PEGylation by the dock and lock (DNL) technique

Abstract

The present invention concerns methods and compositions for forming PEGylated complexes of defined stoichiometry and structure. In preferred embodiments, the PEGylated complex is formed using dock-and-lock technology, by attaching a target agent to a DDD sequence and attaching a PEG moiety to an AD sequence and allowing the DDD sequence to bind to the AD sequence in a 2:1 stoichiometry, to form PEGylated complexes with two target agents and one PEG moiety. In alternative embodiments, the target agent may be attached to the AD sequence and the PEG to the DDD sequence to form PEGylated complexes with two PEG moieties and one target agent. In more preferred embodiments, the target agent may comprise any peptide or protein of physiologic or therapeutic activity. The PEGylated complexes exhibit a significantly slower rate of clearance when injected into a subject and are of use for treatment of a wide variety of diseases.

Claims

1. A PEGylated complex comprising: a) a therapeutic agent attached to a DDD (dimerization and docking domain) moiety, wherein the amino acid sequence of the DDD moiety consists of the N-terminal 44 amino acids from human protein kinase A (PKA) RII; and b) a PEG moiety attached to an AD (anchor domain) moiety wherein the amino acid sequence of the AD moiety is from an AKAP protein; wherein two copies of the DDD sequences bind to one copy of the AD sequence to form a PEGylated complex.

2. The complex of claim 1, wherein the amino acid sequence of the AD moiety is selected from the group consisting of SEQ ID NO:1.

3. The complex of claim 1, further comprising disulfide bonds between the DDD and AD moieties.

4. The complex of claim 1, wherein the therapeutic agent is selected from the group consisting of an enzyme, a cytokine, a chemokine, a growth factor, an antibody and an antigen binding fragment thereof.

5. The complex of claim 1, wherein the therapeutic agent is selected from the group consisting of interferon-, interferon-, interferon-, MIF, HMGB-1 (high mobility group box protein 1), TNF-, G-CSF and GM-CSF.

6. The complex of claim 1, wherein the complex comprises a structure selected from the group consisting of: (i) IMP350: CGQIEYLAKQIVDNAIQQAGC(SS-tbu) -NH.sub.2 (SEQ ID NO:1), (ii) IMP360: CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQ ID NO:1), (iii) IMP362: PEG.sub.20-CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS (SEQ ID NO:1); and (iv) IMP413: PEG.sub.30-CGQIEYLAKQIVDNAIQQAGC(SS-tbu) G-EDANS (SEQ ID NO:1).

7. The complex of claim 1, wherein the therapeutic agent is a cytokine.

8. The complex of claim 1, wherein the therapeutic agent is interferon (IFN)-, G-CSF or erythropoietin.

9. The complex of claim 1, wherein the therapeutic agent attached to the DDD sequence is a fusion protein.

10. The complex of claim 1, wherein the complex comprises one PEG moiety and two therapeutic agents.

11. The complex of claim 1, wherein the therapeutic agent is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, and IL-18.

12. The complex of claim 1, wherein the therapeutic agent is selected from the group consisting of Eotaxin, CCL19, CCL21, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, and Gro-.

13. The complex of claim 1, wherein the therapeutic agent is selected from the group consisting of PDGF, Flt-3 ligand, erythropoietin, thrombopoietin, hGH, CNTF, VEGF, P1GF, insulin, IGF, somatostatin, tissue plasminogen activator, and LIF.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Cartoon illustration of the DNL method. Triangles depict component A, which forms a homodimer (a.sub.2) mediated by the dimerization and docking domain (DDD). The location of free thiol groups (SH) of the engineered cysteine residues is indicated. Octagons depict component B containing an anchor domain (AD) peptide. The DNL reaction results in the generation of a covalent trimeric structure via binding of DDD and AD peptides and subsequent formation of disulfide bridges.

(2) FIG. 2. Cartoon drawing of IMP362. A 20 kDa PEG (starburst), AD2 peptide (helix), EDANS fluorescent tag (oval) and the positions of free sulfhydryl groups (SH) are indicated.

(3) FIG. 3. Cartoon drawing of IMP413. A 30 kDa PEG (starburst), AD2 peptide (helix), EDANS fluorescent tag (oval) and the positions of free sulfhydryl groups (SH) are indicated.

(4) FIG. 4. Analysis of roller bottle production and purification by anti-IFNa immunoblot and ELISA. Samples were diluted as indicated and 5 l were subjected to reducing SDS-PAGE and immunoblot analysis with polyclonal anti-IFNa. The dilution, total volume and fraction analyzed of the total volume (f) for each sample is given. The amount of protein in each band was estimated from standards and divided by f to give the total protein estimate. Total protein measurements determined by ELISA are also given.

(5) FIG. 5. Reducing SDS-PAGE analysis of IFN2b-DDD2 following IMAC purification with Ni-NTA. Two independent preparations of IFN2b-DDD2 (lanes 1 &2), which were eluted from Ni-NTA resin with 250 mM imidazole buffer, and the 30 mM imidazole buffer column wash were resolved by SDS-PAGE under reducing conditions and stained with Coomassie blue. The position of M.sub.r standards and IFN2b-DDD2 (arrow) are indicated.

(6) FIG. 6. Cartoon drawing of 2b-362. IFN2b groups (pentagons), 20 kDa PEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescent tag (oval) are indicted.

(7) FIG. 7. Analysis of CM-FF IEC purification of IFN2b-DDD2-IMP362 by SDS-PAGE with Coomassie Blue staining (A), direct fluorescence imaging (B) and anti-IFN immunoblotting (C). The same gel was used for fluorescence imaging and subsequent Coomassie blue staining. For this gel (A+B), all fractions were concentrated to correspond to the reaction volume (3.5 mL) and 5 l/lane were loaded. Both reduced and non-reduced samples were run as indicated. For the immunoblot (C), all samples were diluted 1:50 from those used for gel A/B. Solid black, grey and open arrow-heads show the positions of IFN2b-DDD2-IMP362, IFN2b-DDD2 and IMP362, respectively. The positions of M.sub.r standards are also indicated.

(8) FIG. 8. Cartoon drawing of 2b-413. IFN2b groups (pentagons), 30 kDa PEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescent tag (oval) are indicted.

(9) FIG. 9. Dose-response curves showing in vitro growth inhibition of Burkitt's lymphoma (Daudi) cells after 4-days in culture in the presence of either rhIFN-2b standard, IFN-2b-DDD2 or 2b-362. MTS dye was added to the plates, which were incubated for 3 h before measuring the OD.sub.490. The % of the signal obtained from untreated cells was plotted vs. the log of the molar concentration. The 50% effective concentration (EC.sub.50) values were obtained by sigmoidal fit non-linear regression using Graph Pad Prism software.

(10) FIG. 10. Evaluation of the pharmacokinetic properties of IFN constructs. Each reagent (test and control) was administered to Swiss-Webster mice at equimolar protein doses as a single bolus i.v. injection of 3 g for rhuIFN-2a, 5 g for PEGINTRON, 11 g for 2b-362, and 13 g for 2b-413. Serum samples were isolated at the times indicated and the serum concentrations of IFN- were determined by ELISA. The M concentration was plotted vs. hours post injection. Data represents the mean value from two mice.

(11) FIG. 11. Evaluation of the therapeutic efficacy of IFN constructs in mice bearing Burkitt's lymphoma (Daudi). Eight-week-old female SCID mice were injected i.v. with 1.510.sup.7 Daudi cells. Groups of 5 mice were administered PEGINTRON, 2b-362 and 2b-413 at doses of 3,500, 7,000 or 14,000 Units once per week for 4 weeks. Therapy commenced 1 day after the Daudi cells were transplanted. Injection times are indicated with arrows. Survival curves and median survival are shown for each group.

(12) FIG. 12. Evaluation of the dosing schedule for therapy of tumor-bearing mice. Eight-week-old female SCID mice were injected i.v. with 1.510.sup.7 Daudi-cells. Groups of 6-7 mice were administered 14,000 IU of either PEGINTRON or 2b-413 via a s.c. injection. Therapy was commenced 1 day after the Daudi cells were administered to the mice. Groups were dosed once a week (q7d4), once every other week (q2wk4) or once every 3 weeks (q3wk4). Injection times are indicated with arrows. All the mice received 4 injections in total. Survival curves and median survival are shown for each group.

(13) FIG. 13. Cartoon drawing of G-CSF-413. G-CSF groups (pentagons), 30 kDa PEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescent tag (oval) are indicted.

(14) FIG. 14. SDS-PAGE and anti-EPO immunoblot analysis of IMAC-purified EPO-DDD2. Proteins were resolved by SDS-PAGE under reducing or non-reducing conditions and gels were stained with Coomassie blue or transferred to PVDF membranes for immunoblot analysis with an anti-EPO monoclonal antibody. The amounts of protein loaded/lane is indicated at the bottom of the lanes. The positions of MW standards and of EPO-DDD2 (arrow) are indicated.

(15) FIG. 15. Cartoon drawings of h679-Fab-DDD2 (A), dimeric EPO-DDD2 (B), which combine to create EPO-679 (C) by the DNL method. The variable and constant domains of h679 Fab (ovals), AD2 and DDD2 helices, EPO groups (pentagons) and free sulfhydryl groups (SH) are indicated.

(16) FIG. 16. SDS-PAGE analysis of EPO-679. Proteins (1 g) were resolved by SDS-PAGE under reducing and non-reducing conditions in the lanes as indicated and gels were stained with Coomassie blue. The positions of MW standards and all relevant species (arrows) are indicated.

(17) FIG. 17. Stimulation of cell growth by EPO-constructs. EPO-responsive TF1 cells (110.sup.4) were cultured for 72 hours in the presence of rhEPO, EPO-DDD2 or EPO-679. The relative viable cell density was determined by MTS assay. The log of the concentration in U/mL is plotted vs. OD.sub.490.

(18) FIG. 18. Cartoon drawing of EPO-413. EPO groups (pentagons), 30 kDa PEG (starburst), AD2 and DDD2 peptides (helices) and EDANS fluorescent tag (oval) are indicted.

DOCK AND LOCK (DNL) METHOD

(19) 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) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). PKA, which plays a central role in one of the best studied signal transduction pathway triggered by the binding of the second messenger cAMP to the R subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymes of PKA are found with two types of R subunits (RI and MI), and each type has and isoforms (Scott, Pharmacol. Ther. 1991; 50:123). 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 (Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561).

(20) Since the first AKAP, microtubule-associated protein-2, was characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984; 81:6723), more than 50 AKAPs that localize to various sub-cellular sites, including plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum, have been identified with diverse structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem. 1991; 266:14188). The amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003; 100:4445). Interestingly, AKAPs will only bind to dimeric R subunits. For human RII, the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216). Thus, the dimerization domain and AKAP binding domain of human RII are both located within the same N-terminal 44 amino acid sequence (Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J. 2001; 20:1651), which is termed the DDD herein.

(21) DDD of Human RII and AD of AKAPs as Linker Modules

(22) We have developed a platform technology to utilize the DDD of human RII and the AD of a certain amino acid sequence as an excellent pair of linker modules for docking any two entities, referred to hereafter as A and B, into a noncovalent complex, which could be further locked into a stably tethered structure through the introduction of cysteine residues into both the DDD and AD at strategic positions to facilitate the formation of disulfide bonds, as illustrated in FIG. 1. The general methodology of the dock-and-lock approach is as follows. Entity A is constructed by linking a DDD sequence to a precursor of A, resulting in a first component hereafter referred to as a. Because the DDD sequence would effect the spontaneous formation of a dimer, A would thus be composed of a.sub.2. Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b. The dimeric motif of DDD contained in a.sub.2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a.sub.2 and b to form a binary, trimeric complex composed of a.sub.2b. This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001; 98:8480) to ligate site-specifically.

(23) By attaching the DDD and AD away from the functional groups of the two precursors, such site-specific ligations are also expected to preserve the original activities of the two precursors. This approach is modular in nature and potentially can be applied to link, site-specifically and covalently, a wide range of substances, including peptides, proteins, nucleic acids, and PEG. The DNL method was disclosed in each of the following U.S. Provisional patent applications: 60/728,292, filed Oct. 20, 2005; 60/751,196, filed Dec. 16, 2005; and 60/782,332, filed Mar. 14, 2006, and U.S. patent application Ser. No. 11/389,358, all incorporated herein by reference in their entirety.

(24) PEGylation by DNL

(25) In a preferred method, the target to be PEGylated is linked to a DDD sequence to generate the DDD module. A PEG reagent of a desirable molecular size is derivatized with a related AD sequence and the resulting PEG-AD module is combined with the DDD module to produce the PEGylated conjugate that consists of a single PEG tethered site-specifically to two copies of the target via the disulfide bonds formed between DDD and AD. The PEG reagents are capped at one end with a methoxy group (m-PEG), can be linear or branched, and may contain one of the following functional groups: propionic aldehyde, butyric aldehyde, ortho-pyridylthioester (OPTE), N-hydroxysuccinimide (NETS), thiazolidine-2-thione, succinimidyl carbonate (SC), maleimide, or ortho-pyridyldisulfide (OPPS). Among the targets that may be of interest for PEGylation are enzymes, cytokines, chemokines, growth factors, peptides, aptamers, hemoglobins, antibodies and fragments. The method is not limiting and a wide variety of agents may be PEGylated using the disclosed methods and compositions.

EXAMPLES

(26) The following examples are provided to illustrate, but not to limit, the claims of the present invention.

Example 1

Generation of PEG-AD2 Modules

(27) Synthesis of IMP350

(28) TABLE-US-00001 (SEQIDNO:1) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH.sub.2MH.sup.+2354

(29) IMP350 was made on a 0.1 mmol scale with Sieber Amide resin using Fmoc methodology on a Protein Technologies PS3 peptide synthesizer. 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, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, 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 and purified by reverse phase (RP)-HPLC.

(30) Synthesis of PEG.sub.20-IMP350

(31) IMP350 (0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20 kDa, Nektar Therapeutics) in 7 mL of 1 M Tris buffer at pH 7.81. Acetonitrile, 1 mL, was then added to dissolve some suspended material. The reaction was stirred at room temperature for 3 h and then 0.0527 g of TCEP was added along with 0.0549 g of cysteine. The reaction mixture was stirred for 1.5 h and then purified on a PD-10 desalting column, which was equilibrated with 20% methanol in water. The sample was eluted, frozen and lyophilized to obtain 0.0924 g of crude PEG.sub.20-IMP350 (MH+23508 by MALDI).

(32) Synthesis of IMP360

(33) TABLE-US-00002 (SEQIDNO:9) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANSMH.sup.+2660

(34) IMP 360 was synthesized on a 0.1 mmol scale with EDANS resin (Nova Biochem) using Fmoc methodology on a Protein Technologies PS3 peptide synthesizer. The Fmoc-Gly-OH was added to the resin manually using 0.23 g of Fmoc-Gly-OH, 0.29 g of HATU, 26 L of DIEA, 7.5 mL of DMF and 0.57 g of EDANS resin (Nova Biochem). The reagents were mixed and added to the resin. The reaction was mixed at room temperature for 2.5 hr and the resin was washed with DMF and IPA to remove the excess reagents. 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, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, 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 and purified by RP-HPLC.

(35) Synthesis of IMP362 (PEG.sub.20-IMP360)

(36) A cartoon diagram of IMP362 is provided in FIG. 2. For synthesis of IMP362, IMP360 (0.0115 g) was mixed with 0.1272 g of mPEG-OPTE (20 kDa, Nektar Therapeutics) in 7 mL of 1 M tris buffer, pH 7.81. Acetonitrile (1 mL) was then added to dissolve some suspended material. The reaction was stirred at room temperature for 4 h and then 0.0410 g of TCEP was added along with 0.0431 g of cysteine. The reaction mixture was stirred for 1 h and purified on a PD-10 desalting column, which was equilibrated with 20% methanol in water. The sample was eluted, frozen and lyophilized to obtain 0.1471 g of crude IMP362 (MH+23713).

(37) Synthesis of IMP413 (PEG.sub.30-IMP360)

(38) A cartoon diagram of IMP 413 is provided in FIG. 3. For synthesis of IMP 413, IMP 360 (0.0103 g) was mixed with 0.1601 g of mPEG-OPTE (30 kDa, Nektar Therapeutics) in 7 mL of 1 M tris buffer at pH 7.81. Acetonitrile (1 mL) was then added to dissolve some suspended material. The reaction was stirred at room temperature for 4.5 h and then 0.0423 g of TCEP was added along with 0.0473 g of cysteine. The reaction mixture was stirred for 2 h followed by dialysis for two days. The dialyzed material was frozen and lyophilized to obtain 0.1552 g of crude IMP413 (MH.sup.+ 34499).

Example 2

Generation of DDD Module Based on Interferon (IFN)-2b

(39) Construction of IFN-2b-DDD2-pdHL2 for Expression in Mammalian Cells

(40) The cDNA sequence for IFN-2b was amplified by PCR, resulting in a sequence comprising the following features, in which XbaI and BamHI are restriction sites, the signal peptide is native to IFN-2b, and 6 His is a hexahistidine tag (SEQ ID NO: 10): XbaI---Signal peptide---IFN2b---6 His---BamHI (6 His disclosed as SEQ ID NO: 10). The resulting secreted protein will consist of IFN-2b fused at its C-terminus to a polypeptide consisting of SEQ ID NO:2.

(41) TABLE-US-00003 (SEQIDNO:2) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEF AVEYFTRLREARA

(42) PCR amplification was accomplished using a full length human IFN2b cDNA clone (Invitrogen Ultimate ORF human clone cat# HORFO1Clone ID IOH35221) as a template and the following oligonucleotides as primers:

(43) TABLE-US-00004 IFNA2XbaILeft (SEQIDNO:3) 5-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTG G-3 IFNA2BamHIright (SEQIDNO:4) 5-GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAA CTTTCTTGC-3

(44) The PCR amplimer was cloned into the pGemT vector (Promega). A DDD2-pdHL2 mammalian expression vector was prepared for ligation with IFN-2b by digestion with XbaI and Bam HI restriction endonucleases. The IFN-2b amplimer was excised from pGemT with XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate the expression vector IFN-2b-DDD2-pdHL2.

(45) Mammalian Expression of IFN-2b-DDD2

(46) IFN-2b-DDD2-pdHL2 was linearized by digestion with SalI enzyme and stably transfected into Sp/EEE myeloma cells by electroporation (see. e.g., U.S. patent application Ser. No. 11/487,215, filed Jul. 14, 2006, incorporated herein by reference). Two clones were found to have detectable levels of IFN-2b by ELISA. One of the two clones, designated 95, was adapted to growth in serum-free media without substantial decrease in productivity. The clone was subsequently amplified with increasing methotrexate (MTX) concentrations from 0.1 to 0.8 M over five weeks. At this stage, it was sub-cloned by limiting dilution and the highest producing sub-clone (95-5) was expanded. The productivity of 95-5 grown in shake-flasks was estimated to be 2.5 mg/L using commercial rIFN-2b (Chemicon IF007, Lot 06008039084) as a standard.

(47) Purification of IFN-2b-DDD2 from Batch Cultures Grown in Roller Bottles

(48) Clone 95-5 was expanded to 34 roller bottles containing a total of 20 L of serum-free Hybridoma SFM with 0.8 M MTX and allowed to reach terminal culture. The supernatant fluid was clarified by centrifugation, filtered (0.2 M). The filtrate was diafiltered into 1 Binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5) and concentrated to 310 mL in preparation for purification by immobilized metal affinity chromatography (IMAC). The concentrate was loaded onto a 30-mL Ni-NTA column, which was washed with 500 mL of 0.02% Tween 20 in 1 binding buffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5. The product was eluted with 110 mL of 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5. Approximately 6 mg of IFN2b-DDD2 was purified. FIG. 4 shows the results of an anti-IFNa immunoblot and ELISA used to quantify IFN2b-DDD2.

(49) Characterization of IFN-2b-DDD2

(50) The purity of IFN-2b-DDD2 was assessed by SDS-PAGE under reducing conditions (FIG. 5). The Coomassie blue-stained gel shows that the batch produced from roller bottles (lane 2) was purer than an earlier batch (lane 1). IFN-2b-DDD2 was the most heavily stained band and accounts for approximately 50% of the total protein. The product resolves as a doublet with an M.sub.r of 26 kDa, which is consistent with the calculated MW of IFN-2b-DDD2-SP (26 kDa). There is one major contaminant with a M.sub.r of 34 kDa and many faint contaminating bands.

Example 3

Generation of PEGylated IFN-2b by DNL

(51) Preparation and Purification of 2b-362 (IFN-2b-DDD2-IMP 362)

(52) A cartoon drawing depicting the structure of 2b-362 having two copies of IFN2b coupled to a 20 kDa PEG is provided in FIG. 6. A DNL reaction was performed by the addition of 11 mg of reduced and lyophilized IMP362 in 10-fold molar excess to 2.25 mg (3.5 ml) of IFN-2b-DDD2 in 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH.sub.2PO.sub.4, 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 solution was loaded onto a 1-mL Hi-Trap CM-FF column (Amersham), which was pre-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.

(53) The conjugation process was analyzed by SDS-PAGE with Coomassie blue staining (FIG. 7A), fluorescence imaging (FIG. 7B) and anti-IFN immunoblotting (FIG. 7C). 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. Under non-reducing conditions, the Coomassie blue-stained gel (FIG. 7A) revealed the presence of a major band at a M.sub.r of 110 kDa (lane 2) in the reaction mixture, which was absent in the unbound (lane 3) or 0.25 M NaCl wash fraction (lane 4), but evident in the 0.5 M NaCl fraction (lane 5). Fluorescence imaging (FIG. 7B), which was used to detect the EDANS tag on IMP362, demonstrates that the 110 kDa band contains IMP362 (lanes 2 and 5) and the presence of excess IMP362 in the reaction mixture (lane 2) and the unbound fraction (lane 3), which does not stain with Coomassie blue. Anti-IFN immunoblotting (FIG. 7C) confirms the association of IFN-2b with the 110 kDa band (lanes 2 and 5). These data together indicate that the DNL reaction results in the site-specific and covalent conjugation of IMP362 with a dimer of IFN-2b. Under reducing conditions, which breaks the disulfide linkage, the components of the DNL structures are resolved. The calculated MW of 2b-362 is 75 kDa, which matches well the mass of 76,728 Da determined by MALDI TOF. The observed discrepancy between the calculated mass and the estimated Mr by SDS-PAGE is due to PEG, which is known to inflate the molecular size when PEGylated products are analyzed by SDS-PAGE or SE-HPLC. Overall, the DNL reaction resulted in a near quantitative yield of a homogeneous product that is >90% pure after purification by cation-exchange chromatography.

(54) Preparation and Purification of 2b-413 (IFN-2b-DDD2-IMP413)

(55) A cartoon drawing depicting the structure of 2b-413 having two copies of IFN2b coupled to a 30 kDa PEG is provided in FIG. 8. 2b-413 was prepared as described immediately above using IMP413 instead of IMP362.

Example 4

Evaluation of the in vitro potency of IFN-2b-DDD2, 2b-362, and 2b-413

(56) In Vitro Anti Proliferative Assay

(57) IFN-2b-DDD2 and 2b-362 were assayed for inhibition of growth of Burkitt's lymphoma (Daudi) cells. Briefly, IFN-2b standard (Chemicon IF007, Lot 06008039084), IFN-2b-DDD2 (batch 010207) and 2b-362 (batch 010807) were each diluted to 500 M in RPMI 1640 media supplemented with 10% FBS, from which three-fold serial dilutions in triplicate were made in 96-well tissue culture plates (50 L sample/well). Daudi cells were diluted to 410.sup.5 cells/mL and 50 L were added to each well (20K/well). The concentration range for each test reagent was 500 M to 0.008 M. After 4 days at 37 C., MTS dye was added to the plates (20 L per well) and after 3 h the plates were read with an Envision plate reader (Perkin Elmer, Boston Mass.) at 490 nm. Dose-response curves were generated (FIG. 9) and 50% effective concentration (EC.sub.50) values were obtained by sigmoidal fit non-linear regression using Graph Pad Prism software (Advanced Graphics Software, Encinitas, Calif.). The calculated EC.sub.50 for IFN2b-DDD2 and 2b-362 were similar (16 pM) and about 5-fold less potent than the IFN-2b standard (EC.sub.504 pM). In a similar experiment the 2b-413 had similar potency as 2b-362.

(58) Anti-Viral Assay

(59) Duplicate samples were analyzed in a viral challenge assay using encephalomyocarditis (EMC) virus on A549 cells by an independent analytical laboratory (PBL Interferon Source, Piscataway, N.J.). 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 Graph Pad Prizm software using a sigmoidal fit (variable slope) non-linear regression. The anti-viral titer was determined by comparison of EC.sub.50 values with that of an IFNa standard. The specific anti-viral activities were calculated at 1.210.sup.8 U/mg and 8.810.sup.6 U/mg for 2b-362 and 2b-413, respectively.

Example 5

In Vivo Evaluation of 2b-413 and 2b-362

(60) Pharmacokinetics

(61) The study was performed in adult female Swiss-Webster mice (35 g). There were 4 different treatment groups of 2 mice each. Each reagent (test and control) was administered at equimolar protein doses (3 g of rhuIFN-2a, 5 g of PEGINTRON, 11 g of 2b-362, and 13 g of 2b-413) as a single bolus i.v. injection. Mice were bled via the retro-orbital method at various time-points (pre-dose, 5-min, 2-, 8-, 24-, 48-, 72-, 96-, and 168-h post-injection). The blood was allowed to clot, centrifuged, and the serum was isolated and stored at 70 C. until assayed for IFN- concentration and subsequent PK-analysis.

(62) Concentrations of IFN- in the serum samples were determined using a human interferon alpha ELISA kit following the manufacturers instructions (PBL Interferon Source). Briefly, the serum samples were diluted appropriately according to the human IFN- standard provided in the kit. An antibody coupled to the microtiter plate wells captures interferon. A second antibody is then used to reveal the bound interferon, which is quantified by anti-secondary antibody conjugated to horseradish peroxidase (HRP) following the addition of Tetramethyl benzidine (TMB) substrate. The plates were read at 450 nm, and the results are shown in FIG. 10.

(63) The PK properties of each agent are summarized in Table 1. As expected, rhIFN-2a had the most rapid clearance from the blood of injected mice. Its clearance was approximately 3-fold faster than the PEGINTRON and more than 13-fold faster than the DNL-IFN reagents. The PEGINTRON was in turn cleared greater than 4-fold faster than 2b-362 or 2b-413. There was little difference in the elimination rates between 2b-362 and 2b-413.

(64) TABLE-US-00005 TABLE 1 Blood Pharmacokinetic Analysis of Interferon-2b Containing DNL Molecules Administered as Intravenous Injections to Naive Swiss-Webster Mice. IFN Elimination Animal Dose C.sub.max T.sub.1/2 T.sub.1/2 AUC.sub.0.08.fwdarw. Rate MRT.sub.0.08.fwdarw. Number (pmol) (pM) (hours) (hours) (h * pM) (1/h) (h) Recombinant Human Interferon-2a Animal No. 1 160 16,411 0.29 10.53 7,011 2.34 0.63 Animal No. 2 160 21,835 0.31 7.14 10,147 2.15 0.78 Mean 160 19,123 0.30 8.84 8,579 2.25 0.71 PEG-INTRON Animal No. 1 160 87,090 0.53 6.29 137,790 0.63 5.42 Animal No. 2 160 105,774 0.43 5.11 150,905 0.70 4.79 Mean 160 96,432 0.48 5.70 144,348 0.67 5.11 IFN-2b-IMP362 Animal No. 1 320 60,833 1.72 7.54 379,462 0.16 9.03 Animal No. 2 320 97,089 1.43 10.14 570,336 0.17 11.56 Mean 320 78,961 1.58 8.84 474,899 0.17 10.30 IFN-2b-IMP413 Animal No. 1 320 152,923 0.69 12.85 1,012,470 0.15 16.75 Animal No. 2 320 100,495 4.03 28.53 1,179,056 0.09 26.56 Mean 320 126,709 2.36 20.69 1,095,763 0.12 21.66

(65) In terms of mean residence time (MRT), there is a clear correlation with size among the various reagents. The 19-kDa rhIFN-2a had a MRT that was 7-fold less than the 31 kDa PEGINTRON (0.7 h versus 5.1 h, respectively), which had a 2-fold lower MRT when compared to the 70 kDa 2b-362 (10.3 h). The MRT for the 80 kDa 2b-413 (21.7 h) was 2-fold longer than 2b-362. Finally, a test for bioequivalence showed that none of the reagents tested were the same in terms of PK, indicating that the differences are genuine (i.e., circulating half-life for 2b-413>2b-362>PEGINTRON>rhIFN-2a).

(66) Anti-Tumor Therapeutic Efficacy

(67) An initial in vivo tumor therapy study demonstrated that the DNL-PEGylated interferons were more potent and longer-lasting compared to PEGINTRON. Eight-week-old female C.B.-17 SCID mice were injected i.v. with a human Burkitt's lymphoma cell-line (Daudi) at 1.510.sup.7 cells per animal. There were 10 different treatment groups of 5 mice each. Equivalent units of activity of PEGINTRON, 2b-362 and 2b-413 were administered once every 7 days via s.c. injection in either the left or right flank at three different doses (3500, 7000, and 14000 Units). Therapy commenced 1 day after the Daudi cells were transplanted.

(68) Mice were observed daily for signs of distress and paralysis. They were weighed weekly. In the event a mouse or mice lost greater than 15% of its body weight (but <20%) it was weighed every 2 days until it either gained back its weight to <15% loss or was sacrificed due to >20% loss. Mice were also terminated when hind-limb paralysis developed or if they became otherwise moribund.

(69) Survival curves generated from this study are shown in FIG. 11. PEGINTRON, 2b-362, and 2b-413 all demonstrated significant improvement in survival when compared to saline control mice (P<0.0016). Except for the 3,500 IU dose of 2b-362, both 2b-413 and 2b-362 were superior to PEGINTRON when administered at equal activity doses (P0.0027). 2b-362 showed more than twice the potency of PEGINTRON. Doses of 7,000 IU and 3,500 IU of 2b-362 were superior to 14,000 IU (P=0.0016) and 7,000 IU (P=0.0027) doses of PEGINTRON, respectively. 2b-413 is more than four times as potent as PEGINTRONsince a 3,500 IU dose of the former was superior to 14,000 IU of the latter (P=0.0027). 2b-413 was significantly better than 2b-362 (P<0.0025) when administered at equivalent doses. However, there were no statistically significant differences among the three doses of 2b-413, even though the 14,000 IU dose resulted in a median survival of 60 days in comparison to the 3,500 IU dose and its 46-day median survival (P=0.1255). The in vivo efficacy observed for 2b-362, 2b-413, and PEGINTRON thus correlate well with the PK data.

(70) The increased bioavailability of 2b-362 and 2b-413 demonstrated by PK analysis contributes to the enhanced in vivo anti-tumor potency of DNL-PEGylated IFN. In turn, these two factors allow for a less frequent dosing schedule used in tumor therapy. This was demonstrated with a similar in vivo tumor therapy study as above, in which equal units of activity of PEGINTRON or 2b-413 were administered with varied dosing schedules. This study was performed in 8-week-old female SCID mice injected i.v. with Daudi 1.510.sup.7 cells. There were 7 different treatment groups of 6-7 mice each. Each reagent (test and control) was administered 14,000 IU via a s.c. injection in either the left or right flank. Therapy was commenced 1 day after the Daudi-cells were administered to the mice. One set of mice was dosed once a week for 4 weeks (q7d4), another dosed on a bi-weekly schedule over 8 weeks (q2wk4), while the third set of mice was dosed once every 3 weeks over 12 weeks (q3wk4). All the mice received a total of 4 injections.

(71) Survival curves generated from this study are shown in FIG. 12. All animals that received either form of interferon at any of the various schedules had significantly improved survival in comparison to saline control mice (P<0.0009). Importantly, all the IFN-IMP413-treated mice had significantly improved survival when compared to those animals treated at the same schedule with PEGINTRON (P<0.0097). Of note, those mice treated every other week with IFN-1MP413 (q2wkx4) not only had significantly improved survival in comparison to those treated with PEGINTRON at the same schedule (MST=>54 days versus 28 days, respectively; P=0.0002), but were also significantly better than those animals treated weekly (q7d4) with PEGINTRON (MST=36.5 days; P=0.0049). Further, survival of mice treated every three weeks with IFN-IMP413 (q3wk4) was significantly better than those treated with PEGINTRON every two weeks (MST=54 days versus 28 days; P=0.002) and approaches significance when compared to those treated weekly with PEGINTRON (P=0.0598).

(72) These studies demonstrate DNL-PEGylation of IFN2b results in improved and long-lasting efficacy, allowing for less frequent dosing. Similar enhancements is realized when this technology is applied to other cytokines (such as G-CSF and EPO), growth factors, enzymes, antibodies, immunomodulators, hormones, peptides, drugs, interference RNA, oligonucleotides, vaccines and other biologically active agents.

Example 6

Generation of DDD Module Based on Granulocyte-Colony Stimulating Factor (G-CSF)

(73) Construction of G-CSF-DDD2-pdHL2 for Expression in Mammalian Cells

(74) The cDNA sequence for G-CSF was amplified by PCR resulting in sequences comprising the following features, in which XbaI and BamHI are restriction sites, the signal peptide is native to human G-CSF, and 6 His is a hexahistidine tag (SEQ ID NO: 10): XbaI---Signal peptide---G-CSF---6 His---BamHI (6 His disclosed as SEQ ID NO: 10). The resulting secreted protein consisted of G-CSF fused at its C-terminus to a polypeptide consisting of SEQ ID NO:5.

(75) TABLE-US-00006 (SEQIDNO:2) KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEF AVEYFTRLREARA

(76) PCR amplification was accomplished using a full-length human G-CSF cDNA clone (Invitrogen IMAGE human cat#97002RG Clone ID 5759022) as a template and the following oligonucleotides as primers:

(77) TABLE-US-00007 G-CSFXbaILeft (SEQIDNO:5) 5-TCTAGACACAGGACCTCATCATGGCTGGACCTGCCACCCAG-3 G-CSFBamHI-Right (SEQIDNO:6) 5-GGATCCATGATGGTGATGATGGTGTGACTTGGGCTGGGCAAGGTGGC GTAG-3

(78) The PCR amplimer was cloned into the pGemT vector. A DDD2-pdHL2 mammalian expression vector was prepared for ligation with G-CSF by digestion with XbaI and Bam HI restriction endonucleases. The G-CSF amplimer was excised from pGemT with XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate the expression vector G-CSF-DDD2-pdHL2.

(79) Mammalian Expression of G-CSF-DDD2

(80) G-CSF-pdHL2 was linearized by digestion with SalI enzyme and stably transfected into Sp/EEE myeloma cells by electroporation. Clones were selected with media containing 0.15 M MTX. Clone #4 was shown to produce 0.15 mg/L of G-CSF-DDD2 by sandwich ELISA.

(81) Purification of G-CSF-DDD2 from Batch Cultures Grown in Roller Bottles

(82) Approximately 3 mg of G-CSF-DDD2 is purified as descried in Example 2. Clone 4 is expanded to 34 roller bottles containing a total of 20 L of Hybridoma SFM with 0.4 M MTX and allowed to reach terminal culture. The supernatant fluid is clarified by centrifugation, filtered (0.2 M), diafiltered into 1 Binding buffer (10 mM Imidazole, 0.5 M NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5 and concentrated. The concentrate is loaded onto a Ni-NTA column, which is washed with 0.02% Tween 20 in 1 binding buffer and then 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5. The product is eluted with 250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH.sub.2PO.sub.4, pH 7.5.

Example 7

Generation of PEGylated G-CSF by DNL

(83) A cartoon drawing depicting the structure of G-CSF-413 having two copies of G-CSF coupled to a 30 kDa PEG is provided in FIG. 13. A DNL reaction is performed by the addition of reduced and lyophilized IMP413 in 10-fold molar excess to G-CSF-DDD2 in PBS. After 6 h at room temperature in the dark, the reaction mixture is purified by immobilized metal affinity chromatography using Ni-NTA.

Example 8

Generation of DDD Module Based on Erythropoeitin (EPO)

(84) Construction of G-CSF-DDD2-pdHL2 for Expression in Mammalian Cells

(85) The cDNA sequence for EPO was amplified by PCR resulting in sequences comprising the following features, in which XbaI and BamHI are restriction sites, the signal peptide is native to human EPO, and 6 His is a hexahistidine tag (SEQ ID NO: 10): XbaI---Signal peptide---EPO---6 His---BamHI (6 His disclosed as SEQ ID NO: 10). The resulting secreted protein consists of EPO fused at its C-terminus to a polypeptide consisting of SEQ ID NO:2.

(86) PCR amplification was accomplished using a full-length human EPO cDNA clone as a template and the following oligonucleotides as primers:

(87) TABLE-US-00008 EPOXbaIleft (SEQIDNO:7) 5-TCTAGACACAGGACCTCATCATGGGGGTGCACGAATGTCC-3 EPOBamHIRight (SEQIDNO:8) 5-GGATCCATGATGGTGATGATGGTGTGACTTTCTGTCCCCTGTCCTGC AG-3

(88) The PCR amplimer was cloned into the pGemT vector. A DDD2-pdHL2 mammalian expression vector was prepared for ligation with EPO by digestion with XbaI and Bam HI restriction endonucleases. The EPO amplimer was excised from pGemT with XbaI and Bam HI and ligated into the DDD2-pdHL2 vector to generate the expression vector EPO-DDD2-pdHL2.

(89) Mammalian Expression of EPO-DDD2

(90) EPO-pdHL2 was linearized by digestion with SalI enzyme and stably transfected into Sp/EEE myeloma cells by electroporation. Clones were selected with media containing 0.15 M MTX. Clones #41, 49 and 37 each were shown to produce 0.5 mg/L of EPO by an ELISA using Nunc Immobilizer Nickel-Chelate plates to capture the His-tagged fusion protein and detection with anti-EPO antibody.

(91) Purification of EPO from Batch Cultures Grown in Roller Bottles

(92) Approximately 2.5 mg of EPO-DDD2 is purified by IMAC from 9.6 liters of serum-free roller bottle culture as described in Example 2. SDS-PAGE and immunoblot analysis indicate that the purified product constitutes approximately 10% of the total protein following IMAC (FIG. 14). Under reducing conditions the EPO-DDD2 polypeptide is resolved as a broad band with a M.sub.r (40-45 kDa) greater than its calculated mass (28 kDa) due to extensive and heterogeneous glycosylation. Under non-reducing conditions the EPO-DDD2 primarily resolves as a disulfide-linked covalent dimer (mediated by DDD2) with a M.sub.r of 80-90 kDa.

Example 9

DNL Conjugation of EPO-DDD2 with a Fab-AD2 Module

(93) h679 is a humanized monoclonal antibody that is highly specific for the hapten HSG (histamine-succinyl-glycine). Production of an h679-Fab-AD2 module, which is depicted in the cartoon drawing in FIG. 15 (A), has been described previously (Rossi et. al, Proc. Natl. Acad. Sci. USA. 2006; 103:6841). A cartoon drawing depicting the dimeric structure of EPO-DDD2 is provided in FIG. 15 (B). A small-scale preparation of EPO-679 (EPO-DDD2h679-Fab-AD2) was made by DNL. EPO-DDD2 (1 mg) was reacted overnight with h679-Fab-AD2 (1 mg) in PBS containing 1 mM reduced glutathione and 2 mM oxidized glutathione. The DNL conjugate was purified by HSG-based affinity chromatography as described previously (Rossi et. al, Proc. Natl. Acad. Sci. USA. 2006; 103:6841). A cartoon drawing depicting the structure of EPO-679 with two EPO moieties and h679-Fab is provided in FIG. 15 (C). Coomassie blue staining of SDS-PAGE gels demonstrated the creation of EPO-679 (FIG. 16). The DNL product, which is resolved as a broad band with a M.sub.r of 150-170 kDa under non-reducing conditions, is highly purified and consists only of the three constituent polypeptides (EPO, h679-Fd-AD2 and h679 Kappa) as demonstrated by SDS-PAGE under reducing conditions.

Example 10

Biological Activity of EPO-DDD2 and EPO-679

(94) EPO-DDD2 and EPO-679 were assayed for their ability to stimulate the growth of EPO-responsive TF1 cells (ATCC) using recombinant human EPO (Calbiochem) as a positive control. TF1 cells were grown in RPMI 1640 media supplemented with 20% FBS without GM-CSF supplementation in 96-well plates containing 110.sup.4 cells/well. The concentrations (units/ml) of the EPO constructs were determined using a commercial kit (Human erythropoietin ELISA kit, Stem Cell Research, Cat#01630). Cells were cultured in the presence of rhEPO, EPO-DDD2 or EPO-679 at concentrations ranging from 900 U/ml to 0.001 U/ml for 72 hours. The viable cell densities were compared by MTS assay using 20 l of MTS reagent/well incubated for 6 hours before measuring the OD490 in a 96-well plate reader. Dose response curves and EC50 values were generated using Graph Pad Prism software (FIG. 17). Both EPO-DDD2 and EPO-679 show in vitro biological activity at approximately 10% of the potency of rhEPO.

Example 11

Generation of PEGylated EPO by DNL

(95) A cartoon drawing depicting the structure of EPO-413 having two copies of EPO coupled to a 30 kDa PEG is provided in FIG. 18. A DNL reaction is performed by the addition of reduced and lyophilized IMP413 in 10-fold molar excess to EPO-DDD2 in PBS. After 6 h at room temperature in the dark, the reaction mixture is purified by immobilized metal affinity chromatography using Ni-NTA.

Example 12

Production of 2-PEG:1-Target Agent Complexes

(96) In alternative embodiments, it is desirable to make PEGylated complexes with a stoichiometry of 2 PEG moieties to 1 target agent. Such PEGylated complexes are readily made by the methods of Examples 1-3 above, by attaching the PEG moiety to the DDD sequence and the active agent to the AD sequence. A PEGylated complex with a 2:1 stoichiometry of PEG to IFN-2b is prepared by a modification of the methods of Examples 1-3. The complex exhibits stability in serum and shows interferon activity that is lower than the PEGylated complex with a 1:2 stoichiometry of PEG to IFN-2b. However, clearance rate for the bi-PEGylated complex is slower than the clearance rate for the mono-PEGylated complex.