A Versatile Method for the Conjugation of Proteins and Peptides to

For the synthesis of pDMAEMA-protein/peptide conjugates, the polymer has to be functionalized, since in ... 2-(Dimethylamino)ethyl methacrylate (DMAEM...
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Bioconjugate Chem. 1999, 10, 687−692

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A Versatile Method for the Conjugation of Proteins and Peptides to Poly[2-(dimethylamino)ethyl methacrylate] W. N. E. van Dijk-Wolthuis,†,‡ P. van de Wetering,† W. L. J. Hinrichs,† L. J. F. Hofmeyer,§ R. M. J. Liskamp,§ D. J. A. Crommelin,† and W. E. Hennink*,† Department of Pharmaceutics, Sorbonnelaan 16, Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, PO Box 80 082, 3508 TB Utrecht, The Netherlands, and OctoPlus BV, Niels Bohrweg 11-13, 2333 CA Leiden, The Netherlands. Received October 28, 1998; Revised Manuscript Received March 29, 1999

Random copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with aminoethyl methacrylate (AEMA) were synthesized by radical polymerization. The amount of incorporated primary amino groups could be controlled by the feed ratio of AEMA to DMAEMA, and was varied from 2 to 6 mol %. Subsequently, protected thiol groups were introduced in a derivatization step with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and subsequent treatment with dithiothreitol (DTT). The obtained thiolated p(DMAEMA-co-AEMA) was conjugated to transferrin (Tf) or the F(ab′) fragment of mAb 323/A3 via a disulfide linkage. Moreover, the maleimide derivative of the nuclear localization signal (NLS) decapeptide Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Asp-NH2 was coupled to the thiolated polymer via a thioether linkage. The coupling efficiency, as determined by GPC (Tf), SDS-PAGE [F(ab′)], or 1 H NMR (NLS peptide) was 90-95% for the Tf conjugate, and more than 95% for the F(ab′) conjugate and the NLS conjugate. The synthetic strategy described in this paper is a universal method for the preparation of conjugates of proteins and peptides with pDMAEMA in particular. This method can possibly be used to synthesize protein-polymethacrylate conjugates in general.

INTRODUCTION

Water-soluble methacrylate/methacrylamide polymers are presently under investigation for the delivery of anticancer drugs (1, 2) and for the delivery of genetic material (3). These polymers can be easily synthesized by the radical polymerization of the corresponding monomers, and their molecular weight can be controlled by the polymerization conditions. To deliver the polymerdrug conjugates or polymer-DNA complexes selectively into the target cell, a homing device (protein, sugar moiety) is often required. These homing devices are covalently coupled to the polymer of interest via established conjugation reactions using functional groups in both the polymer and the protein. Functional groups are intrinsically present in the polymer or can be introduced by copolymerization. Jelinkova et al. reported on the synthesis of conjugates of poly(hydroxypropyl methacrylamide) (HPMA) with monoclonal antibodies (MAb) by reaction of active esters of HPMA with MAb in aqueous solution (4). Besides methods based on active esters, protein conjugates can also be synthesized using methods based on the formation of disulfide bridges. The SPDP method, as first described by Carlsson et al. (5), is successfully and frequently applied for the synthesis of protein-protein conjugates (e.g., R-amylase-urease and ribonuclease-albumin) as well as for the synthesis of polymer-protein conjugates, e.g., pLys-Tf1 conjugates (6) and PEI-protein conjugates (7). Within our group, we are * To whom correspondence should be addressed. Phone: +31 30 253 6964. Fax: +31 30 251 7839. E-mail: W.E.Hennink@ pharm.uu.nl. † Department of Pharmaceutics. ‡ Department of Medicinal Chemistry. § OctoPlus BV.

working on poly[2-(dimethylamino)ethyl methacrylate] (pDMAEMA) as polymeric gene transfer agent. This polymer has good transfection potential as compared to other nonviral systems (pLys, lipofectin) (3). We are therefore interested in the design of systems based on this polymer, which selectively deliver the plasmid DNA into the nucleus of the target cell. It can be anticipated that by using a homing device the cell selectivity will increase and that by introduction of a so-called nuclear localization signal (NLS) peptide, nuclear targeting can probably be accomplished. For the synthesis of pDMAEMA-protein/peptide conjugates, the polymer has to be functionalized, since in pDMAEMA, no groups are available for the covalent linkage of a homing device. The aim of this research is to develop a universal method to couple proteins or peptides to pDMAEMA. Therefore, pDMAEMA was first functionalized with primary amino groups by copolymerization of DMAEMA with a limited amount of 2-aminoethyl methacrylate (AEMA). Next, (part of) the amino groups were translated into thiol groups, using the SPDP method, after which this thiolated polymer was coupled to three different homing devices. First, transferrin was selected as a homing device since this protein is predominantly taken up by proliferating cells (8). Second, a F(ab′) fragment of the mouse monoclonal antibody 323/A3 was 1Abbreviations: AEMA, aminoethyl methacrylate; APS, ammonium peroxydisulfate; DMAEMA, 2-(dimethylamino) ethyl methacrylate; DTT, dithiothreitol; Fmoc, 9-fluorenylmethoxycarbonyl; GPC, gel permeation chromatography; NLS, nuclear localization signal; NMP, N-methylpyrrolidone; pDMAEMA, poly(2-(dimethylamino) ethyl methacrylate); p(DMAEMA-coAEMA), poly(dimethylaminoethyl methacrylate-co-aminoethyl methacrylate); SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate; Tf, transferrin.

10.1021/bc980126+ CCC: $18.00 © 1999 American Chemical Society Published on Web 06/02/1999

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coupled. This antibody is recognized by a Mr 43 kDa membrane glycoprotein, which is highly expressed on a variety of carcinomas (9). Last, thiolated pDMAEMA was coupled via a thioether linkage to a maleimide derivatized NLS peptide. As NLS sequence, the SV40 Slarge T antigen NLS sequence (Gly-Pro-Lys-Lys-Lys-Arg-LysVal-Glu-Asp-NH2 ) was chosen because many NLS peptides contain (a variant of) this sequence (10, 11). MATERIALS AND METHODS

General. Materials. The following compounds were used as received: 2-aminoethyl methacrylate hydrochloride (AEMA, Polysciences Inc., Warrington, PA), ammonium peroxydisulfate (APS, Fluka, >98%), apotransferrin (12) (Sigma, low endotoxin, approximately 98%), dithiothreitol (DTT, Acros, 99%), 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes, Acros, 99%), 1-hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA, peptide grade, Biosolve, The Netherlands), 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids, N-methylpyrrolidone (NMP, peptide grade Biosolve), succinimidyl 4-(p-maleimidophenyl butyrate) (SMPB, Pierce), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia Biotech), and tris(hydroxymethyl)aminomethane (TRIS, Acros, 99+ %). N,N-Dimethylformamide (DMF, peptide synthesis grade, Biosolve, The Netherlands) was dried on molecular sieves before use. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, Fluka, >98% by GC) was purified by distillation under reduced pressure just before use. All other chemicals were of analytical grade. Water was purified by reversed osmosis. The PBS used in this study contained 0.1 M phosphate, pH 7.5, with 0.1 M NaCl, whereas the HBS buffer was composed of 20 mM Hepes, pH 7.4, and 0.155 M NaCl. Methods. Solid-phase peptide syntheses were carried out on an ABI 433A Peptide Synthesizer. The Fmocdeprotection reaction was monitored at 301 nm. Tentagel S RAM resin was purchased from Rapp Polymere (Tu¨bingen, Germany). Fast-atom bombardment (FAB) mass spectrometry (MS) was carried out on a JEOL JMS SX/ SX 102A four-sector mass spectrometer, coupled with a HP-9000 data system. 1H NMR spectra were recorded with a Gemini 300 MHz spectrometer (Varian Associates Inc. NMR Instruments, Palo Alto, CA). Approximately 30 mg of material was dissolved in 0.8 mL of 2H2O. The 2HOH signal at 4.8 ppm was used as the reference line. A pulse length of 4.5 ms (PW90 ≈ 12 ms) was used with a relaxation delay of 15 s. The molecular weights and molecular weight distributions of p(DMAEMA-co-AEMA) and its conjugates were determined by gel permeation chromatography (GPC) with an LC Module 1 Plus system (Waters Associates Inc., Milford, MA) and three thermostated (35 °C) columns in series (Shodex Ollpak KB-802 and KB-80, Showa Denko, Japan), equipped with a model 410 differential refractometer. The mobile phase was 0.1 M Tris (pH 7.2)/0.7 M NaNO3, and the column was calibrated using dextran standards of known molecular weights (Fluka). The amount of free transferrin (Tf) in the p(DMAEMA-co-AEMA)-Tf conjugates was determined by GPC using a TSK 3000 column, eluted with 0.1 M phosphate (pH 6.7)/0.1 M Na2SO4. Conjugated Tf eluted in the void volume of the column (5 min), whereas the retention time of free Tf was 7 min and was quantified via a calibration curve (linear range 0.01-1.0 mg/mL Tf). Purification of derivatized polymer or protein was performed by gel filtration using Sephadex G-25 superfine columns with a void volume of 2.5 mL (PD-10,

van Dijk-Wolthuis et al.

Pharmacia, Sweden). The intrinsic viscosity of p(DMAEMA-co-AEMA) was determined by measuring the specific viscosity of polymer solutions in 0.7 M NaNO3/ 0.1 M Tris, pH 7.2, at 1.0, 0.66, and 0.33% w/v and extrapolating to zero concentration. The analysis was done with an Uhbelohde viscometer at 35 °C. The pDMAEMA concentration was quantified by Bio-Rad protein assay in water at 588 nm. The absorption was corrected for the protein present in the sample, and the calibration curve was linear from 0.5 to 4 mg/mL. SDSPAGE was performed using the mini-PROTEAN II electrophoresis cell (Bio-Rad). The separating gel was 0.75 mm thick and contained 12.5% acrylamide. The gels were stained with Coomassie Blue. Synthesis of Poly(dimethylaminoethyl methacrylate-co-aminoethyl methacrylate) [p(DMAEMA-coAEMA)]. The DMAEMA-based copolymers were synthesized essentially according to Van de Wetering et al. (3). Freshly distilled 2-(dimethylamino)ethyl methacrylate (5.0 mL, 30 mmol) and varying amounts of aminoethyl methacrylate hydrochloride (0.75, 1.5 or 3 mmol) were dissolved in 25 mL of 1.3 M hydrochloric acid, and the pH was adjusted to 3. The solutions were deoxygenated by vacuum and nitrogen purging, and a calculated amount of APS (M/I 25, 50, or 100 mol/mol) was added. After polymerization in a water bath at 60 °C for 16 h, the polymers were purified by extensive dialysis against demineralized water and isolated by lyophilization. Yield: ca. 6 g (95-99%). 1H NMR (2H O): d 0.9-2.4 [5H, several bs, -CH 2 2 CR(CH3)-], 3.0 [6H, bs, -N(CH3)2], 3.45 (small, bs, CH2NH2), 3.6 [2H, bs, (CH3)2NCH2-], 4.4 (2H, bs, -OCH2-). Thiolation of p(DMAEMA-co-AEMA). To a solution of p(DMAEMA-co-AEMA) (25 mg/mL in PBS) was added a calculated amount of SPDP (40 mM in ethanol) under vigorous mixing. After 30 min at room temperature, excess reagent and N-hydroxysuccinimide were removed by gel filtration (PD-10/PBS). The pDMAEMA concentration was quantified by BioRad protein assay in water at 588 nm. The amount of incorporated PDP groups was determined by quantifying 2-mercaptopyridine (pyrSH) at 343 nm, which was released from the PDP groups by treatment of 950 mL of PDP-derivatized polymer with 50 mL of 100 mM DTT in water (molar absorptivity of pyrSH: 8.08 × 103 M-1 cm-1) (5). The thiol groups in p(DMAEMA-co-AEMA-PDP) were deprotected by treatment with excess dithiothreitol (DTT) (about 25 mg/100 mg of derivatized polymer) for 15-30 min at room temperature and subsequent gel filtration (PD-10/PBS), resulting in a solution of 11 mg/mL p(DMAEMA-co-AEMA)-SH (abbreviated as pol-SH). Thiolation of Transferrin. To a solution of transferrin (10 mg/mL) was added a calculated amount of SPDP (40 mM in ethanol) under vigorous mixing. After 30 min at room temperature, excess reagent and Nhydroxysuccinimide were removed by gel filtration (PD10/ PBS). The transferrin concentration was quantified by UV absorbance at 280 nm. The amount of incorporated PDPgroups was determined as described for PDP-derivatized p(DMAEMA-co-AEMA). Preparation of F(ab′) Fragments of the Mouse Monoclonal Antibody 323/A3. The mouse Mab 323/A3 was incubated with pepsin as described before (13) to yield F(ab′)2 fragments. It has been shown that under the selected conditions reduction of the disulfide bond between the heavy chains occurs, while the disulfide bond between the heavy and the light chain stayed intact (13).

Technical Notes

The fragments were incubated at 3 mg/mL with 25 mM DTT in acetate buffer pH 5.5 (100 mM NaAc, 100 mM NaCl, and 1 mM EDTA) for 90 min at room temperature. DTT was removed by gel filtration (PD-10/deoxygenated HBS), yielding 2.1 mg/mL F(ab′) fragments in HBS. The F(ab′) fragments, which contain a thiol group, were used immediately for covalent coupling to PDP-derivatized p(DMAEMA-co-AEMA). Synthesis of the 4-(4-Maleimidophenyl)butyrate Derivative of Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-GluAsp (NLS-MPB). The NLS decapeptide was synthesized by solid-phase synthesis. First, the amino acid Asp was attached to the resin (TentaGel S RAM). Coupling and deprotection cycles were carried out on a ABI 433A Peptide Synthesizer using ABI FastMoc 0.25 mmol protocols (14). Repetitive Fmoc cleavage was accomplished with 20% piperidine in NMP. Peptide couplings were performed using 4 molar equivalents of Fmoc-amino acid, BOP, and HOBt in the presence of DiPEA in NMP for 45 min. The Fmoc was removed by 20% piperidine in NMP. The Fmoc removal and formation of dibenzofulvene-piperidine adduct was followed by UV at 301 nm. A small portion of the resin was used for cleavage of the peptide by stirring in 95% TFA/H2O for 2 h. The resin was removed by filtration, and the filtrate was lyophilized to give the deprotected peptide, which was characterized by HPLC, 1H NMR, and FAB MS (m/z 1183.7 [M + H]+). The remaining peptide on resin (105 mg, 0.017 mmol of peptide) was weighed into a 10 mL flask, the Fmocgroup was removed using a 20% piperidine solution in NMP (stirred for 1 h at ambient temperature), after which the resin was washed with NMP (5×). The resin was suspended in NMP, after which 5 equiv (0.08 mmol) of SMPB, and 3 equiv of DiPEA were added. After 1.5 h stirring, the resin was filtered and washed with NMP. The product, containing a maleimide group, was cleaved from the resin by stirring with 95/5 TFA/water (v/v) for 2 h. The reaction mixture was filtered and the residue was washed with water. The total filtrate was lyophilized, yielding 0.039 g of peptide (0.018 mmol). 1H NMR (2H2O): d 7.02 (s, 2H, maleimide), 7.25 (m, 2H, Ph), 7.40 (m, 2H, Ph). FAB MS: m/z 1424.8 [MH]+. Synthesis of Conjugates. Conjugates of Transferrin with pDMAEMA. The p(DMAEMA-co-AEMA)-Tf conjugate was prepared by addition of 0.67 mL of 15 mg/mL Tf-PDP (2.5 mol of PDP/mol of Tf) to 2.0 mL 10 mg/mL of pol-SH [1 mol of SH/150 mol of monomeric unit; p(DMAEMA-co-AEMA) with 6 mol % AEMA, Mn 30 kDa, Mw 785 kDa (relative to dextran)]. After incubation for 16 h, the low molecular mass compounds were removed from the reaction mixture by gel filtration (PD-10/HBS). The composition of the conjugate was determined by UVanalysis [A280 nm for Tf, Bio-Rad at 588 nm in water for p(DM)AEMA], whereas the conjugation efficiency was determined by quantification of free Tf by GPC chromatography. Conjugates of F(ab′)-Fragment 323/A3 with pDMAEMA. The p(DMAEMA-co-AEMA)-F(ab′) conjugate was prepared by addition of 3.5 mL of 2.1 mg/mL F(ab′) in PBS to 0.88 mL of 17 mg/mL pol-PDP [1 PDP/140 monomeric units; p(DMAEMA-co-AEMA) with 6 mol % AEMA, Mn 30 kDa, Mw 785 kDa (relative to dextran)]. After incubation for 16 h, the low molecular mass compounds were removed from the reaction mixture by gel filtration (PD-10/HBS). The composition of the conjugate was determined by UV-analysis [A280 nm for F(ab′), Bio-Rad at 588 nm for p(DM)AEMA], whereas the conjugation efficiency was determined by SDS-PAGE analysis.

Bioconjugate Chem., Vol. 10, No. 4, 1999 689 Scheme 1. Reaction Scheme of the Synthesis of Poly[2-(dimethylamino)ethyl Methacrylate-co-2-aminoethyl Methacrylate] and Subsequent Thiolation with N-Succinimidyl 3-(2-Pyridyldithio)propionate (SPDP)

Conjugation of NLS-MPB. To 3.5 mL of 12 mg/mL p(DMAEMA-co-AEMA)-SH in PBS [1 SH/50 monomeric units; 6 mol % AEMA, Mn 30 kDa, Mw 785 kDa (relative to dextran)], 0.011 g (0.0077 mmol) of NLS-MPB dissolved in 0.28 mL of water was added. After reacting for 18 h at room temperature, the polymer-peptide conjugate was obtained by gel filtration (PD-10/water) and subsequent lyophilization. RESULTS AND DISCUSSION

Synthesis of pDMAEMA with Primary Amines. To obtain pDMAEMA with primary amino groups, which are necessary for the coupling of ligands, 2-(dimethylamino)ethyl methacrylate (DMAEMA, Scheme 1-1) was copolymerized with aminoethyl methacrylate (AEMA, 2). Since AEMA is only available as its HCl-salt, water was selected as solvent and ammonium peroxydisulfate (APS) as radical initiator (Scheme 1). The pH of the aqueous solution was set at 3-5 with hydrochloric acid in order to minimize saponification of DMAEMA (15) and to prevent reaction of the primary amino group with ester groups. The polymers were obtained in high yield (9599%). The polymer was worked up by dialysis against water at pH 7 and subsequent lyophilization. A 1H NMR spectrum of the copolymer (Figure 1A) showed, besides the signals of the dimethylaminoethyl methacrylate signals, a small signal at 3.45 ppm, which can be ascribed to the methylene protons adjacent to the primary amino group in the aminoethyl methacrylate monomer. The intensity of this peak indeed increased with increasing amounts of AEMA in the feed of the polymerization. However, the amount of incorporated amino groups cannot be determined accurately by NMR, due to partly overlapping signals. For the determination of the amount of incorporated AEMA, the primary amines in the copolymer were quantitatively derivatized with an excess of the thiolating reagent SPDP. Subsequent spectrophotometric analysis of the incorporated amount of PDP-groups yielded an accurate determination of the primary amine content of p(DMAEMA-co-AEMA). Figure 2 shows the influence of the feed ratio of AEMA on the total methacrylate content of the copolymer. It can be seen that the incorporation efficiency of AEMA is about

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Figure 3. Molecular weight by intrinsic viscosity (A) and by GPC (relative to dextran, panel B) of p(DMAEMA-co-AEMA) as a function of the monomer/initiator ratio during synthesis.

Figure 1. 1H NMR spectrum in 2H2O of p(DMAEMA-coAEMA) with 5.4 mol % AEMA (A), p(DMAEMA-co-AEMA)-NLS conjugate (B), and the MPB derivative of the NLS peptide (C).

Figure 4. Correlation between feed ratio of SPDP/AEMA and the incorporation of thiol groups in p(DMAEMA-co-AEMA).

Figure 2. Correlation between feed ratio of AEMA to total methacrylate content and the incorporation of primary amines in p(DMAEMA-co-AEMA).

70% and the primary amine content can be tailored by the amount of AEMA in the feed. The polymerization was carried out at varying ratios of monomer to initiator (M/I ratio). Figure 3 shows the influence of the M/I ratio on the molecular weight (GPC) of p(DMAEMA-co-AEMA) with 6 mol % AEMA. For comparison, the intrinsic viscosity of the polymer is also given. It can be concluded that, as expected, the molecular weight of p(DMAEMA-co-AEMA) increases with increasing M/I ratio. Conjugation of Peptides and Proteins. p(DMAEMA-

co-AEMA) contains primary amino groups which can be used to introduce (masked) thiol groups on the polymer backbone. These thiol groups can subsequently be used to couple a variety of compounds by a disulfide linkage. Therefore, p(DMAEMA-co-AEMA) is first derivatized with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP, Scheme 1, compd 4) in PBS at pH 7.5, yielding p(DMAEMA-co-AEMA) with masked thiol groups (pol-PDP). Subsequent removal of the protecting thiopyridyl group by reaction with 1,4-dithio-2,3-dihydroxybutane (dithiothreitol, DTT) resulted in p(DMAEMA-co-AEMA) with free thiol groups (pol-SH, Scheme 1, compd 6). Figure 4 shows the amount of incorporated dithiopyridine groups as a function of the feed ratio of SPDP to AEMA residues. It can be concluded that the amount of incorporated PDP groups can be easily tailored by varying the ratio of SPDP to AEMA residues in the polymer, and that the incorporation efficiency of SPDP is about 70%. Above 1.4 molar equivalents of SPDP to amino groups, a plateau value was reached, indicating complete derivatization of the amino groups. Both the polymer with the masked and the free thiol groups can be used for conjugation with compounds such as peptides and proteins via a variety of ways. Transferrin can be easily conjugated to p(DMAEMAco-AEMA) after introduction of PDP groups in Tf and subsequent reaction with pol-SH, yielding p(DM)AEMATf conjugate (Scheme 2, compd 8). For conjugation purposes with Tf, p(DMAEMA-co-AEMA) with a rela-

Technical Notes

Bioconjugate Chem., Vol. 10, No. 4, 1999 691

Scheme 2. Coupling of Transferrin (A), a F(ab′) Fragment (B), and NLS-MPB (C) to Derivatized p(DMAEMA-co-AEMA)

tively low amount of (masked) thiol groups [1 SH/150 (DM)AEMA units] was used to prevent possible crosslinking of polymer and protein. Incubation of underivatized Tf with thiolated p(DMAEMA-co-AEMA) did not result in the formation of pol-Tf conjugate, indicating that PDP groups are necessary for conjugation (results not shown). The degree of derivatization of Tf was dependent on the feed ratio of SPDP to Tf and could be tailored from 2.5 to 14 mol of PDP/mol of Tf. The higher the degree of derivatization, the more turbid the conjugation reaction mixture became, indicating the formation of cross-linked products. Therefore, Tf with a relatively low degree of derivatization (2.5 mol of PDP/mol of Tf) was used for conjugation. The conjugation efficiency could be determined by quantifying the amount of nonconjugated Tf by GPC chromatography. Above a ratio of 1:1 mol of PDP/ mol of SH, the conjugation efficiency was 90-95% and was independent of the ratio of Tf-PDP to pol-SH. The conjugate contained 1.0 mg/mL p(DMAEMA-co-AEMA) and 0.41 mg/mL Tf. Monoclonal antibody 323/A3, directed against the EGP-2 receptor on OVCAR-3 cells, was treated with pepsin to yield the corresponding [F(ab′)]2 molecule. After reaction with DTT, the F(ab′) fragment (Scheme 2, compd 9) was subsequently coupled via its free thiol group to p(DMAEMA-coAEMA) with masked thiol groups [p(DM)AEMA-PDP, Scheme 2, compd 5] to yield the p(DM)AEMA-F(ab′) conjugate (Scheme 2, compd 10). The conjugate contained 2.1 mg/mL p(DMAEMA-co-AEMA) and 1.0 mg/mL F(ab′), with a coupling efficiency of more than 95% (SDS-PAGE analysis, results not shown). After synthesis of the NLS peptide, analysis by HPLC, 1 H NMR, and FAB MS indicated that the expected product had been formed. A reaction between the immobilized NLS peptide and SMPB resulted in the formation of the maleimide derivative of the NLS peptide, which was analyzed by FAB MS and 1H NMR. The MS analysis showed the expected mass for the derivatized peptide sequence m/z 1424.8 [MH]+). Figure 1C shows

the 1H NMR of the NLS-MPB. Although the 1H NMR spectrum was too complex for complete elucidation, some specific amino acids (e.g., Val at 0.9 ppm) and the maleimide group (at 7.0 ppm) could be distinguished. The NLS-MPB (Scheme 2, compd 11) was subsequently incubated with thiolated p(DMAEMA-co-AEMA) and purified by gel filtration (PD-10). The yield after lyophilization was 37 mg (70%), and the incorporation efficiency of NLS peptide, determined by 1H NMR, was more than 95% based on SH groups, and 50% based on NLS-MPB. The isolated polymer-NLS conjugate (Scheme 2, compd 12) was characterized with 1H NMR (Figure 1B). Comparison of the NMR spectrum of NLS-MPB (Figure 1C) with the spectrum of the conjugate (Figure 1B) shows that the maleimide signal at 7.0 ppm has disappeared while the phenyl protons of the MPB group are still visible at 7.25 and 7.4 ppm. This and the presence of certain amino acid peaks in the spectrum indicates that the NLS peptide is covalently bound to the polymer. The incorporation efficiency of NLS peptide, estimated by 1H NMR, was more than 95% based on thiol groups in the polymer, and 50% based on NLS-MPB. It has been demonstrated that conjugates held together via disulfide bonds introduced via the SPDP method may be reduced in vivo (16). However, it can be envisaged that protein/polymer conjugates can be synthesized via a more stable thioether linkage essentially using the method described for the NLS-PDMAEMA conjugate. In addition, other methods described in the literature (e.g., as described in ref 17) can also be applied to synthesize PDMAEMA-protein conjugates using essentially the same strategy as described in this paper. CONCLUSIONS

pDMAEMA can be functionalized with primary amines by copolymerization of DMAEMA with low amount of AEMA. The molecular weight and the primary amine content of the resulting polymer can be tailored by the feed ratio of these monomers. Thiol groups can be

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incorporated via a controlled derivatization step. Subsequently, proteins and peptides can be coupled to the polymer with high coupling efficiencies via a disulfide bond or a thioether linkage. LITERATURE CITED (1) Duncan, R. (1992) Drug-polymer conjugates: potential for improved chemotherapy. Anti-Cancer Drugs 3, 175-210. (2) Flanagan, P. A., Duncan, R., Subr, V., Ulbrich, K., Kopeckova, P. and Kopecek, J. (1992) Evaluation of protein-N(2-hydroxypropyl)methacylamide copolymer conjugates as targetable drug-carriers. 2. Body distribution of conjugates containing transferrin antitransferrin receptor antibody or anti-Thy 1.2 antibody and effectiveness of transferrin. J. Controlled Release 18, 25-38. (3) Van de Wetering, P., Cherng, J.-Y., Talsma, H., and Hennink, W. E. (1997) Relation between the transfection efficiency and cytotoxicity of poly((2-dimethylamino)- ethyl methacrylate)-plasmid complexes. J. Controlled Release 49, 59-69. (4) Jelinkova, M., Strohalm, J., Plocova, D., Subr, V., St′astny, M., Ulbrich, K., and Rihova, B. (1998) Targeting of human and mouse T-lymphocytes by monoclonal antibody-HPMA copolymer-doxorubicin conjugates directed against different T-cell surface antigen. J. Controlled Release 52, 253-270. (5) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. Nsuccinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. (6) Wagner, E., Cotton, M., Mechtler, K., Kirlappos, H., and Birnsteil, M. (1991) DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety. Bioconjugate Chem. 2, 226-231. (7) Kircheis, R., Kichler, A., Wallner, G., Kursa, M., Ogris, M, Felzmann, T., Buchberger, M., and Wagner, E. (1997) Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4, 409-418.

van Dijk-Wolthuis et al. (8) Wagner, E., Curiel, D., and Cotton, M. (1994) Delivery of drugs, proteins and genes into cells using transferrin as ligand for receptor-mediated endocytosis. Adv. Drug Del. Rev. 14, 113-136. (9) Edwards, D. P., Grzyb, K. T., Dressler, L. G., Mansel, R. E., Zava, D. T., Sledge Jr., G. W., and McGuire, W. L. (1986) Monoclonal antibody identification and characterization of a Mr 43, 000 membrane glycoprotein associated with human breast cancer. Cancer Res. 4, 1306-1317. (10) Garcia-Bustos, J., Heitman, J., and Hall, M. N. (1991) Nuclear protein localization. Biochim. Biophys. Acta 1071, 83-101. (11) Boulikas, T. (1997) Nuclear localization signal peptides for import of plasmid DNA in gene therapy. Int. J. Oncol. 10, 301-309. (12) Wherever transferrin (or Tf) is mentioned in the text, apotransferrin (i.e., not loaded with iron) is meant. (13) Na¨ssander, U.K., Steerenberg, P. A., de Jong, W. H., van Overveld, W. O. W. M., te Boekhorst, C. M. E., Poels, L. G., Jap, P. H. K., and Storm, G. (1995) Design of immunolipososmes directed against human ovarian carcinoma. Biochim. Biophys. Acta 1235, 126-139. (14) Applied Biosystems Model 433A Peptide Synthesizer User’s Manual, June 1993, version 1.0. (15) Van de Wetering, P., Zuidam, N. J., Van Steenbergen, M. J., Van der Houwen, O. A. G. J., Underberg, W. J. M., and Hennink, W. E. (1998) A mechanistic Study of the Hydrolytic Stability of Poly(2-(dimethyl)aminoethyl methacrylate). Macromolecules 31, 8063-8068. (16) Worrell, N. R., Cumber, A. J., Parnell, G. D., Ross, W. C., and Forrester, J. A. (1986) Fate of an antibody-ricin A Chain conjugate administered to normal rats. Biochem. Pharmacol. 35, 417-423. (17) Arpicco, S., Dosio, F., Brusa, P., Crosasso, P., and Cattel, L. (1997) New coupling reagents for the preparation of disulfide cross-linked conjugates with increased stability. Bioconjugate Chem. 8, 327-337.

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