Identification of CD21-Binding Peptides with Phage Display and

Bioconjugate Chem. 2006, 17, 514−523

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Identification of CD21-Binding Peptides with Phage Display and Investigation of Binding Properties of HPMA Copolymer-Peptide Conjugates Hui Ding,† Wolfgang M. Prodinger,‡ and Jindrˇich Kopecˇek*,†,§ Departments of Pharmaceutics and Pharmaceutical Chemistry, and Bioengineering, University of Utah, Salt Lake City, Utah 84112, and Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Innsbruck, Austria. Received November 3, 2005; Revised Manuscript Received January 19, 2006

Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCR1-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymerP1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.

INTRODUCTION Cancer therapy has entered a new era since the introduction of monoclonal antibodies (mAbs) for the treatment of various cancers (1-4). To date, nearly 10 monoclonal drugs have been approved by the United States Food and Drug Administration; examples include Rituxan (5) (Anti-CD20 mAb), Herceptin (6) (anti-Her2/neu mAb), and Bexxar (7) (131I labeled Anti-CD20 mAb). mAbs alone may affect antitumor activity through the mechanisms of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. mAbs have also been extensively used to specifically deliver cytotoxic agents to tumor sites, due to the highly specific interaction between mAbs and their corresponding tumor antigens (4). Compared with conventional chemotherapy, therapy with immunoconjugates (targeting mAbs conjugated with anticancer drugs) results in significantly enhanced tumor-killing activity with minimal side effects (8). An alternate approach is targeting with receptor-binding peptides (9). Because of their small molecular size, precise control of their chemical makeup and their chemical stability provides a variety of choices to construct sophisticated targeting arrangements, such as graft, starlike, and dendritic structures. Peptide targeting has become feasible due to the advent of combinatorial peptide library techniques (10, 11), such as phage display (12, 13). Phage display is a powerful technique for bioscreening: by engineering the DNA structure of phage, a † Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah. § Department of Bioengineering, University of Utah. ‡ Innsbruck Medical University. * To whom correspondence should be addressed. Phone: (801) 5814532. Fax: (801) 581-3674. E-mail: [email protected].

phage peptide library is constructed, so that millions of different peptides are displayed on the phage capsid and are accessible for interaction with target proteins. Peptides with desired properties can be selected and identified with appropriate screening methods. Phage display has been extensively used for epitope mapping (14), vaccine discovery (15), and ligand identification (16). There are also many successful examples using phage display to identify peptide ligands for cancer targeting (11, 17). Proteins uniquely expressed or overexpressed by cancer cells can provide suitable targets. Although library screening with purified proteins is the most straightforward method, when the target protein is known and has been isolated, successful screening has been reported using targets other than purified proteins, such as in vitro screening with cancer cells (18), tumor tissues (19, 20), and even directly applied to the entire cancer patient’s body (21). With phage display, peptides targeting the CD21 receptor, a surface marker for malignant B cell lymphoma phenotyping, may be selected (22). CD21 receptor (complement receptor 2, CR2) is a 145-kDa transmembrane glycoprotein that is expressed primarily on mature B lymphocytes, epithelial cells, thymocytes, and follicular dendritic cells (23). The extracellular region consists of 15-16 short consensus repeats (SCRs),1 of which SCR1 and SCR2 are responsible for the interaction with several natural ligands, such as C3d, C3dg, and Epstein Barr virus (EBV). The expression of CD21 on B and T lymphocytes is upregulated by infection with the lymphocytotropic virus EBV and HTLV (24, 25). CD21 receptor mediates internalization through endocytosis (26); therefore, it is regarded as a suitable target for the treatment of lymphoma with chemotherapy. Two different forms of CD21 receptors, soluble CD21 (sCD21) (27) and recombinant short CD21 (rsCR2.1-4) (28), were used in this work. sCD21 is a 135-kDa protein generated from shedding

10.1021/bc0503162 CCC: $33.50 © 2006 American Chemical Society Published on Web 03/01/2006

HPMA Copolymer Conjugated with CD21-Binding Peptide

and cleavage of the extracellular portion of CD21 receptor, while rsCR2.1-4 is a recombinant short CD21 receptor containing SCR1-4. Both sCD21 and rsCR2.1-4 are fully functional in interaction with ligands that specifically bind to SCR1-4. Our goal is to select high-affinity peptide ligands of CD21 receptor, determine their biorecognizability, and use them as targeting moieties in a potential anticancer drug delivery system. A well-characterized polymer for delivery of cancer therapeutics is the water-soluble synthetic polymer, poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA). The advantages of using HPMA copolymer as a drug carrier include (29): improved physical properties of polymer-drug conjugates such as enhanced drug solubility, elongated blood circulation time, passive accumulation in solid tumors, and the potential to overcome efflux-pump related multidrug resistance (MDR) (30, 31). To enhance targeting efficiency, it is possible to incorporate several peptide ligands into one HPMA copolymer macromolecule (32), thus increasing the apparent binding affinity through multivalent interactions (33). Targeting peptides have been used previously to direct HPMA copolymers to tumor sites. A nonapeptide (EDPGFFNVE) (34), a portion of EBV virus envelope glycoprotein gp350/220, assumed to be responsible for the binding of EBV virus to CD21 receptor (34), has been incorporated into HPMA copolymer. The cytotoxicity of peptide-targeted HPMA copolymer-doxorubicin conjugates was dependent on the amount of peptide per macromolecule (32, 35, 36). In vitro studies by Nan et al. revealed that cellular uptake of HPMA copolymer increased significantly after it was modified with the peptide, WHYPWFQNWAMA, which targets human squamous cell carcinoma of the head and neck (37). In vivo studies with HPMA copolymer containing the integrin-targeting peptide, KACDCRGDCFCG, demonstrated not only passive accumulation of polymer conjugate at the tumor site, but also active targeting of polymer conjugate mediated by the peptide (38). The work described above suggested that HPMA copolymers equipped with peptides as targeting moieties may work as a promising approach to targeted chemotherapy. In this work, peptide ligands recognizable by CD21 receptor were selected with phage display. Specific binding between selected peptides and CD21 receptor was confirmed by phage ELISA, and the binding affinities were determined with fluorescence quenching. To evaluate the efficiency of targeting lymphoma with selected peptides, we synthesized HPMA copolymer-peptide P1 (RMWPSSTVNLSAGRR) conjugates. Specific interactions were verified between HPMA copolymerpeptide conjugates and surface-bound CD21 receptor using ELISA experiments. We further investigated the effect of the spacer between the peptide and polymer, and the effect of 1 Abbreviations: A, 8-amino-3,6-dioxaoctanoic acid; A2, dimer of A; AIBN, 2,2′-azobisisobutyronitrile; BSA, bovine serum albumin; DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide; ELISA, enzyme linked immunosorbent assay; Fmoc, 9-fluorenylmethoxycarbonyl; HMBA, hydroxymethylbenzoic acid; HPMA, N-(2-hydroxypropyl)methacrylamide; HRP, horseradish peroxidase; NHS, N-hydroxysuccinimide ester; MA, methacryloyl; MA-GG-OH, N-methacryloylglycylglycine; MALDI-TOF MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; NP, EDPGFFNVE; P, HPMA copolymer backbone; PB, HPMA copolymer backbone labeled with a PEG3400-biotin graft; P1, RMWPSSTVNLSAGRR; P2, PNLDFSPTCSFRFGC; P3, GRVPSMFGGHFFFSR; P4, RLAYWCFSGLFLLVC; P5, PVAAVSFVPYLVKTY; PBS, phosphatebuffered saline; PEG, poly(ethylene glycol); PHPMA, polyHPMA; rsCR2.1-4, recombinant short (truncated) CD21 receptor containing SRCs 1-4; SCR, short consensus repeat; sCD21, soluble CD21 receptor; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, 3,3′,5,5′-tetramethylbenzidine; TPBS, PBS containing 0.1% Tween 20.

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peptide content per macromolecule upon surface binding of HPMA copolymer-P1 conjugate.

EXPERIMENTAL PROCEDURES Materials. Escherichia coli strain K91 kan and the fuse-5 15-mer phage library (39) were gifts from Dr. G. P. Smith (University of Missouri, Columbia). HB5 anti-CD21 mAb was a gift from Dr. A. Tang. sCD21 (35) and rsCR2.1-4 (28) were prepared as previously described. 2-Chlorotrityl chloride resin, hydroxymethylbenzoic acid (HMBA) resin, and all N-R-Fmoc protected amino acids were purchased from Novabiochem. Screening of CD21 Specific Binding Phage with fUSE5 15-mer Phage Library. MaxiSorp plate (NUNC, Denmark) was coated with 200 µL (20 µg/mL) of sCD21 receptor at 4 °C overnight and blocked with 4% skim milk for 2 h at room temperature. The plate was then washed with 0.1% Tween/PBS. Before exposing the phage to the receptor, the plate was preblocked by incubation with 2% BSA in PBS for 15 min. Approximately 1012 phage virions were incubated with receptorcoated plate for 3 h. The nonspecific bound phage was removed with stringent washing: washing with TPBS (pH 7.4) 15 times, followed by washing with TPBS (pH 5.0) 5 times, and finally washing with PBS 5 times. The bound phage was eluted with glycine hydrochloride buffer (0.1 M HCl, pH adjusted to 2.2 with glycine). The eluted phage was neutralized with 1 M Trisbuffer (pH 9.1) and amplified. An additional three rounds of selection were performed as described above with decreased phage incubation time: 1 h, 30 and 15 min, respectively. In the fourth round of selection, to minimize the selection of nonspecific bound phage caused by nonspecific elution with glycine hydrochloride buffer, the bound phage was eluted with 4 µg of sCD21 receptor in 200 µL of PBS. After the fourth round of selection, 21 randomly chosen phage clones were tested with preliminary phage ELISA and the DNA of nine phage clones with the highest binding was sequenced using the primer 5′-TCCACAGACAGCCCTCATAG-3′. Phage ELISA and Competitive Phage ELISA. Receptors sCD21 and rsCR2.1-4 (200 ng in 100 µL of PBS) were coated onto a MaxiSorp plate by incubation overnight at 4 °C. The plate was blocked with 2% BSA in PBS for 2 h at room temperature. Phage P1 (∼1 × 107 virion/mL) was incubated in the plate for 1 h at room temperature. The plate was then washed eight times with TPBS. After washing, horseradish peroxidase (HRP)/anti-M13 mAb conjugate (Pharmacia Biotech, Uppsala, Sweden) was diluted 1:5000 with 1% BSA/TPBS and incubated in the plate for 1 h. Again, the plate was washed five times with TPBS, followed by incubation with HRP substrate (ImmunoPure TMB Substrate kit, PIERCE Biotech. Inc.), a mixture of 3,3′,5,5′-tetramethylbenzidine (TMB) and peroxide solution, for 30 min. The reaction was stopped by addition of 100 µL of 1 N H2SO4, and the absorbance was read using a microplate reader at 450 nm and background subtraction at 630 nm. For the competitive phage ELISA, the plate surface was coated with sCD21 and blocked with 2% BSA. Phage P1 was preincubated with different amounts of (competitor) rsCR2.1-4 (0, 0.45, 0.9, 1.35, and 1.8 µg, respectively, in 100 µL of TPBS containing 1% BSA) for 30 min before the mixture was placed in sCD21 coated plate; there, the mixtures of phage and rsCR2.1-4 were incubated with surface-bound sCD21. The detection of bound phage was the same as that used for the phage ELISA. Synthesis of Peptides. Peptides, RMWPSSTVNLSAGRR (P1), PNLDFSPTCSFRFGC (P2), and GRVPSMFGGHFFFSR (P3), were synthesized manually on a solid support of 2-chlorotrityl chloride resin using standard Fmoc chemistry. The synthesized peptides were cleaved from resin with a mixture of TFA/H2O/phenol/TIS (88:5:5:2, v/v/w/v) for 4 h at room

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Figure 1. Structures of peptide monomers.

temperature. All synthesized peptides were purified with RPHPLC and verified with MALDI-TOF MS. MALDI-TOF MS calculated for P1 (MH+) 1717.88, found 1718.25; MALDI-TOF MS calculated for P2 (MH+) 1702.73, found 1702.78; MALDITOF MS calculated for P3 (MH+) 1728.84, found 1728.89. Fluorescent Labeling of rsCR2.1-4. To a solution of rsCR2.1-4 in 200 µL of PBS (0.5 mg/mL), 20 µL of 1 M NaHCO3 (pH 9.0), and 23.3 µL of FITC in DMSO (10 mg/ mL) was added, and the reaction was stirred for 5 h at room temperature in the dark. To remove free FITC, the reaction mixture was diluted to 0.5 mL with PBS and immediately applied to a PD-10 column (Amersham Biosciences) preequilibrated with PBS buffer. The concentration of the fluorescently labeled protein and labeling efficiency were determined using UV spectrometry. Determination of Peptide Binding Constants with Fluorescence Quenching. A series of mixtures of the labeled receptor rsCR2-FITC at 0.02 µΜ, and peptides, P1, P2, and P3, at different concentrations were prepared in 150 µL of PBS. The mixtures were incubated at room temperature for 5 h. The fluorescence intensity of each sample was measured in duplicates with a LS-55 luminescence spectrometer (Perkin-Elmer) with excitation and emission wavelengths of 495 and 515 nm, respectively. The fluorescence background caused by the peptides was negligible within the concentration range used. The association constant was estimated by fitting the quenching data to eq 1 with software KaleidaGraph (version 3.0) (40).

Q)

QmKa[P] 1 + Ka[P]

Q)

F0 - F F

(1)

(2)

where Q is the quenching value defined in eq 2, and Qm represents maximal fluorescence quenching, Ka is the association constant of peptide binding, and [P] is the concentration of free peptide. F0 and F are fluorescence intensities of fluorescently labeled rsCR2.1-4 in the absence and presence of peptide ligands, respectively. Synthesis of Peptide Monomers (MA-GG-P1, MA-A2-P1, MA-GG-NP). Peptide monomers MA-GG-P1, MA-A2-P1 and MA-GG-NP (see structures in Figure 1) were synthesized on HMBA resin using standard Fmoc chemistry. After the last amino acid residue of P1 (or NP) was attached on the resin, the peptides were extended with additional spacers: a GG spacer was added by coupling with MA-GG-OH (N-methacryloylglycylglycine); A2 spacer was added by coupling with two repeats of Fmoc-A (9-fluorenylmethoxycarbonyl-8-amino-3,6-dioxaoctanoic acid, Peptides International) and capped with methacryloyl chloride. All peptide monomers were purified with RPHPLC and were verified with MALDI-TOF MS. MALDI-TOF MS calculated for MA-GG-P1 (MH+) 1898.97, found 1898.93; MALDI-TOF MS calculated for MA-A2-P1 (MH+) 2076.06, found 2076.11; MALDI-TOF MS calculated for MA-GG-NP (M + Na+) 1257.50, found 1257.54.

Synthesis of MA-PEG3400-biotin and biotin-PEG-P1. Synthesis of MA-PEG-biotin. N-(3-Aminopropyl)methacrylamide hydrochloride (MA-NH2) (2.7 mg) was dissolved in 200 µL of DMF and 6.7 µL of N,N-diisopropylethylamine (DIPEA) was added. The temperature of the solution was lowered to 0 °C with an ice bath. Biotin-PEG3400-NHS (PEG, poly(ethylene glycol), mol wt 3400; NHS, N-hydroxysuccinimide ester; Nektar Therapeutics) dissolved in 200 µL of DMF was added dropwise. The reaction was kept at 0 °C for 30 min and then left overnight at room temperature. Then, the crude product was diluted 1:1 with water and MA-PEG3400-biotin purified on a PD-10 column. Molecular weight as determined by MALDI-TOF MS was Mw ) 3.89 kDa; Mn ) 3.86 kDa; Mw/Mn ) 1.01. Synthesis of biotin-PEG-P1. Peptide P1 (5 mg) was dissolved in 0.5 mL of 0.1 M NaHCO3 (pH 8.5). The temperature was lowered to 0 °C with an ice bath. A DMF solution of biotinPEG-NHS (5 mg in 100 µL) was added dropwise to the peptide solution. The reaction was kept 0 °C for 30 min and then left overnight at room temperature. The solvent was removed by evaporation, and the biotin-PEG3400-P1 was purified with RPHPLC. The identity of biotin-PEG3400-P1 was confirmed by MALDI-TOF MS. Molecular weight as determined by MALDITOF MS was Mw ) 5.31 kDa; Mn ) 5.29 kDa; Mw/Mn ) 1.01. Synthesis of Biotin-Labeled HPMA Copolymer-Peptide Conjugates (Figure 2). The synthesis of HPMA copolymerpeptide conjugate PB-GG-P1 (PB is HPMA copolymer backbone labeled with a PEG3400-biotin graft) is described as an example: HPMA (11.7 mg), MA-GG-P1 (10 mg), MA-PEG3400biotin (3 mg) (molar ratio, 93:6:1), and initiator AIBN (1.6 mg) were dissolved in 155 µL of DMSO in a glass ampule. The solution was bubbled with nitrogen for 10 min and polymerized in the sealed ampule at 50 °C for 24 h. After the reaction was finished, the reaction solution was diluted to 3 mL with H2O and dialyzed against water in dialysis tubing with molecular cutoff of 12-14 kDa for 3 days. After dialysis, the polymer water solution was lyophilized. PB, PB-A2-P1, and PB-GG-NP were synthesized similarly, except that PB contains no peptide. The polymers were analyzed with size-exclusion chromatography on an A ¨ KTA FPLC system (Pharmacia). The content of peptide was determined by amino acid analysis, and the content of biotin was determined with an EZ Biotin Quantitation kit (PIERCE Biotech. Inc.). ELISA of HPMA Copolymer-Peptide Conjugates. The receptor rsCR2.1-4 (150 ng) was coated onto a MaxiSorp plate by overnight incubation at 4 °C. The plate was blocked with 4% milk at room temperature for 1.5 h, washed with TPBS, and incubated with HPMA copolymer-peptide conjugates (0.02 µM) for 30 min. Then, the plate was washed eight times with TPBS, followed by incubation with streptavidin/HRP (Zymed Laboratories) for 30 min. The plate was washed 5 times with TPBS; then TMB substrate was added to the plate and incubated for 30 min. The absorbance was read at 450/630 nm. For the inhibitive ELISA of PB-GG-P1, plate surface was coated with rsCR2.1-4 (150 ng) and blocked with 4% milk. Different concentrations of free peptide P1 (0-100 µM) were preincubated with surface-bound rsCR2.1-4 in 100 µL of TPBS for 15 min. Then, PB-GG-P1 was added to each plate well (final concentration 0.02 µM) and incubated for 30 min. The bound PB-GG-P1 was detected as described above.

RESULTS Identification of Peptide Ligands for CD21 Receptor. Peptide ligands for the CD21 receptor were selected with a 15mer phage library based on the phage vector fUSE 5 using sCD21 as the target molecule. To select specifically bound phage and exclude nonspecifically bound phage, a stringent biopanning process was performed including several measures: extensive


HPMA Copolymer Conjugated with CD21-Binding Peptide Table 1. Screening Results of CD21 Receptor Binding Phages selected phage

sequence

occurrence

P1 P2 P3 P4 P5

RMWPSSTVNLSAGRR PNLDFSPTCSFRFGC GRVPSMFGGHFFFSR RLAYWCFSGLFLLVC PVAAVSFVPYLVKTY

4 1 1 2 1

washing with TPBS (pH 7.4) (41), stringent washing with TPBS (pH 5.0) (41), decreasing the incubation time in each round of selection (42), and elution with sCD21 at the last round selection (42). After four rounds of selection, a preliminary phage ELISA of 21 individual phage colonies was performed, and the DNA of the nine phages with the highest binding was sequenced (Table 1). Five different sequences were obtained; four phages shared the sequence RMWPSSTVNLSAGRR (P1) and two shared the sequence RLAYWCFSGLFLLVC (P4). It is interesting to note that two sequences, PNLDFSPTCSFRFGC (P2) and P4, contained two cysteine residues. The distance between the two cysteines are five (P2) and eight (P4) amino acid residues, respectively. Consequently, the formation of cyclic peptide structures with constrained conformations is more probable than cross-linking. Except for P1, all sequences of selected peptides, P2-P4, consisted mostly of hydrophobic residues, which may participate in hydrophobic interactions with the CD21 receptor. However, the five sequences did not show obvious homology with each other. Phage ELISA and Competitive Phage ELISA. The binding affinity of the selected peptides toward the CD21 receptor was first verified by phage ELISA with sCD21 and rsCR2.1-4

Figure 3. The specificity of selected phage with different sequences was confirmed by phage ELISA. The plate surface was coated with two different forms of CD21 receptor, sCD21 and rsCR2.1-4. The absorbance was compared with the control surface without receptor.

(Figure 3). As expected, all the selected phages were found to bind to sCD21 specifically, and all showed little binding to the surface in the absence of receptor. Since rsCR2.1-4 represents only the first four SCRs of the whole CD21 receptor, it was unexpected that all the selected phages bound to rsCR2.1-4, indicating that the binding sites of selected peptides are all localized in this region (Figure 3). Furthermore, the binding of the different phages to sCD21 correlated very well with their binding to rsCR2.1-4. Even though phage P4 showed the highest signal in the presence of CD21 receptors, it also showed significant binding to the surface in the absence of CD21

Figure 2. Structures of HPMA copolymer-P1 conjugates and PEG-P1 conjugate. HPMA copolymer-peptide conjugates consist of three components: an HPMA copolymer backbone, a PEG-biotin graft used as a label for immunoassay, and peptide ligands with spacers of different lengths, GG and A2. Each polymer chain of the HPMA copolymer-peptide conjugates contains several copies of peptide, while each polymer chain of biotin-PEG3400-P1 contains only one peptide.


Figure 4. Competitive phage ELISA. Different amounts of rsCR2.1-4 were used to compete with surface-bound sCD21 for binding to phage P1.

Figure 5. Fluorescence quenching of fluorescein labeled rsCR2.1-4 as a function of peptide concentration. Data are depicted as mean ( standard deviation from duplicate measurements.

receptor, which possibly is a result of the three consecutive phenylalanine residues in its structure. We further verified that the binding sites of selected peptides are within the first four SCRs using competitive phage ELISA. Using phage P1 as an example, whole receptor sCD21 was immobilized on a MaxiSorp plate surface and rsCR2.1-4 was co-incubated with P1 as a competitor to surface-bound sCD21. As shown in Figure 4, with increasing concentration of competitor the number of phage bound to sCD21 decreased, as indicated by the decreasing ELISA signal. The binding of sCD21 with P1 was inhibited by rsCR2.1-4, which confirmed that the binding site of P1 is within first four SCRs. This result provided a basis for using rsCR2.1-4 for the binding constant determination of the selected peptides. Binding Constant Determination with Fluorescence Quenching. The rsCR2.1-4 receptor was fluorescently labeled with FITC, and it was found that the extent of labeling had a significant effect on the fluorescence quenching effect. When the labeling of rsCR2.1-4 was low (F/P ≈ 1), the quenching effect was not apparent (data not shown). When the labeling of rsCR2.1-4 was high (F/P ≈ 5), the quenching effect was pronouncedsover 80% quenching was observed, as shown in Figure 5. Our previous studies of the binding kinetics of peptide LVLLTRE (a peptide derived from one-bead one-compound peptide library, to be published) with rsCR2.1-4-FITC indicated that at least 3 h are needed to reach equilibrium. Therefore, the

Ding et al.

peptides were incubated with the receptor for 5 h, before measuring the fluorescence intensity. The association constants of peptides P1, P2, and P3 were determined to be 2.9 × 106 M-1, 4.7 × 106 M-1, and 2.2 × 106 M-1, respectively (Figure 5). P2 (PNLDFSPTCSFRFGC) contains two cysteine residues and can form a cyclic structure. To evaluate the impact of conformation constrain on biorecognition, the binding affinities of cyclic P2 and linear P2 were compared. Linear P2 in reduced form showed poor aqueous solubility. Its quenching study was carried out with the assistance of a small amount of DMF for solubilization. The binding constant of linear P2 was estimated to be 2 × 106 M-1 (data not shown). Therefore, the cyclic structure of P2 enhanced the binding about 2-fold when compared with linear P2. Synthesis and Characterization of HPMA CopolymerPeptide Conjugates. All HPMA copolymer conjugates were synthesized by copolymerization of monomers of HPMA, peptide, and biotin. The composition of the synthesized polymers is summarized in Table 2. The weight-average molecular weight was determined with size-exclusion chromatography on a Sepharose 6B column calibrated with PHPMA standards. The concentration of the peptide monomers, MA-GG-P1 and MAA2-P1, in the feed was 6 mol %, but only about 1 mol % of peptide-containing comonomer was incorporated into the copolymer. Peptide monomer, MA-GG-NP, possessed a better incorporation efficiency: 2.6 mol % of peptide monomer was incorporated to a content identical to the feed composition. We synthesized PB-GG-NP using a feed composition of 4% MAGG-NP, so that the content of NP in PB-GG-NP was comparable to the content of peptide P1 in conjugates PB-GG-P1 and PBA2-P1. (One may speculate that the difference of incorporation efficiency relates to the length of the peptide; however, more experiments would be needed to verify this hypothesis.) The copolymers were labeled with biotin by incorporation of a small amount of the MA-PEG3400-biotin comonomer. Biotin was used to detect surface-bound conjugates in the ELISA experiments. Only 1 mol % of MA-PEG3400-biotin was used in the monomer feed, and approximately 0.3-0.4% of biotin-containing comonomer was incorporated into the HPMA copolymer (Table 2). This corresponds to about one biotin per macromolecule. ELISA of Peptide HPMA Copolymer Binding to Surfaces with Different Amounts of rsCR2.1-4. Two experiments were performed to evaluate the impact of peptide conjugation with HPMA copolymers on biorecognition by the CD21 receptor, including (a) the dependence of binding on receptor surface density and (b) competitive inhibition with free peptide P1. For the receptor density dependent study, MaxiSorp plate was coated with different amounts of rsCR2.1-4 (0-640 ng), and the binding of PB-GG-P1 (0.02 µM) was detected with ELISA using biotin/streptavidin/HRP as the reporting system. To minimize the nonspecific interaction between plate surface and polymerpeptide conjugate, we tried several blocking agents and found that 4% milk produced the lowest background. We are aware that milk may contain some amount of endogenous biotin, which may interact with the biotin/streptavidin/HRP detection system; however, in our experiment, this did not seem to be a problem. If the endogenous biotin were to interact with the detection system, it can be masked with egg white and milk (43). The binding of PB-GG-P1 to surface-bound receptor was directly related to the receptor density: the higher the receptor density, the higher the amount of surface-bound PB-GG-P1 (Figure 6). These data indicate a specific interaction between PB-GG-P1 and surface-bound receptor. This experiment also provided the binding profile of PB-GG-P1, so that an optimum amount of receptor density could be identified. In the final surface binding experiments, 150 ng of rsCR2.1-4 was used to coat the surface.



Figure 8. ELISA results of different polymer-peptide conjugates binding to surface-bound receptor. Data are depicted as mean ( standard deviation from duplicate measurements. Figure 6. The relationship between the binding of PB-GG-P1 and amount of surface coating receptor. Data are depicted as mean ( standard deviation from duplicate measurements.

Figure 7. Inhibition of PB-GG-P1 binding to surface-bound receptor by free peptide P1. The data represent mean ( standard deviation from duplicate measurements.

Competitive Inhibition of Binding with Free Peptide. To further demonstrate the binding specificity of HPMA copolymerpeptide conjugate and CD21 receptor, we investigated binding of conjugate PB-GG-P1 (0.02 µM) in the presence of different concentrations of free peptide P1 (0-100 µM). Free P1 was able to inhibit the binding of PB-GG-P1 at concentrations higher than 25 µM (Figure 7). However, no apparent inhibition was observed when the concentration of free peptide was below 6.25 µM, and the inhibition was only 25% even at the P1 concentra-

tion of 100 µM. This fact implied that the binding affinity of PB-GG-P1 is much higher than that of free peptide, given that binding concentration of PB-GG-P1 was only 0.02 µM. One could expect that the biorecognition (binding) of the peptide in a polymer conjugate may decrease due to the sterical hindrance of the polymer backbone on the formation of the ligandreceptor complex. However, the binding affinity of the HPMA copolymer-bound peptide was enhanced (Figure 7). The enhanced binding could be ascribed to the effect of multivalent interactions between PB-GG-P1 and surface-bound receptor. Each polymer chain of PB-GG-P1 contained about 4.4 grafted peptides (Table 2A). With the appropriate receptor density on the surface, multivalent interactions between PB-GG-P1 and receptor apparently occurred, resulting in a binding affinity enhanced by 3-4 orders of magnitude. A separate experiment, described below, was devoted to clarify the effect of multivalent interactions on the binding of HPMA copolymer-peptide conjugates. Surface Binding of the Conjugates with Varying Structure. Biotin-labeled HPMA copolymer-P1 conjugates, differing in the structure and length of the spacer separating the peptide and the HPMA copolymer backbone, were synthesized (Table 2A), and their binding to CD21 was evaluated. Two spacers, namely, GG (glycylglycine) and A2 (dimer of 8-amino-3,6dioxaoctanoic acid), and two peptides, P1 and NP were used. As controls, biotin-labeled HPMA copolymer (no peptide) and biotin-labeled PEG-P1 conjugate were evaluated. PB (HPMA copolymer containing no peptide) did not show any binding to the surface with or without receptor. As in experiments described above, HPMA copolymer containing the peptide ligand P1 (PB-GG-P1) demonstrated receptor-specific binding (Figure 8). Polymer conjugate (PB-GG-NP) containing

Table 2. Composition of (A) Various Polymer-Peptide Conjugates and (B) HPMA Copolymer-P1 Conjugates with Different Contents of P1 (A) Various Polymer-Peptide Conjugatesa conjugates PB

PB-GG-P1 PB-A2-P1 PB-GG-NP PEGB-P1

Mwc (kDa)

structure

Pdd

peptide (mol %)e

peptide no./chain

biotin (mol %)f

0 4.4 4.3 5.2 1

0.4 0.36 0.36 0.35 N/A

PHPMA-(PEG-biotin) 63.2 1.7 0 PHPMA-(GG-P1)-(PEG-biotin) 47.1 1.4 1.3 PHPMA-(A2-P1)-(PEG-biotin) 55.6 1.5 1.1 PHPMA-(GG-NP)-(PEG-biotin) 60.3 1.3 1.2 biotin-PEG3400-P1 5.3 1 N/A (B) HPMA Copolymer-P1 Conjugates with Different Contents of P1b

conjugates

feed composition (HPMA/biotin/P1)

Mwc (kDa)

Pdd

peptide (mol %)e

peptide no./chain

biotin (mol %)f

PB-GG-P1A PB-GG-P1B PB-GG-P1C PB-GG-P1D PB-GG-P1E

98.5:1:0.5 98:1:1 97:1:2 95:1:4 93:1:6

67.1 81.1 79.4 65.2 47.1

1.6 1.5 1.6 1.5 1.4

0.07 0.15 0.31 0.98 1.3

0.3 0.8 1.7 4.5 4.4

0.28 0.40 0.37 0.31 0.37

a PB HPMA copolymer backbone labeled with a PEG b B c 3400-biotin graft. P HPMA copolymer backbone labeled with a PEG3400-biotin graft. Mw: weight-average molecular weight as determined with size exclusion chromatography. d Pd: polydispersity, ratio of weight-average molecular weight over number-average molecular weight. e Determined with amino acid analysis. d Determined with EZ Biotin Quantitation kit.


Ding et al.

peptide per polymer chain) and PB-GG-P1E (4.4 peptide per polymer chain) demonstrated extraordinary binding. Although the number of peptide grafts per macromolecule in PB-GG-P1D and PB-GG-P1E was similar, the latter showed much better binding than PB-GG-P1D. This may be explainable by the higher mol % of peptide in PB-GG-P1E (the difference is due to the lower molecular weight of conjugate PB-GG-P1E than that of PB-GG-P1D). The above results demonstrated the importance of multivalent interaction in the binding of HPMA copolymerpeptide conjugates to the CD21 receptor.

DISCUSSION

Figure 9. The binding of HPMA copolymer-P1 conjugates (PB-GGP1A-E; containing different amounts of peptide P1) to the CD21 receptor. For composition of copolymers, see Table 2B. Data are depicted as mean ( standard deviation from duplicate measurements.

a nonapeptide (EDPGFFNVE) ligand of CD21 receptor reported by literature (34), also demonstrated specific binding to CD21 receptor, however, to a lesser degree compared with PB-GGP1. The binding of biotin-labeled PEG-P1 conjugate (PEGB-P1) was also evaluated. PEGB-P1 is composed of a 3400-Da poly(ethylene glycol) macromolecule with one terminus modified with peptide P1 and the other with biotin. However, no appreciable binding of PEGB-P1 to receptor was detected (Figure 8). This conjugate (PEGB-P1) contains only one peptide per macromolecule. Consequently, it interacts with the receptor only in a monovalent manner. In contrast, each polymer chain of PB-GG-P1 has 4-5 copies of peptide P1, thus capable of multivalent interactions with surface-bound receptor. This difference in biorecognition may be attributed to the difference in the binding mode (multivalent vs monovalent interactions). The Effect of Peptide Accessibility on the Binding Affinity. Both PHPMA and the peptide may influence the physical properties of HPMA copolymer-peptide conjugates. PHPMA itself is water-soluble; its conformation in solution is a random coil (44). When hydrophobic moieties are attached to HPMA copolymer side chains, the conformation of the macromolecule in the solution may change as a result of intramolecular and/or intermolecular association of side chains (45). On the other hand, the polymer backbone, due to its constant mobility, may have a shielding effect on the accessibility of peptide to the target. To investigate this possibility, the peptide P1 was conjugated to HPMA copolymer via two different spacers, GG and A2 (Figure 1). Spacer A2 is four times as long as spacer GG. It is also more flexible because the amide bonds limit the flexibility of GG. Therefore, the peptide in PB-A2-P1 may be better exposed and should have more accessibility than that in PBGG-P1. However, no obvious binding differences between PBGG-P1 and PB-A2-P1 were observed (Figure 8). This suggests that in both conjugates, peptide P1 was sufficiently exposed for interaction with the target receptor, and the spacer effect was minimal. Because P1 is a peptide 15 amino acid residues long, the results do not eliminate possible spacer effects using shorter peptides. Demonstration of Multivalent Interaction in Surface Binding. To evaluate the relationship between the amount of peptides per macromolecule and biorecognition, biotin-labeled HPMA copolymers containing different amounts of GG-P1 side chains were synthesized (Table 2B), and their binding properties were determined. When the peptide content of conjugates (PBGG-P1A and PB-GG-P1B) was low, less than one peptide per polymer chain, the binding of the conjugates was negligible (Figure 9). Conjugate PB-GG-P1C (1.7 peptide per polymer chain) showed minimal binding; however, both PB-GG-P1D (4.5

Identification of CD21 Ligands with Phage Display. Two forms of CD21 receptor, soluble CD21 (sCD21) and recombinant short CD21 rsCR2.1-4 were prepared. SCD21 contains the whole extracellular portion of CD21 receptor, while rsCR2.1-4 only the first four SCRs. sCD21, about four times as large as rsCR2.1-4, is expected to have more binding sites than rsCR2.1-4. Therefore, biopanning with whole sCD21 should select peptides not only binding to SCR1-4, but also to SCR5-16. Since the goal was to find suitable peptide ligands for the CD21 receptor, sCD21 was used, instead of rsCR2.14, for phage display, to find the best peptide binders of the whole CD21 receptor. Surprisingly, all peptide ligands selected with sCD21 also bound to SCR1-4 as indicated by phage ELISA with rsCR2.1-4 and competitive phage ELISA, demonstrating that the binding sites of all selected peptides were located within the first four SCRs. Several factors might contribute to this result: SCR1-4 is well exposed and accessible for interaction with phage; SCR1-4 consists of binding sites most suitable for peptide binding; peptides binding outside of SCR1-4 are relatively weaker and cannot withstand stringent screening. Another feature of selection results is the lack of obvious homology among all of the selected peptides, which implies that SCR1-4 is not the only antigenic region, but also a multiantigenic region. It has several distinctive binding sites, which was verified by epitope mapping with different antibodies. Although the epitopes of anti-CD21 antibodies, FE8 (46), OKB7 (47), mAb171 (48), and mAb1048 (48) are all located in SCR12, they are distinct from each other. The binding sites of C3d and EBV have been extensively studied, but the results had been controversial until the X-ray crystallographic structure of C3dCD21 complex was published (49). There is evidence that the binding sites of C3d and EBV are far from each other (50). Therefore, it was not surprising that the selected peptides exhibited no homology. Moreover, the phage display results further confirmed that the CD21 receptor is multi-antigenic. Affinity Analysis by Fluorescence Quenching. Fluorescence spectrometry is most suited for protein-ligand interactions with dissociation constants in the range 10-4 to 10-8 M. The formation of the protein-ligand complex usually induces a conformational change of the protein and an alteration of the microenvironment of the fluorophore. Some fluorescence parameters such as anisotropy and intensity are sensitive to such changes, and the variation of fluorescence intensity upon the formation of protein-ligand complex can be used for affinity analysis (51). By comparing on the crystal structure of both free CD21 (SCR1-2) (50) and the complex (49) of CD21 (SCR1-2) and C3d, it was found that the binding of C3d with CD21 is an induced-fitting process. Not only does the conformation of the local binding site change upon binding, but the global conformation also changes (50). This implies that the CD21 receptor (or at least SCR1-2) is a relatively flexible protein. The conformational change of CD21 receptor upon binding with ligands should be detectable with fluorescence spectrometry. The recombinant short CD21 receptor, rsCR2.1-4, was used



for affinity analysis by fluorescence quenching. It is a much smaller molecule compared to the whole CD21 receptor. With a smaller molecule, the change of fluorescence parameters is more sensitive to conformational change. Although selected peptides are only 15 amino acid residues long, their interaction with rsCR2.1-4 induced enough conformational change to be detected by fluorescence quenching. The dissociation constants of three synthesized peptides were determined to be within the micromolar range, which is typical for most phage display results (11). We are aware that the binding affinity of selected peptides might be affected, to some extent, by the labeling of rsCR2.1-4 with FITC. Synthesis of HPMA Copolymer-Peptide Conjugates. There are two different ways to synthesize HPMA copolymerpeptide conjugates: polymer-analogous reactions and copolymerization. In polymer-analogous reaction, a HPMA copolymer precursor containing, e.g., reactive p-nitrophenyl ester groups at side chain termini is prepared first, followed by the attachment of the peptide to the HPMA copolymer precursor by aminolysis of the active ester (32). However, this approach has some limitations, as the peptide should have only one free amino group to achieve site-specific conjugation. This can be inconvenient because most peptides usually contain more than one reactive group; therefore, protection during synthesis is required. In comparison, copolymerization using peptide-containing monomers with HPMA, in an appropriate solvent, does not present this limitation. Because P1 contains three arginine residues and one amino group reactive toward the active ester of the HPMA copolymer precursor, PB-GG-P1 was prepared by the copolymerization of HPMA, MA-GG-P1, and MA-PEG-biotin in DMSO (a good solvent). Affinity Analysis of Polymer-Bound Peptides. Several different targeting moieties have been incorporated into HPMA copolymers, including lectins (52), antibodies (53), and antibody fragments (54). Successful targeting with peptides will undoubtedly expand the application of HPMA copolymer as an anticancer drug carrier. Therefore, it is necessary to examine the biorecognizability of selected peptides after they are conjugated with HPMA copolymer. We chose peptide P1 for this purpose, since P1 is quite soluble in water, thus avoiding the possible complications caused by inter/intramolecular hydrophobic interactions. When multiple copies of a peptide are incorporated into a polymer-based drug delivery system, the properties of both the peptides and the polymer may change accordingly. On one hand, the targeting efficiency of the peptides may be partially masked by the polymer. On the other hand, the polymer structure may be influenced by the composition of peptides, especially when peptides contain a large number of hydrophobic or charged residues. Therefore, the interaction between the HPMA copolymer-peptide conjugates and the surface-bound receptor was investigated. It was found that conjugation of P1 with HPMA copolymer did not impair its binding affinity toward the CD21 receptor as demonstrated by receptor-dependent surface binding and inhibition experiments using free peptide. On the basis of the binding results of PB-GG-P1 and PB-GG-NP, the selected peptide P1 showed better recognizability than the previously reported nonapeptide (NP) when incorporated into HPMA copolymer-peptide conjugates (32). By changing the spacer from GG to GFLG, the binding affinity of HPMA copolymer-peptide NP conjugates increased as determined by the whole cell binding assay (32, 36), implying that extended spacer length may improve the interaction between the NP and the cell surface receptor. However, in our experiment, the spacer effect (A2 is four times as long as GG) was not significant. It was noted that P1 is a 15-amino-acid peptide and NP is a 9-amino-acid peptide. It was speculated that length

of peptide ligands may account for the spacer effect on different HPMA copolymer-peptide conjugatessthe longer the peptide length, the less the spacer effect. The enhanced surface binding affinity of PB-GG-P1 compared with free peptide and PEGB-P1 suggested multivalent interactions of HPMA copolymer conjugates. Because each PEG chain contained only one peptide, while each HPMA copolymer chain contained 4-5 copies of peptide, HPMA copolymer-peptide conjugates are capable of multivalent interaction with surfacebound receptor. This multivalent effect was also observed in inhibitive ELISA by free peptide: noticeable inhibition was observed only when free peptide concentration was 3-4 orders of magnitude higher than peptide concentration in conjugates. The binding affinity test with peptide conjugates of different peptide contents confirmed the importance of multivalent interaction in the surface binding of HPMA copolymer-peptide conjugates. Also shown in whole cell binding assays, the binding affinity of HPMA copolymer NP conjugates increased with the increase of NP content in the conjugates (32, 36). This observation is directly correlated to our results in this work: multivalent interaction played an important role on the binding of HPMA copolymer not only in our plate surface model but also in the cell surface system. Therefore, HPMA polymerpeptide conjugates are beneficial to recognize a target, which is capable of multivalent interaction. A soluble form of CD21, shed from the cell membrane, may compete with the binding of HPMA copolymer-peptide conjugates to lymphoma cells, thus reducing its targeting specificity. This fact further strengthens the importance of the multivalent capability of HPMA-peptide conjugates; if multivalent interactions are achievable with the membrane-bound receptor, then the competition by the soluble receptor will be significantly minimized.

CONLUSIONS Five peptide sequences were identified as ligands for CD21 receptor using phage display. The binding affinity of selected peptides was confirmed by phage ELISA, and the binding constants of three peptides were determined with fluorescence quenching. Using surface binding studies, it was demonstrated that the peptide RMWPSSTVNLSAGRR (P1) retained its recognizability by the CD21 receptor after its incorporation into HPMA copolymer. The specificity of its recognition was confirmed with competitive inhibition by free peptide. The spacer effect for this peptide in the HPMA copolymer conjugates was minimal. The importance of multivalent interaction for biorecognition of HPMA copolymer-peptide conjugates toward the CD21 receptor was confirmed.

ACKNOWLEDGMENT This work was supported in part by NIH Grants CA88047 and CA51578 from the National Cancer Institute. We thank Dr. G. P. Smith for providing the fUSA-5 15-mer phage library, Dr. A. Tang for valuable discussions and the gift of HB5 antiCD21 mAb, and J. Callahan for carefully revising the manuscript.

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Identification of CD21-Binding Peptides with Phage Display and

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