An in vitro paradigm to assess potential anti-Aβ antibodies for Alzheimer’s disease

Although the amyloid β-protein (Aβ) is believed to play an initiating role in Alzheimer’s disease (AD), the molecular characteristics of the key pathogenic Aβ forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aβ. Here, we combined the use of Aβ-rich aqueous extracts of brain samples from AD patients as a source of human Aβ and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aβ antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aβ antibody, which better protects against forms of Aβ oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aβ therapeutics.


7.4, at 37 o C for 3 days
. Aβ monomers and PFs were stored in working aliquots at -80 o C.
Immediately prior to antibody binding studies, Aβ conformers were thawed and then centrifuged at 16,000 g and 4 o C for 20 minutes, and the supernatants were used.

Generation and selection of the novel murine anti-Aβ mAb 1C22
The covalently stabilized synthetic Aβ dimer, [Aβ1-40S26C]2, readily assemblies to form kinetically trapped protofibrils (PFs) 49,50 and as such provides a convenient form of assembled Aβ (free of both Aβ monomer and fibrils) to use as an immunogen and a tool to identify aggregate-preferring mAbs. Five, 4 month-old BALB/c mice (Charles Rivers Laboratories, Wilmington, MA) and 5, 4 month-old B6D2F1 (Jackson Labs (Bar Harbor, ME) mice were immunized 4 times with ~30 µg/mouse of protofibrillar [Aβ1-40S26C]2 emulsified in aluminium and magnesium hydroxide (Imject ALUM™, Pierce, Rockford, IL) and sera collected 3 days after the final immunization. The presence of antibodies capable of recognizing fresh protofibrillar [Aβ1-40S26C]2 was then assessed using a direct ELISA in which [Aβ1-40S26C]2 PFs were immobilized on the plate and mouse sera then serially diluted across the plate and the amount of antibody bound to the immobilized Aβ determined using a goat anti-mouse IgG (γ specific)biotin conjugate and streptavidin-HRP. All mice produced at least some anti-Aβ response. Seven animals had titers ≥150,000 and these were used to generate hybridomas. Spleens were harvested and fused with SP20 cells according to published methods. Supernatants from more than 7,000 hybridomas were screened for IgGs capable of binding to immobilized [Aβ1-40S26C]2 PFs. Three hundred and seventy-seven positive clones were identified, and of these, 64 retained anti-Aβ reactivity after sub-cloning.
The 31 clones which exhibited the highest levels of binding to [Aβ1-40S26C]2 PFs were grown in 175 cm 2 flasks and 25 of these yielded useful amounts of purified mAbs and were re-analyzed for their ability to bind to immobilized [Aβ1-40S26C]2 PFs. Six mAbs exhibiting EC50 ≥1 nM were then analyzed using a 4-tiered screening procedure ( Figure S1), the goal of which was to identify mAbs that preferential bind non-monomeric forms of Aβ. The results of this screen identified 1C22, an IgG1, as the mAb which exhibited the strongest relative preference for [Aβ1-40S26C]2 PFs.
All animal procedures were performed in accordance with the National Institutes of Health Policy on the Use of Animals in Research and were approved by the Harvard Medical School Standing Committee on Animals or under license from the Department of Health, Republic of Ireland with approval by the animal research ethics committee of University College Dublin.

Production of Fab fragments
Fab fragments were generated using an Fab kit (Thermo Fisher Scientific, Waltham, MA) that contained agarose-immobilized ficin. Briefly, each IgG was diluted to 2 mg/ml in digestion buffer containing 25 mM cysteine (Thermo Fisher Scientific, Waltham, MA) and incubated on a spin column containing agarose-immobilized ficin for 5 hours at 37 o C. Fab fragments were purified from intact antibodies and Fc fragments using agarose-immobilized protein A-agarose spin columns. Finally, Fab fragments were separated from residual F(ab')2 by size exclusion chromatography using a Superdex 75 10/300GL column (GE Healthcare, Uppsala, Sweden) eluted with PBS, pH 7.4. Non-reducing SDS-PAGE and silver stain detection confirmed that the Fab fragments were >90% pure.
Thereafter, the activity of purified Fabs was confirmed by ELISA. Plates were coated with PFs, then test and control Fabs added, and these detected using either biotinylated goat anti-mouse IgG (FCγ, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) or goat anti-mouse Fab specific (Sigma-Aldrich).

Production of induced neurons (iNs) from human induced pluripotent cells (iPSCs)
The YZ1 iPSC line was obtained from UCONN stem cell core and used to prepare neurogenin 2 (Ngn2)-induced human neurons ( 34 and Figure 4). iPSCs were maintained in media containing DMEM/F12, Knockout Serum Replacement, pencillin/streptomycin/glutamine, MEM-NEAA, and 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA) with addition of 10 μg/mL bFGF (Millipore, Billerica, MA) directly prior to media application. Neuronal differentiation was performed via a doxycycline induced Neurogenin 2 system ( 34 , and Figure 4). iPSCs were plated at a density of 95,000 cells/cm 2 for viral infection. Lentiviruses were obtained from Alstem with "ultrapure titres" and used at the following concentrations: pTet-O-NGN2-puro: 0.1 ul/ 50,000 cells; Tet-O-FUW-eGFP: 0.05ul/ 50,000 cells; Fudelta GW-rtTA: 0.11ul/50,000 cells. To induce Neurogenin 2 expression doxycycline is added on "iN day 1" ( Figure 4A) at a concentration of 2 µg/ml. On iN day 2 puromycin is added at 10 mg/ml and is maintained in the media at all times thereafter. On iN day 4, cells were plated at 50,000 cells/well on matrigel (BD Biosciences, San Jose, CA)-coated Greiner 96 well microclear plates and maintained in media consisting of Neurobasal medium (Gibco), Glutamax, 20% Dextrose, MEM NEAA with B27, with BDNF, CNTF, GDNF (PeprpTech, Rocky Hill, NJ) each at a concentration of 10 ng/ml. At designated time points, iNs were characterized using live-cell imaging to monitor neuritic complexity ( Figure 4A-C), and used for immunocytochemistry ( Figure 4D) or Western blotting to assess the expression of neuronal markers ( Figure S4). At iN day 14 neurite number and expression of neural markers had reached near maximal levels. Thus, for experiments investigating the effects of AD brain extracts on neuronal viability iNs were used at iN day 21, a time point when iNs were fully mature.

Western blot and immunocytochemical characterization of induced neurons (iNs)
Cells grown in 96 well plates were lysed by the addition of 50 μl lysis buffer (0.1% SDS, 1% NP-40, 50 mM HEPES, pH 7.4, 2 mM EDTA, 100 mM NaCl, 5 mM Na3VO4, 40 µM p-nitrophenyl phosphate, plus protease inhibitors) and incubated at 4 o C for 30 minutes. Lysates were centrifuged at 15,000 g and 4 o C for 25 minutes in a bench top Eppendorf centrifuge.
Supernatants were collected and protein content determined using a BCA kit (Thermo Fisher Scientific, Waltham, MA). Twenty µg of total protein was loaded in each lane and electrophoresed on pre-cast 15 well 4-12% polyacrylamide Bis-Tris LDS gels (Invitrogen, Carlsbad, CA). Proteins were transferred onto 0.2 µm nitrocellulose at 400 mA for 2 hours and blots incubated overnight at 4°C with primary antibody (Table S1). Membranes were washed three times for 10 minutes with PBST and then incubated in PBS containing 0.02%(w/v) SDS for 1 hour with either goat anti-mouse infrared 800 antibody (Rockland, Gilbertville, PA) diluted 1:15,000 or goat anti-rabbit infrared 700 antibody (Rockland, Gilbertville, PA) diluted 1:5,000 for 1 hour at RT. Membranes were washed three times for 10 minutes with PBST and then a further two times with PBS and immunoreactive bands were visualized using a Li-COR Odyssey infrared imaging system (Li-COR, Lincoln, NE).
The maturity of DIV21 iNs was also assessed using immunocytochemistry and confocal microscopy. Cells were fixed in 4% paraformaldehyde (PFA) and 4% sucrose at room temperature for 15 minutes, and then permeabilized with ice-cold methanol for 3 minutes. Cells were then washed with PBS 3 times and blocked using 5%(w/v) BSA in PBS containing 0.02% sodium azide.
Thereafter, iNs were incubated overnight with primary antibodies (Table 1) at 4 o C. Cells were again washed 3 times with PBS and then incubated for 1 hour at room temperature with fluorescence-conjugated secondary antibodies (AlexaFluor 546 goat anti-mouse; and AlexaFluor 633 goat anti-rabbit). Finally, iNs were washed 3 times with PBS and examined using a Zeiss LSM710 confocal microscope fitted with a 40x air objective (NA: 0.8). Images were captured in a Z-stack manner (5-10 stacks, interval 0.5-1.0 m) and maximal pixel intensity projections were created with averaging of 2 frames set to 1024 x 1024 pixel resolution.

Preparation of human brain extracts
Human tissue was used in accordance with the Partner's Institutional Review Board (Protocol: Walsh BWH 2011). Frozen hemibrains from 2 end-stage AD cases (referred to as AD1 and AD2) were obtained from the Massachusetts ADRC Neuropathology Core, Massachusetts General Hospital). AD1 was a 68 years old female with end-stage AD and fulminant amyloid and neurofibrillary tangle pathology. AD2 was a 69 year old woman who had pathological changes consistent with end-stage AD. Approximately 20 g of cortical gray matter was dissected from each case and this material was then sliced into ~2 g lots with a razor blade and homogenized in artificial cerebrospinal fluid base buffer (aCSF-B) (124 mM NaCl, 2.8 mM KCl, 1.25 mM NaH2PO4, ethylenediaminetetraacetic acid, 1 mM ethyleneglycoltetraacetic acid, 5 μg/ml Leupeptin, 5 μg/ ml Aprotinin, 2 μg/ml Pepstatin, 120 μg/ml Pefabloc and 5 mM NaF) and tissue homogenized in 5 volumes of ice-cold aCSF-B with 25 strokes of a Dounce homogenizer (Fisher, Ottawa, Canada).
Resulting 20% (w/v) homogenates were centrifuged at 200,000 g for 110 minutes and 4°C in a It is possible the manipulations necessary to process tissue may lead to changes in the aggregation state and dynamics of brain-derived Aβ, but this is unavoidable. However, to account for changes that might occur during immunodepletion we included an identical sample that was "mock immunodepleted" using pre-immune serum, and freeze thawing was avoided by aliquotting samples immediately after their preparation and using samples (mock immunodepleted, immunodepleted and control brain) only once. hours. Blots were microwaved in PBS and Aβ detected using the anti-Aβ40 and anti-Aβ42 antibodies, 2G3 and 21F12, and bands visualized using a Li-COR Odyssey infrared imaging system (Li-COR, Lincoln, NE).

MSD Aβ immunoassay
Samples were analyzed for Aβ content using an assay specific for Aβx-42, which preferentially detects Aβ monomers 27,70 . This assay was performed using the Meso Scale Discovery (MSD) platform and reagents from Meso Scale (Rockville, MD). m266 (3 μg/ml) was used for capture and biotinylated 21F12 (1 μg/ml) for detection. Samples, standards and blanks were loaded in triplicate and analyzed as described previously 27,70 . Photomicroscopy was performed on an Olympus BX40 microscope using visible light.
Immunostained slides were photographed at 10x with an Olympus Qcolor camera using Qphoto software with exposure settings held constant and identical throughout. Values for EC50s and IC50s were calculated from antibody binding, capture, and Aβ competition curves as shown in Figures 1 and 2. Values for dissociation constants (KD) and apparent KDs (KAPP) were determined from sensograms that were globally fit to a 1:1 langmuir binding model or by steady state analysis of the binding data as shown in Figure 2. Values are means ± SD, with the number of replicates ≥3.

st screen: Protofibril ELISA
Protofibrils formed from [Ab1-40S26C] 2 were immobilized on the surface of 96 well plates and each purified mAb was serially diluted across the plate and the conc. of mAb that produced 50% maximal binding (EC 50 ) was determined from fitted dilution curves.

nd screen: Monomer ELISA
Ab monomer was isolated using SEC and immediately immobilized on the surface of 96 well plates and each purified mAb was serially diluted across the plate and the conc. of mAb that produced 50% maximal binding (EC 50 ) was determined from fitted dilution curves.

rd screen: competition ELISA
mAbs were tested for their ability to bind to plate-immobilized Ab monomer in the presence of increasing amounts of solution-phase protofibrils or monomer. The amount of mAb was kept constant and binding of mAb to protofibrils was plotted vs. the conc. of competing conformer. The conc. of conformer that caused a 50% reduction in binding to protofibrils (IC 50 ) was determined. Human induced neuron (iN day 21) were treated with medium, or AD2 brain extract mockimmunodepleted (Mock ID) or immunodepleted with the anti-Aβ antiserium AW7 (AW7 ID).
Phase contrast images (top panels) at 0 and 72 hours were analyzed using the IncuCutye NeuroTrack algorithm to identify neurites (middle panels) and the NeuroTrack-identified neurites