HK1148471B - Pegylation by dock and lock (dnl) technique - Google Patents

Description

Pegylation by docking and locking (DNL) techniques

RELATED APPLICATIONS

This application claims priority from U.S. patent application serial No. 11/925408, filed on 26/10/2007, the entire contents of which are incorporated herein by reference.

Background

The efficacy of a therapeutic agent can be enhanced by improving its bioavailability through a variety of means, one of which is pegylation, i.e., the process of chemically linking polyethylene glycol to the therapeutic agent of interest, and the resulting conjugate exhibits an increased serum half-life. Other advantages of pegylated products may also include lower immunogenicity, reduced dosing frequency, increased solubility, enhanced stability and reduced renal clearance. Since the most common reactive sites on proteins (including peptides) for attachment of PEG are the epsilon amino group of lysine and the alpha amino group of the N-terminal residue, earlier pegylation methods resulted in multi-site modifications that not only produced mono-pegylated conjugates consisting of mixtures of positional isomers, such as PEGINTRON^TM(Grace et al, J.biol.chem.2005; 280: 6327) and(Dhalluin et al, Bioconjugate chem.2005; 16: 504), but also to adducts comprising more than one PEG chain. It is reported that site-specific attachment of a single PEG to the alpha amino group of the N-terminal residue is the major product of PEG-acetaldehyde (PEG-ALD) reaction with IFN-beta 1b (Basu et al, Bioconjugate chem.2006; 17: 618) or IFN-beta 1a (Pepinsky et al, J.Pharmacol. Exp. Ther.2001; 297: 1059) at low pH. Similar strategies were applied to the preparation of G-CSF N-terminally linked to PEG (Kinstler et al pharm. Res. 1996; 13: 996) or type I soluble tumor necrosis factor receptor (Kerwin et al Protein Sci.2002; 11: 1825). A solid phase process for N-terminal pegylation of recombinant interferon alpha-2 a has recently been reported (Lee et al, bioconjugate.

Site-directed pegylation of free cysteine residues introduced into a protein of interest using PEG-maleimide (PEG-MAL) has also been achieved for several recombinant constructs, including IL-2(Goodson and Katre, Biotechnology.1990: 8: 343); IFN- α 2(Rosendahl et al, Bioconjugate chem.2005; 16: 200); GM-CSF (Doherty et al, Bioconjugate chem.2005; 16: 1291); scFv (Yang et al, Protein Eng.2003; 16: 761), and minibodies (Kubetzko et al, J.biol.chem; 2006; 201: 35186). A common approach for improving the efficacy of enzyme therapy is to prepare conjugates containing multiple small PEGs, as known for methioninases (Yang et al, Cancer Res.2004; 64: 6673); l-methionine gamma-lyase (Takakura et al, Cancer Res.2006: 66: 2807); arginine deaminase (Wang et al, Bioconjugate chem.2006; 17: 1447); adenosine deaminase (Davis et al, Clin. exp. Immunol. 1981; 46: 649); l-asparaginase (Bendich et al, Clin. exp. Immunol. 1982; 48: 273); and liver catalases (Abuchowski et al, J.biol.chem.1977; 252: 3582).

Bovine serum albumin is also described (Abuchowski et al, J.biol.chem.1977; 252: 3578); hemoglobin (Manjula et al, Bioconjugate chem.2003; 14: 464); visomant (Mosharraf et al, int.J.pharm.2007; 336: 215); small molecule substances, such as inhibitors of integrin α 4 β 1 (Pepinsky et al, J.Pharmacol. exp. Ther.2005; 312: 742); lymphoma targeting peptides (DeNardo et al, Clin. cancer. Res.2003; 9 (Suppl.): 3854 s); PEGylation of anti-VEGF aptamers (Bunka and Stockley, nat. Rev. Microbiol. 2006; 4: 588) and oligodeoxynucleotides (Fisher et al, Drug Metab. Dispos. 2004; 32: 983). However, there is a need for a general method of pegylation that can exclusively produce mono-pegylated conjugates consisting of a single PEG moiety specifically attached to a predetermined position of a candidate agent, and that retains the biological activity of the unmodified counterpart.

Summary of The Invention

Methods and compositions for generating pegylated complexes having a selected number of attached PEG residues attached to selected positions of a candidate agent are disclosed. In a preferred embodiment, the agent is mono-pegylated. In a more preferred embodiment, as detailed below, the target to be pegylated may be attached to a DDD (dimerization and docking domain) sequence and a PEG moiety may be attached to an AD (anchor domain) sequence. Dimers of the DDD sequence bind with high affinity to monomers of the AD sequence, resulting in the formation of mono-pegylated effector partial dimers. The stoichiometry of conjugation and the position of the PEG residues are determined by the specificity of the DDD/AD interaction.

In a more preferred embodiment, the complex may be covalently stabilized by introducing cysteine residues at appropriate positions in the DDD and AD sequences to form disulfide bonds that stabilize the mono-pegylated complex. In other embodiments, the PEG reagent may be capped at one end with a linear or branched methoxy group (m-PEG).

In other preferred embodiments, pegylated complexes prepared by the DNL method exhibit serum clearance rates that are at least one order of magnitude slower than non-pegylated effector moieties. In certain alternative embodiments, a pegylated complex may alternatively be constructed with a PEG moiety attached to the DDD sequence and an effector moiety attached to the AD sequence, resulting in 2 PEG: stoichiometry of 1 effect moiety.

One skilled in the art will recognize that virtually any physiologically or therapeutically active agent to be administered in vivo may be stabilized by pegylation, including, but not limited to, enzymes, cytokines, chemokines, growth factors, peptides, aptamers, hemoglobin, antibodies, and fragments thereof. Exemplary agents include MIF, HMGB-1 (high mobility group box 1), TNF- α, 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, IL-18, IL-19, IL-23, IL-24, CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro- β, Eotaxin (Eotaxin), interferon- α, - β, - λ, G-CSF, GM-1, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin, thrombopoietin, hGH, CNTF, leptin (leptin), oncostatin M, VEGF, EGF, FGF, PlGF, insulin, hGH, calcitonin, factor VIII, IGF, somatostatin, tissue plasminogen activator and LIF.

The mono-pegylated complexes are suitable for use in a variety of therapeutic and diagnostic applications. Methods of using the mono-pegylated compounds may comprise: detection, diagnosis and/or treatment of a disease or other medical condition. Such conditions may include, but are not limited to, cancer, hyperplasia, diabetes, diabetic retinopathy, macular degeneration, inflammatory bowel disease, crohn's disease, ulcerative colitis, rheumatoid arthritis, sarcoidosis, asthma, edema, pulmonary hypertension, psoriasis, corneal graft rejection, neovascular glaucoma, Osler-Webber syndrome, myocardial angiogenesis, plaque neovascularization, restenosis, neointimal formation following vascular trauma, telangiectasia, hemophilia patient's joints, angiofibromas, fibrosis associated with chronic inflammation, pulmonary fibrosis, deep vein thrombosis or wound granulogenesis.

In particular embodiments, the disclosed methods and compositions can be used to treat autoimmune diseases, such as, for example, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyadenylic syndrome, bullous pemphigoid, juvenile diabetes, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangiitis obliterans, Sjogren's syndrome, primary biliary cirrhosis, hashimoto thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tuberculosis of the spinal cord, giant cell arteritis/myalgia, pernicious anemia, accelerated glomerulonephritis, psoriasis or fibrositis.

It is contemplated that any type of tumor and any type of tumor antigen may be targeted. Exemplary types of tumors that can be targeted include acute lymphoblastic leukemia, acute myelogenous leukemia, bile duct cancer, breast cancer, bone cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer.

Brief description of the drawings

FIG. 1 cartoon representation of the DNL method. The triangle represents component A, which forms a homodimer mediated by a Dimerization and Docking Domain (DDD) (a)₂). The position of the free thiol (SH) group of the engineered cysteine residue is indicated. The octagon represents component B containing the Anchor Domain (AD) peptide. The DNL reaction generates a covalent trimeric structure by the binding of DDD to the AD peptide and the subsequent formation of disulfide bridges.

FIG. 2 cartoon representation of IMP 362. The positions of the 20kDa PEG (explosion star), the AD2 peptide (helix), the EDANS fluorescent tag (oval) and the free thiol group (SH) are shown.

FIG. 3 cartoon representation of IMP 413. The positions of 30kDa PEG (explosion star), AD2 peptide (helix), EDANS fluorescent tag (oval) and free thiol (SH) are shown.

FIG. 4 is an analysis of flask production and purification using anti-IFNa immunoblotting and ELISA. Samples were diluted as indicated and subjected to reducing SDS-PAGE with 5. mu.l and immunoblot analysis with polyclonal anti-IFNa. The dilution, total volume, analytical fraction of total volume (f) per sample are given. The amount of protein in each band was estimated from the standard and divided by the total volume to give an estimate of total protein. Total protein measurements by ELISA are also given.

FIG. 5 cartoon representation of α 2 b-362. IFN alpha 2b groups (pentagons), 20kDa PEG (explosion star), AD2 and DDD2 peptide (helices) and EDANS fluorescent tag (ovals) are indicated.

FIG. 6 cartoon representation of α 2 b-413. IFN alpha 2b groups (pentagons), 30kDa PEG (explosion star), AD2 and DDD2 peptide fragments (helices) and EDANS fluorescent tags (ovals) are indicated.

FIG. 7 shows a dose response curve for in vitro growth inhibition of Burkitt lymphoma (Daudi) cells after 4 days of culture in the presence of rhIFN-. alpha.2b standard, IFN-. alpha.2b-DDD 2, or. alpha.2b-362. MTS dye was added to the plate, which was measuring OD₄₉₀Incubation was preceded by 3 h. The obtained signal was plotted as a percentage of untreated cells versus the log of the molar concentration. 50% Effective Concentration (EC) was obtained by sigmoidal fitted nonlinear regression of Graph Pad Prism software₅₀)。

Figure 8. evaluation of pharmacokinetic properties of IFN α constructs. Each reagent (test and control) was administered as a single i.v. injection at an equimolar protein dose to Swiss-Webster mice with 3. mu.g rhuIFN-. alpha.2a and PEGINTRON^TM5 μ g, α 2b-362 11 μ g, and α 2b-413 13 μ g. Serum samples were separated at the indicated times and the serum concentration of IFN-. alpha.was determined by ELISA. ρ M concentration is plotted against hours post injection. Data represent the average from two mice.

Figure 9 evaluation of IFN-a construct therapeutic efficacy in mice bearing burkitt lymphoma (Daudi). 8 week old female SCID mice i.v. injected 1.5x10⁷Daudi cells. Administration of PEGINTRON to a group of 5 mice^TMα 2b-362 and α 2b-413 at doses of 3,500, 7,000 or 14,000 units, once per week for 4 weeks. Treatment started one day after Daudi cell transplantation. The injection time is shown by the arrow. Survival curves and median survival for each group are shown.

Figure 10 evaluation of dosing schedules for treatment of tumor bearing mice. 8 week old female SCID mice i.v. injected 1.5x10⁷Daudi-cells. Groups of 6,7 mice were administered 14,000IU of PEGINTRON via subcutaneous injection^TMOr α 2 b-413. Treatment was started one day after Daudi cells were administered to mice. The groups were administered once a week (q7dx4), once a week (q2wkx4) and once a three week (q3wkx 4). The injection time is indicated by an arrow. All mice received a total of 4 injections. Survival curves and median survival values for each group are shown.

FIG. 11 cartoon representation of G-CSF-413. The G-CSF group (pentagon), 30kDaPEG (explosion star shape), AD2 and DDD2 peptide (helix) and EDANS fluorescent tag (ellipse) are indicated.

FIG. 12 cartoon representation of h679-Fab-DDD2(A), dimeric EPO-DDD2(B), which h679-Fab-DDD2 and polyepo-DDD 2 combine by DNL to produce EPO-679 (C). The h679Fab (oval) variable and constant domains, AD2 and DDD2 helices, EPO groups (pentagons) and free thiol (SH) are indicated.

FIG. 13 stimulation of cell growth by EPO constructs. EPO-responsive TF1 cells (1X 10)⁴) Cultured in the presence of rhEPO, EPO-DDD2 or EPO-679 for 72 hours. Relative viable cell density was determined by MTS analysis. The log value of the U/mL concentration is assigned to the OD₄₉₀And (6) drawing.

FIG. 14 cartoon representation of EPO-413. The EPO group (pentagon), 30kDa PEG (explosion star), AD2 and DDD2 peptides (helix) and EDANS fluorescent tag (oval) are indicated.

FIG. 15 shows the structure of IMP-421.

FIG. 16 Structure of mPEG 2-MAL-40K.

Docking and locking (DNL) method

The DNL method utilizes specific protein/protein interactions that occur between the regulatory (R) subunit of cAMP-dependent Protein Kinase (PKA) and the dockerin domain (AD) of the A-kinase dockerin (AKAP) (Bailie et al, FEBS letters.2005; 579: 3264.Wong and Scott, nat. Rev. MoI. cell biol.2004; 5: 959). PKA was first isolated from rabbit skeletal muscle in 1968 and served as the core in one of the most well studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunit (Walsh et al, J.biol.chem.1968; 243: 3763). The structure of the holoenzyme is composed of two catalytic subunits placed in inactive form by the R subunit (Taylor, J.biol.chem.1989; 264: 8443). The isozymes of PKA were found to have two types of R subunits (RJ and RII), and each type has alpha and beta isoforms (Scott, Pharmacol. Ther.1991; 50: 123). The R subunit is only isolated as a stable dimer, and the dimerization domain has been shown to consist of the first 44 amino terminal residues (Newson et al, nat. struct. biol. 1999; 6: 222). Binding of cAMP to the R subunit results in the release of the active catalytic subunit, which is localized to the selected substrate by the compartmentalization of PKA via its docking with AKAP, to obtain a broad spectrum of serine/threonine kinase activity (Scott et al, J.biol.chem.1990; 265; 21561).

Since the first AKAP, tubulin-2, was characterized in 1984 (Lohmann et al, proc.natl.acad.sci. usa.1984; 81: 6723), 50 AKAP with different structures, located at different subcellular sites including the plasma membrane, actin cytoskeleton, nucleus, mitochondria and endoplasmic reticulum, have been identified in a number of species from yeast to human (Wong and Scott, nat.rev.mol.cell biol.2004; 5: 959). The AD of AKAP to PKA is a 14-18 residue amphipathic helix (Carr et al, J.biol.chem.1991; 266: 14188). The amino acid sequence of AD varies widely between AKAP and has been reported to have binding affinities for RII dimers ranging from 2-90nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100: 4445). Interestingly, AKAP binds only to the dimeric R subunit. For human RII α, AD binds to a hydrophobic surface formed by 23 amino-terminal residues (Collidge and Scott, Trends Cell biol.1999; 6: 216). Thus, the dimerization domain of human RII α and the AKAP binding domain are both located in the same N-terminal 44 amino acid sequence (Newton et al, nat. struct. biol. 1999; 6: 222; Newton et al, EMBO J. 2001; 20: 1651), which is referred to herein as DDD.

DDD of human RII alpha and AD of AKAP as linker Module

We have developed a platform technology that utilizes DDD of human RII α and AD of a particular amino acid sequence as an excellent pair of linker modules to dock any two entities (hereinafter referred to as a and B) into a non-covalent complex that can be further locked into a stable tethered structure by introducing cysteine residues into important sites of both DDD and AD to facilitate disulfide bond formation, as shown in fig. 1. The conventional approach to the "dock-and-lock" approach is as follows. Entity A is constructed by linking the DDD sequence to a precursor of A to give a first component, hereinafter referred to as a. Since the DDD sequence affects the spontaneous formation of dimers, A will be derived from a₂And (4) forming. Entity B is constructed by linking the AD sequence to a precursor of B, resulting in a second component, hereinafter referred to as B. a is₂The dimerization motif of DDD contained in (a) will form a docking site for binding to the AD sequence contained in (b), thereby promoting a₂And b rapidly associate to form a complex comprising a₂b is a binary, trimeric complex. The binding event is made irreversible with a subsequent reaction of covalently fixing the two entities via disulfide bridges, which reaction occurs very efficiently based on an efficient local concentration principle, since the initial binding interaction brings the reactive thiol groups placed on both DDD and AD close (Chimura et al, proc.natl.acad.sci.usa.2001; 98: 8480) for site-specific ligation.

By linking DDD and AD at functional groups remote from the two precursors, it is also expected that these site-specific linkages retain the original activity of the two precursors. This approach is modular in nature and potentially can be used to site-specifically and covalently attach a variety of substances, including peptides, proteins, nucleic acids, and PEG. The DNL process is disclosed in the following U.S. provisional patent applications: 60/728,292 filed on 20/10/2005; 60/751,196 filed on 16/12/2005; and 60/782,332 filed on 3/14/2006; and the following U.S. patent applications: 11/389,358 filed 24/3/2006; 11/391,584 filed on 28/3/2006; 11/478,021 filed on 29.6.2006; 11/633,729 filed on 5.12.2006; and 11/925,408 filed on 26.10.2007.

In a preferred embodiment, as described in the examples below, the effector moiety to be pegylated is a protein or peptide, which can be linked to a DDD or AD unit to form a fusion protein or peptide. Various methods of generating fusion proteins are known, including nucleic acid synthesis, hybridization, and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double-stranded nucleic acids can be inserted into expression vectors for fusion protein production using standard Molecular biology techniques (see, e.g., Sambrook et al, Molecular Cloning, A laboratory Manual, 2 nd edition, 1989). In such preferred embodiments, the AD and/or DDD moieties may be linked to the N-terminus or C-terminus of the effector protein or peptide.

However, the skilled person will recognise that the site of attachment of the AD or DDD site to the effector moiety may vary depending on the chemical nature of the effector moiety and the site of the effector moiety involved in its physiological activity. Site-specific ligation of various effector moieties can be performed using techniques known in the art, such as using divalent crosslinking reagents and/or other chemical conjugation techniques.

Pegylation by DNL

In a preferred method, a target to be pegylated is ligated to a DDD sequence to produce a DDD module. PEG reagents of the desired molecular size are derivatized with the relevant AD sequences, and the resulting PEG-AD module is combined with the DDD module to produce a pegylated conjugate consisting of a single PEG site-specifically linked to two copies of the effector moiety through the disulfide bond formed between DDD and AD. The PEG reagent may have a methoxy capping at one end (m-PEG), may be linear or branched, and may contain one of the following functional groups: propionaldehyde, butyraldehyde, o-pyridyl thioester (OPTE), N-hydroxysuccinimide (NHS), thiazolidine-2-thione, Succinimide Carbonate (SC), maleimide, or o-pyridyl disulfide (OPPS). Effector moieties that may be targets for pegylation include enzymes, cytokines, chemokines, growth factors, peptides, aptamers, hemoglobin, antibodies, and antibody fragments. The method is not limiting and a variety of agents can be pegylated using the disclosed methods and compositions. PEGs of different sizes and derivatized with a variety of reactive moieties are available from commercial sources, as discussed in more detail in the examples below.

Cytokines and other immunomodulators

In certain preferred embodiments, the effector moiety to be pegylated is an immunomodulatory agent. An immunomodulator is an agent that, when present, alters, inhibits or stimulates the immune system of the body. Useful immunomodulators can include cytokines, stem cell growth factors, lymphotoxins, hematopoietic factors, Colony Stimulating Factors (CSFs), Interferons (IFNs), erythropoietins, thrombopoietins, and combinations thereof. Particularly useful are lymphotoxins, such as of Tumor Necrosis Factor (TNF); hematopoietic factors, such as Interleukins (IL); colony stimulating factors, such as granulocyte colony stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF); interferons, such as interferon- α, - β or- γ; and stem cell growth factors, such as the stem cell growth factor designated "S1 factor".

In a more preferred embodiment, the effector moiety to be pegylated is a cytokine, such as lymphokines, monokines, growth factors, and traditional polypeptide hormones. Included among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; a proinsulin; a relaxin hormone; a premenstrual relaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); hepatocyte growth factor; prostaglandins, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-alpha and-beta; mullerian inhibiting substances (mullerian inhibitingsubstances); mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors, such as NGF-beta; platelet growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons, such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage-CSF (M-CSF); interleukins (IL), such as IL-1, IL-1 α, 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-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor (TNF, e.g., TNF- α), and LT. Pegylation of exemplary cytokines is described in examples 2-12.

The amino acid sequences of protein or peptide immunomodulators, such as cytokines, are well known in the art, and any such known sequence may be used in the practice of the present invention. Those skilled in the art know many sources of public information about cytokine sequences. For example, the NCBI database contains protein sequences and encoding nucleic acid sequences for a number of cytokines and immunomodulators, such as erythropoietin (GenBank NM 000799), IL-1 β (GenPept AAH08678), GM-CSF (GenPept AAA52578), TNF- α (GenPept CAA26669), and virtually any of the peptide or protein immunomodulators described above. The identification of the appropriate amino acid and/or nucleic acid sequence for substantially any protein or peptide effector portion of interest is routine for those skilled in the art.

Antibodies and antibody fragments

In other embodiments, the antibody or antigen-binding fragment of the antibody may be pegylated. Antigen-binding antibody fragments are well known in the art, such as F (ab')₂、F(ab)₂Fab', Fab, Fv, scFv and the likeFragments, and any such known fragment may be used. As used herein, an antigen-binding antibody fragment refers to any fragment of an antibody that binds to the same antigen recognized by an intact antibody or a parent antibody. Techniques for preparing AD and/or DDD conjugates of virtually any antibody or fragment of interest are known (e.g., U.S. patent application sequence No. 11/633,729).

Antibodies or fragments thereof that are not conjugated to a therapeutic agent, referred to as "naked" antibodies or fragments thereof, may be used. In alternative embodiments, the antibody or fragment may be conjugated to one or more therapeutic and/or diagnostic agents. A variety of such therapeutic and diagnostic agents are known in the art, as will be discussed in detail below, and any such known therapeutic or diagnostic agent may be used.

Techniques for making monoclonal antibodies against essentially any target antigen are well known in the art. See, e.g., Kohler and Milstein, Nature 256: 495(1975), and Coligan et al (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY (latest protocol IN IMMUNOLOGY), Vol.1, pp.2.5.1-2.6.7 (John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtained by: injecting a composition containing an antigen into a mouse, removing a spleen to obtain a B lymphocyte, fusing the B lymphocyte and a myeloma cell to produce a hybridoma, cloning the hybridoma cell, collecting a positive clone that selects an antibody that produces the antigen, culturing the clone that produces the antibody to the antigen, and isolating the antibody from the hybridoma cell culture.

Monoclonal antibodies can be isolated and purified from hybridoma cultures using a variety of well-established techniques. These separation techniques include affinity chromatography using protein A Sepharose (Sepharose), size exclusion chromatography and ion exchange chromatography. See, e.g., Coligan, pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. See also Baines et al, "Purification of Immunoglobulin G (IgG)", METHODS IN MOLECULARBIOLOGY (molecular biology METHODS), Vol.10, pp.79-104 (Humana Press, Inc.1992).

After initial incubation of the antibodies against the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. The use of antibody components derived from humanized, chimeric or human antibodies avoids potential problems associated with the immunogenicity of murine constant regions.

Chimeric antibodies

Chimeric antibodies are antibodies in which the variable regions of a human antibody have been modified, for example,

a recombinant protein in which a variable region (including a Complementarity Determining Region (CDR)) of a mouse antibody is substituted. When administered to a subject, the chimeric antibody exhibits reduced immunogenicity and increased stability. Conventional techniques for cloning mouse immunoglobulin variable domains are disclosed, for example, in Orlandi et al, proc.nat' l acad.scl USA 86: 3833(1989). Techniques for constructing chimeric antibodies are well known to those skilled in the art. For example, Leung et al, Hybridoma 13: 469(1994), by combining V encoding murine LL2 (an anti-CD 22 monoclonal antibody)_κAnd V_HDNA sequences of the domains and the corresponding human kappa and IgG₁DNA sequence of constant region domain, resulting in LL2 chimera.

Humanized antibodies

Techniques for generating humanized monoclonal antibodies are well known in the art (see, e.g., Jones et al, Nature 321: 522(1986), Riechmann et al, Nature 332: 323(1988), Verhoeyen et al, Science 239: 1534(1988), Carter et al, Proc. Nat' l Acad. Sci. USA 89: 4285(1992), Sandhu, Cr. Rev. Biotech.12: 437 (1992)), and Singer et al, J.Immun.150: 2844 (1993)). Chimeric or murine monoclonal antibodies can be humanized by transferring the mouse CDRs from the heavy and light variable chains of a mouse immunoglobulin to the corresponding human antibody variable domains. The mouse Framework Region (FR) of the chimeric monoclonal antibody was also substituted with the human FR sequence. Since transferring only the mouse CDRs to human FRs often results in a decrease or even loss of antibody affinity, additional modifications may be required to restore the original affinity of the murine antibody. This can be achieved by replacing one or more of certain human residues of the FR regions with their murine counterparts to obtain antibodies with good binding affinity for their epitopes. See, e.g., Tempest et al, Biotechnology 9: 266(1991) and Verhoeyen et al, Science 239: 1534(1988). In general, those human FR amino acid residues other than their murine counterparts and those immediately adjacent to or in contact with one or more CDR amino acid residues will be candidate residues for substitution.

Human antibodies

Methods for producing fully human antibodies using combinatorial approaches or transgenic animals transformed with human immunoglobulin genomes are known in the art (e.g., Mancini et al, 2004, New Microbiol. 27: 315-28; Conrad and Scheller, 2005, comb. chem. high through Screen.8: 117-26; Brekke and Loset, 2003, curr. Opin. Phamacol 3: 544-50). Fully human antibodies can also be constructed by gene or chromosome transfection methods as well as phage display techniques, all of which are known in the art. See, e.g., McCafferty et al, Nature 348: 552-553(1990). Such fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function substantially like endogenous human antibodies in vivo. In certain embodiments, the claimed methods and procedures may use human antibodies produced by these techniques.

In an alternative embodiment, phage display technology can be used for the production of human antibodies (e.g., Dantas-Barbosa et al, 2005, Genet. mol. Res.4: 126-40). Human antibodies may be generated from normal humans or humans exhibiting particular disease states such as cancer (Dantas-barbebosa et al, 2005). An advantage of constructing human antibodies from diseased individuals is that the circulating antibody profile may favor antibodies against disease-associated antigens.

In one non-limiting example of this approach, Dantas-Barbosa et al (2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA is obtained from circulating blood lymphocytes (Id.). Recombinant fabs were cloned from μ, γ and κ chain antibody profiles and inserted into phage display libraries (Id.). RNA was converted to cDNA and Fab cDNA libraries were generated using specific primers for the heavy and light chain immunoglobulin sequences (Marks et al, 1991, J mol. biol. 222: 581-97). Library construction was performed according to Andris-Widhopf et al (2000, in: PhageDisplay Laboratory Manual, Barbas et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pages 9.1 to 9.22). The final Fab fragments were digested with restriction enzymes and inserted into the phage genome to make phage display libraries. Such libraries can be screened by standard phage display methods, as known in the art.

Phage display can be performed in a variety of formats, for a review of them, see, e.g., Johnson and Chiswell, Current Opinion in Structural Biology 3: 5564-571(1993). Human antibodies may also be produced by in vitro activated B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated herein by reference in their entirety. Those skilled in the art will recognize that these techniques are exemplary and that any known method for making and screening human antibodies or antibody fragments may be utilized.

In another alternative embodiment, transgenic animals that have been genetically engineered to produce human antibodies can be used to produce antibodies against virtually any immunological target using standard immunization protocols. Methods for obtaining human antibodies from transgenic mice are disclosed in Green et al, Nature genet.7: 13(1994), Lonberg et al, Nature 5 (55: 856(1994), and Taylor et al, int.Immun.6: 579 (1994). A non-limiting example of such a system is that from Abgenix (Fremont, CA)(e.g., Green et al, 1999, J.Immunol. methods 231: 11-23). In thatAnd similar animals, the mouse antibody genes have been inactivated and replaced with functional human antibody genes, but the remainder of the mouse immune system remains unchanged.

Transformation with germline-configured YACs (Yeast Artificial chromosomes)The YACs contain portions of the human IgH and Ig kappa loci, including most of the variable region sequences, as well as accessory genes and regulatory sequences. The human variable region repertoire can be used to generate antibody-producing B-cells that can be processed into hybridomas by known techniques. Immunising with target antigensHuman antibodies will be produced by normal immune reactions and can be harvested and/or produced by standard techniques as discussed above.A plurality of strains are available, wherein each strain is capable of producing a different class of antibodies. Human antibodies produced transgenically have been shown to have therapeutic potential while retaining the pharmacokinetic properties of normal human antibodies (Green et al, 1999). Those skilled in the art will recognize that the claimed compositions and methods are not limited to useInstead, any transgenic animal that has been genetically engineered to produce human antibodies can be used.

Antibody fragments

Antibody fragments that recognize a particular epitope can be generated by known techniques. Antibody fragments are the antigen-binding portions of antibodies, e.g., F (ab')₂、Fab′、F(ab)₂Fab, Fv, sFv and the like. F (ab')₂The fragments may be produced by pepsin digestion of the antibody molecule, while the Fab' fragments may be produced by reductionF(ab′)₂Disulfide bridge formation of fragments. Alternatively, Fab' expression libraries can be constructed (Huse et al, 1989, Science, 246: 1274-. F (ab)₂Fragments may be generated by papain digestion of antibodies, and Fab fragments may be obtained by disulfide bond reduction.

Single chain Fv molecules (scFv) comprise a VL domain and a VH domain. The VL and VH domains associate to form a target binding site. The two domains are further covalently linked by a peptide linker (L). Methods for making scFv molecules and designing suitable peptide linkers are disclosed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, r.rag and m.whitlow, "singlecain Fvs" (single chain Fv) FASEB roll 9: 73-80(1995) and R.E.bird and B.W.Walker, "Single Chain Antibody Variable Regions" TIBTECH, Vol.9: 132-137(1991).

Antibody fragments can be prepared by proteolysis of full-length antibodies or by expression of DNA encoding the fragment in e. Antibody fragments can be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. Such methods are described, for example, in golden berg, U.S. Pat. nos. 4,036,945 and 4,331,647 and references contained therein. See also Nisonoff et al, Arch biochem. biophysis.89: 230 (1960); porter, biochem.j.73: 119(1959), Edelman et al, IN METHODS IN Enzymatic, Vol.1, p 422 (Academic Press1967), and Coligan, pp.8.1-2.8.10 and 2.10-2.10.4.

Known antibodies

The antibodies used can be obtained commercially from a variety of known sources. For example, a variety of antibody-secreting hybridoma lines are available from the american type culture collection (ATCC, manassas, va). Many antibodies directed against different disease targets, including but not limited to tumor associated antigens, have been deposited with the ATCC and/or have been published with variable region sequences, and can be used in the claimed methods and compositions. See, for example, U.S. patent nos. 7,312,318; 7,282,567, respectively; 7,151,164, respectively; 7,074,403, respectively; 7,060,802, respectively; 7,056,509, respectively; 7,049,060, respectively; 7,045,132, respectively; 7,041,803, respectively; 7,041,802, respectively; 7,041,293, respectively; 7,038,018, respectively; 7,037,498, respectively; 7,012,133, respectively; 7,001,598, respectively; 6,998,468, respectively; 6,994,976, respectively; 6,994,852, respectively; 6,989,241, respectively; 6,974,863, respectively; 6,965,018, respectively; 6,964,854, respectively; 6,962,981, respectively; 6,962,813, respectively; 6,956,107, respectively; 6,951,924, respectively; 6,949,244, respectively; 6,946,129, respectively; 6,943,020, respectively; 6,939,547, respectively; 6,921,645, respectively; 6,921,645, respectively; 6,921,533, respectively; 6,919,433, respectively; 6,919,078, respectively; 6,916,475, respectively; 6,905,681, respectively; 6,899,879, respectively; 6,893,625, respectively; 6,887,468, respectively; 6,887,466, respectively; 6,884,594, respectively; 6,881,405, respectively; 6,878,812, respectively; 6,875,580, respectively; 6,872,568, respectively; 6,867,006, respectively; 6,864,062, respectively; 6,861,511, respectively; 6,861,227, respectively; 6,861,226, respectively; 6,838,282, respectively; 6,835,549, respectively; 6,835,370, respectively; 6,824,780, respectively; 6,824,778, respectively; 6,812,206, respectively; 6,793,924, respectively; 6,783,758, respectively; 6,770,450, respectively; 6,767,711, respectively; 6,764,688, respectively; 6,764,681, respectively; 6,764,679, respectively; 6,743,898, respectively; 6,733,981, respectively; 6,730,307, respectively; 6,720, 15; 6,716,966, respectively; 6,709,653, respectively; 6,693,176, respectively; 6,692,908, respectively; 6,689,607, respectively; 6,689,362, respectively; 6,689,355, respectively; 6,682,737, respectively; 6,682,736; 6,682,734, respectively; 6,673,344, respectively; 6,653,104, respectively; 6,652,852, respectively; 6,635,482, respectively; 6,630,144, respectively; 6,610,833, respectively; 6,610,294, respectively; 6,605,441, respectively; 6,605,279, respectively; 6,596,852, respectively; 6,592,868, respectively; 6,576,745, respectively; 6,572, respectively; 856; 6,566,076, respectively; 6,562,618, respectively; 6,545,130, respectively; 6,544,749, respectively; 6,534,058, respectively; 6,528,625, respectively; 6,528,269, respectively; 6,521,227, respectively; 6,518,404, respectively; 6,511,665, respectively; 6,491,915, respectively; 6,483,930, respectively; 6,482,598, respectively; 6,482,408, respectively; 6,479,247, respectively; 6,468,531, respectively; 6,468,529, respectively; 6,465,173, respectively; 6,461,823, respectively; 6,458,356, respectively; 6,455,044, respectively; 6,455,040, 6,451,310, 6,444,206' 6,441,143; 6,432,404, respectively; 6,432,402, respectively; 6,419,928, respectively; 6,413,726, respectively; 6,406,694, respectively; 6,403,770, respectively; 6,403,091, respectively; 6,395,276, respectively; 6,395,274, respectively; 6,387,350, respectively; 6,383,759, respectively; 6,383,484, respectively; 6,376,654, respectively; 6,372,215, respectively; 6,359,126, respectively; 6,355,481, respectively; 6,355,444, respectively; 6,355,245, respectively; 6,355,244, respectively; 6,346,246, respectively; 6,344,198, respectively; 6,340,571, respectively; 6,340,459, respectively; 6,331,175, respectively; 6,306,393, respectively; 6,254,868, respectively; 6,187,287; 6,183,744, respectively; 6,129,914, respectively; 6,120,767, respectively; 6,096,289, respectively; 6,077,499; 5,922,302, respectively; 5,874,540; 5,814,440, 5,798,229, 5,789, 554; 5,776,456; 5,736,119, respectively; 5,716,595, respectively; 5,677,136, respectively; 5,587,459, respectively; 5,443,953, respectively; 5,525,338, respectively. These are merely exemplary and a variety of other antibodies and hybridomas thereof are known in the art. One skilled in the art will recognize that antibody sequences or antibody secreting hybridomas against virtually any disease-associated antigen can be identified by simply searching ATCC, NCBI, and/or the U.S. patent and trademark office databases for antibodies against a selected disease-associated target of interest. The antigen binding domain of the cloned antibody may be amplified, excised, ligated to an expression vector, transfected into a modified host cell and used for protein production using standard techniques known in the art.

Therapeutic agents

In alternative embodiments, therapeutic agents such as cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, or other agents may be pegylated as described herein. Pegylated forms of therapeutic agents have been disclosed, for example, SN38(ZHao et al, bioconjugate Chem 2008, 10: 849-59), uricase (Biggers and Scheinfeld, Curr Opin Investig Drugs 2008, 9: 422-29), docetaxel (Liu et al, 2008, J Pharm Sci 97: 3274-90), and camptothecin (Haverstick et al, 2007, bioconjugate Chem 18: 2115-21). The drug used may have a drug property selected from the group consisting of antimitotic, antikinase, alkylating, antimetabolite, antibiotic, alkaloid, antiangiogenic, pro-apoptotic agent, and combinations thereof.

Exemplary drugs for use may include 5-fluorouracil, aplidine, azalipine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamicin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, Cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecin (camptothecas), cyclophosphamide, cytarabine, dacarbazine, docetaxel, actinomycin D, daunorubicin, doxorubicin, 2-pyrrolinyl doxorubicin (2P-DOX), cyanomorpholinium doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epiphyllotoxin Epsiloxotin, estrogen receptor binding agents, etoposide (16) VP, Etoposide glucuronide, etoposide phosphate, fluorouracil deoxynucleoside (FUdR), 3 ', 5' -O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide (lenalidomide), folinic acid, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, neomycin, nitrourea, plicamycin (plicomycin), procarbazine, taxol, pentostatin, PSI-341, raloxifene, semustine, streptozotocin, tamoxifen, taxol, temozolomide (aqueous form of DTIC), platinum, thalidomide, thioprine, thiothiuracil, thiothidine, etc, Thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.

Toxins used may include ricin, abrin, alpha toxin, saponin, ribonuclease (rnase), e.g., ranpirnase (onconase), dnase I, staphylococcus (staphylococcus) enterotoxin a, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas (Pseudomonas) exotoxin and Pseudomonas endotoxin.

Chemokines used may include RANTES, MCAF, MIP1- α β, MIP1- β and IP-I0. In certain embodiments, anti-angiogenic agents, such as angiostatin, baculostatin, angiostatin, mastinostatin, anti-VEGF antibodies, anti-PIGF peptides and antibodies, anti-angiogenic factor antibodies, anti-Flk-1 antibodies and peptides, anti-Kras antibodies, anti-cMet antibodies, anti-MIF (macrophage migration inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-beta, thrombospondin, 2-methoxyestradiol, proliferator-related proteins, carboxyamidotriazole, CM101, marimastat, pentosan polysulfate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, linoxamine, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-470, platelet factor 4, or minocycline.

Other useful therapeutic agents may include oligonucleotides, particularly antisense oligonucleotides that are preferably directed against oncogenes and oncogene products such as bcl-2 or p 53.

Conjugation

A variety of techniques and compositions for conjugating small molecules to proteins or peptides, such as AD or DDD peptides, are known and may be used. The therapeutic agent may be attached, for example, to a reduced sulfhydryl group and/or a sugar side chain. The therapeutic agent may be linked to the reduced protein or peptide containing the cysteine residue by disulfide bond formation. Alternatively, such agents can be linked using heterobifunctional crosslinkers such as N-3- (2-pyridyldithio) propionic acid Succinate (SPDP). Yu et al, int.j.cancer 56: 244(1994). The usual techniques for such conjugation are well known in the art. See, e.g., Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING (protein conjugation and cross-LINKING chemistry) (CRCPress 1991); updalacis et al, "Modification of Antibodies by chemical methods" (Antibodies modified by chemical methods), in MONOCLONAL Antibodies: PRINCIPLES AND application (monoclonal antibodies principle and application), Birch et al, (eds.), page 187-230 (Wiley-Liss, Inc. 1995); price, "Production and characterization of Synthetic Peptide-Derived Antibodies" (Production and characterization of Synthetic Peptide-Derived Antibodies) in MONOCLONAL Antibodies: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION (monoclonal antibody PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONs), Ritter et al, (eds.), pp.60-84 (Cambridge University Press 1995).

Therapeutic applications

The compositions described herein are particularly useful in the treatment of various disease states. In a preferred embodiment, the disease may be an autoimmune disease or a cancer, such as a hematopoietic cancer or a solid tumor. Exemplary, non-limiting conditions that can be treated using the disclosed compositions and methods include indolent forms of B-cell lymphoma, aggressive forms of B-cell lymphoma, non-hodgkin's lymphoma, multiple myeloma, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hodgkin's lymphoma, fahrenheit macroglobulinemia, as well as GVHD, cryoglobulinemia, hemolytic anemia, allosensitization, and organ transplant rejection. Also included are autoimmune diseases of class III, such as immune-mediated thrombocytopenia, e.g., acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sjogren's syndrome, multiple sclerosis, sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, rheumatoid arthritis, glandular syndrome, bullous pemphigoid, diabetes, Henoch-Sch nlein purpura, streptococcal post-infection nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Gopasadodture syndrome, thromboangiitis obliterans, primary biliary cirrhosis, Hashimoto thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, Polychondritis, pemphigus vulgaris, Wegener's granuloma, membranous nephropathy, amyotrophic lateral sclerosis, tuberculosis of the spinal cord, giant cell arteritis/myalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.

Solid tumors that may be treated include neuroblastoma, malignant melanoma, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, prostate cancer, lung cancer, renal cancer, colorectal cancer, gastric cancer, bladder cancer, glioma, sarcoma, brain cancer, esophageal cancer, epithelial cancer, osteosarcoma, testicular cancer, liver cancer, and pancreatic cancer.

Reagent kit

Various embodiments may be directed to kits containing components suitable for treating or diagnosing diseased tissue in a patient. An exemplary kit can contain at least a pegylated therapeutic agent as described herein. If the composition-containing components for administration are not formulated for delivery through the digestive tract, such as oral delivery, a device capable of delivering the kit components by some other route may be included. One type of device for applications such as parenteral delivery is a syringe used to inject the composition into the body of a subject. Inhalation devices may also be used. In certain embodiments, the pegylated therapeutic agent can be provided in the form of a prefilled syringe or an autoinjector pen containing a sterile, liquid formulation or a lyophilized product.

The kit components may be packaged together or divided into two or more containers. In some embodiments, the container may be a vial containing a sterile, lyophilized formulation of a composition suitable for reconstitution. The kit may also comprise one or more buffers suitable for reconstituting and/or diluting other reagents. Other containers that may be used include, but are not limited to: a bag, tray, box, tube or similar container. The kit components may be packaged and maintained aseptically in containers. Another component which may be included is instructions to the person using the kit regarding its use.

Examples

The following examples are provided to illustrate but not to limit the claimed invention.

Example 1 Generation of PEG-AD2 Module

Synthesis of IMP350

CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂(SEQ ID NO：1)MH⁺2354

IMP350 including AD2 sequence was prepared using Fmoc method on a Protein Technologies PS3 peptide synthesizer using Sieber Amide resin at a level of 0.1 mmol. Starting from the C-terminus, the protected amino acids used are Fmoc-Cys (t-Buthio) -OH, Fmoc-Gly-OH, Fmoc-Ala-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. Peptides were cleaved from the resin and purified by Reverse Phase (RP) HPLC.

PEG₂₀Synthesis of IMP350

IMP350(0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20kDa, Nektar Therapeutics) in 7 ml of 1M Tris buffer (pH 7.81). Then 1ml of acetonitrile was added to dissolve some of the suspended material. The reaction was stirred at room temperature for 3 hours, then 0.0527 grams of TCEP and 0.0549 grams of cysteine were added. The reaction mixture was stirred for 1.5 hours and then purified on a PD-10 desalting column equilibrated with 20% methanol in water. The sample was eluted, frozen and lyophilized to give 0.0924 g of crude PEG₂₀-IMP350(MH⁺23508，MALDI)。

Synthesis of IMP360

CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS(SEQ ID NO：1)MH⁺2660

IMP360 including the AD2 sequence was prepared using the Fmoc method at a Protein Technologies PS3 polypeptide synthesizer using Fmoc-Gly-EDANS resin at a level of 0.1 mmol. Fmoc-Gly-OH was added to the resin manually using 0.23g of Fmoc-Gly-OH, 0.29g of HATU, 26. mu.L of DIEA, 7.5mL of DMF and 0.57g of EDANS resin (Nova Biochem). These reagents are mixed and added to the resin. The reaction was mixed at room temperature for 2.5 hours and the resin was washed with DMF and IPA to remove excess reagents. Starting from the C-terminus, the protected amino acids used are Fmoc-Cys (t-Buthio) -OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln (Tn) -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 polypeptide was cleaved from the resin and purified by RP-HPLC.

IMP362(PEG₂₀-IMP 360) synthesis

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

IMP 413(PEG₃₀-MP 360) synthesis

The cartoon of IPM413 is provided in FIG. 3. For the synthesis of IMP413, IMP360(0.0103 g) was mixed with 0.1601 g mPEG-OPTE (30kDa, Nektar Therapeutics) in 7 ml 1M Tris buffer (pH 7.81). Acetonitrile (1 ml) was then added to dissolve some of the suspended material. The reaction was stirred at room temperature for 4.5 hours, then 0.0423 g of TCEP and 0.0473 g of cysteine were added. The reaction mixture was stirred for 2 hours and then dialyzed for 2 days. The dialyzed material was frozen and lyophilized to give 0.1552 g of crude IMP413 (MH)⁺34499)。

Synthesis of IMP421

IMP 421 Ac-C-PEG₃-C(S-tBu)GQIEYLAKQIVDNAIQQAGC(S-tBu)G-NH₂(SEQ IDNO：9)

By adding the following amino acids to the resin in the order shownPreparation of the peptide IMP421, MH on TGR resin (487.6 mg, 0.112 mmol)⁺2891：Fmoc-Gly-OH、Fmoc-Cys(t-Buthio)-OH、Fmoc-Gly-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gln(TrQ-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、Fmoc-Cys(t-Buthio)-OH、Fmoc-NH-PEG₃-COOH, Fmoc-Cys (Trt) -OH. The N-terminal amino acid is protected as an acetyl derivative. The peptide was then cleaved from the resin and purified by RP-HPLC to yield 32.7 mg of a white solid.

Synthesis of IMP457

IMP421 (SEQ ID NO: 9, FIG. 15) comprising the AD2 sequence was synthesized by standard chemical methods. To a solution of 15.2 mg (5.26. mu. mol) IMP421(F.W.2890.50) and 274.5 mg (6.86. mu. mol) of mPEG2-MAL-40K in 1ml acetonitrile was added 7 ml 1M Tris (pH 7.8) and allowed to react at room temperature for 3 hours. Excess mPEG2-MAL-40K was quenched with 49.4 mg L-cysteine (FIG. 16), followed by deprotection of S-S-tBu for 1 hour with 59.1 mg TCEP. The reaction mixture was dialyzed overnight at 2-8 ℃ to 5L of 5mM ammonium acetate (pH 5.0) using two 3-12 ml volumes of 10K Slide-A-Lyzer dialysis cartridges (4 ml per cartridge). The next day, three additional 5L buffer changes of 5mM ammonium acetate (pH 5.0) were made, each dialysis lasting at least 2¹/₂h. The purified product (19.4ml) was transferred to 2 20ml scintillation vials, frozen and dried at low pressure to yield 246.7mg of a white solid. MALDI-TOF gave the results for mPEG2-MAL-40K 42,982 and IMP-45745,500.

Example 2 Generation of Interferon (IFN) - α 2 b-based DDD modules

Construction of IFN-. alpha.2b-DDD 2-pdHL2 for expression in mammalian cells

The cDNA sequence of IFN-. alpha.2b was amplified by PCR to give a sequence including the following features, XbaI and BamHI as restriction sites, the signal peptide native to IFN-. alpha.2b, and 6His as 6 histidine tag: XbaI- - -signal peptide- - -IFN alpha 2b- - -6His- - -BamHI. The resulting secreted protein consists of IFN- α 2b linked at its C-terminus to a polypeptide consisting of SEQ ID NO: 2.

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：2)

PCR amplification was performed using the full-length human IFN α 2b cDNA Clone (Invitrogen Ultimate ORF human Clone cat # HORFO1 Clone ID IOH35221) as template and the following oligonucleotides as primers:

IFNA2 Xba I left side

5’-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTGG-3’(SEQ IDNO：3)

IFNA2 BamHI right hand

5’-GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3’(SEQ ID NO：4)

The PCR amplificates were cloned into pGemT vector (Promega). DDD2-pdHL2 mammalian expression vector was prepared by digestion with XbaI and BamHI restriction enzymes for ligation with IFN-. alpha.2b. The IFN-. alpha.2b amplimer was excised from pGemT with XbaI and Bam HI and ligated into DDD2-pdHL2 vector to generate IFN-. alpha.2b-DDD 2-pdHL2 expression vector.

Mammalian expression of IFN-alpha 2b-DDD2

IFN-. alpha.2b-DDD 2-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 2006, 7, 14, which is incorporated herein by reference). 2 clones were found to have detectable levels of IFN-. alpha.2b by ELISA. One of the two clones (designated 95) was adapted to growth in serum-free medium without a significant decrease in productivity. Clones were subsequently amplified over 5 weeks with increasing Methotrexate (MTX) concentrations of 0.1 to 0.8. mu.M. At this stage, it was subcloned by limiting dilution and the highest yielding subclone (95-5) was expanded. The productivity of 95-5 grown in shake flasks was estimated to be 2.5 mg/l using commercially available rIFN-. alpha.2b (Chemicon IF007, Lot 06008039084) as standard.

Purification of IFN-. alpha.2b-DDD 2 in batch cultures grown in spinner flasks

Clone 95-5 was expanded to 34 spinner flasks containing a total of 20L of serum-free hybridoma SFM using 0.8. mu.M MTX and allowed to reach terminal culture. The supernatant was clarified by centrifugation and filtered (0.2. mu.M). The filtrate was washed with 1 Xbinding buffer (10mM imidazole, 0.5M NaCl, 50mM NaH)₂PO₄(pH 7.5)) was diafiltered and concentrated to 310mL of preparation for purification by Immobilized Metal Affinity Chromatography (IMAC). The concentrate was loaded on a 30-ml Ni-NTA column, which was washed with 500 ml of 0.02% Tween 20 in 1 Xbinding buffer, followed by 290 ml of 30mM imidazole, 0.02% Tween 20, 0.5M sodium chloride, 50mM sodium dihydrogen phosphate (pH 7.5). The product was eluted with 110 ml of 250mM imidazole, 0.02% Tween 20, 150mM sodium chloride, 50mM sodium dihydrogen phosphate (pH 7.5). Approximately 6 mg of IFN alpha 2b-DDD2 was purified. Figure 4 shows for quantification of IFN alpha 2b-DDD2 anti IFN alpha immunoblot and ELISA results.

Characterization of IFN-. alpha.2b-DDD 2

The purity of IFN-. alpha.2b-DDD 2 was assessed by SDS-PAGE under reducing conditions (not shown). Coomassie blue stained gels showed that the roller bottle produced a purer batch than the previous batch (not shown). IFN-. alpha.2b-DDD 2 is the heaviest stained band, accounting for approximately 50% of total protein (not shown). The product is isolated as having M_rA26 kDa doublet (doublet), which corresponds to the calculated MW of IFN-. alpha.22 b-DDD2-SP (26 kDa). Here, M is present with a molecular weight of 34kDa_rA major contaminant and many faint bands of contamination (not shown).

Example 3 Pegylation of IFN-. alpha.2b by DNL

Preparation and purification of alpha 2b-362(IFN-a2b-DDD2-IMP362)

Figure 5 is cartoon representation, which shows the alpha 2b-362 structure, alpha 2b-362 with 20kDa polyethylene glycol coupled with two copies of IFN alpha 2 b. The DNL reaction was performed by adding a 10-fold molar excess of 11mg of reduced and lyophilized IMP362 to 2.25 mg (3.5 ml) of IFN-. alpha.2b-DDD 2 dissolved in 250mM imidazole, 0.02% Tween 20, 150mM sodium chloride, 1mM EDTA, 50mM sodium dihydrogen phosphate (pH 7.5). After 6h at room temperature in the dark, the reaction mixture was dialyzed against CM loading buffer (150mM NaCl, 20mM NaAc (pH 4.5)) at 4 ℃ in the dark. The solution was loaded onto a 1ml Hi-Trap CM-FF column (Amersham) pre-equilibrated with CM loading buffer. After sample loading, the column was washed to baseline with CM loading buffer, followed by 15 ml of 0.25M sodium chloride, 20mM sodium acetate (pH 4.5). The pegylated product was eluted with 12.5 ml of 0.5M sodium chloride, 20mM sodium acetate (pH 4.5).

The conjugation process was analyzed by SDS-PAGE stained with Coomassie Brilliant blue, fluorescence imaging and anti-IFN α immunoblotting (not shown). To normalize the samples for direct comparison of protein mass, 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 brilliant blue stained gel showed a 110kDa M in the reaction mixture_rThe main band of (a), which is not present in the unbound or 0.25M sodium chloride-washed fraction, but is evident in the 0.5M sodium chloride fraction (not shown). Fluorescence imaging of the EDANS tag used to detect IMP362 indicated that the 110 kDa-containing band contained IMP362 and the presence of excess IMP362 in the reaction mixture and unbound fraction, which was not stained with coomassie brilliant blue (not shown). anti-IFN alpha immunoblots confirmed the association of IFN-alpha 2b with the 110kDa band (not shown). Together, these data indicate that the DNL reaction results in site-specific and covalent conjugation of IMP362 with the IFN- α 2b dimer. Under reducing conditions that break the disulfide bonds, the components of the DNL structure are separated (not shown). The calculated MW of α 2b-362 is 75kDa, which matches well with 76,728Da as determined by MALDI TOF. The observed deviation between the calculated mass and the Mr assessed by SDS-PAGE is due to PEG, which is known to exaggerate the size of the macromolecule when the pegylated product is analyzed by SDS-PAGE or SE-HPLC. Overall, the DNL reaction resulted in a nearly quantitative yield of homogeneous product that was > 90% pure after purification by cation exchange chromatography (not shown).

Preparation and purification of alpha 2b-457 (IFN-alpha 2b-DDD2-IMP457)

The DNL reaction was performed by adding a 10-fold molar excess of 2.5mg of reduced and lyophilized IMP457 to 1mg (1.7 mL) of IFN-. alpha.2b-DDD 2 dissolved in 250mM imidazole, 0.02% Tween 20, 150mM sodium chloride, 1mM EDTA, 50mM sodium dihydrogen phosphate (pH 7.5). After 60h at room temperature, 1mM oxidized glutathione was added to the reaction mixture, which was left for an additional 2 hours. The mixture was diluted 1: 20 with CM loading buffer (150mM NaCl, 20mM NaAc (pH 4.5)) and titrated to pH4.5 with acetic acid. The solution was loaded onto a 1-mL Hi-Trap CM-FF column (Amersham) pre-equilibrated with CM loading buffer. After sample loading, the column was washed to baseline with CM loading buffer, followed by 15 ml of 0.25M sodium chloride, 20mM sodium acetate (pH 4.5). The pegylated product was eluted with 20ml of 0.5M sodium chloride, 20mM sodium acetate (pH 4.5). A2b-457 is concentrated to 2ml and diafiltered against 0.4M PBS (pH 7.4). The final yield was approximately 1mg of a2b-457 with greater than 90% purity as determined by SDS-PAGE and IFN α ELISA.

Preparation and purification of alpha 2b-413 (IFN-alpha 2b-DDD2-IMP413)

FIG. 6 is a cartoon representation showing the structure of α 2b-413, α 2b-413 possessing two copies of IFN α 2b coupled to 30kDa polyethylene glycol. α 2b-413 was prepared as described immediately above, except that IMP413 was substituted for IMP 362.

Example 4 in vitro potency assessment of IFN-. alpha.2b-DDD 2, alpha.2b-362, and alpha.2b-413

In vitro antiproliferative assay

The growth inhibition of Burkitt lymphoma (Daudi) cells by IFN-. alpha.2b-DDD 2 and. alpha.2b-362 was analyzed. Briefly, IFN-. alpha.2b standards (Chemicon IF007, Lot 06008039084), IFN-. alpha.2b-DDD 2 (batch 010207), and. alpha.2b-362 (batch 010807) were each diluted to 500pM with RPMI1640 medium supplemented with 10% FBS, from which three triplicate serial dilutions (50. mu.L samples/well) were prepared in 96-well tissue culture plates. Daudi cells were diluted to 4X 10⁵Each cell/mL, and eachAdd 50. mu.L (20K/well) to the wells. The concentration of each test agent ranged from 500pM to 0.008 pM. After 4 days at 37 ℃ the MTS dye was added to the plate (20. mu.L/well) and after 3 hours the plate was read with an Envision plate reader (Perkin Elmer, Boston MA) at 490 nm. Dose-response curves were generated (FIG. 7) and 50% Effective Concentrations (EC) were obtained by sigmoidal fitted non-linear regression using Graph Pad Prism Software (advanced graphics Software, Encinitas, Calif.)₅₀). Calculation of IFN alpha 2b-DDD2 and cc2b-362 EC₅₀Similar (. about.16 pM), and their potency is greater than IFN-. alpha.2b standard (EC)₅₀4pM) was about 5 times lower. In a similar experiment, α 2b-413 was similarly effective as α 2 b-362.

Antiviral assays

Duplicate samples were analyzed in a virus challenge assay on a549 cells using Encephalomyocarditis (EMC) virus by a separate analytical laboratory (PBL Interferon Source, Piscataway, NJ). The plates were stained with crystal violet and the OD values were measured spectrophotometrically on a 96-well plate reader after the dye had dissolved. Data were analyzed using Graph Pad Prism software using sigmoidal fit (variable slope) nonlinear regression. By mixing with IFN alpha standard EC₅₀Comparison of values antiviral titers were determined. The specific antiviral activity of α 2b-362 and α 2b-413, respectively, was calculated to be 1.2X 10⁸Unit/mg and 8.8X 10⁶Units per milligram.

Example 5 in vivo evaluation of α 2b-413 and α 2b-362

Pharmacokinetics

The study was performed in adult female Swiss-Webster mice (. about.35 g). There were 4 different treatment groups of 2 mice each. Equimolar protein doses (3. mu.g rhuIFN-. alpha.2a, 5. mu.g PEGINTRON) per reagent (test and control)^TM11 μ g α 2b-362 and 13 μ g α 2b-413) were administered as a single bolus i.v. injection. Mice were bled via the retro-orbital method at various time points (pre-dose, 5 min post-injection, 2-, 8-, 24-, 48-, 72-, 96-, and 168 h). Allowing the blood to clot, centrifuging, separating the serum, and storing at-70 ℃ until IFN- α is assayedConcentration and subsequent PK analysis.

IFN- α concentrations in serum samples were determined using a human interferon- α ELISA kit according to the manufacturer's instructions (PBL Interferon Source). Briefly, serum samples were diluted appropriately according to the human IFN- α standard provided in the kit. The antibody coupled to the wells of the microtiter plate captures interferon. Bound interferon was then visualized with a secondary antibody that was quantitated by an anti-secondary antibody conjugated to horseradish peroxidase (HRP) after addition of Tetramethylbenzidine (TMB) substrate. The plate was read at 450nm and the results are shown in FIG. 8.

The PK profile for each reagent is listed in table 1. As expected, rhIFN- α 2a had the fastest clearance rate from the blood of the injected mice. The clearance rate ratio of PEGINTRON^TMApproximately 3 times faster than the DNL-IFN reagent, more than 13 times faster. PEGINTRON^TMClearance is again more than 4 times faster than α 2b-362 or α 2 b-413. There is only a small difference between the clearance rates of α 2b-362 and α 2 b-413.

With respect to the Mean Residence Time (MRT), there is a clear correlation with the size of the various agents. MRT ratio of 19-kDa rhIFN-. alpha.2a to 31kDa PEGINTRON^TM7-fold less (0.7 h vs. 5.1h, respectively) 31kDa PEGINTRON^TMHas a 2-fold lower MRT when compared to 70kDa alpha 2b-362(10.3 h). The MRT of 80kDa α 2b-413(21.7h) is 2 times longer than α 2 b-362. Finally, bioequivalence testing indicated that none of the test agents were identical in PK, showing a true difference (i.e., circulating half-life of α 2b-413 > α 2b-362 > peginntron^TM＞rhIFN-α2a)。

Antitumor therapeutic efficacy

Preliminary in vivo tumor therapy studies showed that DNL-pegylated interferon is compared to PEGINTRON^TMMore effective and durable. Eight-week old female C.B. -17SCID mice to1.5×10⁷Individual cells/animal human Burkitt lymphoma cell line (Daudi) was i.v. injected. There were 10 different treatment groups of 5 mice each. PEGINTRON was injected every 7 days at 3 different doses (3500, 7000 and 14000 units) on the left or right side s.c^TMα 2b-362 and α 2 b-413. Treatment was started 1 day after Daudi cell transplantation.

Mice were observed daily for signs of distress and paralysis. Their body weights were measured weekly. When a mouse or mice lost more than 15% of body weight (but less than 20%), they were weighed every 2 days until their body weight was either restored to less than 15% loss or sacrificed due to the loss of more than 20% of body weight. Mice were also sacrificed when developing paraplegia or if they became moribund.

The survival curves generated by this study are shown in figure 9. PEGINTRON compared to saline control mice (P < 0.0016)^TMα 2b-362 and α 2b-413 all showed significant improvement in survival. Except for the 3,500IU dose of alpha 2b-362, both alpha 2b-413 and alpha 2b-362 are superior to PEGINTRON when injecting equal active doses^TM(P < 0.0027). Alpha 2b-362 shows an excess over PEGINTRON^TMTwice the effectiveness. The dosages of 7000IU and 3500IU of alpha 2b-362 are respectively superior to PEGINTRON^TMThe 14,000IU (P ═ 0.0016) and 7000IU (P ═ 0.0027) doses. Because the efficacy of alpha 2b-413 at 3500IU dose is better than that of PEGINTRON at 14000IU^TMThe potency of (P ═ 0.0027), so that α 2b-413 is PEGINTRON^TMMore than four times the efficacy of (c). Alpha 2b-413 is obviously better than alpha 2b-362(P < 0.0025) when the same dose is injected. However, there were no statistically significant differences between the three doses of α 2b-413, even though the 14000IU dose resulted in a median survival of 60 days, compared to a median survival of 3500IU dose of 46 days (P ═ 0.1255). Thus, alpha 2b-362, alpha 2b-413 and PEGINTRON were observed^TMEfficacy in vivo correlates well with PK data.

The increased bioavailability of α 2b-362 and α 2b-413 demonstrated by PK analysis promotes an increase in the antitumor efficacy of DNL-pegylated IFN α in vivo. These two, in turnThe factors allow for less frequent dosing regimens in tumor therapy. This was confirmed in a study similar to the in vivo tumor treatment described above, in which PEGINTRON^TMOr α 2b-413 according to different dosing schedules. This study was conducted at 1.5X10⁷Daudi cells were performed in 8-week old female SCID mice injected i.v. There were 7 different treatment groups, 6-7 mice per group. Each reagent (test and control) was administered by s.c. injection of 14,000IU on the left or right side. Treatment began 1 day after Daudi cells were administered to mice. One group of mice was dosed once a week for 4 weeks (q7dx4), another group was dosed on a biweekly schedule for 8 weeks (q2wkx4), and a third group of mice was dosed once every three weeks for 12 weeks (q3wkx 4). All mice received a total of 4 injections.

Fig. 10 shows the survival curves generated by this study. All animals receiving either form of interferon at any of the different regimens had a significant improvement in survival (P < 0.0009) compared to saline control mice. Importantly, and with PEGINTRON^TMSurvival of mice treated with all IFN-IMP413 was significantly improved (P < 0.0097) compared to animals treated with the same regimen. Notably, PEGINTRON was accepted in the same protocol^TMMice treated with IFN-IMP413(q2wkx4) every other week not only significantly improved survival compared to mice treated with IFN-IMP413(q2wkx4) (MST > 54 days and 28 days; P0.0002, respectively), but also significantly higher than mice treated with PEGINTRON weekly (q7dx4)^TMThose treated (MST: 36.5 days; P: 0.0049). Furthermore, the survival of mice treated with IFN-IMP413(q3wkx4) every 3 weeks was significantly better than that of mice treated with PEGINTRON every two weeks^TMTreated mice (MST 54 and 28 days; P0.002) and weekly peginntron^TMThose treated were nearly significant (P-0.0598).

These studies indicate that even though pegylation of the DNL of IFN α 2b results in enhanced and long-term efficacy compared to other pegylated forms of IFN α 2b, which allows for a lower dosing frequency. Similar improvements are achieved when this technology is applied to other cytokines (such as G-CSF and EPO), growth factors, enzymes, antibodies, immunomodulators, hormones, peptides, drugs, interfering RNA, oligonucleotides, vaccines and other bioactive agents.

Example 6 Generation of a granulocyte colony stimulating factor (G-CSF) -based DDD Module

Construction of G-CSF-DDD2-pdHL2 for expression in mammalian cells

The cDNA sequence of G-CSF was amplified by PCR to give a sequence comprising the following features, XbaI and BamHI as restriction sites, the signal peptide native to human G-CSF, and 6His as 6 histidine tag: XbaI- -signal peptide- -G-CSF- -6His- -BamHI. The secreted protein thus obtained consists of G-CSF linked at its C-terminus to a protein consisting of SEQ ID NO: 5.

KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：2)

PCR amplification was performed using a cDNA Clone of full-length human G-CSF (Invitrogen IMAGE humancat #97002 RGclone ID 5759022) as template and the following oligonucleotides as primers:

G-CSF XbaI left

5'-TCTAGACACAGGACCTCATCATGGCTGGACCTGCCACCCAG-3' (SEQ ID NO: 5) G-CSF BamHI-Right

5’-GGATCCATGATGGTGATGATGGTGTGACTTGGGCTGGGCAAGGTGGCGTAG-3’(SEQ ID NO：6)

The PCR amplificates were cloned into pGemT vectors. The DDD2-pdHL2 mammalian expression vector was prepared by restriction enzyme digestion with XbaI and Bam HI for ligation with G-CSF. The G-CSF amplificate was excised from pGemT using XbaI and Bam HI, and ligated into DDD2-pdHL2 vector to generate G-CSF-DDD2-pdHL2 expression vector.

Mammalian expression of G-CSF-DDD2

G-CSF-pdHL2 was linearized by SalI enzymatic digestion and stably transfected into Sp/EEE myeloma cells by electroporation. Clones were selected with medium containing 0.15. mu.M MTX. Clone #4 produced 0.15mg/L of G-CSF-DDD2 as indicated by sandwich ELISA.

Purification of G-CSF-DDD2 in batch cultures grown in roller bottles

Approximately 3 mg of G-CSF-DDD2 was purified as described in example 2. Clone 4 was expanded to 34 spinner flasks containing a total of 20L hybridoma SFM using 0.4. mu.M MTX and allowed to reach terminal culture. The supernatant was clarified by centrifugation, filtered (0.2. mu.M), diafiltered into 1 Xbuffer (10mM imidazole, 0.5M sodium chloride, 50mM sodium dihydrogen phosphate (pH 7.5)) and concentrated. The concentrate was loaded onto a Ni-NTA column, which was washed with 1 Xbuffer in 0.02% Tween 20, then 30mM imidazole, 0.5M sodium chloride, 50mM sodium dihydrogen phosphate (pH 7.5). The product was eluted with 250mM imidazole, 0.02% Tween 20, 150mM sodium chloride, 50mM sodium dihydrogen phosphate (pH 7.5).

Example 7 Generation of pegylated G-CSF by DNL

FIG. 11 is a cartoon representation showing the structure of G-CSF-413, G-CSF-413 possessing two copies of G-CSF coupled to 30kDa polyethylene glycol. The DNL reaction was accomplished by adding a 10-fold molar excess of reduced and lyophilized IMP413 to G-CSF-DDD2 dissolved in PBS. After 6h at room temperature in the dark, the reaction mixture was purified by immobilized metal affinity chromatography using Ni-NTA.

Example 8 Generation of an Erythropoietin (EPO) -based DDD Module

Construction of G-CSF-DDD2-pdHL2 for expression in mammalian cells

The cDNA sequence of EPO was amplified by PCR to give a sequence including the following features, wherein XbaI and BamHI are restriction sites, the signal peptide is native to human EPO, and 6His is a 6 histidine tag: XbaI- -signal peptide- -EPO- -6His- -BamHI. The resulting secreted protein consists of EPO linked at its C-terminus to a polypeptide consisting of SEQ ID NO: 2.

PCR amplification was performed using the full-length human EPO cDNA clone as template and the following oligonucleotides as primers:

EPO XbaI left

5’-TCTAGACACAGGACCTCATCATGGGGGTGCACGAATGTCC-3’(SEQ ID NO：7)

EPO BamHI right

5’-GGATCCATGATGGTGATGATGGTGTGACTTTCTGTCCCCTGTCCTGCAG-3’(SEQ ID NO：8)

The PCR amplificates were cloned into pGemT vectors. The DDD2-pdHL2 mammalian expression vector was prepared for ligation with EPO by digestion with XbaI and Bam HI restriction enzymes. The EPO amplificate was excised from pGemT using XbaI and Bam HI and ligated into DDD2-pdHL2 vector to generate expression vector EPO-DDD2-pdHL 2.

Mammalian expression of EPO-DDD2

EPO-pdHL2 was linearized by SalI enzymatic digestion and stably transfected into Sp/EEE myeloma cells using the electroporation technique. Clones were selected with medium containing 0.15. mu.M MTX. By ELISA using Nunc Immobilizer Nickel-Chemate plates to capture His-tagged fusion proteins and detection with anti-EPO antibody, clones No. 41, No. 49 and No. 37 were each shown to produce EPO at-0.5 mg/L.

Purification of EPO in batch cultures grown in spinner flasks

Approximately 2.5mg of EPO-DDD2 was purified by IMAC from 9.6 liter serum-free spinner flask cultures as described in example 2. SDS-PAGE and immunoblot analysis showed that the purified product after IMAC (not shown) accounted for approximately 10% of the total protein. Under reducing conditions, the EPO-DDD2 polypeptide is separated into larger than its calculated mass (28kDa) with M due to extensive and heterogeneous glycosylation_rA broad band of- (40-45 kDa). Under non-reducing conditions, EPO-DDD2 mainly separates as M with 80-90kDa_rDisulfide-linked covalent dimers (mediated by DDD 2).

Example 9 DNL conjugation of EPO-DDD2 with Fab-AD2 Module

h679 is a humanized monoclonal antibody highly specific for hapten HSG (histamine-succinyl-glycine). The generation of the h679-Fab-AD2 module has been described previously, and is shown in the cartoon representation of FIG. 12A (Rossi et al, Proc. Natl. Acad. Sci. USA.2006; 103: 6841). The cartoon representation in FIG. 12B depicts the dimeric structure of EPO-DDD 2. EPO-679(EPO-DDD2x h679-Fab-AD2) was produced in small amounts by DNL. EPO-DDD2(1mg) was reacted with h679-Fab-AD2(1mg) in PBS containing 1mM reduced glutathione and 2mM oxidized glutathione overnight. The DNL conjugate was purified using HSG-based affinity chromatography as described previously (Rossi et al, Proc. Natl. Acad. Sci. USA.2006; 103: 6841). FIG. 12C depicts the structure of EPO-679 with a cartoon representation, EPO-679 having 2 EPO portions and h 679-Fab. Coomassie blue staining of SDS-PAGE gels confirmed the production of EPO-679 (not shown). The DNL product, which was composed of only 3 constituent polypeptides (EPO, h679-Fd-AD2 and h679K) separated under non-reducing conditions into a broad band with an Mr of 150-170kDa, was highly purified and as demonstrated by SDS-PAGE under reducing conditions (not shown).

Example 10 biological Activity of EPO-DDD2 and EPO-679

The ability of EPO-DDD2 and EPO-679 to stimulate EPO-responsive TF1 cell growth (ATCC) was tested using recombinant human EPO (Calbiochem) as a positive control. TF1 cells were grown in 96-well plates containing RPMI1640 medium supplemented with 20% FBS and no GM-CSF, said 96-well plates containing 1X10⁴Cells/well. The concentration (units/ml) of the EPO construct was determined using a commercially available kit (human erythropoietin ELISA kit, Stem CellResearch, Cat # 01630). The cells were cultured for 72 hours in the presence of rhEPO, EPO-DDD2 or EPO-679 at concentrations ranging from 900U/ml to 0.001U/ml. Comparison of viable cell densities by MTS assay, OD measurement in 96-well plates₄₉₀Previously, the assay was incubated for 6 hours using 20. mu.l MTS reagent/well. Dose response curves and EC₅₀Values were generated using Graph Pad Prism software (FIG. 13). Both EPO-DDD2 and EPO-679 showed in vitro biological activity of about 10% of the potency of rhEPO.

Example 11 Generation of EPO by PEGylation of DNL

FIG. 14 depicts the structure of EPO-413 with two copies of EPO coupled to 30kDa PEG, using a cartoon representation. The DNL reaction was accomplished by adding a 10-fold molar excess of reduced and lyophilized IMP413 to EPO-DDD2 dissolved in PBS. After 6h at room temperature in the dark, the reaction mixture was purified by immobilized metal affinity chromatography using Ni-NTA.

Example 12.2-PEG: production of 1-effector moiety complexes

In alternative embodiments, it is desirable to generate a protein having a 2PEG moiety: 1 effector moiety. Such pegylated complexes are readily formed according to the methods of examples 1-3 above by attaching the polyethylene glycol moiety to the DDD sequence and the active agent to the AD sequence. PEGylation complexes with a 2: 1 stoichiometric ratio of PEG to IFN-. alpha.2b can be prepared by adapting the methods of examples 1-3. The complex is stable in serum and has lower interferon activity than a PEGylated complex with a 1: 2PEG to IFN-alpha 2b stoichiometric ratio. However, the clearance rate of the bis-pegylated complexes is slower than that of the mono-pegylated complexes.

Example 13 production of antibody fragments for PEGylation

Fab antibody fragments can be produced as fusion proteins containing DDD or AD sequences. An independent transgenic cell line can be developed for each Fab fusion protein. Once produced, these modules can be purified (if necessary) or maintained in cell culture supernatant. After production, anything (Fab-DDD)₂The module can be combined with any PEG-AD module, or any Fab-AD module can be combined with any (PEG-DDD)₂The modules are combined to generate a pegylated Fab construct.

DDD1：SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ IDNO：10)

DDD2：CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ IDNO：11)

AD1：QIEYLAKQIVDNAIQQA(SEQ ID NO：12)

AD2：CGQIEYLAKQIVDNAIQQAGC(SEQ ID NO：1)

The plasmid vector pdHL2 was used to generate a variety of antibodies and antibody-based constructs. See Gillies et al, J Immunol Methods (1989), 125: 191-202; losman et al, cancer (Phila) (1997), 80: 2660-6. The bicistronic mammalian expression vector directs the synthesis of heavy and light chains of IgG. For many different IgG-pdHL2 constructs, the vector sequences were essentially identical, with the only differences being in the variable domain (VH and VL) sequences. These IgG expression vectors can be converted into Fab-DDD expression vectors or Fab-AD expression vectors using molecular biological tools known to those skilled in the art. To generate the Fab-DDD expression vector, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain were replaced with a sequence encoding the first 4 residues of the hinge, the Gly-Ser linker of 14 residues, the first 44 residues of human RIIa (called DDD 1). To generate the Fab-AD expression vector, the sequence of AD (referred to as AD1) was synthesized by substituting the hinge, the CH2 and CH3 domains of IgG, with a Gly-Ser linker encoding the first 4 residues, 15 residues of the hinge, and a 17 residue known as AKAP-IS generated using bioinformatics and peptide array techniques and shown to bind with very high affinity (0.4nM) to the RIIa dimer. See Alto, et al proc.natl.acad.sci, u.s.a (2003), 100: 4445-50.

2 shuttle vectors were designed to facilitate the transformation of the IgG-pdHL2 vector into the Fab-DDD 1 or Fab-AD1 expression vectors as described below.

Preparation of CH1

The CH1 domain was amplified by PCR using pdHL2 plasmid vector as template. The left PCR primer consists of upstream (5 ') of the CH1 domain and a SacII restriction endonuclease site, which is 5' of the CH1 coding sequence. The right primer consists of a sequence encoding the first 4 residues of the hinge (PKSC) followed by GGGGS, the last two codons (GS) of which contain a BamHI restriction site.

5 'of CH1 left primer'

5’GAACCTCGCGGACAGTTAAG-3’(SEQ ID NO：13)

CH1+G₄S-Bam Right

5’GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTTGCTGG-3’(SEQ ID NO：14)

The 410bp PCR amplimer was cloned into pGemT PCR cloning vector (Promega, Inc.) and clones inserted in the direction of T7 (5') were selected.

(G₄S)₂Construction of DDD1

Duplex oligonucleotides (designated (G) were synthesized by Sigma Genosys (Haverhill, UK)₄S)₂DDD1) to encode the amino acid sequence of DDD1 located before the 11 residue linker peptide, the first two codons including a BamHI restriction site. The stop codon and the Eag1 cleavage site were attached to the 3' end. The encoded polypeptide sequence is shown below.

GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ ID NO：15)

Two oligonucleotides with a 30 base pair overlap at their 3' end, designated RIIA1-44 top and RIIA1-44 bottom, were synthesized (Sigma Genosys) and combined to include the central 154 base pair of the 174bp DDD1 sequence. The oligonucleotides are annealed and a primer extension reaction is performed with Taq polymerase.

RIIA1-44 top

5’GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGACAG-3’(SEQ ID NO：16)

RIIA1-44 bottom

5’GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCCTG-3’(SEQ ID NO：17)

Following primer extension, the duplexes were PCR amplified using the following primers:

G4S Bam-left

5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQ ID NO：18)

1-44 stop Eag Right

5’-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3’(SEQ ID NO：19)

This amplicon was cloned into pGemT and screened for inserts in the orientation of insert T7 (5'). (G)₄S)₂Construction of AD1

A duplex oligonucleotide (Sigma Genosys) was synthesized and designated (G)₄S)₂-AD1 to encode the amino acid sequence of AD1 preceded by an 11-residue linker peptide, the first two codons of which include a BamHI restriction site. A stop codon and an EagI cleavage site were added to the 3' end. The encoded polypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA(SEQ ID NO：20)

2 complementary overlapping oligonucleotides were synthesized, designated AKAP-IS top and AKAP-IS bottom.

AKAP-IS Top

5′GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGACGGCCG-3′(SEQ ID NO：21

AKAP-IS bottom

5’CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCCGGATCC-3’(SEQ ID NO：22)

The duplexes were amplified by PCR using the following primers:

G4S Bam-left

5’-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3’(SEQ ID NO：23

AKAP-IS Eag Right termination

5’-CGGCCGTCAGGCCTGCTGGATG-3’(SEQ ID NO：24)

The amplificates were cloned into the pGemT vector and the inserts were screened in the orientation of T7 (5').

Connecting CH1 with DDD1

A190 bp fragment encoding the DDD1 sequence was excised from pGemT using BamHI and Not1 restriction enzymes and ligated to the same site on CH1-pGemT to generate the shuttle vector CH1-DDD 1-pGemT.

Connecting AD1 with CH1

A110 bp fragment containing the AD1 sequence was excised from pGemT using BamHI and Not1 restriction enzymes and ligated to the same site on CH1-pGemT to generate the shuttle vector CH1-AD 1-pGemT.

Cloning of CH1-DDD1 or CH1-AD1 into pdHL 2-based vectors

Using this modular design, CH1-DDD1 or CH1-AD1 can be introduced into any IgG construct of the pdHL2 vector. The entire heavy chain constant region was replaced by one of the constructs described above by removing the SacII/Eagl restriction fragment (CH1-CH3) from pdHL2 and replacing it with SacII/Eag1 fragment from CH1-DDD1 or CH1-AD1, respectively, of the corresponding pGemT shuttle vector. Construction of C-DDD1-Fd-hMN-14-pdHL2

The hMN-14 antibody is a humanized CEA-binding antibody comprising the CDR sequences of murine MN-14 (see, e.g., U.S. Pat. No. 6676924). C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for the production of a stable dimer comprising two copies of the fusion protein C-DDD 1-Fab-hMN-14, wherein DDD1 is linked to hMN-14Fab at the carboxy-terminus of CH1 via a flexible peptide spacer. The plasmid vector hMN14(I) -pdHL2 for hMN-14IgG production was converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restriction enzymes to remove the CH1-CH3 domain and insert the CH1-DDD1 fragment cut from the CH1-DDD1-SV3 shuttle vector using SacII and Eagl.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector used to produce C-DDD2-Fab-hMN-14, with the dimerization and docking domain of DDD2 attached to the carboxy-terminus of Fd by a 14 amino acid residue Gly/Ser peptide linker. The secreted fusion protein comprises two identical copies of hMN-14Fab maintained together by non-covalent interactions of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping, complementary oligonucleotides were synthesized, which included the coding sequence for the portion of the linker peptide (GGGGSGGGCG, SEQ ID NO: 25) and residues 1-13 of DDD 2. The oligonucleotides were annealed and fiberized with T4PNK phosphate, resulting in overhang formation at the 5 'and 3' ends, which was suitable for ligation with BamHI and PstI restriction enzyme digested DNA, respectively.

G₄Top of S-DDD2

5’GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3’(SEQ ID NO：26)

G₄Bottom of S-DDD2

5’GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTCCGCCAGACCCGCCACCTCCG-3’(SEQ ID NO：27)

The duplex DNA was ligated with shuttle vector CH1-DDD1-pGemT prepared by BamHI and Pstl digestion to generate shuttle vector CH1-DDD 2-pGemT. A507 bp fragment was excised from CH1-DDD2-pGemT with SacII and EagI and ligated with the IgG expression vector hMN14(I) -pdHL2 prepared by digestion with SacII and EagI. The final expression construct was C-DDD2-Fd-hMN-14-pdHL 2.

Example 14 Pegylation of Fab antibody fragments

The C-DDD2-Fd-hMN14-pdHL2 vector was transfected into Sp/EEE cells (see U.S. patent application Ser. No. 11/487,215) and used to produce C-DDD 2-Fab-hMN-14. The bicistronic expression vector directs the synthesis and secretion of both hMN-14 kappa light chain and C-DDD2-Fd-hMN-14, which combine to form C-DDD2-Fab-hMN 14. C-DDD2-Fab-hMN-14 spontaneously forms dimers, which are equimolar mixtures with IMP362, IMP413 or IMP457, leading to the formation of pegylated C-DDD2-Fab-hMN-14 dimer. The hMN-14Fab portion retains its binding specificity for the CEA antigen. Injection into nude mice bearing CEA-expressing tumors showed that hMN-14F (ab), which is not pegylated₂In contrast, PEGylated C-DDD2-Fab-hMN-14 exhibited a significantly prolonged circulating half-life, enhanced efficacy and reduced dosing frequency of the dosing regimen.

Example 15 formation of C-H-AD2-IgG-pdHL2 expression vector

pdHL2 mammalian expression vectors have been used to mediate the expression of a variety of recombinant iggs. A plasmid shuttle vector was formed to facilitate transformation of any IgG-pdHL2 vector into the C-H-AD2-IgG-pdHL2 vector. The gene for Fc (CH2 and CH3 domains) was amplified using pdHL2 vector as template and the following oligonucleotides as primers.

Left Fc BglII

5’-AGATCTGGCGCACCTGAACTCCTG-3’(SEQ ID NO：28)

Fc Bam-EcoRI right

5’-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3’(SEQ ID NO：29)

The amplificate was cloned into a pGemT PCR cloning vector. The Fc insert was excised from pGemT and ligated with the AD2-pdHL2 vector to generate the shuttle vector Fc-AD2-pdHL 2.

To convert any IgG-pdHL2 expression vector into the C-H-AD2-IgG-pdHL2 expression vector, the 861bp BsrGI/NdeI restriction enzyme fragment was excised from the former and replaced with the 952bp BsrGI/NdeI restriction enzyme fragment excised from the Fc-AD2-pdHL2 vector. BsrGI cleaved within the CH3 domain, NdeI cleaved downstream of the expression cassette (3').

Example 16 production of C-H-AD2-hLL2IgG

Epratuzumab or IgG of hLL2 is a humanized anti-human CD22MAb (see, e.g., U.S. Pat. nos. 5,443,953, 5,789,554, 6,187,287, 7,074,403). Expression vectors for C-H-AD2-hLL2IgG were generated from hLL2IgG-pdHL2 and used to transfect Sp2/0 myeloma cells by electroporation as described in example 15. After transfection, cells were plated in 96-well plates and transgenic clones were selected in media containing methotrexate. The cloned C-H-AD2-hLL2IgG productivity was screened by sandwich ELISA using 96-well plate microtiter plates coated with hLL 2-specific anti-idiotype MAbs and detected with peroxidase conjugated anti-human IgG. Clones were expanded into roller bottles for protein production and C-H-AD2-hLL2IgG was purified from spent medium in a single step using protein A affinity chromatography. SE-HPLC analysis separated 2 protein peaks (not shown). The retention time of the slower eluting peak (8.63 min) was similar to hLL2 IgG. The retention time of the faster eluting peak (7.75 min) was consistent with-300 kDa protein. This peak was later determined to represent the disulfide-linked dimer of C-H-AD2-hLL 2-IgG. During the reaction of DNL this dimer is reduced to the monomeric form. SDS-PAGE analysis indicated that purified C-H-AD2-hLL2-IgG included both a monomeric form of the module and a dimeric form of disulfide linkage (not shown). The protein bands representing these two forms were confirmed by SDS-PAGE under non-reducing conditions; whereas under reducing conditions, all forms were reduced to two bands (heavy chain-AD 2 and kappa chain) representing the component polypeptides. No other contaminating bands were detected.

Example 17 production of C-H-AD2-hA20 IgG

hA20 IgG is a humanized anti-human CD20 MAb (see, e.g., U.S. patent No. 7,154,164). C-H-AD2-hA20 IgG expression vector was generated from hA20 IgG-pDHL2 and used to transfect SP2/0 myeloma cells by electroporation as described in example 15. After transfection, cells were plated in 96-well plates and transgenic clones were selected in media containing methotrexate. The cloned C-H-AD2-hA20 IgG productivity was screened by sandwich ELISA using 96-well plate microtiter plates coated with hA 20-specific anti-idiotype MAbs and detected with peroxidase conjugated anti-human IgG. Clones were expanded into roller bottles for protein production to purify C-H-AD2-hA20 IgG from spent medium using a separate step of protein A affinity chromatography. SE-HPLC and SDS-PAGE analysis gave very similar results to those obtained for C-H-AD2-hLL2IgG in example 16.

Example 18 production of pegylated IgG

C-H-AD2-hLL2IgG or C-H-AD2-bA20 IgG antibody was mixed with PEG-DDD2 to form pegylated hLL2 or hA 20. hLL2 or hA20 retained their binding specificity for CD22 and CD20 antigens, respectively. Injection into nude mice bearing CD 20-or CD 22-expressing tumor cells showed that the pegylated antibodies exhibited a significantly prolonged circulating half-life, enhanced efficacy, and reduced dosing frequency of the dosing regimen, compared to non-pegylated antibodies.

Seq.1isting_IBC118WO-for PCTUS0881085_Chinese.txt

Sequence listing

<110> IBC pharmaceutical Co

<120> Pegylation by docking and latching (DNL) technique

<130>IBC118W03

<140>

<141>

<150>11/925,408

<151>2007-10-26

<160>33

<170> PatentIn version 3.5

<210>1

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>1

Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile

1 5 10 15

Gln Gln Ala Gly Cys

20

<210>2

<211>63

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>2

Lys Ser His His His His His His Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Cys Gly His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu

20 25 30

Gln Gly Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val

35 40 45

Glu Phe Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala

50 55 60

<210>3

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>3

tctagacaca ggacctcatc atggccttga cctttgcttt actgg 45

<210>4

<211>55

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>4

ggatccatga tggtgatgat ggtgtgactt ttccttactt cttaaacttt cttgc 55

<210>5

<211>41

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>5

tctagacaca ggacctcatc atggctggac ctgccaccca g 41

<210>6

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>6

ggatccatga tggtgatgat ggtgtgactt gggctgggca aggtggcgta g 51

<210>7

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>7

tctagacaca ggacctcatc atgggggtgc acgaatgtcc 40

<210>8

<211>49

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>8

ggatccatga tggtgatgat ggtgtgactt tctgtcccct gtcctgcag 49

<210>9

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD RES

<222>(1)..(1)

<223>Cys(S-tBu)

<220>

<221>MOD RES

<222>(21)..(21)

<223>Cys(S-tBu)

<400>9

Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile

1 5 10 15

Gln Gln Ala Gly Cys Gly

20

<210>10

<211>44

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>10

Ser His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr

1 5 10 15

Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala

20 25 30

Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala

35 40

<210>11

<211>45

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>11

Cys Gly His Ile Gln Ile Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly

1 5 10 15

Tyr Thr Val Glu Val Leu Arg Gln Gln Pro Pro Asp Leu Val Glu Phe

20 25 30

Ala Val Glu Tyr Phe Thr Arg Leu Arg Glu Ala Arg Ala

35 40 45

<210>12

<211>17

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>12

Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln

1 5 10 15

Ala

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>13

gaacctcgcg gacagttaag 20

<210>14

<211>53

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>14

ggatcctccg ccgccgcagc tcttaggttt cttgtccacc ttggtgttgc tgg 53

<210>15

<211>55

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>15

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Ile Gln Ile

1 5 10 15

Pro Pro Gly Leu Thr Glu Leu Leu Gln Gly Tyr Thr Val Glu Val Leu

20 25 30

Arg Gln Gln Pro Pro Asp Leu Val Glu Phe Ala Val Glu Tyr Phe Thr

35 40 45

Arg Leu Arg Glu Ala Arg Ala

50 55

<210>16

<211>92

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>16

gtggcgggtc tggcggaggt ggcagccaca tccagatccc gccggggctc acggagctgc 60

tgcagggcta cacggtggag gtgctgcgac ag 92

<210>17

<211>92

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>17

gcgcgagctt ctctcaggcg ggtgaagtac tccactgcga attcgacgag gtcaggcggc 60

tgctgtcgca gcacctccac cgtgtagccc tg 92

<210>18

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>18

ggatccggag gtggcgggtc tggcggaggt 30

<210>19

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>19

cggccgtcaa gcgcgagctt ctctcaggcg 30

<210>20

<211>29

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>20

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Glu Tyr

1 5 10 15

Leu Ala Lys Gln Ile Val Asp Asn Ala Ile Gln Gln Ala

20 25

<210>21

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>21

ggatccggag gtggcgggtc tggcggaggt ggcagccaga tcgagtacct ggccaagcag 60

atcgtggaca acgccatcca gcaggcctga cggccg 96

<210>22

<211>96

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>22

cggccgtcag gcctgctgga tggcgttgtc cacgatctgc ttggccaggt actcgatctg 60

gctgccacct ccgccagacc cgccacctcc ggatcc 96

<210>23

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>23

ggatccggag gtggcgggtc tggcggaggt 30

<210>24

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>24

cggccgtcag gcctgctgga tg 22

<210>25

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>25

Gly Gly Gly Gly Ser Gly Gly Gly Cys Gly

l 5 10

<210>26

<211>73

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>26

gatccggagg tggcgggtct ggcggaggtt gcggccacat ccagatcccg ccggggctca 60

cggagctgct gca 73

<210>27

<211>65

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>27

gcagctccgt gagccccggc gggatctgga tgtggccgca acctccgcca gacccgccac 60

ctccg 65

<210>28

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>28

agatctggcg cacctgaact cctg 24

<210>29

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>29

gaattcggat cctttacccg gagacaggga gag 33

<210>30

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD RES

<222>(21)..(21)

<223>Cys(SS-tBu)

<400>30

Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile

1 5 10 15

Gln Gln Ala Gly Cys

20

<210>31

<211>22

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<220>

<221>MOD RES

<222>(21)..(21)

<223>Cys(SS-tBu)

<220>

<221>MOD RES

<222>(22)..(22)

<223>Gly-EDANS

<400>31

Cys Gly Gln Ile Glu Tyr Leu Ala Lys Gln Ile Val Asp Asn Ala Ile

1 5 10 15

Gln Gln Ala Gly Cys Gly

20

<210>32

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthesis of 6XHis tag

<400>32

His His His His His His

1 5

<210>33

<211>10

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>33

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

Claims (8)

1. A pegylated complex comprising:

a) an effector moiety attached to the dimerization and docking domain DDD sequence; and

b) a PEG moiety linked to the anchor domain AD sequence of the human a-kinase anchor protein AKAP;

wherein 2 DDD sequences bind to 1 AD sequence to form a pegylated complex, the complex further comprising a disulfide bond between the DDD and AD sequences, wherein the amino acid sequence of the DDD is SEQ ID NO: 2,

wherein the effector moiety is selected from the group consisting of an enzyme, a cytokine, a chemokine, a growth factor, a peptide, an aptamer, hemoglobin, an antibody, and an antibody fragment.

2. The complex of claim 1, wherein the PEG moiety has a methoxy cap at one end.

3. The complex of claim 1, wherein the effector moiety is selected from interferon-a, interferon- β, interferon- γ, MIF, HMGB-1 (high mobility group box 1), TNF-a, 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, IL-18, IL-19, IL-23, IL-24, CCL19, CCL21, MCP-1, ranan MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, Gro- β, eotaxin, and mixtures thereof, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin, thrombopoietin, hGH, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, PlGF, insulin, hGH, calcitonin, factor VIII, IGF, somatostatin, tissue plasminogen activator, and LIF.

4. The complex of claim 1, wherein the PEG moiety attached to an AD sequence comprises IMP350, IMP360, PEG₂₀IMP360 or PEG₃₀-IMP360，

Wherein IMP350 is CGQIEYLAKQIVDNAIQQAGC (SS-tbu) -NH₂And is and

IMP360 is CGQIEYLAKQIVDNAIQQAGC (SS-tbu) G-EDANS.

5. The complex of claim 1, wherein the effector moiety is Interferon (IFN) - α 2b, G-CSF, or erythropoietin.

6. A pegylated complex comprising:

a) an effector moiety linked to the AD sequence of the human a-kinase anchor protein AKAP; and

b) a PEG moiety attached to a DDD sequence;

wherein two DDD sequences bind to an AD sequence to form a pegylated complex, wherein the amino acid sequence of the DDD is SEQ ID NO: 2,

wherein the effector moiety is selected from the group consisting of an enzyme, a cytokine, a chemokine, a growth factor, a peptide, an aptamer, hemoglobin, an antibody, and an antibody fragment.

7. A method of pegylating an effector moiety, the method comprising:

a) linking an effector moiety to a DDD sequence;

b) attaching a PEG moiety to the AD sequence of the human A-kinase anchor protein AKAP; and

c) allowing the DDD sequence to bind to the AD sequence to form a pegylated complex comprising two effector moieties-a DDD sequence and a PEG-AD sequence, wherein the amino acid sequence of the DDD is SEQ ID NO: 2,

wherein the effector moiety is selected from the group consisting of an enzyme, a cytokine, a chemokine, a growth factor, a peptide, an aptamer, hemoglobin, an antibody, and an antibody fragment.

8. A method of pegylating an effector moiety comprising:

a) linking an effector moiety to the AD sequence of the human a-kinase anchor protein AKAP;

b) attaching a PEG moiety to the DDD sequence; and

c) allowing the DDD sequence to bind to the AD sequence to form a pegylated complex comprising 1 effector moiety-AD sequence and 2 PEG-DDD sequences, wherein the amino acid sequence of the DDD is SEQ ID NO: 2,

wherein the effector moiety is selected from the group consisting of an enzyme, a cytokine, a chemokine, a growth factor, a peptide, an aptamer, hemoglobin, an antibody, and an antibody fragment.