Multivalent Poly(ethylene glycol)-Containing Conjugates for In Vivo

Keith A. Cockerill, Jeffrey R. Hammaker, Christina A. Kessler, Eric M. Smith, ... of anti-β2GPI antibodies and the treatment of antiphospholipid ...
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Bioconjugate Chem. 2003, 14, 1067−1076

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Multivalent Poly(ethylene glycol)-Containing Conjugates for In Vivo Antibody Suppression David S. Jones,* Michael J. Branks, Mary-Ann Campbell, Keith A. Cockerill, Jeffrey R. Hammaker, Christina A. Kessler, Eric M. Smith, Anping Tao, Huong-Thu Ton-Nu, and Tong Xu La Jolla Pharmaceutical Company, 6455 Nancy Ridge Drive, San Diego, California 92121. Received June 19, 2003; Revised Manuscript Received August 19, 2003

Poly(ethylene glycol) (PEG) was incorporated into multivalent conjugates of the N-terminal domain of β2GPI (domain 1). PEG was incorporated to reduce the rate of elimination of the conjugates from plasma and to putatively improve their efficacy as toleragens for the suppression of anti-β2GPI antibodies and the treatment of antiphospholipid syndrome (APS). Three structurally distinct types of multivalent platforms were constructed by incorporating PEG into the platform structures in different ways. The amount of PEG incorporated ranged from about 5000 g per mole to about 30000 g per mole. The platforms were functionalized with either four or eight aminooxy groups. The conjugates were prepared by forming oxime linkages between the aminooxy groups and N-terminally glyoxylated domain 1 polypeptide. The plasma half-life of each conjugate, labeled with 125I, was measured in both mice and rats. The half-lives of the conjugates ranged from less than 10 min to about 1 h in mice, and from less than 3 h to about 19 h in rats. The ability of five tetravalent conjugates to suppress antidomain 1 antibodies in immunized rats was also measured. Incorporation of PEG in the conjugates significantly reduced the doses required for suppression, and the amount of reduction correlated with the amount of PEG incorporated.

INTRODUCTION

B cell tolerance is a new approach to the treatment of B cell-mediated autoimmune diseases in which autoantibodies are pathogenic. The approach relies on multivalent presentation of B cell epitopes that are devoid of T cell epitopes to suppress the formation of antibodyproducing cells immunospecifically and, therefore, selectively suppress pathogenic antibody formation. More information on the underlying principles of B cell tolerance can be found in recent review articles (1-3). Molecules designed to selectively suppress a pathogenic antibody response are called B cell toleragens. They are composed of multiple B cell receptor epitopes or mimetic epitopes connected to nonimmunogenic multivalent platforms. B cell toleragens have been successfully used to reduce antibody levels in animal models and in humans suffering from autoimmune diseases. Toleragens composed of double-stranded oligonucleotides (dsON) were developed for treating systemic lupus erythematosus (SLE). Those toleragens suppressed anti-dsON antibodies in mice immunized with dsON (4, 5). Human clinical trials have demonstrated suppression of anti-doublestranded DNA antibodies in patients suffering from SLE (6). Toleragens composed of mimetic epitopes of β2GPI are under development for treating antiphospholipid syndrome (APS), a condition in which anti-β2GPI antibodies (also referred to as anti-phospholipid antibodies) are believed to cause thrombotic events. Toleragens prepared using peptide mimics of β2GPI were effective in suppressing anti-peptide antibody formation in an animal model; however, the peptides themselves bound the anti-β2GPI antibodies of only a small subset of patients (7). The * To whom correspondence should be addressed. Tel.: (858) 646-6628. Fax: (858) 626 2845. E-mail: [email protected].

N-terminal domain of β2GPI (domain 1) was recently discovered to recognize a high percentage of (APS) patient antisera (8). A toleragen composed of domain 1 was effective in suppressing anti-domain 1 antibodies in mice immunized with domain 1 (9). Antibody suppression in mice, using both the peptide toleragens and the domain 1 toleragen, required in vitro treatment of primed spleen cells followed by transfer into immune-ablated hosts. The domain 1 toleragen was, however, effective in vivo in rats immunized with domain 1. The difference in in vivo effectiveness of the toleragens in mice and rats was attributed to differential rates of clearance for the two species, and it became apparent that the effectiveness of toleragens could be improved by decreasing their rate of clearance from circulation. Rapid clearance is a problem that is often encountered in the development of polypeptide-based therapeutic drugs that are generally administered parenterally. Frequent dosing of such drugs is not desirable, because it increases the cost of drug and is inconvenient for the patient. Continuous infusion is often necessary to maintain an effective drug concentration. In general, proteins of less than approximately 70 kDa, the size of albumin, will clear from blood by passing through the glomerular membrane in a size dependent manner (10). A solution to rapid kidney clearance of drugs is to increase their physical size. That can be accomplished by attaching the drug to a macromolecule of sufficient size to prevent it from passing through the glomerular membrane (11). There are numerous examples of conjugates of polypeptides and macromolecules that have been prepared to enhance efficacy. Adriamycin and mitomycin C have been attached to 70 kDa dextran to prepare forms of those drugs that are more persistent in the circulation (12, 13). Attachment of poly(ethylene glycol) (PEG) to polypeptides has been widely used to improve their clinical effectiveness (11, 14, 15). In addition to reducing

10.1021/bc034103t CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003

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Figure 1. Three distinct types of PEG-containing conjugate structures.

kidney clearance and providing prolonged in vivo circulation half-lives, “PEGylated” proteins may be less susceptible to proteolysis or clearance by receptor-mediated mechanisms, and they may be less immunogenic and antigenic (16). Other advantageous properties of PEG are its water solubility, its amenability to chemical modification at one or both termini, and its availability in a variety of molecular weight ranges with low polydispersity. Some examples of polypeptides that have been attached to PEG to prolong their circulation half-lives include antibody fragments (17), granulocyte colony stimulating factor (18), bovine hemoglobin (19), growth hormones (20), insulin (21), and interferon R-2a (22). Several PEGylated polypeptides have been approved for treating human diseases. Further examples, of which there are many, may be found in recent review articles (14, 15). This report describes new anti-β2GPI specific toleragens that were constructed from novel PEG-containing platforms designed to provide longer circulatory half-lives and therefore greater efficacy. Our aim was to incorporate PEG into the platforms before attachment of domain 1 in order to avoid nonselective modification of the polypeptide and to provide a well-defined conjugate. The syntheses of three novel structurally distinct types of PEGcontaining platforms are described, as is the use of these platforms to prepare multivalent conjugates of domain 1. The structural varieties of conjugates are shown graphically in Figure 1. Summaries of pharmacokinetic data and in vivo tolerance data are also presented for the new conjugates. EXPERIMENTAL PROCEDURES

Abbreviations. DIPEA, diisopropylethylamine; NHS, N-hydroxysuccinimide; DCC, 1,3-dicylohexylcarbodiimide; CDI, 1,1′-carbonyldiimidazole; HOBt, 1-hydroxybenzotriazole hydrate; Et3N, triethylamine; Boc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid; PBS, phosphate buffered saline; TA/D1, transaminated domain 1. General. Monomethoxypoly(ethylene glycol) benzotriazolyl carbonates of molecular weight 11690 g/mol (mPEG12K-BTC), 5215 g/mol (mPEG5K-BTC), 22334 g/mol (mPEG20K-BTC), 32120 g/mol (mPEG30K-BTC), poly(ethylene glycol) bis(benzotriazolyl) carbonate of molecular weight 21529 g/mol (PEG20K-bis-BTC), and monoBoc-diaminopoly(ethylene glycol) of molecular weight 5094 g/mol (BocNH-PEG(5K)-NH2) were obtained from Shearwater Polymers, Huntsville, AL (now Nektar Therapeutics). N-(Benzyloxycarbonyloxy)succinimide was obtained from Aldrich Chemical Co., Milwaukee, WI. Silica gel chromatography was performed on silica gel (230-400 Mesh ASTM) purchased from Baxter. TLC was

Jones et al.

performed on silica gel plates (EM Separations cat. no. 5554). PBS was prepared by dissolving 175 g of NaCl, 6.5 g of NaH2PO4‚H2O, and 40.9 g of Na2HPO4‚7H2O in H2O and diluting to a final volume of 20 L. Sodium acetate 100 mM pH 4.6 buffer was prepared by dissolving 0.820 g (10 mmol) of sodium acetate in 95 mL of water, adding acetic acid to obtain a pH of 4.6, and adding water to obtain a volume of 100 mL. Tris acetate 100 mmol pH 8.0 buffer was prepared by dissolving 1.21 g of tris(hydroxymethyl)aminomethane in 95 mL of water, adding acetic acid to obtain a pH of 8.0, and adding water to obtain a volume of 100 mL. NMR spectra were recorded on a Bruker AC-300 spectrometer with a broad band probe. Low resolution mass spectra were recorded on a Finnigan LCQ electrospray mass spectrometer or obtained from the Mass Spectroscopy Lab at the Scripps Research Institute, San Diego, CA. High-resolution MALDI mass spectra were obtained from the Mass Spectroscopy Lab at the Scripps Research Institute, San Diego, CA. High-resolution FAB mass spectra were obtained from the Mass Spectrometry Laboratory at the University of California at Berkeley, Berkeley, CA. Optical densities of plate wells were obtained with a Power Wave 340 Microplate Spectrophotometer from Bio-Tek Instruments, Winooski, VT. Gamma counts were measured with a Cobra gamma counter from Packard Instruments, Dowers Grove, IL. Protein concentrations were determined by UV absorbance at 280 nm using a molar extinction coefficient of 9531 L mol-1 cm-1 (1.35 mL mg-1 cm-1) for the domain 1 polypeptide. Nunc Maxisorp Immunoplates were obtained from Nalge Nunc International, Rochester, NY. Recombinant human β2GPI was obtained from Sigma, St. Louis, MO. Nonfat dry milk was produced by Carnation, Solon, OH. Alkaline phosphatase-conjugated goat anti-rat IgG was obtained from Jackson ImmunoResearch, West Grove, PA. Female CD-1 mice were obtained from The Jackson Laboratories, Bar Harbor, ME. Male Sprague-Dawley rats were obtained from Charles River, Hingham, MA. S-Acetyl-6-(N-tert-butyloxycarbonyl)aminooxyhexane-1-thiol, Compound 2. To a solution of 77 mg (0.67 mmol) of potassium thioacetate in 15 mL of acetone was added 209 mg (0.61 mmol) of 6-(N-tert-butyloxycarbonyl)aminooxy-1-iodohexane (23), and the mixture was stirred at room temperature for 18 h. The acetone was removed under vacuum, and the residue was partitioned between 50 mL of CH2Cl2 and 3 × 25 mL of 1 N NaOH. The CH2Cl2 layer was dried (Na2SO4), filtered, and concentrated to a brown oil. Purification by silica gel chromatography (15/85 EtOAc/hexanes) provided 166 mg (94%) of compound 2 as a colorless oil: 1H NMR (CDCl3) δ 1.39 (m, 4H), 1.48 (s, 9H), 1.59 (m, 4H), 2.32 (s, 3H). 2.86 (t, 2H), 3.82 (t, 2H), 7.10 (s, 1H); 13C NMR (CDCl3) δ 25.5, 28.0, 28.4, 28.6, 29.1, 29.5, 30.8, 76.7, 81.6, 157.1, 196.0; HRMS (MALDI) calculated for (M + Na): C13H25NaNO4S: 314.1396. Found: 314.1402. 6-(N-tert-butyloxycarbonyl)aminooxyhexane-1thiol, Compound 3. Compound 2 (50 mg, 172 µmol) and 22 µL (17.4 mg, 85.8 µmol) of tri-n-butylphosphine was placed under nitrogen, and 2 mL of a nitrogen sparged 1 M solution of NaOH in MeOH was added to the mixture. The mixture was stirred for 18 h at room temperature, and 172 µL (180 mg, 3 mmol) of TFA was added. The mixture was partitioned between 25 mL of EtOAc and 3 × 25 mL of 1 N HCl, and the combined aqueous layers were extracted with 25 mL of EtOAc. The combined EtOAc extracts were dried (Na2SO4), filtered, and concentrated. The resulting oil was purified by silica gel chromatography (15/85/0.1 EtOAc/hexanes/MeOH) to

Multivalent Poly(ethylene glycol)-Containing Conjugates

provide 28 mg (65%) of 3 as a colorless oil: 1H NMR (CDCl3) δ 1.32 (t, 1H), 1.40 (m, 4H), 1.49 (s, 9H), 1.62 (m, 4H), 2.53 (d of t, 2H). 3.84 (t, 2H), 7.09 (s, 1H); 13C NMR (CDCl3) δ 24.4, 25.3, 27.8, 28.0, 28.2, 33.7, 76.6, 81.5, 156.8; HRMS (MALDI) calculated for (M + Na): C11H23NaNO3S: 272.1291. Found: 272.1294. Preparation of Compound 5 from Compound 2 without Isolation of Compound 3. Compound 2 (13 mg, 45 µmol) and 6 µL (4.5 mg, 22.3 µmol) of tri-nbutylphosphine was placed under nitrogen, and 3 mL of a nitrogen sparged solution of 4/1 6 N NH4OH/CH3CN was added to the mixture. The mixture was stirred for 1 h at room temperature and concentrated under vacuum. The residue was dissolved in 3 mL of a nitrogen-sparged solution of 10/90 water/CH3CN. The resulting solution of 3 was kept under nitrogen atmosphere, and 10 mg (7.44 µmol) of compound 4 (prepared as described in ref 5) was added, followed by 8 µL (5.77 mg, 44.6 µmol) of DIPEA. The mixture was stirred for 18 h and concentrated under vacuum. The residue was purified by silica gel chromatography (multiple step gradient, 1/99 to 5/95 to 7.5/92.5 to 10/90 to 15/85 MeOH/CH2Cl2) to provide 14 mg (93%) of 5 as a colorless oil: TLC (10/90 MeOH/ CH2Cl2), Rf ) 0.3; 1H NMR (CDCl3) δ 1.51 (m, 92H), 2.22 (t, 8H), 2.54 (t, 8H), 3.19 (m, 24H), 3.38 (brd s, 8H), 3.75 (m, 12H), 3.82 (t, 4H), 4.19 (t, 4H), 7.38 (brd s, 8H), 7.94 (s, 4H); 13C NMR (CDCl3) δ 12.2, 17.2, 18.3, 25.0, 25.2, 26.2, 27.6, 28.0, 28.2, 28.8, 28.9, 32.6, 35.8, 39.3, 42.5, 54.2, 64.3, 69.0, 70.1, 76.2, 81.0, 156.6, 156.7, 169.2, 173.5; mass spectrum (ES) m/z calculated for C92H173N14O26S4 (M + H): 2018. Found: 2018; HRMS (ESI) (M + 2Na)+2 calculated for C92H172Na2N14O26S4: 1031.5617. Found: 1031.5633. 6-(N-tert-Butyloxycarbonyl)aminooxyhexanoic Acid, Compound 7. To a magnetically stirred solution of 7.2 g (26 mmol) of compound 6 (23) in 10 mL of EtOH was added 5 mL of 50% w/w NaOH solution in water. The reaction was done in 5 min, as determined by TLC (1/5 EtOAc/hexanes). The reaction mixture was concentrated, and the residue was partitioned between 20 mL of CH2Cl2 and 20 mL of 1 N HCl solution. The layers were separated, and the aqueous layer was extracted with 6 × 10 mL portions of CH2Cl2. The combined CH2Cl2 layers were dried (MgSO4), filtered, and concentrated to give 6.5 g (99%) of compound 7 as a colorless oil. 1H NMR CDCl3 (δ) 1.44 (m, 2H), 1.48 (s, 9H), 1.66 (m, 4H), 2.37 (t, 2H), 3.85 (t, 2H), 7.20 (s, 1H); 13C NMR CDCl3 (δ) 24.6, 25.5, 27.8, 28.4, 34.0, 76.6, 82.0, 157.5, 179.3; HRMS (MALDI) calculated for (M+ Na): C11H21NaNO5: 270.1312. Found: 270.1309. Synthesis of Compound 8. To a magnetically stirred solution of 1.00 g (4.04 mmol) of compound 7 in 10 mL of CH2Cl2 at 0 °C was added 511 mg (4.44 mmol) of NHS followed by 1.25 g (6.06 mmol) of DCC. The mixture was brought to room temperature within 1 h. The mixture was stirred for 16 h, 750 µL of acetic acid was added, and the mixture was placed in the freezer for 30 min. The solids were removed by filtration, and the filtrate was concentrated to a viscous oil. Purification by silica gel chromatography (15/85 acetone/toluene) provided 1.13 g (81%) of compound 8 as a white amorphous solid: 1H NMR CDCl3 (δ) 1.48 (s, 9H), 1.51 (m, 2H), 1.68 (m, 2H), 1.79 (m, 2H), 2.63 (t, 2H), 2.84 (br. s, 4H), 3.87 (t, 2H), 7.18 (br. s, 1H); 13C NMR CDCl3 (δ) 24.32, 25.07, 25.53, 27.42, 28.16, 30.77, 76.13, 81.51, 156.89, 168.45, 169.15; HRMS (MALDI) calculated for (M + Na): C15H24NaN2O7: 367.1476. Found: 367.1474. Synthesis of Compound 9. A magnetically stirred solution of 1.49 g (11.4 mmol) of 6-aminocaproic acid and

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958 mg (11.4 mmol) of NaHCO3 in 144 mL of water was cooled to 0 °C. To the solution was added 27.8 mL of CH3CN followed by a solution of 1.31 g (3.80 mmol) of compound 8 in 30 mL of CH3CN. The mixture was brought to room temperature within an hour and stirred for 18 h. The mixture was concentrated, and the residue was partitioned between 70 mL of 1 N HCl and 2 × 70 mL of CH2Cl2. The combined CH2Cl2 layers were washed with brine, dried (MgSO4), filtered, and concentrated. Purification by silica gel chromatography 45/55/0.5 acetone/ toluene/acetic acid provided 1.18 g (86%) of compound 9 as a viscous oil. 1H NMR CDCl3 (δ) 1.42 (m, 4H), 1.49 (s, 9H), 1.53 (m, 2H), 1.65 (m, 6H), 2.20 (t, 2H), 3.38 (t, 2H), 2.88 (q, 2H), 3.86 (t, 2H), 5.84 (br t, 1H), 7.67(br s, 1H); 13 C NMR CDCl3 (δ) 24.1, 25.3, 25.4, 26.1, 27.5, 28.2, 29.0, 33.7, 36.4, 76.4, 81.7, 157.2, 173.4, 177.6; HRMS (MALDI) calculated for (M + Na): C17H32NaN2O6: 383.2152. Found: 383.2141. Synthesis of Dimer Cassette, Compound 10. To a magnetically stirred solution of 1.09 g (3.04 mmol) of compound 9 in 11 mL of EtOAc was added 493 mg (3.04 mmol) of CDI. The reaction was stirred under N2 for 1 h, and 160 µL (152 mg, 1.48 mmol) of diethylenetriamine was added followed by 423 µL (307 mg, 3.03 mmol) of Et3N at which time a precipitate formed. The mixture was stirred for 3 h and partitioned between 25 mL of H2O and 200 mL of CH2Cl2. The CH2Cl2 layer was washed with 25 mL of H2O and 25 mL of brine, dried (MgSO4), filtered, and concentrated. Purification by silica gel chromatography (step gradient 17/83/0.5 MeOH/CH2Cl2/ concd NH4OH to 20/80/0.5 MeOH/CH2Cl2/concd NH4OH) provided 851 mg (73%) of compound 10 as a light yellow, glassy solid. 1H NMR CD3OD (δ) 1.38 (m, 8H), 1.46 (s, 9H), 1.49 (m, 4H), 1.62 (m, 12 H), 2.18 (t, 4H), 2.20 (t, 4H), 2.75 (t, 4H), 3.16 (t, 4H), 3.30 (t, 4H), 3.77 (t, 4H); 13 C NMR CD3OD (δ) 26.5, 26.6, 26.8, 27.6, 28.6, 28.8, 30.2, 37.0 (36.98), 37.0 (37.02), 39.7, 40.2, 49.4, 77.2, 81.9, 159.1, 176.0, 176.5; HRMS (MALDI) calculated for (M + H): C38H74N7O10: 788.5491. Found: 788.5488. Compound 12. To a solution of 1.50 g (7.76 mmol) of compound 11 (prepared as described in ref 24) and 1.30 g (15.52 mmol) of NaHCO3 in 20 mL of H2O at 0 °C was added a solution of 2.32 g (9.31 mmol) of N-(benzyloxycarbonyloxy)succinimide dissolved in 20 mL of dioxane. Cooling was discontinued, and the mixture was stirred for 4 h at room temperature. The mixture was acidified to approximately pH 1 with 1 N HCl and partitioned between 20 mL of 1 N HCl and 100 mL of CH2Cl2. The aqueous layer was extracted with four additional 50 mL portions of CH2Cl2. All of the CH2Cl2 extracts were combined, dried (MgSO4), filtered, and concentrated. The residue was dissolved in 30 mL of CH2Cl2 and cooled to 0 °C. To the solution was added 6.26 g (31.0 mmol) of 4-nitrophenylchloroformate followed by addition of 5 mL (4.91 g, 62.1 mmol) of pyridine dropwise over approximately 2 min during which time a precipitate formed. Cooling was discontinued, and the mixture was allowed to stir for 3 h at room temperature. The precipitate was removed by filtration and washed with approximately 20 mL of CH2Cl2. Approximately 1 mL of water was added to the filtrate, and the mixture was stirred for 5 min and shaken with 50 mL of 1 N HCl. The organic layer was collected, and the aqueous layer was extracted with 50 mL of CH2Cl2. The combined organic layers were washed successively with four 25 mL portions of saturated NaHCO3 solution, 25 mL of 1 N HCl, and 25 mL of brine, dried (MgSO4), filtered, and concentrated. Purification by silica gel chromatography (loaded in CH2Cl2 and eluted with 50/50 EtOAc/hexanes) provided 3.81 g (75%) of

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compound 12 as a viscous oil: 1H NMR (CDCl3) δ 3.483.82 (m, 12H), 4.39 (m, 4H), 5.12 (s, 2H), 7.34 (m, 7H), 8.23 (d, 2H); 13C NMR (CDCl3) δ 47.8, 48.2, 67.2, 68.0, 68.1, 68.2, 68.3, 69.6, 69.9, 121.7, 125.2, 127.8, 128.0, 128.5, 136.6, 145.4, 152.4, 155.4, 156.1; HRMS mass spectrum (MALDI) (M + Na) calculated for C30H31NaN3O14: 680.1698. Found 680.1697. Compound 13. A solution of 294 mg (0.45 mmol) of compound 12 in 30 mL of pyridine was added to a flask containing 775 mg (0.98 mmol) of compound 10. Triethylamine (249 µL,181 mg, 1.79 mmol) was added, and the mixture was stirred at room temperature under nitrogen atmosphere for 18 h. Most of the pyridine was removed under reduced pressure, and the residue was partitioned between 100 mL of CH2Cl2 and 3 × 20 mL of 1 N HCl. The CH2Cl2 extract was washed with 20 mL of brine, dried (MgSO4), filtered, and concentrated to a yellow oil. Purification by silica gel chromatography (step gradient: 5/95/0.1 to 10/90/0.1 to 15/85/0.1 MeOH/CH2Cl2/ HOAc) provided 633 mg (72%) of compound 13 as a viscous oil: 1H NMR (CD3OD) δ 1.20-1.52 (m, 24H), 1.43 (s, 36H), 1.60 (m, 24H), 2.16 (m, 16H), 3.12 (m, 8H), 3.33 (m, 20H), 3.50-3.70 (m, 16H), 3.76 (t, 8H), 4.16 (m, 4H), 5.12 (s, 2H), 7.36 (brd s, 5H), 7.93 (m, 4H); 13C NMR (CD3OD) δ 26.6, 26.8, 27.6, 28.7, 28.8, 30.2, 37.0, 38.7, 39.0, 40.2, 40.3, 65.8, 68.3, 70.1, 70.3, 70.5, 77.2, 81.9, 128.8, 129.2, 129.7, 138.1, 158.0, 158.1, 159.1, 176.0, 176.2; HRMS mass spectrum (MALDI) (M + Na) calculated for C94H167NaN15O28: 1977.1997. Found 1977.1899. Compound 14. A solution of 300 mg (0.15 mmol) of compound 13 and 8.8 µL (9.2 mg, 0.13 mmol) of acetic acid in 20 mL of MeOH was purged with H2. To the solution was added 75 mg of 10% palladium on carbon. A hydrogen-filled balloon was attached, and the mixture was stirred for 2 h. The flask was purged with nitrogen, the catalyst was removed by filtration, and the mixture was concentrated to provide 282 mg (quantitative yield) of compound 14 as a gummy solid: 1H NMR (CD3OD) δ 1.38 (m, 16H), 1.48 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H), 2.83 (t, 4H), 3.17 (t, 8H), 3.38 (m, 16H), 3.63 (t, 4H), 3.69 (t, 4H), 3.78 (t, 4H), 4.21 (m, 4H); 13C NMR (CD3OD) δ 26.7, 27.0, 27.8, 28.8, 28.9, 30.3, 37.1, 38.8, 39.1, 40.3, 49.9, 66.0, 70.4, 70.9, 77.3, 82.0, 158.2, 159.2, 176.1, 176.3; mass spectrum (ESI) (M + H)+ calculated for C86H162N15O26: 1821. Found 1821; HRMS (MALDI) (M + Na) calculated for C86H162HN15O26: 1821.1809. Found 1821.1803. Compound 16b. To a solution of 20 mg (11.0 µmol) of compound 14 in 5 mL of DMF was added 103 mg (8.8 µmol) of compound 15b (mPEG12K-BTC) followed by 5 µL (3.6 mg, 35.9 µmol) of Et3N. The mixture was stirred at room temperature for 18 h and concentrated. The residue was purified by silica gel chromatography (multistep gradient; 5/95 to 15/85 to 20/80 MeOH/CH2Cl2) to provide 109 mg (92%) of compound 16b as a waxy solid: 1H NMR (CDCI3) δ 1.37 (m, 16H), 1.49 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H), 3.68 (m, approximately 1056H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m, 4H); mass spectrum (MALDI) average m/z calculated for C610H1208N15O289: 13370. Found: 13711. Compound 16a. This compound was prepared in 69% yield using essentially the same procedure used for the preparation of compound 16b; however, compound 15a (mPEG5K-BTC) was used: 1H NMR (4:1 CDCI3/CD3OD) δ 1.37 (m, 16H), 1.49 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H), 3.68 (m, approximately 468H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m, 4H); mass spectrum (MALDI) average m/z calculated for C316H619N15O142: 6898. Found: 6800.

Jones et al.

Compound 16c. This compound was prepared in 92% yield using essentially the same procedure used for the preparation of compound 16b; however, compound 15c (mPEG20K-BTC) was used: 1H NMR (5:1 CDCI3/CD3OD) δ 1.37 (m, 16H), 1.49 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H), 3.68 (m, approximately 2024H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m, 4H); mass spectrum (MALDI) average m/z calculated for C1094H2175N15O531: 24023. Found: 23400. Compound 16d. This compound was prepared in 54% yield using essentially the same procedure used for the preparation of compound 16b; however, compound 15d (mPEG30K-BTC) was used: 1H NMR (5:1 CDCI3/CD3OD) δ 1.37 (m, 16H), 1.49 (m, 44H), 1.65 (m, 24H), 2.20 (t, 16H), 3.20 (q, 8H), 3.36 (m, 16H), 3.61 (m, 4H), 3.68 (m, approximately 2,900H), 3.84 (t, 8H), 3.91 (m, 4H), 4.23 (m, 4H); mass spectrum (MALDI) average m/z calculated for C1538H3663N15O753: 33818. Found: 33775. Compound 17. To a solution of 54 mg (29.6 µmol) of compound 14, 9 mg (13.5 µmol) of compound 12, and 8 mg (53.9 µmol) of HOBt in 5 mL of anhydrous pyridine was added 15 µL (10.9 mg, 107.8 µmol) of Et3N. The mixture was stirred for 18 h and concentrated to a viscous oil. The residue was purified by HPLC using a 24 mm × 30 cm C18 column (gradient 30% to 60% B over 60 min; A ) H2O/0.1% TFA, B ) CH3CN/0.1% TFA) to provide 17 mg (31%) of compound 17 as a waxy white solid: 1H NMR (CD3OD) δ 1.20-1.51 (m, 48H), 1.43 (s, 72H) 1.60 (m, 48H), 2.19 (t, 32H), 3.14 (t, 16H), 3.32 (m, 32H), 3.50 (m, 12H), 3.59 (m, 12H), 3.64 (m, 12H), 3.78 (t, 16H), 4.18 (m, 12H), 5.12 (s, 2H), 7.32 (m, 5H); 13C NMR (CD3OD) δ 26.6, 26.8, 27.6, 28.6, 28.8, 30.2, 37.0, 38.7, 39.0, 40.2, 65.8, 68.3, 70.2, 70.6, 77.2, 81.9, 128.8, 129.2, 129.3, 129.7, 129.9, 138.2, 157.8, 157.9, 158.0, 159.1, 176.0, 176.2; HRMS (ESI) (M + 3Na)+3 calculated for C190H343Na3N31O60: 1362.8134. Found: 1362.8224. Compound 18. A solution of 9 mg (2.24 µmol) of compound 17 in 10 mL of MeOH was placed under an H2 atmosphere by applying partial vacuum and filling with H2. To the solution was added 10 mg of 10% palladium on carbon, a H2-filled balloon was attached, and the mixture was stirred at room temperature for 2 h. The flask was purged with N2, the catalyst was removed by filtration, and the filtrate was concentrated to provide 6 mg (69%) of compound 18 as a colorless gum: 1H NMR (CD3OD) δ 1.23-1.50 (m, 48H), 1.48 (s, 72H), 1.61 (m, 48H), 2.19 (t, 32H), 3.08 (brd t, 4H) 3.17 (t, 16H), 3.38 (m, 36H), 3.52 (m, 8H), 3.63 (t, 8H), 3.70 (m, 12H), 3.78 (t, 16H), 4.21 (m, 12H); 13C NMR (CD3OD) δ 26.7, 26.9, 27.7, 28.7, 28.9, 30.2, 37.1, 38.8, 39.1, 40.3, 65.9, 70.3, 77.2, 81.9, 158.0, 158.1, 159.1, 176.0, 176.2; mass spectrum (ESI) (M + H)+ calculated for C182H338N31O58: 3887. Found 3887. HRMS (ESI) (M + 3Na)+3 calculated for C182H337Na3N31O58: 1318.1350. Found: 1318.1419. Compound 19. To a solution of 13 mg (3.34 µmol) of compound 18 in 5 mL of pyridine was added 60 mg (2.68 µmol) of compound 15c (mPEG20K-BTC) followed by 5 µL (3.6 mg, 35.9 µmol) of Et3N. The mixture was stirred at room temperature for 18 h and concentrated. The residue was purified by silica gel chromatography (multistep gradient; 10/90 to 15/85 to 20/80 MeOH/CH2Cl2) to provide 45 mg (68%) of compound 19 as a waxy solid: 1 H NMR (CDCI3) δ 1.30 (m, 32H), 1.50 (m overlapping s at 1.48, 88H), 1.67 (m, 48H), 2.24 (t, 32H), 3.23 (m, 16H), 3.41 (m, 32H), 3.65 (m, approximately 2024H), 3.70 (t, 24H), 3.89 (m, 16H), 4.21 (m, 12H); mass spectrum (MALDI) average m/z calculated for C1127H2227N31O531: 24696. Found: 23270.

Multivalent Poly(ethylene glycol)-Containing Conjugates

Compound 21. To a solution of 22 mg (27.3 µmol) of compound 10 in 5 mL of pyridine was added 236 mg (10.9 µmol) of compound 20 (PEG20K-bis-BTC) followed by 8 µL (5.8 mg, 57.4 µmol) of Et3N. The mixture was stirred at room temperature for 18 h and concentrated. The residue was purified by silica gel chromatography (multistep gradient; 5/95 to 10/90 to 15/85 to 20/80 MeOH/ CH2Cl2) to provide 242 mg (96%) of compound 21 as a white solid: 1H NMR (CDCI3) δ 1.35 (m, 16H), 1.48 (m, 44H), 1.61 (m, 24H), 2.20 (m, 16H), 3.22 (m, 8H), 3.523.96 (m, approximately 2000H), 4.23 (m, 4H); mass spectrum (MALDI) average m/z calculated for C1040H2068N14O504: 22836. Found: 23246. Compound 24. To a solution of 3.87 mg (4.85 µmol) of compound 22 (25) in 5 mL of pyridine was added 124 mg (24.2 µmol) of compound 23 (BocNH-PEG(5K)-NH2) and 5 µL (3.63 mg, 35.9 µmol) of Et3N, and the mixture was stirred for 18 h and concentrated under vacuum. The residue was purified by silica gel chromatography (step gradient; 5/95 to 15/85 MeOH/CH2Cl2) to provide 77 mg (77%) of compound 24 as a white solid: 1H NMR (CDCI3) δ 1.48 (s, 36H), 3.32 (m, 16H), 3.52-3.96 (m, approximately 1818H), 4.10 (m, 8H); mass spectrum (MALDI) average m/z calculated for C933H1860N8O464: 20604. Found: 21340. Compound 25. Compound 24 (77 mg, 3.73 µmol) was dissolved in 5 mL of TFA, and the mixture was allowed to stand for 3 h. The TFA was removed under a stream of N2, and the residue was dissolved in 5 mL of CH2Cl2. To the resulting solution was added a solution of 7.72 mg (22.4 µmol) of compound 8 in 5 mL of CH2Cl2 followed by 35 µL (25.4 mg, 251 µmol) of Et3N. (Note: The pH of the mixture should be checked and adjusted accordingly with Et3N to make sure it is basic.) The mixture was stirred under nitrogen for 18 h and partitioned between 50 mL of CH2Cl2 and three 25 mL portions of 1 N HCl. The CH2Cl2 layer was washed with brine, dried (MgSO4), filtered, and concentrated. Purification by silica gel chromatography (step gradient; 5/95 to 10/90 MeOH/CH2Cl2) provided 42 mg (53%) of compound 25 as waxy solid: 1H NMR (CDCI3) δ 1.40 (m, 8H), 1.48 (s, 36H), 1.66 (m, 16H), 2.18 (t, 8H), 3.32 (m, 16H), 3.38-3.89 (m, approximately 1818H), 4.10 (m, 8H), 4.97 (t, 4H), 6.43 (t, 4H), 7.47 (s, 4H); mass spectrum (MALDI) average m/z calculated for C957H1904N12O472: 21122. Found: 21796. Synthesis of Compound 1a (LJP 1027). Compound 5 (4.1 mg, 2.05 × 10-6 mol) was treated with 3 mL of a solution of 1/9 TFA/CH2Cl2 for 2 h at room temperature. The mixture was concentrated to an oil under vacuum, and immediately 200 µL of 0.1 M pH 8.0 tris acetate buffer was added followed by 200 µL of CH3CN. The resulting solution was added to a helium sparged solution of 87 mg (12.1 × 10-6 mol) of TA/D1 in 10.9 mL of 0.1 M pH 4.6 sodium acetate buffer using approximately 0.5 mL of CH3CN to rinse residual material into the reaction mixture. The mixture was allowed to stand for 16 h at room temperature, and the resulting mixture was purified by preparative HPLC (1 in. × 30 cm diphenyl column (Vydak), 12 mL/min, gradient 27% to 45% B over 40 min; A ) H2O/0.1% TFA, B ) CH3CN/0.1% TFA). The fractions containing product were lyophilized to provide 34 mg (55%) of compound 1a as a white powder: mass spectrum (ESI) m/z calculated for C1352H2104N338O370S24: 29783. Found: 29783. Synthesis of Compound 1g (LJP 1082). Compound 21 (318 mg, 13.9 × 10-6 mol) was treated with 16 mL of a solution of 1/1 TFA/CH2Cl2 for 30 min at room temperature. The mixture was concentrated to an oil under vacuum, and immediately 3 mL of 0.1 M pH 8.5 Tris

Bioconjugate Chem., Vol. 14, No. 6, 2003 1071

acetate buffer was added followed by 3 mL of CH3CN. The resulting solution was added to a helium-sparged solution of 532 mg (7.53 × 10-5 mol) of TA/D1 in 35 mL of 0.1 M pH 4.6 sodium acetate buffer using 2 mL of CH3CN to rinse residual material into the reaction mixture. The pH was adjusted to between 4.4 and 4.6 by addition of approximately 2.4 mL of 5 N NaOH solution, and the mixture was allowed to stand for 16 h. The pH was adjusted to 7 by addition of 5 N NaOH solution, and the mixture was dialyzed against water followed by 10 mM pH 7 sodium phosphate solution for approximately 16 h until a conductivity of 1.0 mmho was obtained. The resulting solution was purified in three portions by cation exchange chromatography (2 cm × 17 cm column packed with Toyopearl CM650M (TosoHass); flow rate 8 mL/ minute; gradient 2-13% B 0-120 min; A ) 9/1 10 mM pH 7 sodium phosphate/CH3CN, B ) A + 1 M NaCl). The fractions containing product were partially lyophilized to reduce the volume and dialyzed against water. The desalted solution was lyophilized to provide 413 mg (59%) of compound 1g as a white powdery solid: mass spectrum (MALDI) average m/z calculated for C2300H4000N338O848S20: 50600. Found: 51620. Compound 1b (LJP 1078). Compound 1b was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 6.0/1.0 of TA/D1/compound 16a was used resulting in an overall yield of 55% of compound 1b: mass spectrum (MALDI) average m/z calculated for C1576H2551N339O486S20: 34666. Found: 35025. Compound 1c (LJP 1081). Compound 1c was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 8.0/1.0 of TA/D1/compound 16b was used resulting in an overall yield of 35% of compound 1c: mass spectrum (MALDI) average m/z calculated for C1870H3140N339O633S20: 41143. Found: 41922. Compound 1d (LJP 1077). Compound 1d was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 6.0/1.0 of TA/D1/compound 16c was used resulting in an overall yield of 59% of compound 1d: mass spectrum (MALDI) average m/z calculated for C2354H4107N339O875S20: 51802. Found: 51636. Compound 1e (LJP 1086). Compound 1e was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 6.6/1.0 of TA/D1/compound 16d was used resulting in an overall yield of 31% of compound 1e: mass spectrum (MALDI) average m/z calculated for C2808H5015N339O1102S20: 61803. Found: 61739. Compound 1f (LJP 1084). Compound 1f was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 12.0/1.0 of TA/D1/compound 19 was used resulting in an overall yield of 11% of compound 1f: mass spectrum (MALDI) average m/z calculated for C3710H6219N579O1251S40: 81638. Found: 81893. Compound 1h (LJP 1083). Compound 1h was prepared in a manner essentially similar to that described for compound 1g. A mole ratio of 5.4/1.0 of TA/D1/compound 25 was used resulting in an overall yield of 61% of compound 1h: mass spectrum (MALDI) average m/z calculated for C2217H3836N336O816S20: 48898. Found: 49390. Measurement of Anti-domain 1 Antibodies in Rats. Nunc Maxisorp Immunoplates were coated overnight with 50 µL of 5 µg/mL of recombinant human β2GPI in pH 9.6 carbonate buffer at 4 °C. Subsequent steps were carried out at room temperature. Plates were washed 3× with PBS and then blocked by treating with 250 µL of a 2% solution of nonfat dry milk for 1 h in PBS. Plates were washed 3× with PBS, and then wells were treated for 1 h with 50 µL of serial dilutions of each serum sample in PBS in triplicate. Plates were washed 3× with

1072 Bioconjugate Chem., Vol. 14, No. 6, 2003 Scheme 1a

a Reagents and conditions: (a) pH 4.6 sodium acetate buffer; R ) multivalent platform, x ) 4 or 8, D1 ) domain 1 polypeptide of β2GPI (amino acid sequence RTCPK PDDLP FSTVV PLKTF YEPGE EITYS CKPGY VSRGG MRKFI CPLTG LWPIN TLKCT PR).

PBS, and then the wells were treated for 1 h with 50 µL of alkaline phosphatase-conjugated goat anti-rat IgG diluted 1:2000 in PBS/0.1% BSA. Plates were washed 3× with H2O and developed for 20 min with chromogenic substrate solution consisting of 10 gm of phenolphthalein monophosphate, 97.4 mL of 2-amino-2-methyl-1-propanol, 9.62 mL of H2O, and 21 mL HCl of concd HCl solution. Color development was stopped by adding 50 µL of 0.2 M Na2HPO4 to each well, and optical absorbance at 550 nm was measured. Nominal antibody units were assigned to the standard pool, and the concentrations of anti-domain 1 antibody (units/mL) in test sera were derived from the standard curve. Serum from nonimmunized animals was used as a control, and a pool of sera from immunized animals was used to generate a standard curve of antibody levels at various dilutions. Percent suppression of anti-domain 1 antibody by the various multivalent domain 1 conjugates was calculated by comparison to PBS-treated controls. Measurement of Plasma Half-Life of Conjugates in Rats and Mice. The compounds were radiolabeled with 125I by the iodine monochloride method (26) and injected iv into female CD-1 mice or male SpragueDawley rats. Mouse plasma samples were collected periodically for 1 h, and rat plasma samples were collected for 24 h. The amount of radiolabeled drug was detected using a gamma counter. Pharmacokinetic parameters were calculated using WinNonLin software (Pharsight, Mountain View, CA). Plasma half-lives were calculated from the areas under the time-concentration curves using the formula t1/2 ) ln 2(AUC/C0) assuming a single component first-order elimination at t ) t1/2. RESULTS AND DISCUSSION

Three types of novel PEG-containing platforms were designed and synthesized, and they were used to prepare multivalent conjugates of domain 1. The types of platform differ in the way PEG was incorporated. In one type of platform monomethoxy PEG, which has only one end available for attachment chemistry, was attached to a platform intermediate with a free secondary amine. A second type of platform was prepared in which bivalent PEG is incorporated as an integral part of the platform by branching at each end of the PEG chain. The third type of platform was prepared with PEG incorporated into each of four branching arms of the platform. A nonPEG-containing platform was also prepared. All of the platforms contained either four or eight Bock-protected aminooxy groups. Each of the platforms was converted to a multivalent domain 1 conjugate by a two step process involving removal of the Boc-protecting groups and subsequent reaction of the free aminooxy groups with transaminated domain 1 (TA/D1) as described in Scheme 1. TA/D1 is a derivative of domain 1 that has been N-terminally glyoxylated by a transamination reaction as previously described (9). The non-PEG containing conjugate, compound 1a, was prepared to serve as a non-PEG control. The synthesis began with the preparation of a thiol-containing Boc-

Jones et al. Scheme 2a

a Reagents and conditions: (a) KSCOCH , acetone; (b) tri-n3 butylphosphine, aqueous NaOH, MeOH.

Scheme 3a

a Reagents and conditions: (a) Compound 3, DIPEA, 9/1 H O/ 2 CH3CN; (b) 1/9 TFA/CH2Cl2; (c) transaminated domain 1, pH 4.6 aqueous NaOAc, CH3CN.

Scheme 4a

a Reagents and conditions: (a) NaOH, H O/EtOH; (b) NHS, 2 DCC, CH2Cl2; (c) 6-aminocaproic acid, NaHCO3, H2O, CH3CN; (d) CDI, EtOAc, diethylenetriamine, Et3N.

protected aminooxy linker, compound 3, as described in Scheme 2. The linker was prepared from 6-iodo-N-Boc1-aminooxyhexane (23) by reaction with potassium thioacetate to provide compound 2. Hydrolysis of 2 with ammonium hydroxide provided compound 3. The ammonium hydroxide and solvent were removed, and the crude thiol was used directly in the next step. Reaction of 3 with compound 4 provided the tetravalent Boc-protected aminooxy platform, compound 5, as described in Scheme 3. The Boc protecting groups were removed by treating with trifluoroacetic acid, and TA/ D1 was attached to the resulting free aminooxy groups to provide conjugate 1a (LJP 1027). A series of domain 1 toleragens were prepared with attached monomethoxy PEG of different molecular weights. The synthesis of these molecules began by preparing a bivalent Boc-protected aminooxy linker, compound 10, with a free secondary amine as diagramed in Scheme 4. Compound 6 was prepared as described previously (23), and it was saponified to provide compound 7. Compound 7 was converted to the N-hydroxysuccinimidyl ester, compound 8, which was used to acylate 6-aminocaproic acid to provide compound 9. Treatment of compound 9 with carbonyldiimidazole followed by diethylenetriamine provided the key intermediate, compound 10, also referred to as the dimer cassette. A tetravalent Boc-protected aminooxy scaffold with a free secondary amine attachment site was prepared as

Multivalent Poly(ethylene glycol)-Containing Conjugates

Bioconjugate Chem., Vol. 14, No. 6, 2003 1073

Scheme 5a

a Reagents and conditions: (a) N-(benzyloxycarbonyloxy)succinimide, NaHCO , H O, dioxane; (b) 4-nitrophenylchloroformate, 3 2 pyridine; (c) compound 10, pyridine, Et3N; (d) H2, Pd/C, acetic acid, MeOH; (e) Et3N, DMF; (f) TFA/CH2Cl2; (g) transaminated domain 1 polypeptide, 100 mM pH 4.6 sodium acetate, CH3CN.

described in Scheme 5. Compound 12 was prepared in two steps from bis(2-(2-hydroxyethoxy)ethyl)amine, compound 11, which was prepared as previously described (24). Compound 11 was first treated with N-(benzyloxycarbonyloxy)succinimide to place a Cbz protecting group on the secondary nitrogen atom. The hydroxyl groups were subsequently converted to 4-nitrophenyl carbonate esters using 4-nitrophenyl chloroformate. Compound 12 was reacted with compound 10 to provide compound 13, and the Cbz protecting group was removed by hydrogenolysis to provide compound 14. Compound 14, also referred to as a tetramer cassette, was connected to monomethoxy-PEG of various average molecular weights. Thus reaction of compound 14 with hydroxybenzotriazole esters of methoxy-PEG with average molecular weights of approximately 5000 g/mol, 12000 g/mol, 20000 g/mol, and 30000 g/mol, compounds 15a-d, gave rise to PEGcontaining Boc-protected tetravalent platforms 16a-d. The Boc protecting groups were removed by treating with trifluoroacetic acid, and TA/D1 was attached to the free aminooxy groups to provide conjugates 1b-e. An octavalent analogue was prepared as described in Scheme 6. Condensation of the tetramer cassette, compound 14, with compound 12 provided the Cbz-protected octamer cassette, compound 17. The Cbz protecting group was removed by hydrogenolysis to provide the octamer

cassette, compound 18. The octamer cassette was connected to methoxy-PEG with an average molecular weight of approximately 20000 g/mol using essentially the same chemistry described above for the tetravalent analogues, and the resulting platform was similarly deprotected and conjugated with TA/D1 to provide compound 1f. The second type of PEG containing domain 1 conjugate was prepared as described in Scheme 7. The synthesis began by attaching a dimer cassette to each end of PEG. Thus, the bis(hydroxybenzotriazolyl) carbonate ester of PEG with a molecular weight of approximately 20000 g/mol, compound 20, was treated with 2 equiv of compound 10 to provide compound 21. Compound 21 was deprotected and treated with TA/D1 under standard conditions to provide compound 1g. The third class of PEG-containing conjugates was prepared by incorporation of PEG into each of the linking arms of the platform as described in Scheme 8. This example incorporates a total of approximately 20000 g/mol of PEG with 5000 g/mol of PEG in each arm. Thus, tetravalent 4-nitrophenyl carbonate ester 22 was reacted with mono-Boc-diamino-PEG of approximately 5000 g/mol, compound 23, to provide the branched Boc-protected tetraamine 24. The protecting groups were removed, and the resulting tetraamine was condensed with compound

1074 Bioconjugate Chem., Vol. 14, No. 6, 2003

Jones et al.

Scheme 6a

a Reagents and conditions: (a) HOBt, Et N, pyridine; (b) H , Pd/C, MeOH; (c) 15c, Et N, pyridine; (d) TFA/CH Cl ; (e) transaminated 3 2 3 2 2 domain 1 polypeptide, 100 mM pH 4.6 sodium acetate, CH3CN.

Scheme 7a

a Reagents and conditions: (a) compound 10, Et N, pyridine; (b) TFA/CH Cl ; (c) transaminated domain 1 polypeptide, 100 mM 3 2 2 pH 4.6 sodium acetate, CH3CN.

8 to give the tetravalent aminooxy platform, compound 25. The platform was deprotected and treated with TA/ D1 under standard conditions to provide compound 1h. The pharmacokinetics of several radiolabeled domain 1 conjugates was studied, as was their ability to suppress anti-domain 1 antibodies and the relevant antibody producing cells in vivo. Pharmacokinetics and antibody suppression data is summarized for the tetravalent conjugates presented in this report. The potency of five tetravalent conjugates as toleragens was evaluated for their ability to suppress antibody levels in rats immunized with domain 1. Rats were primed with a conjugate of domain 1 and KLH (9) and adjuvant, treated with toleragen, and boosted.1 Serum samples were col-

lected, and anti-domain 1 antibody levels were measured using an ELISA. Table 1 summarizes the results of the antibody suppression experiments. It is clear that the three 20 kDa PEG conjugates (1d, 1g, and 1h) are required in lower doses to achieve significant antibody 1 Lewis rats (Harlan, Indianapolis, IN) weighing approximately 200 g were immunized ip with 10 µg of domain 1-KLH conjugate in alum with pertussis as adjuvant. Three weeks after the primary immunization, groups of four animals were treated iv with toleragen (0.88, 8.8, or 88 nmol/kg; 2.5, 0.25, and 0.025 mg of polypeptide per kilogram of body weight) or PBS control. Five days after treatment, animals were boosted ip with 10 µg of domain 1-KLH conjugate, and sera samples were collected 7 days after boost.)

Multivalent Poly(ethylene glycol)-Containing Conjugates

Bioconjugate Chem., Vol. 14, No. 6, 2003 1075 Table 2. Plasma t1/2 of Conjugates in Micea and Ratsb

Scheme 8a

a

compound

mice (h)

rats (h)

1a (no PEG) 1b (5K PEG) 1c (12K PEG) 1d (20K PEG) 1e (30K PEG) 1f (20K PEG) 1g (20K PEG) 1h (20K PEG)

0.11 ( .01 0.12 ( .04 0.20 ( .04 0.37 ( .03 1.03 ( .37 0.13 ( .01 0.37 ( .38 0.44 ( .02

1.5 ( .3 6.7 ( .6 7.7 ( .4 8.5 ( .6 18.9 ( .6 7.4 ( .8 9.5 ( .5 8.7 ( .2

Female CD-1;

b

Male Sprague-Dawley.

copies of domain 1 polypeptide from four to eight, however, resulted in a possible increase in clearance. Compound 1f, having 20 kDa PEG, has eight polypeptide domains and appears to clear marginally faster than the other conjugates with 20 kDa PEG. CONCLUSION

a Reagents and conditions: (a) Et N, pyridine; (b) TFA; (c) 3 compound 8, Et3N, CH2Cl2; (d) TFA/CH2Cl2; (e) transaminated domain 1 polypeptide, 100 mM pH 4.6 sodium acetate, CH3CN.

Table 1. Percent Reduction of Anti-domain 1 Antibodies in Immunized Ratsa dose per kg body weight compound

0.88 nmol

8.8 nmol

88 nmol

1a (no PEG) 1b (5K PEG) 1d (20 K PEG) 1g (20 K PEG) 1h (20 K PEG)

32 34 61b 72b 73b

36 73 82b 89b 93b

76b 86b 89b 96b 94b

a Lewis rats; percent reduction compared with PBS treated controls. b p e 0.05. For a brief description of the protocol see footnote 1.

suppression. The 5 kDa PEG conjugate 1b appears to be marginally more effective than 1a, which contains no PEG. The rates of elimination of all of the conjugates were measured in mice and in rats. The conjugates were radiolabeled with 125I using the iodine monochloride method (26), and the plasma concentrations of the compounds were measured in female CD-1 mice and in male Sprague-Dawley rats dosed at 1 mg/kg.2 The shapes of the time vs concentration curves were quite different in mice than in rats. In mice most of the conjugate cleared from plasma during the initial phase, whereas in rats most of the clearance took place during the second phase. The half-lives of the conjugates in both species are reported in Table 2. It is evident that, among the tetravalent conjugates, increases in the amount of PEG correlate with reduction in clearance rates. Increasing the molecular weight by increasing the number of 2 Equivalent pharmacokinetics was demonstrated between the Lewis rats used in the present efficacy studies and the Sprague-Dawley rats.)

We have described the preparation of three novel types of multivalent PEG-containing platforms. The platforms were used to prepare well-defined conjugates of the first domain of β2GPI, and the conjugates were studied for efficacy as B cell toleragens. It is clear that incorporation of PEG into the conjugates improved their effectiveness in suppressing the formation of anti-domain 1 antibodies in rats that were immunized with domain 1. In general as the molecular weight contribution from PEG increased, lower doses of toleragen were required. As the amount of PEG incorporated into the molecules was increased, the amount of suppression at all doses increased. The trend is most noticeable at the medium and low doses (8.8 and 0.88 nmol/kg). The improved efficacy of these conjugates is most likely a result of their persistence in circulation. It is likely that in addition to size-dependent kidney filtration, clearance of the conjugates can take place by other mechanisms such as uptake by various tissues, and incorporation of PEG may also affect the rates of clearance by those mechanisms. Conversely, glomerular filtration rates can depend on properties other than size. Comparison of the clearance rate of compound 1d with that of compound 1f shows that increasing the number of polypeptide epitopes actually appears to result in faster clearance; whereas, one might expect the opposite if size were the only factor affecting clearance. The domain 1 polypeptide is positively charged due to an excess of positively charged amino acids, and it is well-known that charge can affect clearance from circulation. The glomerular filtration rate of anionic IgG has been shown to be slower than that of cationic IgG (28). Similarly, it has been demonstrated that the kidney filtration rate of dextran depends on charge, with positively charged molecules clearing faster than negatively charged molecules (29). It is noteworthy that the spacing of the epitopes does not seem to significantly affect the ability of the conjugates to suppress antibody formation in the immunized rat model in which they were tested. The results with the three tetravalent conjugates (1d, 1g, and 1h), which contain 20000 MW PEG and are quite different in structure, show that they are remarkably similar in both their pharmacokinetic profiles and their ability to suppress antibody formation. Information on the relative abilities of the three structurally distinct tetravalent conjugates (1d, 1g, and 1h) to cluster B cell surface receptors would provide additional insight into the mechanism of B cell tolerance. Future studies will be aimed at further probing the structure activity relationships of

1076 Bioconjugate Chem., Vol. 14, No. 6, 2003

B cell toleragens with the goal of developing improved therapies for autoimmune diseases. ACKNOWLEDGMENT

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