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A Single-Chain Variable Fragment Intrabody Prevents Intracellular

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A Single-Chain Variable Fragment Intrabody Prevents Intracellular View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Apollo The FASEB Journal • Research Communication A single-chain variable fragment intrabody prevents a intracellular polymerization of Z 1-antitrypsin while allowing its antiproteinase activity † ‡ Adriana Ordoñez,*´ Juan Perez,´ ,1 Lu Tan,*,1 Jennifer A. Dickens,*,1 Neda Motamedi-Shad, ‡ ‡ James A. Irving, Imran Haq, Ugo Ekeowa,* Stefan J. Marciniak,* Elena Miranda,§,2,3 ‡ and David A. Lomas ,2,3 *Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, † Cambridge, United Kingdom; Department of Cell Biology, Genetics and Physiology, University of ‡ Malaga, Malaga, Spain; Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; and §Department of Biology and Biotechnologies, “Charles Darwin,” and Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University, Rome, Italy a a ABSTRACT Mutant Z 1-antitrypsin (E342K) accumu- 1-ANTITRYPSIN DEFICIENCY is characterized by the accumula- a lates as polymers within the endoplasmic reticulum (ER) of tion of ordered polymers of mutant 1-antitrypsin within hepatocytes predisposing to liver disease, whereas low the endoplasmic reticulum (ER) of hepatocytes (1). The a levels of circulating Z 1-antitrypsin lead to emphysema by resulting inclusions cause a toxic gain of function that is loss of inhibition of neutrophil elastase. The ideal therapy associated with liver damage, whereas the plasma defi- should prevent polymer formation while preserving in- ciency predisposes to early-onset lung emphysema caused hibitory activity. Here we used mAb technology to identify by the loss of protease inhibition for several serine pro- a interactors with Z 1-antitrypsin that comply with both teases, particularly neutrophil elastase (2, 3). The most a fi requirements. We report the generation of an mAb (4B12) common cause of 1-antitrypsin de ciency is homozygosity a in vitro that blocked 1-antitrypsin polymerization at a 1:1 for the Z allele (E342K), which results in almost 90% of the a molar ratio, causing a small increase of the stoichiometry synthesized 1-antitrypsin being degraded or retained as of inhibition for neutrophil elastase.A single-chain variable polymers within the ER of hepatocytes (1). Preventing the a fragment (scFv) intrabody was generated based on the se- polymerization of Z 1-antitrypsin is an important thera- quence of mAb4B12. The expression of scFv4B12 within peutic goal, but strategies that achieve this while abolishing the ER (scFv4B12KDEL) and along the secretory pathway its inhibitory activity would exacerbate lung disease in a fi a (scFv4B12) reduced the intracellular polymerization of individuals with 1-antitrypsin de ciency. 1-Antitrypsin, as a Z 1-antitrypsin by 60%. The scFv4B12 intrabody also other members of the serine proteinase inhibitor (serpin) a increased the secretion of Z 1-antitrypsin that retained superfamily, exerts its antiproteinase activity by a unique inhibitory activity against neutrophil elastase. MAb4B12 mechanism (4, 5) involving a mobile and exposed reactive recognized a discontinuous epitope probably located in the center loop (RCL). Binding of the target proteinase to the a region of helices A/C/G/H/I and seems to act by altering serpin (e.g., neutrophil elastase to 1-antitrypsin) cleaves protein dynamics rather than binding preferentially to the the serpin at a precise position within the RCL. As a result, native state. This novel approach could reveal new target the RCL inserts into b-sheetA becoming strand 4A, and the sites for small-molecule intervention that may block the proteinase translocates to the lower pole of the 1:1 enzyme: transition to aberrant polymers without compromising a — the inhibitory activity of Z 1-antitrypsin. Ordoñez,´ A., Perez,´ J., Tan, L., Dickens, J. A., Motamedi-Shad, N., 1 These authors contributed equally to this work. Irving,J.A.,Haq,I.,Ekeowa,U.,Marciniak,S.J.,Miranda, 2 These authors contributed equally to this work. E., Lomas, D. A. A single-chain variable fragment intrabody 3 Correspondence: E.M.: Department of Biology and Bio- a technologies, “Charles Darwin,” and Pasteur Institute, Cenci prevents intracellular polymerization of Z 1-antitrypsin while allowing its antiproteinase activity. FASEB J. Bolognetti Foundation, Sapienza University of Rome, P.le 29, 2667–2678 (2015). www.fasebj.org Aldo Moro 5, Rome 00185, Italy. E-mail: mariaelena.mirandabanos@ uniroma1.it; D.A.L.: University College London, 1st Floor Maple House, 149, Tottenham Court Rd., London W1T 7NF, United Key Words: monoclonal antibody • scFv intrabody • serpin Kingdom. E-mail: [email protected] polymer • liver disease This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC Abbreviations: ER, endoplasmic reticulum; GFP, green BY 4.0) (http://creativecommons.org/licenses/by/4.0/) which fluorescent protein; HNE, human neutrophil elastase; HRP, permits unrestricted use, distribution, and reproduction in any horseradish peroxidase; pCMV, cytomegalovirus promoter; medium, provided the original work is properly cited. RCL, reactive center loop; RMSD, root mean square de- doi: 10.1096/fj.14-267351 viation; scFv, single-chain variable fragment; VH, variable This article includes supplemental data. Please visit http:// heavy chain; VL, variable light chain www.fasebj.org to obtain this information. 0892-6638/15/0029-2667 © The Author(s) 2667 Downloaded from www.fasebj.org by (5.69.44.37) on March 03, 2018. The FASEB Journal Vol. 29, No. 6, pp. 2667-2678. inhibitor complex. Critically, insertion of the RCL into production of hybridoma was carried out as described previously b-sheet A is also necessary for serpin polymerization (1, 6, 7). (19). Hybridoma clones were first screened against purified mo- a a Thelastdecadehasseenthedevelopmentofdif- nomeric Z 1-antitrypsin and then for their ability to block Z 1- a antitrypsin polymerization. Selected hybridoma cells were sub- ferent strategies to prevent the polymerization of Z 1- antitrypsin, aimed to therapeutic interventions. Three main cloned by limiting dilution and expanded as cell lines. The resulting antibodies were characterized by ELISA assays as described pre- approaches have been investigated: chemical chaper- fl – – viously (20). Brie y for (1) antigen-mediated ELISA, plates were ones (8 10), peptide analogs of the RCL (11 13), and coated with purified antigen proteins (monomer and polymer Z a m small compounds to prevent the conformational transi- 1-antitrypsin) at 4 g/ml, followed by incubation with hybridoma tion that underlies polymer formation (14). Several in culture media and with a rabbit anti-mouse horseradish peroxidase vitro studies have shown that peptide analogs of the RCL (HRP; Sigma-Aldrich, Dorset, United Kingdom); for (2) 2C1- a are able to block the in vitro polymerization of Z 1-anti- antigen-9C5-HRP sandwich ELISA, plates were coated with our fi a trypsin but result in the loss of inhibitory activity (12, 13), mouse monoclonal antibody speci cfor 1-antitrypsin polymers whereas others have evaluated the effects of chemical (mAb2C1) (20) at 2 mg/ml, followed by incubation with unknown a samples and with mAb 9C5-HRP (0.2 mg/ml) (20); for (3) com- chaperones or small molecules on Z 1-antitrypsin se- cretion in cell models or in vivo, without evidence of the petitive ELISA, plates were coated with the antigen protein at 4 mg/ml and mAbs to be tested were serially diluted followed by functional activity of the secreted protein (8, 14). Finding m a a incubation with mAb 9C5-HRP (0.2 g/ml); and (4) 1-antitrypsin a molecule that binds to 1-antitrypsin and prevents sandwich ELISA was performed as previously described (19, 20) a polymer formation while retaining antiproteinase func- using either our mAb3C11 (for total 1-antitrypsin) or mAb2C1 fi a tionality has thus become an important aim of the eld. (for 1-antitrypsin polymers) as detection antibodies. Over the last few years, intracellularly expressed antibody fragments (intrabodies) have emerged as a powerful ap- Purification of mAb and Fab production proach to modulate the function of targets in different in- tracellular compartments (15). The most common intrabody The full-length antibody was purified from hybridoma cell su- structure is the single-chain variable fragment (scFv), com- pernatant by using HiTrap Protein G Sepharose columns (GE posed of 1 heavy and 1 light variable domains (VH and VL, Healthcare Life Sciences, Waukesha, WI, USA). The Fab frag- fl respectively) linked by the (Gly4Ser)3 synthetic exible pep- ment was produced using the Pierce Mouse IgG1 Fab and F(ab9)2 tide.ThescFvisthesmallestfragmentofanantibodycapable Preparation Kit (Life Technologies, Rockford, IL, USA). of maintaining the antigen-binding specificity, with excellent properties of solubility, stability, and expression in mamma- a lian cells (16). They can be targeted to subcellular compart- Inhibitory activity of 1-antitrypsin ments by incorporating trafficking signals specificfortheER, The stoichiometry of inhibition of M (wild-type) or Z variants of cytosol, nucleus, lysosomes, or mitochondria (17). Intra- a bodies have been used as research and therapeutic agents for 1-antitrypsin with human neutrophil elastase (HNE; Millipore, a variety of protein conformational pathologies such as Alz- Billerica, MA, USA) was determined as described previously (21) ’ ’ ’ in the presence/absence of an 8-fold molar excess of mAb4B12 heimer s,
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