Synthesis, Solution Conformation, and Antibody Recognition of

Comparative ELISA binding studies, using monoclonal antibody raised against the β-amyloid (1−17) peptide, showed that conjugates with T-helper cell...
1 downloads 0 Views 163KB Size
Download PDF
Bioconjugate Chem. 2005, 16, 921−928

921

Synthesis, Solution Conformation, and Antibody Recognition of Oligotuftsin-Based Conjugates Containing a β-Amyloid(4-10) Plaque-Specific Epitope Marilena Manea,† Ferenc Hudecz,‡,§ Michael Przybylski,† and Ga´bor Mezo˜‡,* Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457 Konstanz, Germany, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University, 1518 Budapest, Hungary, and Department of Organic Chemistry, Eo¨tvo¨s L. University, 1518 Budapest, Hungary . Received January 10, 2005; Revised Manuscript Received April 13, 2005

One possible therapeutic approach to treat or prevent Alzheimer’s disease (AD) is immunotherapy. On the basis of the identification of Aβ(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Aβ(4-10) epitope alone or flanked by R- or β-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the β-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Aβ(4-10) B-cell epitope with the β-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Aβ with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.

INTRODUCTION

Alzheimer’s disease (AD), a progressive neurodegenerative disorder of the brain, is the most common form of dementia among the elderly. One of the pathological characteristics of AD is the accumulation and invariant deposition of β-amyloid peptide (Aβ) into extracellular toxic plaques in the brain, which is thought to be the cause of cognitive decline (the β-amyloid hypothesis of AD). One possible therapeutic approach of AD is immunotherapy, via active or passive immunization, with the aim of either increasing the clearance of Aβ from the brain or inhibiting its aggregation to amyloid plaques (13). It has been shown that active immunization with Aβ(1-42) in transgenic mouse models of AD reduces both the accumulation of Aβ plaques in brain and associated cognitive impairment; no adverse effects were observed in these experiments (4-7). However, a therapeutic trial of immunization with Aβ(1-42) in humans was discontinued because several patients developed significant meningo-encephalitic cellular inflammatory reactions (8, 9). An alternative strategy to the active immunization with the complete Aβ peptide might be the use of peptides * Corresponding author. Tel: +36-1-2090-555/1426, Fax: +36-1-372-2620, e-mail: [email protected]. † University of Konstanz. ‡ Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University. § Department of Organic Chemistry, Eo ¨ tvo¨s L. University.

that are not fibrillogenic/toxic and not identical to endogeneous Aβ, which should reduce the likelihood of these side effects (10). Using the epitope excision method based on selective proteolytic digestion of the antigen-antibody complex in combination with high-resolution mass spectrometry (11), the N-terminal Aβ(4-10) sequence has been identified as the core epitope region recognized by therapeutically active antibodies raised against Aβ(1-42) and/or its oligomeric assemblies (12-15). Antibodies against the N-terminal regions of Aβ were able to invoke plaque clearance to the same degree as active immunization (16, 17). It is known that the administration of oligopeptide epitope rarely elicits appropriate immune responses (18). One approach to improve the immunogenicity of small peptides is the appropriate replacement of amino acid residues in the epitope core and/or the alteration of the flanking regions connected to the N- and/or C-terminal of the core (19, 20). Another strategy for increasing the sensitivity of the immunogenic or antigen binding properties is the multiplication of copies of the same or defined number of different B- or T-cell epitopes (19). It has also been shown that general peptides incorporating helper T-cell determinants and B-cell epitopes in multiple copies in branched architecture (topology) were better immunogens (18). To achieve this, epitope peptides can be conjugated to appropriate carrier molecules such as keyhole limpet hemocyanine (KLH), bovine serum albu-

10.1021/bc0500037 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/25/2005

922 Bioconjugate Chem., Vol. 16, No. 4, 2005

min (BSA), tetanus toxoid (TT), and purified protein derivatives (PPD). But these are not applicable in human therapy due to their immunological properties such as immunogenicity, cross-reactivity, and induction of tolerance (21-23). To overcome these drawbacks, synthetic polymers (24), branching lysine core (25), and sequential polypeptide (26) and carriers with discrete molecular mass and sequence (27) were developed and applied with success for preparation of antigens. The first set of conjugates containing the β-amyloid(4-10) epitope peptide was prepared in our laboratories using branched chain polymeric polypeptides with serine (SAK) or glutamic acid (EAK) at the branch terminating position (28). In these constructs the epitope peptide, elongated by CysGly5 at the N- or Gly5Cys at the C-terminus was attached to the chloroacetylated carrier via a thioether bond. A third construct contained polymeric carrier (EAK) and an epitope chimera in which a promiscuous T-helper cell epitope and the Aβ(4-10) epitope were joined by a dipeptide (Ahx-Cys) spacer (FFLLTRILTIPQSLD-AhxC-FRHDSGY). Comparative binding studies of the conjugates with a monoclonal antibody raised against amyloid protein beta-(1-17) showed that the chemical nature of the carrier has a significant influence on the antibody recognition. In addition we found that the presence of a T-helper cell epitope peptide in the conjugate resulted in lower antibody binding. Recently a novel group of sequential oligopeptides based on tuftsin was developed in our laboratory (29). The oligotuftsin derivatives consisting of tandem pentapeptide repeat unit [TKPKG]n (n ) 2, 4, 6, 8) derived from the canine tuftsin sequence TKPK extended by a C-terminal Gly. These compounds are nontoxic and nonimmunogenic and exhibit tuftsin-like biological properties, e.g. immunostimulatory activity and chemotactic activity on monocytes (30, 31). Considering the beneficial biological properties of the oligotuftsin derivatives and also their nonpolymeric structure, we prepared a new set of the Aβ(4-10) epitope conjugates to further investigate the effect of chemical structure on the antibody recognition of covalently attached epitope. These constructs are different from those reported earlier (28). Conjugates presented in this paper contain (a) sequential oligopeptide carrier instead of polymeric one, (b) amide linkage between the epitope and the carrier, and (c) Aβ(4-10) epitopes flanked by R- or β-alanine residues in contrast to the previously described versions, which possessed pentaglycine additions. In this paper we report on the synthesis, structural characterization, and antibody binding properties of a new set of peptide-conjugates containing the Aβ(4-10) epitope with or without R- or β-alanine flanks (FRHDSGY, AAFRHDSGYAA, βAβAFRHDSGYβAβA) and a second generation of the tetratuftsin based carrier without (a) or with (b) promiscuous T-helper cell epitope of a hepatitis B surface antigen (Ac-[TKPKG]4-NH2, AcFFLLTRILTIPQSLD-[TKPKG]4-NH2) (Figure 1). Our results show that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Aβ(4-10) B-cell epitope with β-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Aβ with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.

Manea et al.

Figure 1. Schematic structure of the Aβ(4-10)-tetratuftsin derivative conjugates (A) and their elongated versions by a helper T-cell epitope (B). EXPERIMENTAL PROCEDURES

Materials. All amino acid derivatives and MBHA resin were purchased from Reanal (Budapest, Hungary) or NovaBiochem (Laufelfingen, Switzerland). Scavengers, coupling agents, and cleavage reagents (p-cresol, dithiothreitol (DTT), N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), piperidine, trifluoroacetic acid (TFA), and hydrogen fluoride (HF)) were Fluka (Buchs, Switzerland) products. Acetic anhydride (Ac2O) and solvents for synthesis and purification were delivered from Reanal (Budapest, Hungary). Synthesis of Tetratuftsin Derivative. Ac-[TKPKG]4NH2 (Ac-T20, 1) was synthesized manually on a MBHA resin (1.04 mmol/g capacity) using the Boc/Bzl strategy. The following side-chain protected amino acid derivatives were used: Boc-Lys(ClZ)-OH, Boc-Thr(Bzl)-OH. The protocol was as follows: (i) DCM washing (3 × 0.5 min), (ii) Boc cleavage using 33% TFA in DCM (2+20 min), (iii) DCM washing (5 × 0.5 min), (iv) neutralization with 10% DIEA in DCM (4 × 1 min), (v) DCM washing (4 × 0.5 min), (vi) coupling 3 equiv of Boc amino acid-DCCHOBt in DCM-DMF (4:1 or 1:4 depends on the solubility of amino acid derivatives) mixture (60 min), (vii) DMF washing (2 × 0.5 min), (viii) DCM washing (2 × 0.5 min), (ix) ninhydrin or isatin assay. After completion of the synthesis, the acetylation of the N-terminus was carried out with acetic anhydride (1 mL) and DIEA (1 mL) in DMF for 1 h. The peptide was cleaved from the resin by the aid of liquid HF in the presence of p-cresol and DTT as scavengers (HF-p-cresol-DTT)10 mL: 0.5 g: 0.1 g) for 1.5 h at 0 °C. The crude product was precipitated with diethyl ether; the filtrate was washed three times with diethyl ether and solubilized using 10% acetic acid before freeze-drying. The crude material was purified by RPHPLC on a semipreparative C18 column and characterized by RP-HPLC, MALDI-FT-ICR mass spectrometry (Table 1), and amino acid analysis (data not shown). Tandem Synthesis of Conjugates Composed of Oligotuftsin Derivatives and β-Amyloid(4-10) Epitope Peptides. Conjugates Ac-[TKPK(H-FRHDSGY)G]4NH2 (T20-Aβ(4-10)4,2),Ac-[TKPK(H-AAFRHDSGYAA)G]4NH2 (T20-Aβ(4-10-Ala)4,3),Ac-[TKPK(H-βAβAFRHDSGYβAβA)G]4-NH2 (T20-Aβ(4-10-βAla)4, 4), Ac-FFLLTRILTIPQSLD-[TKPK(H-FRHDSGY)G]4-NH2 (T20-T-cellAβ(4-10)4,5),Ac-FFLLTRILTIPQSLD-[TKPK(H-AAFRHDSGYAA)G]4-NH2 (T20-T-cell-Aβ(4-10-Ala)4, 6), Ac-FFLLTRILTIPQSLD-[TKPK(H-βAβAFRHDSGYβAβA)G] 4 NH2 (T20-T-cell-Aβ(4-10-βAla)4, 7) containing amide bond between the carrier and the epitope peptide were

Bioconjugate Chem., Vol. 16, No. 4, 2005 923

Structure−Activity Relationship of Bioconjugates

Table 1. Characteristics of Tetratuftsin Derivative (Ac-T20) and Epitope Peptide Conjugates HPLC tR (min)a [M + H]+ calcd/found

peptide/conjugate

no.

sequence

Ac-T20 T20(Aβ4-10)4 T20(Aβ4-10-Ala)4 T20(Aβ4-10-βAla)4 T20-T-cell(Aβ4-10)4 T20-T-cell(Aβ4-10-Ala)4 T20-T-cell(Aβ4-10-βAla)4

1 2 3 4 5 6 7

Ac-[TKPKG]4-NH2 Ac-[TKPK(H-FRHDSGY)G]4-NH2 Ac-[TKPK(H-AAFRHDSGYAA)G]4-NH2 Ac-[TKPK(H-βAβAFRHDSGYβAβA)G]4-NH2 Ac-FFLLTRILTIPQSLD[TKPK(H-FRHDSGY)G]4-NH2 Ac-FFLLTRILTIPQSLDTKPK(H-AAFRHDSGYAA)G]4-NH2 Ac-FFLLTRILTIPQSLD[TKPK(H-βAβAFRHDSGYβAβA)G]4-NH2

14.5 18.6 20.7 21.1 34.2 33.5 32.5

2105.2922/2105.2995b 5554.7811/5554.7823c 6691.3747/6691.3538c 6691.3747/6691.3455c 7312.7891/7312.8017c 8449.3827/8449.3996c 8449.3827/8449.4016c

concn (µmol/L Aβ4-10)d 0.16 ( 0.052 0.14 ( 0.031 0.06 ( 0.010 0.28 ( 0.058 0.22 ( 0.035 0.06 ( 0.015

a RP-HPLC column: Phenomenex Synergy C 12 column (250 × 4.6 mm I.D.) with 4 µm silica (80 Å pore size); eluents: 0.1% TFA/water (A), 0.1% TFA/ACN-water 80:20, V/V (B); flow rate: 1 mL/min; gradient: 0 min 0% B, 5 min 0% B, 50 min 90% B. b MALDI and cESI mass spectrometric analyses were performed with a Bruker APEX II FT-ICR instrument equipped with an actively shielded 7T superconducting magnet. d The lowest amount of Aβ(4-10) epitope peptide in µmol/L to obtain an OD ) 1.0 in direct ELISA experiment.

Figure 2. Outline of the synthesis of the Aβ(4-10) epitope peptide-tetratuftsin derivative conjugate (2) obtained by total synthesis.

prepared manually by tandem synthesis on a MBHA resin (1.04 mmol/g capacity) using the Boc/Bzl strategy (Figure 2). The following side-chain protected amino acid derivatives were used: Boc-Lys(Fmoc)-OH, Boc-Lys(ClZ)OH, Boc-Thr(Bzl)-OH, Boc-Asp(OcHex)-OH, Boc-Ser(Bzl)OH, Boc-Arg(Tos)-OH, Boc-Tyr(BrZ)-OH, Boc-His(Bom)OH, and the syntheses were performed according to the protocol described above. After completion of the synthesis of the tetratuftsin derivative and its version elongated by T-cell epitope, the acetylation of the N-terminus was carried out with acetic anhydride (1 mL) and DIEA (1 mL) in DMF for 1 h. After removal of the N-Fmoc protecting group of Lys by 2% DBU, 2% piperidine in DMF for 2+2+5+15 min, the Aβ(4-10) epitope peptide with/without R- or β-alanine flanking region was built up on the -amino group of lysine by the Boc/Bzl strategy. A ratio of 12 equiv of Boc-amino acid derivatives and coupling reagents to the four Lys residues were used. The conjugates were cleaved from the resin by the aid of liquid HF in the presence of p-cresol and L-Cys (HF-p-cresolCys)10 mL:0.5 g:0.1 g) for 1.5 h at 0 °C. After their cleavage from the resin, the crude products (2, 3, and 4) were purified by RP-HPLC on a semipreparative C18 column, while products (5, 6, and 7), on a C4 column. The purified conjugates were characterized by RP-HPLC, ESI-FT-ICR mass spectrometry (Table 1), and amino acid analysis (data not shown). Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Analytical RP-HPLC was

performed on a Knauer (H. Knauer, Bad Homburg, Germany) HPLC system using a Phenomenex Synergy C12, column (250 × 4.6 mm I.D.) with 4 µm silica (80 Å pore size) (Torrance, CA) or Vydac C4 column (250 × 4.6 mm I.D.) with 5 µm silica (300 Å pore size) (Hesperia, CA) as stationary phases. Linear gradient elution (0 min 0% B; 5 min 0% B; 50 min 90% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrilewater (80:20, V/V)) was used at a flow rate of 1 mL/min at ambient temperature. Peaks were detected at λ ) 220 nm. The samples were dissolved in eluent A or in eluent A and B mixture (in case of compounds 5, 6, 7). The crude products were purified on a semipreparative Phenomenex Jupiter C18 column (250 × 10 mm I.D.) with 10 µm silica (300 Å pore size) (Torrance, CA) or Vydac C4 column (250 × 10 mm I.D.) with 10 µm silica (300 Å pore size) (Hesperia, CA). Flow rate was 4 mL/min. The same eluents as described above with an appropriate linear gradient (0 min 10% B; 5 min 10% B; 50 min 55% B in case of conjugates 2, 3, and 4 or 0 min 20% B; 5 min 20% B; 55 min 70% B in case of conjugates 5, 6, and 7) were applied. Amino Acid Analysis. The amino acid composition of the conjugates was determined by amino acid analysis using a Beckman Model 6300 analyzer (Fullerton, CA). Prior to analysis samples were hydrolyzed in 6 M HCl in N2 atmosphere at 110 °C for 24 h. Mass Spectrometry. MALDI-FT-ICR mass spectrometric analysis was performed with a Bruker APEX II

924 Bioconjugate Chem., Vol. 16, No. 4, 2005

Figure 3. ESI-FT-ICR mass spectrum of T20(Aβ4-10-βAla)4 (4). The insert shows the [M + H]+ ion after deconvolution of the ESI-FT-ICR mass spectrum.

FT-ICR instrument equipped with an actively shielded 7T superconducting magnet, a cylindrical infinity ICR analyzer cell, and an external Scout 100 fully automated X-Y target stage MALDI source with pulsed collision gas (Bruker Daltonik, Bremen, Germany). The pulsed nitrogen laser is operated at 337 nm. A 100 mg/mL solution of 2,5-dihydroxybenzoic acid (DHB) (Aldrich, Steinheim, Germany) in acetonitrile:(0.1% trifluoroacetic acid in water) (2:1) was used as a matrix. A 0.5 µL amount of matrix solution and 0.5 µL of sample solution were mixed on the stainless steel MALDI sample target and allowed to dry. Calibration was performed with a standard peptide mixture with an m/z range of approximately 5000 (32). ESI-FT-ICR MS were performed with the same Bruker Daltonik Apex II FT-ICR spectrometer (Bremen, Germany) equipped with a 7.0 T actively shielded superconducting magnet (Magnex, Oxford, UK), an APOLLO (Bruker Daltonik) electrospray ionization source, an API1600 ESI control unit, and a UNIX-based Silicon Graphics O2 workstation. Acquisition of spectra was performed with the Bruker Daltonik software XMASS and corresponding programs for mass calculation, data calibration, and processing. The masses of the singly charged ions were calculated by deconvolution of the multiply charged species (Figure 3). For sample preparation, 50% methanol, 2% acetic acid in water was used (33). Circular Dichroism Spectroscopy. Circular dichroism (CD) spectra were recorded on a Jasco spectropolarimeter, model J-720 (Jasco, Espoo, Finland), at room temperature in quartz cells of 0.05 cm path-length, under constant nitrogen flush. The instrument was calibrated with 0.06% (w/v) ammonium-D-camphor-10-sulfonate (Katayama Chemical, Osaka, Japan) in water. Double distilled water and 1:1 and 9:1 mixtures (v/v) of trifluoroethanol (TFE) (Fluka, Buchs, Switzerland) and water were used as solvents. The concentration of the samples was 100 µM (in case of conjugates) and 200 µM (in case of Ac-T20). The spectra were averages of six scans between λ ) 190 and 260 nm. Results are expressed in terms of mean residue ellipticity (deg cm2 dmol-1) after subtraction of the buffer baseline.

Manea et al.

Enzyme-Linked Immunosorbent Assay (ELISA). ELISA studies were performed using the purified type IgG1 monoclonal antibody (mAb) 6E10 (Chemicon, Temecula, CA), raised against Aβ(1-17) as described (28). Briefly, a standard dilution of the mAb 6E10 (c ) 1 mg/ mL in PBS) and conjugates as coating antigens was used. Ninety-six-well ELISA plates (BioRad, Hercules, CA) were coated overnight at room temperature with 100 µL/ well of conjugates (serial dilutions from 20 µM to 0.0001 µM of Aβ(4-10) epitope concentration). The antigen concentrations were expressed as the Aβ(4-10) epitope concentration, and they were the following: 20 µM, 6.66 µM, 2.22 µM, 0.74 µM, 0.246 µM, 0.082 µM, 0.027 µM, 0.0091 µM, 0.003 µM, 0.001 µM, 0.0003 µM, and 0.0001 µM. After coating, the wells were washed (0.05% Tween20 v/v in PBS, pH ) 7.5), and the nonspecific adsorption sites were blocked (5% BSA w/v in PBS). The mAb 6E10 at c ) 1 µg/µL was diluted 4000 times in 5% BSA (SigmaAldrich, Steinheim, Germany) and added to each well (100 µL). After incubation at room temperature for 2 h and washing steps, 100 µL of peroxidase labeled goat anti-mouse IgG (Jackson Immuno Research, West Grove, PA) diluted 1000 times in 5% BSA was added to each well. After an additional incubation for 2 h and washing three times, 100 µL of o-phenylenediamine dihydrochloride (Merck, Darmstadt, Germany) in sodium phosphatecitrate buffer at c ) 1 mg/mL and 2 µL of 30% hydrogenperoxide (Merck, Darmstadt, Germany) per 10 mL of substrate buffer was added. After 5 min, the absorbance at λ ) 450 nm was measured on a Wallac 1420 Victor2 ELISA Plate Counter (PerkinElmer, Boston, MA). The concentration of the antigen, which gave an OD450 of 1 was calculated (Table 1). RESULTS AND DISCUSSION

Synthesis and Structural Characterization of Aβ(4-10) Epitope Peptide Conjugates. In this work we report on the synthesis and characterization of a new group of synthetic antigens, in which the β-amyloid(410) epitope peptide was attached through an amide bond to two sequential carriers, the tetratuftsin derivative consisting of four units of TKPKG pentapeptide and its analogue elongated by a T-helper cell epitope (FFLLTRILTIPQSLD). Both compounds were prepared by solidphase methodology using Boc-chemistry and orthogonal protecting groups for lysine side-chains: Z(2Cl) in position 2 and Fmoc in position 4 of the pentapeptide unit. The Aβ(4-10) epitope peptide with/without R- or β-alanine flanking regions was connected to the -amino groups of lysine residues after selective removal of the N-Fmoc protecting group under alkaline conditions (Figure 2). After completion of the assembly of the 4879-mer conjugates (2-7), the compounds were cleaved from the resin with HF in the presence of p-cresol and L-Cys. L-Cys was added to scavenge the formaldehyde derived from the Bom protecting group of His during the cleavage procedure. The compounds were purified by HPLC and characterized by RP-HPLC, high-resolution FT-ICR mass spectrometry (Table 1, Figure 3), and amino acid analysis. In contrast to antigens prepared by the attachment of epitopes with polymeric carrier through thioether bond as published by us earlier (28), these new set of conjugates have several advantages: (a) discrete molar mass, (b) structurally identical species, (c) the presence of amide bonds only. In addition these constructs were also different from previous ones since the flanking regions of the Aβ(4-10) epitope were Ala2 or βAla2 dimers instead of pentaglycine.

Bioconjugate Chem., Vol. 16, No. 4, 2005 925

Structure−Activity Relationship of Bioconjugates

Figure 4. CD spectra of the acetylated tetratuftsin derivative (1).

Conformational Analysis by CD Spectroscopy. The conformational preferences of the carrier and epitope peptide conjugates in water and 1:1 and 9:1 TFE:water mixtures (v/v) was analyzed by CD spectroscopy. TFE is known to preferentially stabilize proteins and peptides in a helical conformation (34). The CD spectrum of the acetylated tetratuftsin derivative (1) recorded in water showed a strong negative π-π* band around 198 nm characteristic of unordered structure. The presence of TFE in the solvent mixture did not lead to spectral changes. The CD spectrum measured in 9:1 TFE:water mixture (v/v) reflects the predominance of random coil conformation, characterized by a negative maximum at 198 nm with a small shoulder at approximately 226 nm (Figure 4). Previous studies showed that nonacetylated tetratuftsin is very flexible, adopting a random coil conformation in water, TFE, and 1:1 TFE:water mixture (29). These results indicate that the acetylation of the tetratuftsin derivative does not change its conformational properties in solution. The attachment of β-amyloid(4-10) epitope peptide with/without the R- or β-alanine flanking region to the carrier did not result in significant conformational changes of the carrier (Figure 5). The CD spectra of the Aβ(410) epitope peptide-tetratuftsin derivative conjugates recorded in water showed the presence of a negative maximum and an additional positive band around 220 nm which characterized the unordered structure. Compared to pure water as solvent, the CD spectra in 1:1 and 9:1 TFE:water mixture (v/v) showed only a small shift in the π-π* electronical transitions toward higher wavelengths and the presence of a small negative shoulder at 222 nm (n-π* transition). A positive band around 220 nm could be detected only in case of conjugate 2, and the presence of R- or β-alanine flanking regions in the structure of the conjugates 3 and 4 determined the disappearance of this additional band. The conjugates 5-7 containing in their structure the T-helper cell epitope adopt a random coil conformation in water. Interestingly, in the presence of TFE, they adopt an ordered structure, which is more pronounced by increasing the content of TFE in the solvent mixture (the π-π* electronic transitions were shifted toward a higher wavelength, from 198 nm to 204-205 nm, and also the intensity of the n-π* transition at 225 nm increased) (Figure 6). This indicates that the conformational preferences of the conjugates depend on their

primary structure and that TFE enhances existing propensities and does not induce helix formation arbitrarily. In contrast to the essentially unordered oligotuftsin based carrier, branched chain polymeric polypeptides used for the previous set of antigens (EAK and SAK) have ordered structure (28). We found that in both conjugate families the conformation of the carriers has a significant influence on the CD spectra of the respective conjugates. In case of branched polypeptide conjugates we detected high level of ordered structure (28). Similarly, conjugates with oligotuftsin derivatives displayed essentially unordered conformation (Figures 5 and 6). Antigenicity of the Aβ(4-10) Epitope Peptide Conjugates against an Anti-Aβ-(1-17) Monoclonal Antibody. The β-amyloid(4-10) epitope-tetratuftsin derivative conjugates were compared for binding to an anti-Aβ-(1-17) monoclonal antibody by direct ELISA. The conjugates containing the Aβ(4-10) epitope in four copies or the free carrier were coated to ELISA plates. Subsequently mAb, peroxidase-conjugated second Ab, and OPD substrate were added and the absorbance was measured (Table 1, Figures 7A and 7B). The carrier itself did not show any reactivity with mAb (data not shown). On the basis of ELISA data, the influence of the flanking regions neighboring the B-cell epitope and the presence of T-helper cell epitope peptide in the backbone on the antibody binding were compared. The conjugates in which the B-cell epitope was flanked by two β-alanine residues at both N- and C-termini (4, 7) showed the highest binding activity to the mAb (lowest amount of Aβ(4-10) epitope peptide to obtain an OD ) 1: 0.06 µmol/ L); no differences related to the presence of the T-helper cell epitope in the structure of conjugate 7 could be detected (Table 1). The Aβ(4-10)-tetratuftsin derivative conjugates had similar binding properties, and the presence of R-alanine dimer flanking regions had no significant influence on the antigenicity of B-cell epitope (2, 3). Conjugates 5 and 6, containing the T-helper cell epitope in the polypeptide backbone, bound to the antibody with similar intensities, but their antigenicity was lower in comparison with that of the constructs 2 and 3 (e.g. 0.28 for 5 vs 0.16 for 2). We conclude that in the Aβ(4-10) epitope-tetratuftsin construct, the presence of R-alanine adjacent to the B-cell epitope had no marked effect on the antibody binding, whereas the β-alanine flanking region increased significantly the antibody recognition of Aβ(4-10) epitope. The data presented here suggest that inclusion of appropriate flanks to the B-cell epitope (e.g. β-Ala) may increase the antibody binding of the epitopes attached to the tetratuftsin derivative. Elongation of the carrier backbone by a sequence of amino acids (T-helper cell epitope) only slightly decreased antibody binding to the B-cell epitope (0.16 vs 0.28; 0.14 vs 0.22), but the selection of appropriate flank in the B-cell epitope was able to compensate this effect (0.06 vs 0.06) (Table 1). The coating ability of different conjugates with oligotuftsin-derived or polymer carriers is probably similar, but the coating itself may reduce the flexibility of the antigenic peptides and affect the binding. However, the data suggest that there is no significant influence on antibody recognition. CONCLUSION

In this study we report on the preparation and structural and binding characteristics of synthetic antigen conjugates comprising the β-amyloid(4-10) epitope as

926 Bioconjugate Chem., Vol. 16, No. 4, 2005

Manea et al.

Figure 5. CD spectra of the Aβ(4-10)-tetratuftsin derivative conjugates in water (A) and 1:1 (B) and 9:1 (C) TFE:water mixtures (v/v).

Figure 6. CD spectra of the Aβ(4-10)-tetratuftsin derivative-T-helper-cell epitope conjugates in water (A) and 1:1 (B) and 9:1 (C) TFE:water mixtures (v/v).

lead structures for new type of vaccines against Alzheimer’s disease. Constructs with defined structures, in which the Aβ(4-10) epitope was attached through an amide bond to the tetratuftsin derivative as oligopeptide carrier, were successfully prepared by tandem solid-phase

methodology. The alteration of the flanking regions connected to the N- and C-terminal ends of the Aβ(410)-epitope, e.g. by β-amino acid residues, resulted in increased binding. Comparative ELISA binding studies, using monoclonal antibody raised against the β-amyloid

Structure−Activity Relationship of Bioconjugates

Figure 7. Binding of mouse anti-amyloid protein beta-(1-17) monoclonal antibody to Aβ(4-10) epitope peptide conjugates 2-4 (A) and to conjugates 5-7 (B), as measured by direct ELISA.

(1-17) peptide, showed that conjugates with a T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Aβ(4-10) B-cell epitope with β-alanine dimer flanking regions at both N- and C-termini. Taken together, results suggest that modification of the B-cell epitope peptide from Aβ with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in a T-cell epitope containing synthetic antigen with preserved, and even improved, antibody recognition. These findings can be utilized for the design of further optimized synthetic antigens. Corresponding immunization studies with Aβ(4-10) epitope conjugates based on these second generation tetratuftsin as well as on branched chain polymeric polypeptide (28) carrier are in progress in our laboratories. ACKNOWLEDGMENT

This work was supported by grants from the Hungarian National Science Fund (OTKA No. T032425, T043576) and the Deutsche Forschungsgemeinschaft (TransregioSFB Konstanz-Zuerich and Biopolymer-Mass Spectrometry). We thank Dr. Xiaodan Tian for constructive criticism and discussions of this work. LITERATURE CITED (1) Shenk, D. (2002) Amyloid-β immunotherapy for Alzheimer’s disease: the end of the beginning. Nature Rev. Neurosci. 3, 824-828. (2) Monsonego, A., and Weiner, L. H. (2003) Immunotherapeutic approaches to Alzheimer’s disease. Science 302, 834-838.

Bioconjugate Chem., Vol. 16, No. 4, 2005 927 (3) Selkoe, D. J., and Schenk, D. (2003) Alzheimer’s disease: Moleular understanding predicts amyloid-based therapeutics. Annu. Rev. Pharmacol. Toxicol. 43, 545-584. (4) Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Zhenmei, L., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., and Seubert, P. (1999) Immunization with amyloid-β attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400, 173-177. (5) Janus, C., Pearson, J., McLaurin, J., Mathews, P. M., Jiang, Y., Schmidt, S. D., Chishti, M. A., Horne, P., Heslin, D., French, J., Mount, H. T., Nixon, R. A., Mercken, M., Bergeron, C., Fraser, P. E., St George-Hyslop, P., and Westaway, D. (2000) Aβ peptide immunization reduces behavioral impairment and plaques in a model of Alzheimer’s disease. Nature 408, 979-982. (6) Morgan, D., Diamond, D. M., Gottschall, P. E., Ugen, K. E., Dickey, C., Hardy, J., Duff, K., Jantzen, P., DiCarlo, G., Wilcock, D., Connor, K., Hatcher, J., Hope, C., Gordon, M., and Arendash, G. W. (2000) Aβ peptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature 408, 982-985. (7) Weiner, H. L., Lemere, C. A., Maron, R., Spooner, E. T., Grenfell, T. J., Mori, C.; Issazadeh, S., Hancock, W. W., and Selkoe, D. J. (2000) Nasal administration of amyloid β-peptide decreases cerebral amyloid burden in a mouse model of Alzheimer’s disease. Ann. Neurol. 48, 567-579. (8) Check, E. (2002) Nerve inflammation halts trial for Alzheimer’s drug. Nature 415, 462. (9) Nicoll, A. R. J., Wilkinson, D., Holmes, C., Steart, P., Markham H, and Weller, R. O. (2003) Neuropathology of human Alzheimer disease after immunization with amyloid-β peptide: a case report. Nature Med. 9, 448-452. (10) Sigurdsson, E. M., Scholtzova H., Mehta D. P., Frangione B., and Wisniewski T. (2001) Immunization with a nontoxic/ nonfibrillar amyloid-β homologous peptide reduces Alzheimer’s disease associated pathology in transgenic mice. Am. J. Pathol. 159, 439-447. (11) Macht, M., Fiedler, W., Kurzinger, K., and Przybylski, M. (1996) Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T. Biochemistry 35, 15633-15639. (12) McLaurin, J., Cecal, R., Kierstead, M. E., Tian. X., Phinney, A. L, Manea, M., French, J. E., Lambermon, M. H., Darabie, A. A., Brown, M. E., Janus, C., Chishti, M. A., Horne, P., Westaway, D., Fraser, P. E., Mount, H. T., Przybylski, M., and St George-Hyslop, P. (2002) Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nature Med. 8, 1263-1269. (13) Przybylski, M., McLaurin, J., Cecal, R. E., Kierstead, M. E., Tian, X., Janus, C., Manea, M., Horne, P., Westaway, D., Fraser, P. E., and St George-Hyslop, P. (2002) Lead structures for active immunization against Alzheimer’s disease (AD) upon elucidation of a plaque-specific epitope recognized by therapeutically active antisera from transgenic AD mice. Peptides 2002 (Benedetti, E., and Pedone, C., Eds.) pp 862863, Edizioni Zino, Napoli, Italy. (14) Cecal, R., McLaurin, J., Tian, X., Manea, M., Janus, C., Westaway, D., Fraser, P., St George-Hyslop, P., and Przybylski, M. (2002) Mass spectrometric elucidation of an amyloid plaque-specific epitope: molecular basis for probing the protheolytic degradation mechanism of Alzheimer’s amyloid precursor protein APP. Peptides 2002 (Benedetti, E., and Pedone, C., Eds.) pp 700-701, Edizioni Zino, Napoli, Italy. (15) Bard, F., Barbour, R., Cannon, C., Carretto, R., Fox, M., Games, D., Guido, T., Hoenow, K., Hu, K., Johnos-Wood, K., Khan, K., Kholodenko, D., Lee, C., Lee, M., Motter, R., Nguyen, M., Reed, A., Shenk, D., Tang, P., Vasqzez, N., Seubert, P., and Yednock, T. (2003) Epitope and isotype specificities of antibodies to β-amyloid peptide for protection against Alzheimer’s disease-like neuropathology. Proc. Natl. Acad. Sci. U.S.A. 100, 2023-2028.

928 Bioconjugate Chem., Vol. 16, No. 4, 2005 (16) Bard, F., Cannon, C., Barbour, R., Burke, R.-L., Games, D., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Lieberburg, I., Motter R., Nguyen, M., Soriano, F., Vasquez, N., Weiss, K., Welch, B., Seubert, P., Schenk, D., and Yednock, T. (2000) Peripherally administered antibodies against amyloid β-peptide enter the central nervous system and reduce pathology in a model of Alzheimer disease. Nature Med. 6, 916-920. (17) DeMattos, R. B., Bales, R. K., Cummins, D. J., Dodart, J. K., Paul, S. M., and Holzman, D. M. (2001) Peripheral antiAβ antibody alters CNS and plasma Aβ clearance and decreases brain Aβ burden in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A. 98, 8850-8855. (18) Purcell, A. W., Zeng, W., Mifsud, N. A., Ely, L. K., MacDonald, W. A., and Jackson, D. C. (2003) Dissecting the role of peptides in the immune response: theory, practice and the application to vaccine design. J. Pept. Sci. 9, 255-281. (19) Hudecz, F. (2001) Manipulation of epitope function by modification of peptide structure: a minireview. Biologicals 29, 197-207. (20) Wilkinson, K. A., Vordermeier, M. H., Kajtar, J., Jurcevic, S., Wilkinson, R., Ivanyi, J., and Hudecz, F. (1997) Modulation of peptide specific T cell responses by non-native flanking regions. Mol. Immunol. 34, 1237-1246. (21) Schutze, M. P., Leclerc, C., Jolivet, M., Audibert, F., and Chedid, L. (1985) Carrier-induced epitopic suppression, a major issue for future synthetic vaccines. J. Immunol. 135, 2319-2322. (22) Sakata, S., and Atassi, Z. (1981) Immune recognition of serum albumin- -XIII. Autoreactivity with rabbit serum albumin of rabbit antibodies against bovine or human serum albumins and autoimmune recognition of rabbit serum albumin. Mol. Immunol. 18, 961-967. (23) Herzenberg, L. A., Tokuhisa, T., Herzenberg, L. A. (1980) Carrier-priming leads to hapten-specific suppression. Nature 285, 664-667. (24) Hudecz, F., Votavova, H., Gaa´l, D., Sponar, J., Kajta´r, J., Blaha, K., and Szekerke, M. (1985) Branched polypeptides with poly(L-lysine) backbone: synthesis conformation and immunomodulation. Polymeric Materials in Medication (Gebelein, Ch. G.; Carraher, Ch. E.) pp 263-289, Plenum Press, New York.

Manea et al. (25) Tam, J. P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413. (26) Trudelle, Y., Bracks, A., Delmas, A., Pedoussant, S., and Rivaille, P. (1987) Synthesis of new carrier for immunization: polytuftsin. Int. J. Pept. Protein Res. 30, 54-56. (27) Tsikaris, V., Sakarellos, C., Cung, M. T., Marraud, M., and Sakarellos-Daitiotis, M. (1996) Concept and design of a new class of sequential oligopeptide carriers (SOC) for covalent attachment of multiple antigenic peptides. Biopolymers 38, 291-293. (28) Manea, M., Mezo˜, G., Hudecz, F., and Przybylski, M. (2004) Conjugates containing a b-amyloid plaque specific epitope as lead structure for vaccines against Alzheimer’s disease. Biopolymers 76, 503-511. (29) Mezo˜, G., Kala´szi, A., Reme´nyi, J., Majer, Zs., Hilbert, A Ä ., La´ng, O., Ko˜hidai, L., Barna, K., Gaa´l, and D., Hudecz, F. (2004) Synthesis, conformation and immunoreactivity of new carrier molecules based on repeated tuftsin-like sequence. Biopolymers 73, 645-656. (30) Fridkin, M., and Najjar, V. A. (1989) Tuftsin: its chemistry, biology, and clinical potential. Crit. Rev. Biochem. Mol. Biol. 24, 1-40. (31) Siemion IZ, and Kluczyk A. (1999) Tuftsin: on the 30-year anniversary of Victor Najjar’s discovery. Peptides 20, 645674. (32) Damoc, E., Youhnovski, N., Crettaz, D., Tissot, J. D., and Przybylski, M. (2003) High-resolution proteome analysis of cryoglobulins using Fourier transform-ion cyclotron resonance mass spectrometry. Proteomics 3, 1425-1433. (33) Rossier, J. S., Youhnovski, N., Lion, N., Damoc, E., Reymond, F., Girault, H. H., and Przybylski, M. (2003) Thinchip microspray system for high-performance Fourier transform ion-cyclotron resonance mass spectrometry of biopolymers. Angew. Chem., Int. Ed. 42, 53-58. (34) Buck, M. (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q. Rev. Biophys. 31, 297-355.

BC0500037