Bioconjugate Chem. 2009, 20, 1899–1907
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A New Method to Produce MonoPEGylated Dimeric Cytokines Shown with Human Interferon-r2b Chien-Hsing Chang,*,†,‡ Edmund A. Rossi,† Thomas M. Cardillo,‡ Diane L. Nordstrom,‡ William J. McBride,‡ and David M. Goldenberg§ IBC Pharmaceuticals, Inc., Morris Plains, New Jersey 07950, Immunomedics, Inc., 300 American Road, Morris Plains, New Jersey 07950, and Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey 07109. Received April 17, 2009; Revised Manuscript Received June 24, 2009
We have adapted the dock-and-lock (DNL) method into a novel PEGylation technology using human interferonR2b (IFN-R2b) as an example. Central to DNL is a pair of distinct protein domains involved in the natural association between cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of poly(ethylene glycol) (PEG) to a dimeric form of IFNR2b. The combination of a fusion protein comprising IFN-R2b and the dimerization-and-docking domain (DDD) of PKA with a PEG-derivatized anchoring domain (AD) of an interactive AKAP results in facile formation of a trimeric complex containing two copies of IFN-R2b and a single site-specifically linked PEG chain. Three such monoPEGylated dimers of IFN-R2b have been generated, the first with a 20 kDa linear PEG, referred to as R2b-362, the second with a 30 kDa linear PEG (R2b-413), and the third with a 40 kDa branched PEG (R2b-457). All three retained antiviral and antitumor activity in Vitro and showed improved pharmacokinetic properties in mice, which translated into potent and prolonged therapeutic efficacy in the Daudi human lymphoma xenograft model. We anticipate wide applicability of the DNL method for developing long-acting therapeutics that are dimeric and monoPEGylated with the increased bioavailability allowing for less frequent dosing.
INTRODUCTION Interferons (IFNs ), in the half-century since their discovery, have been found to regulate infections with RNA and DNA viruses, enhance innate and acquired immunity, and affect normal and tumor cell growth and death (1). The therapeutic effectiveness of IFNs has also been validated to date by the approval of IFN-R2 for treating hairy cell leukemia, chronic myelogenous leukemia, malignant melanoma, follicular lymphoma, condylomata acuminata, AIDS-related Kaposi sarcoma, and chronic hepatitis B and C; IFN-β for treating multiple sclerosis; and IFN-γ for treating chronic granulomatous disease and malignant osteopetrosis. Despite a vast body of literature on this group of autocrine and paracrine cytokines, their functions in health and disease are still being elucidated, including more effective and novel forms being introduced clinically (2, 3). 1
* To whom correspondence should be addressed. Tel: (973) 6051330. Fax: (973)-605-1103. E-mail:
[email protected]. † IBC Pharmaceuticals, Inc. ‡ Immunomedics, Inc. § Garden State Cancer Center. 1 Abbreviations: AD, anchoring domain; AKAPs, A-kinase anchoring proteins; AUC, area under curve; BCN3, bicin3; DDD, dimerization and docking domain; DNL, dock and lock; EDANS, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNs, interferons; IL-2, interleukin-2; mPEG, monomethoxyPEG; mPEG2-MAL-40K, a 40 kDa branched PEG with a maleimide; MST, median survival time; MTS, [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]; OPTE, ortho-pyridylthioester; PBST, phosphate-buffered saline containing Tween; PEG, poly(ethylene glycol); PEGINTRON, Peginterferon alfa-2b; PEGASYS, Peginterferon alfa-2a; PKA, cAMP-dependent protein kinase; TCEP, Tris [2-carboxyethyl] phosphine hydrochloride.
As demonstrated by PEGINTRON (4) and PEGASYS (5), the efficacy of IFNs can be enhanced by improving the bioavailability with PEGylation, a process of chemically linking PEG to the therapeutic agent of interest, with the resulting conjugate exhibiting an increased serum half-life (6). Additional advantages of PEGylation in general include reduced immunogenicity, decreased dosing frequency, increased solubility, enhanced resistance to proteolysis, and exclusion of renal clearance. For example, a popular approach for improving the therapeutic efficacy of an enzyme has been to prepare conjugates containing multiple PEGs of small size, as known for methioninase (7), L-methione γ-lyase (8), arginine deiminase (9), adenosine deaminase (10), L-asparaginase (11), and liver catalase (12). PEGylations of nonenzyme proteins, such as bovine serum albumin (13); small molecules, such as inhibitors of integrin R4β1 (14); anti-VEGF aptamer (15), and oligodeoxynucleotides (16), have also been described. Because the most common reactive sites on proteins (including peptides) for attaching PEG are the ε-amino groups of lysine and the R-amino group of the N-terminal residue, most methods of PEGylation result in modification of multiple sites, yielding not only monoPEGylated conjugates consisting of mixtures of positional isomers (4, 5), but also adducts comprising more than one PEG chain. Nevertheless, site-specific attachment of a single PEG to the R-amino group of the N-terminal residue was reported to be the predominant product upon reacting PEGaldehyde at low pH with IFN-β1b (17) or IFN-β1a (18). Similar strategies were applied to prepare N-terminally linked PEG to G-CSF (19) or type I soluble necrosis factor receptor (20). Sitedirected PEGylation of an endogenous or engineered free cysteine residue in a target protein has also been demonstrated with PEG-maleimide for G-CSF (21), hemoglobin (22), IL-2 (23), IFN-R2 (24), GM-CSF (25), EPO (26), scFv (27), Fab (28), and miniantibodies (29). Moreover, the feasibility of reversible or releasable PEGylation, wherein covalently attached
10.1021/bc9001773 CCC: $40.75 2009 American Chemical Society Published on Web 09/08/2009
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Figure 1. Schematics of IMP457, R2b-DDD2, and R2b-457. Ribbons indicate 40 kDa branched PEG; red helix, AD2; blue helix, DDD2; SH, free sulfhydryl groups of engineered cysteine residues.
PEG can be cleaved in ViVo, has been achieved with a variety of degradable linkages as exemplified by linking a 40-kDa PEGSH to IFN-R2 with a 2-sulfo-9-fluorenylmethoxycarbonylcontaining bifunctional reagent (30), by attaching linear or branched PEG-BCN3 to lyzosome or IFN-β1b (31), or by conjugating PEG to lysozyme via a dithiobenzyl urethane linkage (32). Recently, an innovative strategy for site-specific PEGylation of disulfide bonds has been reported (33), but its use may be limited to only those proteins with native disulfide bonds that are suitably oriented for such modification. The dock-and-lock (DNL) method developed by us (34, 35) provides a unique approach to build multivalent, multifunctional complexes of defined composition and retained bioactivity. The key to the DNL method is the exploitation of the specific protein-protein interactions occurring in nature between the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (36, 37). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has R and β isoforms (38). The R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (39). The AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (40). Interestingly, AKAPs will only bind to dimeric R subunits. For human RIIR, the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (41). Thus, the dimerization domain and AKAP binding domain of human RIIR are both located within the same N-terminal 44 amino acid sequence (39, 42), which is termed the dimerization and docking domain (DDD) herein. Because an entity derivatized with the DDD (termed the DDD-module) is expected to self-associate to form a dimer, thereby creating a docking site for an entity derivatized with the AD (termed the AD-module), combining a DDD-module with an AD-module results in the formation of a noncovalent complex containing three entities, which can be stably tethered by introducing cysteine residues into the DDD and AD at strategic positions, as demonstrated with the DDD2 and AD2 (34). Here, with a DDD2-module of IFN-R2b expressed as a fusion protein and three synthetic AD2-modules of different mPEG, we describe the successful adaptation of the DNL method into a novel PEGylation technology, which has the salient feature of monoPEGylating a dimeric form of IFN-R2b at a specific site that is outside the sequence of native IFN-R2b (Figure 1).
EXPERIMENTAL PROCEDURES Generation of IMP360. The AD2-peptide with an EDANS at its carboxyl terminus to provide a fluorescent tag was termed
Chang et al.
IMP360 and made at 0.1 mmol scale with EDANS resin (Novabiochem, EMD Biosciences, Inc., San Diego, CA) on a PS3 peptide synthesizer (Protein Technologies, Tucson, Az) using Fmoc methodology. Starting from the C-terminus, the protected amino acids used were Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, FmocGln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-LeuOH, Fmoc-Tyr(But)-OH, Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Cys(Trt)-OH. The peptide was cleaved from the resin, purified by RP-HPLC, and determined to have a molecular mass of 2660 Da. The sequence of IMP360 is CGQIEYLAKQIVDNAIQQAGC(SStbu)-EDANS. Generation of IMP362 and IMP413. The two linear PEGAD2 modules were prepared by coupling IMP360 to mPEGOPTE (Nectar Therapeutics, San Carlos, CA) of 20 kDa or 30 kDa, resulting in IMP362 or IMP413, respectively. The sulfhydryl group of the N-terminal cysteine of IMP360 displaces pyridine-2-thiol from mPEG-OPTE and forms a thioester bond to mPEG. A subsequent reaction of the adjacent amino group with the carbonyl group of the thioester leads to the formation of a more stable amide bond and the regeneration of the sulfhydryl group. To prepare IMP362, IMP360 (11.5 mg) was mixed with 20 kDa mPEG-OPTE (127 mg) in 7 mL of 1 M Tris-HCL, pH 7.8. Acetonitrile (1 mL) was added to dissolve some suspended material. The reaction was stirred at room temperature for 4 h to effect the attachment of mPEG to the amino-terminal cysteine via an amide bond. Subsequently, 41 mg of Tris [2-carboxyethyl] phosphine hydrochloride (TCEP) and 43 mg of cysteine were added to deprotect the remaining cysteine. The reaction mixtures were stirred for 1 h and desalted using PD-10 columns, which had been equilibrated with 20% methanol in water. The samples were lyophilized to obtain approximately 150 mg of IMP362 (MH+ 23 713). IMP413 (MH+ 34 499) was made similarly using 30 kDa mPEG-OPTE (190 mg). Generation of IMP421 and IMP457. The AD2-containing peptide (IMP421, MH+ 2891, Figure 2) was made for derivatizing mPEG2-MAL-40K (Nectar Therapeutics) to obtain the branched PEG-AD2 module (IMP457). IMP421 was synthesized on NovaSyn TGR resin (487.6 mg, 0.112 mmol) by adding the following amino acids to the resin in the order shown: FmocGly-OH, Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-AlaOH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, FmocVal-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH, FmocGlu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-GlyOH, Fmoc-Cys(t-Buthio)-OH, Fmoc-NH-PEG3-COOH, FmocCys(Trt)-OH. The peptide was then cleaved from the resin and purified by RP-HPLC to yield 32.7 mg of a white solid. The N-terminal cysteine of IMP421 has its amino group protected as an acetyl derivative and provides a sulfhydryl group to react with the maleimide of mPEG2-MAL-40K. IMP457 was made as follows. To a solution of IMP421 (15.2 mg, 5.26 µmol) and mPEG2-MAL-40K (274.5 mg, 6.86 µmol) in 1 mL of acetonitrile was added 7 mL of 1 M Tris pH 7.8. After 3 h at room temperature, the excess mPEG2-MAL-40K was quenched with L-cysteine (49.4 mg), followed by S-S-tBu deprotection over 1 h with TCEP (59.1 mg). The resulting solution was dispensed into two 10K Slide-A-Lyzer dialysis cassettes (4 mL in each) and dialyzed overnight at 2-8 °C against 5 L of 5 mM ammonium acetate, pH 5.0. Following three additional 5 L buffer changes, the dialyzed solution (19.4 mL) was transferred into two 20 mL scintillation vials, frozen,
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Figure 2. Schematic structure of IMP421.
and lyophilized to yield a white solid (246.7 mg). The molecular mass as determined by MALDI-TOF was 42 982 for mPEG2MAL-40K (Supporting Information Figure S1A) and 45 500 for IMP457 (Supporting Information Figure S1B). Generation of r2b-DDD2. All restriction enzymes were purchased from New England Biolabs (Beverly, MA). A fulllength human IFN-R2b cDNA clone (Invitrogen Ultimate ORF human clone cat# HORF01Clone ID IOH35221) was used as a template for amplification by PCR to generate a sequence comprising XbaIsSPsIFN-R2bs6 HissBamHI, where XbaI/ BamHI, SP, and 6 His are restriction sites, the signal peptide native to IFN-R2b, and a hexahistidine tag, respectively. The following oligonucleotides were synthesized by Sigma Genosys (Haverhill, UK) and used as primers. IFNA2 XbaI left 5′-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG-3′ IFNA2 BamHI right 5′-GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3′ PCR reactions were performed using Amplitaq polymerase (Applied Biosystems, Foster City, CA) and a Perkin-Elmer (Wellesley, MA) GeneAmp PCR system 9600. The PCR amplimer was cloned into the pGemT vector (Promega) from which the IFN-R2b amplimer was excised with XbaI and BamHI and ligated to a DDD2-pdHL2 mammalian expression vector (34) previously digested with XbaI and BamHI to generate IFNR2b-DDD2-pdHL2, the expression vector for R2b-DDD2. IFNR2b-DDD2-pdHL2 (30 µg) was linearized by digestion with SalI and stably transfected by electroporation (450 V, 25 µF) into Sp/ESF myeloma cells (2.8 × 106 cells), an engineered derivative of Sp2/0 that can be grown and transfected in serumfree medium. Transfectants were cultured in Hybridoma SFM (Invitrogen, Carlsbad, CA) supplemented with 0.2 µM methotrexate (Calbiochem, La Jolla, CA) in 96-well plates, and screened for IFN-R2b expression with a human interferon alpha ELISA kit (PBL Interferon Source, Piscataway, NJ) following the manufacturers’ instructions. R2b-DDD2 was purified from terminal batch cultures of a select clone grown in roller bottles. Briefly, the supernatant fluid was clarified by centrifugation, filtered through 0.2 µm membranes, diafiltered into 1× binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM NaH2PO4, pH 7.5), concentrated 20-fold, and loaded onto a Ni-NTA column (Qiagen). After washing with 0.02% Tween 20 in 1× binding buffer and then 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl, 50 mM NaH2PO4, pH 7.5, R2b-DDD2 was eluted with 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH2PO4, pH 7.5, and stored in the same buffer at 2-8 °C until needed. The mass of R2b-
DDD2 was determined by MALDI-TOF to be 52 185, indicating a dimer. The monomeric form of R2b-DDD2 consists of IFNR2b fused at its carboxyl-terminus to a 63-residue peptide of KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA. Preparation and Purification of r2b-362, r2b-413, and r2b-457. Conjugations of R2b-DDD2 with IMP362, IMP413, and IMP457 were performed to generate PEGylated R2b-362, R2b-413, and R2b-457, respectively. In general, a 10-fold molar excess of reduced and lyophilized IMP362, IMP413, or IMP457 was added to 2.25 mg of R2b-DDD2 in 3.5 mL of 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH2PO4, pH 7.5. After 6 h at room temperature in the dark, the reaction mixture was dialyzed against CM loading buffer (150 mM NaCl, 20 mM NaAc, pH 4.5) at 4 °C in the dark. The PEGylated product was purified by cation exchange chromatography using a 1 mL Hi-Trap CM-FF column (Amersham), which was equilibrated with CM loading buffer. After sample loading, the column was washed with CM loading buffer to baseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM NaAc, pH 4.5. The PEGylated product was eluted with 12.5 mL of 0.5 M NaCl, 20 mM NaAc, pH 4.5. SDS-PAGE, Fluorescence Imaging, and Immunoblot. SDS-PAGE analyses were performed using 4-20% gradient Tris-glycine gels (Cambrex Bio Science Rockland, Rockland, ME). To normalize the samples for direct protein mass comparison, each fraction eluted from the CM-FF column was concentrated to 3.5 mL to match the reaction volume. Samples were diluted with an equal volume of 2× sample buffer (2% SDS, 5% glycerol, 62.5 mM Tris-HCl, pH 6.8) and heated to 95 °C before loading 10 µL per lane. For reducing gels, 5% of β-2-mercaptoethanol was included in the sample buffer. Gels were imaged by direct fluorescence to visualize bands containing the EDANS tag using a Kodak Image Station 4000R before staining with Coomassie blue to visualize the protein bands. Duplicate gels were transferred to PVDF membranes for immunoblot analysis. Blots were probed with rabbit antiinterferon alpha polyclonal antibody (AB1434, Chemicon International) diluted 1:1000 in 1% BSA-PBST. Signal was detected with HRP-conjugated goat antirabbit IgG (Jackson Immunoresearch) and Pierce ECL Western Blotting Substrate (Pierce Biotechnology, Rockford, IL). Antiviral Assay. The reduction of viral cytopathic effect (CPE) was determined by an independent laboratory (PBL Interferon Source, Piscataway, NJ) using encephalomyocarditis virus (EMCV) to infect human lung epithelial A549 cells. Plates were stained with crystal violet and the OD was measured by spectrophotometry on a 96-well plate reader following solubilization of the dye. The data were analyzed with GraphPad Prism
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software (Advanced Graphics Software, Encinitas, CA) using a sigmoidal fit (variable slope) nonlinear regression. The antiviral titer was determined by comparison of EC50 values with that of an IFNR standard. Antiproliferative Assay. Each test article was diluted to 500 pM in RPMI 1640 media supplemented with 10% FBS, from which triplicate, 3-fold serial dilutions were made in 96-well tissue culture plates (50 µL/well). Daudi cells were diluted to 4 × 105 cells/mL and 50 µL was added to each well (20K/well). After 4 days at 37 °C, viable cell densities were determined by MTS using a CellTiter 96 Cell Proliferation Assay (Promega, Madison, WI). Dose-response curves were generated and EC50 values were obtained by sigmoidal fit nonlinear regression using GraphPad Prism software. Specific activities were determined by comparison of EC50 values with PEGINTRON using the specific activity provided with the product insert. Reporter Gene Assay. The iLite Human Interferon Alpha Cell-Based Assay Kit (PBL Interferon Source) was used to determine specific activities following the manufacturers’ suggested protocol. Briefly, samples were diluted in 1% BSA-PBS to 5, 1.25, and 0.3125 ng/mL for R2b-362 and R2b-413; to 10, 2.5, and 0.625 ng/mL for R2b-457 and PEGASYS; and to 1, 0.25, and 0.0625 ng/mL for PEGINTRON. Each dilution was assayed in triplicate and incubated overnight with the supplied cells. Specific activities were extrapolated from a standard curve generated with the supplied standard. Pharmacokinetics in Mice. The initial study evaluating R2b362, R2b-413, PEGINTRON, and rhIFN-R2a (Chemicon IF007, lot 06008039084) was performed in 4 groups of adult female Swiss-Webster mice (∼35 g) with 2 animals in each group. Each test article was administered as a single bolus i.v. injection at mole-equivalent dose: rhIFN-R2a, 3 µg; PEGINTRON, 5 µg; R2b-362, 11 µg; and R2b-413, 13 µg. Mice were bled via the retro-orbital method at various time points (predose, 5 min, and 2, 8, 24, 48, 72, 96, and 168 h postinjection). The subsequent study evaluating R2b-457, R2b-413, PEGINTRON, and PEGASYS was performed in 4 groups of adult male Swiss-Webster mice (∼40 g) with 4 animals in each group. Each test article was administered as a single s.c. injection at mole-equivalent dose (100 pmol) in equal volumes (250 µL). Mice were bled via the retro-orbital method at various time points (30 min, and 2, 8, 24, 48, 72, and 96 h postinjection). For each study, the blood was allowed to clot, centrifuged, and the serum removed for storage at -70 °C until analysis. Concentrations of IFN-R in the serum samples were determined using a human interferon alpha ELISA kit (PBL Interferon Source) following the manufacturers’ instructions. Briefly, the serum samples were diluted appropriately according to the human IFN-R standard provided in the kit. The antibody immobilized to the microtiter plate wells captures interferon. A second antibody is then used to reveal the bound interferon, which is quantified by antisecondary antibody conjugated to horseradish peroxidase (HRP) following the addition of tetramethyl benzidine. The plates were read at 450 nm. The amount of IFN in the serum for each animal was used to determine the various PK parameters. These data were analyzed with WinNonLin PK software (v 5.1; Pharsight Corp.; Mountain View, CA) using noncompartmental analysis (representing the best-fit model for the data). In ViWo Antitumor Efficacy. All four studies were performed with the approval of the local animal care and use committee in eight-week-old female SCID mice injected i.v. with 1.5 × 107 Daudi cells per animal. Therapy commenced 1 day after the Daudi transplantation. Mice were observed daily for signs of distress and paralysis. They were weighed weekly. In the event a mouse lost greater than 15% of its body weight (but
