Specific Amyloid β Clearance by a Catalytic Antibody Construct

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JBC Papers in Press. Published on February 27, 2015 as Manuscript M115.641738 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.641738 Amyloid β Clearing Catabody

Specific Amyloid β Clearance by a Catalytic Antibody Construct* Stephanie A. Planque,1 Yasuhiro Nishiyama,1 Sari Sonoda,1 Yan Lin,2 Hiroaki Taguchi,1 Mariko Hara,1 Steven Kolodziej,1 Yukie Mitsuda,1 Veronica Gonzalez,2 Hameetha B. R. Sait,2 Ken-ichiro Fukuchi,3 Richard J. Massey,4 and Robert P. Friedland,5 Brian O’Nuallain,6 Einar M. Sigurdsson2,7 & Sudhir Paul1,7 From 1The Chemical Immunology Research Center, Department of Pathology and Laboratory Medicine, University of Texas–Houston Medical School, Texas 77030, USA; 2Departments of Neuroscience, Physiology and Psychiatry, New York University School of Medicine, New York, NY 10016, USA; 3 Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, IL 61605, USA; 4Covalent Bioscience Inc., Houston, TX 77054, USA; 5Department of Neurology, University of Louisville School of Medicine, Louisville, KY 40202, USA; 6Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis St, Boston, MA 02115, USA. 7

Address correspondence and reprint requests to Dr. Sudhir Paul, Department of Pathology and Laboratory Medicine, Chemical Immunology Research Center, University of Texas Medical School at Houston, 6431 Fannin, Houston, Texas 77030, USA; Ph: 713-500-5347; Fax: 713-500-0730; E-mail address: [email protected]. For mouse amyloid clearance studies, address correspondence to Dr. Einar Sigurdsson, Department of Neuroscience and Physiology, New York University School of Medicine, 450 East 29th Street, New York, New York 10016, USA; Ph: 212-263-3913; Fax: 212-2632160; E-mail address: [email protected].  rapidly, with no reactivity to the Aβ precursor protein, transthyretin amyloid aggregates or irrelevant proteins containing the catabodysensitive Aβ dipeptide unit. The catabody dissolved preformed Aβ aggregates and inhibited Aβ aggregation more potently than an Aβ-binding IgG. Intravenous catabody treatment reduced brain Aβ deposits in a mouse Alzheimer disease model without inducing microgliosis or microhemorrhages. Specific Aβ hydrolysis appears to be an innate immune function that could be applied for therapeutic Aβ removal.

CAPSULE Background: Naturally-occurring catalytic antibodies (catabodies) can hydrolyze peptide bonds. Result: A catabody engineered from innate immunity principles hydrolyzed amyloid β (Aβ) specifically, dissolved Aβ aggregates and cleared brain Aβ deposits without evident toxicity. Conclusion: The catabody could potentially be developed as a therapy for Alzheimer disease. Significance: The innate catabody repertoire may be a source of useful catabodies to toxic amyloids.

INTRODUCTION According to the “amyloid hypothesis”, soluble and fibrillar amyloid β peptide (Aβ)8 aggregates contribute causally in the pathogenesis of Alzheimer’s disease (AD). The aggregates activate microglial inflammatory processes, exert direct neurotoxic effects, and disrupt the brain anatomic architecture (1). In addition to deposits of Aβ1-42 (Aβ42) that damage the brain parenchyma, accumulation of Aβ1-40 (Aβ40) in blood vessel walls causes microvasculature-related

SUMMARY Classical immunization methods do not generate catalytic antibodies (catabodies) but recent findings suggest that the innate antibody repertoire as a rich catabody source. We describe the specificity and amyloid β (Aβ) clearing effect of a catabody construct engineered from innate immunity principles. The catabody recognized the Aβ C-terminus noncovalently and hydrolyzed Aβ 1

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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Running title: Amyloid β Clearing Catabody

Amyloid β Clearing Catabody

Catalytic antibodies (catabodies) hold potential for digesting the antigen into harmless soluble fragments with no dependence on accessory inflammatory cells. Conventional immunization procedures based on acquired immunity principles do not produce catabodies with hydrolytic rates sufficient for medical use. Recent studies suggest that catalysis is an innate property of the germline immunoglobulin variable (V)-domains that have developed over the course of Darwinian evolution (12). Degradation of several self-antigens by catabodies in autoimmune disease was reported (12,13). The innate Vdomain repertoire expressed prior to contact with an antigen is very large, containing diverse light and heavy chain V domains (VL and VH domains) that hold potential for specific recognition of individual antigenic epitopes. We reported the catalytic immunoglobulin V-domain (IgV) construct 2E6 isolated from a human IgV library (14). Here we present evidence showing that the IgV degrades and clears Aβ specifically with no evidence of microglial activation or microhemorrhages.

The IgVs were purified from bacterial periplasmic extracts by binding of the C-terminal His6 tag to metal affinity columns followed by acid elution (pH 5.0, designated aIgV), yielding the 30 kDa intact IgV and its 18 kDa fragment in electrophoresis gels (14). Unfractionated culture supernatants were prepared by centrifugation (5,000xg, 30 min) of IgV-secreting and control bacteria grown to equivalent density as before (~0.8 A600 units). Diagnostic anion exchange chromatography of culture supernatants (33 mL) was in a neutral pH buffer (MonoQ HR 5/5 FPLC column, 1 mL/min GE Healthcare; 20 min 0-1 M NaCl gradient in 50 mM Tris-HCl, pH 7.4, 0.1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate (CHAPS)). The partially fractionated IgV 2E6 preparation was obtained at a larger scale by two cycles of anion exchange FPLC at neutral pH (designated nIgV; from 1 Liter

MATERIALS AND METHODS

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AntibodiesIgV clones 2E6 and MMF6 were isolated from an IgV library cloned in pHEN2 vector from peripheral blood lymphocytes of 3 lupus patients without amyloid disease (14). The two IgVs contain the same C-terminal VL2 domain and different N-terminal VL1 domains (respectively, GenBank accession numbers FJ231715 and KF018653). The mutant IgV 2E6 gene was synthesized by Mutagenex Inc (Hillsborough, NJ) by replacing the VL1 domain framework regions (FRs; Kabat residues 1-23, 3549, 57-88 and 98-107) with the corresponding FRs of IgV MMF6 VL1 domain and cloned into pHEN2 vector (NcoI/XhoI sites). IgV expression in culture supernatants after 24 h induction with isopropyl-βD-thiogalactopyranoside was 1.5-5.4 mg/L. Aβbinding IgG1 59 contains the VL-VH domain pair of a single chain Fv fragment that binds Aβ42 aggregates and clears brain Aβ deposits in a transgenic mouse model (15). The polymerase chain reaction-amplified VL and VH domain cDNAs of the single chain Fv were cloned in pTT5 expression vector adjacent to the murine κ and γ1 constant domain segments, respectively, and coexpressed transiently in HEK 293 cells (16,17). Assembled IgG1 59 in tissue culture supernatant (~0.6 mg IgG/L) was concentrated 10X, purified using immobilized Protein A, dialyzed against 10 mM sodium phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl (PBS), and the protein concentration was determined (16).

neuroinflammation and compromised blood-brain barrier (BBB) integrity (2), resulting in cerebral amyloid angiopathy (CAA) in nearly all AD patients (3). Intravenous administration of brain penetrating Aβ-binding monoclonal IgGs was proposed for AD therapy (4-6). Such IgGs exert competing favorable and unfavorable effects (7,8). While the Aβ-IgG immune complexes are cleared via the Fc-receptor dependent uptake pathway by phagocytic cells (the microglia) (4), the activated cells release inflammatory mediators and neurotoxic factors (5,9). Moreover, Aβ-binding monoclonal IgGs clear parenchymal Aβ42, but they induce increased Aβ40 deposition in blood vessel walls, enhancing the incidence of microhemorrhages and CAA (6,7) thought to be correlated with cognitive impairments (10). Reminiscent of the exogenous IgG effect, the appearance of Aβ-binding autoantibodies in the cerebrospinal fluid of AD patients correlates with exacerbated CAA (11).

Amyloid β Clearing Catabody

volumes of these markers: aprotinin (6,512 Da), vasoactive intestinal peptide (3,326 Da), N-tertbutoxycarbonyl-O-benzyl-Glu-Ala-Arg-7-amino4-methylcoumarin (721 Da) and Ala-7-amino-4methylcoumarin (246 Da). Apparent KM and turnover number (kcat) were estimated from initial hydrolysis rates measured at a constant 125I-Aβ40 amount (~300,000 cpm) mixed with increasing Aβ40 concentrations (0.1 nM–25 µM), assuming an IgV mass of 30 kDa (14). Aβ42 (90 µg) was labeled with 125I using 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril-coated tubes (Pierce; Rockford, IL) and free 125I was removed (Sep-Pak Vac C18 cartridge; Waters, Milford, MA). The acetonitrile-eluted 125I-Aβ42 was dried, mixed with non-radiolabeled Aβ42 (1 mg), re-treated with HFIP for 1 h, lyophilized and stored (-80°C). After reconstitution in dimethylsulfoxide, the 125I-Aβ42 (5 mM) was diluted to a concentration of 50 µM in PBS, incubated for 92 h (37°C), and fibrillar Aβ42 was collected by centrifugation (16,000xg, 20 min). The radioactivity content of fibrillar 125I-Aβ42 was 2.3x107 CPM/µmol Aβ. Fibrillar 125I-Aβ42 (2.2 nmol/0.1 mL) was incubated with nIgV samples in PBS containing 0.1 mM CHAPS. Formic acid was added to 60% v/v in the supernatants collected by centrifugation (16,000xg, 20 min) and FPLC-gel filtration in formic acid was conducted as before. The pelleted 125I-Aβ42 was dissolved in PBS containing 60% formic acid and analyzed by FPLC gel filtration similarly.

Proteolysis assaysSynthetic Aβ40 from EZBiolab (Carmel, IN) treated with 1,1,1,3,3,3hexafluoro-2-propanol (HFIP, Sigma-Aldrich) was radiolabeled with 125I at Tyr10. Aβ40 forms particulate aggregates slowly compared to Aβ42. Routine hydrolysis tests were conducted using 125IAβ40 (~30,000 cpm) treated with nIgV or aIgV preparations (3-24 h) in PBS containing 0.1 mM CHAPS and 0.1% bovine serum albumin, followed by trichloroacetic acid precipitation to separate the intact peptide from fragments (14). Reported values are means±s.d. (2 or 3 replicates). Formic acid was added to 60% v/v prior to FPLCgel filtration of 125I-Aβ40 (50,000 cpm; ~0.2 nM) digested with IgVs (2.5 µg/mL, 24 h; Superdex peptide column, GE Healthcare, run in 60% formic acid; 0.5 mL fractions). The nominal peptide mass was computed from the retention

For epitope identification, non-radioactive Aβ40, Aβ1-16, Aβ17-42 or Aβ19-40 (Anaspec, Fremont, CA) were included as competitive inhibitors in the 125I-Aβ40 hydrolysis reaction mixture (0.2 μg/mL nIgV 2E6; 3 h, 37°C). Hydrolysis of soluble FLAG-tagged APP751 (83 kDa, 0.1 μM; Origene Technologies, Rockville, MD), glutathione-S-transferase (GST)-tagged amphiphysin (153 kDa, 0.1 μM) and GST-tagged zinc finger protein 154 (43 kDa, 0.1 μM; Abnova, Taipei, Taiwan) treated with 2.5 μg/mL nIgV (24 h) was estimated by SDS electrophoresis and staining with horseradish peroxidase-conjugated anti-FLAG or anti-GST IgG antibodies (SigmaAldrich) (16). Minor low-mass protein bands visible in the amphiphysin reaction mixtures in addition to the major intact amphiphysin band 3

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culture supernatant concentrated 25-fold on a 10 kDa Pellicon 2 membrane followed by dialysis against chromatography buffer). The concentrated culture supernatant contained 160 mg total protein and 1.1 mg IgV 2E6, determined, respectively, by the microBCA method and dot-blotting with antibody to the c-myc-peptide tag (14). The first FPLC cycle was in the pH 7.4 chromatography buffer as before (Hitrap Q FF column, GE Healthcare; 3 mL/min). The unbound fraction containing the anti-c-myc reactive IgV 2E6 (retention volume, 4-16 mL) was subjected to the second FPLC cycle on the same column at more alkaline pH using a 0-1 M NaCl gradient over 20 min (50 mM Tris-HCl, 0.1 mM CHAPS adjusted to pH 8.0 with Tris base). Anti-amyloid tests were done using the resultant bound nIgV 2E6 fraction (protein content 1.8 mg, IgV content 0.11 mg). The culture supernatant from control bacteria harboring empty pHEN2 vector was fractionated identically by two chromatography cycles. Control IgV MMF6 eluted in the bound fraction from the first chromatography cycle conducted as described for nIgV 2E6 (retention volume 44.1-56.2 mL, protein content 3.8 mg, nIgV MMF6 content 0.11 mg). nIgV 2E6 requires bound divalent metal for maintenance of the catalytic activity (18). To avoid diluting the protein, catalytically inactive nIgV 2E6 was prepared by dialysis for 24 h against PBS containing 0.1 mM CHAPS and 10 mM EDTA, followed by dialysis against the same buffer without EDTA (to remove the chelator).

Amyloid β Clearing Catabody

Anti-amyloid assaysPre-aggregated fibrillar Aβ42 (starting Aβ42 peptide concentration 20 µM; prepared as described for 125I-Aβ42) was treated with nIgVs (24 h, 37°C) in PBS, 0.1 mM CHAPS and 1% dimethysulfoxide. ThT (5 µM) was added and fluorescence emission was measured after 30 min. Inhibition of fibrillization was determined similarly by treating non-aggregated Aβ42 (20 µM) with the nIgVs. The data were corrected for background ThT fluorescence of the same antibody without Aβ42 (
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