Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity

Molecular Immunology 46 (2009) 532–540

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Complementarity determining regions of an anti-prion protein scFv fragment orchestrate conformation specificity and antiprion activity Andreas Müller-Schiffmann a,1 , Benjamin Petsch b,1 , S. Rutger Leliveld a,c , Janine Muyrers a , Agnieska Salwierz d , Christian Mangels e , Stephan Schwarzinger e , Detlev Riesner d , Lothar Stitz b , Carsten Korth a,∗ a

Department Neuropathology, Heinrich Heine University Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany Friedrich Loeffler Institute, Tübingen, Germany c Institute for Neuroscience and Biophysics 2, FZ Jülich, Germany d Institute for Physical Biology, Heinrich Heine University Düsseldorf, Germany e Department of Biopolymers, University Bayreuth, Germany b

a r t i c l e

i n f o

Article history: Received 20 June 2008 Received in revised form 16 July 2008 Accepted 17 July 2008 Available online 30 October 2008 Keywords: Prions Monoclonal antibody Conformation specificity Complementarity determining regions Recombinant antibodies Retro-inverso d-peptides

a b s t r a c t The prion protein, PrP, exists in several stable conformations, with the presence of one conformation, PrPSc , associated with transmissible neurodegenerative diseases. Targeting PrP by high-affinity ligands has been proven to be an effective way of preventing peripheral prion infections. Here, we have generated bacterially expressed single chain fragments of the variable domains (scFv) of a monoclonal antibody in Escherichia coli, originally raised against purified PrPSc that recognizes both PrPC and PrPSc . This scFv fragment had a dissociation constant (KD ) with recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM, as demonstrated by a mouse prion bioassay. A peptide corresponding to the complementarity determining region 3 of the heavy chain (CDR3H) selectively bound PrPSc but had lost antiprion activity. However, synthesis and application of an improved peptide mimicking side chain topology of CDR3H while exhibiting increased protease resistance, a retro-inverso d-peptide of CDR3H, still bound PrPSc and reinstated antiprion activity. We conclude that (1) scFvW226 is so far the smallest polypeptide with bioassay confirmed antiprion activity, and (2) differential conformation specificity and bioactivity can be regulated by orchestrating the participation of different CDRs. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Prion diseases are unique, transmissible, neurodegenerative diseases since the infectious agent consists solely of an alternative conformational isoform of the host-encoded prion protein, PrPSc , that replicates without a nucleic acid (Prusiner, 1982, 1998; Safar et al., 2005). Replication is thought to occur by induction of the

Abbreviations: aFFF, asymmetric field-flow fractionation; BBB, blood–brain barrier; CD, circular dichroism; CDR, complementarity determining region; CDR3H, third CDR of the heavy chain; (d-)riCDR3H, retro-inverso d-peptide analogue to CDR3H; NHS, N-hydroxysuccinimide; PrP, prion protein; PrPC , cellular isoform of PrP; PrPSc , misfolded isoform of PrP; scFv, single chain fragments of the variable domains; RML, Rocky Mountain Laboratory mouse-adapted scrapie strain (Chandler et al., 1961); SPR, surface plasmon resonance; TAMRA, tetramethylrhodamine; VH, heavy chain variable domain; VL, light chain variable domain; W226-Hc, recombinant heavy chain domain of W226. ∗ Corresponding author. Tel.: +49 211 811 6153; fax: +49 211 811 7804. E-mail address: [email protected] (C. Korth). 1 These authors contributed equally. 0161-5890/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2008.07.023

infectious conformation in the normal prion protein PrPC (Prusiner, 1982). The different stable conformations, or “conformers”, of PrP have pioneered the general concept of protein conformational diseases within the neurodegenerative diseases by stating that a misfolded or misprocessed protein is causative in the pathogenesis of the disease (Prusiner, 2001; Taylor et al., 2002). Structural analysis of these conformers has been difficult due to either their instability or insolubility. Therefore, it has been essential to generate ligands that are specific for the misfolded proteins, in particular to be able to study them in their cellular environment (Leliveld and Korth, 2007). Moreover, the development of a diverse set of specific ligands has also spurred the idea that soluble protein oligomers rather than insoluble (amyloidal) deposits are key to several neurodegenerative processes. For example, conformationspecific monoclonal antibodies (mABs) have been raised against PrPSc (Korth et al., 1997; Paramithiotis et al., 2003) or to A␤ oligomers which are major pathogenic conformers in Alzheimer disease (Kayed et al., 2003). Ultimately, these ligands are meant to detect the disease-associated conformers in tissues or body fluids as a method of identifying asymptomatic or early stage individuals

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at risk of developing disease (Kuhn et al., 2005; Lesne et al., 2006; Luibl et al., 2006; Nazor et al., 2005). So far, there is no pharmacotherapy of neurodegenerative diseases aimed at intervening with the fundamental biological causes of these diseases. Active or passive immunization approaches targeting disease-associated A␤ conformers in the case of Alzheimer disease (Schenk et al., 1999) or shielding the “normal” substrate conformer PrPC in the case of prion diseases (White et al., 2003) have been performed in mouse models of these diseases. While anti-A␤ mABs seem to pass the blood–brain barrier (BBB) and prevent A␤ aggregation (Bard et al., 2000), anti-PrP antibodies have only been successful after peripheral (intraperitoneal) inoculation, where they could act on peripheral sites of replication (Heppner et al., 2001; White et al., 2003) indicating their inability to pass the BBB. In order to improve the BBB permeability of anti-PrP antibodies and thus enhance their therapeutic applicability, we cloned single chain fragments of the variable region (scFv) derived from a mAB that recognizes both PrPC and PrPSc and exhibits high antiprion activity. These scFvs were abundantly expressed in the periplasm of E. coli in a soluble and active form. In an effort to further minimize their size, we showed that the complementarity determining region 3 of the heavy chain (CDR3H) alone bound PrPSc in a conformation-specific manner, but lost antiprion activity. Intending to design a peptide mimicking the side chain topology of the parent l-peptide while being more resistant to proteolytic degradation, we synthesized a retro-inverso (d-)riCDR3H peptide. This peptide reinstated antiprion activity, demonstrating that these 17 amino acid-containing peptides, too, have potential as diagnostic and therapeutic agents in prion diseases.

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5 -TTTTGCCGGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3 (VH reverse) or 5 -AAAGGATCCGACATTGTGATGACCCAGTCT-3 (VL forward) and 5 -AAAAGCGGCCGCGGATACAGTTGGTGCAGCATC-3 (VL reverse). PCR products were digested with NgoMIV (VH ) or BamHI (VL ) and ligated to the NgoMIV and BamHI site of an oligonucleotide coding for a (Gly4 Ser)3 linker domain (Huston et al., 1988). An 800 bp fragment corresponding to the correct ligation product was eluted from an agarose gel and amplified using the VH forward and VL reverse primer. The product was cut with NcoI and EagI and ligated into the prokaryotic expression vector pET22b (Novagen), allowing the expression with an N-terminal pelB leader sequence and a C-terminal His6 -tag (see Fig. 1). In addition, a cmyc tag was cloned into the EagI/XhoI sites between the scFv and the His6 -tag (see Fig. 1). For construction of only the heavy chain domain (W226-Hc), VH was amplified with appropriate primers allowing the cloning via NcoI and EagI into pET22b-Myc/His6 . For eukaryotic expression of scFvW226, the combined scFvW226 cDNA was amplified with a 5 -primer including a IgG␬-leader sequence (Donofrio et al., 2005) and ligated via HindIII/EcoRI (W226) into pCDNA 3.1 (Invitrogen). 2.2. Peptides CDR3H corresponding to the sequence NH2 –YFCARWNWERDAMDYWG–COOH (one letter amino acid code; Korth et al., 2008) and the retro-inverso d-peptide [(d-)riCDR3H] corresponding to the sequence NH2 –gwydmadrewnwracfy–COOH (one letter amino acid code, small letter convention for d-peptides; Korth et al., 2008) were synthesized either unlabeled or N-terminally linked to 6-carboxy-tetramethylrhodamine (TAMRA) by JPT Peptide Technology (Berlin, Germany).

2. Materials and methods 2.3. Protein expression and purification 2.1. Constructs W226 hybridoma secreting IgG1 mAB and recognizing both PrPC and PrPSc had been generated by standard fusion procedure of myleoma cells with splenocytes from a PrP knockout mouse (Büeler et al., 1992) immunized with purified mouse PrPSc (B.P. and L.S., unpublished). To prepare a single chain Fv construct (scFv), mRNA purified from W226 hybridoma was used for PCR amplification with the following primer set: 5 AAAACCATGGCGGAGGTCCAGCTGCAGCAGTC-3 (VH forward) and

Expression of scFvW226 or W226-Hc was induced in BL21 (␭DE3) Rosetta (EMD, Novagen Brand, Madison, WI): bacteria were grown at 37 ◦ C to high density (OD600 > 2.0) in a 2 L-fermentor (MoBiTec, Göttingen, Germany) and cooled down on ice before induction with 0.5 mM IPTG at 15 ◦ C over night. Cell pellets were lyzed in 20 mM Tris pH 8.0, 1% TX-100, 500 mM NaCl, 5 mM imidazole, 20 mM MgCl2 , 1 mM PMSF, 1 mg/mL lysozyme and 500 U DNase. Lysates were cleared by centrifugation and soluble protein in the supernatant was purified via Ni-NTA columns (Qiagen,

Fig. 1. Scheme of scFvW226-derived constructs. Shown is a schematic drawing of the used antibody fragments (Korth et al., 2008). The variable heavy and light chain domains of W226, connected by a flexible (Gly4 Ser)3 -linker domain (scFv) and W226-Hc were cloned into the prokaryotic expression vector pET22b, allowing the secretion of the antibody fragments into the periplasm by an N-terminal pELB leader sequence. In addition, C-terminal cmyc- and His6 -tags were included. The sequence of the heavy chain CDR3 in l-(CDR3H) or retro-inverso form ((d-)riCDR3H) is shown beneath.

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Hilden, Germany). After loading, the column was washed with 10 column volumes (CV) 20 mM Tris pH 8.0, 500 mM NaCl, 1% TX-100, 5 mM imidazole, 10 CV 20 mM Tris pH8.0, 500 mM NaCl, 1% TX-100, 20 mM imidazole and 10 CV 20 mM Tris pH 8.0, 1000 mM NaCl, 5 mM imidazole. Bound proteins were eluted with 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole yielding a purity of about 60% for scFvW226 and 90% for W226-Hc. Eluted scFvW226 was further purified to >95% purity by affinity chromatography employing recombinant mouse PrP (Korth et al., 1999) coupled to N-hydroxy succinimide (NHS)-sepharose (Amersham, USA) according to manufacturer’s recommendations. From the affinity column, scFvW226 was eluted with 50 mM glycine pH 2.5 and immediately neutralized with 100 mM Tris pH 8.8. Finally, purified antibody fragments were dialyzed twice against PBS. The mass of scFvW226 was measured by mass spectrometry and found to be identical to the calculated one (data not shown). 2.4. Pull-down experiments scFvW226, W226-Hc, riCDR3H and CDR3H were coupled to NHS-sepharose. 10% mouse brain homogenates prepared from C57BL/6 or RML-infected C57BL/6 mice (Chandler, 1961) were diluted 1:10 in 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 0,3% sarcosyl and precleared by centrifugation for 15 min at 22,000 × g. 1 mL thereof was incubated with 20 ␮L of loaded beads at 4 ◦ C over night. In a positive control experiment, 5 ␮g of recombinant mouse PrP in the same buffer was used. After incubation, beads were washed twice in IP1-buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 0,5% DOC), IP2-buffer (50 mM Tris pH 7.5, 500 mM NaCl, 0,1% NP40, 0,05% DOC) and IP3-buffer (50 mM Tris pH 7.5, 0.1% NP40, 0.5% DOC). Where necessary, beads were also incubated with 20 ␮g/mL Proteinase K (Merck, Darmstadt, Germany) in 20 ␮L IP3 buffer prior elution of bound PrP with 2× loading buffer at 95 ◦ C. The eluates were separated on a 4–20% Tris–HCl gel (Biorad, USA) and PrP was detected by Western blot using full-length mAb W226. 2.5. Circular dichroism (CD) analyses Far-UV CD spectra (195–250 nm) were recorded using a Jasco J-810 spectrometer. Sample conditions: 3 ␮M protein in 20 mM NaPO4 pH 7.5, 0.2 mM EDTA at room temperature (2 mm cuvette). Scan conditions: 20 nm/min scan speed, 100 mdeg sensitivity, 0.2 nm pitch, 1 nm bandwidth, 2 s response time, 40 accumulations. 2.6. Asymmetric field-flow fractionation (aFFF) In brief, aFFF is molecular sizing technique that separates particles (1 nm to 100 ␮m) in a laminar flow channel, based on differences in diffusion coefficient (Giddings, 1993). aFFF does not use a stationary phase with a large surface area, such as gel filtration, and is therefore less likely to underestimate the amount of aggregate or oligomer due to variable adsorption to the matrix. Here, the following system was used: Eclipse 2 equipped with HELEOS, Optilab Rex (Wyatt Technologies, USA) and a multiple wavelength detector (Agilent, USA). Software: Eclispe 2.5 and Astra 5.3.1.4. Conditions: scFvW226 was separated in 10 mM Tris–HCl pH 8, 50 mM NaCl, 1 mM EDTA with a 1 mL/min channel flow, using a 490 ␮m spacer and 5 kDa MWCO cellulose membrane. Flow scheme: sample inject (50 ␮L, 75 ␮g) → focussing (2 min, 3 mL/min cross-flow (VX)) → 1st elution phase (20 min, 2 mL/min linear VX) → 2nd phase (5 min 2.0–0.15 mL/min VX gradient) → 3rd phase (5 min VX off). Following the Zimm model for light scattering, the average molecular weight (MW) of each scFvW226 peak was calculated from the multi-angle laser light scattering data

(HELEOS, 18 angles) and the protein concentration as supplied by the refractive index (Wyatt, 1993). This combination of measurements allows direct molecular weight determination of the eluted material. 2.7. Surface plasmon resonance (SPR) analysis Binding kinetics were determined on a Biacore 1000 (GE Healthcare). Recombinant full-length mouse PrP (0.1 ␮M) was diluted in 10 mM NaOAc, pH 4.5 and immobilized on a EDC/NHS activated CM5 chip at a flow rate of 5 ␮L/min. After immobilization and blocking with ethanolamine a steady signal of about 500 RU was obtained. All kinetic SPR analyses were run at a 5 ␮L/min flow rate using PBS buffer. Antibody fragments were injected at different concentrations ranging from 10 to 1000 nM. Association and dissociation phases were recorded for 180 and 600 s respectively. After each cycle, the surface was regenerated with a 180 s pulse of NaOH ranging from 1 to 50 mM. Kinetic data were calculated using BIAevaluation 4.1 software according to a 1:1 (Langmuir) binding model. Three experiments with independent PrP coatings were performed. 2.8. Alanine scan of the W226 epitope Peptides corresponding to helix 1 of human PrP were purchased from Panatecs GmbH (Tübingen, Germany). Solutions for fluorescence spectroscopy were prepared in 10 mM sodium phosphate, pH 7.1, and centrifuged to remove any insoluble particles. Concentrations of stock solutions were determined with a spectrophotometer using molar absorption coefficients ε280 of 5500, 1490 and 125 cm−1 for each tryptophane, tyrosine, and cystine residue, respectively (Pace et al., 1995). Fluorescence experiments were carried out at 25 ◦ C on a Fluorolog-3-␶ (Spex-Horiba-Jobin Yvone, Germany) instrument equipped with an automatic titration device. The concentration of scFvW226 was 0.5 ␮M in a 1 cm path length quartz cell with a magnetic stir bar, helix 1 peptides were loaded into the automated titration device at concentrations of 500 ␮M. Tryptophane fluorescence was excited at 295 nm with a bandwidth of 2.5 nm and detected at 350 nm with a bandwidth of 5 nm. Peptide solutions were automatically dispensed into the observation cell and stirred for 45 seconds prior to data acquisition (20 data points a 0.5 s each). Typically, 21 titration steps to final peptide concentrations of 25 ␮M were carried out, except for the mutant peptides S143A, Y145A, and R151A, for which 13 titration steps were carried out. Duplicate titrations were carried out for the wild type sequence and for mutants D147A and R148A in order to obtain estimates for the experimental error. Using the program Grafit (Erithacus Software Ltd., U.K.) the data were fit to the equation for single site binding (Goodrich and Kugel, 2006). F=Fmin +

(p + L + KD −



(p + L + KD )2 − 4 × p × L) × (Fmax − Fmin ) 2×p

where F is the observed fluorescence, Fmin the fluorescence of free scFvW226, Fmax the fluorescence of the complex of scFvW226 with huPrP-helix 1 peptide, p the concentration of scFvW226, L the concentration of huPrP-helix 1 peptide, and KD the dissociation constant. 2.9. PrPSc inhibition assay 2.9.1. PrPSc inhibition by purified antibody fragments from E. coli ScN2a cells (Bosque and Prusiner, 2000; Butler et al., 1988) were grown in MEM, supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 ␮g/mL streptomycin and 10% FCS.

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Fig. 2. Purification and characterization of scFvW226. (a) Coomassie-stained gel of the purification process involving metal affinity chromatography (IMAC) and subsequently PrP affinity chromatography (samples after single purification steps as indicated). The final fraction after elution from the PrP affinity column is pure (arrow). (b) Asymmetric field-flow fractionation analysis of scFvW226. Purified scFvW226 consisted of approximately 82% (w/w) monomers and 16% dimers. The largest particles that we could detect were probably tetramers (∼120 kDa, ≤2%). Black line/scale on the right side, elution time; grey line/scale on the left, molecular weight of eluted multimers (logarithmic scale). (c) Quantitation of scFvW226-PrP binding by surface plasmon resonance spectroscopy. Recombinant mouse PrP was immobilized on a CM5-chip. Different concentrations of scFvW226 were injected at a flow rate of 5 ␮L/min. Association and dissociation curves were recorded for 180 and 600 s, respectively. After each cycle, surface was regenerated with 50 mM NaOH. Curve fitting calculations of three independently PrP-coated chips yielded a KD of approximately 2 nM (X2 = 16.2). (d) Western blot of an immunoprecipitation of normal (N) and RML scrapie-infected (Sc) mouse brain homogenates with immobilized scFvW226. Starting material is blotted on the left panel. Normal homogenate is pulled down, and the pulled-down material form scrapie brains is protease-resistant indicating that scFvW226 recognizes both PrPC and PrPSc .

For treatment, ScN2a cells were seeded in 60 mm dishes and incubated with antibody fragments for 7 days. After 3 days, medium/antibody fragments were changed. Cells were lyzed in 500 ␮L lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5% TX-100, 0.5% DOC) and equal amounts of lysates (determined by measuring the protein concentration) were treated with proteinase K (20 ␮g/mL) for 30 min at 37 ◦ C. After stopping protease digestion with 100 ␮M PMSF, PrPSc in 400 ␮L lysis buffer was pelleted at 100,000 × g in a TLA-55 rotor in an Optima ultracentrifuge (BeckmanCoulter, USA). PrPSc was detected after separation on a 4–20% Tris–HCl gel (Biorad, USA) by Western blot using mAb W226.

2.9.2. PrPSc inhibition by antibody fragments recombinantly expressed in cells ScN2a cells were split in 60 mm dishes the day before transfection to obtain 50% confluency and 1.3 ␮g pcDNA plasmid encoding scFvW226 or control scFv was transfected with HiPerfect (Qiagen, Germany) according to manufacturer’s instructions. After 4 days, cells either were lyzed and analyzed for PrPSc as described above or they were transferred to a 100 mm dish and incubated for additional 3 days before lysis. In addition, non-infected N2a cells were transfected in the described way and, after 4 days, conditioned medium was transferred to freshly seeded ScN2a cells which subsequently were incubated for another 4 days.

Table 1 Alanine scan of helix 1

2.10. Bioassay

Position

Amino acid sequence

KD (nM)

wt. S143 D144 Y145 E146 D147 R148 Y149 R151 E152 N153 M154 H155 R156

SDYEDRYYRENMHRY ADYEDRYYRENMHRY SAYEDRYYRENMHRY SDAEDRYYRENMHRY SDYADRYYRENMHRY SDYEARYYRENMHRY SDYEDAYYRENMHRY SDYEDRAYRENMHRY SDYEDRYYAENMHRY SDYEDRYYRANMHRY SDYEDRYYREAMHRY SDYEDRYYRENAHRY SDYEDRYYRENMARY SDYEDRYYRENMAAY

410 871 769 n.d. 1983 515 n.d. 1951 1128 n.d. 698 391 594 405

Mutated amino acids are in bold letters. n.d., no detectable binding.

Two separate treatment experiments for determining the presence of prions after scFvW226 treatment by inoculation in tg20 mice (Fischer et al., 1996) were performed: ScN2a cells were grown in 60 mm dishes and treated with 10, 30, 100 or 300 nM scFvW226 for 10 days with two splittings and scFvW226 renewals. In a second experiment, ScN2a cells grown in 60 mm dishes and treated with either 320 nM scFvW226 or W226-Hc for 1 week. Treatment was then discontinued and after 3 weeks (and two splittings), cells were collected by scraping, washed in PBS, counted and resuspended in 100 ␮L PBS, followed by five cycles of freeze/thawing. For both experiments, 20 ␮L of lysates corresponding to 0.8 or 2.8 × 105 cells, respectively, were injected into the cerebrum of five tg20 mice for each treatment condition or untreated control. Mice were terminated after onset of characteristic symptoms and

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the presence of prions was confirmed by Western blot (data not shown). 2.11. ScN2a cell immunofluorescence staining Live ScN2a cells were washed with PBS and, in one condition, preincubated with medium containing 100 ␮M scFvW226 for 30 min at RT. Subsequently, 1 ␮M of undigested or trypsindigested TAMRA-labeled (d-)riCDR3H or CDR3H was added. Trypsin-digestion was carried out with 100 ␮g trypsin for 3 h at 37 ◦ C. After incubation with labeled peptides of 3 h, cells were fixed with 4% paraformaldehyde and washed three times with PBS before inspection. 3. Results 3.1. ScFvW226 construction, expression, and characterization

Fig. 3. ScFvW226 clears prions from scrapie-infected cells. (a) Western blot of protease K-treated ScN2a cell lysates treated either by transfection with recombinant IgG␬-scFvW226 or by transfer with supernatant from non-infected, IgG␬-scFvW226-transfected N2a cell conditioned medium. ScN2a cells were either (from left to right) untreated, transfected with empty vector (pCDNA 3.1), transfected with control IgG␬-scFv19B10, or IgG␬-scFvW226 for 4 or 7 days. In addition, conditioned medium of N2a cells transfected with control IgG␬-scFv19B10, or IgG␬scFvW226 was used on ScN2a cells for 4 days. The blot clearly demonstrates that all scFvW226-containing constructs are antiprion active. Full-length mAB W226 was used for detection. (b) Western blot of ScN2a cell lysates treated with recombinant scFvW226 from E. coli. ScN2a cells were treated with different concentrations of scFvW226 as indicated, for 1 week. Subsequently, treatment was discontinued for either 1 week (upper panel) or 3 weeks (“set-off experiment”; lower panel). As controls, quinacrine (1000 nM) and full-length mAB W226 (16 nM) were used. The blot demonstrates a permanent, dose-dependent, prion-clearing effect of recombinant scFvW226 in E. coli. Full-length mAB W226 was used for detection. (c) Western blot of ScN2a cell lysates treated with recombinant scFvW226 from E. coli to determine the minimal prion-clearing concentration. ScN2a cells were treated with different concentrations of scFvW226 as indicated for 1 week. At a minimal concentration of 4 nM, scFvW226 cleared prions. Full-length mAB W226 was used for detection.

The original mAB W226 was derived from a hybridoma cell line generated after immunization with purified mouse PrPSc (B.P. and L.S., unpublished). For the IgG1 subtype mAB, a monovalent dissociation constant (KD ) with recombinant PrP had been determined to be 0.5 nM by surface plasmon resonance. Binding to both PrPC and PrPSc was confirmed by immunoprecipitation. The variable light and heavy chains were cloned as an scFv into the pelB containing pET22b vector, including an N-terminal pelB leader sequence, a (Gly4 Ser)3 -spacer (Huston et al., 1988) between H and L chain, and a C-terminal cmyc and His6 -tag (see Fig. 1). The pelB leader sequence was intended to target the protein to the periplasm, thereby increasing the chances of disulfide formation and correct folding of the scFv. The net yield of expression was approximately 10 mg soluble protein per liter of bacterial culture. scFvW226 was purified by immobilized metal affinity chromatography (IMAC) and affinity chromatography using recombinant mouse PrP covalently attached to sepharose by amine linkage (Fig. 2a). Like other scFvs (Pledger et al., 1999) the far-UV circular dichroism spectrum of scFvW226 showed it to contain mostly ␤-structure: a secondary structure estimate yielded 6% ␣-helix, 45% ␤-sheet, 11% ␤-turn and 39% loop residues (Lobley et al., 2002). Purified scFvW226 had a well-defined size distribution, consisting of 82% monomers, 16% dimers and a trace of higher order oligomers, as determined by asymmetric fieldflow fractionation (Fig. 2b). Gel filtration analysis of scFvW226 did give comparable results (data not shown). Measured by SPR, the KD of scFvW226 with recombinant mouse PrP was determined to be approximately 2 nM, i.e. only four times lower than for the full-length mAB (Fig. 2c). This KD could be an over-estimate to the intrinsic affinity of the scFv due to avidity effects of dimeric or multimeric scFvW226 species (Fig. 2b). The formation of stable dimers in scFvW226 commonly results from the swapping of heavy and light chain domains between two scFv monomers, thus creat-

Table 2 Bioassays of treated ScN2a cells in tg20 mice Construct

Number of ScN2a cells (×105 )

Dosage of fragment (nM)

Treatment time (days)

Number of sick n/n0

Incubation time (days)

PBS scW226 scW226 scFvW226 scFvW226

0.8 0.8 0.8 0.8 0.8

– 10 30 100 300

10 10 10 10 10

5/5 0/5 0/5 0/5 0/5

130 ± 20

PBS scFvW226 W226-Hc

2.8 2.8 2.8

– 300 300

21 21 21

5/5 0/5 5/5

75 ± 2 78 ± 3

ScN2a cells were treated with scFv in different concentrations, and for different times; even at the lowest concentration (10 nM) scFvW226 cleared prions completely. PBS treatment (control) or treatment with 320 nM W226-Hc did not clear prions.

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Fig. 4. Differential conformation specificity and antiprion activity of complementarity determining region 3 of the heavy chain (CDR3H) domain as an l-peptide and as a retro-inverso d-peptide ((d-)riCDR3H). (a) Western blot of PK-digested ScN2a cell lysates untreated (n.t.), treated with 1 ␮M quinacrine (Q), with recombinant heavy chain domain (W226-Hc) from E. coli, synthetic (d-)riCDR3H, or l-CDR3H at the concentrations indicated. Only (d-)riCDR3H had antiprion activity at 4 ␮M concentration. (b) Schematic surface representation of the linear sequences corresponding to CDR3H peptides with the l-peptide at the top and the d-peptide below. Residues shown in blue and red are basic and acidic, respectively. It can clearly be seen that the positions of the side-chain functionalities are conserved despite reversal of the amino acid sequence. Colors for atom positions are light red = O, light blue = N, grey = C, white = H, yellow = S. The model was generated with HyperChem 8.0 software (HyperCube, USA). (c) Western blot of pull-down experiments from normal (N) or scrapie-infected (Sc) mouse brain homogenates with immobilized L-CDR3H or (d-)riCDR3H. Whereas scFvW226 could precipitate both PrPC and PrPSc (see Fig. 2d), the CDR3H region pulled down only PrPSc in a conformation-specific manner, as did the (d-)riCDR3H peptide.

ing a non-covalent ‘diabody’ (Carmichael et al., 2003). As a result, the epitope-binding sites would be on opposite ends of the diabody (6–8 nm). Assuming that both sites are still functional, they could only bind two adjacent PrP molecules (3.5 nm for the globular C-terminus (Riek et al., 1996)) when these were in close proximity on the chip surface. Knowing that full-length PrP was coated at 500 RUs, this would correspond to between one and two PrP molecules per 100 nm2 (assuming 1 RU–1 pg protein/mm2 ). It is furthermore unlikely that two highly basic PrP molecules (pI ∼ 10, pH 4.5) would attach in close proximity on a negatively charged dextran-coated Biacore chip. Therefore, the contribution of double binding events due to the presence of scFvW226 dimers is deemed to be negligible for the fitting of binding curves. Changing PrP from ligand to analyte is hampered by the fact that PrP is poorly soluble in Tris- or phosphate-buffered saline pH 7–8, meaning that any soluble material will have a heterogeneous size distribution (Leliveld et al., 2008). In addition, the important activity of scFvW226 to be determined was its antiprion activity which would likely be favored by any avidity effects. ScFvW226 retained binding activity to recombinant mouse PrP after incubation at 60 ◦ C or in 90% serum at 37 ◦ C for 72 h, indicating a high stability under physiological conditions (data not shown). ScFvW226 retained the binding characteristics from its full-length ancestor in that it immunoprecipitated both PrPC and PrPSc from brain homogenates (Fig. 2d).

The epitope of scFvW226 was mapped to the linear sequence WEDRYYREN (one letter amino acid code), corresponding to residues 145–153 in helix 1 of PrP, using a PepSpot peptide array (JPT Peptides, Germany) similar to what has been used before for a similar purpose (Korth et al., 1997, 1999). Briefly, on this peptide array, 13 amino acid-long peptides covering the entire mouse PrP sequence with 11 amino acids overlapping are spotted; the epitope was defined to contain those amino acids common to all immunoreactive peptides. Systematic mutagenesis of the epitope using peptides based on human PrP by an alanine scanning revealed the significance of residues Y145, R148 and E152 for binding (Table 1). 3.2. ScFvW226 antiprion activity Next, we probed scFvW226 for antiprion activity. When we transfected scFvW226 cloned behind the IgG␬-signal sequence (Donofrio et al., 2005) for secretion by ScN2a cells we observed a clear time-dependent antiprion effect (Fig. 3a; compare 4 days after transfection with 7 days after transfection). Similarly, when scFvW226 was expressed in non-infected N2a cells and the supernatant of the conditioned medium was transferred to untransfected ScN2a cells after 4 days, prions were cleared (Fig. 3a). A control scFv derived from an antibody recognizing only a subpopulation of PrP (J.M. and C.K, unpublished) failed to be antiprion active.

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Fig. 5. Staining of ScN2a cells with tetramethylrhodamine (TAMRA)-labeled l-CDR3H and (d-)riCDR3H. ScN2a cells were incubated with 1 ␮M (d-)riCDR3H-TAMRA (a) or l-CDR3H-TAMRA (d) for 3 h and after washing with PBS, bound peptides were detected by fluorescence microscopy. To demonstrate the specificity of binding, ScN2a cells were preincubated with 100 ␮M scFvW226 30 min before addition of peptides (b and e). Digestion with trypsin demonstrated protease resistance of (d-)riCDR3H (c) vs. L-CDR3H (f).

When we treated ScN2a cells with purified, bacterially expressed scFvW226, we observed prion-clearing effects with concentrations >3.2 nM (Fig. 3b). These antiprion effects were permanent since 3 weeks after discontinuation of scFvW226 administration, no PK-resistant immunoreactivity corresponding to PrPSc reappeared (Fig. 3b, lower panel). The lowest effective concentration that would clear prions was narrowed down to 4 nM (Fig. 3c). As treatment control, quinacrine was used, a small heterocyclic molecule that is antiprion active through its effects on cellular lipid distribution (Korth et al., 2001; Klingenstein et al., 2006). When lysates of ScN2a cells that had been treated with scFv for either 10 days or 3 weeks were inoculated into tg20 indicator mice, scFvW226 concentrations as low as 10 nM were demonstrated to abolish prions from ScN2a cells (Table 2), thus confirming the results from Fig. 3. 3.3. Miniaturization of scFvW226 There have been several examples where scFvs could be broken down further without losing full biological activity (Bourgeois et al., 1998; Colby et al., 2004; Jackson et al., 1999; Kim et al., 2006). For anti-PrP antibodies, the heavy chain of mAB 6H4 has been shown to be sufficient for antiprion activity in vivo (Heppner et al., 2001). However, the heavy chain variable domain of scFvW226 alone – containing all three CDRs – failed to clear prions from ScN2a cells at concentrations where the entire scFvW226 would (Fig. 4a). When we expressed each of the CDRs separately as cmycand His6 -tagged fragments in E. coli, only the third CDR of the heavy chain domain (CDR3H) exhibited weak binding to recombinant mouse PrP by ELISA (data not shown). This is in agreement with previous studies that showed that CDR3H alone – being the most variable region among all CDRs (Shirai et al., 1996) – exhibited

weak binding to the epitope (Feng et al., 1998; Heap et al., 2005; Monnet et al., 1999). However, neither the E. coli-expressed CDR3H nor its tag-free synthetic equivalent showed antiprion activity (Fig. 4a). 3.4. A retro-inverso (d-) peptide of CDR3H is antiprion active The retro-inverso d-peptide analogues of l-peptides can mimic the side chain topology of the l-peptide provided that binding depends on side chain interactions and not on the peptide backbone (Van Regenmortel and Muller, 1998). d-Peptides are resistant to the l-specific proteases in vivo, and, accordingly, often have a dramatically increased half life time in vivo (Briand et al., 1995; Guichard et al., 1994; Levi et al., 2000). Since binding of CDR3H to PrP depended on bulky and/or charged residues (Table 1; namely, Y145, R148, and E152; Fig. 4b), we tested a retro-inverso d-peptide analogue to CDR3H (termed (d-)riCDR3H) for binding to PrP and biological effects on PrP conversion. Surprisingly, (d-)riCDR3H was antiprion active at concentrations of 4 ␮M where CDR3H had no activity (Fig. 4a). To further characterize the differences that determine the differential antiprion activity of (d-)riCDR3H as opposed to CDR3H, we performed live immunofluorescence stainings of ScN2a cells with TAMRA-labeled (d-)riCDR3H and CDR3H (Fig. 5). While (d-)riCDR3H exhibited staining all over the cell, CDR3H stained only intracellular compartments, likely after its endocytosis. Both stainings could be competed by scFvW226 indicating that both (d-)riCDR3H and CDR3H bound to the same antigens, i.e. PrP. As expected, only CDR3H could be digested by trypsin (Fig. 5). These findings indicated that the differential antiprion activity of (d-)riCDR3H was likely due to different in vivo characteristics like an increased half life time or differential subcellular targeting.

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3.5. CDR3H and (d-)riCDR3H are conformation-specific ligands of PrPSc To investigate whether the CDR peptides had maintained PrPbinding characteristics, we performed pull-down experiments with sepharose-immobilized peptides of brain homogenates from normal and RML-infected mice. Surprisingly, CDR3H and (d-)riCDR3H pulled down only PrP from scrapie-infected homogenates (Fig. 4c) that after digestion with PK revealed partial protease resistance indicating that this conformer was PrPSc (data not shown). Thus, compared to scFvW226, CDR3H peptides changed binding specificity and acquired conformation-specific binding to PrPSc . This binding seemed side-chain mediated since both CDR3H and (d-)riCDR3H bound PrPSc , although the l-peptide a little stronger (Fig. 4c). Attempts to measure affinities of the peptides to PrP by SPR were limited due to the small molecular size of the peptides. Nevertheless, using SPR on recombinant mouse PrP-coated chips, we estimated the KD s of both peptides to be in the range of 1–10 ␮M (data not shown). Likewise, determination of affinities by fluorescence resonance transfer of recombinant mouse PrP with TAMRA-labeled synthetic CDR3H and (d-)riCDR3H equally yielded only low affinities in the one-digit micromolar range with a slightly higher affinity of (d-)riCDR3H for PrP than CDR3H (data not shown). The conformation specificity of CDR3H and (d-)riCDR3H for PrPSc could explain the difficulties of determining KD s with recombinant mouse PrP which is thought to resemble PrPC rather than PrPSc .

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PrPSc degradation rate in ScN2a cells has been proposed as a mechanism of action (Peretz et al., 2001). This mechanism is likely to also apply for the permanent curative effects of the present antibody fragments, either by targeting both PrPC and PrPSc (for scFvW226) or for exclusive targeting of PrPSc (for d-riCDR3H). Although direct targeting of PrPSc to influence conversion is the most intuitive strategy for an antiprion therapeutical intervention, to our knowledge (d-)riCDR3H is the first polypeptide ligand specific for PrPSc which has been shown to be antiprion active. Thus, besides scFvW226 itself, (d-)riCDR3H is therefore a candidate therapeutic or diagnostic agent for prion disorders. The original immunogen for full-length mAB W226 in PrP knockout mice had been sodium phosphotungstic acid-precipitated PrPSc (B.P. and L.S., unpublished; Safar et al., 1998) and, accordingly, it was not surprising that resulting mAbs would recognize PrPSc in addition to PrPC . In the present studies, we have been able to identify CDR3H as a domain within scFvW226 that accounts for specific binding to PrPSc . The other CDRs of scFvW226 seem to enable additional binding of the PrPC conformation. CDR3H would be the core domain for PrPSc binding whereas the other CDRs would modulate this binding behavior to include binding to the PrPC conformation. Thus, CDR3 is a good building block for further engineering of prionspecific ligands. It is tempting to speculate that such a regulation of specificity to epitope conformations could also take place in CDRs recognizing proteins other than PrP. Eventually, such an orchestration of CDRs is a mechanism selected for by B cells for generating conformation-specific mABs. Acknowledgements

4. Discussion In this paper, we presented a high-affinity, antiprion active scFv that could be expressed in high yields as a soluble protein when targeted to the periplasmic space in E. coli. Its reliable antiprion activity makes scFvW226 a candidate for treating prion infections in vivo. To our knowledge, this ca. 30 kDa protein is the smallest polypeptide fragment whose antiprion activity has been confirmed by bioassays, followed by antiprion active Fab fragments that are about twice as big (Peretz et al., 2001). The ease of recombinant modifications of scFvs will enable further modifications, if necessary, for shuttling the PrP-binding fragment across the BBB, and targeting it to the subcellular sites of action in the CNS and peripheral sites of replication. Since the CDR3H is the most variable of all CDRs, there is a good chance that in many antibodies this region will reveal some binding to the antigen itself, although at weaker affinity (Feng et al., 1998; Heap et al., 2005; Monnet et al., 1999). In the case of the CDR3H peptides, the antibody-derived polypeptide changed its epitope such that an all-PrP recognizing scFvW226 became a PrPSc conformation-specific ligand. The affinities of both polypeptides to mouse recombinant PrP could thus not be compared by a convenient assay. Since (d-)riCDR3H was almost equally effective in pulling down PrPSc as CDR3, PrPSc -specific docking of CDR3 was indeed predominantly side chain-based rather than backbone-related. A look at the linearized model structure of these exposed side chains makes such similarities plausible (Fig. 4b). On the PrP side of this binding interface, the charged residues of helix 1 have been found critical for conversion and PrPC –PrPSc interactions (Lau et al., 2007; Moroncini et al., 2004; Norstrom and Mastrianni, 2006). Helix 1 is both a common antigenic site of PrPC and PrPSc (Korth et al., 1997). It is therefore easily conceivable that interactions of a ligand with helix 1, even if weak, could substantially interfere with PrP conversion and hence, prion replication. Accordingly, impeding with the PrPC –PrPSc interaction on the background of a constant normal

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