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* 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
1Correspondence: Department of Neuroscience, Mayo Clinic Jacksonville, Birdsall 210, 4500 San Pablo Rd., Jacksonville, FL 32224, USA. E-mail tgolde{at}mayo.edu
SPECIFIC AIMS
Our aims were to provide a quantitative framework in which to analyze proposed mechanisms of action of anti-Aßbeta; immunotherapy by 1) examining the in vivo binding properties, pharmacokinetics, brain penetrance, and alterations in Aßbeta; levels after a single peripheral dose of anti-Aßbeta; monoclonal antibodies (mAbs) to young non-Aßbeta; depositing APP mice; and 2) estimating the total daily turnover of Aßbeta;.
PRINCIPAL FINDINGS
1. Peripheral administration of anti-mAb creates a stable mAb:Aßbeta; complex in the plasma
Previous studies established that Aßbeta; has a very short half-life in the plasma. Changes in Aßbeta; levels, the in vivo binding properties, and plasma half-life of the mAb itself were examined after passive immunization with an anti-Aßbeta; mAb. Five-hundred micrograms (
1600 pmol) of biotinylated mAb9 were administered intraperitoneally to 3 month old nondepositing female Tg2576 mice. After mAb9 administration, Aßbeta;40 and Aßbeta;42 levels in the plasma increased, on average, 15-fold and 25-fold, respectively, and then slowly decreased over a 2 wk period of time (Fig. 1
A). We also measured the amount of biotinylated mAb9:Aßbeta; complexes in plasma. The mAb9:Aßbeta;40 complex reached its highest value of 450 pmol mAb9 bound to Aßbeta;40 per ml of plasma after 6 h (Fig. 1B
). Size exclusion column chromatography of mouse plasma, collected 1 day post-mAb9 injection, shows that most plasma Aßbeta; that accumulates after mAb9 treatment is present in high molecular weight fractions with peak levels found in the peak fractions in which unbound mAb9 elutes. (Fig. 1C
). Finally, when plasma from biotinylated mAb9 injected mice is precipitated with streptavidin beads and subjected to Western blot analysis, an increase in a 4 kDa Aßbeta; species is observed (Fig. 1D
)
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When 500 µg (1600 pmol) of biotinylated mAb9 are administered to 3 month old female nontransgenic littermates of the Tg2576 mice, 800 pmol mAb9/ml plasma, can be detected in the plasma 1 day later (Fig. 1E
). To directly determine if binding of the mAb9 to Aßbeta; prolongs the half-life of Aßbeta;, we injected a preformed complex of biotinylated mAb9 (500 µg, 1600 pmol) and human Aßbeta; 40 (3200 pmol) into young nontransgenic mice. The mAb9:Aßbeta; 40 complex was detected as described previously. Within 6 h, 500 pmol/ml of the complex are detected and the complex, like the unbound antibody (Ab), is cleared slowly with a half-life of 5–7 days (Fig. 1F
). Thus, in contrast to endogenous Aßbeta;, the mAb9:Aßbeta; 40 complex has a prolonged half-life, demonstrating that the binding of the mAb to Aßbeta; does not result in the formation of a classic immune complex that would be rapidly cleared. Finally, such data show that binding of mAb9 to Aßbeta; prevents the Aßbeta; from being rapidly turned over.
2. Effects of acute immunization with anti-Aßbeta; mAb on Aßbeta; levels in the brains of Tg2576 and BRI-Aßbeta;42B mice
To determine if alterations in brain Aßbeta; occur after peripheral immunization, we examined brain Aßbeta; levels in young nondepositing female Tg2576 mice after intraperitoneal administration of 500 µg of biotinylated mAb9. Despite the marked accumulation of plasma Aßbeta;, there is no appreciable change in the levels of GuHCl-extractable brain Aßbeta; (Fig. 2
A). Soluble, TBS-extractable Aßbeta; levels increase slightly after peripheral administration (Fig. 2B
). Radio-immunoprecipitation assay (RIPA), a moderately denaturing detergent mix, extracts a higher level of Aßbeta; than TBS but lower levels than GuHCl. RIPA-extractable Aßbeta; decreases slightly after immunization by up to 20% of control or 10 pmol/g (Fig. 2C
). No statistically significant decrease in RIPA-soluble Aßbeta; 40 levels is detected up to 14 days after the single mAb administration (Fig. 2D
). Moreover, the slight decrease, observed 24 h after the single mAb administration, is not additive; continuous weekly administration of 500 mg mAb for 4 wk results in similar slight nonsignificant decrease in RIPA-soluble Ab levels (Fig. 2E
).
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The same amount of biotinylated mAb9 was administered to a low-expressing BRI-Aßbeta;42B line, which only expresses Aßbeta; 42, and has at least 5-fold lower levels of total brain Aßbeta; and 100 fold lower plasma levels relative to Tg2576 mice. After mAb9 administration, we again observe a rapid increase in Aßbeta; levels in the plasma, no significant change in total brain Aßbeta; levels extracted by GuHCl, a slight nonsignificant increase in TBS-extractable brain Aßbeta;42 levels, and a slight nonstatistically significant decrease in RIPA-extractable Aßbeta;42. The magnitude of these changes is similar to those seen in Tg2576 mice, indicating that these small effects after mAb administration are not influenced by the relative amount of plasma or brain Aßbeta; in the different transgenic lines. Such data essentially exclude a mass action mechanism of action.
After administration via intraperitoneally injection of 500 µg (1600 pmol) biotinylated mAb9 to nontransgenic mice, we can detect 1.0 ± 0.08 fmol/mg of biotinylated mAb9 6 h postinjection, which is approximately 300 fmol per brain or 0.02% of the total amount of the mAb administered. Such data place an upper limit on the amount of mAb9 present in the brain at the time the plasma mAb levels are near maximal.
3. Effects of anti-Aßbeta; mAb on CSF Aßbeta; and clearance of mAb9:Aßbeta; complexes from the brain
Six hours post-mAb injection, an increase in Aßbeta; levels is observed in CSF collected from the cisterna magna. CSF Aßbeta; levels decrease by 24 h. After intracerebroventricular injection of a preformed complex of 5 µg (160 pmol) of biotinylated mAb9 and 320 pmol of Aßbeta; into the ventricles, the mAb9:Aßbeta; complex is detected in CSF collected from the cisterna magna within 30 min. By 3 h, the levels are dramatically decreased and at 24 h no complex is detectable. Low levels of complex appear in plasma by 30 min and appear relatively stable up to 72 h post-injection. Such data suggest that the complex is rapidly cleared from the CSF, and at least some of this clearance is via export into the vasculature. However, once in the peripheral blood the complex is stable.
4. Additional anti-Aßbeta; mAbs have similar effects on Aßbeta; 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 mice are common to the other anti-Aßbeta; mAb characterized in our previous studies and have been shown to reduce Aßbeta; deposition after peripheral administration, we administered 500 µg biotinylated anti-Aßbeta;1–16 mAb3, anti-Aßbeta;42 mAb42.2 and anti-Aßbeta;40 mAb40; 1 to 3 month old Tg2576 mice. All mAbs caused an increase in total Aßbeta; levels in plasma, but no effect was observed on the brain RIPA-soluble Aßbeta;. Aßbeta; levels in CSF were also increased on administration of all three mAbs, although the dynamics of this increase vary between the mAbs.
CONCLUSIONS AND SIGNIFICANCE
Our data show that in mice 1) binding of mAbs to Aßbeta; significantly prolongs the half-life of plasma Aßbeta; from minutes to days, 2) very little free anti-Aßbeta; mAb actually enters the brain or CSF, 3) anti-Aßbeta; mAb:Aßbeta; complexes are rapidly cleared from the brain, 4) passive administration of these anti-Aßbeta; mAbs has little effect on total steady state predeposition brain Aßbeta; levels, and 5) neither the amount of mAb relative to the amount of Aßbeta; or type of anti-Aßbeta; mAb significantly influence the overall changes in Aßbeta; induced acutely after passive immunization. The changes in brain, CSF, and plasma Aßbeta; levels and anti-Aßbeta; levels are summarized the schematic in Fig. 3
.
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Such data when viewed in the context of the total amount of Aßbeta; turnover per day (which in Tg2576 mice we estimate at 0.25 nmol/day in the brain and 4 nmol per day in the periphery) have important implications with respect to design and interpretation of future studies using active or passive immunotherapy as a treatment or preventive strategy for AD. There is simply insufficient mAb in the brain to significantly influence bulk metabolism of monomeric Aßbeta;, and we find no evidence that total Aßbeta; levels are influenced by mAb administration. If anti-Aßbeta; mAbs work directly on Aßbeta;, they must either alter some select pool or species of Aßbeta; that is present at low abundance and critical for deposition.
In summary, this quantitative evaluation of the acute effects of anti-Aßbeta; mAb delivery to nondepositing AD mouse models serves to illustrate that the mechanisms by which such mAbs might alter amyloid deposition and reverse behavioral deficits remain enigmatic. Though it remains possible that very small acute changes in total Aßbeta; levels (<10%), which are difficult to measure experimentally, account for the long-term effects of immunization, it seems more likely that the mAbs are either 1) working on a select pool or conformer of Aßbeta; within the brain that is required for deposition or 2) indirectly influencing Aßbeta; deposition in the brain. Given the potential clinical promise of both passive and active approaches, additional studies that attempt to understand how mAbs, or active vaccination with Aßbeta;, attenuates AD-like pathologies are warranted.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6463fje
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