Disaggregation of Amyloid Plaque in Brain of Alzheimer's Disease

Aug 7, 2013 - Anti-amyloid antibodies (AAA) are under development as new therapeutics that disaggregate the amyloid plaque in brain in Alzheimer's ...
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Disaggregation of Amyloid Plaque in Brain of Alzheimer’s Disease Transgenic Mice with Daily Subcutaneous Administration of a Tetravalent Bispecific Antibody That Targets the Transferrin Receptor and the Abeta Amyloid Peptide Rachita K. Sumbria,† Eric Ka-Wai Hui,‡ Jeff Zhiqiang Lu,‡ Ruben J. Boado,†,‡ and William M. Pardridge*,† †

Department of Medicine, UCLA, Los Angeles, California 90024, United States ArmaGen Technologies, Inc., Calabasas, California 91302, United States

‡

ABSTRACT: Anti-amyloid antibodies (AAA) are under development as new therapeutics that disaggregate the amyloid plaque in brain in Alzheimer’s disease (AD). However, the AAAs are large molecule drugs that do not cross the blood−brain barrier (BBB), in the absence of BBB disruption. In the present study, an AAA was re-engineered for receptor-mediated transport across the BBB via the endogenous BBB transferrin receptor (TfR). A single chain Fv (ScFv) antibody form of an AAA was fused to the carboxyl terminus of each heavy chain of a chimeric monoclonal antibody (mAb) against the mouse TfR, and this produced a tetravalent bispecific antibody designated the cTfRMAb-ScFv fusion protein. Unlike a conventional AAA, which has a plasma half-time of weeks, the cTfRMAb-ScFv fusion protein is cleared from plasma in mice with a mean residence time of about 3 h. Therefore, a novel protocol was developed for the treatment of one year old presenilin (PS)-1/amyloid precursor protein (APP) AD double transgenic PSAPP mice, which were administered daily subcutaneous (sc) injections of 5 mg/kg of the cTfRMAb-ScFv fusion protein for 12 consecutive weeks. At the end of the treatment, brain amyloid plaques were quantified with confocal microscopy using both Thioflavin-S staining and immunostaining with the 6E10 antibody against Abeta amyloid fibrils. Fusion protein treatment caused a 57% and 61% reduction in amyloid plaque in the cortex and hippocampus, respectively. No increase in plasma immunoreactive Abeta amyloid peptide, and no cerebral microhemorrhage, was observed. Chronic daily sc treatment of the mice with the fusion protein caused no immune reactions and only a low titer antidrug antibody response. In conclusion, reengineering AAAs for receptor-mediated BBB transport allows for reduction in brain amyloid plaque without cerebral microhemorrhage following daily sc treatment for 12 weeks. KEYWORDS: blood−brain barrier, Alzheimer’s disease, amyloid, transferrin receptor, monoclonal antibody

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INTRODUCTION The dementia of Alzheimer’s disease (AD) correlates with the deposition of amyloid plaque in brain.1,2 The plaque is formed by the 40−43 amino acid Abeta peptide.3,4 AD drug development is aimed at disaggregation and clearance of the Aβ amyloid plaque in brain. The most potent amyloid disaggregation agents are anti-amyloid antibodies (AAA), particularly antibodies that target the amino terminal portion of the Aβ peptide.5 Amino terminal-directed AAAs have been developed for the treatment of humans with AD, such as bapineuzumab6 or gantenerumab.7 Conventional monoclonal antibody (mAb) drugs, such as AAAs, are large molecule therapeutics that do not cross the blood−brain barrier (BBB), in the absence of BBB disruption. The brain volume of distribution (VD) of the AAA in vivo is no greater than the brain plasma volume,8 which is indicative of a lack of BBB penetration. Alternatively, AAAs may be re-engineered for BBB penetration, wherein the AAA is engineered as a fusion protein with a BBB molecular Trojan horse.8 The latter also is a mAb, which targets an endogenous BBB receptor transport system, such as the insulin receptor or the transferrin receptor (TfR). © 2013 American Chemical Society

When the neurotherapeutic is a mAb, and the BBB molecular Trojan horse is a mAb, the problem is the engineering of a bispecific antibody (BSA) that retains high affinity for both the target antigen in brain, such as the Abeta amyloid plaque, and the BBB receptor, such as the insulin receptor or TfR. The retention of high affinity binding for both antibodies is possible with the engineering of a tetravalent BSA, where both antibody domains of the BSA retain bivalency for the target.8 For BBB drug delivery of an AAA in a mouse model of AD, an amino terminal-directed AAA was re-engineered as a single chain Fv (ScFv) antibody, and the ScFv domain was fused to the carboxyl terminus of each heavy chain of a chimeric mAb against the mouse TfR; this fusion protein is designated the cTfRMAb-ScFv.9 The structure of the cTfRMAb-ScFv fusion protein is shown in Figure 1. The fusion antibody is composed of 3 domains: (a) the head of the molecule binds the TfR on Received: Revised: Accepted: Published: 3507

June 14, 2013 August 1, 2013 August 7, 2013 August 7, 2013 dx.doi.org/10.1021/mp400348n | Mol. Pharmaceutics 2013, 10, 3507−3513

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protein do not provide adequate exposure of the brain to the amyloid immunotherapeutic. A recent study shows that cTfRMAb-based fusion proteins are absorbed into the plasma following subcutaneous (sc) administration, and that the brain concentration of the fusion protein after sc injection is comparable to the brain concentration after iv administration.16 Therefore, the present study was designed to treat AD transgenic mice with daily sc administration of the cTfRMAbScFv fusion protein. Following 12 weeks of daily sc treatment, the amyloid plaque burden in brain was measured with confocal microscopy. The presence of cerebral microhemorrhage was examined with Prussian blue histochemistry. The formation of antidrug antibodies (ADA) against the cTfRMAb-ScFv fusion protein was quantified with a sandwich ELISA. Owing to the requirement for daily administration of the fusion protein, it was necessary to produce >100 mg of the cTfRMAb-ScFv fusion protein. Therefore, the present study describes production of the fusion protein by stably transfected Chinese hamster ovary (CHO) cells propagated in a fed batch bioreactor in serum free medium.

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EXPERIMENTAL SECTION Production of Fusion Protein. A stably transfected Chinese hamster ovary (CHO) line expressing the cTfRMAbScFv fusion protein was produced as described previously.9 The CHO line was propagated in a 50 L Wave Bioreactor under fedbatch conditions. The volume of medium and cells was expanded from 10.5 L at day 1 to 24.0 L at day 10 when the bioreactor was terminated. The cells were maintained in HyClone SFM4CHO-Utility serum free medium (SFM) plus 80 nM methotrexate, used as a selection agent, and antibiotics (penicillin G, streptomycin). The density of viable cells was 2.2 million cells/mL at day 1, peaked at 7.8 million cells/mL at day 7, and was 4.8 million cells/mL at day 10. The percent cell viability, as determined by trypan blue dye exclusion, was 95% at day 1 and 84% at day 10. The medium was supplemented with glucose, glutamine, and sodium carbonate, and maintained at 36 °C. Mouse IgG levels in the bioreactor medium were determined by ELISA and increased from 2.6 mg/L at day 1 to 28 mg/L at day 9. Following depth filtration, the cTfRMAbScFv fusion protein was purified from the feedstock with a 25 mL column of protein G Sepharose 4 FF as described previously.9 Batches of 4−7 L of conditioned medium were applied to the protein G column, and the fusion protein was eluted with 0.1 M glycine, pH = 2.8, followed by neutralization with 1 M Tris base. The neutralized fusion protein solution was diluted to a protein concentration of 0.35 mg/mL with the addition of 10 mM Tris/0.15 M saline/pH = 7.0, clarified by 0.22 μm ultrafiltration, aliquoted to 5 mL/tube, followed by storage at −70 °C. The purity and identity of the cTfRMAbScFv fusion protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mouse IgG Western blotting as described previously.9 Treatment of AD Transgenic Mice. PSAPP AD transgenic mice were purchased from Jackson Laboratories (Bar Harbor, ME). The hemizygous male mice (strain B6C3-Tg APPswe, PSEN1dE9, 85Dbo/Mmjax, stock 004462) were 12 months old at the start of the 3-month study. Mice were treated either with saline (8 mice) or with the cTfRMAb-ScFv fusion protein (7 mice). The fusion protein (5 mg/kg) was administered daily subcutaneously for 12 consecutive weeks. Mice were weighed weekly. At the end of 12 weeks, mice were euthanized, and the brain was removed for measurement of

Figure 1. The cTfRMAb-ScFv fusion protein is a bispecific antibody formed by fusion of a single chain (ScFv) anti-amyloid antibody to the carboxyl terminus of each heavy chain of the chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), which is designated the cTfRMAb. The variable region of the heavy chain (VH) and the variable region of the light chain (VL) of the cTfRMAb are derived from a rat IgG directed against the mouse TfR.9 The constant (C) region of the cTfRMAb heavy chain is composed of the CH1, hinge, CH2, and CH3 regions, which are all derived from the C-region of mouse IgG1.9 The C-region of the cTfRMAb light chain is derived from the mouse kappa C-region.9 The VH and VL regions of the antiamyloid ScFv are derived from a mouse antibody directed against the amino terminal domain of the Abeta amyloid peptide.8

the mouse BBB, which causes influx from blood to brain; (b) the tail of the fusion antibody binds the Abeta amyloid plaque, which induces plaque disaggregation; and (c) the midsection of the fusion antibody, the CH2-CH3 region, binds the Fc receptor (FcR), which causes efflux from brain back to blood. The FcR, particularly the neonatal FcR, or FcRn, is expressed on the BBB and acts as a reverse transport system to mediate the efflux of IgG molecules from brain to blood.10,11 Conventional AAAs are cleared slowly from plasma following systemic injection, and the plasma half-time is 3−4 weeks.12,13 Consequently, mouse models of AD are typically treated with systemic injections of AAAs given only once per week.14 In contrast, receptor-mediated AAAs are rapidly cleared from plasma. The cTfRMAb-ScFv fusion protein is cleared from plasma with a mean residence time of 2.9 h in the mouse, owing to uptake via the TfR in peripheral organs.9 Owing to this rapid clearance from plasma, there is little rationale for a weekly administration of the cTfRMAb-ScFv fusion protein in a mouse model of AD. In a prior study, AD transgenic mice were treated with twice-weekly intravenous (iv) injections of the cTfRMAbScFv fusion protein.15 Given the rapid clearance from blood, even twice-weekly iv injections of the cTfRMAb-ScFv fusion 3508

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immunoreactive Aβ1−42 and Aβ1−40 by ELISA, for Abeta immunofluorsescence (IF) and Thioflavin-S staining, and for assessment of cerebral microhemorrhage by Prussian blue histochemistry. Plasma Aβ1−40 and Aβ1−42 were measured on the terminal plasma by separate sandwich ELISAs (Invitrogen, Carlsbad, CA). Fluorescent Microscopy of Brain. The right cerebral hemisphere with the cerebellum was placed in cold 4% paraformaldehyde in phosphate buffered saline (PBS: 0.01 M Na2HPO4, 0.15 M NaCl, pH 7.0) for immersion fixation at 4 °C for 24 h. The tissue was incubated for 24 h each in 10%, 20%, and 30% sucrose sequentially for cyroprotection, frozen in powdered dry ice, and stored at −70 °C until sectioning. The tissue was embedded in Tissue-Tek OCT medium, and 10 μm sagittal frozen sections including the cortex and hippocampus were prepared on a cryostat (Micron Instruments, San Marcos, CA) at −20 °C and stored in PBS with 0.01% sodium azide at 4 °C. For visualization of amyloid deposition by immunofluorescence, sections were washed in PBS for 5 min and treated with 70% formic acid for 10 min at room temperature (RT). Sections were then washed in deioinized water for 5 min and blocked with 0.5% bovine serum albumin (BSA)/0.3% Triton X 100 in 0.01 M PBS/7.4 for 1 h at RT, and labeled with1 μg/ mL of Alexafluor 488-conjugated 6E10 mAb (Covance SIG39347; Dedham, MA) overnight at 4 °C. The 6E10 mAb is directed against the amino terminal portion of the Abeta amyloid peptide, and preferentially binds Abeta fibrils.17 Following washing, the sections were mounted using Vectamount aqueous mounting media (Vector Laboratories # H-5501, Burlingame, CA) and coverslipped. A separate series of tissue sections were stained with Thioflavin-S, which stains mature amyloid plaques.18 Brain sections were washed sequentially with 70% and 80% ethanol for 1 min followed by incubation in 1% Thioflavin-S solution in 80% ethanol for 15 min. After sequential washing with 70% and 80% ethanol for 1 min, sections were washed with distilled water for 5 min and coverslipped using Vectamount aqueous mounting media. Quantification of 6E10 and Thioflavin-S Plaque Staining. All images were acquired using the Leica SP2 1PFCS confocal microscope in the UCLA Advanced Light Microscopy/Spectroscopy Shared Facility. For quantification of cortical 6E10 and Thioflavin-S staining, two serial sections per mouse were taken, and two fields (1.5 mm × 1.5 mm) with maximum plaque were randomly chosen within the cortex for each section (total of 4 fields per mouse). For quantification of hippocampal 6E10 and Thioflavin-S staining, two serial sections per mouse were taken, and the hippocampus (1.5 mm × 1.5 mm) was imaged for each section (a total of 2 fields per mouse). All the images were taken at 10× magnification, and each brain section was scanned serially through a thickness of 5.2 μm for plaque quantification. The scanned images were quantified with ImageJ software (version 1.46r), and the following parameters were determined: (a) number of plaques/ mm2; (b) plaque positive area expressed as a percentage of analyzed area; and (c) average plaque size (μm2). Cerebral Microhemorrhage. Microhemorrage in brain was detected with Prussian Blue histochemistry. The fixed sections were treated with 2% potassium ferrocyanide in 2% HCl in Coplin jars for 30 min at RT. Slides were counterstained with Mayer’s hematoxylin. Prussian blue staining was semiquantitatively scored in 3 sections/mouse as follows: 0 (no microhemorrhage or reactivity detected in the entire brain section); 1 (≤2 microhemorrhages/section), 2 (3 to 10

microhemorrhages/section), or 3 (≥11 microhemorrhages/ section); and the average across each mouse was determined.19 Antidrug Antibody ELISA. Plasma was collected at 0, 4, 8, and 12 weeks of the study for measurement of antidrug antibody (ADA) by ELISA, as described previously.15 The detector agent of the assay is biotinylated cTfRMAb-ScFv antibody.15 The capture agent of the assay is either the cTfRMAb-ScFv fusion protein, the cTfRMAb alone, or the mouse IgG1κ isotype control antibody.15 The antibody titer is defined as the optical density (OD) per μL of undiluted mouse plasma. Statistical Analysis. Data are represented as mean ± SEM, and statistical analysis was performed using GraphPad Prism 5.0. Statistically significant differences in 6E10 and Thioflavin-S staining between groups were estimated using Student’s t test. A p < 0.05 was considered to be significant.

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RESULTS No mice developed signs of immune reactions, and there was no difference in body weight between the treatment groups during the course of the 12 week study. The body weight of the saline treated mice was 30.8 ± 1.7 and 27.2 ± 1.1 g at the beginning and the end of the study. The body weight of the cTfRMAb-ScFv fusion protein treated mice was 31.2 ± 1.8 and 27.9 ± 1.7 g at the beginning and the end of the study. A representative image for the Thioflavin-S fluorescent microscopy is shown in Figure 2 panels A and B for cortex from mice treated with saline and cTfRMAb-ScFv fusion protein, respectively, and in panels C and D for hippocampus from mice treated with saline and cTfRMAb-ScFv fusion protein, respectively. A representative image for the 6E10 immunofluorescent microscopy is shown in panels Figure 2 panels E and F for cortex from mice treated with saline and cTfRMAbScFv fusion protein, respectively, and in panels G and H for hippocampus from mice treated with saline and cTfRMAb-ScFv fusion protein, respectively. The results of the quantitation of the Abeta amyloid burden in cortex and hippocampus are given in Table 1 for both the Thioflavin-S and 6E10 studies. The percent of brain area occupied by Abeta plaque was reduced 57% and 49% in cortex as determined by 6E10 labeling and Thioflavin-S labeling, respectively. The percent of brain area occupied by Abeta plaque was reduced 61% and 43% in hippocampus as determined by 6E10 labeling and Thioflavin-S labeling, respectively. Significant reductions in amyloid burden were observed for all parameters for both cortex and hippocampus and for both 6E10 and Thioflavin-S labeling (Table 1). Cerebral microhemorrhage was determined by Prussian blue staining and scored on the basis of hemorrhages/section (Experimental Section). There were scant hemorrhages/section in either treatment group, and the brain sections were scored as 0.33 ± 0.01 and 0.22 ± 0.01 for the saline and cTfRMAb-ScFv fusion protein treatment group, respectively. Brain and plasma Aβ1−40 and Aβ1−42 were determined with separate sandwich ELISAs, and the results are given in Table 2. There was no change in the brain immunoreactive Aβ1−40 and Aβ1−42 in the fusion protein treatment group (Table 2). There was no change in plasma Aβ1−40 and Aβ1−42 in the two treatment groups (Table 2). The ADA formed against the cTfRMAb-ScFv fusion protein was measured by ELISA at 0, 4, 8, and 12 weeks of treatment in 1:50 dilutions of plasma taken from individual mice (Figure 3A). The ADA titer was intermediate at 4 weeks of daily sc 3509

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Table 2. Brain and Plasma Concentration of Immunoreactive Aβ1−40 and Aβ1−42a treatment

a

parameter

units

saline

cTfRMAb-ScFv

brain Aβ1−40 plasma Aβ1−40 brain Aβ1−42 plasma Aβ1−42

ng/mgp pg/mL ng/mgp pg/mL

63 ± 6 825 ± 81 220 ± 14 30,000 ng/g brain as the AD transgenic mouse ages from 4 weeks to 26 weeks.21 In another study, the 3511

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(6) Rinne, J. O.; Brooks, D. J.; Rossor, M. N.; Fox, N. C.; Bullock, R.; Klunk, W. E.; Mathis, C. A.; Blennow, K.; Barakos, J.; Okello, A. A.; Rodriguez Martinez de Llano, S.; Liu, E.; Koller, M.; Gregg, K. M.; Schenk, D.; Black, R.; Grundman, M. 11C-PiB PET assessment of change in fibrillar amyloid-β load in patients with Alzheimer’s disease treated with bapineuzumab: a phase 2, double-blind, placebocontrolled, ascending-dose study. Lancet Neurol. 2010, 9, 363−372. (7) Bohrmann, B.; Baumann, K.; Benz, J.; Gerber, F.; Huber, W.; Knolfach, F.; Messer, J.; Oroszlan, K.; Rachenberger, R.; Richter, W. F.; Rothe, C.; Urban, M.; Bardroff, M.; Winter, M.; Nordstedt, C.; Loetscher, H. Gantenerumab: A novel human anti-Aβ antibody demonstrates sustained cerebral amyloid-β binding and elicits cellmediated removal of human amyloid-β. J. Alzheimer’s Dis. 2012, 28, 49−69. (8) Boado, R. J.; Zhang, Y.; Xia, C. F.; Pardridge, W. M. Fusion antibody for Alzheimer’s disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation. Bioconjugate Chem. 2007, 18 (2), 447−455. (9) Boado, R. J.; Zhou, Q. H.; Lu, J. Z.; Hui, E. K.; Pardridge, W. M. Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor. Mol. Pharmaceutics 2010, 7, 237−244. (10) Zhang, Y.; Pardridge, W. M. Mediated efflux of IgG molecules from brain to blood across the blood-brain barrier. J. Neuroimmunol. 2001, 114, 168−172. (11) Schlachetzki, F.; Zhu, C.; Pardridge, W. M. Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier. J. Neurochem. 2002, 81, 203−206. (12) Siemers, E. R.; Friedrich, S.; Dean, R. A.; Gonzales, C. R.; Farlow, M. R.; Paul, S. M.; DeMattos, R. B. Safety and changes in plasma and cerebrospinal fluid amyloid β after a single administration of an amyloid β monoclonal antibody in subjects with Alzheimer disease. Clin. Neuropharmacol. 2010, 33, 67−73. (13) Black, R. S.; Sperling, R. A.; Safirstein, B.; Motter, R. N.; Pallay, A.; Nichols, A.; Grundman, M. A single ascending dose study of bapineuzumab in patients with Alzheimer disease. Alzheimer Dis. Assoc. Disord. 2010, 24, 198−203. (14) Bard, F.; Cannon, C.; Barbour, R.; Burke, R. L.; Games, D.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Lieberburg, I.; Motter, R.; Nguyen, M.; Soriano, F.; Vasquez, N.; Weiss, K.; Welch, B.; Seubert, P.; Schenk, D.; Yednock, T. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat. Med. 2000, 6, 916−919. (15) Zhou, Q. H.; Fu, A.; Boado, R. J.; Hui, E. K.-H.; Lu, J. Z.; Pardridge, W. M. Receptor-mediated Abeta amyloid antibody targeting to Alzheimer’s disease mouse brain. Mol. Pharmaceutics 2011, 8, 280− 285. (16) Sumbria, R. K.; Zhou, Q.-H.; Hui, E.K.-W.; Lu, J. Z.; Boado, R. J.; Pardridge, W. M. Pharmacokinetics and brain uptake of an IgGTNF decoy receptor fusion protein following intravenous, intraperitoneal, and subcutaneous administration in mice. Mol. Pharmaceutics 2013, 10, 1425−1431. (17) Perdivara, I.; Deterding, L.; Moise, A.; Tomer, K. B.; Przybylski, M. Determination of primary structure and microheterogeneity of a βamyloid plaque-specific antibody using high-performance LC-tandem mass spectrometry. Anal. Bioanal. Chem. 2008, 391, 325−336. (18) Ly, P. T.; Cai, F.; Song, W. Detection of neuritic plaques in Alzheimer’s disease mouse model.J. Visualized Exp. 2011, 53, DOI: 10.3791/2831. (19) Kumar-Singh, S.; Pirici, D.; McGowan, E.; Serneels, S.; Ceuterick, C.; Hardy, J.; Duff, K.; Dickson, D.; Van Broeckhoven, C. Dense-core plaques in Tg2576 and PSAPP mouse models of Alzheimer’s disease are centered on vessel walls. Am. J. Pathol. 2005, 167, 527−43. (20) Wang, A.; Das, P.; Switzer, R. C.; Golde, T. E.; Jankowsky, J. L. Robust amyloid clearance in a mouse model of Alzheimer’s disease provides novel insights into the mechanism of amyloid-β immunotherapy. J. Neurosci. 2011, 31, 4124−4136.

there was no neutralization of either the plasma clearance of the fusion protein or the brain uptake of the fusion protein.32 This safety study showed that chronic cTfRMAb fusion protein administration caused no changes in body weights, organ histology, or 23 metabolic tests, including serum iron and total iron binding capacity.32 The present study shows that cTfRMAb fusion proteins can be safely administered on a daily basis for a sustained treatment period of at least 3 months. In summary, the present study provides support for the reengineering of AAA therapeutics for AD as tetravalent bispecific antibodies (BSA), which both target an endogenous BBB transporter, such as the TfR, and target the Abeta amyloid peptide (Figure 1). While both conventional AAAs and receptor-mediated BSAs cause a reduction in brain amyloid plaque, chronic treatment with the receptor-mediated BSA causes no elevation in plasma Abeta peptide, and no cerebral microhemorrhage (Table 1). Therefore, receptor-mediated BSAs for AD may have more favorable therapeutic indices than conventional AAA therapeutics. The PK profile of receptor-mediated immune therapy is very different from passive immune therapy, as the plasma T1/2 is measured in hours vs weeks for receptor-mediated vs passive immune therapy, respectively (Table 3). The rapid clearance of the receptor-mediated fusion protein from plasma necessitates the development of a daily sc treatment protocol. The present study shows that receptor-mediated BSA therapeutics for AD may be safely administered by daily sc injections of the fusion protein.

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AUTHOR INFORMATION

Corresponding Author

*ArmaGen Technologies, Inc., 26679 Agoura Road, Suite 100 Calabasas, CA 91302. Phone: 818-252-8200. Fax: 818-2528214. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): Drs Boado, Hui, and Lu are employees of, and Dr. Pardridge is a consultant of, ArmaGen Technologies.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Institutes of Health National Institute of Aging [R01AG032244].

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REFERENCES

(1) Cummings, B. J.; Cotman, C. W. Image analysis of beta-amyloid load in Alzheimer’s disease and relation to dementia severity. Lancet 1995, 346 (8989), 1524−1528. (2) Naslund, J.; Haroutunian, V.; Mohs, R.; Davis, K. L.; Davies, P.; Greengard, P.; Buxbaum, J. D. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA, J. Am. Med. Assoc. 2000, 283 (12), 1571−1577. (3) Glenner, G. G.; Wong, C. W. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984, 120 (3), 885−890. (4) Masters, C. L.; Simms, G.; Weinman, N. A.; Multhaup, G.; McDonald, B. L.; Beyreuther, K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. U.S.A. 1985, 82 (12), 4245−4249. (5) Solomon, B.; Kippel, R.; Frankel, D.; Hanan-Aharon, E. Disaggregation of Alzheimer β-amyloid by site-directed mAb. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4109−4112. 3512

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(21) Chishti, M. A.; Yang, D. S.; Janus, C.; Phinney, A. L.; Horne, P.; Pearson, J.; Strome, R.; Zuker, N.; Loukides, J.; French, J.; Turner, S.; Lozza, G.; Grilli, M.; Kunicki, S.; Morissette, C.; Paquette, J.; Gervais, F.; Bergeron, C.; Fraser, P. E.; Carlson, G. A.; George-Hyslop, P. S.; Westaway, D. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J. Biol. Chem. 2001, 276, 21562−21570. (22) Racke, M. M.; Boone, L. I.; Hepburn, D. L.; Parsadainian, M.; Bryan, M. T.; Ness, D. K.; Piroozi, K. S.; Jordan, W. H.; Brown, D. D.; Hoffman, W. P.; Holtzman, D. M.; Bales, K. R.; Gitter, B. D.; May, P. C.; Paul, S. M.; DeMattos, R. B. Exacerbation of cerebral amyloid angiopathy-associated microhemorrhage in amyloid precursor protein transgenic mice by immunotherapy is dependent on antibody recognition of deposited forms of amyloid β. J. Neurosci. 2005, 25, 629−636. (23) Wilcock, D. M.; Alamed, J.; Gottschall, P. E.; Grimm, J.; Rosenthal, A.; Pons, J.; Ronan, V.; Symmonds, K.; Gordon, M. N.; Morgan, D. Deglycosylated anti-amyloid-beta antibodies eliminate cognitive deficits and reduce parenchymal amyloid with minimal vascular consequences in aged amyloid precursor protein transgenic mice. J. Neurosci. 2006, 26, 5340−5346. (24) Jancsó, G.; Domoki, F.; Sántha, P.; Varga, J.; Fischer, J.; Orosz, K.; Penke, B.; Becskei, A.; Dux, M.; Tóth, L. Beta-amyloid (1−42) peptide impairs blood-brain barrier function after intracarotid infusion in rats. Neurosci. Lett. 1998, 253, 139−141. (25) Su, G. C.; Arendash, G. W.; Kalaria, R. N.; Bjugstad, K. B.; Mullan, M. Intravascular infusions of soluble β−amyloid compromise the blood-brain barrier, activate CNS glial cells and induce peripheral hemorrhage. Brain Res. 1999, 818, 105−117. (26) Wilcock, D. M.; Colton, C. A. Immunotherapy, vascular pathology, and microhemorrhages in transgenic mice. CNS Neurol. Disord. Drug Targets 2009, 8, 50−64. (27) Sperling, R.; Salloway, S.; Brooks, D. J.; Tampieri, D.; Barakos, J.; Fox, N. C.; Raskind, M.; Sabbagh, M.; Honig, L. S.; Porsteinsson, A. P.; Lieberburg, I.; Arrighi, H. M.; Morris, K. A.; Lu, Y.; Liu, E.; Gregg, K. M.; Brashear, H. R.; Kinney, G. G.; Black, R.; Grundman, M. Amyloid-related imaging abnormalities in patients with Alzheimer’s disease treated with bapineuzumab: a retrospective analysis. Lancet Neurol. 2012, 11, 241−249. (28) Ostrowitzki, S.; Deptula, D.; Thurfjell, L.; Barkhof, F.; Bohrmann, B.; Brooks, D. J.; Klunk, W. E.; Ashford, E.; Yoo, K.; Xu, Z. X.; Loetscher, H.; Santarelli, L. Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab. Arch. Neurol. 2012, 69, 198−207. (29) Bard, F.; Fox, M.; Friedrich, S.; Seubert, P.; Schenk, D.; Kinney, G. G.; Yednock, T. Sustained levels of antibodies against Aβ in amyloid-rich regions of the CNS following intravenous dosing in human APP transgenic mice. Exp. Neurol. 2012, 238, 38−43. (30) Lee, H. J.; Engelhardt, B.; Lesley, J.; Bickel, U.; Pardridge, W. M. Targeting rat anti-mouse transferrin receptor monoclonal antibodies through blood-brain barrier in mouse. J. Pharmacol. Exp. Ther. 2000, 292, 1048−1052. (31) Greenblatt, D. J.; Sethy, V. H. Benzodiazepine concentrations in brain directly reflect receptor occupancy: studies of diazepam, lorezepam, and oxazepam. Psychopharmacology 1990, 102, 373−378. (32) Zhou, Q.-H.; Boado, R. J.; Lu, J. Z.; Hui, E.K.-W.; Pardridge, W. M. Chronic dosing of mice with a transferrin receptor monoclonal antibody-GDNF fusion protein. Drug Metab. Dispos. 2011, 39, 1149− 1154.

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