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.

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NeurobiologyofDisease
AnEffector-ReducedAnti-?-Amyloid(A?)Antibodywith
UniqueA?BindingPropertiesPromotesNeuroprotection
andGlialEngulfmentofA?
OskarAdolfsson,1MariaPihlgren,1NicolasToni,1YvanVarisco,1AnnaLuciaBuccarello,1KatiaAntoniello,1
SophieLohmann,1KasiaPiorkowska,1ValerieGafner,1JasvinderK.Atwal,2JaniceMaloney,2MarkChen,2
AlvinGogineni,2RobbyM.Weimer,2DeborahL.Mortensen,2MichelFriesenhahn,2CaroleHo,2RobertPaul,2
AndreaPfeifer,1AndreasMuhs,1andRyanJ.Watts2
1ACImmuneSA,1015Lausanne,Switzerland,and2Genentech,SouthSanFrancisco,California94080
Passiveimmunizationagainst?-amyloid(A?)hasbecomeanincreasinglydesirablestrategyasatherapeutictreatmentforAlzheimer’s
disease (AD). However, traditional passive immunization approaches carry the risk of Fc? receptor-mediated overactivation of micro-
glialcells,whichmaycontributetoaninappropriateproinflammatoryresponseleadingtovasogenicedemaandcerebralmicrohemor-
rhage.Here,wedescribethegenerationofahumanizedanti-A?monoclonalantibodyofanIgG4isotype,knownasMABT5102A(MABT).
AnIgG4subclasswasselectedtoreducetheriskofFc?receptor-mediatedoveractivationofmicroglia.MABTboundwithhighaffinityto
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
transgenicmice.WhencomparedwithahumanIgG1wild-typesubclass,containingthesameantigen-bindingvariabledomainsandwith
equalbindingtoA?,MABTshowedreducedactivationofstress-activatedp38MAPK(p38mitogen-activatedproteinkinase)inmicroglia
and induced less release of the proinflammatory cytokine TNF?. We propose that a humanized IgG4 anti-A? antibody that takes
advantageofauniqueA?bindingprofile,whilealsopossessingreducedeffectorfunction,mayprovideasafertherapeuticalternativefor
passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of
vasogenicedema,eveninApoE4carriers.
Introduction
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?
immunotherapywilllikelybemostbeneficialbytargetingmulti-
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.,
2009).
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-
tibodyresponders(Vellasetal.,2009).Activeimmunizationwith
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
ReceivedSept.15,2011;revisedMay11,2012;acceptedMay17,2012.
Authorcontributions:O.A.,M.P.,N.T.,D.L.M.,C.H.,R.P.,A.P.,A.M.,andR.J.W.designedresearch;O.A.,N.T.,Y.V.,
A.L.B.,K.A.,S.L.,K.P.,V.G.,J.K.A.,J.M.,M.C.,A.G.,R.M.W.,D.L.M.,M.F.,C.H.,andR.P.performedresearch;D.L.M.,
M.F.,A.P.,A.M.,andR.J.W.contributedunpublishedreagents/analytictools;O.A.,M.P.,N.T.,A.L.B.,K.A.,K.P.,V.G.,
J.K.A.,J.M.,M.C.,A.G.,R.M.W.,D.L.M.,M.F.,C.H.,R.P.,A.P.,A.M.,andR.J.W.analyzeddata;O.A.,M.P.,A.M.,and
R.J.W.wrotethepaper.
WethankYonglianWu,KunPeng,GloriaMeng,JingLi,andKhanhPhamforkeyinsightsanddata.WethankBob
Kellyforgenerationofantibodyvariants.WethanktheABACUSStudyGroup,includingDrs.AdamBoxer,Malgorzata
Franczak,JeromeGoldstein,MichaelHarris,MelindaLantz,JacoboMintzer,KylePatrick,JoelRoss,BethSafirstein,
FrancoSicuro,LouiseTaber,GeraldTramontano,AlexanderWhite,andDonaldRoyall.
O.A.,M.P.,N.T.,Y.V.,A.L.B.,K.A.,S.L.,K.P.,V.G.,A.P.,andA.M.arefull-timeemployeesofACImmuneSA.J.K.A.,
J.M.,M.C.,A.G.,R.M.W.,D.L.M.,M.F.,C.H.,R.P.,andR.J.W.arefull-timeemployeesofGenentech.
Correspondenceshouldbeaddressedtoeitherofthefollowing:Dr.RyanJ.Watts,DepartmentofNeuroscience,
Genentech,1DNAWay,SouthSanFrancisco,CA94080,E-mail:rwatts@gene.com;orDr.AndreasMuhs,ACImmune
SA,PSE-EPFL,1015Lausanne,Switzerland,E-mail:andreas.muhs@acimmune.com.
DOI:10.1523/JNEUROSCI.4742-11.2012
Copyright©2012theauthors 0270-6474/12/329677-13$15.00/0
TheJournalofNeuroscience,July11,2012 • 32(28):9677–9689 • 9677

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onpassiveimmunizationwithantibodiestargetingA?.Although
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.,
2009).
Here,wedescribeahumanizedanti-A?monoclonalantibody
[MABT5102A(MABT)]thattargetsdifferentA?assemblystates
and contains a human IgG4 backbone with reduced effector
function (van der Zee et al., 1986; Tao et al., 1991). MABT effec-
tivelyreducesA?1–42-inducedneuronaldeathandnotablypro-
motes microglial A? engulfment, but has a significantly reduced
capacity to activate microglial Fc?Rs when compared with an
IgG1 subclass.
To directly assess the potential improvement in safety profile,
MABTwastestedinasingledose,dose-escalationstage,followed
byarandomizedplacebo-controlled,double-blind,parallelmul-
tidose (MD) stage phase I clinical study. Patients were also ran-
domized in the MD stage by ApoE status, as previous studies
showedthatApoE4carriersareathigherriskofdevelopingvaso-
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.
MaterialsandMethods
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
andmeningeswereremoved,andcorticeswerecutintosmallpiecesand
dissociatedwithenzymaticdisruptionat37°Cindissociationbuffer(pa-
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
coatedglasscoverslips,into24-wellplates.CellsweremaintainedinNeu-
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
37°Cand5%CO2.Following1hand30mininculture,themediumwas
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
culturingcellsfor6dinvitro,unlessstatedotherwise.Enrichedmicroglia
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
minat1000?g,andthenresuspendedinprewarmedDMEMcontaining
high glucose containing 10% FCS and penicillin/streptomycin (micro-
glia media). The cell suspension was next transferred to a T75 tissue-
cultureflaskandkeptinahumidifiedincubatorat37°Cand5%CO2for
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
than60%ofcellsstainedpositiveforIba1,withnocellsstainingforboth
GFAP and Iba1. Pure microglia were prepared from postnatal day 3
CX3CR1-GFPmice(TheJacksonLaboratory).Cortexandhippocampus
wasdissectedandtrituratedinDMEMcontaininghighglucoseusinga10
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-
munizingmicewithavaccinepreparedaspreviouslydescribed(Muhset
al., 2007). For in vivo efficacy, both single-transgenic hAPP(V717I)and
double-transgenichAPP(V717I)/PS1female8-to9-month-oldmicewere
administered 10 mg/kg purified mMABT once weekly, 2 or 14 times,
respectively.BrainA?plaqueloadwasevaluatedbythioflavin-Sstaining
in the hAPP(V717I)mice, and memory performance by novel object rec-
ognition test using the hAPP(V717I)/PS1 mice.
A?1–42aggregationassay.A?1–42peptide(Bachem)wasdissolvedin
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-
uumdried,andstoredat?80°CasmonomericA?1–42peptidefilm.For
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
110?Msolutionin27%DMSOandPBS.Thissolutionwasthenallowed
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
Fc?RswasmeasuredusingELISA.HumanFc?Rs(Genentech)arefusion
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-
D265A,orIgG1controlmAbswerepreparedintheassaybuffercontain-
ing10%BlockerCaseininPBS(Pierce).Dilutedsampleswereappliedas
monomericformsforthehigh-affinityreceptor(Fc?RIa),ormultimeric
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
testmAbatanapproximatemolarratioof3:1.Theplateswereincubated
with Fc?Rs at room temperature for 2 h. Plates were washed three times
withwashbuffercontainingPBSand0.05%Tween20aftereachincuba-
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-
dine(Kirkegaard&PerryLaboratories)servedasasubstrate.Plateswere
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

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lecular Devices). Binding curves were generated by plotting the mean
absorbancevaluesfromduplicatesofsampledilutionsagainsttherespec-
tive sample absorbance.
PreparationoftoxicA?1–42oligomers.A?1–42peptide(Bachem)film
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
A?1–42oligomersfor30mininserum-freecellculturemediumat37°C
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-
methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol(SB239063),a
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
treatment,cellsgrownin96-wellplates(Costar)wereincubatedwiththe
MTT dye solution and the generation of a blue formazan product was
measured by reading absorbance at 570 and 690 nm using a microplate
reader(Tecan).Resultsarepresentedasapercentageincreaseinsurvival
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
intotal,andwerefedonday3andonthedayoftreatment.Fortreatment,
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
control.Onday9,following3or4doftreatment,neuronswerefixedand
stainedwithanti-TuJ1antibodyat1:1000dilution.FITC-orAlexaFluor
488-labeledsecondaryantibodieswereusedtovisualizeTuJ1?microtu-
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
thenfixedwith4%paraformaldehydefor20min.Afterthoroughwashes,
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
10%normalgoatserumfor1hatroomtemperature.Afteranovernight
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
measuredincellbodiesdelineatedbysaturatedepifluorescencepictures.
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
A?1–42.PuremicroglialculturesfromCX3CR1-GFPP3pupsweretreated
with1?g/mllipopolysaccharide(Sigma-Aldrich),10?MA?1–42oligomers
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
werefixedwith4%paraformaldehyde,washed,permeatedwithcoldmeth-
anol, blocked with 10% goat serum in PBS, and stained with rabbit anti-
phospho-p38MAPKantibody(CellSignalingTechnology).
In vivo imaging of amyloid plaques. Cranial windows were implanted
above the somatosensory cortex of 10-month-old transgenic hAPP(V717I)/
PS1femalemice,aspreviouslydescribed(Trachtenbergetal.,2002;Holt-
maatetal.,2009),2weeksbeforetheinitialimagingsession.Twenty-four
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
headpost.Imageswerecollectedviaatwo-photonlaser-scanningmicro-
scope (Ultima In Vivo Multiphoton Microscopy System; Prairie Tech-
nologies) using a Ti:sapphire laser (MaiTai DeepSee Spectra Physics;
Newport)tunedto820nmdelivering?30mWtotheback-focalplaneof
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
regionofinterestdrawnaroundagivenplaque.Backgroundisdefinedas
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
poly-L-lysine-coatedsix-wellcellcultureplates(Costar)andusedatDIV
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
ice-coldPBS,harvestedusingacellscraper,andlysedinbufferconsisting
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 ?
SDorSEM,asindicated.DatawereanalyzedbyStudent’sttest,one-way
ANOVA followed by Tukey’s post hoc multiple comparisons, or Wilcox-
on’sranksumnonparametrictestwhenappropriate.Avalueofp?0.05
was taken to indicate a statistically significant difference.
Results
GenerationofahumanizedIgG4antibody,MABT,thatbinds
multipleA? conformations
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

Page 4

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-
transgenichAPP(V717I)
transgenic hAPP(V717I)/PS1 female mice.
When compared with vehicle control,
treatment with mMABT reduced plaque
load in mMABT-treated mice by 31%,
andimprovedmemoryperformancewith
a32%increaseinrecognitionindexasob-
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
hAPP(V717I)transgenicmice(Fig.1E,top)
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
brains.
anddouble-
InhibitionofA?assemblyand
disaggregationofpreformed
protofibrillarA?peptidesby MABT
ThebindingepitopeofMABTwasmapped
to amino acids 12–23 of A?1–42, and
therefore overlaps with the main hydro-
phobic cationic segment of A?1–42
responsiblefortheself-association,subse-
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
A?1–42.Thispromptedustoevaluatethe
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
in?-sheets(LeVine,1993).WhenwecomparedMABTtoacon-
trol anti-A? mAb directed against the N terminus of A?1–42,
andthusnotoverlappingwiththecoreaminoacidsthatformthe
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
assaysarebasedontheabilityofThTtobindtoextended?-sheets
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-
sultsinthisassay,namelythatMABTpreventedtheself-assembly
Figure1.
invivoefficacystudyadministeringmMABTtohAPP(V717I)miceshowedareductioninA?plaqueload(leftpanel)andimproved
memoryperformance(rightpanel).Resultsshowmeans(?95%CI).B,C,AnA?ELISAwasusedtocomparethebindingtohuman
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
A?1–42assemblystates,showingequalbindingforoligomers,monomers,andfibers.E,MABTbindsA?plaquespresentinbrain
sectionsfromtransgenichAPP(V717I)mice(toppanels)andhumanADtemporalneocorticalsections(bottompanels).F,Invitro
functionality was shown by the ability of MABT to impede A?1–42 aggregation (left panel), and to disassemble preformed
A?1–42aggregates(rightpanel)inaThT-basedassay,withanA?1–42toMABTmolarratioof10:1.Ananti-A?IgGmAbwith
aN-terminalepitopewasusedascontrol.Resultsshowthemean(?SD)ofthreeindependentexperiments.*p?0.05;**p?
0.01.G,MABTwasalsotestedinanA?1–42self-assemblyassaythatdoesnotrelyonThTfluorescenceuponbindingtomulti-
mericA?assemblies,asdescribedinMaterialsandMethods.Themeans(?SEM)oftwoassaysareshown.
Theanti-A?MABTmAbbindswithhighaffinitytodifferentA?peptidesandhasantiaggregationproperties.A,An
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Page 5

of A?1–42 in a dose-dependent manner (Fig. 1G). These data
suggest that an antibody directed against the mid-domain of A?,
yetmaintainingbindingtoaggregatedA?,mayprovidethemost
robust inhibitory effect on A?1–42 fibril elongation and/or ag-
gregation relative to antibodies targeting other domains of A?.
MABTneutralizestheneurotoxiceffectsofA?oligomers
in vitro
WenextinvestigatedtheeffectsofMABTinaprimarycellculture
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
cultureswith2.5or5?MA?1–42oligomersover24hresultedin
a reduction in metabolic activity as measured by mitochondrial
oxidationofMTT(Fig.2A),anindicatorofcellviability.Acom-
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
confirmtheseresults,ATPreleasewasmeasuredusingalumines-
cence assay, showing a similar neuropro-
tective effect of MABT (Fig. 2B). To
furtherassesstheeffectofMABTonA?1–
42-mediated neurodegeneration, mouse
embryoniccorticalneuronswerecultured
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,
rightpanel).Fortheseassays,amixtureof
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
degeneration.
A?1–42oligomerinteractionwith
neuronsisinhibitedby MABT
A? peptides, especially aggregation inter-
mediates (Bateman et al., 2007), are
knowntoassociatewithvariouslipidsand
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-
pliedtomixedcorticalculturesfor30min
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
theinteractionofA?1–42oligomerswith
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-
tureswiththisdirectlylabeledA?1–42peptidefurthersupported
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
profilesthatresembledmicroglia(Fig.3E).Thismaybeexpected
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
investigation.
Figure2.
fromP1ratsweretreatedwith2.5or5?MA?1–42oligomerswithorwithout100?g/mlofMABToranIgGcontrolmAb.
An MTT assay was used to determine cell viability as described in Materials and Methods. The means (?SEM) of five
independentexperimentsareshown.*p?0.05;**p?0.01.B,Inacomparableassay,butmeasuringATPproductionas
amarkerofmetabolicactivity,cellsweretreatedwith10?MA?1–42oligomerswithorwithout200?g/mlMABT.Results
show the means (?SEM) of two independent assays. C, Neurotoxicity following extended A?1–42 oligomer treatment
wastestedbymorphologicalanalyses.Cellsasaboveweretreatedfor4dwith10?MA?1–42oligomers,withorwithout
50 ?g/ml of MABT, and then stained for TuJ1 (green) and DAPI (blue). D, Silver-stained SDS-PAGE showing the highly
cytotoxicA?oligomerpreparationsconsistingofamixtureofA?1–42oligomerassemblies,ranginginsizefromdimers
andtrimerstohigher-molecular-weightmultimers.
TheMABTinhibitscytotoxicityofA?1–42oligomersonprimarymixedcorticalcultures.A,Mixedcorticalcells
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Page 6

Aroleforeffectorfunctionin
microglialuptakeofA? oligomers
We next explored the relationship be-
tween MABT/A?1–42 complex forma-
tionandmicroglialuptakeofA?1–42.To
verify that A?1–42 oligomers bound to
MABT are indeed taken up by microglia,
confocalimagingontreatedmixedneuro-
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
APPtransgenicmice(Hickmanetal.,2008).
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-
Talbooetal.,2008).However,uptakeofan-
ti-A?/A?complexesbymicrogliaandFc?R
activationmaytriggerthesecellstobecome
activated.
Inadditiontoovercomingthedirectcy-
totoxicityofA?1–42oligomersonneurons,
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
mutation(MABT-IgG1-D265A)thatdra-
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
becameenrichedinIba1?microglia(Fig.4D).Wealsoobserved
that, in cell cultures treated with A?1–42 in combination with
MABT-IgG1,microgliahadmorecondensednucleiandbrighter
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
not shown).
WenextcomparedthedifferentMABTvariantsfortheirabil-
ity to reverse A?1–42 oligomer-mediated toxicity in mixed pri-
mary cortical cultures. Functional Fc?R binding activity, present
forboththeMABTandMABT-IgG1mAbs,wasrequiredforfull
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
fullrescue,theenhancedbindingoftheMABT-IgG1backboneto
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.
Figure3.
treatedwith2?MA?1–42oligomers,withorwithout100?g/mloftheMABToranIgGcontrolfor30min.Thepanelsfromleft
torightshowthefollowing:treatmentwithbuffercontrol,A?1–42oligomers,andA?1–42oligomerswithMABT.GreenisTuJ1,
redisanti-A?(clone6E10),andblueisDAPI.Thebottomrowshowsallthreemarkers,whereasthetoprowshowsonlyA?and
DAPI.ThetwoinsetsillustratethebindingofA?1–42oligomerstoneurites(left)andtheinhibitionofthisbindingbytheMABT
mAb(right).B,Quantitativemeasuresoffluorescenceareshownfor30minand18htreatments.Meanresults(?SEM)oftwo
experiments are shown. C, A?1–42 tagged with HyLite Fluor-488 verifies that MABT inhibits binding of A?1–42 to neurites.
CorticalculturesfromP1ratsweretreatedasdescribedforA,exceptthatA?1–42taggedwithHyLiteFluor488wasused.Green
isA?1–42andblueisDAPI,withonerepresentativeexperimentshown.D,Greenfluorescencewasquantified,withthemean
(?SD)ofthetwoindependentexperimentsshown.E,UpontreatmentwithMABT,A?1–42oligomersappearedtobetakenup
bycellsresemblingmicroglia.GreenisA?1–42andblueisDAPI.
A?1–42oligomerbindingtoneuritesisreducedbytheanti-A?IgG4mAb.A,MixedcorticalcellsfromP1ratswere
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Page 7

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
Figure4.
MABTaretakenupintomicroglia.A?1–42oligomersareshowninred,andblueisDAPI.Anapical-to-distalslicewasobtainedfromathree-dimensionalimagerenderedfromZ-stacks.Confocal
imagingwasdoneonmixedcorticalcellslabeledforA?(red)andDAPI(blue)asdescribedinMaterialsandMethods.B,C,Microgliawereidentifiedandscannedforthemaximumfluorescence
intensitythroughaseriesofconfocalstacksrepresentingintracellularlocations(B),andthetotalareaoffluorescentsignalaboveaminimumthresholdwasquantified(C).Eachmarkonthegraph
representsthetotalareaofA?stainingwithinasinglecell.Aminimumof20cellswasanalyzedforeachtreatmentcondition.Datawerecomparedusingone-wayANOVAfollowedbyTukey’spost
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
A?1–42,andblueisDAPI.E,DifferentialbindingtoFc?RIIIa-V158wasverifiedinabindingassay.F,Ananti-A?mAbrequireseffectorfunctionviaFc?Rbindingforfullprotectiveeffectagainst
A?1–42oligomertoxicity.MixedcorticalcellsfromP1ratsweretreatedwithA?1–42oligomers,withorwithoutMABT,MABT-IgG1-D265A,MABT-IgG1,oranIgG1controlmAb.Thegraphshows
themean(?SEM)percentageincreaseincellsurvivalcomparedwithA?1–42oligomertreatedcells,fromfiveindependentexperiments.Statisticalanalysiswasdoneusingone-wayANOVA
followedbyTukey’sposthocmultiplecomparison.
A?1–42oligomersaretakenupviaanFcR-mediatedmechanismintomicrogliauponMABTtreatment.A,ConfocalimagingwasusedtoshowthatA?1–42oligomerscomplexedto
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Page 8

generated used human backbones, we were unable to conduct
chronicdosingstudiesasiscommonlydoneformurineantibod-
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
profile(Fig.4E).Thus,usinginvivolive-imagingpreparationsin
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-
ume;?0.036,?0.034,and?0.034).AseconddoseofMABTwas
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-
ingthatMABTcaninduceA?removal,presumablyviamicroglia
uptake.
Reducedmicrogliaresponsewith MABT
ToidentifydownstreammediatorsofA?1–42oligomer-induced
toxicity,weexaminedanumberofcandidatesignalingpathways.
Wefocusedoureffortsonexaminingp38MAPK,whichhasbeen
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
shown)andreachedamaximumat30min.Aftercombiningwith
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.
Astheseinitialassaysmeasuredthetotalp38MAPKactivityin
mixed cortical cultures, including both neuronal and glial cells,
we wanted to know whether the p38MAPK activity detected
whencellsweretreatedwiththecombinationofA?1–42oligom-
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-
firmtheactivationofp38MAPKinmicroglia,wetreatedpurified
microglia in the same way. Under these conditions, A?1–42/IgG
complex-mediatedp38MAPKactivationinmicrogliawasreadily
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
A?1–42oligomersalone,p38MAPKactivationdoesplayarolein
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.Thus,MABThasamoredesirableprofilecomparedwith
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-
Figure5.
visualizedbyinvivotwo-photonmicroscopy,andtrackedovermultipleweeks.B,Therelativechangeinplaquevolumeovertimeplottedasfoldincreasefromtheinitialimagingsession.Onaverage,
theindividualplaquesizedecreasedinvolumeaftersystemicdosingwithMABT(14plaques,2animals).
SystemicdosingofMABTmodulatesindividualamyloidplaquesinvivo.A,AmyloidplaquesinhAPP(V717I)/PS1animalswerelabeledwithmethoxy-X04injectedintraperitoneally,
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Figure 6.
increasesp38MAPKactivationoverthatshownforA?1–42oligomer-treatedmicroglia.MixedcorticalcellsfromP1ratsweretreatedwith10?MA?1–42oligomersfor30minwithorwithout100
?g/mlMABT,MABT-IgG1-D265A,orMABT-IgG1wildtype.AnIgG1mAbnotbindingtoA?wasusedascontrol.Theactivityofp38MAPKwasmeasuredbyaphosphospecificELISAasdescribedin
MaterialsandMethods.Themean(?SEM)offourindependentexperimentsisshown.Statisticalanalysiswasdoneusingone-wayANOVAfollowedbyTukey’sposthocmultiplecomparison.B,
Activationofp38MAPKwithA?1–42oligomerscomplexedwithmAbisspecifictomicroglia.Cellsweretreatedasdescribedaboveandthenstainedforphospho-p38MAPK(green),Iba1(red),and
DAPI(blue).Thepanelsfromlefttorightshowthefollowing:treatmentwithbuffercontrol,A?1–42oligomers,andA?1–42oligomerswithMABT.C,PuremicrogliafromCX3CR1-GFPmicewere
usedtoverifymicroglia-specificp38MAPKactivity.Thetoppanelsshowphospho-p38MAPKimmunofluorescence(red),whereasthebottompanelsareamergeofphospho-p38andGFP(green)on
aphasecontrastimage.D,p38MAPKactivityisrequiredforthefullprotectiveeffectofMABTagainstA?1–42oligomertoxicity.MixedcorticalcellsfromP1ratsweretreated(Figurelegendcontinues.)
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

Page 10

ers; thus, our efforts were focused on evaluating p38MAPK acti-
vation and TNF? secretion as consistent measures of microglial
activation.
ReducedriskofvasogenicedemawithMABTobservedina
phaseIclinical study
AphaseI,randomized,placebo-controlled,double-blind,multi-
centerclinicalstudywasconductedwiththeprimaryobjectiveto
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
genotypedandrandomizedinthemultidosestudytoensurethat
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
withahumanIgG4backbonewithreducedeffectorfunction,can
besafelydosedinhumansatrelativelyhighlevels,thusimproving
CNS exposure. Additional clinical trials are underway to assess
the efficacy of MABT (crenezumab).
Discussion
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-
apyforAD.Amajorthemeofthesafetyfindingsfromthesetrials,
particularly with passive immunotherapy, is disruption of the
BBBasobservedbymagneticresonanceimagingshowingsignsof
vasogenic edema and/or microhemorrhages (Salloway et al.,
2009). Mechanisms underlying these dose-limiting observations
havebeenproposed(forreview,seeWelleretal.,2009).Aprom-
inent immunotherapy-specific hypothesis is that the immune
functions of the particular anti-A? antibody and/or its binding
propertiesarecentraltotheriskassociatedwiththeseBBBinteg-
rityfindings(Wilcocketal.,2004,2006).Ithasalsobeenhypoth-
esized that these safety findings are inseparable from the
mechanisms driving efficacy of immunotherapy (Weller et al.,
2009);however,theevidencetosupportthisclaimislacking.For
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,
thesestudiesdidnotdirectlytesttheabilityoftheseantibodiesto
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-
apyapproachwouldcombinetheessentialA?bindingproperties
to maximize efficacy with the appropriate immune modulation
to limit safety findings.
Weshowthatanti-A?antibodyeffectorfunctioncanbemod-
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
multipleformsofA?,includingoligomericforms,whileinhibit-
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-
sitionthisanti-A?immunotherapycandidatetotestbothmech-
anisms of efficacy and safety in the clinic. Indeed, phase I data
presentedinthisreportareconsistentwiththishypothesis,show-
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-
sogenicedemaasotherhumanIgG1anti-A?antibodies(Carlson
et al., 2011). This is likely because solanezumab does not recog-
nize aggregated A? and thus does not bind vascular amyloid and
elicitanimmuneresponsecausingdisruptionoftheblood–brain
barrier.
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-
tion13and14ofA?arenecessaryforcellmembranebindingand
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
treatmentwithMABTinanunidentifiedcelltypenotresembling
a neuron. Further investigation identified this cell type as micro-
glial, based on Iba1 staining and confirmed in studies using pu-
rifiedCX3CR1-GFPmicroglia.Wewereintriguedbytheseinitial
observations, as MABT is an IgG4 antibody, which has reduced
Fc?R binding properties compared with a wild-type IgG1 and
4
(Figure legend continued.)
?g/mlMABTorMABT-IgG1-D265A,inthepresenceorabsenceof1?MSB239063,ap38MAPK-
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
ANOVAfollowedbyTukey’sposthocmultiplecomparison.E,IgG1islesseffectiveinreducing
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
threeindependentexperimentsisshown.Statisticalanalysiswasdoneusingone-wayANOVA
followedbyTukey’sposthocmultiplecomparison.
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

Page 11

may therefore be less effective at promoting A? engulfment (for
review,seeNimmerjahnandRavetch,2006).Thus,toexplorethe
relative contribution of IgG isotype and associated effector func-
tiontoA?engulfmentbymicroglia,wegeneratedtwoadditional
variantsofMABTwithequalbindingaffinitytoA?,butonewith
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.
ThesedatasuggestthateffectorfunctionmaybenecessaryforA?
engulfment;however,modestbindingtoFc?Rsmaybesufficient
to obtain the desired microglial uptake while limiting activation
and a proinflammatory response. Furthermore, we confirmed
thatMABTcouldreduceA?pathologyinvivousingliveimaging
in hAPP(V717I)/PS1 transgenic mice to assess plaque dynamics
after dosing.
Unlike Fc?R binding, neonatal Fc receptor (FcRn) binding is
notsubstantiallydifferentbetweenIgG4,IgG1,andIgG1-D265A.
Although it is notable that altering Fc? receptor binding alone is
sufficienttoalterabilityofmicroglialengulfmentofA?,thisdoes
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
Figure7.
weekly(MDphase)intravenousadministrationincreasedinproportiontodoseandwerecharacterizedbyslowclearance(?3ml?d?1?kg?1)andalonghalf-life(18–23d).D,Increasesin
plasmatotalA?1–40andtotalA?1–42correlatedwellwithserumMABTconcentrationfollowingsingleorweeklyadministrationintheSDphase;serumMABTandplasmatotalA?sampleswere
collectedjustbeforeMABTadministrationthroughstudyday112.IntheMDphase,sampleswerecollectedjustbeforeMABTadministrationonday0,at1hand7daftereachdoseandto119dafter
lastdosethroughstudyday140.SerumMABTconcentrationsweredeterminedusingaquantitativeELISAwithalimitofdetection(LOD)of0.0225?g/ml.TotalplasmalevelsofA?1–40and
A?1–42weredeterminedusinganelectrochemiluminescenceassaywithaLODof0.01and0.125nM,respectively.A?1–42wasnotdetectableinpatientsgivenplacebo(oratbaselineinpatients
givenMABT)soisnotshown.
MABTphaseIclinicalstudyresults.A,PhaseIclinicalstudydesign.B,Actualstudypopulation.C,TheserumMABTconcentration–timeprofilesfollowingbothsingle(SDphase)or
Adolfssonetal.•Effector-ReducedAnti-A?Antibodies J.Neurosci.,July11,2012 • 32(28):9677–9689 • 9687

Page 12

FcRn-dependent pathway (Deane et al., 2005). Thus, in addition
to the IgG4-dependent ability of MABT to drive microglial en-
gulfmentofA?,itisalsoreasonabletoproposethatMABTcould
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
bindingplaysaroleintheabilityofMABTtopromotemicroglial
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-
pendingonyetpoorlyunderstoodfactors(forreview,seeRanso-
hoffandPerry,2009).Thep38MAPKpathwayplaysacentralrole
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?
treatmentandfoundthatMABT-IgG1withfulleffectorfunction
produced the greatest activation of p38MAPK in microglia. We
alsotestedfordifferentialeffectsonTNF?release,adownstream
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
function.Althoughthereisawelldocumentedroleofp38MAPK
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-
plainwhythelowp38MAPKactivitymeasuredfrommixedneu-
ronalculturestreatedwithA?andMABTappearstoberequired
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
effectorfunctionofananti-A?antibodyisnotnecessaryforneu-
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?
IgG4effector-reducedantibody,wasselectedforitsinvitroandin
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
elicitaproinflammatoryreactionaroundvascularamyloid.Sup-
ported by these preclinical data, a single and multiple dose, mul-
ticenter, randomized, placebo-controlled, double-blind phase I
studytoassesssafety,pharmacokinetics,andpharmacodynamics
was conducted in patients with mild-to-moderate AD. Magnetic
resonanceimagingresultsfromthisstudyareconsistentwiththe
hypothesisproposedherein,asnopatientsshowedsignsofvaso-
genic edema after either single or multiple doses. Moving for-
ward,largerclinicalstudiesareunderwaytofullytesttheefficacy
and safety of crenezumab in AD patients.
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