title: Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody.
abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expansion in the number of polyglutamine-encoding CAG repeats in the gene that encodes the huntingtin (htt) protein. A property of the mutant protein that is intimately involved in the development of the disease is the propensity of the glutamine-expanded protein to misfold and generate an N-terminal proteolytic htt fragment that is toxic and prone to aggregation. Intracellular antibodies (intrabodies) against htt have been shown to reduce htt aggregation by binding to the toxic fragment and inactivating it or preventing its misfolding. Intrabodies may therefore be a useful gene-therapy approach to treatment of the disease. However, high levels of intrabody expression have been required to obtain even limited reductions in aggregation. We have engineered a single-domain intracellular antibody against htt for robust aggregation inhibition at low expression levels by increasing its affinity in the absence of a disulfide bond. Furthermore, the engineered intrabody variable light-chain (V(L))12.3, rescued toxicity in a neuronal model of HD. We also found that V(L)12.3 inhibited aggregation and toxicity in a Saccharomyces cerevisiae model of HD. V(L)12.3 is significantly more potent than earlier anti-htt intrabodies and is a potential candidate for gene therapy treatment for HD. To our knowledge, this is the first attempt to improve affinity in the absence of a disulfide bond to improve intrabody function. The demonstrated importance of disulfide bond-independent binding for intrabody potency suggests a generally applicable approach to the development of effective intrabodies against other intracellular targets.
authors.affiliation: Department of Chemical Engineering and Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Colby
authors.forename: Yijia
authors.initials: Y
authors.lastname: Chu
authors.forename: John P
authors.initials: JP
authors.lastname: Cassady
authors.forename: Martin
authors.initials: M
authors.lastname: Duennwald
authors.forename: Helen
authors.initials: H
authors.lastname: Zazulak
authors.forename: Jack M
authors.initials: JM
authors.lastname: Webster
authors.forename: Anne
authors.initials: A
authors.lastname: Messer
authors.forename: Susan
authors.initials: S
authors.lastname: Lindquist
authors.forename: Vernon Martin
authors.initials: VM
authors.lastname: Ingram
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity; D002460:Cell Line; D019020:Directed Molecular Evolution; D004220:Disulfides; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000378:metabolism / Q000628:therapy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000633:toxicity; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D015202:Protein Engineering; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012441:Saccharomyces cerevisiae
chemical_list: D000906:Antibodies; D004220:Disulfides; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins