AbstractOwing to their specificity and high-affinity binding, monoclonal antibodies have potential as positron emission tomography (PET) radioligands and are currently used to image various targets in peripheral organs. However, in the central nervous system, antibody uptake is limited by the blood鈥揵rain barrier (BBB). Here we present a PET ligand to be used for diagnosis and evaluation of treatment effects in Alzheimer鈥檚 disease. The amyloid 尾 (A尾) antibody mAb158 is radiolabelled and conjugated to a transferrin receptor antibody to enable receptor-mediated transcytosis across the BBB. PET imaging of two different mouse models with A尾 pathology clearly visualize A尾 in the brain. The PET signal increases with age and correlates closely with brain A尾 levels. Thus, we demonstrate that antibody-based PET ligands can be successfully used for brain imaging. IntroductionPositron emission tomography (PET) imaging of amyloid 尾 (A尾) deposits in the brain has rapidly advanced in recent years. The introduction of the radioligand [11C]PIB (ref. 1), a derivate of thioflavin-T, was an important development for diagnosis of Alzheimer鈥檚 disease (AD), as [11C]PIB amyloid imaging detects AD pathology early in the course of disease2 and helps distinguishing AD from other types of dementia3,4. [11C]PIB, and analogues of PIB, detect amyloid plaques, mainly consisting of insoluble fibrils of A尾 (ref. 1). However, the load of insoluble A尾 does not correlate well with disease progression5,6. Soluble A尾 is a better marker of disease status7,8,9,10, and many therapeutic as well as diagnostic efforts are currently targeting soluble A尾 aggregates, for example, oligomers and protofibrils11,12,13,14,15,16,17, which are strongly implicated as the cause of synaptic failure and neurodegeneration in AD9,10,18,19,20,21,22,23. This new focus highlights the pressing need for an imaging agent that can visualize soluble A尾 aggregates. The development of small molecular PET radioligands often suffers from nonspecific binding of the radioligand, and further, low ability to discriminate between different forms of a protein. Radioligands based on antibodies have recently been introduced in clinical use for various peripheral antigens primarily related to cancer24. Antibodies have the advantage that they can be developed to bind a specific form of a protein, but their use as PET radioligands for targets in the central nervous system (CNS) is hampered by their low brain penetration. However, whereas a high enough brain uptake is essential to achieve a PET signal, the specific-to-nonspecific binding, expected to be very high for a monoclonal antibody compared with small molecules, may be equally important.Our previously developed conformation-selective monoclonal antibody mAb158 displays a distinctive selectivity for soluble A尾 protofibrils as compared with monomeric A尾. It binds preferentially to soluble protofibrils over mature, insoluble fibrils, but without affinity for the A尾 protein precursor (A尾PP)25,26,27,28. These characteristics make the antibody suitable to selectively target soluble A尾 aggregates in vivo, and its humanized version, BAN2401, (hereafter referred to as h158), is currently studied in a phase 2b clinical trial as an anti-A尾 therapy against AD. In a previous study we showed that 72鈥塰 after administration, the brain concentrations of iodine-125-labelled mAb158 ([125I]mAb158) was significantly increased in a transgenic AD mouse model (tg-ArcSwe, harbouring the Arctic (E693G) and Swedish (KM670/671NL) A尾PP mutations29) compared with non-transgenic littermates (wild type; WT)27. However, total levels of [125I]mAb158 in the brain were rather moderate, which is anticipated for antibodies in general30.Different strategies have been applied to increase the brain uptake of antibodies and other large molecules. Among them is receptor-mediated transcytosis, where the antibody is fused to a molecule that specifically binds to a blood鈥揵rain barrier (BBB) expressed receptor, for example, the transferrin or insulin receptor, which enables active transport across the BBB (Fig. 1a). This technique was pioneered by Pardridge and colleagues31 in the 1990s, and has recently been implemented in AD therapy to decrease production32,33 or increase degradation34 of A尾.Figure 1: Generation of bispecific fusion protein 8D3-F(ab鈥?2-h158.(a) Schematic picture of TfR-mediated transcytosis. (b) SDS鈥揚AGE displaying F(ab鈥?2-h158, TfR antibody 8D3 and the fusion protein consisting of F(ab鈥?2-h158 and 8D3. (c) Inhibition ELISA demonstrating the fusion protein鈥檚 selective binding to A尾 protofibrils (IC50 1.0鈥塶M) over fibrils (IC50 12鈥塶M) and monomers (IC50 240鈥塶M). The fusion protein showed retained binding to TfR (d) and A尾 protofibrils (e) after 125I labelling, as demonstrated with ELISA (absorbance values on the left y axis and radioactivity on the right y axis). Representative images from triplicate experiments are shown in c鈥?b>e. conc., concentration; c.p.m., counts per minute; IC50, median inhibitory concentration; OD, optical density; SDS鈥揚AGE, SDS鈥損olyacrylamide gel electrophoresis.Full size imageIn the present study, a F(ab鈥?2 fragment of h158 is chemically fused to a transferrin receptor (TfR) antibody35 with the aim to create a PET ligand for specific imaging of soluble A尾 protofibrils. Brain retention of the generated bispecific fusion protein increases 15-fold, compared with F(ab鈥?2-h158. We then show by PET imaging that the brain distribution of 124I-labelled fusion protein correlates closely with the age-dependent increase of A尾 pathology in the brains of two transgenic mouse models with AD-like pathology. This new radioligand has the potential to become an important diagnostic tool in AD and furthermore, the study demonstrates that bispecific radioligands based on antibodies can be applied in medical imaging of proteins associated with CNS disorders.ResultsEngineering of an A尾鈥揟fR bispecific fusion proteinA PET ligand with a fairly short systemic half-life is desired since a rapid elimination from the blood increases the specific signal compared with the background, derived from the blood volume of the studied tissue (about 5% in brain) and decreases the radiation dose for the patient. Therefore, a F(ab鈥?2 fragment was generated by enzymatic cleavage of h158, reducing its systemic half-life to 鈭?/span>2鈥塰 in both tg-ArcSwe and WT mice, compared with 11 days for mAb158 (ref. 27). The A尾-binding properties of F(ab鈥?2-h158 were unchanged, compared with the parent antibody. To increase brain uptake of F(ab鈥?2-h158, it was chemically conjugated to the anti-TfR antibody 8D3 (ref. 36), which has been widely used to increase the brain uptake of large molecules34,37,38,39,40.The 8D3-F(ab鈥?2-h158 fusion protein was sufficiently pure for the intended purpose (Fig. 1b). The difference in fusion protein binding in solution to A尾 monomer, protofibril and fibril, reflected by their ability to inhibit the fusion protein鈥檚 binding to an A尾-coated enzyme-linked immunosorbent assay (ELISA) plate, was very similar to what we have previously reported for mAb158 (refs 25, 27), with median inhibitory concentration values of 240鈥塶M for monomers, 1.0鈥塶M for protofibrils and 12鈥塶M for fibrils (Fig. 1c). Furthermore, it retained its binding to both TfR and A尾 protofibrils also after radioiodination (Fig. 1d,e).Increased brain uptake by TfR-mediated transcytosisThe pharmacokinetic blood profile of the radioiodinated fusion protein [125I]8D3-F(ab鈥?2-h158 was the same in tg-ArcSwe and WT mice displaying a half-life of 11鈥塰 (Fig. 2a). For successful PET imaging, it is important to have a high brain uptake of the ligand in relation to its concentration in blood. Therefore, the brain-to-blood ratio (Kp) of the fusion protein was assessed by ex vivo studies at three different time points. Radioactivity was measured in blood and saline-perfused brains of 12-month-old tg-ArcSwe and WT mice, killed 4, 24 and 72鈥塰 after intraperitoneal (i.p.) injection of [125I]8D3-F(ab鈥?2-h158. At 4鈥塰 Kp was low (0.02卤0.01), with no difference between tg-ArcSwe and WT mice. Kp had increased at 24鈥塰 and was significantly different (P 0.05) between the groups. At 72鈥塰 post injection, Kp had further increased to 0.44卤0.10 (P 0.001) in tg-ArcSwe, while the WT mice were stable on a low level (Fig. 2b). The fusion protein transport into the brain was assessed in 18-month-old mice at 4 and 2鈥塰, corresponding to Cmax of the fusion protein and the F(ab鈥?2 fragment in blood, respectively, and measured as per cent of injected dose per gram brain tissue (% ID per g). At this point, the fusion protein was taken up ninefold more than [125I]F(ab鈥?2-h158, with no difference between tg-ArcSwe and WT mice (Fig. 2c). When studied 72鈥塰 post injection, that is, at the highest Kp of the fusion protein, it showed a 15-fold higher brain retention in tg-ArcSwe mice than the F(ab鈥?2 fragment and a greater difference between tg-ArcSwe and WT mice (Fig. 2d).Figure 2: In vivo blood pharmacokinetics and brain distribution of fusion protein in tg-ArcSwe and WT mice.(a) The bispecific fusion protein (triangles, n=6) showed an increased half-life (11鈥塰) compared with unmodified F(ab鈥?2-h158 (circles, n=3; 2鈥塰). (b) The brain-to-blood concentration ratio of the fusion protein increased over time in tg-ArcSwe mice ( 12 months, n=18) while remaining fairly constant in WT mice (n=14). (c) Comparison of brain distribution of fusion protein (n=7) and unmodified F(ab鈥?2-h158 (n=5) at respective Cmax. At this time point the increase in brain distribution mainly reflected increased transport across the BBB as the increase was observed both in tg-ArcSwe and WT mice ( 18 months). (d) Comparison of brain distribution of fusion protein (n=10) and unmodified F(ab鈥?2-h158 (n=6) 72鈥塰 post injection. At this time point the differences observed between tg-ArcSwe and WT mice ( 18 months) reflected binding to A尾 protofibrils. The symbols and error bars indicate group mean卤s.d. from experiments (a). Each symbol represents one animal, line and error bars indicate group mean卤s.d. (b鈥?b>d). *P 0.05, ***P 0.001 and NS is nonsignificant by two-way analysis of variance followed by Bonferroni鈥檚 post hoc test (b).Full size imageTo assess whether the transport across the BBB may be mediated by the Fc receptor, 10- to 12-month-old WT and tg-ArcSwe mice were injected with 125I-labelled 8D3 or a Fab fragment of 8D3 (Fab-8D3), which lacks the Fc fragment, and ex vivo brain uptake was measured 4鈥塰 post injection. Fab-8D3 was equally well distributed to the brain as 8D3, strongly indicating that the high brain concentrations observed with 8D3 and the fusion protein were due to TfR-mediated transcytosis and independent of the Fc domain of the 8D3 antibody (Fig. 3a). As an additional control, a fusion protein consisting of 8D3 and a F(ab鈥?2 fragment of an antibody (Synagis; specific for the respiratory syncytial virus) of the same IgG isoform as h158, but lacking a specific target in the brain, was also generated. The generated 8D3-F(ab鈥?2-Synagis fusion protein retained its binding to TfR, but did not bind to A尾 protofibrils (Fig. 3b,c).Figure 3: Brain distribution of 8D3 and generation of an A尾 irrelevant fusion protein.(a) Brain distribution of 8D3 (n=3) and Fab-8D3 (n=6) in 10- to 12-month-old WT and tg-ArcSwe mice 4鈥塰 post injection, demonstrating that the TfR-mediated transcytosis of 8D3 is Fc-independent. (b) SDS鈥揚AGE displaying F(ab鈥?2-Synagis, TfR antibody 8D3 and the fusion protein consisting of F(ab鈥?2-Synagis and 8D3. (c) TfR ELISA demonstrating that the irrelevant fusion protein binds to TfR in vitro. (d) In vivo blood pharmacokinetics of irrelevant fusion protein in tg-ArcSwe (n=2) and WT mice (n=4): the irrelevant fusion protein showed a similar half-life as the 8D3-F(ab鈥?2-h158 fusion protein (11鈥塰). (e) Comparison of irrelevant fusion brain concentration at 4鈥塰 (Cmax), demonstrating an increased transport across the BBB in WT mice (n=2), and at 72鈥塰 post injection, where the low concentration of irrelevant fusion protein in both tg-ArcSwe (n=2) and WT (n=2) mice reflects the lack of target in the brain. The symbols and error bars indicate group mean卤s.d. (d). Each symbol represents one animal (a and e). SDS鈥揚AGE, SDS鈥損olyacrylamide gel electrophoresis.Full size imageEx vivo studies were also performed with the irrelevant fusion protein [125I]8D3-F(ab鈥?2-Synagis, which had a similar half-life in blood as [125I]8D3-F(ab鈥?2-h158. Similar to 8D3, the brain retention of the irrelevant fusion protein was elevated in WT mice 4鈥塰 post injection. At 72鈥塰 post injection, the brain retention of the irrelevant fusion protein was the same in tg-ArcSwe and WT mice (Fig. 3d,e) and of the same magnitude as observed with [125I]8D3-F(ab鈥?2-h158 in WT mice.These experiments demonstrated that compared with F(ab鈥?2-h158, the fusion protein transport into the brain was markedly increased in both tg-ArcSwe and WT mice. Hence, the carrier-mediated transcytosis was not dependent on animal type or age. However, in WT animals, lacking A尾 protofibrils, the fusion protein was washed out from the brain when systemic concentrations decreased. Similarly, [125I]8D3-F(ab鈥?2-Synagis, which lacks a protofibril-binding domain, was eliminated from the brains of both WT and tg-ArcSwe mice when the blood concentration decreased. As a consequence of the fairly rapid systemic elimination of the fusion protein, Kp increased in tg-ArcSwe mice over time, but not in WT mice. Moreover, when compared with the whole antibody mAb158 (ref. 27), Kp of the fusion protein was more than 20-fold increased 72鈥塰 post injection.Brain retention follows A尾 pathology in transgenic miceThe brain retention of the fusion protein was measured ex vivo 72鈥塰 post injection in saline-perfused brains of tg-ArcSwe and WT mice at 4, 12 and 18 months of age to follow the course of A尾 pathology. Brain levels of soluble A尾 protofibrils as well as total (formic acid soluble) A尾40 and A尾42 were determined in the same mice. We have previously established that total A尾, measured by ELISA, closely matches A尾 plaque load as determined with immunohistochemistry in tg-ArcSwe mice27. There was a significant difference in brain retention of the fusion protein between tg-ArcSwe and WT mice at 12 months (3.5-fold) and 18 months (6.8-fold), as well as a trend towards increased brain concentrations also in the 4-month group (1.3-fold) (Fig. 4a and Table 1). The WT animals showed constant brain concentrations of fusion protein regardless of age. Both soluble A尾 protofibrils and total A尾40 and A尾42 increased over time in tg-ArcSwe mice, displaying elevated levels of A尾 protofibrils already at 4 months (Fig. 4b). The increase over time in soluble A尾 protofibril levels correlated closely with the brain concentrations of fusion protein (Fig. 4c), while total A尾 concentrations increased with a higher rate (Fig. 4d) suggesting that the A尾 targeted by the fusion protein was mainly of soluble origin. To rule out the possibility that the ligand might be stuck in the endothelium and not transported into the brain, staining for the endothelial marker CD31 in combination with nuclear track emulsion was used to visualize the fusion protein in the brain. The fusion protein was found in the brain parenchyma without accumulation in capillaries in 18-month-old tg-ArcSwe mice (Fig. 4e) but to some extent clustered around the periphery of congophilic amyloid deposits, suggesting it was bound to a halo of soluble A尾 aggregates, which has been reported to surround the fibrillar core of amyloid plaques41 (Fig. 4f).Figure 4: Age-dependent retention of fusion protein in brain.(a) Ex vivo brain retention expressed as % ID per g brain tissue in tg-ArcSwe (n=17) and WT (n=15) mice of different ages 72鈥塰 post injection. (b) A尾 protofibril levels in brain tissue obtained from tg-ArcSwe mice of different ages. (c) Pearson鈥檚 correlation analysis of brain concentrations of fusion protein 72鈥塰 post injection and concentration of A尾 protofibrils in brain tissue of tg-ArcSwe mice. Each diamond represents one animal. (d) Total A尾40 and A尾42 levels in brain tissue from tg-ArcSwe mice of different ages. (e) Nuclear track emulsion and CD31 staining. The fusion protein was not accumulated in capillaries (red arrows) but to a large extent reached the brain parenchyma (black arrows). (f) Nuclear track emulsion and Congo staining revealed that the fusion protein was also located around insoluble amyloid deposits. Scale bar, 50鈥壩糾. Each symbol represents one animal, the line and error bars (a鈥?b>d) indicate group mean卤s.d. ***P 0.001 and NS is nonsignificant by two-way analysis of variance followed by Bonferroni鈥檚 post hoc test (a). Representative images from triplicate experiments are shown in e and f.Full size imageTable 1 Ex vivo brain concentration of [125I]8D3-F(ab鈥?2-h158 at 72鈥塰 post injection.Full size tableIn vivo PET imaging of A尾 pathology in transgenic miceNext, to evaluate the fusion protein as a PET ligand, it was labelled with 124I, a positron-emitting radionuclide with a half-life of 4.2 days. Tg-ArcSwe and WT mice of different ages (4, 8, 12 and 18 months) as well as tg-Swe mice (12 and 18 months), with a delayed age at onset of pathology compared with tg-ArcSwe29,42, were injected i.p. with 鈭?/span>15鈥塎Bq [124I]8D3-F(ab鈥?2-h158 and PET scanned for 60鈥塵in 72鈥塰 post injection. A尾 pathology could be clearly visualized in the 12- and 18-month-old tg-ArcSwe mice while there was no signal in the WT mice (Fig. 5a). In tg-Swe mice, no specific signal was recorded at 12 months, whereas a strong signal was seen at 18 months, confirming that the signal seen in tg-ArcSwe mice was not an effect of the Arctic A尾PP mutation. In a subset of the animals, ex vivo autoradiography was performed, as a comparison to the results of brain distribution seen in the PET images. Clinical PET data, including studies using [11C]PIB, is often quantified as the relative concentration of the PET ligand in a region of interest to that of a reference region. When analysing the present PET data from the whole brain, cortex, hippocampus, striatum and thalamus using the cerebellum as a reference region, the obtained ratio followed the disease progression in all studied regions (Fig. 5b). The cerebellum displayed a low PET signal in all animals at all ages (Fig. 5a) confirming its suitability as a reference region. Deiodination of the fusion protein, measured in plasma after PET scanning, was 5%.Figure 5: Fusion protein PET imaging in transgenic and WT mice.(a) Comparison of PET images obtained during 60鈥塵in, 72鈥塰 post injection of [124I]8D3-F(ab鈥?2-h158 from representative WT, tg-ArcSwe and tg-Swe mice of different ages, demonstrating the progression of A尾 pathology. Ex vivo autoradiography brain images from the same animals are displayed above PET images for comparison of brain distribution. (b) PET image-based quantification of brain distribution of the fusion protein relative to that in cerebellum for WT, tg-ArcSwe, tg-Swe and tg-伪-syn mice in the different age groups (each symbol represents one animal). (c) PET image of 18-month-old tg-伪-syn mouse, 72鈥塰 post injection of [124I]8D3-F(ab鈥?2-h158. (d) PET images obtained with the irrelevant [124I]8D3-F(ab鈥?2-Synagis 72鈥塰 post injection, with ex vivo autoradiography brain images from the same animals displayed above, demonstrating low and equal brain uptake in 18-month-old WT and tg-ArcSwe mice. Representative PET images are shown in a,c and d. Number of animals included in each group is shown in b.Full size imageTo verify that neurodegenerative pathology characterized by protein aggregation per se did not lead to increased retention of [124I]8D3-F(ab鈥?2-h158, 18-month-old (Thy-1)-h[A30P]伪-synuclein transgenic mice (hereafter referred to as tg-伪-syn) were also PET scanned according to the same protocol. The tg-伪-syn mice, like the WT mice, displayed no signal in the brain (Fig. 5c). In addition, to confirm that the differences in brain retention observed between AD and WT mice were not due to increased binding of the 8D3 moiety in the brains of tg-ArcSwe and tg-Swe mice, 18-month-old tg-ArcSwe and WT mice were injected with 鈭?/span>15鈥塎Bq of the irrelevant fusion protein [124I]8D3-F(ab鈥?2-Synagis and PET scanned for 60鈥塵in 72鈥塰 post injection. No accumulation in brain could be observed in either mouse type (Fig. 5d), and when quantified, the concentration ratios in all studied brain regions in relation to cerebellum were around 1 (Fig. 5b), confirming that the higher brain concentrations observed with [124I]8D3-F(ab鈥?2-h158 in tg-ArcSwe and tg-Swe compared with WT mice were indeed due to the A尾 protofibril-binding moiety F(ab鈥?2-h158.PET imaging correlates with soluble A尾 protofibrilsBrains from the mice subjected to PET imaging were analysed for Tris-buffered saline (TBS)-soluble A尾 protofibrils and total (formic acid-soluble) A尾40 and A尾42. Cerebellum and the rest of the brain were analysed separately to enable a direct comparison with PET results. As displayed in Fig. 6a, cerebellum contained overall very low levels of A尾 protofibrils, but more importantly, no increase was seen with increased age in either tg-ArcSwe or tg-Swe mice in direct analogy with PET results. In the rest of the brain, A尾 protofibril levels increased with age after 8 months in tg-ArcSwe, showing elevated levels at 12 months, that is, at the same age as A尾 was detectable with PET, that were further increased at 18 months. The same pattern was observed for the tg-Swe animals, although the increase was seen between 12 and 18 months. Hence, there was no overlap in A尾 protofibril levels between PET-positive and PET-negative animals, which allows to define a PET detection limit completely separating the groups (Fig. 6a). For total A尾40 and A尾42, the pattern was different in tg-ArcSwe mice, with a marked increase in brain A尾 concentrations already at 8 months, continuing up to 12 months and then forming a plateau. Interestingly, unlike soluble A尾 protofibril levels, total A尾40 and A尾42 levels increased with age also in the cerebellum, though with several months delay (Fig. 6b,c). The tg-Swe model showed a large increase in total A尾 at 18 months compared with 12 months, but low levels in the cerebellum at both ages, possibly because of its later onset of A尾 pathology (Fig. 6b,c). For comparison with the PET data, a brain/cerebellum ratio of the three different A尾 species was plotted against the brain/cerebellum PET ratio described above (Fig. 6d鈥揻). The tg-Swe animals were excluded from the A尾40 and A尾42 ratio graphs since they had extremely high ratios, caused by the low A尾 levels in cerebellum. Whereas a strong correlation was seen between the A尾 protofibril and PET ratios, the total A尾 ratios did not correlate with PET data.Figure 6: A尾 levels in brain and cerebellum and correlation with PET data.(a) A尾 protofibril (PF) levels in brain tissue obtained from tg-ArcSwe (n=13) and tg-Swe (n=4) mice of different ages; total A尾40 (b) and A尾42 (c) levels in brain and cerebellum from tg-ArcSwe and tg-Swe mice of different ages; Pearson鈥檚 correlation analysis of brain/cerebellum PET ratio and brain/cerebellum concentration ratio of soluble A尾 protofibrils in tg-ArcSwe and tg-Swe mice (d) and of total A尾40 (e) and A尾42 (f) in tg-ArcSwe mice. Each symbol represents one animal previously subjected to PET scanning; the line and error bars (a鈥?b>c) indicate group mean卤s.d.Full size imageComparison with [11C]PIBA subset of the mice that underwent PET scanning with the fusion protein was also imaged with [11C]PIB to compare the two ligands in mice with different A尾 pathology (Fig. 7). The 8- and 12-month-old transgenic animals showed similar [11C]PIB retention in brain as WT animals. The 18-month-old tg-ArcSwe and tg-Swe mice showed some [11C]PIB retention, mainly in cortical regions, but the brain/cerebellum ratio was in general lower than the ratio obtained with the fusion protein and did not reflect the disease progression to the same extent as the fusion protein did. Radioactivity concentration (corrected for injected dose) of [11C]PIB and [124I]8D3-F(ab鈥?2-h158 was similar in 18-month-old tg-ArcSwe brain (Fig. 7a), while tg-Swe mice showed 60% higher concentration of [124I]8D3-F(ab鈥?2-h158 than of [11C]PIB (Fig. 7b).Figure 7: Comparison of PET imaging with [11C]PIB- and 124I-labelled fusion protein in transgenic and WT mice.PET images obtained during 60鈥塵in 72鈥塰 after injection of [124I]8D3-F(ab鈥?2-h158, or during 20鈥塵in, starting 40鈥塵in after injection of [11C]PIB. Transverse and sagittal views of one representative 18-month-old tg-ArcSwe (a), tg-Swe (b) and WT (c) mouse with the two radioligands. (d) PET image-based quantification of brain distribution of [11C]PIB relative to that in cerebellum for tg-ArcSwe, tg-Swe and WT mice in the different age groups (each symbol represents one animal). Representative PET images are shown in a鈥?b>c. Number of animals included in each group is shown in d.Full size imageDiscussionSeveral new drug compounds intended for treatment of AD have entered clinical phase II and III studies, but there are limited possibilities to measure their effects on the molecular level in vivo. All existing amyloid PET radioligands bind to the 尾-sheet structure of insoluble fibrillar A尾. Evidence today points to soluble forms of aggregated A尾 being better correlated with disease severity and soluble A尾 oligomers and protofibrils are likely to cause the synaptic failure that eventually leads to dementia. The present study shows for the first time an in vivo PET image of A尾 pathology acquired with an antibody-based radioligand (Fig. 5). This was achieved by a combination of TfR-mediated transcytosis to increase BBB penetration and the use of a well-characterized A尾 protofibril selective antibody, which ensures that A尾 aggregates are selectively targeted. Although mAb158 binds, albeit with a lesser affinity, to A尾 fibrils and monomers in vitro25,27 (Fig. 1c), evidence suggests that it primarily targets soluble A尾 protofibrils in vivo. When administered to tg-ArcSwe mice with plaque pathology, mAb158 selectively reduced brain levels of A尾 protofibrils, without altering the plaque pathology43, unlike other A尾 antibodies of the same isotype44. This is probably because soluble A尾 protofibrils, which are already favoured by mAb158, are more accessible to the antibody when it enters the brain parenchyma than the insoluble fibrils deposited in plaques. This is also reflected in Fig. 4e,f, where in vivo administered fusion protein appears in the parenchyma and around the periphery of plaques, where A尾 oligomerization has been reported to occur41.Further, the PET signal and brain retention of the generated bispecific fusion protein increased with age (Fig. 5), that is, with progression of A尾 pathology in the tg-ArcSwe and tg-Swe mouse models. Tg-ArcSwe mice exhibit very dense A尾 plaque pathology, similar to that of the human AD brain, with an onset around 6 months of age42, and an almost linear increase of soluble A尾 protofibrils with age (Figs 4 and 6). Tg-Swe mice display less dense plaques42 with a later onset and a more rapid increase in A尾 pathology (Fig. 6). Hence, at 12 months of age, higher concentrations of [124I]8D3-F(ab鈥?2-h158 was observed in the brains of tg-ArcSwe compared with tg-Swe mice, while the opposite was observed at 18 months. The difference between the two animal models was also evident in the [11C]PIB scans. [11C]PIB binds poorly to the loose unstructured plaques in tg-Swe mice brain and thus [11C]PIB concentrations were lower than [124I]8D3-F(ab鈥?2-h158 in tg-Swe mice, while the concentration of the two radioligands was similar in tg-ArcSwe brain. Brain retention of the fusion protein correlated closely with brain concentrations of soluble A尾 protofibrils in both animal models (Fig. 4), while total A尾 levels, reflecting the plaque pathology, increased at a higher rate, suggesting that the fusion protein preferably binds to soluble A尾 protofibrils. Furthermore, low radioactivity was detected with PET in the cerebellum in all age groups, while it increased with age in the rest of the brain. Similarly, while the level of soluble A尾 protofibrils increased with age in the whole brain, cerebellum displayed a low and constant level of protofibrils over time. Thus, the PET brain/cerebellum radioactivity concentration ratio, which is often used in clinical PET as a read-out measure, correlated with the brain/cerebellum ratio of soluble A尾 protofibrils (Fig. 6). Such a correlation was not seen for total A尾, since both A尾40 and A尾42 increased with age in the cerebellum. These results do not per se exclude that the radioligand may also to some extent bind to fibrillar A尾 in the brain, possibly in the shape of diffuse deposits.Taken together, our findings suggest that brain retention of the fusion protein measured with PET reflects the progression of A尾 pathology. This approach could become an important diagnostic tool to predict disease stage in AD patients, likely including the group of patients displaying mainly diffuse plaque pathology45 that are diagnosed falsely as non-AD with the available amyloid PET radioligands46. Treatment strategies to reduce soluble A尾 aggregates are today studied in late-phase clinical trials, but biomarkers for evaluating the effects of such treatments need to be improved. A PET radioligand visualizing soluble A尾 aggregates could bring substantial benefit in assessing emerging A尾-reducing treatments.To our knowledge this is the first successful study using an antibody-based PET ligand for a CNS application. Since antibodies are very specific binders, the drawback of large unspecific binding often experienced with small molecular PET ligands (including [11C]PIB and analogues) will most likely be avoided with antibody-based ligands. This study therefore also demonstrates the feasibility of antibody-based in vivo imaging of proteins involved in other neurodegenerative disorders, for example, Parkinson鈥檚 disease or frontotemporal lobar degeneration, for which small molecular PET ligands are currently lacking.MethodsAntibody fragmentationF(ab鈥?2-h158 was generated by cleavage of a humanized variant, BAN2401, of the mouse monoclonal antibody mAb158, selectively binding to A尾 protofibrils, that is, soluble A尾 aggregates larger than about 100鈥塳D, eluting in the void volume on a Size Exclusion Superdex 75 column25. The bacterial enzyme IdeS (ref. 47), manufactured and distributed as FabRICATOR (Genovis AB, Lund, Sweden), was used to cleave the antibody. This enzyme cleaves human IgG at a specific site just below the hinge region, producing a homogenous preparation of F(ab鈥?2 fragments. A F(ab鈥?2 fragment of an antibody against respiratory syncytial virus, Synagis (Palivizumab; 530300, Apoteket AB, Solna, Sweden/MedImmune, Gaithersburg, MD, USA), was generated according to the same method. Fab-8D3 was generated from the TfR antibody 8D3 (ref. 36) (MCA2474, AbD Serotec, Oxford, UK) by papain cleavage, according to the manufacturer鈥檚 protocol (Pierce, Rockford, IL, USA).All fragments were purified with CaptureSelect Fc (multi-species) Affinity Resin (Thermo Fisher Scientific, Stockholm, Sweden) to remove Fc fragments and non-cleaved antibody from the preparation. Purity and size of the fragments were evaluated with SDS鈥揚AGE under non-reducing conditions. Briefly, samples were mixed with Laemmli buffer, loaded onto a Novex pre-cast 10鈥?0% Tris-tricine polyacrylamide gel (Invitrogen, Carlsbad, CA) and run at 125鈥塚 for 90鈥塵in, followed by 3 脳 15-min wash in water, and staining with Page Blue (Fermentas, Vilnius, Lithuania) according to the manufacturer鈥檚 instructions.Generation of bispecific fusion proteinsF(ab鈥?2-h158 or F(ab鈥?2-Synagis and 8D3 were chemically conjugated with the Solulink technology (Solulink protein conjugation kit; Solulink, San Diego, CA, USA), where each of the conjugated proteins is modified with one of two linkers, which bind specifically to each other to form a permanent bond. F(ab鈥?2-h158 or F(ab鈥?2-Synagis (3.0鈥塵g鈥塵l鈭?) was labelled with succinimidyl-4-formylbenzamide (S-4FB) using a 4.5-fold molar excess, and 8D3 (3.0鈥塵g鈥塵l鈭?) was labelled with the complimentary succinimidyl-6-hydrazino-nicotinamide (S-HyNic) using a 6-fold molar excess. The labelled proteins were mixed in a 1.5:1 (F(ab鈥?2:8D3) molar ratio and reacted for 2鈥塰 in room temperature in the presence of 10鈥塵M aniline, which catalyses the reaction. To purify the fusion protein, the preparation was incubated for 1鈥塰 with CaptureSelect Fc (multi-species) Affinity Matrix to specifically deplete the preparation of unconjugated F(ab鈥?2 fragments, which lack the Fc domain. After elution from the resin with 0.1鈥塎 glycine-HCl, pH 2.5, the preparation was neutralized with 1鈥塎 Tris and incubated for 1鈥塰 with CaptureSelect IgGCH1 (human) Affinity Matrix (Thermo Fisher Scientific), which specifically binds to the constant domain 1 of human IgG heavy chain, thus depleting the sample of unconjugated 8D3. The purified fusion protein was eluted with 0.1鈥塎 glycine-HCl, pH 2.5, and analysed with SDS鈥揚AGE as above.A尾 inhibition ELISATo assess the 8D3-F(ab鈥?2-h158 fusion protein鈥檚 binding to different A尾 species in solution, an inhibition ELISA was performed as previously described25. The 96-well plates (Corning Inc., Corning, NY, USA) were coated at +4鈥壜癈 for 2鈥塰, with 45鈥塶g per well of A尾 protofibrils and blocked for 1鈥塰 with 1% bovine serum albumin (BSA) in PBS. In the meantime, the fusion protein (0.5鈥塶M) was incubated with serially diluted A尾 monomers, protofibrils or fibrils in a non-binding 96-well plate (Greiner, Kremsm眉nster, Austria) for 1鈥塰 on a shaker and then transferred to the A尾 protofibril-coated plate, where it was incubated for 15鈥塵in. Fusion protein bound to the plate was detected by a 1-h incubation with 1:2,000 diluted horseradish peroxidase (HRP)-conjugated anti-human-IgG-F(ab鈥?2 (109-036-006, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Signals were developed with K blue aqueous TMB substrate (Neogen Corp., Lexington, KY, USA) and read with a spectrophotometer at 450鈥塶m. All A尾 and antibody dilutions were made in ELISA incubation buffer (PBS with 0.1% BSA, 0.05% Tween and 0.15% Kathon). A尾 preparations were made as previously described27 and the (insoluble) fibril preparation was subjected to a brief sonication (30鈥塻) before the analysis to break-up large fibril assemblies and ensure it would stay in suspension.TfR鈥揂尾 protofibril ELISAThe generated 8D3-F(ab鈥?2-h158 fusion protein was tested with ELISA for retained binding to TfR and A尾, respectively, before and after radioiodination. The 96-well plates (Corning Inc.) were coated at +4鈥壜癈 overnight with 10鈥塶g per well of murine TfR (Sinobiological, Beijing, China) or 25鈥塶g per well of streptavidin (Sigma, St. Louis, MO, USA). After 1鈥塰 blocking with 1% BSA in PBS, biotinylated A尾 protofibrils, 4.5鈥塶g per well, prepared as previously described27, were added to streptavidin-coated wells and incubated for 30鈥塵in on a shaker. A serial dilution of the fusion protein, 125I-labelled or non-labelled, was added to TfR- or streptavidin/A尾 protofibril-coated wells, incubated for 2鈥塰 on a shaker and detected by a 1-h incubation with HRP-conjugated 1:2,000 diluted anti-mouse-IgG-F(ab鈥?2 (TfR coat) or anti-human-IgG-F(ab鈥?2 (streptavidin/A尾 protofibril coat) (115-036-006 and 109-036-006, Jackson ImmunoResearch Laboratories). Signals were developed and read as above. All antibody dilutions were made in ELISA incubation buffer. The 8D3-F(ab鈥?2-Synagis fusion protein was tested for retained binding to TfR according to the same method.AnimalsThree transgenic models maintained on a C57BL/6 background were used: the tg-ArcSwe model harbouring the Arctic (A尾PP E693G) and Swedish (A尾PP KM670/671NL) mutations; the tg-Swe model with only the Swedish mutation; and tg-伪-syn mice. Tg-ArcSwe mice show elevated levels of soluble A尾 protofibrils already at a very young age and abundant and rapidly developing plaque pathology starting at around 6 months of age29,42,48. Tg-Swe mice have a later onset of plaque pathology starting at 10鈥?2 months of age and then increasing with age42. Both males and females were used and littermates were used as control animals (WT). As further controls, tg-伪-syn mice displaying overexpression of human 伪-synuclein (protein involved in Parkinson鈥檚 disease)49 were used in the PET experiments. The animals were housed with free access to food and water in rooms with controlled temperature and humidity in an animal facility at Uppsala University.RadiochemistryDirect radioiodination of the four proteins F(ab鈥?2-h158, 8D3, 8D3-F(ab鈥?2-h158 and 8D3-F(ab鈥?2-Synagis with iodine-125 (125I) for ex vivo experiments and iodine-124 (124I) for PET experiments was performed using Chloramine-T (ref. 50). The method is based on electrophilic attack of the phenolic ring of tyrosine residues by in situ oxidized iodine. Briefly, for 125I labelling, 250 pmoles of antibody/fragment or 65 pmoles of fusion proteins (assumed Mw (molecular weight) 270鈥塳Da), 125I stock solution (PerkinElmer Inc., Waltham, MA, USA) and 5鈥壩糶 Chloramine-T (Sigma Aldrich, Stockholm, Sweden) were mixed in PBS to a final volume of 110鈥壩糽. The reaction was allowed to proceed for 90鈥塻 and subsequently quenched by addition of double molar excess of sodium metabisulfite (Sigma Aldrich) and dilution to 500鈥壩糽 in PBS. Fab-8D3 had to be modified with Bolton Hunters reagent (Sulfo-SHPP)51 (Pierce) before radioiodination to introduce extra phenolic rings for the iodine to target. Fab-8D3 (1鈥塵g鈥塵l鈭? in PBS) was incubated for 30鈥塵in with 100 脳 molar excess of sulfo-SHPP (50鈥塵M in H2O) and purified from unbound sulfo-SHPP with a Zeba mini desalting column, Mw cutoff 7鈥塳Da (Pierce). Modified Fab-8D3 was radiolabelled as above.For 124I labelling, 58鈥壩糽 124I stock solution (Perkin-Elmer Inc.) was pre-incubated for 10鈥塵in with 12鈥壩糽 50鈥壩糓 NaI, before addition of 260 pmoles of fusion proteins and 40鈥壩糶 Chloramine-T in PBS to a final volume of 450鈥壩糽. The reaction was allowed to proceed for 120鈥塻 and subsequently quenched by addition of 80鈥壩糶 of sodium metabisulfite in PBS. The radiolabelled proteins were purified from free iodine and low-molecular weight components with a disposable NAP-5 size exclusion column, Mw cutoff 5鈥塳Da (GE Healthcare AB, Uppsala, Sweden), according to the manufacturer鈥檚 instructions, and eluted in 1鈥塵l of PBS. The yield was calculated based on the added radioactivity and the radioactivity in the purified radioligand solution. Labelling was always performed 2鈥塰 before each study. Affinity for A尾 protofibrils and/or TfR was tested with ELISA on the same day as the labelling and the start of the study.[11C]PIB was synthesized as previously described1.Injected radioactivity, specific activity, labelling reaction yields for F(ab鈥?2-h158, 8D3, Fab-8D3, 8D3-F(ab鈥?2-h158, 8D3-F(ab鈥?2-Synagis and [11C]PIB are given in Table 2.Table 2 Radioligand concentrations and injected doses.Full size tableDeiodination of [124I]8D3-F(ab鈥?2-h158 was assessed in the injection solution before the experiment and in plasma samples from injected animals after PET experiments, by separation with Zeba mini desalting columns, where free iodine was trapped in the column.Ex vivo studiesMice were anaesthetized with isoflurane at 2, 4, 24 or 72鈥塰 after a single i.p. injection of [125I]F(ab鈥?2-h158, [125I]8D3, [125I]Fab-8D3, [125I]8D3-F(ab鈥?2-h158 or 8D3-F(ab鈥?2-Synagis. A blood sample was obtained from the heart followed by intracardiac perfusion with 50鈥塵l physiological saline during 2鈥塵in. Following perfusion, brains were isolated and the left hemisphere immediately frozen. The right hemisphere was either immediately frozen (for ex vivo autoradiography) or left to incubate in 4% paraformaldehyde for 24鈥塰, before it was immersed in a sucrose gradient (10, 20 and 30%) in PBS for cryoprotection (for immunohistochemistry) before being sectioned on a cryostat at 鈭?0鈥壜癈.In addition to the terminal blood samples obtained in all animals, blood samples (8鈥壩糽) were obtained from the tail vein for a subset of animals also at 0.5, 1, 2, 3, 4, 6, 8, 24, 48 and 72鈥塰 after injection.Radioactivity in blood samples and in the frozen brain hemisphere was measured with a 纬-counter (1480 Wizard, Wallac Oy, Turku, Finland). The brain and blood concentrations, quantified as % ID per g tissue, were calculated as follows:% ID per g=measured radioactivity per gram brain tissue (or blood)/injected radioactivity.In addition, the brain-to-blood (Kp) concentration ratio was calculated as follows:Kp=measured radioactivity per gram brain tissue/measured radioactivity per gram blood.The number of transgenic and WT animals included in the ex vivo and PET studies and injected radioactivities of the different radioligands are given in Table 2. Values for % ID per g and Kp are summarized in Table 1.Positron emission tomographyIn vivo brain distribution of [124I]8D3-F(ab鈥?2-h158 in tg-ArcSwe (n=13), tg-Swe (n=4), WT (n=13) and tg-伪-syn (n=4) mice and of [124I]8D3-F(ab鈥?2-Synagis in tg-ArcSwe (n=2) and WT (n=1) was visualized with PET during 60鈥塵in 72鈥塰 post i.p. administration of respective radioligand. The day before injection of 124I-labelled fusion protein, animals were given water supplemented with 0.2% NaI to reduce thyroidal uptake of 124I. A subset of the animals, tg-ArcSwe (n=6), tg-Swe (n=4) and WT (n=4), were investigated with PET during 20鈥塵in, starting 40鈥塵in after intravenous administration of [11C]PIB, 1 week before the [124I]8D3-F(ab鈥?2-h158 scans.At every scanning occasion, the animal was placed in the gantry of the animal PET/computed tomography (CT) scanner (Triumph Trimodality System, TriFoil Imaging, Inc., Northridge, CA, USA) and scanned in list mode followed by a CT examination for 3鈥塵in (field of view=8.0鈥塩m). Mice were scanned in a random order.The PET data were reconstructed using a maximum likelihood expectation maximization (MLEM) two-dimensional algorithm (10 iterations). The CT raw files were reconstructed using filter back projection. All subsequent processing of the PET and CT images were performed in imaging software Amide 1.0.4 (ref. 52). The CT scan was manually aligned with a T2-weighted, magnetic resonance imaging-based mouse brain atlas53 containing outlined regions of interests for hippocampus, striatum, thalamus, cortex and cerebellum. The PET image was then aligned with the CT, and thus, the magnetic resonance imaging atlas was also aligned with the PET data. The PET data shown in Figs 5a,c,d and 7a鈥揷 are summed images, that is, representing the average activity during the whole PET scan. The PET data were quantified as a concentration ratio of the radioactivity in five regions of interest (whole brain, cortex, hippocampus, thalamus and striatum) to that in cerebellum.Biochemical and histopathological analysesBrain concentrations of soluble A尾 and total A尾 were measured as described previously18. In short, mice were saline perfused and the left hemisphere of each animal was homogenized at a 1:5 weight:volume ratio in TBS with complete protease inhibitors (Roche), using a tissue grinder with Teflon pestle (2 脳 10 strokes on ice). A volume of 400鈥壩糽 of each sample was mixed with 400鈥壩糽 TBS and centrifuged for 1鈥塰 at 16,000g to obtain a preparation of soluble proteins. The supernatants were aliquoted and stored at 鈭?0鈥壜癈 until analysis. To obtain a preparation including insoluble proteins found in amyloid plaques, 270鈥壩糽 of the original TBS extract was mixed with 730鈥壩糽 of concentrated formic acid, to a final formic acid concentration of 70%, followed by homogenization and centrifugation as above.In the A尾 protofibril ELISA, 96-well plates were coated overnight with 200鈥塶g per well mAb158, and blocked with 1% BSA in PBS. TBS extracts were diluted 1:25 and incubated overnight at +4鈥壜癈, followed by detection with biotinylated mAb158 (0.5鈥壩糶鈥塵l鈭?) and streptavidin-HRP (1:2,000; Mabtech AB). Signals were developed with K blue aqueous TMB substrate (Neogen Corp., Lexington, KY, USA) and read with a spectrophotometer at 450鈥塶m.For ELISA measurement of A尾x-40 and A尾x-42, 96-well plates were coated overnight with 100鈥塶g per well of polyclonal rabbit anti-A尾40 or anti-A尾42 (Agrisera, Ume氓, Sweden), and blocked with 1% BSA in PBS. Formic acid extracts were neutralized with 2鈥塎 Tris and diluted 500鈥?0,000 times depending on A尾 content and incubated overnight at +4鈥壜癈, followed by detection with biotinylated mAb1C3 (0.5鈥壩糶鈥塵l鈭?)25,26 and streptavidin-HRP (1:5,000; Mabtech AB). Signals were developed and read as above. All sample and secondary antibody dilutions were made in ELISA incubation buffer (PBS with 0.1% BSA, 0.05% Tween and 0.15% Kathon).CD31 immunohistochemistry and nuclear track emulsion autoradiography were performed on semi-adjacent sections (20鈥壩糾) of the right hemisphere, immunostained for the endothelial marker CD31. First, sections were washed in PBS and incubated with 3% H2O2 and 10% methanol in water for 15鈥塵in. Nonspecific binding was then blocked using 3% BSA in PBS-Tween (0.1%) for 1鈥塰, followed by an overnight incubation with 0.5鈥壩糶鈥塵l鈭? rat anti-mouse CD31 (550274, BD Biosciences, San Jose, CA, USA) at 4鈥壜癈. The sections were then incubated with 5鈥壩糶鈥塵l鈭? biotinylated goat anti-rat (BA-9400, Vector Laboratories Inc., Burlingame, CA) for 1鈥塰 at room temperature, followed by PBS washes and a 45-min incubation with avidin/biotin complex (Vector Laboratories). The staining was then visualized with a 3-min 3,3鈥?diaminbenzidine (DAB) development, for some sections followed by immersion in Congo red for 45鈥塵in. Sections were then dehydrated in ethanol (70, 95 and 100%) and left to air dry.Immediately after CD31 and Congo staining, sections were immersed for 3鈥塻 in Ilford K5 emulsion (40鈥壜癈), left to air dry for 2鈥塰 and stored at +4鈥壜癈 protected from light. Sections were then developed with Ilford photographic reagents, briefly counterstained with haematoxylin, dehydrated with ethanol and xylene, and mounted with DPX mounting medium.Ex vivo autoradiographyAfter PET imaging, a subset of the [124I]8D3-F(ab鈥?2-h158-injected mice and the [124I]8D3-F(ab鈥?2-Synagis-injected mice was selected for ex vivo autoradiography directly after PET imaging. Following saline perfusion, the right hemisphere was instantly frozen and cryosectioned (20鈥壩糾). Two sections from each animal and 124I-abelled standards of known radioactivity were placed in an X-ray cassette and exposed to positron-sensitive phosphor screens (MS, MultiSensitive, PerkinElmer, Downers grove, IL, USA) for 4 days. The plates were scanned in a Cyclone Plus Imager system (Perkin Elmer) at a resolution of 600 dots per inch. The resulting digital images were normalized to the standards and converted to a false colour scale (Royal) with ImageJ for comparison with PET images.StatisticsResults reported are presented as mean卤s.d. Data were analysed with two-way analysis of variance followed by Bonferroni鈥檚 post hoc test. In cases where the variance was different in the different groups (variance was in general larger in tg-ArcSwe mice than in WT mice), data were log-transformed before analysis to adhere with the analysis of variance assumption of equal variance. 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Ed茅n for technical and experimental assistance, to BioArctic Neuroscience AB for providing a F(ab鈥?2 fragment of the humanized variant (BAN2401) of mAb158 and to P. O鈥機allaghan for comments on the manuscript. This work was supported by grants from the Swedish Research Council (#2012-1593, #2012-2172), Alzheimerfonden, Hj盲rnfonden, Svenska Lundbeck-stiftelsen, 脜hl茅n-stiftelsen, Stohnes stiftelse and Uppsala Berzelii Technology Centre for Neurodiagnostics.Author informationAffiliationsDepartment of Public Health and Caring Sciences/Geriatrics, Uppsala University, Rudbeck Laboratory, Uppsala, 75185, SwedenDag Sehlin,聽Xiaotian T. Fang,聽Linda Cato,聽Lars Lannfelt聽 聽Stina Syv盲nenDepartment of Medicinal Chemistry, Preclinical PET Platform, Uppsala University, Uppsala, 75123, SwedenGunnar AntoniPET Centre, Uppsala University Hospital, Uppsala, 75185, SwedenGunnar AntoniAuthorsDag SehlinView author publicationsYou can also search for this author in PubMed聽Google ScholarXiaotian T. FangView author publicationsYou can also search for this author in PubMed聽Google ScholarLinda CatoView author publicationsYou can also search for this author in PubMed聽Google ScholarGunnar AntoniView author publicationsYou can also search for this author in PubMed聽Google ScholarLars LannfeltView author publicationsYou can also search for this author in PubMed聽Google ScholarStina Syv盲nenView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsD.S. and S.S. designed the project; L.L. and G.A. contributed to the study design; D.S. generated the fusion protein and performed in vitro binding experiments; D.S., X.T.F., L.C. and S.S. performed all brain uptake and PET studies; X.T.F. performed immunohistochemistry experiments; D.S. and S.S. analysed data and wrote the manuscript, with valuable input from X.T.F., L.C., G.A. and L.L.Corresponding authorCorrespondence to Stina Syv盲nen.Ethics declarations Competing interests L.L. is a founder of BioArctic Neuroscience and has shares in the company. The remaining authors declare no competing financial interests. Rights and permissions This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article鈥檚 Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and PermissionsAbout this articleCite this articleSehlin, D., Fang, X., Cato, L. et al. Antibody-based PET imaging of amyloid beta in mouse models of Alzheimer鈥檚 disease. Nat Commun 7, 10759 (2016). https://doi.org/10.1038/ncomms10759Download citationReceived: 11 June 2015Accepted: 19 January 2016Published: 19 February 2016DOI: https://doi.org/10.1038/ncomms10759 Rebecca Faresj枚, Gillian Bonvicini, Xiaotian T. Fang, Ximena Aguilar, Dag Sehlin Stina Syv盲nen Fluids and Barriers of the CNS (2021) Klara Kulenkampff, Adriana-M. Wolf Perez, Pietro Sormanni, Johnny Habchi Michele Vendruscolo Nature Reviews Chemistry (2021) Takahiro Morito, Ryuichi Harada, Ren Iwata, Yiqing Du, Nobuyuki Okamura, Yukitsuka Kudo Kazuhiko Yanai Scientific Reports (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.