BloodâBrain Barrier Penetrating Biologic TNF-Î± Inhibitor for

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Blood-brain barrier penetrating biologic TNF-# inhibitor for Alzheimer's disease Rudy Chang, Jillian Knox, Jae Chang, Aram Derbedrossian, Vitaly Vasilevko, David Cribbs, Ruben J. Boado, William M. Pardridge, and Rachita Sumbria Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 17 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017

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Molecular Pharmaceutics

Blood-brain barrier penetrating biologic TNF-α inhibitor for Alzheimer’s disease Rudy Chang1, Jillian Knox2, Jae Chang1, Aram Derbedrossian1, Vitaly Vasilevko3, David Cribbs3, Ruben J. Boado4, William M. Pardridge4, and Rachita Sumbria1,5*. 1

Department of Biopharmaceutical Sciences, School of Pharmacy, Keck Graduate

Institute, Claremont, CA, USA; 2

Department of Neuroscience, Claremont McKenna College, Claremont, CA, USA;

3

Institute for Memory Impairments and Neurological Disorders, University of California,

Irvine, CA, USA; 4

ArmaGen, Inc., Calabasas, CA, USA;

5

Department of Neurology, University of California, Irvine, CA, USA.

*Address correspondence to: Rachita K. Sumbria, PhD Department of Biopharmaceutical Sciences School of Pharmacy, Keck Graduate Institute Claremont, CA, 91711 Tel: (909) 607-0319 Fax: (909) 607-9826 Email: [email protected]

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Abstract Objective: Tumor necrosis factor alpha (TNF-α) driven processes are involved at multiple stages of Alzheimer’s disease (AD) pathophysiology and disease progression. Biologic TNF-α inhibitors (TNFIs) are the most potent class of TNFIs but cannot be developed for AD since these macromolecules do not cross the blood-brain barrier (BBB). A BBB-penetrating TNFI was engineered by the fusion of the extracellular domain of the type II human TNF receptor (TNFR) to a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), designated as the cTfRMAb-TNFR fusion protein. The cTfRMAb domain functions as a molecular Trojan horse, binding to the mouse TfR and ferrying the biologic TNFI across the BBB via receptor-mediated transcytosis. The aim of the study was to examine the effect of this BBB-penetrating biologic TNFI in a mouse model of AD. Methods: 6 month old APPswe, PSEN1dE9 (APP/PS1) transgenic mice were treated with either saline (n=13), the cTfRMAb-TNFR fusion protein (n=12) or etanercept (nonBBB-penetrating biologic TNFI; n=11) 3 days per week intraperitoneally. After 12 weeks of treatment, recognition memory was assessed using the novel object recognition task, mice were sacrificed, and brains were assessed for amyloid beta (Aβ) load, neuroinflammation, BBB damage and cerebral microhemorrhages. Results: The cTfRMAb-TNFR fusion protein caused a significant reduction in brain Aβ burden (both Aβ peptide and plaque), neuroinflammatory marker ICAM-1, and a BBB disruption marker, parenchymal IgG, and improved recognition memory in the APP/PS1 mice. Fusion protein treatment resulted in low antidrug-antibody formation with no signs


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of either immune reaction or cerebral microhemorrhage development with chronic 12 week treatment. Conclusion: Chronic treatment with the cTfRMAb-TNFR fusion protein, a BBBpenetrating biologic TNFI, offers therapeutic benefits by targeting Aβ pathology, neuroinflammation and BBB-disruption, overall improving recognition memory in a transgenic mouse model of AD. Key words: blood−brain barrier, biologic TNF-α inhibitor, Alzheimer’s disease, transferrin receptor, monoclonal antibody



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Introduction Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by extracellular deposition of amyloid beta (Aβ)-containing senile plaques, intracellular neurofibrillary tangles in the brain and neuronal cell loss.1 AD is the most common cause of dementia and the sixth leading cause of death in the United States, with more than 5 million Americans currently living with it.2 Development of anti-Aβ agents has been at the forefront of AD research, albeit with limited success in mid- to late-stage clinical development.3 Tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine primarily produced by activated microglia/macrophages, plays a central role in AD pathogenesis.4 Many clinical and animal studies have demonstrated a link between excess TNF-α levels in the brain and AD.5-6 Excess TNF-α levels in the brain disrupt microglia mediated clearance of Aβ,7 increase production of Aβ,8-9 cause synaptic dysfunction,10 and accelerate disease progression and cognitive decline.11 Further, a single nucleotide polymorphism in the TNF-α gene is associated with age at onset of AD in certain populations.12 TNF-α driven processes are thus involved at multiple stages of AD pathophysiology and disease progression, and targeting TNF-α may offer a multi-target therapeutic approach for AD. The protective effects of biologic TNF-α inhibitors (TNFIs), including etanercept, a TNF-α decoy receptor (TNFR)-Fc fusion protein, infliximab, a TNF-α monoclonal antibody, and dominant negative TNFIs have been demonstrated in experimental,13-15 and clinical AD studies.16-17 In these studies the biologic TNFIs were administered either via the intracranial13, intra-thecal14-16 or perispinal17 route since these large molecules do not cross the blood-brain barrier (BBB).18 The perispinal route, 4 ACS Paragon Plus Environment

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which is a subcutaneous injection in the back, does not result in consistent brain drug delivery,19 since the BBB must still be penetrated. Treatment of chronic neurodegenerative diseases like AD necessitates long-term treatment, which makes intra-cranial or intra-thecal routes of administration neither practical nor safe. Systemic administration of etanercept failed to improve cognitive decline in AD patients,20 however inhibited neural TNF-α and improved Aβ induced memory impairment in control mice administered Aβ aggregates by intra-thecal injection.21 This inconsistency in the effect of systemic etanercept in clinical and experimental AD can be explained by the large dose (30 mg/kg) of etanercept used in the experimental study compared with a much lower dose (0.7 mg/kg) of etanercept used in the clinical study, as lower doses of systemic etanercept were not protective in experimental AD.20-21 Taken together, if the existing biologic TNFIs are to be developed for AD, these agents should be reengineered to enable BBB penetration. Biologic TNFIs can be re-engineered for BBB penetration by engineering fusion proteins of the TNFI with a molecular Trojan horse (MTH).22 A BBB penetrable biologic TNFI has been developed by engineering a fusion protein of the extracellular domain of the type II human TNFR and a chimeric monoclonal antibody against the mouse transferrin receptor (cTfRMAb). The fusion protein is designated as the cTfRMAb-TNFR fusion protein.23 The cTfRMAb domain of the fusion protein binds to the BBB TfR and ferries the TNFR across the BBB using a receptor mediated transcytosis approach, and the TNFR domain of the fusion protein sequesters excess TNF-α in the brain. Previous studies have shown that the cTfRMAb-TNFR fusion protein and etanercept bind to TNFα with high affinity with KD values of 374 ± 77 and 280 ± 80 pM, respectively.24 The 5 ACS Paragon Plus Environment


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cTfRMAb-TNFR fusion protein rapidly enters the mouse brain in therapeutically relevant amounts following intravenous, subcutaneous and intraperitoneal administration.23 Further, the cTfRMAb-TNFR fusion protein is protective in mouse models of ischemic stroke,25 and Parkinson’s disease,24 and comparable treatment with etanercept (nonBBB penetrating biologic TNFI) is not therapeutic in either of these neurological diseases because etanercept does not cross the BBB.18 Given the role of TNF-α in AD and the brain penetration of the cTfRMAb-TNFR fusion protein, the aim of the current study was to examine the effect of this BBBpenetrating TNFI on AD pathology. For this, 6 month old male APPswe, PSEN1dE9 (APP/PS1) transgenic mice were treated with either saline, the cTfRMAb-TNFR fusion protein or etanercept 3 days per week intraperitoneally. After 12 weeks of treatment, recognition memory was assessed using the novel object recognition task, and brains were assessed for Aβ load, neuroinflammation, BBB disruption and cerebral microhemorrhage development.


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Experimental Section Fusion protein. The cTfRMAb-TNFR fusion protein was produced in stably transfected Chinese hamster ovary cells and purified by protein G affinity chromatography as described previously.25 Etanercept was obtained from International Laboratory USA (San Francisco, USA). The cTfRMAb-TNFR fusion protein and etanercept were formulated in 100 mM glycine, 150 mM NaCl, 28 mM Tris, 0.01% Polysorbate 80, pH=6.42, sterile filtered and stored at either 4˚C or -80˚C till use. Mouse treatment. All animal procedures were approved by University of California, Irvine Institutional Animal Care and Use Committee and were carried out in compliance with University Laboratory Animal Resources regulations. Mice were provided constant access to food and water, and maintained on a 12-h light/12-h dark cycle. Male heterozygous APP/PS1 mutant mice (strain B6C3-Tg APPswe, PSEN1dE9, 85Dbo/Mmjax, stock 004462, Jackson Laboratories, Bar Harbor, ME) were 6 months old at the start of the study and were injected intraperitoneally (i.p.) three days a week, for twelve weeks either with saline (n=13), the cTfRMAb-TNFR fusion protein, 3 mg/kg (n=12) or etanercept, 3 mg/kg (n=11). Based on amino acid sequence, the TNFR domain comprises 49% of etanercept and 26% of the cTfRMAb-TNFR fusion protein. Plasma was collected at 0, 4, 8, and 12 weeks for anti-drug antibody (ADA) measurement by ELISA, as described previously.24 Mice were observed daily, weighed weekly and carefully monitored after each injection for decreased locomotor activity as a sign of an immune reaction. Following 12 weeks of injections, mice were anesthetized with a lethal dose of Nembutal (150 mg/kg, i.p.), cardiac perfusions were performed using ice cold phosphate buffer saline (PBS; 0.01 M Na2HPO4, 0.15 M NaCl, KCl, 7 ACS Paragon Plus Environment


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KPO4, pH 7.0) for 5 min to clear the cerebral vasculature, and the brains were harvested. Cryosectioning. The right cerebral hemispheres was immersion-fixed with 4% paraformaldehyde in PBS for 24h followed by serial incubation for 24h each in 10%, 20% and 30% sucrose solution at 4˚C, frozen using powdered dry ice and stored at 80ºC until cryosectioning. Frozen hemispheres were mounted in Tissue-Tek OCT compound (Fisher Scientific, Hampton, NH) and sliced into 20µm-thick sagittal sections, at -20ºC using a cryostat (Micron Instruments, San Marcos, CA). Free floating sections were stored in PBS with 0.01% sodium azide at 4˚C. Three sections per mouse, approximately 600 µm apart, were used for histochemistry as described below. Amyloid beta (Aβ) fluorescence microscopy. Thioflavin-S (Thio-S) staining to stain mature plaques and 6E10 MAb immunofluorescence to stain Aβ peptide was performed as described previously.26 Briefly, for Thio-S staining, tissue sections mounted onto positively charged microscope slides (Fisherbrand, Hampton, NH) were washed sequentially with 70% and 80% ethanol for 1 min followed by incubation in 1% Thio-S (Sigma Aldrich, St. Louis, MO) solution in 80% ethanol for 15 min. This was followed by sequential washing with 70% and 80% ethanol for 1 min each. For Aβ immunofluorescence, free floating sections were washed in PBS for 5 min and treated with 70% formic acid for 10 min at room temperature (RT). Sections were blocked with 0.5% bovine serum albumin (BSA) in PBS containing 0.3% TritonX-100 for 1h at RT, and incubated with 1µg/mL of Alexa Fluor 488-conjugated 6E10 MAb (BioLegend; San Diego, CA) overnight at 4°C. All the sections (6E10 and Thio-S) received a final wash with distilled water for 5 min and were cover slipped using Vectamount aqueous 8 ACS Paragon Plus Environment

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mounting media (Vector Laboratories, Burlingame, CA) and sealed with nail polish. Slides were stored at 4˚C until imaging. Fluorescent staining was examined using a Leica SP5 Confocal Microscope. For each brain section, two regions in the cortex and the entire hippocampus were scanned serially through a thickness of 5 µm (z-stack depth) and imaged at 10x magnification. Digitized images were analyzed using the NIH Image J software (version 1.46r) and the following parameters were determined by an observer blinded to the experimental groups: (a) number of positive stains/mm2 of tissue analyzed; (b) stain positive area expressed as a percentage of analyzed area; and (c) average stain size (µm2). Neuroinflammation, blood-brain barrier disruption and cerebral microhemorrhage assessment. To determine the effect of TNF-α neutralization on neuroinflammation and blood-brain barrier (BBB) disruption, ICAM-1 (inflammationinducible protein intercellular adhesion molecule-1; marker of inflammation), Iba1 (ionized calcium binding adaptor molecule 1; macrophage/microglial marker), CD11b (cluster of differentiation molecule 11b; microglial marker), and brain parenchymal IgG (marker of BBB disruption) immunohistochemistry were performed as described previously.27 For ICAM-1, Iba-1 and IgG immunohistochemistry, brain sections were incubated in 0.5% hydrogen peroxide in 0.1 M PBS containing 0.3% Triton-X100 (PBST) for 30 min at room temperature to block endogenous peroxidase activity. After washing with PBST, sections were incubated for 30 min with PBST containing 2% BSA to block non-specific protein binding. For IgG and ICAM-1 immunohistochemistry, sections were then incubated overnight at 4°C with a rabbit anti-mouse IgG antibody (1:200 dilution; JacksonImmunoResearch, West Grove, PA) and rabbit monoclonal



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antibody against ICAM-1 (1:500 dilution; Abcam, Cambridge, MA). After washing with PBST, sections were incubated at RT for 1h with biotinylated anti-rabbit IgG (1:500 dilution; JacksonImmunoResearch, West Grove, PA), followed by 1h incubation at RT with ABC complex according to manufacturer instructions (Vector Laboratories, Burlingame, CA). Sections were developed with 3,3'-diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA). For Iba-1 immunohistochemistry, sections were labeled with biotin-conjugated rabbit anti-Iba1 (1:200 dilution, Wako Chemicals USA, Richmond, VA), incubated for 1h at RT with ABC complex according to manufacturer instructions (Vector Laboratories, Burlingame, CA) and developed using the ImmPACT AEC Peroxidase (HRP) Substrate kit as per the vendors instructions (Vector Labs, Burlingame, CA). After washing, sections were mounted on slides using a Vectamount aqueous mounting media (Vector labs, Burlingame, CA), coverslipped, and sealed with clear nail polish. Three cortical images per brain section were acquired at 10X magnification using a light microscope (Motic, British Columbia, Canada) and the total positive immunoreactive area was quantified using NIH Image J software by an observer blinded to the experimental groups. Immunopositive area was expressed as % of total analyzed area. For CD11b immunofluorescence, sections were rinsed in PBS and placed in sodium citrate antigen-retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6.0) at 9095ºC for 15 min. After washing in distilled water and blocking in 5% BSA in PBS for 1h at RT, section were labeled with 5 µg/mL Alexa Fluor 594 anti-mouse/human CD11b (BioLegend, San Diego, CA) overnight at 4˚C. Sections received a final wash with distilled water for 5 min and were cover slipped using Vectamount aqueous mounting


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media (Vector Laboratories, Burlingame, CA) and sealed with nail polish. Fluorescent staining was examined using a Leica SP5 Confocal Microscope. Two regions in the cortex were scanned serially through a thickness of 5 µm per brain and were imaged at 10x magnification. Digitized images were analyzed using the NIH Image J software (version 1.46r) and the immunopositive area expressed as a % of analyzed area was determined. To determine if chronic treatment with the cTfRMAb-TNFR fusion protein and etanercept results in cerebral microhemorrhage development, brain sections were stained with hematoxylin and eosin (H&E) to detect cerebral microhemorrhage, as described previously.27 Briefly, brain sections subbed onto glass slides were washed in distilled water for 5 min, followed by dipping in Mayer’s hematoxylin (Fisher Scientific, Hampton, NH) for 10 min. After consecutive rinses in tap water, Scott’s tap water and tap water, slides were dipped in Eosin Y (Sigma Aldrich, St. Louis, MO) for 10 min. Sections were washed sequentially in 95% and 100% ethanol and were coverslipped with Permount (Fisher Scientific, Hampton, NH) after air drying. The entire brain section was examined to detect cerebral microhemorrhages and representative images were captured at a 20X magnification using a light microscope. Aβ(1-42) ELISA. Each left cerebral hemisphere, without the cerebellum, was placed in pre-chilled cryovials and frozen at -80ºC till homogenization. Frozen left cerebral hemisphere was pulverized on dry ice and the powered brain was homogenized in a Potter-Elvehjem (PE) homogenizer using 10 volumes of homogenization buffer (50 mM Tris-Cl, pH 7.6; 150 mM NaCl, 1 tab/10 ml of Roche complete EDTA-free Mini protease inhibitor, 5 mM EDTA, 2 mM 1,10-phenanthroline; 11 ACS Paragon Plus Environment


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TBS buffer). The homogenate was centrifuged at 100,000xg for 1h at 4ºC and the pellet was homogenized in a PE homogenizer using 10 volumes of homogenization buffer (5 M guanidine HCl, 0.05 M Tris, pH=8.0) and placed on a shaker for 3h at RT. The homogenate was then centrifuged at 20,800xg for 15 minutes at RT. A 50 µL aliquot of the supernatant was diluted to 2.5 mL in a dilution buffer (0.02 M Tris, 0.15 M NaCl, 1 mM EDTA, 1% Triton X-100, pH=7.5) and frozen at -80ºC till further analysis. An aliquot was used for protein assay using the bicinchoninic acid (BCA) kit (Pierce Chemical Co., Rockford, IL) and measurement of immunoreactive Aβ(1-42) by sandwich ELISA using the BetaMark™ β-Amyloid x-42 ELISA Kit (TMB) (BioLegend, San Diego, CA). The aliquot was further diluted 1:45 and 1:150 for the BCA protein and Aβ(1-42) sandwich ELISA, respectively. Anti-drug antibody formation ELISA. Anti-drug antibody (ADA) formation against the cTfRMAb-TNFR fusion protein and etanercept in mouse plasma was detected using a sandwich ELISA, as described previously.24 The cTfRMAb-TNFR fusion protein was the capture agent and biotinylated cTfRMAb-TNFR fusion protein was the detector agent in the sandwich ELISA. Briefly, 100µL per well of 2.5µg/mL of the capture agent was plated in 96-well plates, and incubated overnight at 4ºC. The wells were blocked with PBSB (0.01 M Na2HPO4/0.15 M NaCl/1% BSA/pH7.4) for 1h at RT and washed with PBSB. Plasma samples (diluted 1:50 in PBS; 100µL/well) were added to the wells and incubated for 1h at RT followed by washing with PBSB. Wells were incubated with 25ng/well of biotinylated cTfRMAb-TNFR fusion protein (detector agent) for 1h at RT followed by washing with PBSB. Wells were then incubated with 100 µL/well of 5 µg/mL of streptavidin-peroxidase conjugate (Vector Laboratories, Burlingame, CA) and


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incubated for 30min at RT. After washing with PBSB, wells were incubated with ophenylenediamine-H2O2 developing solution (Sigma Aldrich, St. Louis, MO) 15min in the dark at RT and the reaction was stopped by adding 100 µL of 1M HCl per well. Absorbance was measured at 492 (A492) and 650 (A650) nm. The A650 was subtracted from the A492. Sample (A492 − A650) value was corrected with the PBSB blank (A492 − A650) value. Novel object recognition test. Cognitive testing was assessed at the end of the study (12 weeks after the study start; age of mice = 9 months) using the novel object recognition (NOR) test. NOR is a simple test that assesses learning and memory in rodents based on their spontaneous tendency to explore a novel object more than a familiar object, and is well established in the APP/PS1 mice.28 Mice either explore the novel object more than the familiar object (> 50%), indicating recognition memory for the familiar object, or explore both the objects (novel and familiar) for the same amount of time (50% each), indicating lack of recognition memory for the familiar object.28 The test consisted of a 3 day protocol. Habituation phase: On days 1 and 2, each mouse was placed in a white box (40 cm x 40 cm with 36 cm walls) and allowed to explore the field for 5 min. Training and testing phase: On day 3, during the training phase, each mouse was placed in the white box with two identical objects (object 1 and object 2) that were placed at two opposite positions in the box equidistant from the corners and allowed to explore the objects for 10 min. Object exploration time was defined as the time that each mouse either sniffed (nose within 2 cm from the object) or pawed the object. Mice that had an exploration time of < 5 sec per object were excluded from the analysis. Location preference index in the training phase was estimated to rule out the influence



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of the object location on the recognition test and was estimated as follows: Location preference index = [Time spent exploring one of the identical objects/(Time exploring the identical object pair)] × 100%. Recognition memory was tested 1 hour after the training phase by exposing the mouse to one familiar (object 1) and one new object (object 2) for 10 min, and recognition index in the testing phase were estimated as follows: Recognition Index = [Time exploring novel object/(Time exploring novel object + Time exploring familiar object)]×100%. Statistical Analysis. Data were represented as mean ± SEM and all statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA), unless otherwise stated. One-way ANOVA with Bonferroni’s post hoc test for equal variances or Games-Howell for unequal variances (Minitab, State College, PA) were used to compare more than two groups. NOR data was compared using the onesample t test with a hypothesized mean = 50%. A two-tailed p-value of

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