The two affibody binders used in this study, ZAβ42cc_1-His6 and ZAβ42cc_5-His6 were generated toward protofibrils of Aβ42CC, an engineered mimic of wild-type Aβ42 protofibrils (described in ref. 39) (39). The affibodies displayed affinities in the low nanomolar range for Aβ42CC protofibrils (KD = 1.7 ± 0.6 nM), determined by surface plasmon resonance, and also detected wild type Aβ42 protofibrils in ELISA (39).
In the present study, each of these two constructs were genetically fused to a BBB-shuttle, a single-chain fragment variable (scFv) of 8D3 (see “Cloning and protein production”). The four affibody constructs are hereafter referred to as Z5, scFv8D3-Z5, Z1 and scFv8D3-Z1 in this study (Table I). The measured molecular masses of Z5, scFv8D3-Z5, Z1 and scFv8D3-Z1 were in agreement with the expected theoretical values (Supplementary Fig. 1). However, the sample with scFv8D3-Z5 contained one additional protein of unknown origin, which we failed to remove in the purification (Supplementary Fig. 1). scFv8D3-Z5 was still included in the study with the assumption that the additional protein would not interfere with the functionality of scFv8D3-Z5, as binding to Aβ was retained indicated by Aβ protofibril ELISA. The aim was to study brain uptake and binding to Aβ in the brain of the affibodies and fused affibodies.
Cloning and Production of Proteins
The genetic sequences for the scFv8D3-fusion proteins were cloned into the pQMCF1 vector (Icosagen Cell Factory OU, Tartu, Estonia) (40), containing a CMV promoter and an N-terminal CD33 signal peptide for secretion of the produced proteins. The 8D3 sequence was formatted into a single-chain fragment variable in the heavy to light chain direction, and connected by an 18 amino-acid glycine/serine-rich flexible linker (NSSGTTAASGSSGGSSSGAC). The 8D3 scFv was linked via a 10 amino-acid linker (NGAPGGGGSTSC) to the N-terminus of either of the amyloid beta protofibril-binding affibody molecules Z1 and Z5, respectively. Genetic sequences for control affibody molecules Z1 and Z5 were cloned into the pET26b( +) vector (Novagen), introducing a C-terminal His6-tag. The resulting plasmids (pQMCF1[scFv8D3-Z1-His6], pQMCF1[scFv8D3-Z5-His6], pET26b( +)[Z1-His6] and pET26b( +)[Z5-His6] were sequence verified by Sanger sequencing (Microsynth AG, Balgach, Switzerland).
pQMCF1 plasmids, containing the scFv8D3-fused proteins, were transfected into Chinese Hamster Ovary (CHO) EBNALT 85 cells for production using the Icosagen QMCF technology (40). Cells were cultivated for 13 days, followed by recovery of the fusion proteins from the cell culture supernatants. pET26b( +) vectors, containing the genes encoding non-scFv-fused proteins were transformed by heat shock into Escherichia coli BL21 Star (DE3) cells (Novagen, Madison, WI, USA) for production, according to preciously described protocol (41). Overnight-cultivated cells were harvested by centrifugation and lysed by sonication. All proteins were purified by immobilized metal affinity chromatography (IMAC) using a HisPur™ Cobalt resin (Thermo Fisher Scientific, Rockford, USA) under native conditions. Eluted proteins were buffer-exchanged to PBS using PD-10 desalting columns (GE Healthcare Life Sciences, Uppsala, Sweden). The protein concentration was determined by absorbance measurement at 280 nm. The molecular weight and purity of the purified proteins were subsequently confirmed using SDS-PAGE (NuPAGE™ 4–12% Bis–Tris gels) and MALDI mass spectrometry (4800 MALDI-TOF).
All four affibodies were radiolabeled by direct iodination using the chloramine T method (42). Briefly, 20–40 μg affibody was mixed with 125I stock solution (Perkin-Elmer Inc Waltham, MA, USA). Thereafter, 5 μg Chloramine-T (Sigma Aldrich, Stockholm, Sweden) in PBS was added to a final volume of 110 μl. The reaction was incubated for 90 s at room temperature, and quenched with 10 μg sodium metabisulfite (Sigma Aldrich). The product was diluted to 500 μl with PBS, and separated from free-iodine in a PBS-equilibrated NAP-5 size exclusion column (Cytiva, Uppsala, Sweden). The product was eluted with a total volume of 1 ml PBS.
Pierce Pre-Coated Iodination Tubes (ThermoFischer, Rockford, IL, USA) was used as an alternative to the Chloramine T method to achieve milder reaction conditions (43). First, 1 ml PBS was used to wet the iodination tube. 125I stock solution (Perkin-Elmer) was added together with PBS to the iodination tube to a total volume of 40 μl. The tube was incubated for 6 min on a shaker at room temperature to activate the iodine. 10–20 μg affibody was prepared in Protein LoBind tubes (Eppendorf). Thereafter, 20 μl activated iodine was added to each affibody, followed by a 10 min incubation at room temperature on a shaker (600 rpm). After the incubation, the product was diluted with PBS to 500 μl and purified as described above.
In Vitro Validation of Radiolabeled Affibodies
The affibody binding to Aβ protofibrils and mTfR1 before and after radiolabeling was assessed with indirect ELISA in comparison with di-scFv3D6-8D3 (37). In short, 96-well half area plates (Corning Inc.) were coated with 250 nM Aβ-PF (Innovagen) in PBS or 2 µg/ml mTfR1 (BioArctic, Stockholm, Sweden) in PBS and incubated at 4°C overnight. The plates were blocked with 1% BSA in PBS. The affibodies were serially diluted from 250 nM (and di-scFv3D6-8D3 from 50 nM) and incubated overnight at 4°C. The Aβ-PF plates were incubated for 1 h with HRP-conjugated anti-His-Tag antibody (Proteintech Goup INC., IL, USA), while mTfR1 plates were incubated first for 1 h with Goat-Anti-Affibody IgG (Affibody AB, Stockholm, Sweden) followed by 1 h incubation with Rabbit-Anti-Goat-HRP (ThermoFischer). Signals were developed with K Blue Aqueous TMB substrate (Neogen Corp., Lexington, KY, USA) and the reaction stopped after 10 min with 1 M H2SO4. The plates were analyzed at 450 nm in a spectrophotometer. Affibody and secondary antibody sample dilutions were made in ELISA incubation buffer (PBS, 0.1% BSA, 0.05% Tween-20).
Sandwich ELISA was used to determine concentrations of affibodies after radiolabeling. Half area 96-well plates (Corning Inc.) coated with 0.5 μg/ml Goat-Anti-Affibody IgG (Affibody AB, Stockholm, Sweden) were incubated overnight in at 4°C. After 1 h of blocking with 1% BSA in PBS, affibody samples were serially diluted from 250 nM, followed by incubation overnight at 4°C. The plates were incubated with HRP-conjugated anti-His-Tag antibody (Proteintech) for 1 h incubation on a shaker. The signal was developed and plates read as described above.
Two AβPP transgenic mouse models were used in this study to investigate the in vivo retention of the affibody constructs: tg-Swe, harbouring the Swedish (APP KM670/671NL) APP mutation, and tg-ArcSwe with a combination of the Arctic (APP E693G) and the Swedish APP mutations (tg-ArcSwe), both maintained on a C57BL/6 background. The mutation in tg-Swe mice leads to increased production of Aβ, and a late onset (at 12 months) of Aβ pathology, followed by rapid progression. In tg-ArcSwe mice, increased Aβ production in combination with an aggregation-prone Aβ mutated species produces an earlier onset (at 6 months) of pathology with dense Aβ-plaques (44). These two models are suitable to study the variation in Aβ pathology, from plaques to soluble oligomers. Another reason to use both models was that tg-ArcSwe produces a mutated form of Aβ (Arctic mutation, within the Aβ sequence), while tg-Swe only produces wild-type Aβ (Swedish mutation, outside the Aβ sequence). C57BL/6 WT mice were used as control animals and to study brain delivery of the proteins. All animals were between 17–24 months old and both males and females (n = 67, f = 44 m = 19) were used for the experiments. The animals were housed in an approved animal facility at the Uppsala University with ad libitum access to food and water. All described procedures were approved by the Uppsala Country Animal Ethics board (5.8.18–13,350/17) following the legislation and regulations of the Swedish Animal Welfare Agency and European Communities Council Directive of 22 September 2010 (2010/63/EU).
In Vitro Autoradiography
Cryosections, 20 μm, were prepared from brains of old (2 years) wild type and tg-ArcSwe mice and mounted on Superfrost Plus glass slides (Menzel Gmboltion, Braunschweigh, Germany). The frozen sections were adjusted to room temperature for 1 h, and blocked with 1% BSA in PBS for 1 h. The sections were incubated in PBS for 5 min, followed by the addition 4 nM of [125I]I-Z5 or [125I]I-Z1 (0.9 MBq/nmol), and 1 nM of [125I]I-scFv8D3-Z5 or [125I]I-scFv8D3-Z1 (3–4 MBq/nmol) in PBS for overnight incubation at 4°C. The sections were washed 3 × 15 min in cold 0.1% BSA-PBS-buffer, 20 s in dH2O, and dried under constant air flow at RT for 1 h, then exposed to a phosphor imaging plate (MS, MultiSensitive, PerkinElmer, Downers Grove, IL, USA) for 3 h. The plate was scanned and digitalized at 600 dpi in a Cyclone Plus phosphor imager (PerkinElmer). Images were converted with the “Royal” lookup table and the intensity was adjusted individually for each protein in ImageJ. Regions of interest (ROIs) were quantified using the Integrated Density (IntDen) measurement in triplicate brain sections.
Ex Vivo Study 125I-Labeled Affibodies
Mice were intravenously injected with 125I-labeled affibodies via the tail vein. The animals were injected with either 0.49 ± 0.20 MBq of [125I]I-Z5, 0.43 ± 0.18 MBq of [125I]I-scFv8D3-Z5, 0.76 MBq ± 0.13 MBq of [125I]I-Z1 or 0.88 ± 0.34 MBq of [125I]I-scFv8D3-Z1. To investigate the blood pharmacokinetic profiles, blood samples of 8 μl were collected from the tail vein at time points between 30 min after administration and euthanization, i.e. at 2 h or 24 h post injection. At euthanization, transcardial perfusion under isoflurane anesthesia with 40 ml saline for 2.5 min was used to remove blood from the brain and organs before isolation. Subsequently, the brain was harvested and dissected into the right hemisphere, left cerebrum and left cerebellum. The brain samples were immediately frozen at -80°C. Lung, liver, kidney, heart, pancreas, spleen, femoral bone, skull bone and submandibular gland (smg) were isolated to study the biodistribution of the radiolabeled affibodies. The radioactivity of all samples was measured with a γ-counter (2480 Wizard™, Wallac Oy PerkinElmer, Turku, Finland). Antibody concentrations were expressed as percent of injected dose per gram tissue (%ID/g) or percent of injected dose corrected for body weight (bw) of the animal (%ID/g/bw). The reported brain concentrations were measured in the cerebrum of the left hemisphere.
Ex Vivo Autoradiography
The frozen right hemispheres from [125I]I-affibody-injected animals were sectioned sagittally (20 μm) with a cryostat (CM1850, Leica Biosystems, Nussloch, Germany) and mounted on Superfrost Plus glass slides (Menzel Gmboltion). Duplicate sections from each animal together with a standard of 125I with known radioactivity were exposed to a phosphor imaging plate (MS, Multisensitive, PerkinElmer, Downers Grove, IL, USA) for 7 days. The plates were scanned in a Cyclone Plus phosphor imager (PerkinElmer) at 600 dots per inch.
Sagittal brain cryosections from [125I]I-affibody-injected mice were fixed in ice-cold MeOH for 10 min and washed 2 × 5 min in PBS. Double CD31/Aβ40 staining was performed on selected WT, tg-Swe and tg-ArcSwe brain sections by the following procedure:
The sections were blocked for 1 h with 5% Normal Goat Serum, followed by a wash in PBS. The primary antibodies rat-α-mouse CD31 (BD, #553,370) and rabbit-α-Aβ40 (Agrisera, Umeå, Sweden) or 6E10 (Nordic Biosite, Täby, Sweden) for di-scFv3D6-8D3, was applied to the sections which were then incubated overnight at 4°C with slow shaking. After incubation, the sections were washed in PBS and secondary antibody goat-α-rat (Alexa 488) and goat-α-rabbit (Alexa 647) was added for 1 h with slow shaking, followed by a PBS wash. The sections were stored in PBS until the nuclear track emulsion procedure (described below) was performed on the same day.
Neuronal marker, rabbit-anti-mouse NeuN (Abcam, ab177487, Cambridge, UK), and Aβ staining with 6E10 (Nordic Biosite) was used for the brain section to illustrate pathology in Fig. 2b.
Nuclear Track Emulsion Autoradiography
Nuclear track emulsion autoradiography experiments were done in darkness as previously described (6). In brief, ILFORD K5 emulsion was prepared in a 40°C water bath according to manufacturer’s instructions. The immunofluorenscently stained sections were immersed in the emulsion for 5 s and left to air dry for 2 h, then incubated in darkness for 4 weeks at 4°C. The sections were developed according to the manufacturers’ instructions and dehydrated in increasing EtOH concentration gradient (70%, 95%, 100%) and mounted with Pertex (Histolab). Images of the developed emulsion and CD31-immunofluorescent stained sections were acquired with a Zeiss Observer Z.1 microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) and processed equally using the ZEN software. An inverted lookup table was applied to the brightfield channel, resulting in white emulsion puncta instead of black.
Data is presented as mean ± standard deviation, if not stated otherwise. All calculations were done in Prism v. 9.2.0 (GraphPad Software, Inc.). Saturation binding curves were used to fit the ELISA data and estimate the KD values for the affibodies. One-way analysis of variance with Bonferroni correction was used to compare 2 h brain uptake of the affibodies. Quantification of in vitro autoradiography sections and 24 h brain retention were analyzed with unpaired t-tests to compare tg-ArcSwe and WT brain sections.