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Preparation and biological evaluation of polymerizable antibody Fab′ fragment targeted polymeric drug delivery system
Journal of Controlled Release 74 (2001) 263–268 www.elsevier.com / locate / jconrel
Preparation and biological evaluation of polymerizable antibody Fab9 fragment targeted polymeric drug delivery system ˇ ´ a,b , J. Kopecek ˇ a,b , * Z.-R. Lu a , J.-G. Shiah a , P. Kopeckova a
Department of Pharmaceutics and Pharmaceutical Chemistry /CCCD, 30 S 2000 E Rm 301, University of Utah, Salt Lake City, UT 84112, USA b Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
Abstract A new polymerizable antibody Fab9 fragment with a PEG spacer (MA-PEG-Fab9) was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab9 possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab9 fragment MA-Fab9 with a short spacer. The MA-PEG-Fab9 was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce 6 producing an Fab9 targeted HPMA copolymer-Mce 6 conjugate. The number and weight average molecular weights of the copolymer were 164 000 and 271 000 Da, respectively. About two MA-PEG-Fab9 fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab9 targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate. 2001 Elsevier Science B.V. All rights reserved. Keywords: HPMA copolymer; Antibody fragment; Mesochlorin e 6 ; Drug targeting
1. Introduction The incorporation of antibodies and their fragments to polymeric drug delivery systems may significantly enhance the targeting ability of the polymeric drug delivery systems by the combination of passive targeting with active targeting [1,2]. Recently, we have designed and prepared a new targeted polymeric drug delivery system for mesochlorin e 6 (Mce 6 ), a photodynamic anticancer drug, using the polymerizable Fab9 fragment (MA-Fab9) [3]. OV-TL 16 antibody [4], which recognizes the *Corresponding author. Tel.: 11-801-581-4532; fax: 11-801581-3674. ˇ E-mail address: [email protected] (J. Kopecek).
OA-3 antigen expressed on most human ovarian carcinomas, was used to prepare the polymerizable antibody Fab9 fragments. We have previously reported on the synthesis and in vitro studies of cytotoxicity of the MA-Fab9 targeted HPMA copolymer-Mce 6 conjugate toward OVCAR-3 human ovarian carcinoma cells [3]. This study has indicated that the MA-Fab9 targeted delivery system was recognized and internalized more efficiently by the OVCAR-3 human ovarian carcinoma cells and exhibited a higher cytotoxicity (IC 50 2.6 mM) than the non-targeted system (IC 50 230 mM) [3]. Here, we re-designed the structure of the spacer between the polymerizable double bond and the Fab9 fragment. Poly(ethylene glycol) was used as the spacer to improve the water-solubility of the spacer and to
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00332-7
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avoid using organic solvent for dissolving the spacer in the conjugation reaction. The organic solvent may denature the antibody fragments. The long spacer may reduce the steric effect of the Fab9 molecule on the reactivity of the polymerizable Fab9 fragments in copolymerization. A new Fab9 targeted HPMA copolymer-Mce 6 conjugate was prepared and preliminary investigations of its in vivo antitumor activity has been completed.
2. Experimental
2.1. Synthesis of monomer ( I) Boc-NH-PEG-NH 2 (Mw 53400, confirmed by MALDI-TOF mass spectrometry) was purchased from Shearwater Polymers (Huntsville, AL). BocNH-PEG-NH 2 (347 mg, Mw 53400) and MA-GGONp [5] (52 mg, excess) were dissolved in 10 ml THF with stirring, and DMAP (15 mg) was added. The reaction mixture was stirred for 3 days at r.t. The solvent was removed under vacuum. The residue was washed with ether giving a colorless solid. The solid was dissolved in 5.0 ml CF 3 COOH, and stirred for 45 min for Boc deprotection. The solution was added to ether dropwise to precipitate the product (monomer (I)). The colorless solid product was collected by filtration, washed with ether, and dried under vacuum. Yield: 340 mg (95%). The average molecular weight of monomer (I) was 3500 Da as determined by MALDI-TOF MS. 1 H NMR (ppm, CDCl 3 ): 7.48 (sh, 3H, NH 1 3 ); 7.35, 7.15, 7.05 (sh, 1H each, NH); 5.81, 5.37 (d, 2H, =CH 2 ); 4.00 (d, 2H, CH 2 of Gly); 3.92 (d, 2H, CH 2 of Gly); 3.61 (s, CH 2 of PEG); 1.90 (s, 3H, CH 3 ).
2.2. Synthesis of monomer ( II) The monomer (I) (340 mg) was reacted with N-hydroxysuccinimide N-maleimidocaproate [6] (100 mg, excess) in 15 ml THF in the presence of DMAP (30 mg). The reaction mixture was stirred at r.t. for 24 h. Any solid was removed by filtration, and the solvent was removed under vacuum. The polymer was purified by SEC on a Sephadex LH-20 column, eluted with methanol. The polymer fraction was collected, concentrated and precipitated in ether.
Yield: 230 mg (68%). The average molecular weight of the monomer (II) was 3700 Da as determined by MALDI-TOF mass spectrometry. 1 H-NMR (ppm, CDCl 3 ): 7.30 (sh, 2H, NH); 7.05, 6.95 (sh, 1H each, NH); 6.65 (s, 2H, CH=CH); 5.81, 5.38 (d, 2H, =CH 2 ); 4.00 (d, 2H, CH 2 of Gly); 3.92 (d, 2H, CH 2 of Gly); 3.61 (s, CH 2 of PEG); 3.36 (t, 2H, CH 2 ); 2.14 (t, 2h, CH 2 ); 1.90 (s, 3H, CH 3 ); 1.88 (m, 2H, CH 2 ); 1.62 (m, 2H, CH 2 ); 1.25 (m, 2H, CH 2 ).
2.3. Preparation of MA-PEG-Fab9 The OV-TL 16 antibody was produced by a hybridoma cell line in the CELLMAX bioreactor with serum free medium (Gibco). F(ab9) 2 and Fab9 fragments were prepared as previously described [3]. The freshly prepared Fab9 (96 mg) was reacted with 35 mg monomer (II) at 48C overnight. The MAPEG-Fab9 was purified by SEC on a Superdex 200 column (HR 16 / 60) eluted with Tris buffer (pH 7.4). The MA-PEG-Fab9 was concentrated by ultrafiltration with Centricon-30 concentrators (Mw cut off5 30 000). Yield: 55 mg.
2.4. Copolymerization of HPMA, MA-Mce6 and MA-PEG-Fab9 MA-Gly-Phe-Leu-Gly-Mce 6 (MA-Mce 6 ) [3] (8.6 mg, 7.9 mmol) was dissolved in 1.0 ml Tris buffer (pH 8.5) and mixed with HPMA [7] (32 mg, 0.22 mmol), VA-044 (gift from Wako Pure Chemical Industries, Japan) (19 mg), and MA-PEG-Fab9, (50 mg, 1.0 mmol) in 5 ml Tris buffer. The mixture was saturated with nitrogen, and sealed in an ampoule. The reaction mixture was incubated at 378C for 3h. The copolymer was separated from unreacted HPMA, MA-Mce 6 and initiator by SEC on a Sephadex G-25 column. The copolymer was also separated from MA-PEG-Fab9 by SEC on a Superose 6 column (HR 16 / 60). The number average and weight average molecular weights of the copolymer were 164 000 and 271 000, respectively, as determined by SEC (Superose 6, HR 16 / 60) equipped with a light-scattering detector (MiniDawn, Wyatt Technology, Santa Barbara, CA). The determination of the content of Mce 6 and Fab9 was the same as previously described [3].
Z.-R. Lu et al. / Journal of Controlled Release 74 (2001) 263 – 268
2.5. Antitumor activity of the Fab9 targeted HPMA copolymer-Mce6 conjugate ( PHPMA-Fab9 -Mce6 ) Female nu / nu athymic mice (5–6 weeks old; 16–18 g; purchased from Simonsen Laboratories, Gilroy, CA, USA) were acclimated to a typical animal laboratory environment for 2 weeks before receiving any treatment. The xenografts of human ovarian OVCAR-3 carcinoma were obtained by subcutaneously implanting 2310 6 OVCAR-3 cells from cell culture in nude mice, as previously described [8,9]. Experiments were not initiated until a consistent growth rate and a minimum tumor volume of 20 mm 3 was achieved. The animals were cared for following the guidelines of an approved protocol from the Institutional Animal Care and Use Committee, University of Utah. The antitumor activity of the PHPMA-Fab9-Mce 6 conjugate was evaluated in nude mice bearing human ovarian OVCAR-3 carcinoma xenografts. The mice received an i.v. administration of conjugate solution at a Mce 6 equivalent dose of 1.5 mg / kg. The tumors
265
received a light dose of 220 J / cm 2 (200 mW/ cm 2 ) from a KTP dye-laser (650 nm) 18 h after drug administration. Tumor growth was monitored by measuring the tumor volume using a digital caliper.
3. Results and discussion The spacer previously used in the preparation of the polymerizable Fab9 fragments showed a poor water solubility and a considerable amount of DMSO had to be used for conjugation with Fab9 [3]. It was also observed that the reactivity of polymerizable Fab9 fragments in the copolymerization with HPMA increased with the length of the spacer between the double bond and the Fab9 fragment [10]. Here, we designed a new spacer for the MA-Fab9 by using poly(ethylene glycol) (PEG) with molecular weight of 3400 Da. The procedure for the synthesis of the monomers with a PEG spacer is shown in Scheme 1. A new polymerizable Fab9 fragment, MA-PEG-Fab9, was prepared by the conjugation of the C-terminal
Scheme 1.
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SH group of Fab9 fragment to the maleimido group of monomer (II). The Fab9 fragment of OV-TL 16 was prepared as previously described [3]. The Fab9 targeted HPMA copolymer-Mce 6 conjugate was prepared by the copolymerization of HPMA and a monomer containing Mce 6 [3] with the MA-PEGFab9 in the presence of initiator, 2,29-azobis[2-(2imidazolin-2-yl)propane] (VA-044). A non-targeted HPMA copolymer containing Mce 6 (PHPMA-Mce 6 ) was prepared as a control. The monomer (I) was characterized by 1 H NMR and MALDI-TOF mass spectrometry. The molecular weight of monomer (I) was 3500 Da as determined by MALDI-TOF mass spectrometry. The mass increment of each peak in the mass spectrum of monomer (I) was 82 Da as compared with that of Boc-NHPEG-NH 2 , which corresponded to the mass increase after conjugating to a MA-Gly-Gly residue and the deprotection. The molecular weight of monomer (I) was 3700 Da and the mass increment of each peak in the mass spectrum of monomer (II) was 191 Da as compared with monomer (I), which corresponded to the mass increase after conjugating to the maleimido group. The mass spectrum also showed high conversion to monomers (I) and (II). Double bond protons of the methacryloyl group and maleimido group were
identified in the 1 H NMR spectrum of monomer (I). The number and weight average molecular weights of the Fab9 targeted HPMA copolymer-Mce 6 (PHPMA-Fab9-Mce 6 ) conjugate were 164 000 and 271 000 Da, respectively, as determined by SEC equipped with a light-scattering detector. The content of Fab9 fragment and Mce 6 in the copolymer was determined by UV spectroscopy after purification and lyophilization (Fig. 1). The molar ratio of Fab9:Mce 6 :HPMA was 1:9:95 as calculated based on the mass balance and the content of Fab9 and Mce 6 determined by UV spectroscopy. There were on average two Fab9 fragments incorporated in each copolymer, as estimated from the measured number average molecular weight and the calculated molecular weight of the copolymer. The MA-PEG-Fab9 possessed a higher reactivity in the copolymerization with HPMA than the MAFab9 with a short spacer. Most of MA-PEG-Fab9 fragments were incorporated in the copolymer as shown by the SEC analysis of the reaction mixture. The number of the antibody Fab9 fragments incorporated in the copolymer increased due to the reduced steric hindrance of the MA-PEG-Fab9 as a result of using PEG as the spacer. About one Fab9 fragment was copolymerized into the copolymer for the MA-
Fig. 1. UV spectra of Mce 6 , PHPMA-Mce 6 and PHPMA-Fab9-Mce 6 (in PBS buffer, pH 7.4).
Z.-R. Lu et al. / Journal of Controlled Release 74 (2001) 263 – 268
Fab9 with a short spacer, and the conversion of MA-Fab9 was low [3]. The MA-PEG-Fab9 has a much larger hydrodynamic volume of than the Fab9 because of the large hydrodynamic volume of PEG as shown by the SEC. The in vitro cytotoxicity of the MA-Fab9 targeted HPMA copolymer-Mce 6 conjugate against OVCAR3 human ovarian carcinoma cells was previously investigated [3]. The MA-Fab9 targeted conjugate was internalized much more efficiently than the nontargeted polymeric conjugate and exhibited much higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells. Based on these results, we performed a preliminary investigation on the in vivo antitumor activity of the MA-PEG-Fab9 targeted HPMA copolymer-Mce 6 conjugate in nude mice bearing OVCAR-3 human carcinoma xenografts. The antitumor efficacy of photodynamic treatment of OVCAR-3 xenografts in nude mice with nontargeted and OV-TL 16 Fab9 fragment targeted copolymer-Mce 6 conjugates is shown in Fig. 2. The
267
results indicated that one photodynamic treatment of HPMA copolymer-Mce 6 conjugate (Mce 6 equivalent dose of 1.5 mg / kg) was not able to inhibit tumor growth significantly when compared with controls. On the other hand, tumor growth in mice that received Fab9 targeted Mce 6 conjugate was obviously suppressed. It appears that one PDT treatment with the targeted conjugate can inhibit the tumor growth for approximately 3 weeks. The enhanced antitumor efficacy may probably be attributed to both antibody targeting and the enhanced permeability and retention (EPR) effect [1,2,8,9,11]. The extensive in vivo antitumor activity of the MA-Fab9 targeted HPMA copolymer-Mce 6 is still ongoing.
4. Conclusions A new polymerizable Fab9 fragment (MA-PEGFab9) was prepared by using PEG as the spacer. The conjugation of PEG to Fab9 increased the hydro-
Fig. 2. Antitumor efficacy in photodynamic therapy with non-targeted and OV-TL 16 Fab9 fragment targeted PHPMA-Mce 6 conjugates toward OVCAR-3 xenografts in nude mice; control (♦), one treatment with PHPMA-Mce 6 (m), and one treatment with PHPMA-Fab9-Mce 6 (d).
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dynamic volume of the Fab9 fragment conjugate as compared with that of Fab9 fragment. The results of the preliminary in vivo investigation indicate that the concept of using polymerizable Fab9 fragments for drug targeting is effective and provides a new paradigm for the synthesis of targeted polymeric drug delivery systems.
Acknowledgements This research was supported in part by NIH grant CA51578 from the National Cancer Institute. We thank Dr. L. Poels (University of Nijmegen, The Netherlands) for the generous gift of the hybridoma producing OV-TL 16 antibody.
References ˇ ˇ ´ T. Minko, Z.-R. Lu, HPMA [1] J. Kopecek, P. Kopeckova, copolymer-anticancer drug conjugates: design, activity, and mechanism of action, Eur. J. Pharm. Biopharm. 50 (2000) 61–81. [2] H. Maeda, L.M. Seymour, Y. Miyamoto, Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vitro, Bioconjug. Chem. 3 (1992) 351– 362. ˇ ´ J. Kopecek, ˇ [3] Z.-R. Lu, P. Kopeckova, Polymerizable Fab9 antibody fragments for targeting of anticancer drugs, Nat. Biotechnol. 17 (1999) 1101–1104.
[4] O. Boerman, L. Massuger, K. Makkink, C. Thomas, P. Kenemans, L. Poels, Comparative in vitro binding and biodistribution in tumor-bearing athymic mice of antiovarian carcinoma monoclonal antibodies, Anticancer Res. 10 (1990) 1289–1296. ˇ ´ J. Strohalm, K. Ulbrich, B. [5] J. Kopecek, P. Rejmanova, ´ ´ V. Chytry, ˘ J.B. Lloyd, R. Duncan, Synthetic polyRıhova, meric drugs. US Pat. 5,037,883, Aug. 6, 1991. [6] A.S. Kalgutkar, B.C. Crews, L.J. Marnett, Design, synthesis and biological evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases, J. Med. Chem. 39 (1996) 1692–1703. ¨ ´ ˇ [7] J. Kopecek, H. Bazilova, Poly[N-(2-hydroxypropyl)methacrylamide]. 1. Radical polymerization and copolymerization, Eur. Polym. J. 9 (1973) 7–14. ˇ [8] J.G. Shiah, Y. Sun, C.M. Peterson, J. Kopecek, Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e 6 and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts, J. Control. Release 61 (1999) 145–157. ˇ [9] J.G. Shiah, Y. Sun, C.M. Peterson, R.C. Straight, J. Kopecek, Antitumor activity of N-(2-hydroxypropyl)methacrylamide copolymer-mesochlorin e 6 and adriamycin conjugates in combination treatments, Clin. Cancer Res. 6 (2000) 1008– 1015. ˇ ´ J. Kopecek, ˇ [10] Z.-R. Lu, P. Kopeckova, Polymerizable Fab9 antibody fragments, Proc. Int. Symp. Control. Release Bioact. Mater. 25 (1998) 788–789. ˇ´ P. Kopeckova, ˇ ´ Y. Sun, C.M. Peterson, [11] J.G. Shiah, M. Dvorak, ˇ J. Kopecek, Biodistribution and antitumor efficacy of longcirculating N-(2-hydroxypropyl)methacrylamide copolymerdoxorubicin conjugates in nude mice, Eur. J. Cancer 37 (2001) 131–139.
Preparation and biological evaluation of polymerizable antibody Fab9 fragment targeted polymeric drug delivery system ˇ ´ a,b , J. Kopecek ˇ a,b , * Z.-R. Lu a , J.-G. Shiah a , P. Kopeckova a
Department of Pharmaceutics and Pharmaceutical Chemistry /CCCD, 30 S 2000 E Rm 301, University of Utah, Salt Lake City, UT 84112, USA b Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
Abstract A new polymerizable antibody Fab9 fragment with a PEG spacer (MA-PEG-Fab9) was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab9 possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab9 fragment MA-Fab9 with a short spacer. The MA-PEG-Fab9 was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce 6 producing an Fab9 targeted HPMA copolymer-Mce 6 conjugate. The number and weight average molecular weights of the copolymer were 164 000 and 271 000 Da, respectively. About two MA-PEG-Fab9 fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab9 targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate. 2001 Elsevier Science B.V. All rights reserved. Keywords: HPMA copolymer; Antibody fragment; Mesochlorin e 6 ; Drug targeting
1. Introduction The incorporation of antibodies and their fragments to polymeric drug delivery systems may significantly enhance the targeting ability of the polymeric drug delivery systems by the combination of passive targeting with active targeting [1,2]. Recently, we have designed and prepared a new targeted polymeric drug delivery system for mesochlorin e 6 (Mce 6 ), a photodynamic anticancer drug, using the polymerizable Fab9 fragment (MA-Fab9) [3]. OV-TL 16 antibody [4], which recognizes the *Corresponding author. Tel.: 11-801-581-4532; fax: 11-801581-3674. ˇ E-mail address: [email protected] (J. Kopecek).
OA-3 antigen expressed on most human ovarian carcinomas, was used to prepare the polymerizable antibody Fab9 fragments. We have previously reported on the synthesis and in vitro studies of cytotoxicity of the MA-Fab9 targeted HPMA copolymer-Mce 6 conjugate toward OVCAR-3 human ovarian carcinoma cells [3]. This study has indicated that the MA-Fab9 targeted delivery system was recognized and internalized more efficiently by the OVCAR-3 human ovarian carcinoma cells and exhibited a higher cytotoxicity (IC 50 2.6 mM) than the non-targeted system (IC 50 230 mM) [3]. Here, we re-designed the structure of the spacer between the polymerizable double bond and the Fab9 fragment. Poly(ethylene glycol) was used as the spacer to improve the water-solubility of the spacer and to
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00332-7
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avoid using organic solvent for dissolving the spacer in the conjugation reaction. The organic solvent may denature the antibody fragments. The long spacer may reduce the steric effect of the Fab9 molecule on the reactivity of the polymerizable Fab9 fragments in copolymerization. A new Fab9 targeted HPMA copolymer-Mce 6 conjugate was prepared and preliminary investigations of its in vivo antitumor activity has been completed.
2. Experimental
2.1. Synthesis of monomer ( I) Boc-NH-PEG-NH 2 (Mw 53400, confirmed by MALDI-TOF mass spectrometry) was purchased from Shearwater Polymers (Huntsville, AL). BocNH-PEG-NH 2 (347 mg, Mw 53400) and MA-GGONp [5] (52 mg, excess) were dissolved in 10 ml THF with stirring, and DMAP (15 mg) was added. The reaction mixture was stirred for 3 days at r.t. The solvent was removed under vacuum. The residue was washed with ether giving a colorless solid. The solid was dissolved in 5.0 ml CF 3 COOH, and stirred for 45 min for Boc deprotection. The solution was added to ether dropwise to precipitate the product (monomer (I)). The colorless solid product was collected by filtration, washed with ether, and dried under vacuum. Yield: 340 mg (95%). The average molecular weight of monomer (I) was 3500 Da as determined by MALDI-TOF MS. 1 H NMR (ppm, CDCl 3 ): 7.48 (sh, 3H, NH 1 3 ); 7.35, 7.15, 7.05 (sh, 1H each, NH); 5.81, 5.37 (d, 2H, =CH 2 ); 4.00 (d, 2H, CH 2 of Gly); 3.92 (d, 2H, CH 2 of Gly); 3.61 (s, CH 2 of PEG); 1.90 (s, 3H, CH 3 ).
2.2. Synthesis of monomer ( II) The monomer (I) (340 mg) was reacted with N-hydroxysuccinimide N-maleimidocaproate [6] (100 mg, excess) in 15 ml THF in the presence of DMAP (30 mg). The reaction mixture was stirred at r.t. for 24 h. Any solid was removed by filtration, and the solvent was removed under vacuum. The polymer was purified by SEC on a Sephadex LH-20 column, eluted with methanol. The polymer fraction was collected, concentrated and precipitated in ether.
Yield: 230 mg (68%). The average molecular weight of the monomer (II) was 3700 Da as determined by MALDI-TOF mass spectrometry. 1 H-NMR (ppm, CDCl 3 ): 7.30 (sh, 2H, NH); 7.05, 6.95 (sh, 1H each, NH); 6.65 (s, 2H, CH=CH); 5.81, 5.38 (d, 2H, =CH 2 ); 4.00 (d, 2H, CH 2 of Gly); 3.92 (d, 2H, CH 2 of Gly); 3.61 (s, CH 2 of PEG); 3.36 (t, 2H, CH 2 ); 2.14 (t, 2h, CH 2 ); 1.90 (s, 3H, CH 3 ); 1.88 (m, 2H, CH 2 ); 1.62 (m, 2H, CH 2 ); 1.25 (m, 2H, CH 2 ).
2.3. Preparation of MA-PEG-Fab9 The OV-TL 16 antibody was produced by a hybridoma cell line in the CELLMAX bioreactor with serum free medium (Gibco). F(ab9) 2 and Fab9 fragments were prepared as previously described [3]. The freshly prepared Fab9 (96 mg) was reacted with 35 mg monomer (II) at 48C overnight. The MAPEG-Fab9 was purified by SEC on a Superdex 200 column (HR 16 / 60) eluted with Tris buffer (pH 7.4). The MA-PEG-Fab9 was concentrated by ultrafiltration with Centricon-30 concentrators (Mw cut off5 30 000). Yield: 55 mg.
2.4. Copolymerization of HPMA, MA-Mce6 and MA-PEG-Fab9 MA-Gly-Phe-Leu-Gly-Mce 6 (MA-Mce 6 ) [3] (8.6 mg, 7.9 mmol) was dissolved in 1.0 ml Tris buffer (pH 8.5) and mixed with HPMA [7] (32 mg, 0.22 mmol), VA-044 (gift from Wako Pure Chemical Industries, Japan) (19 mg), and MA-PEG-Fab9, (50 mg, 1.0 mmol) in 5 ml Tris buffer. The mixture was saturated with nitrogen, and sealed in an ampoule. The reaction mixture was incubated at 378C for 3h. The copolymer was separated from unreacted HPMA, MA-Mce 6 and initiator by SEC on a Sephadex G-25 column. The copolymer was also separated from MA-PEG-Fab9 by SEC on a Superose 6 column (HR 16 / 60). The number average and weight average molecular weights of the copolymer were 164 000 and 271 000, respectively, as determined by SEC (Superose 6, HR 16 / 60) equipped with a light-scattering detector (MiniDawn, Wyatt Technology, Santa Barbara, CA). The determination of the content of Mce 6 and Fab9 was the same as previously described [3].
Z.-R. Lu et al. / Journal of Controlled Release 74 (2001) 263 – 268
2.5. Antitumor activity of the Fab9 targeted HPMA copolymer-Mce6 conjugate ( PHPMA-Fab9 -Mce6 ) Female nu / nu athymic mice (5–6 weeks old; 16–18 g; purchased from Simonsen Laboratories, Gilroy, CA, USA) were acclimated to a typical animal laboratory environment for 2 weeks before receiving any treatment. The xenografts of human ovarian OVCAR-3 carcinoma were obtained by subcutaneously implanting 2310 6 OVCAR-3 cells from cell culture in nude mice, as previously described [8,9]. Experiments were not initiated until a consistent growth rate and a minimum tumor volume of 20 mm 3 was achieved. The animals were cared for following the guidelines of an approved protocol from the Institutional Animal Care and Use Committee, University of Utah. The antitumor activity of the PHPMA-Fab9-Mce 6 conjugate was evaluated in nude mice bearing human ovarian OVCAR-3 carcinoma xenografts. The mice received an i.v. administration of conjugate solution at a Mce 6 equivalent dose of 1.5 mg / kg. The tumors
265
received a light dose of 220 J / cm 2 (200 mW/ cm 2 ) from a KTP dye-laser (650 nm) 18 h after drug administration. Tumor growth was monitored by measuring the tumor volume using a digital caliper.
3. Results and discussion The spacer previously used in the preparation of the polymerizable Fab9 fragments showed a poor water solubility and a considerable amount of DMSO had to be used for conjugation with Fab9 [3]. It was also observed that the reactivity of polymerizable Fab9 fragments in the copolymerization with HPMA increased with the length of the spacer between the double bond and the Fab9 fragment [10]. Here, we designed a new spacer for the MA-Fab9 by using poly(ethylene glycol) (PEG) with molecular weight of 3400 Da. The procedure for the synthesis of the monomers with a PEG spacer is shown in Scheme 1. A new polymerizable Fab9 fragment, MA-PEG-Fab9, was prepared by the conjugation of the C-terminal
Scheme 1.
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SH group of Fab9 fragment to the maleimido group of monomer (II). The Fab9 fragment of OV-TL 16 was prepared as previously described [3]. The Fab9 targeted HPMA copolymer-Mce 6 conjugate was prepared by the copolymerization of HPMA and a monomer containing Mce 6 [3] with the MA-PEGFab9 in the presence of initiator, 2,29-azobis[2-(2imidazolin-2-yl)propane] (VA-044). A non-targeted HPMA copolymer containing Mce 6 (PHPMA-Mce 6 ) was prepared as a control. The monomer (I) was characterized by 1 H NMR and MALDI-TOF mass spectrometry. The molecular weight of monomer (I) was 3500 Da as determined by MALDI-TOF mass spectrometry. The mass increment of each peak in the mass spectrum of monomer (I) was 82 Da as compared with that of Boc-NHPEG-NH 2 , which corresponded to the mass increase after conjugating to a MA-Gly-Gly residue and the deprotection. The molecular weight of monomer (I) was 3700 Da and the mass increment of each peak in the mass spectrum of monomer (II) was 191 Da as compared with monomer (I), which corresponded to the mass increase after conjugating to the maleimido group. The mass spectrum also showed high conversion to monomers (I) and (II). Double bond protons of the methacryloyl group and maleimido group were
identified in the 1 H NMR spectrum of monomer (I). The number and weight average molecular weights of the Fab9 targeted HPMA copolymer-Mce 6 (PHPMA-Fab9-Mce 6 ) conjugate were 164 000 and 271 000 Da, respectively, as determined by SEC equipped with a light-scattering detector. The content of Fab9 fragment and Mce 6 in the copolymer was determined by UV spectroscopy after purification and lyophilization (Fig. 1). The molar ratio of Fab9:Mce 6 :HPMA was 1:9:95 as calculated based on the mass balance and the content of Fab9 and Mce 6 determined by UV spectroscopy. There were on average two Fab9 fragments incorporated in each copolymer, as estimated from the measured number average molecular weight and the calculated molecular weight of the copolymer. The MA-PEG-Fab9 possessed a higher reactivity in the copolymerization with HPMA than the MAFab9 with a short spacer. Most of MA-PEG-Fab9 fragments were incorporated in the copolymer as shown by the SEC analysis of the reaction mixture. The number of the antibody Fab9 fragments incorporated in the copolymer increased due to the reduced steric hindrance of the MA-PEG-Fab9 as a result of using PEG as the spacer. About one Fab9 fragment was copolymerized into the copolymer for the MA-
Fig. 1. UV spectra of Mce 6 , PHPMA-Mce 6 and PHPMA-Fab9-Mce 6 (in PBS buffer, pH 7.4).
Z.-R. Lu et al. / Journal of Controlled Release 74 (2001) 263 – 268
Fab9 with a short spacer, and the conversion of MA-Fab9 was low [3]. The MA-PEG-Fab9 has a much larger hydrodynamic volume of than the Fab9 because of the large hydrodynamic volume of PEG as shown by the SEC. The in vitro cytotoxicity of the MA-Fab9 targeted HPMA copolymer-Mce 6 conjugate against OVCAR3 human ovarian carcinoma cells was previously investigated [3]. The MA-Fab9 targeted conjugate was internalized much more efficiently than the nontargeted polymeric conjugate and exhibited much higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells. Based on these results, we performed a preliminary investigation on the in vivo antitumor activity of the MA-PEG-Fab9 targeted HPMA copolymer-Mce 6 conjugate in nude mice bearing OVCAR-3 human carcinoma xenografts. The antitumor efficacy of photodynamic treatment of OVCAR-3 xenografts in nude mice with nontargeted and OV-TL 16 Fab9 fragment targeted copolymer-Mce 6 conjugates is shown in Fig. 2. The
267
results indicated that one photodynamic treatment of HPMA copolymer-Mce 6 conjugate (Mce 6 equivalent dose of 1.5 mg / kg) was not able to inhibit tumor growth significantly when compared with controls. On the other hand, tumor growth in mice that received Fab9 targeted Mce 6 conjugate was obviously suppressed. It appears that one PDT treatment with the targeted conjugate can inhibit the tumor growth for approximately 3 weeks. The enhanced antitumor efficacy may probably be attributed to both antibody targeting and the enhanced permeability and retention (EPR) effect [1,2,8,9,11]. The extensive in vivo antitumor activity of the MA-Fab9 targeted HPMA copolymer-Mce 6 is still ongoing.
4. Conclusions A new polymerizable Fab9 fragment (MA-PEGFab9) was prepared by using PEG as the spacer. The conjugation of PEG to Fab9 increased the hydro-
Fig. 2. Antitumor efficacy in photodynamic therapy with non-targeted and OV-TL 16 Fab9 fragment targeted PHPMA-Mce 6 conjugates toward OVCAR-3 xenografts in nude mice; control (♦), one treatment with PHPMA-Mce 6 (m), and one treatment with PHPMA-Fab9-Mce 6 (d).
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dynamic volume of the Fab9 fragment conjugate as compared with that of Fab9 fragment. The results of the preliminary in vivo investigation indicate that the concept of using polymerizable Fab9 fragments for drug targeting is effective and provides a new paradigm for the synthesis of targeted polymeric drug delivery systems.
Acknowledgements This research was supported in part by NIH grant CA51578 from the National Cancer Institute. We thank Dr. L. Poels (University of Nijmegen, The Netherlands) for the generous gift of the hybridoma producing OV-TL 16 antibody.
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