Peripherally administered antibodies against amyloid ??-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease

Abstract

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

Full text

916 NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000
ARTICLES
One hallmark of Alzheimer disease is the accumulation of amy-
loid β-peptide in the brain and its deposition as plaques. Mice
transgenic for an amyloid β precursor protein (APP) mini-gene
driven by a platelet-derived (PD) growth factor promoter
(PDAPP mice), which overexpress one of the disease-linked mu-
tant forms of the human amyloid precursor protein, show many
of the pathological features of Alzheimer disease, including ex-
tensive deposition of extracellular amyloid plaques, astrocytosis
and neuritic dystrophy
1,2
. Active immunization of PDAPP mice
with human amyloid β-peptide reduces plaque burden and its
associated pathologies
3
. Several hypotheses have been pro-
posed regarding the mechanism of this response
4,5
. Here we re-
port that peripheral administration of antibodies against
amyloid β-peptide, was sufficient to reduce amyloid burden.
Despite their relatively modest serum levels, the passively ad-
ministered antibodies were able to enter the central nervous
system, decorate plaques and induce clearance of preexisting
amyloid. When examined in an ex vivo assay with sections of
PDAPP or Alzheimer disease brain tissue, antibodies against
amyloid β-peptide triggered microglial cells to clear plaques
through Fc receptor-mediated phagocytosis and subsequent
peptide degradation. These results indicate that antibodies can
cross the blood–brain barrier to act directly in the central ner-
vous system and should be considered as a therapeutic ap-
proach for the treatment of Alzheimer disease and other
neurological disorders.
We administered antibodies against amyloid β-peptide (Aβ) by
intraperitoneal injection to mice transgenic for an Aβ precursor
protein (APP) mini-gene driven by a platelet-derived (PD)
growth factor promoter (PDAPP mice). In the first experiment,
8- to 10-month-old heterozygous PDAPP mice (n = 8) received
either PBS, one of two different mouse monoclonal antibodies
against Aβ (10D5 or 21F12) or a polyclonal immunoglobulin
(Ig) fraction obtained from mice immunized with the 42-
amino-acid form of Aβ (pabAβ
1–42
). The mice received weekly
injections of antibody for six months, maintaining a constant
antibody serum concentration throughout the course of the ex-
periment. We used quantitative image analysis to determine
amyloid burden and enzyme-linked immunoassay (ELISA) to
determine Aβ
42
levels in the cortex, as described
3
. Relative to
control-treated mice, the polyclonal immunoglobulin fraction
against Aβ and one of the monoclonal antibodies (10D5) re-
duced plaque burden by 93% and 81%, respectively (Fig. 1a, P <
0.005). There were similar reductions in cortical levels of Aβ
42
by ELISA measurements (6200, 4890 and 13800 ng/g tissue for
10D5, pabAβ
1–42
and PBS, respectively). Although 21F12 seemed
to have a relatively modest effect on plaque burden, there was
no substantial reduction as determined by ELISA measure-
ments (13,580 ng/g). The effect of pabAβ
1-42
on plaque burden
was demonstrated in micrographs of brain sections obtained
from mice having the median level of plaque burden within
their respective groups. Most of the diffuse deposits and many
of the larger compacted plaques were absent in mice treated
with pabAβ
1–42
compared to those in the control groups.
In a second study, we repeated 10D5 treatment and tested two
additional antibodies against Aβ, 3D6 and 16C11 (Fig.1c).
Control groups received either PBS or an irrelevant isotype-
matched antibody (TM2a). The experimental design was the
same as in the previous study except for the age of the mice
(11.5–12 months old). Once again, after 6 months of treatment,
10D5 reduced plaque burden by greater than 80% compared
with that of either the PBS or isotype-matched antibody controls
(P = 0.003). Treatment with 3D6 was equally effective, producing
an 86% reduction in plaque burden (P = 0.003). In contrast,
16C11 failed to have any effect on plaque burden. Unlike ac-
tively immunized mice, mice receiving exogenous antibodies
against Aβ did not demonstrate a T-cell proliferative response to
Aβ when their splenocytes were examined in vitro, indicating
that a T-cell response is not required for amyloid plaque clear-
ance (data not shown).These results indicate that in the absence
of T-cell immunity, antibodies against Aβ peptide are sufficient
to decrease amyloid deposition in PDAPP mice.
To determine whether the peripherally administered antibod-
ies against Aβ had entered the central nervous system (CNS) to
act directly on plaques, we examined brain sections taken from
saline-perfused mice at the end of the second study. We exposed
unfixed cryostat brain sections to a fluorochrome-tagged reagent
against mouse immunoglobulin. Plaques within the brains of
the 10D5- and 3D6-treated groups were strongly ‘decorated’ with
antibody, whereas there was no staining in the 16C11-treated
group (Fig. 2a, top). To show the full extent of plaque deposi-
tion, we immunostained serial sections of each brain with an ex-
ogenous antibody against Aβ, followed by the secondary reagent
(Fig. 2a, bottom). After peripheral administration, 10D5 and 3D6
had gained access to most plaques within the CNS where they
Peripherally administered antibodies against amyloid
β-peptide enter the central nervous system and reduce
pathology in a mouse model of Alzheimer disease
FRÉDÉRIQUE BARD, CATHERINE CANNON, ROBIN BARBOUR, RAE-LYN BURKE, DORA GAMES,
H
ENRY GRAJEDA
, TERESA GUIDO, KANG HU, JIPING HUANG, KELLY JOHNSON-WOOD,
K
AREN KHAN
, DORA KHOLODENKO, MIKE LEE
, IVAN LIEBERBURG, RUTH MOTTER,
M
INH NGUYEN
, FERDIE SORIANO, NICKI VASQUEZ
, KIM WEISS, BRENT WELCH
, PETER SEUBERT,
D
ALE SCHENK & TED YEDNOCK
Elan Pharmaceuticals, 800 Gateway Boulevard, South San Francisco, California 94080, USA
Correspondence should be addressed to F.B.; email: fbard@elanpharma.com
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NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000 917
ARTICLES
may have directly triggered amyloid clearance. It is likely that
16C11 also had access to the plaques but was unable to bind (de-
scribed below).
We undertook a separate study to determine if antibody treat-
ment resulted in the clearance of preexisting amyloid or simply
prevented formation of new plaques. We administered 3D6 or
control antibody to 13-month-old heterozygous PDAPP mice,
then examined the brains for total plaque burden after 3 and 35
days of treatment. Although there was no obvious change in the
number of large plaques over the short treatment period (Fig.
2b), there seemed to be a reduction in diffuse amyloid and small
aggregates of Aβ (Fig. 2b, inset). By image analysis of the frontal
cortex, nearly 60% of the small plaques and diffuse amyloid, cor-
responding to pixels with intermediate to low intensity, had
been eliminated during the intervening 32 days of treatment
(Fig. 2c; P = 0.001). These results confirm that the antibody treat-
ment triggered clearance of preexisting amyloid.
To further examine the effect of antibodies on plaque clear-
ance, we established an ex vivo assay in which primary microglial
cells were cultured with unfixed cryostat sections of either
PDAPP mouse or human Alzheimer disease (AD) brains. After 24
hours, we fixed, permeabilized and immunostained the cultures
to follow the fate of amyloid β-peptide, and visualized the exoge-
nous microglial cells with a nuclear stain. In PDAPP brain sec-
tions assayed in the presence of 16C11 (one of the antibodies
against Aβ that was not efficacious in vivo), amyloid β-protein
plaques remained intact and there was no phagocytosis. In con-
trast, after culture of adjacent sections in the presence of 10D5,
amyloid deposits were mostly eliminated and the microglial cells
showed many phagocytic vesicles containing Aβ (Fig. 3a, top).
We obtained identical results with AD brain sections: 10D5 in-
duced phagocytosis of AD plaques, whereas 16C11 was inactive
(Fig. 3a, bottom). Furthermore, the assay was equally effective
with either mouse or human microglial cells, and with mouse,
rabbit or primate antibodies against Aβ (data not shown).
By comparison of six different antibodies tested in both sys-
tems, the ex vivo assay was predictive of in vivo efficacy (Table
1). Antibodies 10D5, 3D6 and pabAβ
1–42
were all active in the ex
vivo assay and demonstrated efficacy in vivo. In contrast,
16C11, 21F12 and the control antibody TM2a were inactive in
Fig. 2 After peripheral administration, 10D5 and 3D6 enter the CNS, bind to Aβ plaques and
trigger amyloid clearance.
a
, Cryostat sections of brains from treated mice were exposed directly
to fluorescently conjugated goat anitbody against mouse immunoglobulin (top). Serial sections
from the same brains were exposed to 10D5 before GaM-Cy3 reagent to show full extent of
plaque burden (bottom). Scale bar represents 100 µm. Anti-Aβ, antibody against Aβ.
b
, Total
plaque burden in the hippocampus and cortex in mice treated for 3 and 35 d with 3D6. The
plaque burden is greatly reduced by 3D6 and 10D5 compared with that of 16C11.Scale bar rep-
resents 120 µm. Insets, high-powered magnification of individual large plaques within the cortex
of the corresponding section (scale bar represents 20µm). There is a lack of amyloid fibrils and
small aggregates surrounding the plaque in the day-35 section.
c
, Quantification of diffuse amy-
loid and small plaques shows a 60% reduction between the 3- and 35-day treatment groups (n =
10/group; P = 0.001).
a b
c
Fig. 1 Aβ burden in the frontal cortex of PDAPP mice is reduced after 6
months of treatment with antibodies against Aβ. a and c, Percentage of
frontal cortex area occupied by Aβ deposits shown in individual mice sorted
by treatment group (n = 8). Horizontal lines indicate median values. b, Brain
sections of the cortex and hippocampus obtained from mice with the median
level of plaque burden in their respective groups. Scale bar represents 250 µm.
a cb
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918 NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000
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both assays. Comparison of other antibody characteristics
showed that whereas 16C11 and 21F12 bound to aggregated
synthetic Aβ peptide with high avidity, they were unable to
bind to plaques in unfixed brain sections. This result is consis-
tent with the inability of these two antibodies to decorate
plaques after in vivo administration (as shown for 16C11 in Fig.
2a, top) and explains their inability to trigger plaque clearance.
We found no correlation between antibody efficacy and affin-
ity for soluble Aβ: 3D6 and the inactive antibodies captured
soluble Aβ with moderate to high affinity, whereas 10D5 could
not capture the soluble peptide. These results indicate that
recognition and clearance of deposited Aβ is an important
component of antibody efficacy in vivo and may be more im-
portant than a mechanism involving the prevention of plaque
deposition by recognition of soluble Aβ.
We then used confocal microscopy to confirm that Aβ was in-
ternalized during the course of the ex vivo assay (Fig. 3b). In the
presence of control antibody, the exogenous microglial cells
(red) remained in a confocal plane above the tissue section and
contained no phagocytic vesicles, whereas Aβ (green) remained
in plaques within the tissue plane (Fig. 3b, top). In the presence
of 10D5, nearly all plaque amyloid was contained in vesicles
within the exogenous microglial cells (Fig. 3b, bottom). This re-
sult is consistent with the earlier finding of intracellular Aβ im-
munoreactivity within macrophage and microglial cells in the
CNS of immunized mice
3
. To determine the fate of the internal-
ized peptide, we assessed 10D5-treated cultures by western blot
analysis (Fig. 3b, bottom right). At 1 hour, when no phagocytosis
had yet occurred, reaction with a polyclonal antibody against Aβ
showed a strong 4-kDa band, corresponding to Aβ peptide. Aβ
immunoreactivity decreased at day 1 and was absent by day 3.
There was no degradation of Aβ staining in cultures with control
IgG1. Thus, in contrast to what has been reported for Aβ with
other microglial scavenging pathways
6,7
, antibody-mediated
phagocytosis of Aβ led to its degradation.
To determine if phagocytosis in the ex vivo assay was mediated
by Fc, we prepared F(ab′)
2
fragments of 3D6. Although the anti-
body fragments retained their full ability to react with plaques
(Fig. 3c, bottom), they were unable to trigger microglial cell
phagocytosis (Fig. 3c, top). In addition, phagocytosis induced by
the whole 3D6 antibody was in-
hibited by antibodies specific for
Fc receptors on either human or
mouse microglial cells (data not
shown). These results indicate that
in vivo clearance of Aβ occurred
through Fc receptor-mediated
phagocytosis.
In summary, we have shown
that passively administered anti-
bodies against Aβ peptide reduced
the extent of plaque deposition in
a mouse model of Alzheimer dis-
Fig. 3 Fc receptor-mediated phagocytosis of Aβ in an ex vivo assay.
a
,
Mouse microglia cultured with unfixed cryostat sections of PDAPP (top)
or AD (bottom) brain in the presence of antibodies against Aβ. In the
presence of 10D5, Aβ (red) localizes to vesicles within the membrane
boundaries of microglial cells; in the presence of 16C11, plaques remain
intact and there is no evidence of cellular staining. Scale bar represents
10 µm.
b
, Confocal microscopy shows that in the presence of control
IgG1 (top), microglial cells (red) are in a confocal plane above the tissue
section and Aβ (green) remain in plaques within the tissue plane (differ-
ent planes of the same field are shown here). In the presence of 10D5
(bottom left), nearly all Aβ is localized within the microglial cells. Bottom
right, western blot analysis with a polyclonal antibody against Aβ shows
complete degradation of Aβ within 3 d (left margin, molecular size
marker).
c
, Fc receptor-mediated phagocytosis of Aβ, shown by ex vivo
assay in the presence of intact or F(ab′)
2
fragments of 3D6 (top). Scale
bar represents 50 µm. Aβ plaques in consecutive sections are equally
‘decorated’ with intact 3D6 and its F(ab′)
2
fragments (bottom). Scale bar
represents 300 µm.
Table 1 The ex vivo assay as predictor of in vivo efficacy.
Antibody Isotype Avidity for Affinity for Binding to Ex vivo In vivo
aggregated soluble Aβ efficacy efficacy
Aβ (pM) Aβ (nM) plaques
monoclonal
3D6 IgG2b 470 < 30 + + +
10D5 IgG1 43 no capture + + +
16C11 IgG1 90 110 – – –
21F12 IgG2a 500 80 – – –
TM2a IgG1 – – – – –
PabAβ
1–42
mix 600 nd + + +
The affinity of 10D5 was too low to capture soluble Aβ and could not be measured in this assay. nd, not done.
PDAPP
mouse brain
AD
Brain
16C11 10D5
a b
IgG110D5
c
Intact antibody F (ab′)
2
Ex vivo
Immunostaining
1 hr
1 day
3 days
3.4kD
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NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000 919
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ease. Antibody entry into the CNS was not due to abnormal leak-
age of the blood–brain barrier, as there was no increase in vascu-
lar permeability in PDAPP mice. In addition, the concentration
of endogenous immunoglobulins in the brain parenchyma of
aged PDAPP mice was the same as in nontransgenic mice, repre-
senting 0.1% of the antibody concentration in serum (regardless
of isotype; data not shown). Similar findings have been pub-
lished for antibody levels in human cerebrospinal fluid
8
. These
studies indicate that monoclonal antibodies are able to enter the
CNS at therapeutically relevant levels, and that antibodies may
be considered not only for the treatment of Alzheimer disease,
but possibly for other CNS disorders as well.
Methods
Immunization procedures. Monoclonal antibodies against Aβ were raised
against synthetic peptide fragments derived from different regions of Aβ,
and coupled to a carrier protein as described
9,10
. Polyclonal immune sera
were pooled from 100 mice that had been immunized with full length
Aβ
1–42
, and the immunoglobulin fraction was isolated by standard ammo-
nium sulfate precipitation. After purification, all antibodies were dialyzed
against PBS and had a final endotoxin level of < 1 endotoxin unit (EU), as-
measured by the limulus amoebocyte gel clot assay (Associates of Cape
Cod, Cape Cod, Massachusetts). Antibody titers in serum were determined
weekly as described
3
.
Ex vivo assay. Cryostat sections (10 µm in thickness) of PDAPP mouse or
human AD brains (postmortem interval, less than 3 h) were ‘thaw-
mounted’ onto polylysine-coated, round glass coverslips and placed in
wells of 24-well tissue culture plates. The coverslips were washed twice with
assay medium consisting of hybridoma-serum free medium (H-SFM; Life
Technologies) plus 1% FBS, glutamine, penicillin/streptomycin, and 5
ng/ml recombinant mouse GM-CSF granulocyte–monocyte colony-stimu-
lating factor)(R&D Systems, Minneapolis, Minnesota). Antibodies (control
or against Aβ) were added at a 2X concentration (5 µg/ml final) for 1 h.
Microglial cells were then seeded at a density of 0.8 × 10
6
cells/ml assay
medium. In some experiments, medium contained 10 µg/ml soybean
trypsin inhibitor (Life Technologies). The cultures were maintained in a hu-
midified incubator at 37 °C in an atmosphere of 5% CO
2
for 24 h or longer.
After incubation, cultures were fixed with 4% paraformaldehyde and per-
meabilized with 0.1% Triton-X100. Sections were stained with biotinylated
3D6, followed by streptavidin/Cy3 conjugate (Jackson ImmunoResearch,
West Grove, Pennsylvania). Cultures were observed with an inverted fluo-
rescent microscope (TE300; Nikon, Melville, New York) and photomicro-
graphs were taken with a SPOT digital camera using SPOT software
(Diagnostic Instruments, Sterling Heights, Michigan). For western blot
analysis, cultures were extracted with 8 M urea, diluted 1:1 in reducing
tricine sample buffer, and loaded onto a 16% tricine gel (Novex, San Diego,
California). After transfer onto Immobilon, blots were exposed to 5 µg/ml
pabAβ
42
, followed by horseradish peroxidase-conjugated antibody against
mouse, and were developed with an ECL kit (Amersham).
Microglia culture. Microglial cells were obtained from cerebral cortices of
neonate DBA/2N mice (1–3 days old). Cortices were mechanically dissoci-
ated in Hanks’ balanced salt solution with 50 µg/ml DNase I (both from
Sigma). Dissociated cells were filtered through a 100 µm cell strainer
(Falcon, Heidelberg, Germany) and centrifuged at 200g for 5 min. Pellets
were resuspended in growth medium (high-glucose DMEM, 10% FBS and
25 ng/ml recombinant mouse granulocyte–monocyte colony-stimulating
factor), and cells were plated at a density of two brains per T-75 plastic cul-
ture flask. After 7–9 d, the flasks were rotated at 200 rpm using a Lab-Line
orbital shaker with a 19-mm orbit for 2 h at 37 °C. Cell suspensions were
centrifuged at 200g and resuspended in assay medium.
Acknowledgments
We thank K. Hoenow for technical support.
RECEIVED 5 JUNE; ACCEPTED 20 JUNE 2000
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