Insights into the mechanisms of action of anti-A antibodies in Alzheimer's disease mouse models

The FASEB Journal • FJ Express Full-Length Article

Insights into the mechanisms of action of anti-A�antibodies in Alzheimer’s disease mouse models

Yona Levites,* Lisa A. Smithson,* Robert W. Price,* Rachel S. Dakin,* Bin Yuan,†

Michael R. Sierks,† Jungsu Kim,* Eileen McGowan,* Dana Kim Reed,*Terrone L. Rosenberry,* Pritam Das,* and Todd E. Golde*,1

*Departments of Neuroscience and Pharmacology, Mayo Clinic, Mayo Clinic College of Medicine,Jacksonville, Florida, USA; and †Department of Chemical and Materials Engineering, Arizona StateUniversity, Tempe, Arizona, USA

ABSTRACT A number of hypotheses regarding howanti-A� antibodies alter amyloid deposition have beenpostulated, yet there is no consensus as to how A�immunotherapy works. We have examined the in vivobinding properties, pharmacokinetics, brain pen-etrance, and alterations in A� levels after a singleperipheral dose of anti-A� antibodies to both wild-type(WT) and young non-A� depositing APP and BRI-A�42mice. The rapid rise in plasma A� observed afterantibody (Ab) administration is attributable to prolon-gation of the half-life of A� bound to the Ab. Only aminiscule fraction of Ab enters the brain, and despitedramatic increases in plasma A�, we find no evidencethat total brain A� levels are significantly altered.Surprisingly, cerebral spinal fluid A� levels transientlyrise, and when Ab:A� complex is directly injected intothe lateral ventricles of mice, it is rapidly cleared fromthe brain into the plasma where it remains stable. Whenviewed in context of daily turnover of A�, these dataprovide a framework to evaluate proposed mechanismsof A� attenuation mediated by peripheral administra-tion of an anti-A� monoclonal antibody (mAb) effectivein passive immunization paradigm. Such quantitativedata suggest that the mAbs are either indirectly enhanc-ing clearance of A� or targeting a low abundanceaggregation intermediate.—Levites, Y., Smithson, L. A.,Price, R. W., Dakin. R. S., Yuan, B., Sierks, M. R., Kim,J., McGowan, E., Reed, D. K., Rosenberry, T. L., Das,P., Golde, T. E. Insights into the mechanisms of actionof anti-A� antibodies in Alzheimer’s disease mousemodels. FASEB J. 20, E2002–E2014 (2006)

Key Words: immune complex � amyloid deposits

There is compelling evidence that aggregation andaccumulation of A� play a pivotal role in the develop-ment of Alzheimer’s disease (AD). Numerous strategiesto prevent A� aggregation and accumulation are beingevaluated as ways to treat or prevent AD, and a selectnumber of these are now entering the clinic (1).Preclinical studies in APP transgenic mice demonstratethe therapeutic potential of altering A� deposition byinducing a humoral immune response to fibrillar A�42

(fA�42) or passively administering anti-A� mAbs (2, 3).A human clinical trial of active immunization withfA�42�QS-21 adjuvant (AN-1792) was halted due to ameningio-encephalitic-like presentation in �6% of in-dividuals (4–6). No definitive data regarding the na-ture of the meningio-encephalitic presentation havebeen published, but the leading hypothesis, supportedby some recent experimental data, is that it was attrib-utable to an autoreactive T cell response against A�(7). Reports of individuals enrolled in the nowdiscontinued phase II trial suggest that those subjectswho developed robust anti-A� amyloid Ab titers didshow some clinical benefit relative to subjects thatdid not develop robust titers (4, 8, 9). Moreover, ananecdotal report of a small phase II study of ADpatients administered human intravenous infusion ofimmunoglobulin containing anti-A� Abs showedslight improvement in ADAS-cog after administration(10). Because of fears of the possible side effects ofactive vaccination, passive immunization with hu-manized anti-A� mAbs is being vigorously pursued asan alternative approach. One humanized anti-A�mAb is in a phase II trial (http://www.elan.com/research%5Fdevelopment/Alzheimers), and it islikely that additional humanized anti-A� mAbs willbe tested in humans in the very near future. Thus,animal modeling studies and preliminary humandata suggest that efforts to develop better activevaccination and passive immunization strategies arewarranted.

Despite multiple studies examining various parame-ters that may predict the efficacy of active or passiveanti-A� immunotherapy in mice, there is still no con-sensus on how either form of A� immunotherapy works(11, 12). As passive administration of anti-mAb antibod-ies works as effectively as active immunization in APPmice, it is generally acknowledged that it is the anti-A�Ab response that mediates the effects of active immu-nization. The polyclonal response to active vaccination

1 Correspondence: Department of Neuroscience, MayoClinic Jacksonville, Birdsall 210, 4500 San Pablo Rd., Jackson-ville, FL 32224, USA. E-mail: [email protected]

doi: 10.1096/fj.06-6463fje

E2002 0892-6638/06/0020-2002 © FASEB

with fibrillar A� peptides generates multiple Abs withvarying degrees of binding specificity for soluble A�,preamyloid aggregates, and A� amyloid (3, 13–17).Thus, it is potentially misleading to use active vaccina-tion studies to predict precisely which type of Ab ismechanistically associated with efficacy. Nevertheless,in certain active and passive immunization studies, theefficacy of immunization correlates with the ability ofthe immune serum or anti-A� mAbs to recognize A�deposited as amyloid (3, 14, 18). In contrast, certainmAbs that are effective at reducing A� loads and otherAD-like pathologies do not bind A� amyloid (19). Suchdata raise the possibility that the binding properties ofthe mAbs with respect to various forms of A� have notbeen sufficiently characterized to enable identificationof the common target or that there may be multipleways in which anti-A� antibodies can influence amyloiddeposition and other AD-like pathologies.

The amount of A� deposited when immunization isinitiated, the APP mouse model used, the methodologyused to measure differences in amyloid loads, and theproperties of the mAbs used for passive immunizationall affect the outcome of passive immunization (2, 3, 13,14, 20). In PDAPP mice, different types of A� plaquesare reported to be more easily altered by immunization(21). After immunization of PDAPP and other APPmice with abundant diffuse plaques, much larger re-ductions in immunohistochemical amyloid loads arereported than biochemical loads as measured byELISA, suggesting a preferential reduction of diffuseA� deposits (2, 19, 22). Moreover, at least in terms ofpercent reduction in amyloid loads, most studies showmuch more dramatic effects in mice initially treatedwhen they have only minimal plaque deposition (13,18, 23). Even more puzzling, some mAbs that work wellwhen administered before plaque deposition do notwork at all once plaque deposition has begun, whereasother mAbs work in both settings (18). Finally, in somereports it can be shown that intact mAb is not requiredfor efficacy: Fab and scFv fragments work (24, 25;Levites and Golde, unpublished observations), as wellas A� binding proteins/gangliosides (26). Such datasuggest that the binding of A� by Ab, but not effectorfunctions of the Ab, are required for efficacy.

A great deal of debate with respect to mechanismalso centers on peripheral vs. central action of ananti-A� Ab (19, 27). The peripheral sink hypothesisproposes that binding of A� in the blood enhancesefflux from the brain. Several studies have shown thatplasma A� levels increase dramatically after both activeand passive A� immunotherapy and that at least someof the A� in the plasma is complexed to the anti-A�antibodies (19, 27), but it is not clear whether thisperipheral binding accounts for a decrease in braindeposition. Another issue relates to whether microgliaactivation contributes to efficacy; current data on therole of microglia are inconsistent. Several groups re-port transient or stable enhancements of microglialactivation associated with A� removal; others do not (2,28–30). At least in Tg2576 APP mice, a role for

enhanced phagocytosis of mAb:A� complexes via theFcR has been ruled out (30). In postmortem humantissue from several AD patients who had received theAN-1792 vaccine, A� laden microglia are noted in areaswhere A� clearance is hypothesized to have occurred(6, 31).

To provide a quantitative framework in which toanalyze proposed mechanisms of action of anti-A�immunotherapy, we have measured the acute effects onplasma, cerebrospinal fluid (CSF), and brain A� after asingle intraperitoneal dose of several anti-A� mAbs. Forthe initial studies, we chose an anti-A�1–16 IgG2a,(mAb9; ref 18). mAb9 recognizes both monomeric andaggregated A� and A� amyloid. We also performed amore limited set of studies using an anti-A�1–16 IgG1(mAb3), an anti-A�x-40 IgG1 (mAb40.1), and an anti-A�x-42 (mAb42.2) (18). To avoid potential confoundsintroduced by the presence of A� deposits, these studieswere conducted in young APP or BRI-A�42 transgenicmice prior to A� deposition (32, 33). We also measuredmAb9 levels and estimated the half-life of the mAb9:A�complex in nontransgenic mice. We chose these mAbs forstudy, as we have previously shown that 500 �g of eachchronically administered intraperitoneally every 2 wk cansignificantly attenuates A� deposition (18). These datashow that in mice 1) binding of mAbs to A� significantlyprolongs the half-life of plasma A� from minutes to days,2) very little free anti-A� mAb actually enters the brain orCSF, 3) anti-A� mAb:A� complexes are rapidly clearedfrom the brain, 4) that passive administration of theseanti-A� mAbs has little effect on total steady-state prede-position brain A� levels, and 5) that neither the amountof mAb relative to the amount of A� or type of anti-A�mAb significantly influence the overall changes in A�induced acutely after passive immunization. Such data,when viewed in the context of the total amount of A�turnover in a human or mouse per day, have importantimplications with respect to design and interpretation offuture studies using active or passive immunotherapy as atreatment or preventive strategy for AD.

MATERIALS AND METHODS

Antibodies

The anti A�1–16 specific mAb9 (IgG2a) and mAb3 (IgG1)used for immunizations as well as anti-A�40 specific mAb40.1(IgG1) and anti-A�42 specific mAb42.2 (IgG1) used forELISAs were characterized previously (18). Biotinylation wasperformed according to the manufacturer. Briefly, 0.27 �molof sulfo-NHS-LC-biotin (Pierce, Rockford, IL) was added to2 mg mAb9 or mouse IgG and incubated for 2 h at RT,followed by purification of labeled protein over desaltingcolumn. 4G8, human A�17–14 epitope was obtained fromSignet (Dedham, MA). Mouse IgG was obtained fromEquitech-Bio Inc. (Kerrville, TX).

Mice

All animal husbandry procedures performed were approvedby the Mayo Clinic Institutional Animal Care and Use Com-

E2003ANTI-A� IMMUNOTHERAPY: MECHANISMS OF ACTION

mittee in accordance with National Institutes of Healthguidelines under protocol A34602. Tg2576 mice and BRI-A�42B mice were generated and confirmed by genotyping asdescribed previously (32, 33). All animals were housed 3–5 toa cage and maintained on ad libitum food and water with a12 h light/dark cycle.

Binding kinetics

Affinity measurements were performed using a BIAcore Xbiosensor (BIAcore Inc., Piscataway, NJ). A CM5 sensor chip(BIAcore) was activated as recommended by the manufac-turer using an equimolar mix of N-hydroxysuccinimide(NHS) and N-ethyl-N�-(dimethylaminopropyl)carbodiimide(EDC), immobilized with 50 �l of a capture Ab (BR100514,100 �g/ml in 10 mM NaAcatate, pH 4.8), and then blockedwith ethanolamine; 70 �l of the mAb (diluted in runningbuffer (HBS-EP) at 100 �g/ml) were injected onto theimmobilized chip. The association and dissociation rate con-stants (ka and kd) were determined using an A� concentra-tion range with HBS-EP [0.01 M HEPES, 0.15 M NaCl, 3 mMEDTA, 0.005% (v/v) surfactant P20, pH 7; BIAcore, Uppsala,Sweden] as a running buffer at a flow rate of 10 �l/min. Thesensor surface was regenerated using 10 mM Glycine-HCl, pH1.5. Kinetic parameters were evaluated using BIA evaluation3.1 software (BIAcore).

Passive immunizations

Young Tg2576 mice or nontransgenic controls (3 month old,n�4 per group) were given a single intraperitoneal dose of500 �g (1600 pmol) biotinylated mAb9. Control mice re-ceived biotinylated mouse IgG or PBS.

Intracerebroventricular injections

For stereotactic intracerebroventricular injections, nontrans-genic mice (females, 3 monthold, n�2 per group) wereinjected with preformed complex of 50 �g (�160 pmol) ofbiotinylated mAb9 and �320 pmol of A� in the left cerebralventricle. On the day of the surgery, mice were anesthetizedwith isoflurane (5% induction and 3% maintenance) andplaced in a stereotactic apparatus. A midsagittal incision wasmade to expose the cranium, and a hole was drilled to theafter coordinates taken from bregma: A/P, �0.4 mm; L, �1.0mm. A 26 gauge needle attached to a 10 �l syringe waslowered 1.8 mm dorsoventral, and a 4 �l injection was madeover 10 min. The incision was closed with surgical staples, andthe mice were killed at various time points after the surgery.

Measurement of mAb9, A� or A�40:mAb9 complexesin plasma

Groups of female Tg2576 mice or their nontransgenic litter-mates were immunized with biotinylated mAb9 and plasmawas collected at various time points. Control mice receivedbiotinylated mouse IgG or PBS. To measure the A�40-biotinylated mAb9 complex in the plasma capture, ELISA wasused with an Ab against free end of A�40 peptide, mAb40.1(2.5 �g/well), as capture and Neutravidin-HRP, 1:2000, asdetection. For standards, we saturated mAb40.1-coated platewith A� (5 �g/well), applied increasing amounts of biotinyl-ated mAb9, and detected with Neutravidin-HRP. Control PBSinjected plasma was spiked with 500 �g mAb9 to determinethe basal levels of A� capable to bind mAb in the plasma. Todetermine the level of total A�40, we used mAb40 as captureand 4G8, 1:2000, as detection. In non-Tg mice, levels of

biotinylated mAb9 were determined by direct ELISA withA�40 (5 �g/well) as capture and Neutravidin-HRP as detec-tion. Additionally, 1 ml plasma pooled from 3 mice 24 h afterthe administration of biotinylated mAb9 or biotinylatedmouse IgG was fractionated on a 1 � 30 cm Superose 6 PC3.2/30 column (Amersham Biosciences, Piscataway, NJ). Su-perose columns were routinely pretreated with a bolus of BSA(50 mg) in running buffer to block nonspecific bindingfollowed by a wash with at least 4 column volumes of runningbuffer. A�40 in each fraction was measured using captureELISA as described above.

ELISA analysis of extracted A� from the brain

At the time of death, the brains of mice were divided bymidsagittal dissection, and both hemibrains were used forbiochemical analysis. One hemibrain was homogenized inTBS with CompleteTM protease inhibitors (150 mg/ml wetwt) while the other hemibrain was homogenized in radio-immunoprecipitation assay (RIPA; 50 mM Tris-HCl pH 7.4,150 mM NaCl, 1% Triton x-100, 1% Sodium deoxycholate,0.1% SDS) with CompleteTM protease inhibitor. Homoge-nates were than centrifuged at 20,000 g for 1 h at 4°C, theresultant supernatant was collected, representing the TBS- orRIPA-soluble fraction, respectively. Additionally, a hemibrainwas homogenized in guanidinium extraction buffer (GuHCl,5M Guanidine and 50 mM Tris-HCl) and incubated at roomtemperature for 4 h, representing GuHCl fraction. The aftermAbs against A� were used in the sandwich capture ELISA:for brain A�40, mAb40.1 capture and 4G8-HRP detection; forbrain A�42, mAb42.2 capture and 4G8-HRP detection. Todetermine the amount of biotinylated mAb in the brain,direct ELISA with A�40 as capture and Neutravidin-HRP asdetection was used.

Collection of cerebrospinal fluid

The procedure was performed according to that described byVogelweid et al. (34). Briefly, mice were anesthetized with2.5% Avertin IP. The fur of the animal was clipped and placedin ventral recumbence over a gauze roll (attached to a13�10�6 cm support) allowing the head to lie at a 45 degreeangle. A small strip of transpore tape was used to hold thehead in place. A midline incision starting at the base of thepinnae and continuing for �1 cm caudal was made with a #10blade. Iris scissors were used to separate the muscle layers ofthe “pocket” �2 mm below the caudal edge of the occipitalbone down to atlas. The underlying layers were bluntlyseparated with microdissecting forceps and retracted withbull clamps to visualize the dura mater, an opaque triangular-shaped membrane. If microhemorrhaging occurred duringdissection, the window was blotted gently with an absorbenttriangle to clear the area. An 18 gauge needle was guided togently pierce the dura mater over the cisterna magna fol-lowed by immediate replacement with a pulled pipette (andaspirating bulb) to collect the CSF. The CSF was transferredto a gas tight screw cap vial and stored at –80C.

Measurement of A� and A�40-monoclonal Ab complexin CSF

To measure the A�40-biotinylated mAb9 complex in the CSFcapture, ELISA was used with an Ab against free end of A�40peptide, mAb40.1 as capture, and Neutravidin-HRP as detec-tion. To determine to level of total A� we used mAb40.1 ascapture and 4G8-HRP as detection.

E2004 Vol. 20 December 2006 LEVITES ET AL.The FASEB Journal

Statistical analysis

One-way ANOVA followed by the Dunnet’s multiple compar-ison test was performed using the GraphPad Prism version 4software.

RESULTS

Peripheral administration of anti-A� mAb creates astable mAb:A� complex in the plasma

Previous studies have established that A� has a veryshort half-life in the plasma. When free A� is injectedintravenously into the animal, it is cleared with ahalf-life of 10 min (35, 36). Such data are consistentwith our finding that intraperitoneal administration ofa single 20 mg/kg of dose of a -secretase inhibitor toTg2576 mice can reduce plasma A� by 80% within 1 hand by �98% within 5 h, indicating that even endoge-nous plasma A� has a short half-life (data not shown).To study changes in A� levels induced by passiveimmunization with an anti-A� mAb as well as the in vivobinding properties and plasma half-life of the mAbitself, 500 �g (�1600 pmol) of biotinylated mAb9 wasadministered intraperitoneally to 3 month old nonde-positing female Tg2576 mice. Plasma A� levels wereanalyzed by capture ELISA over an extended timecourse. To ensure that the biotinylated mAb9, whichrecognizes A�1–16, did not interfere with detection ofA� by ELISA, A� was captured with end specific anti-A�mAbs and detected with HRP-conjugated 4G8, whichrecognizes a nonoverlapping epitope on A�. In pilotstudies with synthetic A� standards, mAb9 did notinterfere with A� detection in end specific capture 4G8detection ELISAs. After biotinylated Ab9 administra-tion, within 1 day after administration, A�40 in theplasma increased �15-fold, from �50 pmol/ml inuntreated mice to almost 750 pmol/ml and and A�42levels increased �25-fold, from �2 pmol/ml in un-treated mice to almost 55 pmol/ml, respectively.Plasma A� levels then slowly decreased over an ex-tended period of time to near basal levels by 14 days(Fig. 1A). To examine the extent to which mAb bindingof A� causes an increase in plasma A�, we detectedbiotinylated mAb9:A� complexes in plasma using amodified ELISA. The biotinylated mAb9:A� complex iscaptured with an A�40 specific mAb and the complexdetected with Neutravidin-HRP. The amount of thebiotinylated mAb9:A�40 complex reached its highestvalue of �450 pmol mAb9 bound to A�40 per ml ofplasma after 6 h (Fig. 1B). The complex appears to bequite stable with a half-life of �7 days. Although thedifference in standardization methods between ELISAmeasurements of plasma A� and plasma biotinylatedmAb9:A� complexes introduce some uncertainty withrespect to the levels of “total” plasma A� relative to thelevel of biotinylated mAb9:A�, a comparison of thepeak levels of total A� and mAb:A� complex wouldsuggest that the majority of A� is bound to the mAb.

Consistent with these data, we find that preclearing theplasma with protein A/G removes �90% of the ELISAsignal (data not shown). Size exclusion column chro-matography of mouse plasma collected 1 d post mAb9injection shows that most of the plasma A� that accu-mulates after mAb9 treatment is present in a high

Figure 1. A� and mAb levels after passive immunization withmAb9. A) 3 month old Tg2576 mice were dosed i.p. with 500�g (1600 pmol) biotinylated mAb9. A� levels were measuredat different time points by ELISA with end-specific anti-A�40mAb (Ab40.1) as capture and 4G8-HRP as detection. B)Levels of A� bound by biotinylated mAb9 in plasma weremeasured at different time points by ELISA using mAb40.1 ascapture and Neutravidin-HRP as detection. n � 4 per group,*P 0.001 vs. control. C) Plasma from Tg2576 mice dosedwith biotinylated mAb9 or biotinylated mouse IgG was frac-tionated by size-exclusion chromatography. Levels of A� ineach fraction were measured by ELISA. D) A�:biotinylatedmAb complex in plasma of treated Tg2576 mice was immu-noprecipitated with streptavidin beads, dissolved in SDS-Pagesample buffer and subjected to a 12% Bis-Tris electrophoresisgel. A� was detected by mAb9 (1:1000). E, F) 3 month oldnontransgenic mice were dosed i.p with 500 �g (1600 pmol)biotinylated Ab9 (E) or with complex of �1600 pmol biotin-ylated mAb9 and �3200 pmol A�40 (F). E) BiotinylatedmAb9 levels in the plasma were measured by direct ELISA(see methods). F) Levels of A� bound by biotinylated mAb inplasma were measured at different time points by ELISA. n �4 per group, *P 0.01, **P 0.001 vs. control.


molecular weight fraction with a peak level in a fractionthat corresponds to the peak fraction in which un-bound mAb9 elutes (Fig. 1C). In the plasma from themice injected with biotinylated mouse IgG, the levels inmost fractions are much lower and A� appears to bebroadly distributed presumably because, as previouslyreported, it is bound to numerous serum proteins(37–39). Finally, when plasma from biotinylated mAb9injected mice is precipitated with Streptavidin beadsand subjected to Western blot analysis, an increase in a4 kDa A� species is observed (Fig. 1D)

The half-life of an IgG2a Ab in mouse plasma hasbeen reported to be �1 wk (40). When 500 �g (�1600pmol) of biotinylated mAb9 are administered to 3month old female nontransgenic littermates of theTg2576 mice, �800 pmol mAb9/ml plasma can bedetected in the plasma 1 d later. The biotinylated mAb9is quite stable and appears to have a half-life of 5–7 d(Fig. 1E). Collectively, such data suggest that the in-crease in A� levels is attributable to binding andstabilization of A� by the anti-A� mAb. To directlydetermine if binding of the mAb9 to A� prolongs thehalf-life of A�, we administered via i.p. injection apreformed complex of biotinylated mAb9 (500 �g,�1600 pmol) and human A�40 (�3200 pmol) intoyoung nontransgenic mice. The mAb9:A�40 complexwas detected as described previously. As mAb9 does notrecognize mouse A�, these studies are not confoundedby mAb interaction with endogenous mouse A�. Within6 h, �500 pmol/ml of the complex is detected and thecomplex, like the unbound Ab, is cleared slowly with ahalf-life of �5–7 d (Fig. 1F). Thus, in contrast toendogenous A�, the mAb9:A�40 complex has a pro-longed half-life. In addition, these studies suggest thatthe binding of the mAb to A� does not result in theformation of a classic immune complex that would berapidly cleared. Finally, such data suggest that inplasma the tight binding of mAb9 to A� (Kd is esti-mated by surface plasmon resonance to be �3.5e-9 M)prevents the bound A� from being rapidly turned over.

Effects of acute immunization with anti-A� mAbon A� levels in the brains of Tg2576and BRI-A�42B mice

To determine if alterations in brain A� occur afterperipheral immunization, we examined the effects onbrain A� in young female Tg2576 mice for up to 2 wkafter intraperitoneal administration of 500 �g of bio-tinylated mAb9. To reduce interference from vascularA� and mAb9, we extensively perfused the mice withPBS prior to brain harvest. A�40 and A�42 levels weremeasured by ELISA in separate TBS, RIPA, and 5 Mguanadinium hydrochloride (GuHCl) fractions. Inthese studies and as previously reported, GuHCL ex-tracts the highest levels of A� from the brain (41), anddespite the marked accumulation of plasma A� at the 6and 24 h time points, there is no appreciable change inthe levels of GuHCl-extractable brain A�40 or A�42(Fig. 2A). TBS extracts presumably reflect levels of

soluble A� and contain much lower amounts of A�than are present in the GuHCl extract (A�40 �6–7%and A�42 �2–3% of GuHCL extract A� levels). TBS-extractable A�40 and A�42 increase slightly after pe-ripheral administration, though the absolute level ofincrease is small, �5–10% of control values, and doesnot reach statistical significance by ANOVA (Fig. 2B).RIPA, a moderately denaturing detergent mix, extractsa higher level of A�40 and A�42 than TBS but lowerlevels than GuHCl (A�40 �25–30% and A�42 �8–10%of the GuHCL extract A� levels). RIPA-extractable A�decreases slightly after immunization by 20% of controlor �10 pmol/g (Fig. 2C). No statistically significantdecrease in RIPA-soluble A�40 levels is detected up to14 d after a single mAb administration (Fig. 2D).Moreover, the slight decrease observed 24 h after thesingle mAb administration is not additive, since contin-uous weekly administration of 500 mg mAb for 4 wkresults in similar slight but not significant decrease inRIPA-soluble Ab levels (Fig. 2E).

Tg2576 mice make large amounts of A� bothperipherally and in the brain. In nondepositingTg2576 mice, this A� is rapidly turned over. Thehalf-life of A� in brain is estimated to be 1–2 h(42– 44). Indeed, our studies on mAb9 binding toplasma A� in Tg2576 mice suggest that after periph-eral immunization, mAb9 is saturated with A� within6 –12 h of administration. Thus, the small changes inbrain A� observed in Tg2576 mice immediately aftermAb9 administration might be amplified if moremAb were administered or if the same amount ofmAb was administered to a transgenic mouse, whichproduces much lower levels of A�. Because we werealready delivering an amount of mAb that was nearthe maximal tolerated dose, we administered thesame amount of biotinylated mAb9 to a low express-ing BRI-A�42B line (32). This line of BRI-A�42Bmice only expresses A�42 and has �5-fold lowerlevels of total brain A� and �100 fold lower plasmalevels relative to Tg2576 mice. At 3 months of age,these mice do not have detectable A� deposits. AftermAb9 administration we again observe a rapid in-crease in A� levels in the plasma from �0.5 pmol/mlin untreated mice to �7 pmol/ml at 3 h and �30pmol/ml 1 d after immunization (Fig. 3A). Theamount of the biotinylated mAb9:A�42 complexincreases in parallel (data not shown). There was nosignificant change in total brain A�42 levels ex-tracted by GuHCl (Fig. 3B), a slight, nonsignificantincrease in TBS-extractable brain A�42 levels (totalincrease �15%; Fig. 3C), and a slight nonstatisticallysignificant decrease in RIPA-extractable A�42 (�25%;Fig. 3D). The magnitude of these changes is similarto that seen in Tg2576 mice, indicating that the smalleffects induced by mAB9 administration are notinfluenced to any great extent by the relative amountof plasma or brain A� in the different transgeniclines.


Brain levels of mAb9 after acute peripheraladministration of anti-A� mAb

In previous studies we failed to detect anti-A� mAbbinding to plaques after peripheral anti-A� mAb ad-ministration using immunohistochemical techniques.Others, however, have reported that, consistent withprevious reports of blood brain barrier (BBB) pen-etrance of Abs, a small fraction of anti-A� mAbs canpenetrate the BBB (if quantified levels are 0.1% oftotal dose; refs 3, 27, 45). After administration viaintraperitoneal injection of 500 �g (1600 pmol) biotin-ylated mAb9 to nontransgenic mice, we can detect1.0 � 0.08 fmol/mg of biotinylated mAb9 6 h postin-jection, which is �300 fmol per brain or �0.02% of thetotal amount of the Ab administered. The levels of Abfall by 24 h to 0.53 � 0.06 fmol/mg and by 2 wk thelevels are 0.06 � 0.01 fmol/mg. Even lower levels ofmAb9 were detected in the Tg2576 brain (data notshown). Despite extensive perfusion, it is impossible todetermine whether these trace amounts of mAb9 are

truly in the brain or simply stuck to the cerebral vessels;multiple attempts to detect the mAb in situ in the brainsections using immunohistochemical techniques gavenegative results. In any case, such data place an upperlimit on the amount of mAb9 present in the brain at thetime the plasma mAb levels are near maximal.

Effects of anti-A� mAb on CSF A� and clearanceof mAb9:A� complexes from the brain

We also examined the levels of A� and biotinylatedmAb9:A� complexes in the CSF after intraperitonealadministration of mAb9 to Tg2576 mice. Six hourspost-mAb injection, a 6-fold increase in A�40 and a2-fold increase in A�42 levels is observed in CSF collectedfrom the cisterna magna. This result contrasts with plasmaA� levels, which peak at 6 h post mAb injection andremain at a relatively stable baseline over 24–72 h (Fig.1A); CSF A� levels decrease rapidly toward controllevels by 24 h (Fig. 4A). Low levels of biotinylatedmAb9:A� complexes are also detected in the CSF, and

Figure 2. Effects of mAb9 on A� levels in the brains of Tg2576 mice. Three month old Tg2576 mice were dosed with 500 �gbiotinylated mAb9. 6 h–14 days later mice were perfused with PBS, and A� levels in GuHCl (A), TBS (B), and RIPA (C, D) brainextracts were detected by ELISA using end-specific anti-A�40 mAb40.1 or anti-A�42 mAb42.2 as capture and 4G8-HRP asdetection. n � 4 per group. E) 3-month-old Tg2576 mice were dosed with 500 �g biotinylated mAb9 every week for 4 wk. Micewere killed 24 h after the final mAb admisntration. A� levels in RIPA brain extracts were detected by ELISA. n � 4 per group.


change in parallel with A� levels (Fig. 4B). Unlike inplasma, where A� levels are roughly comparable to thelevels of mAb9 bound to A�, in CSF there is �50-foldmore A� than mAb bound to it. One possible explana-tion for this high ratio of A� to mAb would be that mAbis bound to an A� aggregate in CSF. The concentrationof the mAb9:A� complex in the plasma remain un-changed during this period, suggesting there may berapid export of the mA�9:A� complex from the CSF.To explore this possibility, we injected intracerebroven-tricular a preformed complex of 5 �g (�160 pmol) ofbiotinylated mAb9 and �320 pmol of A�. After injec-tion into the ventricles, the biotinylated mAb9:A�complex is detected in CSF collected from the cisternamagna within 30 min. By 3 h, the levels are dramaticallydecreased and at 24 h no complex is detectable (Fig.4C). In contrast, the low levels of complex appear inplasma by 30 min and appear relatively stable up to 72 hpost injection. Such data suggest that even though theanti-A� mAb:A� complex has a long-half-life in theplasma, the complex is rapidly cleared from the CSF,

and at least some of this clearance is via export into thevasculature.

Additional anti-A� mAbs have similar effects on A�levels in plasma, brain and CSF of Tg2576 mice

To determine if the observed dynamics in plasma, CSF,and brain after an acute dose of mAb in TG2576 miceare common to the other anti-A� mAb characterized inour previous studies and shown to reduce A� deposi-tion after peripheral administration, we injected 500 �gbiotinylated anti-A�1–16 mAb3, anti-A�42 mAb 42.2,and anti-A�40 mAb40.1 to 3 month old Tg2576 mice(18). Like mAb9, mAb3 administration results in an�7-fold increase in A�40 and �20-fold increase in A�42levels in plasma (Fig. 5A), but only a slight, nonsignif-icant decrease in A�40 levels in RIPA-soluble brainextracts and no effect on RIPA-soluble A�42 levels (Fig.

Figure 4. Effects of mAb9 on A� levels in the CSF. Threemonth old Tg2576 mice were dosed with 500 �g (1600 pmol)biotinylated Ab9, i.p. A) Total levels of A�40 and A�42 in theCSF were measured using Ab40.1 or Ab42.2 as capture and4G8 as detection. B) Levels of A�40 bound by mAb in the CSFwere measured after 6 or 24 h by capture ELISA using Ab40.1as capture and Neutravidin-HRP as detection. n � 4, *P 0.01, **P 0.001 vs. control. Data is shown from a singleexperiment. Similar data were seen in 2 other independentstudies. C) 3 month old Tg2576 mice were injected with 50 �g(160 pmol) biotinylated mAb9 bound to �320 pmol A�,intracerebroventricular. Levels of A� bound by mAb inplasma and CSF were measured by capture ELISA. n � 2.

Figure 3. Effects of mAb9 on A� levels in the plasma andbrains of BRI-A�42B mice. Three month old BRI-A�42B micewere dosed with 500 �g biotinylated mAb9; 6 and 24 h latermice were bled and perfused with PBS. A� levels in plasma(A) as well as in GuHCl (B), TBS (C), and RIPA (D) brainextracts were detected by ELISA using end-specific anti-A�42mAb42.2 as capture and 4G8 mAb as detection. n � 4 pergroup.


5B). mAb40.1 and mAb42.2 are end-specific antibodiesthat have been shown to selectively bind A�40 andA�42, respectively, in vivo. To avoid interference by theend-specific mAbs present in the plasma, in the ELISAswe only measured total A� levels using mAb9 as captureand mAb 4G8-HRP as detection. Both end-specifcmAbs caused an increase in total A� levels in plasma 6and 24 h after the injection. Higher levels of plasma A�accumulated after administration of mAb40.1, thenmAb42.2, presumably because the mAbs are end-spe-cific; thus the “total” A� level reflects the relativeabundance of these species in the plasma. No effect wasobserved on the brain RIPA-soluble A� (Fig. 5D and E).A� levels in CSF were also increased on administrationof all three mAbs, although the dynamics of thisincrease vary between the antibodies (Fig. 5C and F).

DISCUSSION

These data provide a quantitative framework inwhich to consider possible mechanisms of action of

an anti-A� mAb with respect to decreasing A� depo-sition. Before fully exploring the possible implica-tions of the current findings, it is important toconsider the data in light of estimates of the totalamounts of A� that are synthesized and cleared inTg2576 mice in a similar time period (see Table 1).There is extensive evidence that A� is rapidly metab-olized in vivo (35, 36, 42– 44). In the brain ofnondepositing Tg2576 and other APP mice, thehalf-life is estimated to be 2 h, and in plasma thehalf-life is estimated to be �5–10 min. As measuredby our ELISA systems, the steady-state levels of A� inthe GuHCL extract from Tg2576 brains is �100pmol/gm and the steady-state level in plasma is �50pmol/ml. Assuming a conservative 2 h half-life forbrain A� and total brain wt of �0.4 g, �10 pmol ofA� are cleared from the brain every hour and �240pmol of brain A� are cleared every day. Again,assuming a conservative 10 min half-life and a plasmavol of �1 ml, �25 pmol of A� are cleared every 10min and �3600 pmol of plasma A� are cleared per

Figure 5. Effects of three anti-A� antibodies mAb3, mAb42.2 and mAb 40.1 on A� levels on A� levels in plasma, brains and CSFof Tg2576 mice. Three month old Tg2576 mice were dosed with 500 �g biotinylated mAb3, mAb42.2 and mAb 40.1; 6 and 24 hlater plasma and CSF were extracted and mice were subsequently perfused with PBS. For mAb3, A�40 and A�42 levels in theplasma (A), RIPA brain extracts (B) and CSF (C) were detected by ELISA using mAb42.2 (Ab42) or mAb40.1 (Ab40) as captureand 4G8 mAb as detection. To avoid possible interference, only total A� levels were measured in plasma, brain and CSF ofmAb40.1 and mAb 42.2 treated mice using ELISA with mAb9 as capture and 4G8 mAb as detection (D–F ), n � 4 per group,*P 0.05, **P 0.01 vs. control.


day. Thus, in Tg2576 mice we estimate total A�turnover at �4 nmol per day, with a great deal moreA� cleared in the periphery.

The 500 �g dose of anti-A� mAbs that we have usedin these studies and our previous long-term studiesrepresents �1600 pmol of mAb and would be pre-dicted, if 100% bioavailable, to maximally bind �3200pmol of A� (18). A more likely estimate is that afterintraperitoneal injection no more than �50% of theanti-A� mAb is bioavailable in the plasma. Because thebinding of mAb9 to A� significantly extends the half-life of plasma A� without altering the half-life of themAb, it is clear that mAb9 binding of plasma A� couldsequester the total amount of A� normally cleared inthe periphery for a maximum of 12–24 h. Our datashowing that the biotinylated mAb9:A� complexreaches peak levels (of �500 pmol of mAb9) 6–12 hafter intraperitoneal administration are entirely consis-tent with these estimates. Thus, the large increase inplasma A� observed after peripheral administration iseasily attributable to peripheral binding of A� by themAbs.

Consistent with previous studies of mAb penetranceinto the brain, our current studies indicate that theamount of anti-A� mAb that enters the brain is 0.1%of the total mAb injected (3, 27, 45). If we assume 50%bioavailability, then the maximum amount of anti-A�mAb that can reach the brain using the 500 �g dose is�1.6 pmol. To be able to influence A� deposition, it isreasonable to assume that the mAb must be free andnot have previously bound A� in the periphery. AsmAb9, which is our most effective anti-A� mAb inlong-term peripheral immunization studies, is saturatedin vivo by A� within 6–12 h and appears to have ahalf-life of �3 h in the CSF, it would seem that theupper limit of A� bound by the mAb in the brainbefore saturation would be �6.4 pmol, which is equalto the amount of mAb cleared from the brain in theinitial 12 h after mAb dosing (assuming two bindingsites for A� per mAb). If, as we calculate above, theamount of A� cleared from the brain is �240 pmol perday, then the maximum amount of A� the mAb couldsequester and potentially clear is 3–4% of the brainA� produced in a day. As our empirical data show thatthe amount of anti-A� mAb in the brain is in fact evenless then the 1.6 pmol estimate, it is likely that the mAbwould bind an even smaller percentage of the totalbrain A� produced per day. Although it is more diffi-

cult to precisely determine the level of anti-A� mAbpresent after active immunization, our previous studiessuggest that the steady-state level of anti-A� IgG in miceimmunized with fibrillar A�42 is �10 �g/ml (13). Aslong as the half-life of the endogenously producedanti-A� antibodies is not altered by A� binding, therelative binding capacity of the total pool of polyclonalanti-A� after active immunization will be at least anorder of magnitude less then the binding capacity ofthe 500 �g bolus of mAb9. Thus, only trace amounts offree anti-A� Ab are likely to cross the BBB after activeimmunization and would be predicted to have minimalimpact on steady-state A� levels in nondepositing mice.

Anti-A� mAb binding to A� in the plasma has beenproposed to create a peripheral sink that enhancesclearance of A� from the brain (19, 27). If such amechanism was at work, it should be possible to seereduced levels of total A� in the brain after peripheraladministration of the mAb. Moreover, the decreasewould be maximal during the time in which freeanti-A� mAb is present in the plasma. In Tg2576 micedosed with 500 �g of mAb9, the mAb is fully boundwith A� by 6–12 h. At no time point post-dosing do weobserve significant changes in total GuHCl extractableA� levels in the brain. Perplexingly, we do note subtle,but not statistically significant, changes in the TBS- andRIPA-extractable A� levels at 6 h post-dosing withmAb9, mAb3 and mAb40.1. TBS-extractable A� levelsseem to increase whereas RIPA-extractable levels tran-siently decrease at 6 h, but return to control levels by24 h and remain stable for 2 wk. Moreover, the effect ofmAb9 administration is not additive, repeated dosingdoes not result in a larger decrease in brain levels.When an identical dose of mAb9 was delivered toBRI-A�42B mice, which, before deposition, have muchlower plasma and brain A� levels then Tg2576 mice, weobserved similar trends: a small decrease in the RIPA-extractable pool of A� at 6 and 24 h and a smallincrease in the TBS extractable pool of A�. Given thatthe stoichiometry between the injected mAb and A� isquite different in Tg2576 and BRI-A�42B mice, onewould expect that if the subtle alterations in brain A�were attributable to a simple mass action effect of themAb on total A�, much larger changes in the RIPA andTBS pools of brain A� would be observed in theBRI-A�42B mice.

The dynamic changes in A� levels and mAb levels aresomewhat different between the CSF and plasma, and

TABLE 1. Estimates of steady-state A� levels, half-life, daily turnover, and accumulation in Tg2576 mice and humans

Steady-state level T 1/2 Daily turnover A� Accumulated

Tg2576 micePlasma 50 nMa 10 min (35,36) �4 nmolb

Brain 100 pmol/ga 2 ha �250 pmol 10 nmol (21 m)HumanPlasma 200 pm(60) 10 min (61) �50 nmolc

Brain � 5 pmol/g (59) 7 h (62) �20 nmold 10 �mola,c

a This study. b Mouse brain weight is estimated �400 mg, mouse plasma volume �1–1.5 ml. Turnover calculation example: (50 nmol/l /1–1.5 ml plasma) � 24 h / 2 � 10 min �4 nmol. c Human plasma volume is estimated �3.5 L. d Human brain weight is estimated �1300 g.


also differ between the mAbs. Plasma A� sequesteredby mAb9 rises rapidly and then stays stable for severaldays. In contrast, after mAb9 dosing, CSF A� risesrapidly and declines to near baseline levels by 24 h.Similarly, the mAb9:A� complex levels remain stable inthe plasma between 6 and 24 h, but the levels of thecomplex in the CSF decrease. Moreover, brain mAb9levels decrease by �50% between 6 and 24 h of time.Such data are consistent with a recent report showingthat intraparenchymal injections of the anti-A� mAb4G8 result in rapid clearance of A� from the brain (46).Our data, at least with mAb9, suggest that there is notnecessarily a simple constant equilibrium between themAb:A� complex in plasma and brain or CSF. If thiswas the case, then the CSF and brain changes shouldparallel changes in plasma. The fact that the initial risein CSF A� and mAb:A� complex is observed at the 6 htime point but that both A� and mAb:A� are decreasedin the CSF by 24 h could be explained by severalmechanisms. It is possible that 1) the permeability ofthe BBB is altered acutely after the mAb dosing allow-ing more mAb to penetrate the brain and CSF initiallyor that 2) the presence of a bound or unbound mAb inthe brain activates a clearance mechanism that altersthe equilibrium between the mAb in the CSF andblood. It is also apparent that the dynamics betweenplasma A� and CSF A� may differ from one mAb toanother. In contrast to mAb9, other mAbs cause CSFA� levels to continue to increase from 6 to 24 h orremain relatively constant over this time period. In anycase, these data, together with data from others, show-ing that mAbs injected into the brain or CSF are rapidlycleared into the periphery suggest that an unboundanti-A� mAb could enter the brain, bind A�, and thenclear it into the periphery (45, 46). Given the tinyamounts of mAb that get into the brain, it remainsdifficult to envisage how such a mechanism couldinfluence “total” A� levels sufficiently to alter A� dep-osition.

Long-term peripheral anti-A� mAb delivery has beenshown to reduce A� deposition in the brain of multipledifferent APP mouse models (3, 18, 19, 22, 23). Becauseof differences in transgene expression levels and pat-terns, there are large differences in the relative levels ofplasma and brain A� in these different mouse models.For example, in Tg2576 mice, plasma A� levels are over100-fold higher then in PDAPP mice, whereas thedifferences in brain A� levels, prior to deposition, arethought to be 3-fold. Despite these differences, long-term studies of mAbs with similar binding propertieshave similar overall effects on the extent of A� reduc-tion. Such data would argue that binding of plasma A�by the Ab is unlikely to have a significant impact interms of the ability of the mAb to reduce A� deposition.

It is not possible to use the current data to completelyexclude certain hypothesis as to how passive immuni-zation with anti-A� reduces amyloid deposition. Thedata do, however, provide a framework in which toconsider the potential contribution of various mecha-nisms that have been proposed to account for attenu-

ation of A� deposition. Very little mAb gets in thebrain; thus, if the mAbs are acting centrally, they aredoing so at substoichiometric levels. There is simplyinsufficient mAb in the brain to significantly influencebulk metabolism of A�, and we find no evidence thattotal A� levels are influenced by mAb administration. Ifanti-A� mAbs work directly on A�, they must eitheralter some select pool or species of A� that is present atlow abundance and critical for deposition. Perhaps theconsistent but small, nonsignificant decrease in RIPAsoluble A� reflects changes in this pool? Solublepreamyloid aggregates (oligomers, ADDLs, and proto-fibrils) are present at low levels and may representcritical intermediates in the aggregation pathway (47–50). Although there is no direct evidence that anti-A�antibodies influence preamyloid aggregates in vivo,there is a lot of circumstantial evidence to suggest theydo, including our current data (20, 51–53). Further-more, mAbs can influence A� aggregation in vivo atsubstoichiometric levels, suggesting that they couldpreferentially recognize aggregated or even unaggre-gated A� present in certain conformations that arecritical for subsequent aggregate formation (54). It isalso possible that small amounts of mAb binding to A�in the brain or vasculature could trigger a change thatindirectly influences A� clearance. There is evidencethat in certain APP mouse models, some anti-A� mAbscan enhance phagocytosis of A� by microglia, but inother studies there is no evidence that this is the case(14, 22, 30). Our current data do seem to indicate thatthere are some potential alterations in the transport ofmAb:A� out of the brain after peripheral administra-tion; it is unclear, though, how this alteration couldinfluence A� deposition. It is certainly possible that thesubtle, and opposite, shifts in the RIPA- and TBSextractable pools of A� seen 6–24 h post administra-tion are attributable to experimental variance, al-though the fact that they are seen in two differentmodels would suggest this is not the case. If real, thesechanges do suggest that anti-A� mAbs are likely to bealtering intracerebral A� metabolism in some enig-matic fashion.

Anti-A� binding to plasma A� results in the forma-tion of a stable mAb:A� complex. Though the bindingdoes greatly extend the half-life of the bound A�, itdoes not appear to significantly impact the half-life ofthe mAb, indicating that a classic immune complex isnot formed and rapidly cleared. During the time period(0–12 h post anti-A� mAb9 administration) when sig-nificant amounts of free anti-A� mAb are presumablyentering the blood, there is no detectable effect ontotal brain A� levels in a GuHCL extract. In fact, CSFlevels of A� rise, an effect possibly attributable to mAbbinding A� and prolongation of its half-life in the CSF.Such data would suggest that peripheral anti-A� mAbsdo not enhance total bulk flow of A� across the BBB. Asnoted previously, it is of course impossible to discountthe possibility that the peripheral effects of the mAbbinding to plasma A� alter clearance of some select


pool or species of A� that is critical for A� accumula-tion in the brain.

Anti-A� mAbs can improve behavioral deficits in APPtransgenic mice in the absence of significant effects onamyloid deposition and also improve behavioral deficitswithin a time period that would likely preclude majoreffects on A� loads (20, 51, 55). The mAb-inducedbehavioral improvement has been postulated as attrib-utable to alterations in A� efflux from the brain orbinding of small “neurotoxic” oligomers within thebrain. However, given the large effects on A� in theplasma and the potential vasoactive properties ofplasma A�, it is possible that sequestering of plasma A�by the mAb could attenuate purported vasoconstrictiveeffects of A� within the cerebral vasculature, therebyimproving cerebral blood flow and performance incertain cognitive tasks (56, 57). If anti-A� antibodiesare in fact working through some low abundanceaggregation intermediate, it may be that mAb bindingto nonaggregated A� in the blood or brain mightactually reduce the efficacy of passive immunotherapy.Most of the anti-A� mAb will bind unaggregated A� inthe plasma and be unable to further influence A�metabolism.

At least one humanized monoclonal anti-A� mAb isin human clinical trials, and it is likely that additionalanti-A� mAbs will be tested in humans over the nextseveral years. These studies in mice serve to highlightsome issues that could significantly influence the out-come of the human trials. Assuming that humansmetabolize A� in the brain and plasma at similar ratesto mice, it is possible to estimate both the daily turnoverof A� in the brain and the plasma of humans. As shownin Table 1, these estimates indicate that humans turnover �50 nmol and �20 nmol of A� in the plasma andbrain, respectively, per day. If one were to administeran anti-mAb with the same binding properties as themAb used in these mouse studies, and scale dosing toaccount for differences in body size (�2000 fold in-crease), one would administer at least 1 g of mAb perdose to humans. If one scaled dosing based on therelative levels of A� turnover in the brain of humansper day (�80-fold more than in Tg2576 mice), onewould administer �40 mg of mAb. However, if the mAbworked by binding plasma A�, one might need onlyincrease the relative dose by �6–7 fold, as the Tg2576mice relative to their plasma volume have much higherlevels of A�.

An additional issue to be considered when thinkingabout the levels of mAb needed to confer efficacy has todo with the amount and type of deposited A� presentin the brain. In mice, mAbs that bind A� amyloid areeffective at preventing A� deposition but are less effec-tive at clearing preexisting A� deposits, especially amy-loid deposited in neuritic dense core plaques (13, 18,21). In 21 month old Tg2576 mice, it is possible toestimate that the amount of A� accumulated in thebrain is �10 nmol (equivalent to the A� turned over inthe brain in 40 d) (58). If only a few pmoles of freemAb reach the brain per dose of anti-A� mAb, then it

would seem virtually impossible for the mAb to haveany significant direct effect on the deposits themselves.In the typical human AD brain, on average �10 �molof A� accumulate (equivalent to the A� turned over in�250 d) (59). Thus, there is �1000 times the amountof A� present in the AD brain as in a mouse brain withextensive plaque pathology. Does this mean that toeffectively dose a human with AD one would need todeliver 1000 times more mAb to the brain than isdelivered to a mouse?

In summary, this quantitative evaluation of the acuteeffects of anti-A� mAb delivery to AD mouse modelsprior to deposition serves to illustrate that the mecha-nisms by which such mAbs might alter amyloid deposi-tion and reverse behavioral deficits remain enigmatic.Though it remains possible that very small acutechanges in total A� levels (10%), which are difficultto measure experimentally, account for the long-termeffects of immunization, it seems more likely that themAbs are either 1) working on a select pool or con-former of A� required for deposition or 2) indirectlyinfluencing A� deposition in the brain. Given thepotential clinical promise of both passive and activeapproaches, additional studies that attempt to under-stand how mAbs, or active vaccination with A�, attenu-ates AD-like pathologies are warranted.

These studies were funded by the NIH/NIA to T. Golde(AG18454) and to E. McGowan. (AG022595–01). Additionalresources from the Mayo Foundation provided by a gift fromRobert and Clarice Smith were used to support the Tg2576mouse colony that provided the mice used in these studies. Y.Levites was supported by John Douglas French Alzheimer’sFoundation fellowship. P. Das and Y. Levites were supportedby a Robert and Clarice Smith Fellowship.

REFERENCES

1. Golde, T. E. (2003) Alzheimer disease therapy: Can the amyloidcascade be halted? J. Clin. Invest. 111, 11–18

2. Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H.,Guido, T., Hu, K., Huang, J., Johnson-Wood, K., et al. (1999)Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse [see comments]. Nature400, 173–177

3. Bard, F., Cannon, C., Barbour, R., Burke, R. L., Games, D.,Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., etal. (2000) Peripherally administered antibodies against amyloidbeta-peptide enter the central nervous system and reducepathology in a mouse model of Alzheimer disease. Nat. Med. 6,916–919

4. Gilman, S., Koller, M., Black, R. S., Jenkins, L., Griffith, S. G.,Fox, N. C., Eisner, L., Kirby, L., Boada Rovira, M., Forette, F.,and Orgogozo, J. M. (2005) Clinical effects of A{beta} immuni-zation (AN1792) in patients with AD in an interrupted trial.Neurology 64, 1553–1562

5. Orgogozo, J. M., Gilman, S., Dartigues, J. F., Laurent, B., Puel,M., Kirby, L. C., Jouanny, P., Dubois, B., Eisner, L., Flitman, S.,Michel, B. F., Boada, M., Frank, A., and Hock, C. (2003)Subacute meningoencephalitis in a subset of patients with ADafter Abeta42 immunization. Neurology 61, 46–54

6. Nicoll, J. A., Wilkinson, D., Holmes, C., Steart, P., Markham, H.,and Weller, R. O. (2003) Neuropathology of human Alzheimerdisease after immunization with amyloid-beta peptide: a casereport. Nat. Med. 9, 448–452


7. O’Toole, M., Janszen, D. B., Slonim, D. K., Reddy, P. S., Ellis,D. K., Legault, H. M., Hill, A. A., Whitley, M. Z., Mounts, W. M.,et al. (2005) Risk factors associated with beta-amyloid(1–42)immunotherapy in preimmunization gene expression patternsof blood cells. Arch. Neurol. 62, 1531–1536

8. Fox, N. C., Black, R. S., Gilman, S., Rossor, M. N., Griffith, S. G.,Jenkins, L., and Koller, M. (2005) Effects of A{beta} immuniza-tion (AN1792) on MRI measures of cerebral volume in Alzhei-mer disease. Neurology 64, 1563–1572

9. Hock, C., Konietzko, U., Streffer, J. R., Tracy, J., Signorell, A.,Muller-Tillmanns, B., Lemke, U., Henke, K., Moritz, E., Garcia,E., Wollmer, M. A., Umbricht, D., de Quervain, D. J., Hofmann,M., Maddalena, A., Papassotiropoulos, A., and Nitsch, R. M.(2003) Antibodies against beta-amyloid slow cognitive decline inAlzheimer’s disease. Neuron. 38, 547–554

10. Dodel, R. C., Du, Y., Depboylu, C., Hampel, H., Frolich, L.,Haag, A., Hemmeter, U., Paulsen, S., Teipel, S. J., Brettschnei-der, S., et al. (2004) Intravenous immunoglobulins containingantibodies against beta-amyloid for the treatment of Alzheimer’sdisease. J. Neurol. Neurosurg. Psychiatry 75, 1472–1474

11. Schenk, D. (2002) Opinion: Amyloid-beta immunotherapy forAlzheimer’s disease: the end of the beginning. Nat. Rev. Neurosci.3, 824–828

12. Das, P., and Golde, T. E. (2002) Open peer commentaryregarding Abeta immunization and CNS inflammation by Pasi-netti et al. Neurobiol. Aging 23, 671

13. Das, P., Murphy, M. P., Younkin, L. H., Younkin, S. G., andGolde, T. E. (2001) Reduced effectiveness of Abeta1–42 immu-nization in APP transgenic mice with significant amyloid depo-sition. Neurobiol. Aging 22, 721–727

14. Bard, F., Barbour, R., Cannon, C., Carretto, R., Fox, M., Games,D., Guido, T., Hoenow, K., Hu, K., Johnson-Wood, K., et al.(2003) Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer’s disease-likeneuropathology. Proc. Natl. Acad. Sci. U. S. A. 100, 2023–2028

15. Hock, C., Konietzko, U., Papassotiropoulos, A., Wollmer, A.,Streffer, J., Rotz, R. V., G, G. D., Moritz, E., and Nitsch, R. (2002)Generation of antibodies specific for beta-amyloid by vaccina-tion of patients with Alzheimer disease. Nat. Med. 8, 1270–1275

16. Lee, M., Bard, F., Johnson-Wood, K., Lee, C., Hu, K., Griffith,S. G., Black, R. S., Schenk, D., and Seubert, P. (2005) Abeta42immunization in Alzheimer’s disease generates Abeta N-termi-nal antibodies. Ann. Neurol. 58, 430–435

17. Sigurdsson, E. M., Scholtzova, H., Mehta, P. D., Frangione, B.,and Wisniewski, T. (2001) Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzhei-mer’s disease-associated pathology in transgenic mice. Am. J.Pathol. 159, 439–447

18. Levites, Y., Das, P., Price, R. W., Rochette, M. J., Kostura, L. A.,McGowan, E. M., Murphy, M. P., and Golde, T. E. (2006)Anti-Abeta42- and anti-Abeta40-specific mAbs attenuate amyloiddeposition in an Alzheimer disease mouse model. J. Clin. Invest.116, 193–201

19. DeMattos, R. B., Bales, K. R., Cummins, D. J., Dodart, J. C., Paul,S. M., and Holtzman, D. M. (2001) Peripheral anti-A betaantibody alters CNS and plasma A beta clearance and decreasesbrain A beta burden in a mouse model of Alzheimer’s disease.Proc. Natl. Acad. Sci. U. S. A. 98, 8850–8855

20. Dodart, J. C., Bales, K. R., Gannon, K. S., Greene, S. J.,DeMattos, R. B., Mathis, C., DeLong, C. A., Wu, S., Wu, X.,Holtzman, D. M., and Paul, S. M. (2002) Immunization reversesmemory deficits without reducing brain Abeta burden in Alz-heimer’s disease model. Nat. Neurosci. 5, 452–457

21. Bussiere, T., Bard, F., Barbour, R., Grajeda, H., Guido, T., Khan, K.,Schenk, D., Games, D., Seubert, P., and Buttini, M. (2004) Mor-phological characterization of Thioflavin-S-positive amyloidplaques in transgenic Alzheimer mice and effect of passive Abetaimmunotherapy on their clearance. Am. J. Pathol. 165, 987–995

22. Wilcock, D. M., Rojiani, A., Rosenthal, A., Levkowitz, G., Sub-barao, S., Alamed, J., Wilson, D., Wilson, N., Freeman, M. J.,Gordon, M. N., and Morgan, D. (2004) Passive amyloid immu-notherapy clears amyloid and transiently activates microglia in atransgenic mouse model of amyloid deposition. J. Neurosci. 24,6144–6151

23. Brendza, R. P., Bacskai, B. J., Cirrito, J. R., Simmons, K. A., Skoch,J. M., Klunk, W. E., Mathis, C. A., Bales, K. R., Paul, S. M., Hyman,B. T., and Holtzman, D. M. (2005) Anti-Abeta antibody treatment

promotes the rapid recovery of amyloid-associated neuritic dystro-phy in PDAPP transgenic mice. J. Clin. Invest. 115, 428–433

24. Bacskai, B. J., Kajdasz, S. T., McLellan, M. E., Games, D.,Seubert, P., Schenk, D., and Hyman, B. T. (2002) Non-Fc-mediated mechanisms are involved in clearance of amyloid-betain vivo by immunotherapy. J. Neurosci. 22, 7873–7878

25. Tamura, Y., Hamajima, K., Matsui, K., Yanoma, S., Narita, M.,Tajima, N., Xin, K. Q., Klinman, D., and Okuda, K. (2005) TheF(ab)�2 fragment of an Abeta-specific monoclonal antibodyreduces Abeta deposits in the brain. Neurobiol. Dis. 20, 541–549

26. Matsuoka, Y., Saito, M., LaFrancois, J., Gaynor, K., Olm, V.,Wang, L., Casey, E., Lu, Y., Shiratori, C., Lemere, C., and Duff,K. (2003) Novel therapeutic approach for the treatment ofAlzheimer’s disease by peripheral administration of agents withan affinity to beta-amyloid. J. Neurosci. 23, 29–33

27. DeMattos, R. B., Bales, K. R., Cummins, D. J., Paul, S. M., andHoltzman, D. M. (2002) Brain to plasma amyloid-beta efflux: ameasure of brain amyloid burden in a mouse model of Alzhei-mer’s disease. Science 295, 2264–2267

28. Wilcock, D. M., DiCarlo, G., Henderson, D., Jackson, J., Clarke,K., Ugen, K. E., Gordon, M. N., and Morgan, D. (2003)Intracranially administered anti-Abeta antibodies reduce beta-amyloid deposition by mechanisms both independent of andassociated with microglial activation. J. Neurosci. 23, 3745–3751

29. Wilcock, D. M., Gordon, M. N., Ugen, K. E., Gottschall, P. E.,DiCarlo, G., Dickey, C., Boyett, K. W., Jantzen, P. T., Connor,K. E., Melachrino, J., et al. (2001) Number of Abeta inoculationsin APP�PS1 transgenic mice influences antibody titers, micro-glial activation, and congophilic plaque levels. DNA Cell Biol. 20,731–736

30. Das, P., Howard, V., Loosbrock, N., Dickson, D., Murphy, M. P.,and Golde, T. E. (2003) Amyloid-beta immunization effectivelyreduces amyloid deposition in FcRgamma-/- knock-out mice.J. Neurosci. 23, 8532–8538

31. Masliah, E., Hansen, L., Adame, A., Crews, L., Bard, F., Lee, C.,Seubert, P., Games, D., Kirby, L., and Schenk, D. (2005) Abetavaccination effects on plaque pathology in the absence ofencephalitis in Alzheimer disease. Neurology 64, 129–131

32. Levites, Y., Das, P., Price, R. W., Rochette, M. J., Kostura, L. A.,McGowan, E. M., Murphy, M. P., and Golde, T. E. (2005)Anti-Abeta42 and Anti-Abeta40 specific monoclonal antibodiesattenuate amyloid deposition in an Alzheimer’s disease mousemodel. J. Clin. Invest. 116, 193–201

33. Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y.,Younkin, S., Yang, F., and Cole, G. (1996) Correlative memorydeficits, Abeta elevation, and amyloid plaques in transgenicmice [see comments]. Science 274, 99–102

34. Vogelweid, C. M., and Kier, A. B. (1988) A technique forcollection of cerebrospinal fluid from mice. Lab. Animal Science38, 91–92

35. Hone, E., Martins, I. J., Fonte, J., and Martins, R. N. (2003)Apolipoprotein E influences amyloid-beta clearance from themurine periphery. J. Alzheimers Dis. 5, 1–8

36. Ghiso, J., Shayo, M., Calero, M., Ng, D., Tomidokoro, Y., Gandy,S., Rostagno, A., and Frangione, B. (2004) Systemic catabolismof Alzheimer’s Abeta40 and Abeta42. J. Biol. Chem. 279, 45897–45908

37. Chauhan, V. P., Ray, I., Chauhan, A., and Wisniewski, H. M.(1999) Binding of gelsolin, a secretory protein, to amyloidbeta-protein. Biochem. Biophys. Res. Commun. 258, 241–246

38. Kuo, Y. M., Kokjohn, T. A., Kalback, W., Luehrs, D., Galasko,D. R., Chevallier, N., Koo, E. H., Emmerling, M. R., and Roher,A. E. (2000) Amyloid-beta peptides interact with plasma pro-teins and erythrocytes: implications for their quantitation inplasma. Biochem. Biophys. Res. Commun. 268, 750–756

39. Biere, A. L., Ostaszewski, B., Stimson, E. R., Hyman, B. T.,Maggio, J. E., and Selkoe, D. J. (1996) Amyloid beta-peptide istransported on lipoproteins and albumin in human plasma.J. Biol. Chem. 271, 32916–32922

40. Vieira, P., and Rajewsky, K. (1986) The bulk of endogenouslyproduced IgG2a is eliminated from the serum of adult C57BL/6mice with a half-life of 6–8 days. Eur. J. Immunol. 16, 871–874

41. Lanz, T. A., Fici, G. J., and Merchant, K. M. (2005) Lack of specificamyloid-beta(1–42) suppression by nonsteroidal anti-inflamma-tory drugs in young, plaque-free Tg2576 mice and in guinea pigneuronal cultures. J. Pharmacol. Exp. Ther. 312, 399–406


42. Cirrito, J. R., May, P. C., O’Dell, M. A., Taylor, J. W., Parsada-nian, M., Cramer, J. W., Audia, J. E., Nissen, J. S., Bales, K. R.,Paul, S. M., et al. (2003) In vivo assessment of brain interstitialfluid with microdialysis reveals plaque-associated changes inamyloid-beta metabolism and half-life. J. Neurosci. 23, 8844–8853

43. Maggio, J. E., Stimson, E. R., Ghilardi, J. R., Allen, C. J., Dahl,C. E., Whitcomb, D. C., Vigna, S. R., Vinters, H. V., Labenski,M. E., and Mantyh, P. W. (1992) Reversible in vitro growth ofAlzheimer disease beta-amyloid plaques by deposition of labeledamyloid peptide. Proc. Natl. Acad. Sci. U. S. A. 89, 5462–5466

44. Dovey, H., Varghese, J., and Anderson, J. P. (2000) Functionalgamma-secretase inhibitors reduce beta-amyloid peptide levelsin the brain. J. Neurochem. 76, 1–10

45. Banks, W. A., Pagliari, P., Nakaoke, R., and Morley, J. E. (2005)Effects of a behaviorally active antibody on the brain uptake andclearance of amyloid beta proteins. Peptides 26, 287–294

46. Deane, R., Sagare, A., Hamm, K., Parisi, M., LaRue, B., Guo, H., Wu,Z., Holtzman, D. M., and Zlokovic, B. V. (2005) IgG-assisted age-dependent clearance of Alzheimer’s amyloid beta peptide by theblood-brain barrier neonatal Fc receptor. J. Neurosci. 25, 11495–11503

47. Georganopoulou, D. G., Chang, L., Nam, J. M., Thaxton, C. S.,Mufson, E. J., Klein, W. L., and Mirkin, C. A. (2005) Nanopar-ticle-based detection in cerebral spinal fluid of a soluble patho-genic biomarker for Alzheimer’s disease. Proc. Natl. Acad. Sci.U. S. A. 102, 2273–2276

48. Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C.,Freed, R., Liosatos, M., Morgan, T. E., Rozovsky, I., Trommer,B., Viola, K. L., et al. (1998) Diffusible, nonfibrillar ligandsderived from Abeta1–42 are potent central nervous systemneurotoxins. Proc. Nat. Acad. Sci. U. S. A. 95, 6448–6453

49. Caughey, B., and Lansbury, P. T. (2003) Protofibrils, pores,fibrils, and neurodegeneration: separating the responsible pro-tein aggregates from the innocent bystanders. Annu. Rev. Neu-rosci. 26, 267–298

50. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Rowan, M. J., andSelkoe, D. J. (2002) Amyloid-beta oligomers: their production,toxicity and therapeutic inhibition. Biochem. Soc Trans. 30,552–557

51. Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y.,Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J.,et al. (2000) A beta peptide immunization reduces behaviouralimpairment and plaques in a model of Alzheimer’s disease.Nature 408, 979–982

52. Cleary, J. P., Walsh, D. M., Hofmeister, J. J., Shnkar, G. M.,Kuskowski, M. A., Selkoe, D. J., and Ashe, K. H. (2005) NaturalOligomers of the amyloid-beta peptide specifically disrupt cog-nitive function. Nat. Neurosci. 8, 79–84

53. Lee, E. B., Leng, L. Z., Zhang, B., Kwong, L., Trojanowski, J. Q.,Abel, T., and Lee, V. M. (2006) Targeting amyloid-beta peptide(Abeta) oligomers by passive immunization with a conforma-

tion-selective monoclonal antibody improves learning andmemory in abeta precursor protein (APP) transgenic mice.J. Biol. Chem. 281, 4292–4299

54. Taylor, B. M., Sarver, R. W., Fici, G., Poorman, R. A., Lutzke,B. S., Molinari, A., Kawabe, T., Kappenman, K., Buhl, A. E., andEpps, D. E. (2003) Spontaneous aggregation and cytotoxicity ofthe beta-amyloid Abeta1–40: a kinetic model. J. Protein Chem. 22,31–40

55. Lee, E. B., Leng, L. Z., Zhang, B., Kwong, L., Trojanowski, J. Q.,Abel, T., and Lee, V. M. (2005) Targeting Abeta oligomers bypassive immunization with a conformation selective monoclonalantibody improves learning and memory in APP transgenicmice. J. Biol. Chem. 281, 4292–4299

56. Su, G. C., Arendash, G. W., Kalaria, R. N., Bjugstad, K. B., andMullan, M. (1999) Intravascular infusions of soluble beta-amyloid compromise the blood- brain barrier, activate CNS glialcells and induce peripheral hemorrhage. Brain Res. 818, 105–117

57. Paris, D., Town, T., Parker, T., Humphrey, J., and Mullan, M.(2000) beta-Amyloid vasoactivity and proinflammation in micro-glia can be blocked by cGMP-elevating agents. Ann. N. Y. Acad.Sci. 903, 446–450

58. Kawarabayashi, T., Younkin, L. H., Saido, T. C., Shoji, M., Ashe,K. S., and Younkin, S. G. (2001) Age-dependent changes inbrain, CSF, and plasma amyloid protein in the Tg2576 trans-genic mouse model of Alzheimer’s disease. J. Neurosci. 21,372–381

59. Gravina, S. A., Ho, L., Eckman, C. B., Long, K. E., Otvos, L., Jr.,Younkin, L. H., Suzuki, N., and Younkin, S. G. (1995) Amyloidbeta protein (A beta) in Alzheimer’s disease brain. Biochemicaland immunocytochemical analysis with antibodies specific forforms ending at A beta 40 or A beta 42(43). J.Biol. Chem. 270,7013–7016

60. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M.,Suzuki, N., Bird, T., D., Hardy, J., Hutton, M., Kukull, W., et al.(1996) Secreted amyloid beta-protein similar to that in thesenile plaques of Alzheimer’s disease is increased in vivo by thepresenilin 1 and 2 and APP mutations linked to familialAlzheimer’s disease. Nat. Med.. 2, 864–870

61. Siemers, E., Skinner, M., Dean, R. A., Gonzales, C., Satterwhite,J., Farlow, M., Ness, D., and May, P. C. (2005) Safety, tolerability,and changes in amyloid beta concentrations after administra-tion of gamma-secretase inhibitor in volunteers. Clin. Neurophar-macol. 28, 126–132

62. Bateman, R. J., Munsell, L. Y., Morris, J. C., Swarm, R., Yara-sheski, K. E., and Holtzman, D. M. (2006) Human amyloid-betasynthesis and clearance rates as measured in cerebrospinal fluidin vivo. Nat. Med. 12, 856–861

Received for publication May 8, 2006.Accepted for publication July 24, 2006.


The FASEB Journal • FJ Express Summary

Insights into the mechanisms of action of anti-A�antibodies in Alzheimer’s disease mouse models

Yona Levites,* Lisa A. Smithson,* Robert W. Price,* Rachel S. Dakin,* Bin Yuan,†

Michael R. Sierks,† Jungsu Kim,* Eileen McGowan,* Dana Kim Reed,*Terrone L. Rosenberry,* Pritam Das,* and Todd E. Golde*,1

*Departments of Neuroscience and Pharmacology, Mayo Clinic College of Medicine, Jacksonville,Florida, USA; and †Department of Chemical and Materials Engineering, Arizona State University,Tempe, Arizona, USA

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6463fje

SPECIFIC AIMS

Our aims were to provide a quantitative framework inwhich to analyze proposed mechanisms of action ofanti-A� immunotherapy by 1) examining the in vivobinding properties, pharmacokinetics, brain pen-etrance, and alterations in A� levels after a singleperipheral dose of anti-A� monoclonal antibodies(mAbs) to young non-A� depositing APP mice; and 2)estimating the total daily turnover of A�.

PRINCIPAL FINDINGS

1. Peripheral administration of anti-mAb creates astable mAb:A� complex in the plasma

Previous studies established that A� has a very shorthalf-life in the plasma. Changes in A� levels, the in vivobinding properties, and plasma half-life of the mAbitself were examined after passive immunization with ananti-A� mAb. Five-hundred micrograms (�1600 pmol)of biotinylated mAb9 were administered intraperitone-ally to 3 month old nondepositing female Tg2576 mice.After mAb9 administration, A�40 and A�42 levels inthe plasma increased, on average, �15-fold and �25-fold, respectively, and then slowly decreased over a 2 wkperiod of time (Fig. 1A). We also measured the amountof biotinylated mAb9:A� complexes in plasma. ThemAb9:A�40 complex reached its highest value of �450pmol mAb9 bound to A�40 per ml of plasma after 6 h(Fig. 1B). Size exclusion column chromatography ofmouse plasma, collected 1 day post-mAb9 injection,shows that most plasma A� that accumulates aftermAb9 treatment is present in high molecular weightfractions with peak levels found in the peak fractions inwhich unbound mAb9 elutes. (Fig. 1C). Finally, whenplasma from biotinylated mAb9 injected mice is precip-itated with streptavidin beads and subjected to Westernblot analysis, an increase in a 4 kDa A� species isobserved (Fig. 1D)

When 500 �g (�1600 pmol) of biotinylated mAb9are administered to 3 month old female nontransgeniclittermates of the Tg2576 mice, �800 pmol mAb9/mlplasma, can be detected in the plasma 1 day later (Fig.1E). To directly determine if binding of the mAb9 toA� prolongs the half-life of A�, we injected a pre-formed complex of biotinylated mAb9 (500 �g, �1600pmol) and human A� 40 (�3200 pmol) into youngnontransgenic mice. The mAb9:A� 40 complex wasdetected as described previously. Within 6 h, �500pmol/ml of the complex are detected and the com-plex, like the unbound antibody (Ab), is cleared slowlywith a half-life of �5–7 days (Fig. 1F). Thus, in contrastto endogenous A�, the mAb9:A� 40 complex has aprolonged half-life, demonstrating that the binding ofthe mAb to A� does not result in the formation of aclassic immune complex that would be rapidly cleared.Finally, such data show that binding of mAb9 to A�prevents the A� from being rapidly turned over.

2. Effects of acute immunization with anti-A� mAbon A� levels in the brains of Tg2576and BRI-A�42B mice

To determine if alterations in brain A� occur afterperipheral immunization, we examined brain A� levelsin young nondepositing female Tg2576 mice afterintraperitoneal administration of 500 �g of biotinylatedmAb9. Despite the marked accumulation of plasma A�,there is no appreciable change in the levels of GuHCl-extractable brain A� (Fig. 2A). Soluble, TBS-extract-able A� levels increase slightly after peripheral admin-istration (Fig. 2B). Radio-immunoprecipitation assay(RIPA), a moderately denaturing detergent mix, ex-tracts a higher level of A� than TBS but lower levelsthan GuHCl. RIPA-extractable A� decreases slightly

1 Correspondence: Department of Neuroscience, MayoClinic Jacksonville, Birdsall 210, 4500 San Pablo Rd., Jackson-ville, FL 32224, USA. E-mail [email protected]

doi: 10.1096/fj.06-6463fje

2576 0892-6638/06/0020-2576 © FASEB

after immunization by up to 20% of control or �10pmol/g (Fig. 2C). No statistically significant decrease inRIPA-soluble A� 40 levels is detected up to 14 days afterthe single mAb administration (Fig. 2D). Moreover, theslight decrease, observed 24 h after the single mAbadministration, is not additive; continuous weekly ad-ministration of 500 mg mAb for 4 wk results in similarslight nonsignificant decrease in RIPA-soluble Ab levels(Fig. 2E).

The same amount of biotinylated mAb9 was admin-istered to a low-expressing BRI-A�42B line, which only

expresses A� 42, and has at least �5-fold lower levels oftotal brain A� and �100 fold lower plasma levelsrelative to Tg2576 mice. After mAb9 administration, weagain observe a rapid increase in A� levels in theplasma, no significant change in total brain A� levelsextracted by GuHCl, a slight nonsignificant increase inTBS-extractable brain A�42 levels, and a slight non-statistically significant decrease in RIPA-extractableA�42. The magnitude of these changes is similar tothose seen in Tg2576 mice, indicating that these smalleffects after mAb administration are not influenced bythe relative amount of plasma or brain A� in thedifferent transgenic lines. Such data essentially excludea mass action mechanism of action.

After administration via intraperitoneally injection of500 �g (1600 pmol) biotinylated mAb9 to nontrans-genic mice, we can detect 1.0 � 0.08 fmol/mg ofbiotinylated mAb9 6 h postinjection, which is approxi-mately �300 fmol per brain or �0.02% of the totalamount of the mAb administered. Such data place anupper limit on the amount of mAb9 present in thebrain at the time the plasma mAb levels are nearmaximal.

3. Effects of anti-A� mAb on CSF A� and clearanceof mAb9:A� complexes from the brain

Six hours post-mAb injection, an increase in A� levels isobserved in CSF collected from the cisterna magna.CSF A� levels decrease by 24 h. After intracerebroven-tricular injection of a preformed complex of 5 �g(�160 pmol) of biotinylated mAb9 and �320 pmol ofA� into the ventricles, the mAb9:A� complex is de-tected in CSF collected from the cisterna magna within30 min. By 3 h, the levels are dramatically decreasedand at 24 h no complex is detectable. Low levels ofcomplex appear in plasma by 30 min and appearrelatively stable up to 72 h post-injection. Such datasuggest that the complex is rapidly cleared from theCSF, and at least some of this clearance is via exportinto the vasculature. However, once in the peripheralblood the complex is stable.

4. Additional anti-A� mAbs have similar effects onA� levels in plasma, brain, and CSF of Tg2576 mice

To determine if the observed dynamics in plasma, CSFand brain after an acute dose of mAb in TG2576 miceare common to the other anti-A� mAb characterized inour previous studies and have been shown to reduce A�deposition after peripheral administration, we admin-istered 500 �g biotinylated anti-A�1–16 mAb3, anti-A�42 mAb42.2 and anti-A�40 mAb40; 1 to 3 month oldTg2576 mice. All mAbs caused an increase in total A�levels in plasma, but no effect was observed on thebrain RIPA-soluble A�. A� levels in CSF were alsoincreased on administration of all three mAbs, al-though the dynamics of this increase vary between themAbs.

Figure 1. A� and mAb levels in plasma of Tg2576 mice afteracute passive immunization. A) 3 month old Tg2576 micewere dosed ip with 500 �g (1600 pmol) biotinylated mAb9.A� levels (A) or levels of A� bound by biotinylated mAb9 (B)were measured at different time points by ELISA. C) Plasmafrom Tg2576 mice dosed with biotinylated mAb9 or biotinyl-ated mouse IgG was fractionated by size-exclusion chroma-tography. Levels of A� in each fraction were measured byELISA. D) A�:biotinylated mAb complex in the plasma oftreated Tg2576 mice was immunoprecipitated with streptavi-din beads and A� was detected by mAb9. E and F) 3 monthold nontransgenic mice were dosed ip with 500 �g (1600pmol) biotinylated Ab9 (E) or with complex of �1600 pmolbiotinylated mAb9 and �3200 pmol A�40 (F). BiotinylatedmAb9 levels (E) or levels of A� bound by biotinylated mAb(F) in plasma were measured by direct ELISA; n � 4 pergroup, *P � 0.001, **P � 0.01 vs. control.

2577ANTI-A� IMMUNOTHERAPY: MECHANISMS OF ACTION

CONCLUSIONS AND SIGNIFICANCE

Our data show that in mice 1) binding of mAbs to A�significantly prolongs the half-life of plasma A� fromminutes to days, 2) very little free anti-A� mAb actuallyenters the brain or CSF, 3) anti-A� mAb:A� complexesare rapidly cleared from the brain, 4) passive adminis-tration of these anti-A� mAbs has little effect on totalsteady state predeposition brain A� levels, and 5)neither the amount of mAb relative to the amount ofA� or type of anti-A� mAb significantly influence theoverall changes in A� induced acutely after passiveimmunization. The changes in brain, CSF, and plasmaA� levels and anti-A� levels are summarized the sche-matic in Fig. 3.

Such data when viewed in the context of the totalamount of A� turnover per day (which in Tg2576 micewe estimate at �0.25 nmol/day in the brain and 4 nmolper day in the periphery) have important implicationswith respect to design and interpretation of futurestudies using active or passive immunotherapy as atreatment or preventive strategy for AD. There is simplyinsufficient mAb in the brain to significantly influencebulk metabolism of monomeric A�, and we find noevidence that total A� levels are influenced by mAbadministration. If anti-A� mAbs work directly on A�,they must either alter some select pool or species of A�that is present at low abundance and critical for depo-sition.

In summary, this quantitative evaluation of the acuteeffects of anti-A� mAb delivery to nondepositing ADmouse models serves to illustrate that the mechanismsby which such mAbs might alter amyloid depositionand reverse behavioral deficits remain enigmatic.

Though it remains possible that very small acutechanges in total A� levels (�10%), which are difficultto measure experimentally, account for the long-termeffects of immunization, it seems more likely that themAbs are either 1) working on a select pool or con-former of A� within the brain that is required fordeposition or 2) indirectly influencing A� depositionin the brain. Given the potential clinical promise ofboth passive and active approaches, additional studiesthat attempt to understand how mAbs, or active vacci-nation with A�, attenuates AD-like pathologies arewarranted.

Figure 2. Effects of acute immunizationwith anti-A� Ab on A� levels in the brainsof Tg2576 mice; 3 month old Tg2576mice were dosed with 500 �g biotinylatedmAb9. 6 h–14 days later mice were per-fused with PBS and A� levels in GuHCl(A), TBS (B), and RIPA (C and D) brainextracts were detected by ELISA usingend-specific anti-A�40 mAb40.1 or anti-A�42 mAb42.2 as capture and 4G8-HRPas detection; n � 4 per group. E) 3month old Tg2576 mice were dosed with500 �g biotinylated mAb9 every week for4 wk. Mice were killed 24 h after the finalmAb admisntration. A� levels in RIPAbrain extracts were detected by ELISA;n � 4 per group.

Figure 3. Schematic diagram. Changes in A� and mAb afteranti-A� immunotherapy. General assumptions that weremade in regards to an average young Tg 2576 mouse: plasmavol.: 1 ml, CSF vol.: 20 �l, and brain wt: 400 mg.

2578 Vol. 20 December 2006 LEVITES ET AL.The FASEB Journal

Insights into the mechanisms of action of anti-A antibodies in Alzheimer's disease mouse models

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