An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ.
ABSTRACT Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.
disease (AD). However, traditional passive immunization approaches carry the risk of Fc? receptor-mediated overactivation of micro-
multiple forms of A?, protected against A?1–42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic A? oligomers by
microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP(V717I)/PS1
and induced less release of the proinflammatory cytokine TNF?. We propose that a humanized IgG4 anti-A? antibody that takes
passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of
Alzheimer’s disease (AD) is the most common form of neurode-
generation and is exemplified by debilitating dementia. It is pro-
posed that ?-amyloid (A?) peptides, the proteolytic products of
amyloid precursor protein, are toxic and causative in AD, con-
tributing to memory loss and neurodegeneration (Selkoe, 2002).
The A?1–42 peptide is believed to be the most toxic species,
present in various conformational forms (Bitan et al., 2003;
Cleary et al., 2005; Shankar et al., 2007). Evidence suggests that
some degree of A?1–42 oligomerization is necessary for neuro-
toxicity (Walsh et al., 2002; Kayed et al., 2003; Jan et al., 2011).
Furthermore, multiple soluble assembly forms of A?1–42 are
thought to be both required and sufficient to disrupt neuronal
function and subsequent learning and memory (Cleary et al.,
2005; Townsend et al., 2006; Poling et al., 2008).
Structural alterations and oligomerization of A?1–42 result
in a multifaceted dynamic equilibrium of small protofibrillar in-
termediates in which early oligomeric species act as seeds for
fibrillar plaques (Bitan et al., 2003) and thus are of great interest
as the primary targets of anti-A? therapeutics. A passive anti-A?
ple A?1–42 assemblies, including soluble oligomers (Walsh et
al., 2005), and other A? peptide aggregates that contribute to
early events in the A?1–42 oligomerization process (Frenkel et
al., 1998; Lambert et al., 1998; Lee et al., 2006; Spires-Jones et al.,
An active immunization approach using an A?1–42 vaccine
was cut short due to safety concerns (Orgogozo et al., 2003), yet
some modest long-term functional benefits were reported in an-
A? carries the risk of adverse immunological responses, leading
to inflammation such as meningoencephalitis (Orgogozo et al.,
2003), and also lacks the ability to regulate response level and
duration. To mitigate these risks, drug development has focused
TheJournalofNeuroscience,July11,2012 • 32(28):9677–9689 • 9677
safer, passive immunization may induce antibody–antigen com-
plexes that fully engage Fc? receptors (Fc?Rs) on microglia that
may provoke adverse proinflammatory reactions, possibly lead-
ing to blood–brain barrier (BBB) disruption observed as vaso-
genic edema and/or cerebral microhemorrhage (Salloway et al.,
and contains a human IgG4 backbone with reduced effector
function (van der Zee et al., 1986; Tao et al., 1991). MABT effec-
motes microglial A? engulfment, but has a significantly reduced
capacity to activate microglial Fc?Rs when compared with an
To directly assess the potential improvement in safety profile,
tidose (MD) stage phase I clinical study. Patients were also ran-
domized in the MD stage by ApoE status, as previous studies
genic edema (Sperling et al., 2012). Consistent with our hypoth-
esis, MABT showed no signs of vasogenic edema at doses as high
as 10 mg/kg single dose, or 5 mg/kg MD over four doses. Phar-
macokinetic and pharmacodynamic analysis demonstrated a
dose-proportional increase in exposure to MABT and a robust
elevation in plasma total A? levels, which correlated well with
serum MABT concentrations, thus confirming that MABT en-
gaged A? in humans.
Cell culture preparation. Rat primary cortical cultures were prepared
from Sprague Dawley rats (Charles River Laboratories) of either sex at
postnatal day 1, as described by Meberg and Miller (2003). Cerebellum
pain, CaCl2, EDTA, and HEPES; all from Invitrogen). DNase (Invitro-
gen) was added for 10 min. Following dissociation, dispersed cortical
neurons were plated onto poly-L-lysine (0.01%; molecular weight,
150,000–300,000; Sigma-Aldrich)-coated 6-well, 24-well, or 96-well tis-
sue culture plates. For immunocytochemistry, cells were grown on
robasal media (Invitrogen) without phenol red, with the addition of
L-glutamine (2 mM; Sigma-Aldrich), B27 supplement (Invitrogen), and
penicillin/streptomycin (Sigma-Aldrich) in a humidified incubator at
replaced with astrocyte-conditioned medium. After further 4 d in cul-
ture, cell proliferation was blocked by treatment with cytosine arabino-
side at 2.5 ?M (Invitrogen). Under these culture conditions, 20% of cells
were identified as neurons by NeuN/DAPI staining (data not shown).
Experiments using mixed cortical cells were generally performed after
prepared from cortex and hippocampus were harvested as described for
cortical cultures above. Cortex and hippocampus were put in DMEM
containing high glucose and homogenized by pipetting with a 10 ml
pipette and then with a syringe. The homogenate was centrifuged for 3
high glucose containing 10% FCS and penicillin/streptomycin (micro-
glia media). The cell suspension was next transferred to a T75 tissue-
1 week. The flask was shaken to separate microglia from adherent cells,
and collected and washed in DMEM. The resulting cells were resus-
pended in 1 ml of microglia medium, counted, and plated at 5 ? 104
cells/well. To verify microglial enrichment, cells were stained with the
astroglial and microglial markers GFAP and Iba1, respectively. Greater
GFAP and Iba1. Pure microglia were prepared from postnatal day 3
ml pipette, and then with an 18 gauge needle. The homogenate was
centrifuged for 3 min at 1000 ? g, and then resuspended in prewarmed
DMEM containing high glucose, 10% FBS, and penicillin/streptomycin
(microglia media). The cell suspension was next transferred to a T75
tissue culture flask and kept in a humidified incubator at 37°C and 5%
CO2for 7–10 d. Microglia were isolated by shaking, collected, and
washed in DMEM. The resulting cells were resuspended in 1 ml of mi-
croglia medium, counted, and plated on tissue culture-treated glass
chamber slides at 5 ? 104cells/well for use in experiments.
Generation of anti-A? antibodies and in vivo efficacy studies. The
disulfide-stabilized IgG4 anti-A? monoclonal antibody (mAb) MABT is
a humanized form of a mouse IgG2b mAb (mMABT) generated by im-
al., 2007). For in vivo efficacy, both single-transgenic hAPP(V717I)and
administered 10 mg/kg purified mMABT once weekly, 2 or 14 times,
in the hAPP(V717I)mice, and memory performance by novel object rec-
ognition test using the hAPP(V717I)/PS1 mice.
1,1,1,3,3,3-hexafluoro-2-propanol, sonicated, and shaken overnight at
room temperature. Aliquots were then dried under a flow of argon, vac-
inhibition of aggregation, antibodies were prediluted in PBS and then
added to nonsiliconized incubation tubes containing the following: 10
?M thioflavin-T (ThT) (Sigma-Aldrich), 33 ?M A?1–42 peptide film,
and 8.2% DMSO, with a final 10:1 molar ratio of A?1–42 to antibody.
Incubation was done for 24 h at 37°C, and the spectrofluorescence (ex-
citation, 440 nm; emission, 485 nm) read in six replicates in a black
384-well plate (PerkinElmer) in a microplate reader (Tecan). For disag-
gregation of preaggregated A?1–42, the A?1–42 film was made up as a
to aggregate at 37°C for 24 h, after which the following were added:
prediluted antibody with a final 10:1 molar ratio of A?1–42 to antibody
and 10 ?M ThT. This solution was then incubated for additional 24 h at
37°C, after which spectrofluorescence was measured as described above.
Inhibition of aggregation and disaggregation is expressed as mean per-
centage (?SEM) inhibition or disaggregation, respectively.
Fc?R binding. The binding of test antibodies to a panel of human
proteins containing the extracellular domain of the receptor ?-chain
with a Gly/6xHis/glutathione S-transferase (GST) polypeptide tag at the
C terminus. A mAb with a human IgG1 framework was used as the
positive control (IgG1 control) in this experiment. Plates were coated
with a mouse monoclonal anti-GST antibody (Genentech) in a 0.05 M
sodium carbonate buffer, pH 9.6, overnight at 4°C. After blocking with
an assay buffer containing PBS, 0.5% BSA, and 0.05% Tween 20, the
plates were incubated with Fc?Rs at room temperature for 1 h. Human
Fc?Rs were immobilized to the plate via interaction with the anti-GST
coating. Serial dilutions of anti-A? MABT, MABT-IgG1, MABT-IgG1-
forms for the low-affinity receptors (Fc?RIIa, Fc?RIIb, and Fc?RIIIa).
The multimeric forms of test antibodies were generated by cross-linking
F(ab?)2fragment of goat anti-human ?-chain (MP Biomedicals), with
with Fc?Rs at room temperature for 2 h. Plates were washed three times
tion step. The antibodies bound to the Fc?Rs were detected with horse-
radish peroxidase-conjugated F(ab?)2fragment of goat anti-human
F(ab?)2(Jackson ImmunoResearch Laboratories). Tetramethylbenzi-
incubated at room temperature for 15–20 min to allow color develop-
ment. The reaction was terminated with 1 M H3PO4, and absorbance at
450 nm with reference at 650 nm was measured on a plate reader (Mo-
9678 • J.Neurosci.,July11,2012 • 32(28):9677–9689 Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
lecular Devices). Binding curves were generated by plotting the mean
tive sample absorbance.
was prepared as described above. A 165 ?g aliquot of peptide film was
resuspended in 7 ?l of DMSO, 85 ?l of PBS, and 9 ?l of 2% SDS and
incubated for 6 h at 37°C. Then, 300 ?l of water was added, and after an
overnight incubation at 37°C, A?1–42 oligomers were precipitated with
900 ?l of 33% methanol 4% acetic acid solution for 1 h at 4°C, centri-
fuged at 16,200 ? g for 10 min. Supernatant was removed and A?1–42
oligomers were dried before being resuspended in Na2HPO4/NaCl solu-
tion for a final concentration of 1 ?g/?l.
A?1–42 cellular toxicity assays. The cytotoxicity of A?1–42 oligomers
was tested on mixed cortical cultures at day in vitro 5 (DIV 5). All anti-
bodies, at a final concentration of 100 ?g/ml, were coincubated with
before treatment of cells. For some experiments, mixed cortical cultures
were pretreated for 1 h with 1 ?M trans-4-[4-(4-fluorophenyl)-5-(2-
p38MAPK inhibitor, before treatment with A?1–42 oligomers. Cell via-
bility was performed by standardized 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction assays (Promega),
following the manufacturer’s instructions. Briefly, for the last 3 h of
MTT dye solution and the generation of a blue formazan product was
measured by reading absorbance at 570 and 690 nm using a microplate
over A?1–42 oligomer-treated cells. The ability of the MABT mAb to
protect neurons from A?1–42 oligomer-induced degeneration was also
assessed in an in vitro assay using immunofluorescence. Embryonic day
17.5 mouse cortical neurons were isolated, dissociated, and cultured in
vitro in Neurobasal media with B27 supplement. A?1–42 was prepared
as described above for A?1–42 monomeric peptide film, after which 10
?l of DMSO was added to dissolve the peptide. Then, 78.6 ?l of Ham’s
F12 media was added and the A?1–42 peptide solution at 25 ?M was
incubated at 4°C for 48 h before cell treatment. Cells were grown for 9 d
A?1–42 at 2 ?M with or without MABT at 50 ?g/ml was added at day 5
or day 6, with DMSO-F12 alone at the same volume used for the vehicle
bules using fluorescence microscopy.
Immunohistochemistry. Paraffin-mounted temporal lobe brain sec-
tions (20 ?m) from an AD patient and from an age-matched non-AD
control (Tissue Solutions) were used for immunohistochemistry stain-
ing. Deparaffinized sections were subjected to antigen retrieval using
formic acid and then labeled with 50 ?g/ml MABT as the primary anti-
body. A goat anti-human biotinylated IgG was used as a secondary anti-
body. Staining was done with diaminobenzidine (Dako) and mounting
using Eukitt mounting medium. Images were acquired on a LSM 700
inverted microscope from Zeiss (Carl Zeiss).
Immunocytochemistry and confocal imaging. Cells were grown on glass
coverslips. Following treatment, cells were quickly washed with PBS and
cells were immersed in 100% methanol for 10 min at ?20°C. They were
then washed again and incubated in a blocking solution, PBS containing
incubation with the primary antibody, cells were washed and incubated
for 2 h with the secondary antibody, and then washed and mounted on
glass slides using ProLong Gold antifade reagent (Invitrogen). Epifluo-
rescence and confocal images were acquired on a LSM 700 inverted mi-
croscope from Zeiss AG, using a 63? lens. Fluorescence intensity was
Z-stacks were rendered into a three-dimensional image using ImageJ
1.42 (National Institutes of Health; freeware) from which an apical-to-
distal slice containing the labeled proteins was obtained. Cells treated
with HyLite Fluor 488-tagged A?1–42 were treated in the same way
except no primary or secondary antibodies were used to label for
alone or in combination with 100 ?g/ml anti-A? MABT, MABT-IgG1,
MABT-IgG1-D265A, control IgG1, or antibodies alone for 30 min. Cells
anol, blocked with 10% goat serum in PBS, and stained with rabbit anti-
In vivo imaging of amyloid plaques. Cranial windows were implanted
above the somatosensory cortex of 10-month-old transgenic hAPP(V717I)/
hours before each imaging session, animals were peripherally injected
with 10 mg/kg methoxy-X04 intraperitoneally to visualize individual
amyloid plaques (Klunk et al., 2002) and immediately before imaging
injected intravenously with AngioSense680 (VisEn Medical) to visualize
blood vessels. For each imaging session, animals were anesthetized with
an isoflurane–oxygen mixture and mounted to the microscope using a
scope (Ultima In Vivo Multiphoton Microscopy System; Prairie Tech-
nologies) using a Ti:sapphire laser (MaiTai DeepSee Spectra Physics;
a 40?, NA 0.8 objective lens (Olympus Imaging). The pattern of the
vasculature was used to reproducibly position the mouse relative to the
objective from day-to-day enabling individual amyloid plaques to be
imaged over many weeks. The volumes of individual plaques were esti-
mated by summing the number of pixels above background within a
the mean pixel intensity plus 2 SDs within a region of interest drawn
adjacent to an amyloid plaque. Following the fourth and eighth imaging
session, animals were dosed intraperitoneally with 60 mg/kg MABT.
Phospho-p38MAPK ELISA. Rat cortical cultures were seeded onto
5. Unless otherwise indicated, cells were treated with 2 ?M A?1–42 oli-
gomers with or without mAb at 100 ?g/ml for 30 min. In some assays,
cells were pretreated for 1 h with the p38MAPK inhibitor SB239063.
Anisomycin was used as a positive control. Treatments were stopped by
placing cells on ice and aspirating the medium. Cells were washed with
of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM sodium
orthovanadate, 1% Triton X-100, and containing protease and phos-
phatase inhibitor cocktails. Protein concentration was determined by
the BCA assay (Pierce). For semiquantitative measure of p38MAPK
activation, a rat phospho-p38MAPK colorimetric ELISA kit was used
(Cell Signaling Technology), following manufacturer’s instructions.
Plates were read on a spectrophotometric microplate reader (Tecan)
at a 370 nm.
TNF? release. Rat cortical cultures enriched for microglia (?60%
Iba1?of total DAPI?cells) were treated with 10 ?M A?1–42 oligomers
with or without 100 ?g/ml antibodies for 6 or 24 h. Lipopolysaccharide
(Sigma-Aldrich) at 1 ?g/ml was used as a positive control stimulus. Cell
supernatants were removed at the indicated time points, passed through
a 0.2 ?m filter, and tested for TNF? with a Quantikine rat TNF?/
TNFSF1A (R&D Systems), following the manufacturer’s instructions.
Statistical analysis. All statistical analyses were done using GraphPad
Prism, version 5 (GraphPad Software). Data are presented as means ?
ANOVA followed by Tukey’s post hoc multiple comparisons, or Wilcox-
was taken to indicate a statistically significant difference.
Murine anti-A? monoclonal antibodies were generated by im-
munizing mice with an A? peptide antigen using a liposomal
vaccine formulation as previously described (Muhs et al., 2007;
Hickman et al., 2011). Several criteria were used to select candi-
date antibodies, including the ability to bind multiple A? species
Adolfssonetal.•Effector-ReducedAnti-A?Antibodies J.Neurosci.,July11,2012 • 32(28):9677–9689 • 9679
and to inhibit A?1–42 assembly into
small cytotoxic peptide aggregates. A
monoclonal murine mAb with an IgG2b
backbone (mMABT) was selected for in
vivo efficacy studies using both single-
transgenic hAPP(V717I)/PS1 female mice.
When compared with vehicle control,
treatment with mMABT reduced plaque
load in mMABT-treated mice by 31%,
served in a novel object recognition test
(Fig. 1A). The mMABT was further affin-
ity matured and humanized onto an IgG4
backbone (referred to as MABT). To test
the binding of MABT to A? in vitro, we
made a series of different A?1–42 prepa-
rations. The binding of MABT was mea-
sured by ELISA, and similar to the
mMABT (Fig. 1B), was shown to be
highly comparable among the different
A? peptides (Fig. 1C) or A?1–42 assem-
bly states (Fig. 1D).
MABT was subsequently tested for
binding to A? plaques in the brains of
transgenic mice expressing the human
amyloid precursor protein (hAPP(V717I))
and to amyloid plaques in human AD
brain sections. Amyloid plaques in both
and in AD brain (Fig. 1E, bottom) were
immunodecorated with the MABT mAb.
Together, these data provide evidence of
MABT binding to both soluble A? oli-
gomers and A? aggregates present in AD
to amino acids 12–23 of A?1–42, and
therefore overlaps with the main hydro-
phobic cationic segment of A?1–42
quent oligomerization, and the core of
A?1–42 ?-sheet assembly (Pike et al.,
1993; Esler et al., 1996; Haass and Selkoe,
2007). We therefore hypothesized that
MABT would inhibit A?1–42 assembly
and possibly dissociate preaggregated
effects of MABT on in vitro A?1–42 ag-
gregation using ThT, a dye that does not impede with amyloid
assembly but fluoresces upon binding to amyloid aggregates rich
trol anti-A? mAb directed against the N terminus of A?1–42,
self-assembly domain, MABT demonstrated 83% greater inhibi-
tory effect on A?1–42 aggregation in a ThT assay (Fig. 1F, left
panel). Similarly, an 80% greater dissociation of preaggregated
A?1–42 peptide was observed, when compared with the control
N terminus anti-A? mAb (Fig. 1F, right panel). These in vitro
of the A?1–42 peptide (LeVine, 1993). Therefore, to verify that
this was not an artifact due to potential mAb-mediated displace-
ment of ThT binding to ?-sheet rich A?1–42, we performed an
assay that does not rely on ThT fluorescence, but rather on the
capacity of labeled A?1–42 to aggregate or self-assemble onto
immobilized unlabeled A?1–42. We obtained comparable re-
and murine A?1–42 and human A?1–40, for mMABT (B) and MABT (C). D, MABT was also tested for binding to different
functionality was shown by the ability of MABT to impede A?1–42 aggregation (left panel), and to disassemble preformed
9680 • J.Neurosci.,July11,2012 • 32(28):9677–9689 Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
of A?1–42 in a dose-dependent manner (Fig. 1G). These data
suggest that an antibody directed against the mid-domain of A?,
robust inhibitory effect on A?1–42 fibril elongation and/or ag-
gregation relative to antibodies targeting other domains of A?.
model using cytotoxic A?1–42 oligomers. Primary cortical cul-
tures from P1 rats were grown and treated with free A?1–42
oligomers or oligomers bound to MABT. Treatment of cortical
a reduction in metabolic activity as measured by mitochondrial
plete rescue from toxicity was observed for A?1–42 oligomer
concentration up to 5 ?M in the presence of MABT (MABT to
A?1–42 molar ratio of 1:7.5) compared with control IgG. To
cence assay, showing a similar neuropro-
tective effect of MABT (Fig. 2B). To
42-mediated neurodegeneration, mouse
for 6 d, and treated with A?1–42 with or
without MABT for 4 d. Control cultures
showed healthy morphology (Fig. 2C, left
panel). Treatment with A?1–42 for 4 d
resulted in robust neurite degeneration
(Fig. 2C, center panel). Cells treated with
the combination of A?1–42 and MABT
appeared similar to control cells (Fig. 2C,
cytotoxic A?1–42 oligomer assemblies
was used, ranging in size from dimers
and trimers to higher-molecular-weight
multimers (Fig. 2D). These results show
that MABT was able to protect primary
neurons from A?1–42 oligomer-induced
A? peptides, especially aggregation inter-
mediates (Bateman et al., 2007), are
proteins present in cell membranes. We
hypothesized that MABT may exert its
neuroprotective effects by reducing bind-
ing of A?1–42 oligomers to neurons. To
test this idea, an immunofluorescent
staining for membrane-bound A? was
performed. A?1–42 oligomers were ap-
or 18 h, after which cultures were stained
for A? and the neuron-specific class III
?-tubulin, TuJ1. We observed that treat-
ment of cortical neurons with A?1–42
oligomers resulted in A? binding to neu-
rons, particularly localized to neuritic
processes (Fig. 3A, middle panels and in-
sets). Cotreatment with MABT blocked
neurons. This effect was readily apparent as early as 30 min (Fig.
3A,B) and remained for at least 18 h of treatment (Fig. 3B).
Although we used an N-terminal anti-A? mAb (clone 6E10) to
stain for A?1–42 oligomers in our assay (Fig. 3A), thus reducing
the potential for interference between MABT and the detection
mAb used for staining, we confirmed these results using HiLyte
Fluor-488 fluorescently labeled A?1–42. Treating cortical cul-
the conclusion that MABT reduced binding of A?1–42 to neu-
ronal processes in primary cortical cultures (Fig. 3C,D).
Observations from immunofluorescent studies also indicated
that, in the presence of MABT, there was a shift in A?1–42 oli-
gomer association away from neuronal processes toward cellular
for an antigen–antibody complex binding to microglia through
an Fc?R-mediated mechanism (Clark, 1997), but was unex-
pected for the effector-reduced MABT, which required further
An MTT assay was used to determine cell viability as described in Materials and Methods. The means (?SEM) of five
show the means (?SEM) of two independent assays. C, Neurotoxicity following extended A?1–42 oligomer treatment
50 ?g/ml of MABT, and then stained for TuJ1 (green) and DAPI (blue). D, Silver-stained SDS-PAGE showing the highly
Adolfssonetal.•Effector-ReducedAnti-A?AntibodiesJ.Neurosci.,July11,2012 • 32(28):9677–9689 • 9681
We next explored the relationship be-
tween MABT/A?1–42 complex forma-
verify that A?1–42 oligomers bound to
MABT are indeed taken up by microglia,
nal cultures was performed. When com-
pared with cells treated with A?1–42
oligomers alone, we found that MABT
mediated rapid uptake of A?1–42 oli-
gomers into cellular profiles likely to be
microglia (Fig. 4A). This was readily ap-
parent as early as 30 min following treat-
ment. Microglia play a crucial role in
uptake and degradation of A?, a function
that is postulated to be compromised in
Relative to anti-A? immunotherapy, it has
been proposed that one possible mecha-
nism whereby A? plaques are cleared is
through the Fc?R binding properties of
anti-A? bound to A? (Koenigsknecht-
a therapeutic anti-A? antibody ideally
would have a reduced proinflammatory re-
sponse. We therefore compared MABT to
antibodies carrying the same antigen bind-
ing sequences, but harboring different IgG
backbones with variable Fc?R binding af-
finities, and therefore different microglia
activating potential. These included a
wild-type human IgG1 with full Fc?R
binding capacity (MABT-IgG1), and a
human IgG1 backbone carrying a D265A
matically reduces Fc?R binding (Shields
et al., 2001). All of the backbone variants tested bound with sim-
ilar affinity to A?1–42, as verified using surface plasmon reso-
nance (data not shown). The ability of these different mAb
backbones to internalize A?1–42 oligomers into microglia was
then compared using confocal imaging on A?1–42 oligomer-
treated primary cortical microglia. We found that A?1–42 oli-
gomer internalization correlated well with Fc?R binding, with
MABT-IgG1 ? MABT ? MABT-IgG1-D265A (Fig. 4B,C). To
verify that microglia are indeed the cells taking up A?1–42 com-
plexed to mAbs, we repeated the study using HiLyte Fluor 488-
tagged A?1–42 and costained for the microglial marker Iba1.
Upon binding to either MABT or MABT-IgG1, tagged A?1–42
that, in cell cultures treated with A?1–42 in combination with
Iba1 staining, features suggesting greater antigen/antibody-
mediated microglial activation.
To verify the differences in Fc?R binding between MABT
backbone variants, we measured the binding of the different
cross-linked IgG mAb to Fc?RIIIa-V158 and, as expected,
found a hierarchy of binding such that MABT-IgG1 ?
MABT ? MABT-IgG1-D265A (Fig. 4E). Comparable results
were obtained with other members of the Fc?R family (data
ity to reverse A?1–42 oligomer-mediated toxicity in mixed pri-
mary cortical cultures. Functional Fc?R binding activity, present
reversal of A?1–42 oligomer-mediated toxicity (Fig. 4F). The
MABT-IgG1-D265A mAb, which lacks Fc?R binding function-
ality, showed only a nonsignificant trend toward reversal of
A?1–42 oligomer-mediated cellular toxicity. Perhaps surpris-
ingly, the MABT-IgG1 wild-type mAb, which bears greater Fc?R
binding affinity compared with the IgG4 MABT, trended toward
a smaller protective effect when compared with MABT. We hy-
pothesized that, while binding to microglial Fc?Rs is needed for
Fc?Rs compared with that of a MABT may result in undesired
microglia activation, which may translate into reduced overall
protection against A?1–42 oligomer-mediated neurotoxicity.
experiments are shown. C, A?1–42 tagged with HyLite Fluor-488 verifies that MABT inhibits binding of A?1–42 to neurites.
9682 • J.Neurosci.,July11,2012 • 32(28):9677–9689Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
Therefore, in addition to having the ideal A? binding properties,
optimizing the level of microglial activation may be crucial in
developing an anti-A? therapeutic antibody with the desirable
safety and efficacy properties.
Having observed that MABT can efficiently promote A? en-
gulfment by microglia in vitro, we assessed whether systemic ad-
ministration of MABT in hAPP(V717I)transgenic mice could
induce amyloid plaque removal in vivo. As the antibodies we
hoc multiple comparison. Means (?SEM) are shown. D, Microglia (Iba1?) was verified as the cell type taking up A?1–42 complexed to MABT. Red is Iba1, green is HyLite Fluor 488-labeled
Adolfssonetal.•Effector-ReducedAnti-A?AntibodiesJ.Neurosci.,July11,2012 • 32(28):9677–9689 • 9683
generated used human backbones, we were unable to conduct
ies, both because human antibodies can robustly induce an anti-
therapeutic antibody response and because there is not a murine
version of human IgG4 that replicates the Fc receptor binding
a short course of dosing, we confirmed the ability of MABT to
promote glial engulfment of A? by analyzing individual plaques
in 10-month-old hAPP(V717I)/PS1 transgenic mice over several
weeks (Fig. 5A). On average, individual plaques increased in vol-
ume over the initial 3 week imaging period (Fig. 5B, average
week-to-week normalized change in volume; 0.156, 0.060, and
0.081). Following a single peripheral MABT administration (60
mg/kg, i.p.), plaque volume decreased over the 3 week period
after dosing (average week-to-week normalized change in vol-
administered to a single animal and seven plaques were followed
by imaging, three of which were completely removed by week 9.
These observations are consistent with our in vitro data suggest-
shown to contribute to neurotoxicity and microglial activation
(Li et al., 2004; Wang et al., 2004). We first examined p38MAPK
activation in primary mixed cortical cultures treated with
A?1–42 oligomers alone, or in combination with the anti-A?
MABT, MABT-IgG1, MABT-IgG1-D265A, or a control IgG1
that does not bind to A?. When cells were treated with A?1–42
oligomers, p38MAPK was activated within 15 min (data not
the different mAbs, only MABT-IgG1, carrying the IgG1 wild-
type backbone and thus having the greatest binding affinity to
Fc?R, significantly increased the A?1–42 oligomer-induced
p38MAPK activity above A?1–42 baseline levels, as shown by a
phospho-p38MAPK-specific ELISA (Fig. 6A). Since the various
anti-A? antibodies bind with similar affinity to A?1–42, the
MABT-IgG1 mAb should neutralize toxic A?1–42 oligomers to
the same degree as MABT. However, the greater Fc?R binding
affinity of the IgG1 backbone may result in microglia activation
that can be detrimental to cells that are highly susceptible to the
actions of A?1–42 oligomers, such as neurons. MABT com-
plexed to A?1–42 oligomers did not reduce the A?1–42
oligomer-induced p38MAPK activity, but rather showed a trend
toward higher activity, possibly reflecting the partial Fc?R acti-
vation by this antibody.
mixed cortical cultures, including both neuronal and glial cells,
we wanted to know whether the p38MAPK activity detected
ers and MABT was specific to microglia. Cells were treated as
previously, but this time phospho-p38MAPK activity was exam-
ined by immunofluorescence staining along with the microglia
marker Iba1. Upon treatment with A?1–42 oligomers com-
plexed to MABT or MABT-IgG1 mAbs, ?93% of cells staining
positive for phospho-p38MAPK were Iba1?(Fig. 6B). To con-
microglia in the same way. Under these conditions, A?1–42/IgG
identified (Fig. 6C).
To address the contribution of p38MAPK activation to
A?1–42 oligomer-mediated neurotoxicity, we next treated cells
with a second-generation p38MAPK-specific inhibitor, and then
with A?1–42 oligomers alone or in combination with either the
MABT or the low-Fc?R binding MABT-IgG1-D265A mAb. Un-
expectedly, the MABT-mediated increase in MTT signal was re-
duced to that of MABT-IgG1-D265A in presence of the
p38MAPK inhibitor, indicating a reduction in MABT-mediated
rescue function upon p38MAPK inhibition (Fig. 6D). As pre-
dicted, p38MAPK inhibition had no effect on cells treated with
A?1–42 oligomers complexed with the MABT-IgG1-D265A
mAb. This indicates that, although the MABT mAb does not
significantly induce p38MAPK levels over those seen with
MABT-mediated neuroprotection. The cellular target of this ac-
tivity in the mixed culture system is not known.
To link the increased microglia activity more directly to a
downstream proinflammatory readout, we measured TNF? re-
lease by primary cell cultures enriched for microglia (?61%
Iba1?; data not shown). The release of proinflammatory TNF?
by enriched microglia when treated with A?1–42 oligomers was
reduced in the presence of all anti-A? mAbs tested (Fig. 6E).
However, the greatest effect was observed in the presence of
MABT-IgG1, combining both the neuroprotective effects with
the ability to promote A? engulfment by microglia with limited
microglial activation. Similar results were observed when evalu-
ating other canonical cytokines; however, these cytokines were
not consistently upregulated by the addition of our A? oligom-
9684 • J.Neurosci.,July11,2012 • 32(28):9677–9689Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
MABT reduces microglial activation as measured by p38MAPK activity and TNF? secretion. A, When complexed to A?1–42 oligomers, the IgG1 wild-type backbone significantly
Adolfssonetal.•Effector-ReducedAnti-A?Antibodies J.Neurosci.,July11,2012 • 32(28):9677–9689 • 9685
ers; thus, our efforts were focused on evaluating p38MAPK acti-
vation and TNF? secretion as consistent measures of microglial
determine the safety and tolerability of intravenous MABT in
patients with mild-to-moderate AD. Secondary objectives of this
study were to characterize pharmacokinetics and A? pharmaco-
dynamics after single and multiple doses of MABT in AD pa-
tients. The study consisted of a single-dose (SD), dose escalation
stage, followed by a randomized placebo-controlled, double-
blind, parallel multidose (MD) stage (Fig. 7A). The main patient
selection criteria included diagnosis of AD (National Institute of
Neurological and Communicative Disorders and Stroke and the
Alzheimer’s Disease and Related Disorders Association criteria),
mini-mental state examination score of 15–26 (inclusive) at
screening, and a 50–86 years of age range. The actual patient
population included in the study are outlined in Figure 7B.
No patients receiving MABT developed vasogenic edema, in
either the single or multidose study. Importantly, patients were
at least 40% of enrolled patients were ApoE4 carriers (48% were
carriers), as these patients were previously shown to be at higher
risk of developing vasogenic edema (Salloway et al., 2009). The
serum concentration–time profiles for MABT following both a
single dose (0.3–10 mg/kg) and four weekly doses (0.5–5 mg/kg)
increased in proportion to dose and were characterized by slow
clearance (?3 ml ? d?1? kg?1) and a long half-life (18–23 d)
(Fig. 7C). A dose-dependent elevation in plasma total A? levels
was observed following single or weekly intravenous administra-
tion, which correlated well with serum MABT concentrations
(Fig. 7D), thus suggesting substantial target engagement in vivo.
These data suggest that an anti-A? antibody targeting a wide
array of A? assembly states, including aggregated A?, combined
CNS exposure. Additional clinical trials are underway to assess
the efficacy of MABT (crenezumab).
Recent clinical results using both active and passive immuniza-
tion to target A? in AD patients have identified a number of
challenges (Orgogozo et al., 2003; Lee et al., 2005; Salloway et al.,
2009); thus, great emphasis has been placed on developing alter-
native strategies toward a safer and more effective immunother-
particularly with passive immunotherapy, is disruption of the
vasogenic edema and/or microhemorrhages (Salloway et al.,
2009). Mechanisms underlying these dose-limiting observations
inent immunotherapy-specific hypothesis is that the immune
functions of the particular anti-A? antibody and/or its binding
esized that these safety findings are inseparable from the
mechanisms driving efficacy of immunotherapy (Weller et al.,
example, previous studies have shown that completely effector-
less murine anti-A? antibodies are capable of reducing plaque
load in mice, while reducing the risks associated with vascular
damage (Carty et al., 2006; Wilcock et al., 2006). Nevertheless,
promote microglial engulfment of A?. Also, these studies were
conducted with antibodies that only bind to the less toxic
C-terminal region of A?1–40. Ideally, an anti-A? immunother-
to maximize efficacy with the appropriate immune modulation
to limit safety findings.
ulated to maintain A? engulfment properties, while limiting ac-
tivation of microglia that may ultimately be deleterious to the
vascular and nervous systems. Furthermore, targeting a unique
epitope on A? has allowed us to identify an antibody that binds
ing aggregation and promoting disaggregation of A?. Together,
the reduced effector function of MABT, a humanized anti-A?
IgG4 antibody, and its unique A? binding profile exclusively po-
anisms of efficacy and safety in the clinic. Indeed, phase I data
ing that, at dose levels and exposure ?10-fold compared with
other anti-A? mAbs on human IgG1 backbones (Salloway et al.,
2009; Ostrowitzki et al., 2012), MABT showed no signs of vaso-
genic edema. Interestingly, however, solanezumab is also on a
human IgG1 backbone, but has not shown the same level of va-
et al., 2011). This is likely because solanezumab does not recog-
nize aggregated A? and thus does not bind vascular amyloid and
Having identified an anti-A? antibody with in vivo efficacy
and unique binding properties, we set out to investigate the abil-
ity of MABT to inhibit A?-mediated cellular toxicity. A recent
study has demonstrated that the two adjacent histidines at posi-
uptake (Poduslo et al., 2010). These histidine residues overlap
with the epitope of MABT, thus predicting the results of A?
cellular binding studies in which we observe robust inhibition of
A? binding to neurons. In addition to blocking A? interaction
with neurons, we see that toxicity mediated by A? oligomers is
also inhibited; thus, MABT is neuroprotective to toxicity medi-
ated by A?.
In the process of evaluating A?-mediated cellular toxicity in
mixed neuronal culture systems, which include neurons, micro-
glia, and astrocytes, we observed an accumulation of A? after
a neuron. Further investigation identified this cell type as micro-
glial, based on Iba1 staining and confirmed in studies using pu-
observations, as MABT is an IgG4 antibody, which has reduced
Fc?R binding properties compared with a wild-type IgG1 and
(Figure legend continued.)
specific inhibitor. A cytotoxicity assay using the MTT readout was performed after 24 h. The
mean (?SEM) of four experiments is shown. Statistical analysis was done using one-way
A?1–42-mediated proinflammatory release by microglia as measured by TNF? levels from
conditioned media of enriched microglia following 24 h of treatment. The mean (?SEM) of
with 10 ?M A?1–42 oligomers alone or together with 100
9686 • J.Neurosci.,July11,2012 • 32(28):9677–9689 Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
may therefore be less effective at promoting A? engulfment (for
relative contribution of IgG isotype and associated effector func-
full effector function (MABT-IgG1) and one with no effector
function (MABT-IgG1-D265A). Using these reagents, we ob-
served that MABT-IgG4 was nearly as effective as the IgG1
variant at promoting A? engulfment. In contrast, MABT-IgG1-
D265A showed modest-to-little promotion of A? engulfment.
to obtain the desired microglial uptake while limiting activation
and a proinflammatory response. Furthermore, we confirmed
in hAPP(V717I)/PS1 transgenic mice to assess plaque dynamics
Unlike Fc?R binding, neonatal Fc receptor (FcRn) binding is
Although it is notable that altering Fc? receptor binding alone is
not rule out the possibility that completely effectorless anti-A?
antibodies may be efficacious in vivo via a FcRn-mediated mech-
anism. Indeed, previous studies have shown that effectorless an-
tibodies are efficacious in vivo (Carty et al., 2006; Wilcock et al.,
2006); furthermore, an FcRn-driven mechanism of anti-A? ac-
tion has been directly investigated, suggesting that A?/anti-A?
complexes are cleared across the blood–brain barrier via an
Adolfssonetal.•Effector-ReducedAnti-A?Antibodies J.Neurosci.,July11,2012 • 32(28):9677–9689 • 9687
FcRn-dependent pathway (Deane et al., 2005). Thus, in addition
to the IgG4-dependent ability of MABT to drive microglial en-
use FcRn to enhance its activity in vivo.
Interestingly, MABT-IgG4 and MABT-IgG1 were both neu-
roprotective; however, the antibody that had no remaining Fc?R
binding, MABT-IgG1-D265A, was less protective to A?-
mediated cellular toxicity. Even if full effector function via Fc?R
engulfment of A?, MABT-IgG1 did not demonstrate better neu-
roprotection and may have even shown a trend toward reduced
efficacy. We therefore hypothesize that the robust effector func-
tion of an anti-A? IgG1 antibody, when complexed to A?, may
aberrantly initiate or augment a proinflammatory response that
is detrimental for neuronal cell survival.
There is substantial evidence that microglia are robustly acti-
vated in many neurodegenerative diseases, including AD, and
that activation may be either neuroprotective or neurotoxic de-
in the signaling network responsible for the upregulation of pro-
inflammatory cytokines in microglia, such as TNF?, and regu-
lates their biosynthesis by multiple mechanisms (Simon et al.,
1985; J. C. Lee et al., 1994; Gallagher et al., 1997; Y. B. Lee et al.,
2000). We therefore assayed for p38MAPK response after A?
produced the greatest activation of p38MAPK in microglia. We
response to p38MAPK activation; all MABT variants tested re-
duced the release of TNF? by microglia; however, this effect was
significantly blunted for MABT-IgG1, which has full effector
in stress-induced and proinflammatory signaling, there is also
evidence for a supportive function of p38MAPK in A? phagocy-
tosis (Doyle et al., 2004) and nonamyloid processing of APP
(Bandyopadhyay et al., 2006). These previous findings may ex-
for full rescue from toxicity, as the neuroprotective effect of
MABT was significantly reduced in the presence of a p38MAPK
inhibitor. Considering the entirety of these data comparing var-
ious IgG isotypes and subsequent effector function by assaying
neuroprotection and microglial activation, we propose that full
roprotective effects and is likely deleterious when considering
enhanced microglial activation as measured by p38MAPK activ-
ity and relative TNF? release.
MABT (also known as crenezumab), a humanized anti-A?
vivo efficacy, including ability to protect neurons from A?
oligomer-induced toxicity. MABT was also selected for its ability
to promote microglial engulfment of A? without aberrantly ac-
tivating microglia. Based on these preclinical data, we hypothe-
size that MABT would have a reduced risk of vascular-related
findings, which are likely a consequence of an anti-A? antibody
binding aggregated A? and maintaining full effector function to
ported by these preclinical data, a single and multiple dose, mul-
ticenter, randomized, placebo-controlled, double-blind phase I
was conducted in patients with mild-to-moderate AD. Magnetic
genic edema after either single or multiple doses. Moving for-
and safety of crenezumab in AD patients.
Bandyopadhyay S, Hartley DM, Cahill CM, Lahiri DK, Chattopadhyay N,
Rogers JT (2006) Interleukin-1alpha stimulates non-amyloidogenic
pathway by alpha-secretase (ADAM-10 and ADAM-17) cleavage of APP
in human astrocytic cells involving p38 MAP kinase. J Neurosci Res
Bateman DA, McLaurin J, Chakrabartty A (2007) Requirement of aggrega-
tion propensity of Alzheimer amyloid peptides for neuronal cell surface
binding. BMC Neurosci 8:29.
Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, Teplow DB
(2003) Amyloid beta-protein (Abeta) assembly: Abeta 40 and Abeta 42
oligomerize through distinct pathways. Proc Natl Acad Sci U S A
Prevalence of asymptomatic vasogenic edema in pretreatment Alzhei-
ezumab. Alzheimers Dement 7:396–401.
Carty NC, Wilcock DM, Rosenthal A, Grimm J, Pons J, Ronan V, Gottschall
PE, Gordon MN, Morgan D (2006) Intracranial administration of
deglycosylated C-terminal-specific anti-Abeta antibody efficiently clears
amyloid plaques without activating microglia in amyloid-depositing
transgenic mice. J Neuroinflamm 3:11.
Clark MR (1997) IgG effector mechanisms. Chem Immunol 65:88–110.
Ashe KH (2005) Natural oligomers of the amyloid-beta protein specifi-
cally disrupt cognitive function. Nat Neurosci 8:79–84.
Deane R, Sagare A, Hamm K, Parisi M, LaRue B, Guo H, Wu Z, Holtzman
DM, Zlokovic BV (2005) IgG-assisted age-dependent clearance of Alz-
heimer’s amyloid-? peptide by the blood–brain barrier neonatal Fc re-
ceptor. J Neurosci 25:11495–11503.
S, Suzuki N, Modlin RL, Yeh WC, Lane TF, Cheng G (2004) Toll-like
receptors induce a phagocytic gene program through p38. J Exp Med
Esler WP, Stimson ER, Ghilardi JR, Lu YA, Felix AM, Vinters HV, Mantyh
PW, Lee JP, Maggio JE (1996) Point substitution in the central hydro-
phobic cluster of a human beta-amyloid congener disrupts peptide fold-
ing and abolishes plaque competence. Biochemistry 35:13914–13921.
Frenkel D, Balass M, Solomon B (1998) N-terminal EFRH sequence of Alz-
heimer’s beta-amyloid peptide represents the epitope of its anti-
aggregating antibodies. J Neuroimmunol 88:85–90.
Gallagher TF, Seibel GL, Kassis S, Laydon JT, Blumenthal MJ, Lee JC, Lee D,
Boehm JC, Fier-Thompson SM, Abt JW, Soreson ME, Smietana JM, Hall
RF, Garigipati RS, Bender PE, Erhard KF, Krog AJ, Hofmann GA, Shel-
drake PL, McDonnell PC, et al. (1997) Regulation of stress-induced cy-
tokine production by pyridinylimidazoles; inhibition of CSBP kinase.
Bioorg Med Chem 5:49–64.
HaassC,SelkoeDJ (2007) Solubleproteinoligomersinneurodegeneration:
Hickman DT, Lo ´pez-Deber MP, Ndao DM, Silva AB, Nand D, Pihlgren M,
Giriens V, Madani R, St-Pierre A, Karastaneva H, Nagel-Steger L, Will-
bold D, Riesner D, Nicolau C, Baldus M, Pfeifer A, Muhs A (2011)
Sequence-independent control of peptide conformation in liposomal
vaccines for targeting protein misfolding diseases. J Biol Chem
Hickman SE, Allison EK, El Khoury J (2008) Microglial dysfunction and
defective ?-amyloid clearance pathways in aging Alzheimer’s disease
mice. J Neurosci 28:8354–8360.
Holtmaat A, Bonhoeffer T, Chow DK, Chuckowree J, De Paola V, Hofer SB,
Hu ¨bener M, Keck T, Knott G, Lee WC, Mostany R, Mrsic-Flogel TD,
Nedivi E, Portera-Cailliau C, Svoboda K, Trachtenberg JT, Wilbrecht L
(2009) Long-term, high-resolution imaging in the mouse neocortex
through a chronic cranial window. Nat Protoc 4:1128–1144.
A, Lashuel HA (2011) Abeta42 neurotoxicity is mediated by ongoing
J Biol Chem 286:8585–8596.
9688 • J.Neurosci.,July11,2012 • 32(28):9677–9689 Adolfssonetal.•Effector-ReducedAnti-A?Antibodies
Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW,
Glabe CG (2003) Common structure of soluble amyloid oligomers im-
plies common mechanism of pathogenesis. Science 300:486–489.
Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP,
Debnath ML, Holt DP, Wang Y, Hyman BT (2002) Imaging Abeta
plaques in living transgenic mice with multiphoton microscopy and
methoxy-X04, a systemically administered Congo red derivative. J Neu-
ropathol Exp Neurol 61:797–805.
Koenigsknecht-Talboo J, Meyer-Luehmann M, Parsadanian M, Garcia-
Alloza M, Finn MB, Hyman BT, Bacskai BJ, Holtzman DM (2008)
Rapid microglial response around amyloid pathology after systemic
anti-A? antibody administration in PDAPP mice. J Neurosci 28:
Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M,
Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch
CE, Krafft GA, Klein WL (1998) Diffusible, nonfibrillar ligands derived
from Abeta1–42 are potent central nervous system neurotoxins. Proc
Natl Acad Sci U S A 95:6448–6453.
Lee EB, Leng LZ, Lee VM, Trojanowski JQ (2005) Meningoencephalitis as-
sociated with passive immunization of a transgenic murine model of
Alzheimer’s amyloidosis. FEBS Lett 579:2564–2568.
Targeting amyloid-beta peptide (Abeta) oligomers by passive immuniza-
Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, Mc-
Nulty D, Blumenthal MJ, Heys JR, Landvatter SW (1994) A protein ki-
nase involved in the regulation of inflammatory cytokine biosynthesis.
Lee YB, Schrader JW, Kim SU (2000) p38 map kinase regulates TNF-alpha
production in human astrocytes and microglia by multiple mechanisms.
LeVine H 3rd (1993) Thioflavine T interaction with synthetic Alzheimer’s
disease beta-amyloid peptides: detection of amyloid aggregation in solu-
tion. Protein Sci 2:404–410.
Li R, Yang L, Lindholm K, Konishi Y, Yue X, Hampel H, Zhang D, Shen Y
(2004) Tumor necrosis factor death receptor signaling cascade is re-
quired for amyloid-? protein-induced neuron death. J Neurosci
Meberg PJ, Miller MW (2003) Culturing hippocampal and cortical neu-
rons. Methods Cell Biol 71:111–127.
Greferath R, Kolonko N, Nagel-Steger L, Riesner D, Brady RO, Pfeifer A,
Nicolau C (2007) Liposomal vaccines with conformation-specific amy-
loid peptide antigens define immune response and efficacy in APP trans-
genic mice. Proc Natl Acad Sci U S A 104:9810–9815.
NimmerjahnF,RavetchJV (2006) Fcgammareceptors:oldfriendsandnew
family members. Immunity 24:19–28.
P, Dubois B, Eisner L, Flitman S, Michel BF, Boada M, Frank A, Hock C
(2003) Subacute meningoencephalitis in a subset of patients with AD
after Abeta42 immunization. Neurology 61:46–54.
Ostrowitzki S, Deptula D, Thurfjell L, Barkhof F, Bohrmann B, Brooks DJ,
Klunk WE, Ashford E, Yoo K, Xu ZX, Loetscher H, Santarelli L (2012)
with gantenerumab. Arch Neurol 69:198–207.
Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW (1993) Neu-
rodegeneration induced by ?-amyloid peptides in vitro: the role of pep-
tide assembly state. J Neurosci 13:1676–1687.
GL, Kandimalla KK (2010) HH domain of Alzheimer’s disease Abeta
provides structural basis for neuronal binding in PC12 and mouse corti-
cal/hippocampal neurons. PLoS One 5:e8813.
PolingA,Morgan-PaisleyK,PanosJJ,KimEM,O’HareE,ClearyJP,Lesne ´ S,
Ashe KH, Porritt M, Baker LE (2008) Oligomers of the amyloid-beta
cedures. Behav Brain Res 193:230–234.
Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli,
specialized responses. Annu Rev Immunol 27:119–145.
Salloway S, Sperling R, Gilman S, Fox NC, Blennow K, Raskind M, Sabbagh
M, Honig LS, Doody R, van Dyck CH, Mulnard R, Barakos J, Gregg KM,
Clinical Trial Investigators (2009) A phase 2 multiple ascending dose
Selkoe DJ (2002) Alzheimer’s disease is a synaptic failure. Science
Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini
BL (2007) Natural oligomers of the Alzheimer amyloid-? protein in-
duce reversible synapse loss by modulating an NMDA-type glutamate
receptor-dependent signaling pathway. J Neurosci 27:2866–2875.
Shields RL, Namenuk AK, Hong K, Meng YG, Rae J, Briggs J, Xie D, Lai J,
Stadlen A, Li B, Fox JA, Presta LG (2001) High resolution mapping of
the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc
gamma RIII, and FcRn and design of IgG1 variants with improved bind-
ing to the Fc gamma R. J Biol Chem 276:6591–6604.
Simon PL, Laydon JT, Lee JC (1985) A modified assay for interleukin-1
(IL-1). J Immunol Methods 84:85–94.
Sperling R, Salloway S, Brooks DJ, Tampieri D, Barakos J, Fox NC, Raskind
M, Sabbagh M, Honig LS, Porsteinsson AP, Lieberburg I, Arrighi HM,
Morris KA, Lu Y, Liu E, Gregg KM, Brashear HR, Kinney GG, Black R,
Grundman M (2012) Amyloid-related imaging abnormalities in pa-
tients with Alzheimer’s disease treated with bapineuzumab: a retrospec-
tive analysis. Lancet Neurol 11:241–249.
Spires-Jones TL, Mielke ML, Rozkalne A, Meyer-Luehmann M, de Calignon
A, Bacskai BJ, Schenk D, Hyman BT (2009) Passive immunotherapy
rapidly increases structural plasticity in a mouse model of Alzheimer
disease. Neurobiol Dis 33:213–220.
Tao MH, Canfield SM, Morrison SL (1991) The differential ability of hu-
terminal sequence of the CH2 domain. J Exp Med 173:1025–1028.
Townsend M, Shankar GM, Mehta T, Walsh DM, Selkoe DJ (2006) Effects
of secreted oligomers of amyloid beta-protein on hippocampal synaptic
plasticity: a potent role for trimers. J Physiol 572:477–492.
Trachtenberg JT, Chen BE, Knott GW, Feng G, Sanes JR, Welker E, Svoboda
K (2002) Long-term in vivo imaging of experience-dependent synaptic
plasticity in adult cortex. Nature 420:788–794.
van der Zee JS, van Swieten P, Aalberse RC (1986) Inhibition of comple-
ment activation by IgG4 antibodies. Clin Exp Immunol 64:415–422.
Vellas B, Black R, Thal LJ, Fox NC, Daniels M, McLennan G, Tompkins C,
(2009) Long-term follow-up of patients immunized with AN1792: re-
duced functional decline in antibody responders. Curr Alzheimer Res
Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS, Rowan
MJ,SelkoeDJ (2002) Naturallysecretedoligomersofamyloidbetapro-
Walsh DM, Klyubin I, Shankar GM, Townsend M, Fadeeva JV, Betts V,
PodlisnyMB,ClearyJP,AsheKH,RowanMJ,SelkoeDJ (2005) Therole
of cell-derived oligomers of Abeta in Alzheimer’s disease and avenues for
therapeutic intervention. Biochem Soc Trans 33:1087–1090.
Wang Q, Walsh DM, Rowan MJ, Selkoe DJ, Anwyl R (2004) Block of long-
term potentiation by naturally secreted and synthetic amyloid ?-peptide
in hippocampal slices is mediated via activation of the kinases c-Jun
protein kinase as well as metabotropic glutamate receptor type 5. J Neu-
Weller RO, Boche D, Nicoll JA (2009) Microvasculature changes and cere-
on therapy. Acta Neuropathol 118:87–102.
Morgan D (2004) Passive immunotherapy against Abeta in aged
amyloid deposits in spite of increased vascular amyloid and microhem-
orrhage. J Neuroinflamm 1:24.
Wilcock DM, Alamed J, Gottschall PE, Grimm J, Rosenthal A, Pons J, Ronan
V, Symmonds K, Gordon MN, Morgan D (2006) Deglycosylated anti-
amyloid-? antibodies eliminate cognitive deficits and reduce parenchy-
mal amyloid with minimal vascular consequences in aged amyloid
precursor protein transgenic mice. J Neurosci 26:5340–5346.
Adolfssonetal.•Effector-ReducedAnti-A?AntibodiesJ.Neurosci.,July11,2012 • 32(28):9677–9689 • 9689