Self-Assembled Peptides Exposing Epitopes Recognizable by Human

and nonimmunogenic and have been frequently used as anticancer drug .... The solution was cooled to 0 °C. A solution of methacryl- oyl chloride (1.1 ...
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Bioconjugate Chem. 2000, 11, 363−371

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Self-Assembled Peptides Exposing Epitopes Recognizable by Human Lymphoma Cells Aijun Tang,† Chun Wang,‡ Russell Stewart,‡ and Jindrˇich Kopecˇek*,†,‡ Departments of Pharmaceutics and Pharmaceutical Chemistry/CCCD and of Bioengineering, University of Utah, Salt Lake City, Utah 84112. Received October 11, 1999; Revised Manuscript Received January 19, 2000

A bifunctional N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer containing nitrilotriacetic acid (NTA) and benzophenone (BP) groups was synthesized by free-radical copolymerization of HPMA, 2-methacrylamidobutyl nitrilotriacetic acid (MABNTA), and 4-methacrylamido benzophenone (MABP) using 2,2′-azobisisobutyronitrile (AIBN) as initiator. A His-tagged coiled coil stem loop peptide containing a tridecapeptide (TDP) epitope (GFLGEDPGFFNVE) in the loop region (CCSL-TDP) was designed and synthesized genetically by expressing an artificial gene in Escherichia coli BL21 (DE3). The peptide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), size-exclusion chromatography (SEC), and circular dichroism (CD) spectroscopy. Surfaces containing self-assembled CCSL-TDP peptide were prepared by first covalently grafting poly(HPMA-co-MABNTA-co-MABP) onto polystyrene (PS) surface by UV irradiation, then charging the surface with nickel through NTA groups, and finally attaching the CCSL-TDP peptide through Ni-histidine chelation. The modified PS surfaces with and without self-assembled CCSLTDP peptide were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Cell attachment studies with human Burkitt’s lymphoma Raji B cells showed that the cells selectively bound to the self-assembled CCSL-TDP peptide surfaces, but not to the surfaces of PS, PS with grafted copolymer, and PS with grafted copolymer and self-assembled coiled coil peptide with similar structure but without the epitope. This indicates that the cell attachment was mediated by the CCSL-TDP peptide, most probably by the TDP epitope region. The CCSL peptide self-assembly presented here may represent a feasible model of exposing epitopes for biorecognition studies.

INTRODUCTION

In recent years, it has been well accepted that the use of polymeric drug delivery systems is an efficient approach for improving cancer chemotherapy. The covalent binding of anticancer drugs to water soluble polymer carriers can enhance the tumor accumulation and specificity of drug action by the enhanced permeation and retention (EPR) effect (1, 2). N-(2-Hydroxypropyl)methacrylamide (HPMA)1 copolymers are water soluble and nonimmunogenic and have been frequently used as anticancer drug carriers (reviewed in ref 1). Clinical data demonstrated that the maximum tolerated dose of HPMA * To whom correspondence should be addressed. Phone: (801) 581-4532. Fax: (801) 581-3674. E-mail: Jindrich.Kopecek@ m.cc.utah.edu. † Department of Pharmaceutics and Pharmaceutical Chemistry/ CCCD. ‡ Department of Bioengineering. 1 Abbreviations: AIBN, 2,2′-azobisisobutyronitrile; BP, benzophenone; BSA, bovine serum albumin; CCSL, coiled coil stem loop; CD, circular dichroism; DOX, doxorubicin (adriamycin); HBS, Hepes-buffered saline; HPMA, N-(2-hydroxypropyl)methacrylamide; IMAC, immobilized metal affinity chromatography; MABNTA, 2-methacrylamidobutyl nitrilotriacetic acid; MABP, 4-methacrylamido benzophenone; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NP, nonapeptide, EDPGFFNVE; NTA, nitrilotriacetic acid; PS, polystyrene; SEC, size-exclusion chromatography; SSIMS, static secondary ion mass spectrometry; TDP, tridecapeptide, GFLGEDPGFFNVE; TOF-SIMS, time-of-flight secondary ion mass spectrometry; XPS, X-ray photoelectron spectroscopy.

copolymer-doxorubicin (DOX) conjugates was 3-4 times higher than that of free DOX (3). In addition, HPMA copolymer-bound DOX has the potential to overcome the P-glycoprotein type of multidrug resistance (4), inhibit DNA repair, replication, and biosynthesis and most significantly, to induce apoptosis and lipid peroxidation when compared to free DOX (5, 6). To further increase the specificity and therapeutic efficacy of the HPMAdrug conjugates, our group and collaborators have investigated various compounds such as carbohydrates (7) and antibodies (8, 9) for use as targeting moieties to achieve biorecognizability of the conjugates. In addition to antibodies and carbohydrates, receptor-binding epitopes are another type of targeting moiety we are trying to utilize in targetable HPMA copolymer-based drug delivery systems. Polyvalent interactions are important in biological systems. Such interactions can be collectively much stronger than corresponding monovalent interactions (10). Similar polyvalency effects were found in synthetic systems as well. The biorecognition of HPMA copolymers containing side chains terminated in N-acylated galactosamine by the asialoglycoprotein receptor was reported to depend on bound ligand amount (7). Polyvalent interactions (cooperative binding) were also observed in the inhibition of virus-mediated agglutination of erythrocytes by polyacrylamides containing pendent sialoside groups (11), the recognition of HPMA copolymers containing fucosylamine residues by lectin (12), and mouse lymphoma binding of short peptides fused with coiled coil assembly domains into a multivalent binding molecule

10.1021/bc990133d CCC: $19.00 © 2000 American Chemical Society Published on Web 04/04/2000

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(13). A main advantage of using small epitopes as targeting moieties is the possibility of incorporating multiple epitope molecules into one macromolecule, which contributes to cooperative interactions. In addition, the use of relatively small epitopes may facilitate the transcompartmental transport compared to that of the large antibody-containing conjugates. A nonapeptide sequence (NP) EDPGFFNVE is located close to the N terminus of the Epstein-Barr virus (EBV) main envelope protein gp350/220. This NP epitope was demonstrated to be involved in the EBV attachment to CD21 receptor on human B lymphocytes (14). CD21/EBV receptor is also expressed on T cells (15, 16). It was found that the amount of CD21 receptor on Raji lymphoblastoid cells is 10-20-fold higher than on normal B cells (17). It also appears that EBV receptor is overexpressed on malignant T cells (such as Molt-4) than on normal T cells (18). Our group and collaborators investigated the possibility of using the NP epitope as targeting moiety in HPMA copolymer-DOX conjugates for potential treatment of human lymphoma (19-21). It was found that the apparent binding constants of the NP epitopecontaining conjugates to T cells were about two times higher compared to B cells. This modified biorecognition may be explained by the structure of the spacers, i.e, their composition and length. More importantly, the HPMA copolymer(GFLG)-NP-DOX conjugates showed a significantly increased cytotoxicity to the experimental lymphoma cell line when compared to nontargeted conjugates, and the polyvalency effect was also observed. In addition, the targeted conjugate was not toxic to peripheral blood lymphocytes (PBL) at a concentration lethal to T lymphoblastic cells (19). Studies of the in vivo targeted delivery of HPMA copolymer-DOX conjugates to HSB-2 human leukemia T cells on a scid mouse model showed that the targeted conjugates selectively killed T cells while the nontargeted conjugate did not (20). Similar data were obtained on CCRF-CEM human T-cell leukemia model in scid mice (21). It appears that small synthetic epitopes represent a new category of targeting moieties for polymer-based targetable drug delivery systems. A coiled coil is a slightly left-handed super-helix formed from two or more right-handed R-helices wound around each other (22). The ability of coiled coils to form stable, specific oligomers has been frequently used to engineer novel proteins. Coiled coil structures taken from native proteins or designed de novo have been used to construct highly avid antibodies (13, 23), epitope-displaying scaffolds (24-26), and other novel chimeric proteins with biological and therapeutic importance (27-30). Myszka and Chaiken have applied the basic principle of forming stable coiled coils to design a coiled coil stem loop (CCSL) peptide that has an antiparallel intramolecular coiled coil structure. Their study showed that the RGD sequence incorporated in the loop region is recognizable by the fibrinogen receptor GPIIbIIIa (24). Our long-term goal is to study the structure-biorecognition relationship of motifs including the NP epitope so as to select the optimal oligopeptide(s) for use as targeting moieties in HPMA copolymer-anticancer drug conjugates for the treatment of human lymphoma. To this end, we designed, on one hand, a His-tagged coiled coil stem loop peptide containing a tridecapeptide (TDP) epitope (GFLGEDPGFFNVE) in the loop region (CCSLTDP) and synthesized the peptide by genetic engineering methods. On the other hand, we designed and prepared a complexed Ni2+-containing surface by covalently grafting onto polystyrene substrate a HPMA copolymer con-

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taining nitrilotriacetic acid (NTA) group through which the Ni2+ was charged. Upon applying to this surface, the CCSL-TDP peptide would, theoretically, self-assemble through His-Ni chelation in a well-organized manner to expose the receptor-binding epitope moieties on the surface. The biorecognizability of the TDP epitope by human lymphoma cells was studied using Raji B cell line. EXPERIMENTAL PROCEDURES

Materials. Chemicals. N-Z-L-lysine, bromoacetic acid, 5% Pd/C (Fluka, Milwaukee, WI), and 4-aminobenzophenone (Aldrich, Milwaukee, WI) were used without purification. Methacryloyl chloride (Aldrich, Milwaukee, WI) was purified by distillation, and 2,2′-azobisisobutyronitrile (AIBN) (Alfa, Danvers, MA) was purified by recrystallization from ethanol. HPMA was prepared as previously described (31). Enzymes. EcoRI, NdeI, ApaI, T4 polynucleotide kinase, and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA). Cell Lines. Raji B cell line (Burkitt’s lymphoma, human) was obtained from the American Type Culture Collection (ATCC, Rockville, Maryland). Raji cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). Cells were grown at 37 °C in a humidified atmosphere of 5% (v/v) CO2 in air. Falcon 1008 polystyrene dishes (35 × 10 mm, Becton Dickinson, Oxnard, CA) were used as the substrates for the surface modifications and the containers for cellbinding studies. Synthesis of Poly(HPMA-co-MABNTA-co-MABP). Synthesis of 2-Methacrylamidobutyl Nitrilotriacetic Acid (MABNTA). As described by Schmitt et al. (32), NR,NRbis[carboxymethyl]-N-[benzyloxycarbonyl]-L-lysine (NZ-NTA derivative) was prepared from bromoacetic acid and N-benzyloxycarbonyl-L-lysine, and this N-Z-NTA derivative was then hydrogenated using 5% Pd/C to obtain NR,NR-bis[carboxymethyl]-L-lysine (NTA derivative). This NTA derivative (2.62 g, 10 mmol) was dissolved in 15 mL of 2 N NaOH and a small amount of the polymerization inhibitor t-octylpyrocatechine was added. The solution was cooled to 0 °C. A solution of methacryloyl chloride (1.1 mL, 11.3 mmol) in 10 mL of methylene chloride was added dropwise to this solution while stirring, and with a slight delay, 10 mL of 1 N NaOH was added. The temperature was kept between -10 and 0 °C during the reaction. After addition, the reaction mixture was stirred at room temperature for an additional 2 h. The organic layer was removed, and the aqueous solution was washed with diethyl ether. The aqueous solution was acidified with 6 M HCl to pH 2, and the product was extracted with ethyl acetate (15 × 15 mL). The organic layers were combined and dried over anhydrous magnesium sulfate overnight. The drying agent was filtered off and the solvent was removed by rotary evaporation. The crude product was recrystallized from tetrahydrofuran and dried in vacuo: yield 70%; mp 105-107 °C; TLC Rf 0.78 in methanol/water (4/1). Anal. (C14H22N2O7) C, H, N. 1H NMR (DMSO-d6, 200 MHz): δ 1.3-1.6 [m, 6H, (CH2)3], δ 1.8 (s, 1H, CH3), δ 3.1 (m, 2H, CONH-CH2), δ 3.3 [t, 3H, CH2-CH(COOH)-], δ 3.5 [s, 4H, N(CH2COOH)2], δ 5.3 and 5.6 (s and s, 2H, CH2)), δ 7.6 (t, 1H, CONH), δ 12.4 (s, 3H, 3COOH). Synthesis of 4-Methacrylamido benzophenone (MABP). 4-Aminobenzophenone (ABP) (0.60 g, 3 mmol) was dissolved in 10 mL of methylene chloride and cooled to 0 °C. Na2CO3 (0.32 g, 3 mmol) and a small amount of

Self-Assembled Peptide Exposing Epitopes

Figure 1. (A) amino acid sequence of the CCSL-TDP peptide containing the tridecapeptide (TDP) epitope in the loop region. (B) cDNA sequence encoding the CCSL-TDP peptide.

polymerization inhibitor were added. A solution of methacryloyl chloride (0.29 mL, 3 mmol) in 5 mL of methylene chloride was added dropwise to the above mixture while stirring, and the reaction temperature was kept between -10 and 0 °C during the reaction. After addition was completed, the reaction mixture was stirred at room temperature for an additional 2 h. The inorganic salt formed was filtered off, and the filtrate was kept at 4 °C overnight. The small amount of solid formed was again filtered off and the filtrate was evaporated to remove most solvent. The residual oily liquid crystallized at 4 °C. The crude product was recrystallized from 2-propanol to obtain white crystals. Molecular weight calculated, 265.33; observed in electrospray ionization mass spectrometry, 265.31; mp 120-122 °C; TLC Rf 0.38 in ethyl acetate/hexane (4/6). 1H NMR (DMSO-d6): δ 1.98 (s, 3H, CH3), δ 5.58 and 5.85 (s and s, 2H, CH2)), δ 7.50-7.90 (m, 9H, C6H5-CO-C6H4-), δ 10.15 (s, 1H, -CONH). Copolymerization of HPMA, MABNTA, and MABP. Poly(HPMA-co-MABNTA-co-MABP) was prepared by radical precipitation polymerization in acetone as previously described (33). Briefly, HPMA (75 mol %), MABNTA (20 mol %), and MABP (5 mol %) were dissolved in acetone at a total monomer concentration of 12.5 wt %, and the polymerization initiator AIBN was added at a concentration of 0.6 wt %. The copolymerization was allowed to proceed at 50 °C for 24 h, and the copolymer precipitated out as white solid. The copolymer was purified by dissolving it in methanol and reprecipitating it into acetone. The content of NTA group determined by titration was 1.02 µmol/mg copolymer. Design and Expression of a Coiled Coil Stem Loop (CCSL) Peptide CCSL-TDP. The peptide (Figure 1A) was designed based on the structure proposed by Myszka et al. (24). The double-stranded cDNA (Figure 1B), which encods the CCSL-TDP peptide, was chemically synthesized as six single-stranded oligonucleotides of 64-75 nucleotides long. The complementary strands were annealed to form three duplexes (1-3). Duplex 2 was phosphorylated using T4 polynucleotide kinase. A modified expression vector pRSETB (34) was digested

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with NdeI and EcoRI and purified, and to the two restriction enzyme sites the three duplexes were ligated. The construct was identified by digestion with ApaI, and the correctness was verified by direct sequencing. Escherichia coli BL21 (DE3) competent cells (Novagen, Madison, WI) were transformed with the recombinant vector, and the target peptide expression was induced by isopropyl β-thiogalactoside (IPTG). The peptide was purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA resin (Qiagen, Santa Clarita, CA) and analyzed by SDS-PAGE for purity. Characterization of the CCSL-TDP Peptide. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). MALDI-TOF MS analysis was performed on a MALDI-TOF mass spectrometer (Voyager-DE STR Biospectrometry Workstation, PerSpective Biosystems, Inc., Framingham, MA). Circular Dichroism (CD) Spectroscopy. CD spectra were measured in PBS (pH 7.4) or PBS containing 50% trifluoroethanol (TFE) at 4 °C on an AVIV 62DS CD spectrometer equipped with a thermoelectric temperature control system. The wavelength scans were performed using a 0.1-cm cell, a 1-nm bandwidth, and a 5-s averaging time and were repeated three times, averaged, and corrected for buffer contribution. For temperatureinduced denaturation-renaturation experiments, the CD signal at 222 nm was monitored over a temperature range of -4 to 70 °C. The temperature was varied in 2 °C increments, and the sample was equilibrated for 5 min before the 60-s data point averaging. Size-Exclusion Chromatography (SEC). The CCSLTDP peptide was analyzed on a Pharmacia FPLC system, using an HR10/30 Superdex 75 column and PBS (pH 7.4) as elution buffer. The peptide was dissolved in PBS (pH 7.4) buffer at a concentration of 0.17 and 0.14 mg/mL (or 23.4 and 18.8 µM), and the solutions were filtered through a 0.2 µm filter. The elution of the protein was monitored by UV absorbance at 280 nm and the refractive index. Construction of a Self-Assembled CCSL-TDP Surface. A poly(HPMA-co-MABNTA-co-MABP) solution in PBS (1 mL of 0.1 mg/mL) was added to a Falcon 1008 polystyrene dish and it was exposed to 366 nm UV irradiation (1.9 mW/cm2) for 1 h. The dish was washed with water and ethanol and again with water to remove unbound copolymer. The dish with grafted copolymer was treated with 1 mM NaOH to neutralize the carboxylic groups followed by 40 mM NiSO4 to charge the nickel. After washing, a CCSL-TDP solution (1 mL of 50 µg/mL) in HBS (pH 7.4) was added to the dish and incubation was allowed to proceed for 1 h at room temperature. Characterization of the Surfaces. Static Contact Angles. Static contact angle measurements were performed on polystyrene (PS) before and after copolymer grafting with an NRL C. A. Goniometer (Mountain Lakes, NJ) equipped with a video camera imaging system. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed on a VG Scientific 220i surface analysis system (East Grinstead, U.K.). The XPS spectra and surface composition were determined for PS, PS with grafted copolymer (PS-cop), PS with grafted copolymer and complexed Ni (PS-cop-Ni), and PS with grafted copolymer and self-assembled CCSL-TDP peptide (PScop-Ni-CCSLTDP). Time-of-Flight Secondary Ion Mass Spectrometry (TOFSIMS). Static SIMS analyses were performed on a CAMECA/IONTOF model TOF-SIMS IV system (Muenster, Germany) with a 3-pA, 25-keV Ga+ ion beam.

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Charge compensation was achieved with a low-energy flood gun. The signal acquisition time was 2 min for all samples. Cell-Binding Study. Cell-binding study was performed on PS dish without surface modification and on the dishes with modified surfaces, including PS with grafted copolymer, PS with grafted copolymer and selfassembled coiled coil peptide TagEK42 (34), and PS with grafted copolymer and self-assembled CCSL-TDP peptide. All dishes were incubated with HBS-2% BSA for 1 h at room temperature to block nonspecific interactions. Raji B lymphoblastoid cells (5 × 105 cells suspended in 1 mL of HBS-2% BSA) were added to the CCSL-TDPcoated dish and control dishes and incubated at 4 °C for 1 h. After washing five times with HBS, cells were fixed by 3% p-formaldehyde. Cell attachment was observed by microscopy using a Nikon ECLIPSE E800 Microscope (S&M Microscopes, Colorado Springs, CO) and an image/ analysis system Image-Pro Plus (Media Cybernetics, Silver Spring, MD). The effect of cell density in the suspension on the surface attachment of Raji cells was investigated by using three different cell densities, i.e., 2 × 105, 5 × 105, and 1 × 106 cells/mL. Cell attachment experiments were also performed with Raji cells at the same density (5 × 105 cells/mL) on different surfaces with adsorbed peptide: PS with adsorbed peptide (PS-CCSLTDP), PS with grafted copolymer and adsorbed peptide (PS-cop-CCSLTDP), PS with grafted and NaOH neutralized copolymer and adsorbed peptide (PS-cop-Na-CCSLTDP), and PS with grafted copolymer and self-assembled CCSL-TDP peptide (PScop-Ni-CCSLTDP). RESULTS

Characterization of the CCSL-TDP peptide. The molecular weight of the CCSL-TDP peptide determined by MALDI-TOF MS was 7277.0, which agrees very well with the theoretical value of 7277.1. The secondary structure of the CCSL-TDP peptide was characterized by CD spectroscopy. The CD spectra (Figure 2A) indicated that the CCSL-TDP peptide possessed a certain degree of R-helical content and a fairly large amount of random coil structure at 4 °C in PBS buffer. Upon adding the helix-inducing solvent TFE, the ellipticity minimum at 222 nm increased about 2-fold, indicating that the R-helical content was twice of that seen in the absence of TFE. The thermal unfolding of the peptide suggested a largely cooperative structural transition (Figure 2B), yet the broad range of transition and the low midpoint transition temperature (Tm ≈ 20 °C) are indicative of the presence of loose structures and a relatively low overall stability. Two peptide concentrations were used in the SEC analysis. A single peptide peak was observed for both concentrations. Using a calibration curve based on elution times of four globular proteins (GCSF 18.8 kDa, carbonate anhydrase 29 kDa, ovalbumin 45 kDa, and albumin 66 kDa), the molecular mass was estimated as 13.1 kDa. Surface Characterization. The static water contact angles were 82.8 ( 0.4° for PS substrate and 50.4 ( 0.7° for PS with grafted copolymer, indicating a large increase in surface wettability upon modification. XPS measurements were performed on a control PS surface and on surfaces after subsequent modifications. The spectra are shown in Figure 3 (PS spectrum not shown). The presence of the N1s peak in the spectrum of the PS-cop clearly indicates the attachment of the

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Figure 2. (A) CD spectra of 28 µM CCSL-TDP peptide in PBS (pH 7.4) (open circles) and PBS containing 50% TFE (filled circles) at 4 °C. (B) Temperature dependence of the secondary structure of CCSL-TDP peptide: heating (filled circles), cooling (open circles).

Figure 3. XPS spectra of three surfaces: PS-cop (A), PS-copNi (B), and PS-cop-Ni-CCSLTDP (C).

copolymer to the surface since no N peak was observed for the PS control. The appearance of the Ni2p3 peak in the PS-cop-Ni implies that the Ni2+ was charged to the surface. SSIMS measurements were performed on three surfaces: PS, PS-cop-Ni, and PS-cop-Ni-CCSLTDP, and the spectra of them were compared. Shown in Figure 4A are the positive ion spectra of the PS-cop-Ni (panel I) and PS-cop-Ni-CCSLTDP (panel II). All characteristic peaks listed in the literature for polystyrene (35) were observed here for the PS surface (spectra not shown) with correct relative magnitudes. The presence of the peaks with

Self-Assembled Peptide Exposing Epitopes

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Figure 4. (A) SIMS spectra of two surfaces: PS-cop-Ni (panel I), and PS-cop-Ni-CCSLTDP (panel II). Peaks indicative of some ions from the copolymer are labeled with chemical formulas, and peaks indicative of some of the amino acids are labeled with one-letter abbreviations. (B) Column chart: the relative intensities of relevant peaks in the spectra from the two surfaces, PS-cop-Ni (filled column) and PS-cop-Ni-CCSLTDP (open column).

increased relative intensities in panel I, such as CH3+ (m/z 15), C3H5+ (m/z 41), and C4H7+ (m/z 55), further verified the attachment of copolymer. As is seen from the spectra shown in panel II, some peaks indicative of amino acid residues (36) are present with increased relative intensities. Those peaks include the peak at m/z 30 contributed by glycine, lysine, etc., the peak at m/z 44 contributed by alanine, serine, and glutamic acid, the peak at m/z 72 contributed by valine, and the peak at m/z 86 contributed by leucine, etc. To more clearly present the differences in relative intensities of the relevant peaks for modified PS with and without selfassembled CCSL-TDP peptide, a column chart was graphed in Figure 4B. Biorecognition of the Self-Assembled Peptide by Lymphoma Cells. Figure 5A shows the pictures of Raji cell attachment to different surfaces. Raji cells attached significantly to the self-assembled CCSL-TDP peptide surface, whereas no cell attachment occurred on the control surfaces of PS, PS-cop, and PS-cop-Ni-TagEK42, where TagEK42 is a coiled coil peptide with similar structure [RGHHHHHHGMASMTGGQQMGRDLYDDDDKDP(VSSLESK)6] to CCSL-TDP but without the epitope (34). Figure 5B shows the pictures of cell attachment with increasing cell densities. The higher the cell density in the incubation suspension, the greater the number of cells attached. Shown in Figure 5C are pictures of cell attachment on different surfaces with adsorbed CCSL-TDP peptide. Cells attached to the selfassembled CCSL-TDP peptide surface in a similar manner as before, but attached to the two PS surfaces modified with grafted copolymer and adsorbed peptide

in smaller numbers. There was almost no cell attachment to the PS with adsorbed peptide. DISCUSSION

The combination of receptor-binding epitopes with synthetic water-soluble polymer-drug conjugates to produce targetable polymeric anticancer drugs recognizable by a subset of lymphocytes may yield new potent macromolecular therapeutics effective in the treatment of human lymphomas. The targeting potential of the NP epitope has been evaluated (19-21); however, detailed studies are needed to find the optimal epitope-receptor pair for the best targeting. While phage display appears to be the most suitable technique for screening a large amount of peptide sequences (37), a simplified coiled coil stem loop (CCSL) scaffold may be a better choice as a conformational constraining matrix to present a known epitope sequence to immunocompetent cells for structurebiorecognition studies. The purpose of the current work was to demonstrate the feasibility of preparing a CCSL peptide that would assemble on the surface of a solid substrate and expose an epitope recognizable by lymphoma cells. The rationale of the epitope display and receptor recognition is schematically shown in Figure 6. Figure 6A shows the overall research design, and Figure 6B shows our current approach to assembling the peptide. The synthetic copolymer poly(HPMA-co-MABNTA-coMABP) was designed to contain two types of functional groups, nitrilotriacetic acid (NTA) group and benzophenone (BP) group. NTA is a tetradentate ligand that forms stable bonds with hexacoordinate Ni2+. In fact, Ni-

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Figure 5. (A) Raji cell attachment to different surfaces: (1) unmodified polystyrene (PS), (2) PS with grafted copolymer (PS-cop), (3) PS with grafted copolymer and self-assembled coiled coil peptide (TagEK42) without the epitope (PS-cop-Ni-TagEK42), (4) PS with grafted copolymer and self-assembled CCSL-TDP peptide displaying the tridecapeptide (TDP) epitope (PS-cop-Ni-CCSLTDP). (B) Raji cell attachment to surfaces containing self-assembled CCSL-TDP peptide at different cell densities: (1) 2 × 105, (2) 5 × 105, (3) 1 × 106 cells/mL. (C) Raji cell attachment to different surfaces containing adsorbed CCSL-TDP peptide: (1) PS with adsorbed peptide (PS-CCSLTDP), (2) PS with grafted copolymer and adsorbed peptide (PS-cop-CCSLTDP), (3) PS with grafted and NaOH neutralized copolymer and adsorbed peptide (PS-cop-Na-CCSLTDP), (4) PS with grafted copolymer and self-assembled CCSL-TDP peptide (PS-cop-Ni-CCSLTDP).

NTA resin has been commercially used in IMAC to purify proteins containing a hexahistidine tag. The BP group is photoreactive (38) and serve to covalently attach the copolymer onto a polystyrene substrate. The grafting of the copolymer was visualized by a significant increase in the water wetability of the surface (the static water contact angle changed from 83° before grafting to 50° after grafting). The NTA group, on the other hand, was used to chelate Ni2+. The Ni2+ here serves as the bridge between the synthetic polymer and the CCSL-TDP peptide. We hypothesized that if the peptide interacts specifically with the copolymer-modified surface, i.e., through Ni-histidine chelation, a well organized peptide monolayer should form which exposes the biorecognizable epitope. In our design, we chose a coiled coil peptide to present the receptor-binding epitope. The coiled coil structure is a commonly found folding motif in many native proteins.

The most prominent feature of the coiled coil is the heptad repeats [(abcdefg)x]. One heptad constitutes two turns, each covering three and a half residues. Among them, residues “a” and “d” are hydrophobic, while others are usually polar or ionic residues. The coiled coil structure is mainly stabilized by the hydrophobic interactions between helices. Interactions between the ionic residues “e” and “g” are important in specific association among helices. Even though they make only a relatively small contribution to the stabilization of the coiled coil structure, the ion pairs are thought to be important in dictating parallel or antiparallel packing (24-26, 39). Myszka and Chaiken (24) designed a 56-residue polypeptide which folded into a stable intramolecular antiparallel coiled coil, referred to as coiled coil stem loop (CCSL). Leucines and valines were used as hydrophobic residues, and glutamic acids and lysines as the ionic ones. Expanding their concept, we designed a 70-residue CCSL

Self-Assembled Peptide Exposing Epitopes

Figure 6. (A) Schematic representation of the general approach to studying the biorecognition of displayed epitopes by lymphoma cells; (B) Current approach to our research design.

peptide (CCSL-TDP) (Figure 1A). The modifications include a 3-heptad stem, a 21-residue loop containing a tridecapeptide (TDP) sequence, a 6-His tag at the Nterminus, and tyrosines at both ends of the stems. The His-tag served to provide means for peptide purification and chelation during self-assembling. Tyrosines were used as chromophores for determining peptide concentration and may be used for radiolabeling. We synthesized the CCSL-TDP peptide by genetic engineering methods. In the design of the cDNA, we incorporated restriction enzyme sites of NdeI and EcoRI at 5′ and 3′ ends of the coding strand, respectively, so that the DNA segment could be easily inserted into a plasmid vector digested with these two enzymes. Sites of restriction enzymes such as ApaI served to detect the insertion of the DNA segment. The KasI and ApaI sites corresponding to the

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hinge regions of the stem loop peptide were specially designed for easy modification of the loop region (Figure 1B). The CD analyses showed a low R-helical content and a low midpoint transition temperature of the peptide. This may be due to the insufficient ability of the three heptad repeats in each of the stem regions to form a stable coiled coil structure. The length of the coiled coil stem is a major factor in determining the overall stability of the peptide, and it was reported that the helicity of the peptides increased with increasing chain length in a cooperative manner (40). The relatively long and randomly structured loop is a significant contributing factor as well. Moreover, the presence of ionic residues (E and K) at the stem-loop junctions as well as the terminal tyrosines may impair the overall stability of the secondary structure. On the other hand, despite the lower content of ordered secondary structure, the thermal unfolding/folding process of this peptide was completely reversible as demonstrated by the overlapping temperature profiles (Figure 2B), which seems to suggest a high specificity for forming such a structure. In addition, the R-helical content in the presence of 50% TFE increased to about 44% as estimated from the observed ellipticity at 222 nm, which is close to the theoretical value of about 60% (42 helix-forming residues and 28 others). This indicates that the designed sequence of the CCSL-TDP stem region has the potential of forming an R-helical structure. Our approach to the establishment of the peptide selfassembly includes the photografting of a copolymer containing Ni2+-chelating groups and subsequent immobilization of the His-tagged CCSL-TDP peptide in the presence of Ni2+. This peptide immobilization method was similar to the approach of Sigal et al. (41) that NTA-NiHis chelation was employed. However, instead of using a self-assembled monolayer (SAM) formed from alkanethiols on gold surface, we used a polystyrene substrate and a bifunctional HPMA copolymer to constitute the intermediate layer. The generation of the copolymer layer was based on the photoactivity of the benzophenone group. This surface grafting can be conveniently performed on PS Petri dishes, which can be used as such for subsequent modifications and biorecognition studies. The HPMA copolymer chains attached to the substrate surface here may be representative of the environment seen by the target cells during conjugate-cell biorecognition with HPMA copolymer conjugates. The use of a coiled coil scaffold for presenting the biorecognition sequence may be beneficial in providing a constrained conformation mimicking protein domain for the relatively long TDP epitope. The extent of the conformational constraint on the epitope can be modified by varying the structure of the spacers between the stems and the epitope. This model system may be useful for the optimization of peptides that would be most suitable as targeting moieties in HPMA copolymer conjugates. The surface containing a self-assembled CCSL-TDP peptide and control surfaces were analyzed by both X-ray photoelectron spectroscopy (XPS) and static secondary ion mass spectrometry (SSIMS). XPS and SSIMS are the dominant techniques among others for the surface analysis of real-world polymer systems because they can probe surface chemistry with a high degree of discrimination. They are also highly complementary and are increasingly used together in surface analysis (42). XPS is a surface sensitive technique that provides a quantitative elemental analysis. For each surface, the atomic percentage (at. %) for each element was calculated

370 Bioconjugate Chem., Vol. 11, No. 3, 2000

Tang et al.

Table 1. Comparison of the Atomic Ratios of Selected Elements on Different Surfaces in XPS Analyses surfaces

N/C

π*/C

PS-cop PS-cop-Ni PS-cop-Ni-CCSLTDP

0.0235 0.0274 0.0739

0.0358 0.0368 0.0189

from the integrated areas of corresponding peaks after correcting for the different photoelectric cross sections of the orbitals. While the absolute at. % may vary among different measurements for the same sample due to experimental variations, the atomic ratios are always comparable among different samples. Table 1 lists the N/C and π*/C (π* is attributed to the benzene shake-up satellite) ratios of the three surfaces. The N/C and π*/C ratios are similar for PS-cop and PS-cop-Ni, which may indicate that the surface copolymer layer is quite uniform in thickness. If this is true, the significant increase in N/C and decrease in π*/C ratios can be explained by attachment of CCSL-TDP peptide. SSIMS is used to probe a material’s chemical structure through the mass spectral analysis of its molecular fragments. It is more surface sensitive (∼20 Å sampling depth) and more chemically selective than XPS because it can provide detailed molecular information. Mantus et al. (36) used SSIMS to analyze proteins adsorbed to biomaterial surfaces and established a spectral interpretation protocol by examining homopolymers of 16 amino acids, which allows for the assignment of peaks unique to various amino acids. Figure 4A, panel II, and Figure 4B showed the presence of some amino acid peaks for the surface containing self-assembled CCSL-TDP peptide. These ions are dominantly immonium ions which have the generic structure of H2N+dCH-R. These data suggested the attachment of the peptide on the surface. In the cell attachment study, the Raji cells selectively bound to the surface containing self-assembled CCSLTDP peptide but not to control surfaces (Figure 5A). This indicates that there are no specific interactions between the cells and the polystyrene substrate, the copolymer, or a coiled coil peptide that does not contain the epitope. Therefore, the cell attachment was mediated by the CCSL-TDP peptide, most probably by the TDP epitope in the loop region. Not surprisingly, when the cell density was increased, more cells were available to interact with the exposed surface ligands, thus more cells attached to the self-assembled CCSL-TDP surface (Figure 5B). To test the role of the TDP loop region and the specificity of cell interaction with the CCSL-TDP peptide, a cell attachment study was also performed on different surfaces with adsorbed CCSL-TDP peptide (Figure 5C). In the absence of nickel, the CCSL-TDP peptide probably interacts with the copolymer surfaces mainly through nonspecific interactions rather than Ni-His chelation involving the His-tag. Since the peptide was not well oriented to expose the loop region on the surface, fewer cells attached. The absence of cells on the PS-CCSL surface may indicate that the nonspecific interaction between the peptide and polystyrene results in the TDP region being buried rather than being exposed on the surface. This is not unreasonable considering that the loop region is slightly more hydrophobic than the stem regions where the hydrophobic residues tend to be located in the interface of the R-helices. In summary, the current data seem to suggest that the CCSL peptide self-assembly on a solid substrate may represent a feasible model for displaying epitopes for biorecognition studies.

ACKNOWLEDGMENT

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