Structural differences of amyloid-β fibrils revealed by antibodies from phage display

BMC Biotechnology

Patrick Droste³  André Frenzel⁷  Miriam Steinwand⁵  Thibaut Pelat²  Philippe Thullier⁴  Michael Hust⁶  Hilal Lashuel¹  Stefan Dübel⁶ 

[1] SV-BMI, Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, École Polytechnique Fédérale de Lausanne, Station 19, Lausanne, 1015, Switzerland

[2] Current address: BIOTEM Parc d’Activités Bièvre Dauphine, 885, rue Alphonse Gourju, Apprieu, 38140, France

[3] Current address: Celerion Switzerland AG, Allmendstrasse 32, Fehraltorf, 8320, Switzerland

[4] Institut de recherche Biomédicale des Armées (IRBA-CRSSA); Département de Microbiologie; Unité de biotechnologie des anticorps et des toxines, La Tronche Cedex, France

[5] Current address: Delenex Therapeutics AG, Wagistrasse 27, Schlieren, 8952, Switzerland

[6] Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Spielmannstr.7, Braunschweig, 38106, Germany

[7] YUMAB GmbH, Rebenring 33, Braunschweig, 38106, Germany

关键词: scFv;    Immune library;    Phage display;    Beta-amyloid;    Abeta;    Aβ;    Alzheimer’s disease;   

Others  :  1216688
DOI  :  10.1186/s12896-015-0146-8

 received in 2014-11-27, accepted in 2015-04-20,  发布年份 2015

【 摘 要 】

Background

Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aβ and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of β-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of Aβ.

Results

Here we report the generation and characterization of a variety of amyloid-β specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aβ42 peptides. These antigens were used for the generation of two Aβ42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-β. Three of the obtained antibodies target the N-terminal part of Aβ42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aβ42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis.

Conclusion

We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti β-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.

【 授权许可】

   
2015 Droste et al.

【 图 表 】

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【 参考文献 】

• [1]Fitzpatrick AL, Kuller LH, Lopez OL, Kawas CH, Jagust W. Survival following dementia onset: Alzheimer’s disease and vascular dementia. J Neurol Sci. 2005; 229–230:43-9.
• [2]Goedert M, Wischik CM, Crowther RA, Walker JE, Klug A. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau. Proc Natl Acad Sci U S A. 1988; 85:4051-5.
• [3]Kosik KS, Joachim CL, Selkoe DJ. Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U S A. 1986; 83:4044-8.
• [4]Glenner GG, Wong CW. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun. 1984; 120:885-90.
• [5]Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K. Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer’s disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 1985; 4:2757-63.
• [6]Selkoe DJ, Abraham CR, Podlisny MB, Duffy LK. Isolation of low-molecular-weight proteins from amyloid plaque fibers in Alzheimer’s disease. J Neurochem. 1986; 46:1820-34.
• [7]Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D et al.. Isolation and quantification of soluble Alzheimer’s beta-peptide from biological fluids. Nature. 1992; 359:325-7.
• [8]Harper JD, Lansbury PT. Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu Rev Biochem. 1997; 66:385-407.
• [9]Jarrett JT, Berger EP, Lansbury PT. The carboxy terminus of the. beta. amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s disease. Biochemistry (Mosc). 1993; 32:4693-7.
• [10]Garzon-Rodriguez W, Sepulveda-Becerra M, Milton S, Glabe CG. Soluble Amyloid Aβ-(1–40) exists as a stable dimer at low concentrations. J Biol Chem. 1997; 272:21037-44.
• [11]Roher AE, Chaney MO, Kuo Y-M, Webster SD, Stine WB, Haverkamp LJ et al.. Morphology and toxicity of Aβ-(1–42) dimer derived from neuritic and vascular Amyloid deposits of Alzheimer’s disease. J Biol Chem. 1996; 271:20631-5.
• [12]Bitan G, Fradinger EA, Spring SM, Teplow DB. Neurotoxic protein oligomers–what you see is not always what you get. Amyloid Int J Exp Clin Investig Off J Int Soc Amyloidosis. 2005; 12:88-95.
• [13]Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ et al.. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci. 2005; 8:79-84.
• [14]Dahlgren KN. Oligomeric and fibrillar species of Amyloid-beta peptides differentially affect neuronal viability. J Biol Chem. 2002; 277:32046-53.
• [15]Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A et al.. A specific amyloid-β protein assembly in the brain impairs memory. Nature. 2006; 440:352-7.
• [16]Walsh DM, Klyubin I, Fadeeva JV, Cullen WK, Anwyl R, Wolfe MS et al.. Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature. 2002; 416:535-9.
• [17]Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB. Amyloid β-protein fibrillogenesis detection of a protofibrillar intermediate. J Biol Chem. 1997; 272:22364-72.
• [18]Jan A, Adolfsson O, Allaman I, Buccarello A-L, Magistretti PJ, Pfeifer A et al.. Aβ42 Neurotoxicity is mediated by ongoing nucleated polymerization process rather than by discrete A 42 species. J Biol Chem. 2010; 286:8585-96.
• [19]Ahmed M, Davis J, Aucoin D, Sato T, Ahuja S, Aimoto S et al.. Structural conversion of neurotoxic amyloid-β(1–42) oligomers to fibrils. Nat Struct Mol Biol. 2010; 17:561-7.
• [20]Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat Neurosci. 2012; 15:349-57.
• [21]Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid β-peptide. Nat Rev Mol Cell Biol. 2007; 8:101-12.
• [22]Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW et al.. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 2003; 300:486-9.
• [23]Kim T, Vidal GS, Djurisic M, William CM, Birnbaum ME, Garcia KC, et al. Human LilrB2 Is a β-Amyloid Receptor and Its Murine Homolog PirB Regulates Synaptic Plasticity in an Alzheimer’s Model. Science. 2013;341:1399–1404.
• [24]Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M et al.. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A. 1998; 95:6448-53.
• [25]Walsh DM, Klyubin I, Fadeeva JV, Rowan MJ, Selkoe DJ. Amyloid-beta oligomers: their production, toxicity and therapeutic inhibition. Biochem Soc Trans. 2002; 30:552-7.
• [26]Nelson PT, Braak H, Markesbery WR. Neuropathology and cognitive impairment in Alzheimer disease: a complex but coherent relationship. J Neuropathol Exp Neurol. 2009; 68:1-14.
• [27]Fox NC, Freeborough PA, Rossor MN. Visualisation and quantification of rates of atrophy in Alzheimer’s disease. Lancet. 1996; 348:94-7.
• [28]Johnson KA, Fox NC, Sperling RA, Klunk WE. Brain imaging in Alzheimer disease. Cold Spring Harb Perspect Med. 2012; 2:a006213.
• [29]Marshall GA, Monserratt L, Harwood D, Mandelkern M, Cummings JL, Sultzer DL. Positron emission tomography metabolic correlates of apathy in Alzheimer disease. Arch Neurol. 2007; 64:1015-20.
• [30]Shaw LM, Vanderstichele H, Knapik-Czajka M, Clark CM, Aisen PS, Petersen RC et al.. Cerebrospinal fluid biomarker signature in Alzheimer’s disease neuroimaging initiative subjects. Ann Neurol. 2009; 65:403-13.
• [31]Buckholtz NS. Perspective: in search of biomarkers. Nature. 2011; 475:S8.
• [32]Colledge NR, Walker BR, Ralston S, Davidson S. Davidson’s principles and practice of medicine. Churchill Livingstone/Elsevier, Edinburgh; New York; 2010.
• [33]Doody RS. Therapeutic standards in Alzheimer disease. Alzheimer Dis Assoc Disord. 1999; 13 Suppl 2:S20-6.
• [34]Ferris SH. Evaluation of memantine for the treatment of Alzheimer’s disease. Expert Opin Pharmacother. 2003; 4:2305-13.
• [35]Pelat T, Hust M, Hale M, Lefranc M-P, Dübel S, Thullier P. Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity. BMC Biotechnol. 2009; 9:60. BioMed Central Full Text
• [36]Laffly E, Danjou L, Condemine F, Vidal D, Drouet E, Lefranc M-P et al.. Selection of a Macaque Fab with framework regions like those in humans, high affinity, and ability to neutralize the Protective Antigen (PA) of Bacillus anthracis by binding to the segment of PA between residues 686 and 694. Antimicrob Agents Chemother. 2005; 49:3414-20.
• [37]Pelat T, Hust M, Laffly E, Condemine F, Bottex C, Vidal D et al.. High-affinity, human antibody-like antibody fragment (Single-Chain Variable Fragment) neutralizing the Lethal Factor (LF) of Bacillus anthracis by inhibiting protective Antigen-LF complex formation. Antimicrob Agents Chemother. 2007; 51:2758-64.
• [38]Schütte M, Thullier P, Pelat T, Wezler X, Rosenstock P, Hinz D et al.. Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus. PLoS One. 2009; 4:e6625.
• [39]Rülker T, Voß L, Thullier P, O’ Brien LM, Pelat T, Perkins SD et al.. Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in vitro and in vivo. PLoS One. 2012; 7: Article ID e37242
• [40]Hülseweh B, Rülker T, Pelat T, Langermann C, Frenzel A, Schirrmann T et al.. Human-like antibodies neutralizing Western equine encephalitis virus. mAbs. 2014; 6:718-27.
• [41]Miethe S, Rasetti-Escargueil C, Liu Y, Chahboun S, Pelat T, Avril A et al.. Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library. mAbs. 2014; 6:446-59.
• [42]Avril A, Froude J, Mathieu J, Pelat T, Thullier P. Isolation of antibodies from Non-human primates for clinical use. Curr Drug Discov Technol. 2014; 11:20-7.
• [43]Bradbury ARM, Sidhu S, Dübel S, McCafferty J. Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol. 2011; 29:245-54.
• [44]Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A et al.. A human scFv antibody generation pipeline for proteome research. J Biotechnol. 2011; 152:159-70.
• [45]Jäger V, Büssow K, Wagner A, Weber S, Hust M, Frenzel A et al.. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC Biotechnol. 2013; 13:52. BioMed Central Full Text
• [46]Lee M, Bard F, Johnson-Wood K, Lee C, Hu K, Griffith SG et al.. Aβ42 immunization in Alzheimer’s disease generates Aβ N-terminal antibodies. Ann Neurol. 2005; 58:430-5.
• [47]Lemere CA, Beierschmitt A, Iglesias M, Spooner ET, Bloom JK, Leverone JF et al.. Alzheimer’s disease Aβ vaccine reduces central nervous system Aβ levels in a non-human primate, the Caribbean Vervet. Am J Pathol. 2004; 165:283-97.
• [48]Vasilevko V, Pop V, Kim HJ, Saing T, Glabe CC, Milton S et al.. Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta. Neurobiol Dis. 2010; 39:301-10.
• [49]Bard F, Barbour R, Cannon C, Carretto R, Fox M, Games D et al.. Epitope and isotype specificities of antibodies to beta -amyloid peptide for protection against Alzheimer’s disease-like neuropathology. Proc Natl Acad Sci U S A. 2003; 100:2023-8.
• [50]Miller DL, Currie JR, Mehta PD, Potempska A, Hwang Y-W, Wegiel J. Humoral immune response to fibrillar beta-amyloid peptide. Biochemistry (Mosc). 2003; 42:11682-92.
• [51]Bacskai BJ, Kajdasz ST, McLellan ME, Games D, Seubert P, Schenk D et al.. Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J Neurosci Off J Soc Neurosci. 2002; 22:7873-8.
• [52]Dornieden S, Müller-Schiffmann A, Sticht H, Jiang N, Cinar Y, Wördehoff M et al.. Characterization of a single-chain variable fragment recognizing a linear epitope of Aβ: a biotechnical tool for studies on Alzheimer’s disease? PLoS ONE. 2013; 8:e59820.
• [53]Howlett DR, Perry AE, Godfrey F, Swatton JE, Jennings KH, Spitzfaden C et al.. Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans. Biochem J. 1999; 340:283-9.
• [54]Ida N, Hartmann T, Pantel J, Schröder J, Zerfass R, Förstl H et al.. Analysis of heterogeneous A4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay. J Biol Chem. 1996; 271:22908-14.
• [55]Johnson-Wood K, Lee M, Motter R, Hu K, Gordon G, Barbour R et al.. Amyloid precursor protein processing and Aβ42 deposition in a transgenic mouse model of Alzheimer disease. Proc Natl Acad Sci U S A. 1997; 94:1550-5.
• [56]Meinhardt J, Sachse C, Hortschansky P, Grigorieff N, Fändrich M. Abeta(1-40) fibril polymorphism implies diverse interaction patterns in amyloid fibrils. J Mol Biol. 2009; 386(3):869-77.
• [57]Habicht G, Haupt C, Friedrich RP, Hortschansky P, Sachse C, Meinhardt J et al.. Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Aβ protofibrils. Proc Natl Acad Sci. 2007; 104:19232-7.
• [58]Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M et al.. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Mol Neurodegener. 2007; 2:18. BioMed Central Full Text
• [59]O’Nuallain B, Wetzel R. Conformational Abs recognizing a generic amyloid fibril epitope. Proc Natl Acad Sci. 2002; 99:1485-90.
• [60]Frenkel D, Katz O, Solomon B. Immunization against Alzheimer’s β-amyloid plaques via EFRH phage administration. Proc Natl Acad Sci U S A. 2000; 97:11455-9.
• [61]Legleiter J, Czilli DL, Gitter B, DeMattos RB, Holtzman DM, Kowalewski T. Effect of different Anti-Aβ antibodies on Aβ Fibrillogenesis as assessed by atomic force microscopy. J Mol Biol. 2004; 335:997-1006.
• [62]McLaurin J, Cecal R, Kierstead ME, Tian X, Phinney AL, Manea M et al.. Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4–10 and inhibit cytotoxicity and fibrillogenesis. Nat Med. 2002; 8:1263-9.
• [63]Robert R, Dolezal O, Waddington L, Hattarki MK, Cappai R, Masters CL et al.. Engineered antibody intervention strategies for Alzheimer’s disease and related dementias by targeting amyloid and toxic oligomers. Protein Eng Des Sel. 2009; 22:199-208.
• [64]Solomon B, Koppel R, Hanan E, Katzav T. Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide. Proc Natl Acad Sci U S A. 1996; 93:452-5.
• [65]Hilbich C, Kisters-Woike B, Reed J, Masters CL, Beyreuther K. Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer’s disease beta A4 peptides. J Mol Biol. 1992; 228:460-73.
• [66]Moreth J, Mavoungou C, Schindowski K. Passive anti-amyloid immunotherapy in Alzheimer’s disease: What are the most promising targets? Immun Ageing A. 2013; 10:18. BioMed Central Full Text
• [67]Prins ND, Scheltens P. Treating Alzheimer’s disease with monoclonal antibodies: current status and outlook for the future. Alzheimers Res Ther. 2013; 5:56. BioMed Central Full Text
• [68]Sato T, Kienlen-Campard P, Ahmed M, Liu W, Li H, Elliott JI et al.. Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42. Biochemistry (Mosc). 2006; 45:5503-16.
• [69]Jan A, Gokce O, Luthi-Carter R, Lashuel HA. The ratio of monomeric to aggregated forms of Aβ40 and Aβ42 is an important determinant of Amyloid-β aggregation, fibrillogenesis, and toxicity. J Biol Chem. 2008; 283:28176-89.
• [70]“USDA Animal Welfare Act (AWA). 7 U.S.C. 2131 et seq., as amended and Health Research Extension Act of 1985 (‘“Animals in Research”’). 1985.”
• [71]Guide for the Care and Use of Laboratory Animals. 8th ed. National Academies Press (US), Washington (DC); 2011.
• [72]Pelat T, Hust M, Thullier P. Obtention and engineering of non-human primate (NHP) antibodies for therapeutics. Mini Rev Med Chem. 2009; 9:1633-8.
• [73]Frenzel A, Kügler J, Wilke S, Schirrmann T, Hust M. Construction of human antibody gene libraries and selection of antibodies by phage display. Methods Mol. Biol. Clifton NJ. 2014; 1060:215-43.
• [74]Fuchs M, Kampfer S, Helmsing S, Spallek R, Oehlmann W, Prilop W et al.. Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B. BMC Biotechnol. 2014; 14:68. BioMed Central Full Text
• [75]Durocher Y, Perret S, Kamen A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002; 30:E9.
• [76]Frank R. Spot-synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron. 1992; 48:9217-32.
• [77]Frank R. The SPOT-synthesis technique: synthetic peptide arrays on membrane supports—principles and applications. J Immunol Methods. 2002; 267:13-26.
• [78]Tomaselli S, Esposito V, Vangone P, van Nuland NAJ, Bonvin AMJJ, Guerrini R et al.. The alpha-to-beta conformational transition of Alzheimer’s Abeta-(1–42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding. Chembiochem Eur J Chem Biol. 2006; 7:257-67.