Presentation of Epitopes on Genetically Engineered Peptides and

Biomacromolecules 2002, 3, 421-431

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Presentation of Epitopes on Genetically Engineered Peptides and Selection of Lymphoma-Targeting Moieties Based on Epitope Biorecognition Aijun Tang† and Jindrˇich Kopecˇ ek*,†,‡ Department of Pharmaceutics and Pharmaceutical Chemistry and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112 Received September 7, 2001; Revised Manuscript Received March 11, 2002

A His-tagged coiled coil stem loop peptide with stable secondary structure was designed and biosynthesized. A series of oligopeptides related to the EBV envelope glycoprotein 350/220 N-terminal nonapeptide as potential CD21 receptor-binding epitopes were engineered into the loop region of the peptide scaffold. It was shown that these peptides had a stable R-helical coiled coil structure and assumed a monomeric form in PBS. Biorecognition of the epitopes was studied by immobilizing the epitope-containing peptides on complexed Ni2+-containing surfaces through His-Ni2+ chelation and incubating with purified soluble CD21 receptor or CD21+ cells. The results showed that the potential epitopes bound to CD21 and CD21+ cells at different affinities depending on oligopeptide structures. This approach allows for the evaluation of epitope biorecognizabilities and the selection of optimal oligopeptides among sequences for use as targeting moieties in the design of new lymphoma-targeting polymeric drug carriers. Introduction Water-soluble polymer-based drug carriers have been widely used for studying the delivery of anticancer drugs.1 One of the water-soluble polymer systems, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates, has been shown to provide improved solubility, prolonged circulation time, decreased nonspecific toxicity, and the potential to overcome multidrug resistance to hydrophobic, low molecular weight anticancer drugs.2 Various targeting moieties such as antibodies3,4 and carbohydrates5 have been incorporated in the polymer-drug conjugates to increase their specificity and efficacy. We are interested in using receptor-binding epitopes as biorecognition sites to mediate specific interactions of the conjugates with receptor-bearing cells. The main advantages of using small synthetic epitopes as targeting moieties include the following: possible multivalent interactions6 as a result of incorporating multiple epitope molecules to each macromolecule chain and easier transcompartmental transport due to the relative small size7 of the epitope-containing conjugates (compared to large antibody-containing conjugates). The CD21 receptor, also called type two complement receptor (CR2), is a 145 kDa glycoprotein consisting of 15 or 16 short consensus repeats (SCR) of 60-70 amino acids in the extracellular region, a transmembrane domain and an intracytoplasmic tail.8,9 CD21 is expressed on mature B cells,10 follicular dendritic cells,11 peripheral blood T lymphocytes,12 and thymocytes.13 It has been shown that the * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah. ‡ Department of Bioengineering, University of Utah.

CD21 receptor was overexpressed on malignant cells relative to normal cells.14,15 Epstein-Barr virus (EBV), a human herpesvirus, binds to the CD21 receptor on human B cells through the viral main envelope glycoprotein 350/220.16,17 A nonapeptide (NP) sequence EDPGFFNVE, located close to the N-terminus of gp350/220 (NH2‚‚‚T(11)IESLIHLTGE(21)DPGFFNVEIP(31)EFPFYPTC‚‚‚COOH),18 was identified as the epitope involved in EBV binding to CD21+ B cells.19 Therefore, CD21 may be exploited as a potential target for the delivery of anticancer drugs utilizing the NP epitope as a targeting moiety. Previous work in our laboratory studied the recognition of NP-containing HPMA copolymer conjugates by lymphocytes.20 It was shown that the binding affinity depended on the epitope content in the conjugates. In addition, the binding seemed to be affected by the length of the conjugation spacers, “GG” and “GFLG”. The varied biorecognition might be a result of difference in steric hindrance from the different spacer lengths, or due to differences in the actual functioning epitopes, “GG-NP” vs “GFLG-NP”. Here, we investigated the biorecognition of some NP-related sequences, such as NP with its flanking oligopeptides in native gp350/220, to find potential targeting moieties useful in polymer-drug conjugate systems. This will allow us to verify the concept of synthetic epitopemediated birecognition of polymer conjugates and to design new polymer carriers for delivery of anticancer drugs to human lymphoma cells. In a previous study, we developed a model system based on a coiled coil stem loop (CCSL) peptide self-assembled on a solid substrate for the presentation of an epitope incorporated in the loop region of the peptide.21,22 CD21+ Raji B cell binding experiments showed that CCSL peptide assembly on a solid substrate was a feasible model to display

10.1021/bm015606+ CCC: $22.00 © 2002 American Chemical Society Published on Web 04/12/2002

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Figure 1. Structure of peptide CCSL-II-A. (A) Amino acid sequence and coding strand of the cDNA. Important unique restriction enzyme sites are underlined and in bold. (B) Helical wheel of the stem region. One-letter abbreviations for amino acids are used. All “e” and “g” positions are occupied by “E” in “E-region”, and by “K” in “K-region”. Solid arrows indicate hydrophobic interactions in the coiled coil interface, and dashed arrows indicate electrostatic interactions between the two coiled coil strands. Table 1. Loop Sequences of CCSL-II Peptides

a

CCSL-II peptides

loopsa (design features)

A B C D E F G H K M N

GAGGGFLGEDPGFFNVEGGGP (NP + GFLG: GFLG-NP) GAGGEDPGFFNVEGGGP (NP only) GAGGGLTGEDPGFFNVEGGGP (NP + upstream tetrapeptide “GLTG”) GAGGEDPGFFNVEIPEFGGGP (NP + downstream tetrapeptide “IPEF”) GAGGGLTGEDPGFFNVEIPEFGGGP (NP + upstream and downstream tetrapeptides) GAGGEFGLDPGNFVEGFGGGP (scrambled “GFLG-NP”) GAGGGLTGEDPGGGGP (truncated NP) GAGGEDPGFFNVEMGWGGP (NP with a downstream M) GAGGMEDPGFFNVEGGWGP (NP with an upstream M) GAGGP (no NP) GAGGRGP (no NP)

NP, scrambled GFLG-NP, and truncated NP sequences are in bold. “M” residue as CNBr cleavable site in the loops of peptides M and N is underlined.

epitopes for biorecognition studies. However, this first peptide maintained a low R-helical content and an unstable secondary structure. Structural stability had to be achieved first, before the incorporation of NP-related epitopes into the peptide scaffold could be used to study the structurebiorecognition relationship of the presented epitopes. In this paper, we report the design, biosynthesis, and physicochemical characterization of a series of stable CCSL peptides containing NP-related epitopes in their loop regions and evaluation of the epitope recognition by the soluble CD21 receptor protein and CD21+ cells. Materials and Methods Design of Epitope-Containing Coiled Coil Stem Loop Peptides. A 122-residue peptide was designed (designated as CCSL-II-A, where “II” represents CCSL peptide series II to discriminate it from series I CCSL peptides containing a shorter stem) (Figure 1). This peptide contains an R-helixforming stem consisting of six and one-half heptad repeats in each of the two helical regions (from now on, the N-terminal helix will be referred to as the “E-region” and the C-terminal helix as the “K-region”) (Figure 1B), a C-terminal His-tag, and a loop containing the same trideca-

peptide sequence (“GFLG-NP”) used in previous work.21 A series of peptides containing the same stem and His-tag, but different loops, were then designed as shown in Table 1. Construction of Expression Vectors. The starting plasmid vector used was a kind gift from C. Wang (currently at MIT). Restriction enzymes (Nhe I, Sph I, EcoR I, Kas I, Apa I), mung bean nuclease, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA). Escherichia coli strain DH5R (Gibco BRL, Life Technologies, Grand Island, NY) was used for plasmid amplification. Oligonucleotides were synthesized by the University of Utah DNA/Peptide Facility (Salt Lake City, UT). The starting vector, containing a gene encoding part of the E-region of peptide CCSL-II-A, was digested with Nhe I and Sph I (Figure 2A). A synthetic gene (Figure 2B) encoding the remaining part of the E-region, the loop, and part of the K-region of the peptide was assembled from ten oligonucleotides and inserted into the Nhe I and Sph I sites of the vector. The resulting recombinant plasmid vector was then digested with Sph I and EcoR I, and into these sites a second gene (Figure 2B) assembled from four oligonucleotides was inserted encoding the remaining part of the K-region and the His-tag of the peptide. The second recombinant plasmid

Presentation of Epitopes

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Figure 2. Gene cloning of CCSL-II peptides: (A) cloning strategy; (B) artificial genes for major part of peptide A; (C) genes encoding the loops of peptides B-N (BamH I site underlined).

vector was further digested with Sph I. After removing the 3′-overhangs with mung bean nuclease, the blunt-ended linearized vector was self-ligated to give the final plasmid DNA for peptide A (Figure 2A). Recombinant plasmid DNAs encoding all other CCSL-II peptides were obtained by replacing the gene for the loop

of peptide A with genes for the loops of these peptides. Each gene was assembled from two oligonucleotides (Figure 2C) and inserted into the Kas I/Apa I sites of recombinant plasmid pRSETB-Kan-CCSL-II-A (Figure 2A). Protein Expression and Purification. E. coli BL21(DE3)pLysS competent cells (Novagen, Madison, WI) were

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transformed with the recombinant plasmids encoding CCSLII peptides and cultured in LB medium containing kanamycin (Fisher Biotech, Fair Lawn, NJ) and chloramphenicol (Sigma, St. Louis, MO) at 37 °C in an incubator shaker (Innova 4080, New Brunswick Scientific, Edison, NJ). When the cell density reached an OD value of about 1, protein expression was induced by adding isopropyl β-thiogalactoside (Sigma) to a final concentration of 0.4 mM. Incubation was continued overnight before cells were harvested. The CCSL-II peptides were purified by immobilized metal affinity chromatography under native conditions.23 Bacterial cells, resuspended in Tris buffer (20 mM Tris, 0.5 M NaCl, pH 6.9), were lysed by sonication on a sonic dismembrator (Fisher Scientific model 550, Pittsburgh, PA) and centrifuged. The supernatants were loaded onto columns packed with NiNTA agarose resin (Qiagen, Valencia, CA) equilibrated in Tris buffer. After washing, peptides were eluted with Tris buffer containing 250 mM imidazole. The eluted fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions containing the target peptides were combined, concentrated in an Amicon filter device (Amersham Pharmacia Biotech, Piscataway, NJ) using YM3 ultrafiltration membranes (Millipore Corp., Bedford, MA), desalted on PD-10 columns (Amersham Pharmacia Biotech), and lyophilized. Cyanogen Bromide (CNBr) Cleavage. Peptides H and K containing a methionine residue in the loop regions were cleaved by CNBr.24 Peptides were mixed with CNBr in 70% formic acid at a CNBr/methionine molar ratio of 100, and the reactions were stirred at room temperature for 24 h. After the addition of two volumes of DI water, the mixtures were lyophilized. Each cleavage was confirmed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (data not shown). Circular Dichroism (CD) Spectroscopy. CD measurements of the CCSL-II peptides were performed on an Aviv 62DS CD spectrometer (Lakewood, NJ) equipped with a thermoelectric temperature control system. Lyophilized peptides were dissolved in PBS (pH 7.4) and the molar concentrations were determined using Micro BCA protein assay kit (Pierce, Rockford, IL). Peptide solutions were scanned three times from 250 to 195 nm using a 0.1 cm optical path length quartz cuvette, 1 nm steps and 5-second averaging time. The averaged spectra were smoothed after subtracting the buffer background using the “Star 3.0 Stationary” data processing program. The effect of helix-inducing agent trifluoroethanol (TFE) on R-helical content was tested on peptide A. A stock solution in PBS was diluted 1:1 (v/v) into PBS and TFE to obtain solutions of equal molar concentrations of peptide A in PBS and PBS-50% TFE. The CD spectra were recorded as described above. Temperature-induced unfolding profiles of the CCSL-II peptides were measured at 222 nm from apparent temperatures of 25-100 °C (24-90 °C calibrated by a digital thermocouple) at 5 °C per step, 60 s averaging time, and 5 min equilibration time for each temperature. The reverse profiles were also recorded to test the reversibility of the unfolding processes using the same setups for the heating

Tang and Kopecˇ ek

experiments. The effect of denaturing agent on temperatureinduced unfolding was tested on peptide A by adding GuHCl to a final concentration of 0.5 M. To determine the effect of CNBr cleavage on the secondary structure of the peptides, CD spectra were recorded for intact and CNBr-cleaved peptide K (CCSL-II-K and CCSLII-K/CNBr) at concentrations from 5 to 80 µM. Concentrations outside this range were beyond the measuring capabilities of the instrument. Temperature-induced unfolding and the refolding processes were also recorded for peptides K and K/CNBr. Analytical Ultracentrifugation. Sedimentation equilibrium experiments were performed on a Beckman Optima XL-A analytical ultracentrifuge with peptide CCSL-II-K in PBS at three concentrations (77.5, 38.8, and 19.4 µM). The samples were centrifuged to equilibrium with a rotor speed of 35 000 rpm at 20 °C and their absorbances were recorded at 280 nm against buffer references. The collected data were processed and analyzed using the nonlinear regression program NONLIN.25 Preparation of HB5 Affinity Column. THB-5 hybridoma cells (ATCC, Rockville, MD) were initially cultured in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT) at 37 °C in a humidified incubator containing 5% CO2 in air. These were then gradually adapted to a serum-free medium, Hybridoma-SFM (Gibco BRL). Cell culture supernatant was collected, filtered sequentially through 0.45 µm and 0.2 µm filters, and loaded onto a protein G affinity column (Protein G Sepharose 4 Fast Flow, Amersham Pharmacia Biotech) equilibrated in binding buffer (10 mM NaH2PO4/Na2HPO4, 150 mM NaCl, 10 mM EDTA, pH 7.0). After the column was washed with binding buffer, HB5 was eluted in 0.5 M acetate buffer (pH 3), which was immediately neutralized with 1 M Tris (pH 9). An HB5 affinity column was prepared by immobilizing HB5 on a 1-mL Hitrap NHS-activated column (Amersham Pharmacia Biotech) as recommended by the manufacturer. HB5 in elution buffer was dialyzed against coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) and concentrated. After washing the 2-propanol protecting solvent from the NHS-activated column with 0.1 N HCl, HB5 solution was incubated in the column for 30 min at room temperature. The excess NHS groups were then deactivated by incubating with ethanolamine buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and acetate buffer (0.1 M acetate, 0.5 M NaCl, pH 4.0) sequentially. The coupling efficiency was 75%, as estimated by desalting the eluted coupling buffer after the reaction over a PD-10 column and measuring the UV absorbance at 280 nm. Purification of Soluble CD21 (sCD21). Raji B lymphoblastoid cells (human Burkitt’s lymphoma, ATCC), initially cultured in RPMI 1640 medium containing 10%FBS in cell culture flasks, were harvested and seeded to a Cellmax capillary bioreactor cartridge (Spectrum Laboratories, Laguna Hills, CA) and then gradually adapted to a 1% serum medium. Cell suspension was harvested from the extracapillary space every 2 days and supernatant collected. The supernatant was filtered sequentially through a paper filter,


Figure 3. Analysis of sCD21 by dot blotting (A) and SDS-PAGE on a 10-15% gradient gel (B).

a 1 µm glass fiber filter, and 0.45 and 0.2 µm plastic filters. The clear solution was loaded onto the HB5 affinity column equilibrated in PBS at a flow rate of 0.3 mL/min. After the column was washed with normal PBS, and then PBS containing 0.5 M NaCl, sCD21 was eluted with 0.1 M glycine-HCl (pH 2.5)26 containing protease inhibitors (protease inhibitor cocktail, Sigma). The eluted fractions were immediately neutralized with 1 M Tris (pH 9) and analyzed by dot blotting for HB5 reactivity (Figure 3A) using a modified Qiagen immunodetection protocol. Protein solutions were spotted on prewetted PVDF membrane (Bio-Rad, Hercules, CA) and allowed to dry. After being washed in TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.5), the membrane was incubated with TBS-3%BSA to block nonspecific binding. The membrane was washed in TBSTween-Triton buffer (TBS containing 0.05% (v/v) Tween 20 and 0.2% (v/v) Triton X-100) and then in TBS. It was then incubated with 1 µg/mL HB5 in TBS-3%BSA, and after additional washings, incubated with antimouse IgG (whole molecule) alkaline phosphatase conjugate (Sigma) diluted 1:10 000 in TBS-3%BSA. After final washings with TBSTween-Triton buffer, the membrane was stained with a staining solution (BCIP/NBT tablet (Sigma) dissolved in DI water) and the reaction stopped by rinsing in water. The identity of the protein was verified by SDS-PAGE analysis (Figure 3B). Fractions containing purified sCD21 were dialyzed against PBS before use. ELISA. CCSL-II peptide solutions in PBS (50 µL of 5 µM) were incubated in Ni-NTA HisSorb strip wells (Qiagen) at 4 °C overnight. After being washed with PBS, the CCSL-II peptide coated wells were incubated with gentle shaking with purified sCD21 (50 µL of 10 µg/mL in PBS) for 3 h at room temperature followed by incubation with HB5 (50 µL of 5 µg/mL in PBS-0.2%BSA) for 1 h. The wells were rinsed with TBS, and incubated with 50 µL of antimouse IgG (whole molecule) alkaline phosphatase conjugate in TBS-0.2%BSA at a 1:10 000 dilution for another 1 h. The wells were then washed three times with TBS, and 100 µL of BluePhos microwell phosphatase substrate (KPL, Gaithersburg, MD) was added to each well. Absorbance at 630 nm was measured on a microplate reader (Bio-Rad model 3550). Inhibition of sCD21 binding to CCSL-II peptides was performed by incubating sCD21 with a synthetic peptide CEDPGFFNVE (CNP) (University of Utah DNA/Peptide Facility) at 100 µM prior to incubation in the


CCSL-II peptide coated wells. To further test the dose dependence of the inhibition, various concentrations of CNP were used in the inhibition studies with peptide D. Cell Binding. Cell binding studies were performed using CD21+ Raji cells. The CCSL-II peptides were immobilized in Ni-NTA HisSorb strip wells as described above. The wells were then incubated with PBS-2%BSA at 37 °C for 1 h to block nonspecific interactions. Raji cells were harvested from RPMI 1640-10%FBS and washed once with DPBS (Sigma). The cells were resuspended in DPBS-0.5%BSA at a density of 1 × 106 cells/mL, and 1 × 105 cells (100 µL) were dispensed into each well and incubated at 37 °C in a cell culture incubator for 1 h. The cell suspensions were removed, and the wells were washed twice with PBS containing 0.1 mg/mL each of CaCl2 and MgCl2. The cells were fixed with 3% paraformaldehyde for 20 min at room temperature. Cell staining was performed as described by Chemicon (Temecula, CA) CytoMatrix cell adhesion assay protocol. The attached cells were stained with 100 µL of 0.2% crystal violet (Sigma) in 10% ethanol for 15 min at room temperature. After extra dye solutions were removed, the wells were gently washed four times with PBS. The absorbed dye was solubilized with 100 µL of a 50/50 mixture of 0.1 M NaH2PO4 (pH 4.5)/50% ethanol and the absorbance at 570 nm was measured on a Bio-Rad microplate reader. In parallel inhibition studies, the cells were preincubated with anti-CD21 mAbs HB5 and FE8, and a synthetic peptide CNP (10 µg/106 cells) before incubating in the CCSL-II peptide coated wells. Hybridoma cell culture supernatant containing FE8 was a kind gift from Dr. W. M. Prodinger (University of Innsbruck, Innsbruck, Austria). FE8 was purified by Protein G affinity chromatography as described above on a 1-mL HiTrap Protein G column (Amersham Pharmacia Biotech). Statistical Analysis. One-way analysis of variance was performed on peptide-sCD21 and peptide-Raji cell binding data using SigmaStat 2.0. The results showed that there was a statistically significant difference in the mean values (P < 0.001) for both sets of data. A Student-Newman-Keuls (SNK) test was then used for all pairwise multiple comparisons to identify the different peptide(s). “Dunnett’s method” was used for multiple comparisons vs a control group (peptide M) to test the differences of other peptides from peptide M. Results Biosynthesis of CCSL-II Peptides. All of the CCSL-II peptides have the same amino acid sequences in their stem regions, and contain different loops of NP-related epitopes. First, a recombinant DNA for peptide A was constructed by sequential insertions of synthetic genes. After the identity of the construct was confirmed by direct sequencing, the gene encoding the loop of peptide A was replaced by synthetic genes for the loops of all other peptides. All genes except for those encoding the loops of peptides M and N contain a unique restriction enzyme site of BamH I for convenient identification of the positive clones. In DNA constructs for peptides M and N, the original Kas I and Apa I sites were removed so that the positive clones could be easily recog-

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Figure 4. Analysis of protein expression and purification by SDS-PAGE on 20% gels. For each peptide, lane 1 is crude cell lysate, lane 2 is Ni-NTA affinity column flow-through, and lane 3 is eluted pure CCSL-II peptide. Table 2. Analysis of CCSL-II Peptides by MALDI-TOF MS CCSL-II peptides

theoretical Mw

measured Mw


12 355 11 980 12 309 12 467 12 795 12 355 11 671 12 298 12 298 10 831 10 987

12 357 11 999 12 308 12 468 12 796 12 357 11 666 12 297 12 303 10 833 10 996

nized by negative responses in digestions with the two enzymes. The correctness of all constructs was verified by DNA sequencing. These peptides were expressed in E. coli and only the soluble forms were purified on Ni-NTA affinity columns. SDS-PAGE analysis showed that they were expressed at moderate levels and appeared as single bands after affinity purification (Figure 4). The final yields of these peptides were about 3-5 mg per liter of culture media. Peptide identities were confirmed by MALDI-TOF MS (Table 2). Amino acid composition was analyzed for peptide A [residue (theoretical value, measured value)]: A (19, 18.7); D + N (4, 4.2); E (14, 14.6); F (3, 3); G (11, 11.3); H (6, 5.9); K (12, 11.3); L (15, 14.6); S (22, 19.4); V (12, 12); Y (1, 0.9). Secondary Structures of CCSL-II Peptides and Their Thermal Stability. Circular dichroism spectroscopy was used to analyze the secondary structures of the peptides and their temperature dependence. All peptides had a similar CD spectrum with two negative peaks at 222 and 208 nm and an ellipticity minima ratio of about 1 (Figure 5A with peptide A as an example). These are typical characteristics for R-helical coiled coil structures.27 Furthermore, the R-helical content did not change in the presence of R-helix-inducing agent TFE (Figure 5A), implying the formation of the helical structure of the peptide to its full capacity. The temperature-induced change in the secondary structures of the CCSL-II peptides was monitored at 222 nm. All peptides showed similar temperature profiles with no obvious unfolding, with over 70% of the helical structures retained at 90 °C (Figure 5B with peptide A as an example). After cooling to room temperature, nearly 100% of the loss of

Table 3. Summary of the Results from CD Analysis ellipticity ratios CCSL-II peptides

R-helicity (%)

θ222/θ208

θ90 °C/ θ24 °Ca

θ24 °C, cooling/ θ24 °C, heatingb


69.8 63.4 60.2 76.0 66.8 67.9 62.9 81.5 69.9 74.2 79.0

1.02 1.02 1.02 0.98 1.05 1.01 1.07 1.03 1.03 1.07 1.05

0.83 0.83 0.80 0.78 0.82 0.81 0.74 0.86 0.84 0.87 0.81

0.99 0.95 0.96 0.93 0.96 0.97 0.87 1.02 1.00 0.99 0.98

a This ratio describes the thermal stability of the peptide secondary structure. b This ratio describes the reversibility of the thermal unfolding.

secondary structures was recovered, indicating a complete reversibility of the thermal unfolding processes (Table 3). The only exception in this case was peptide G, which showed a slight hysteresis. Significant unfolding of the peptides was only seen with the presence of denaturing agent (Figure 5B). The effect of CNBr cleavage on peptide secondary structures was also analyzed by CD spectroscopy (Figure 6A). The R-helical content did not change with peptide concentrations in a range of 5-80 µM for both intact and CNBr cleaved peptide K (CCSL-II-K and CCSL-II-K/CNBr). However, peptide K/CNBr showed significant unfolding at high temperatures while peptide K showed a stable secondary structure similar to other peptides. Despite the apparent melting of peptide K/CNBr, the secondary structure was fully regained after cooling to room temperature (Figure 6B). Association of CCSL-II Peptides in Solution. Peptide K was used to illustrate the association state of CCSL-II peptides in solutions. The analytical ultracentrifugation data from three concentrations of peptide K fit well to an ideal single species model (Figure 7) with an apparent molecular mass of 13.3 kDa. This value is quite close to the theoretical molecular weight of 12.3 kDa, which is indicative of a predominantly monomeric form of the peptide in PBS. The raw data were also fitted with a monomer-dimer model. However, it did not result in a “better fit”. In addition, a



Figure 5. Secondary structure and thermal stability of peptide CCSL-II-A by CD spectroscopy. (A) CD spectra in PBS and PBS-50%TFE. (B) Temperature profiles without or with 0.5 M GuHCl.

Figure 6. Effect of CNBr cleavage on CCSL-II peptide secondary structure: (A) R-helical content as a function of concentration and (B) temperature profiles for CCSL-II-K and CCSL-II-K/CNBr.

Figure 7. Analysis of peptide CCSL-II-K (77.5 µM in PBS) by analytical ultracentrifugation at 20 °C, 35 000 rpm.

relatively large apparent association constant of 616 µM suggested the absence of significant aggregation or association.

Peptide-sCD21 Interactions by ELISA. In general, there are no dramatic differences in the relative binding affinities among the peptides in sCD21 binding experiments. Peptides D and F showed the highest sCD21 affinities relative to other peptides (In SNK test, P < 0.05 for peptides D and F vs all other peptides except for the pair D vs F). Scrambling the NP sequence (peptide F vs A) seemed to facilitate binding to sCD21. On the other hand, truncating the NP sequence (peptide G vs C), addition of a “GFLG” or “GLTG” sequence to the N-terminus of the NP (peptide A vs B or C vs B), and addition of tetrapeptides at both N and C termini (peptide E vs B) resulted in no statistically significant differences in relative CD21 binding affinities. However, complete removal of the NP sequence (peptides M and N) resulted in an apparent decrease in binding affinities (statistical analysis indicated P < 0.05 for all peptides (except N) vs peptide M, and peptides M and N were not significantly different). CNBr cleavage had little effect on binding affinities of peptides H and K to sCD21 (Figure 8A). The peptide-sCD21 interactions were inhibited by a synthetic peptide CNP (Figure 8A), and this inhibition seemed to be dose-dependent in experiments with peptide D (Figure 8B).

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Figure 8. Recognition of the epitopes presented on CCSL-II peptides by sCD21 receptor. (A) Binding of sCD21 or sCD21 preincubated with 100 µM synthetic peptide CNP (CEDPGFFNVE) to immobilized CCSL-II peptides. The dashed line represents the background absorbance for peptides M and N. Column heights represent the mean values, and error bars represent the standard deviations (n ) 4 for peptides A-G, and n ) 3 for all other peptides for sCD21 only; n ) 2 for sCD21 + CNP). Asterisks (/) next to columns D and F represent P < 0.05 when compared to other peptides. (B) Inhibition of CCSL-II-D-sCD21 interaction by CNP at different concentrations. Data points are depicted by means and error bars ((standard deviations) (n ) 2).

Figure 9. Recognition of the epitopes presented on CCSL-II peptides by CD21+ Raji cells alone (series 1: n ) 2 for peptides H & K/CNBr, and n ) 3 for all other peptides; asterisk (/) above column D represents P < 0.05 when compared to other peptides), or Raji cells preincubated with 10 µg/106 cells of anti-CD21 mAb HB5 (series 2: n ) 2), synthetic peptide CNP (series 3: n ) 2), and anti-CD21 mAb FE8 (series 4: n ) 2). Column heights represent the mean values, and error bars represent the standard deviations.

Peptide-Raji Cell Interactions. CD21+ Raji B cells were used to study peptide-cell interactions (Figure 9). Peptide D bound most avidly to CD21-bearing Raji cells (In SNK test, P < 0.05 for peptide D vs all other peptides). Peptide F, however, did not show a high apparent binding affinity to Raji cells relative to other peptides as in the above receptor binding studies. Similar to the observations from peptidesCD21 binding experiments, there were no statistically significant differences in Raji cell binding affinities among peptides A, B, C, and E. However, truncating the NP sequence (peptide G vs C) seemed to decrease the binding affinity in this case. Control peptide M showed the lowest cell binding affinity relative to other peptides (P < 0.05 for peptides M vs all other peptides except peptides G and N). CNBr cleavage did not result in any change in peptide K-Raji cell interactions; however, it did significantly decrease the binding of Raji cells to peptide H.

Potential inhibitors studied were anti-CD21 mAbs HB5 and FE8 as well as the synthetic peptide CNP. In general, mAb HB5 and the synthetic peptide CNP had no effect on peptide-Raji cell interactions. In contrast, mAb FE8 seemed to inhibit the binding of Raji cells to the peptides (Figure 9). Discussion The incorporation of small peptides in conformationally constrained scaffolds has been widely used previously to facilitate binding to target proteins.28 Different naturally occurring and de novo designed proteins and peptides29 with various constraining secondary structures30 have been used as scaffolds for engineering new binding sites on peptides/ proteins. A coiled coil stem loop (CCSL), an intramolecular antiparallel coiled coil, has been proposed as a potential



Figure 10. Schematic representations of (A) oriented immobilization of a His-tagged CCSL peptide on a solid substrate through His-Ni2+ chelation and (B) conversion of intramolecular coiled coil into intermolecular coiled coil upon CNBr cleavage of peptides H and K.

structure-simplified and conformationally constrained scaffold into which biorecognition sites can be inserted.31 On the basis of this concept, we designed His-tagged CCSL peptides for the presentation of epitopes incorporated in the loop regions of the peptides. Such CCSL peptides may serve three functions: (1) provide a certain degree of conformational constraint on the incorporated epitopes, (2) facilitate oriented peptide immobilization through His-Ni2+ chelation for favorable presentation of the loop regions (Figure 10A), and (3) extend the epitope sequences away from the surface for optimal accessibility. Our previous study21 demonstrated the feasibility of using coiled coil stem loop peptide assembly on a solid substrate as a model of displaying epitopes for biorecognition studies. However, the lack of stable secondary structure in our first peptide made it necessary to design a new CCSL peptide scaffold with a stable secondary structure. Design and Genetic Engineering of Stable CCSL Peptides for Epitope Presentation. To increase the probability of forming a stable coiled coil structure, our previously designed peptide CCSL-TDP21 was modified by expanding the length of the R-helix-forming stem from the original three to six and one-half heptad repeats. The half heptad structure consisting of “VAAL” at each of the stem-loop junctions served as hydrophobic “clamps” to secure the coiled coil conformation. A special design feature of the new scaffold was the incorporation of a pair of asparagines in the hydrophobic interface (Figure 1B). The polar interactions of the buried asparagine residues were intended to impart specificity to the formation of two-stranded coiled coils at the expense of stability and help the “in-register” arrangement of the coiled coil strands.32,33 The C-terminal His-tag was connected to the end of the K-region and was used as a handle for both peptide purification (by metal affinity chromatography) and immobilization through His-Ni2+ chelation. A series of NP-related epitopes were inserted into the newly designed peptide scaffold to obtain a group of CCSLII peptides (Table 1). Except for the N- and C-terminal spacers of the epitopes, “GAGG” and “GGGP”, where

restriction enzyme sites (Kas I and Apa I) were embedded in cDNAs, peptide A has the NP and an N-terminal “GFLG”, which was the spacer used previously for covalent attachment of NP to the HPMA copolymer backbone.20 Peptide B has the NP sequence only. Peptide C has the NP and a tetrapeptide sequence “GLTG” similar to the tetrapeptide “HLTG” upstream of the NP in native gp350/220, and D has the NP plus the tetrapeptide sequence “IPEF” downstream of the NP in native gp350/220, while E has the NP with both the upstream and downstream tetrapeptides.10 We replaced the original “H” residue in the NP upstream tetrapeptide in gp350/220 with a “G” in our design to avoid interference of the histidine with His-tag immobilization of the peptides. These peptides were designed to study the effect of NP-flanking sequences in native gp350/220 on its biorecognition. Peptide F has a scrambled “GFLG-NP” sequence, and G has a truncated NP sequence. These two peptides were designed to elucidate the importance of the NP amino acid sequence on biorecognition. Peptides H and K have a CNBr cleavable site, methionine, in their loop regions. CNBr cleavage (methionyl bond) released one end of the epitope, conferring it with greater conformational flexibility. It also resulted in a change of the coiled coil conformation from the original intramolecular to an intermolecular one (Figure 10B). A “W” residue was incorporated into both peptides H and K as a chromatic handle. In peptides M and N, the NP sequence was completely removed. The difference between the two peptides was that N had an additional positively charged residue, an arginine. Peptide N was designed to resolve solubility problems that might occur to peptide M due to its overall electroneutrality. All peptides, including peptide M, were expressed at a moderate level in E. coli. These CCSL-II peptides were shown to have a stable coiled coil structure. One feature of intramolecular coiled coil structure is the concentration independence of the R-helical content.34 This was indeed observed for our CCSLII peptides (Figure 6A for CCSL-II-K). However, similar concentration independence of R-helicity was found for a CNBr-cleaved peptide (K/CNBr) at micromolar concentra-

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tions. This observation was different from a literature finding showing that R-helicity dropped significantly with decreasing peptide concentrations for an intermolecular coiled coil of three and one-half heptad repeats.34 However, it is known that coiled coil stability depends on chain length.35 Coiled coils containing five heptad repeats were shown to form highly stable structures with dissociation constants in the nanomolar range.36 Therefore, it was not surprising that intermolecular coiled coils of six and one-half heptad repeats in our study possessed significant R-helical content at micromolar concentrations. Peptide K was used for analysis of peptide association in PBS due to its convenient chromatographic properties. Sedimentation equilibrium experiments indicated a monomeric peptide structure. The absence of association/aggregation favors single-point attachment and uniform distribution of peptide molecules on the solid substrate, as well as increasing accessibility of the epitope region. Biorecognition of NP-Related Epitopes in CCSL Peptides. Biorecognition of the epitopes by sCD21 and CD21+ Raji cells was studied. Soluble CD21 corresponds to the extracytoplasmic SCR domains of the CD21 receptor with an apparent molecular size of 135 kDa.37 It is known that SCRs 1-2 of CD21 contain the binding sites for both EBV envelope glycoprotein 350/220 and CD21’s natural ligand C3dg,38 an activation product of the third component of the complement system (C3). The NP sequence was originally identified as a CD21 receptor binding epitope19 from studies on the synthetic peptides corresponding to two regions in gp350/220 that share a high similarity in amino acid sequence with C3dg.16,17 One study, based on binding of synthetic peptide-bound microspheres to CD21+ Raji cells, showed that a C3d segment having similar amino acid sequence to the NP epitope played an important role in the C3d-CD21 interactions.39 However, a conflicting observation in a study using alanine-substituted variants of human C3 indicated that this previously proposed CD21 binding site on C3dg only played a minor role in mediating C3dg-CD21 interactions.40 The X-ray crystal structure of human C3d demonstrated that the CD21 binding site on C3d was probably located on a concave surface where predominantly acidic residues formed an extended pocket.41 Mutation studies further indicated that acidic residues located on the two opposite sides of the acidic pocket were important CD21-contacting sites in C3d, and the adjacent hydrophobic residues also made an important contribution.42 On the other hand, it has been shown that two peptide segments on SCRs 1-2 of CD21 containing basic residues constituted the binding sites for C3dg.43 These basic residues seemed to form an extensive positively charged surface that might be involved in C3dg binding.41 In addition, it was shown that the CD21-C3dg binding was ionic-strength-dependent, implying the contributions from charged residue interactions.44 The peptide-sCD21 binding experiments showed that two peptides, D and F, had higher receptor binding affinities relative to other peptides (P < 0.05). There is no statistically significant difference between the two although peptide D has a loop sequence of “NP-IPEF” while peptide F contains a scrambled “GFLG-NP” sequence. This observation seemed

Tang and Kopecˇ ek

to agree with literature findings41,42 suggesting that acidic as well as hydrophobic residues might be important factors in binding of protein/peptide ligands to the CD21 receptor. It was not clear why peptide E, with both the upstream and downstream NP-flanking tetrapeptides, did not show a comparable binding affinity to sCD21 as peptide D. One possible explanation is that a peptide sequence may require both the key residues and the appropriate linear and spatial arrangements of these residues for favorable interactions. It is also possible that some amino acid residues had modifying effects on ligand-receptor interactions. In summary, our data showed no pronounced differences in receptor binding affinities when the peptide sequence was varied. Since the epitope modifications were all based on a core sequence, the NP epitope, mild changes in receptor binding affinities are perhaps not surprising. In addition, the relative low affinity of the epitopes to CD21 receptor as well as the relative high background for control peptides (which may be associated with the assay method) probably reduced the “sensitivity” of this system to detect differences in relative CD21 binding affinities of the epitopes. The absence of effect due to CNBr cleavage on peptidesCD21 interactions seemed to indicate that conformational constraint at both termini of the peptides was not essential for peptide recognition. This may indicate that one-end attachment of peptide ligands to polymer drug carriers in future studies is not a factor affecting biorecognition of the resulting conjugates. Synthetic peptide CNP was originally used for conjugation to maleimide-activated lipids through the terminal cysteine residue (unpublished data). The dose-dependent inhibition of sCD21-peptide interactions by synthetic peptide CNP indicated that the NP was a CD21 ligand that bound to a similar site on sCD21 as other potential epitopes and inhibited the binding of sCD21 to these epitopes at high concentrations. The observation that CNBr cleavage had no effect on Raji cell-peptide K interactions but significantly decreased Raji cell binding to peptide H is quite interesting. A possible reason is disruption of the intermolecular coiled coil conformation, followed by removal of the epitope sequence attached to the non-His-tagged E-region from the surface. In contrast, the epitope sequence attached to the His-tagged K-region remained on the surface regardless of whether the intermolecular coiled coil structure was present (Figure 10B). It was not clear how the intermolecular structure could be disrupted in peptide H/CNBr. It might be related to low local concentrations of the peptide helical strands, binding of the epitope to cells, as well as the shear force applied during washing process in cell binding experiments. It was shown that anti-CD21 mAb HB5 binds to SCRs 3-4 of CD21,45 while mAb FE8 binds to SCRs 1-2 of CD21.46 Our observation that HB5 had little effect on Raji cell-CCSL-II peptide interactions while FE8 inhibited Raji cell binding to CCSL-II peptides verified the binding of NPrelated epitopes to SCRs 1-2 of CD21 receptor. The synthetic peptide CNP inhibited CCSL-II peptide-sCD21 interactions but failed to inhibit Raji cell binding. This observation is consistent with the literature findings19 that


one N-terminal peptide of gp350/220 efficiently blocked the binding of recombinant gp350/220 and C3dg to CD21+ B cells only when it was in BSA-conjugated multimeric forms. Conclusion A series of coiled coil stem loop peptides with different loops consisting of NP-related epitopes were designed and synthesized by genetic engineering methods. A thermally stable secondary structure for all peptides indicated that the resulting coiled coil stem loop peptides act as a scaffold useful for the incorporation and presentation of epitope sequences as long as 25 amino acid residues. The incorporation of epitope sequences in this peptide scaffold and oriented immobilization of the epitope-containing peptides on solid substrates provided a useful means of presenting the epitopes for evaluation of their biorecognizabilities and selection of optimal epitope sequences. The selected peptide sequences will be used as targeting moieties in HPMA copolymeranticancer drug conjugates. Biological activities of these conjugates will be evaluated to identify potential lymphomatargeting polymeric drugs. Acknowledgment. This work was supported by NIH Grant CA88047, and A.T. was supported in part by The University of Utah Graduate Research Fellowship. We thank Dr. L. Joss (University of Utah) for assistance in sedimentation equilibrium experiments, Dr. W. M. Prodinger (University of Innsbruck, Innsbruck, Austria) for providing cell culture supernatant containing FE8, and Dr. S. Kern (University of Utah) for help with statistical analysis. We are grateful to the following Kopecˇek group colleagues: J. Callahan for valuable discussions in hybridoma cell culture and use of the artificial capillary bioreactor system and Dr. C. Wang (currently at MIT) for his kind gift of the starting cloning vector. References and Notes (1) Putnam, D.; Kopecˇek, J. AdV. Polym. Sci. 1995, 122, 55. (2) Kopecˇek, J.; Kopecˇkova´, P.; Minko, T.; Lu, Z. Eur. J. Pharmaceutics Biopharm. 2000, 50, 61. (3) R ˇ ´ıhova´, B.; Kopecˇkova´, P.; Strohalm, J.; Rossmann, P.; Veˇtvicˇka, V.; Kopecˇek, J. Clin. Immunol. Immunopathol. 1988, 46, 100. (4) Omelyanenko, V.; Kopecˇkova´, P.; Gentry, C.; Shiah, J. G.; Kopecˇek, J. J. Drug. Targeting 1996, 3, 357. (5) Duncan, R.; Seymour, L. C.; Scarlett, L.; Lloyd, J. B.; Rejmanova´, P.; Kopecˇek, J. Biochim. Biophys. Acta 1986, 880, 62. (6) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 2754. (7) Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4607. (8) Moore, M. D.; Cooper, N. R.; Tack, B. F.; Nemerow, G. R. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9194. (9) Weis, J. J.; Toothaker, L. E.; Smith, J. A.; Weis, J. H.; Fearon, D. T. J. Exp. Med. 1988, 167, 1047. (10) Tedder, T. F.; Clement, L. T.; Cooper, M. D. J. Immunol. 1984, 133, 678.

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Presentation of Epitopes on Genetically Engineered Peptides and

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