3 bound to Aß1-40 with high affinity and to TfR with moderate affinity. Di-scFv [124I]3D6-8D3 was injected in two transgenic mouse models overexpressing human Aß and wild-type control mice and PET scanned at 14, 24 or 72â¯h after injection. Di-scFv [124I]3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking Aß. This difference was observed from 24â¯h onwards, and at 72â¯h, 18 months old transgenic animals, with high load of Aß pathology, displayed SUVR of 2.2-3.5 in brain while wild-type showed ratios close to unity. A subset of the mice were also scanned with [11C]PIB. Again wt mice displayed ratios of unity while transgenes showed slightly, non-significantly, elevated SUVR of 1.2, indicating improved sensitivity with novel di-scFv [124I]3D6-8D3 compared with [11C]PIB. Brain concentrations of di-scFv [124I]3D6-8D3 correlated with soluble Aß (pâ¯<â¯0.0001) but not with total Aß, i.e. plaque load (pâ¯=â¯0.34). We have successfully created a small bispecific antibody-based radioligand capable of crossing the BBB, subsequently binding to and visualizing intrabrain Aß in vivo. The radioligand displayed better sensitivity compared with [11C]PIB, and brain concentrations correlated with soluble neurotoxic Aß aggregates.
Assuntos
Doença de Alzheimer/diagnóstico por imagem , Peptídeos beta-Amiloides/análise , Encéfalo/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Doença de Alzheimer/metabolismo , Amiloide/análise , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos/química , Autorradiografia/métodos , Barreira Hematoencefálica/metabolismo , Modelos Animais de Doenças , Humanos , Radioisótopos do Iodo , Camundongos Transgênicos , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismoRESUMO
Almost 50 million people worldwide are affected by Alzheimer's disease (AD), the most common neurodegenerative disorder. Development of disease-modifying therapies would benefit from reliable, non-invasive positron emission tomography (PET) biomarkers for early diagnosis, monitoring of disease progression, and assessment of therapeutic effects. Traditionally, PET ligands have been based on small molecules that, with the right properties, can penetrate the blood-brain barrier (BBB) and visualize targets in the brain. Recently a new class of PET ligands based on antibodies have emerged, mainly in applications related to cancer. While antibodies have advantages such as high specificity and affinity, their passage across the BBB is limited. Thus, to be used as brain PET ligands, antibodies need to be modified for active transport into the brain. Here, we review the development of radioligands based on antibodies for visualization of intrabrain targets. We focus on antibodies modified into a bispecific format, with the capacity to undergo transferrin receptor 1 (TfR1)-mediated transcytosis to enter the brain and access pathological proteins, e.g. amyloid-beta. A number of such antibody ligands have been developed, displaying differences in brain uptake, pharmacokinetics, and ability to bind and visualize the target in the brain of transgenic mice. Potential pathological changes related to neurodegeneration, e.g. misfolded proteins and neuroinflammation, are suggested as future targets for this novel type of radioligand. Challenges are also discussed, such as the temporal match of radionuclide half-life with the ligand's pharmacokinetic profile and translation to human use. In conclusion, brain PET imaging using bispecific antibodies, modified for receptor-mediated transcytosis across the BBB, is a promising method for specifically visualizing molecules in the brain that are difficult to target with traditional small molecule ligands.
Assuntos
Anticorpos/genética , Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Engenharia de Proteínas , Animais , Anticorpos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Humanos , Transporte ProteicoRESUMO
Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the TribodyTM format, for PET imaging of soluble amyloid-beta (Aß) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the Aß protofibril selective antibody mAb158. Five TribodyTM proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and Aß protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human Aß. The systemic pharmacokinetics were similar with half-lives in blood of around 9h for all three ligands. Brain concentrations at 2h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for A1, A2 and A3 respectively), except in the cerebellum, an area largely devoid of Aß pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with Aß pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.
Assuntos
Peptídeos beta-Amiloides , Emaranhados Neurofibrilares , Neuroimagem/métodos , Tomografia por Emissão de Pósitrons/métodos , Doença de Alzheimer/diagnóstico por imagem , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos/química , Especificidade de Anticorpos , Autorradiografia , Barreira Hematoencefálica/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Humanos , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Transferrina/imunologia , Receptores da Transferrina/metabolismoRESUMO
Methods to investigate blood-brain barrier transport and pharmacologically active drug concentrations in the human brain are limited and data translation between species is challenging. Hence, there is a need to further develop the read-out of techniques like positron emission tomography (PET) for studying neuropharmacokinetics. PET has a high translational applicability from rodents to man and measures total drug concentrations in vivo. The aim of the present study was to investigate the possibility of translating total drug concentrations, acquired through PET, to unbound concentrations, resembling those measured in the interstitial fluid by microdialysis sampling. Simultaneous PET scanning and brain microdialysis sampling were performed in rats throughout a 60min infusion of [N-methyl-11C]oxycodone in combination with a therapeutic dose of oxycodone and during a 60min follow up period after the end of infusion. The oxycodone concentrations acquired with PET were converted into unbound concentrations by compensating for brain tissue binding and brain intracellular distribution, using the unbound volume of distribution in brain (Vu,brain), and were compared to microdialysis measurements of unbound concentrations. A good congruence between the methods was observed throughout the infusion. However, an accumulating divergence in the acquired PET and microdialysis data was apparent and became more pronounced during the elimination phase, most likely due to the passage of radioactive metabolites into the brain. In conclusion, the study showed that PET can be used to translate non-invasively measured total drug concentrations into unbound concentrations as long as the contribution of radiolabelled metabolites is minor or can be compensated for.
Assuntos
Analgésicos Opioides/farmacocinética , Microdiálise/métodos , Oxicodona/farmacocinética , Tomografia por Emissão de Pósitrons/métodos , Animais , Barreira Hematoencefálica/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (Aß) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to different extents and the specific and unspecific binding was studied in vitro. Next, cationized F(ab')2-h158 was labelled with iodine-125 and its brain distribution and pharmacokinetics was studied in mice. Cationization did not alter the in vitro affinity to Aß protofibrils, but increased the unspecific binding somewhat. Ex vivo experiments revealed a doubling of brain concentrations compared with unmodified F(ab')2-h158 and in vivo imaging with single photon emission computed tomography (SPECT) showed that the cationized F(ab')2-h158, but not the unmodified F(ab')2-h158 could be visualized in the brain. To conclude, cationization is a means to increase brain concentrations of therapeutic antibodies or fragments and may facilitate the use of antibodies/fragments as imaging biomarkers in the brain.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Carbodi-Imidas/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Animais , Cátions/administração & dosagem , Reagentes de Ligações Cruzadas/química , Camundongos , Camundongos Transgênicos , Distribuição Tecidual/efeitos dos fármacosRESUMO
BACKGROUND: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). RESULTS: The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aß) aggregates in vivo. CONCLUSIONS: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.