WO2013020079A2 - Conjugates of an il-11 moiety and a polymer - Google Patents

Conjugates of an il-11 moiety and a polymer Download PDF

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WO2013020079A2
WO2013020079A2 PCT/US2012/049582 US2012049582W WO2013020079A2 WO 2013020079 A2 WO2013020079 A2 WO 2013020079A2 US 2012049582 W US2012049582 W US 2012049582W WO 2013020079 A2 WO2013020079 A2 WO 2013020079A2
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moiety
conjugate
water
conjugates
soluble polymer
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PCT/US2012/049582
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WO2013020079A3 (en
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Deborah H. Charych
Sean M. Culbertson
Ping Zhang
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Nektar Therapeutics
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • one or more embodiments of the present invention relate generally to the field of conjugates comprising an IL-1 1 moiety (i.e., a moiety having at least some activity similar to human IL-1 1) and a water-soluble, non-peptidic polymer.
  • the invention relates to (among other things) the fields of compositions comprising conjugates, methods for synthesizing conjugates, and methods of administering a
  • Interleukin 1 1 (“IL-1 1 ,” sometimes referred to as adipogenesis inhibitory factor) is a cytokine that interacts with a variety of hematopoietic and non-hematopoietic cell types. For example, IL-11 has been shown to increase the number of platelets in blood.
  • IL-11 acts to promote epithelial growth of damaged epithelial tissues located in the gastrointestinal tract. See Schwertschlag et al. (1999) Leukemia 13: 1307-1315. Also, IL-1 1 has been shown to have immunomodulatory effects on both macrophages and T cells, which result in reduced inflammatory responses. Trepicchio et al. (1999) J. Clin. Invest. 104(1 1): 1527-1537. Thus, IL-1 1 exhibits a myriad of effects, some of which have been proposed as being clinically useful.
  • Oprelvekin is a form of human recombinant IL-11 ("rhIL-11 ”) marketed under the NEUMEGA brand (Wyeth Pharmaceuticals Inc., Philadelphia PA) and is indicated for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in adult patients with nonmyeloid malignancies who are at high risk of severe thrombocytopenia. Following administration, rhIL-11 is rapidly cleared. Takagi et al. (1995) J. Pharmacol. Exp. Ther. 275:537-543. As a result, administration of oprelvekin typically occurs once daily via a single subcutaneous injection in the abdomen, thigh, hip or upper arm. Daily injections are often inconvenient and potentially painful, which is suboptimal.
  • conjugates of IL-1 1 having improved characteristics and profiles.
  • one or more embodiments of the present invention is therefore directed to such conjugates as well as compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art.
  • a conjugate comprising a residue of an IL-11 moiety covalently attached to a water-soluble polymer.
  • a conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein the residue of the IL-11 moiety is covalently attached to the water-soluble polymer via a releasable linkage.
  • a conjugate comprising a residue of an IL-11 moiety covalently attached to a water-soluble polymer, wherein the IL-11 moiety is free of cysteine residues.
  • a conjugate comprising a residue of an IL-1 1 moiety covalently attached to a branched water-soluble polymer, wherein the branched water-soluble polymer lacks a lysine residue,
  • a conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein an amine of the IL-11 moiety is covalently attached to the water-soluble polymer via a linkage other than an amide linkage.
  • a conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein an amine of the IL-11 moiety is covalently attached to the water-soluble
  • composition comprising a conjugate as described herein along with a pharmaceutically acceptable excipient.
  • a method for delivering a conjugate comprising the step of subcutaneously administering to the patient a composition comprised of a conjugate of a residue of an IL-1 1 moiety and a water-soluble polymer.
  • Figure 1 is a representation of a chromatogram following reverse phase HPLC analysis of [mPEG2-ru-NHS, 40kDa]-[rIL-l 1] conjugate soution, as further described in Example 1.
  • Figure 2 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-ButyrALD, 20kDa]-[rIL-l 1] conjugate solution, as further described in Example 2.
  • Figure 3 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG2-ru-ButyrALD, 40kDa]-[rIL-l 1] conjugate solution, as further described in Example 3.
  • Figure 4 is a representation of a typical chromatogram following cation exchange chromatography of [mPEG-ButyrALD, 40kDa]-[rIL-l l] conjugates, as further described in Example 3.
  • Figure 5 is a representation of the results following SDS-PAGE analysis of the reaction mixture prepared in accordance with the procedures set forth in Example 4 and Example 6.
  • Figure 6 is a representation of a chromatogram following SEC-HPLC analysis of [C2-PEG2-FMOC-NHS, 40kDa]-[rIL-l 1] conjugate solution, as further described in Example 5.
  • Figure 7 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-HP-ALD]-[rIL-l 1] conjugate solution, as further described in Example 7.
  • Figure 8 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-MAHP-ALD]-[rIL-l 1 ] conjugate solution, as further described in Example 8,
  • Figure 9 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-PipHP-ALD]-[rIL-l 1] conjugate solution, as further described in Example 9.
  • Figure 10 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-ALD20K]-[rIL-l l] conjugate solution, as further described in Example 10.
  • Figure 11 is a representation of a typical chromatogram following cation exchange chromatography of [C2-PEG2-FMOC-20 ]-[rIL-l 1] conjugates, as further described in Example 11.
  • Figure 12 depicts non-linear regression curves of stable conjugates tested in the IL-1 1 bioassay as further described in Example 14.
  • Figure 13 depicts non-linear regression curves of releasable conjugates tested in the IL-1 1 bioassay as further described in Example 14.
  • Figure 14 is a plot of the TNFoc production in response to acute LPS challenge in mice pretreated with a test article, as further described in Example 5.
  • PEG polyethylene glycol
  • poly(ethylene glycol) poly(ethylene glycol)
  • PEGs for use in accordance with the invention comprise the following structure "-(OCH 2 CH 2 ) n - M where (n) is 2 to 4000.
  • PEG also includes
  • PEG includes structures having various terminal or “end capping” groups and so forth.
  • PEG also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2 CH 2 - repeating subunits.
  • the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below.
  • end-capped and “terminally capped” are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.
  • the end-capping moiety comprises a hydroxy or Ci_ 2 o alkoxy group, more preferably a Cj.io alkoxy group, and still more preferably a Ci -5 alkoxy group.
  • examples of end-capping moieties include alkoxy (e.g. , methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like.
  • the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH 3 0(CH 2 CH 2 0) n - and CH3(OCH 2 CH 2 ) n -].
  • the end-capping group can also be a silane.
  • the end-capping group can also advantageously comprise a detectable label.
  • the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector.
  • suitable labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, and the like.
  • Suitable detectors include photometers, films,
  • the end-capping group can also advantageously comprise a phospholipid.
  • a phospholipid When the polymer has an end-capping group comprising a phospholipid, unique properties are imparted to the polymer and the resulting conjugate.
  • exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines.
  • Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine,
  • the end-capping group may also include a targeting moiety, such that the polymer - as well as anything, e.g., an IL-1 1 moiety, attached thereto ⁇ can preferentially localize in an area of interest.
  • Non-naturally occurring with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature.
  • a non-naturally occurring polymer may, however, contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.
  • water soluble as in a "water-soluble polymer” is any polymer that is soluble in water at room temperature.
  • a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering.
  • a water-soluble polymer will preferably be at least about 35%) (by weight) soluble in water, more preferably at least about 50%> (by weight) soluble in water, still more preferably about 70%> (by weight) soluble in water, and still more preferably about 85%) (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water.
  • Molecular weight in the context of a water-soluble polymer can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques.
  • the polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.
  • active when used in conjunction with a particular functional group, refer to a functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive” or “inert” group).
  • spacer moiety refers to a bond or an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymeric reagent and an IL-1 1 moiety or an electrophile or nucleophile of an IL-11 moiety.
  • the spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage.
  • a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of an IL-1 1 moiety and a water-soluble polymer can be attached directly or indirectly through a spacer moiety).
  • Alkyl refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.
  • “Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i ' -butyl, and /-butyl.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.
  • Cycloalkylene refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.
  • Alkoxy refers to an -OR group, wherein R is alkyl or substituted alkyl, preferably C 1-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).
  • substituted refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C 3 - 8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.
  • “Substituted aryl” is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).
  • Noninterfering substituents are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.
  • Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms.
  • Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl” includes heteroaryl.
  • Heteroaryl is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
  • Heterocycle or “heterocyclic” means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.
  • Substituted heteroaryl is heteroaryl having one or more noninterfering groups as substituents.
  • Substituted heterocycle is a heterocycle having one or more side chains formed from noninterfering substituents.
  • An "organic radical” as used herein shall include akyl, substituted alkyl, aryl, and substituted aryl.
  • Electrophile and “electrophilic group” refer to an ion or atom or collection of atoms, which may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.
  • Nucleophile and nucleophilic group refers to an ion or atom or collection of atoms, which may be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center or an electrophile.
  • a “physiologically cleavable” or “hydrolyzable” or “degradable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.
  • a “releasable linkage” is a covalent linkage that cleaves under physiological conditions at a rate that is clinically useful and includes, for example and without limitation, hydrolyzable bonds.
  • An "enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.
  • a “hydrolytically stable” linkage or bond refers to a chemical bond, typically a covalent bond, which is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time.
  • hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like.
  • a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • therapeutically effective amount are used interchangeably herein to mean the amount of a polymer-(lL-l 1) moiety conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated IL-11 moiety) in the bloodstream or in the target tissue.
  • the precise amount will depend upon numerous factors, e.g., the particular IL-1 1 moiety, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one of ordinary skill in the art, based upon the information provided herein.
  • Multi-functional means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different.
  • Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, and can contain, for example, a number satisfying one or more of the following ranges: from 3-50 functional groups; from 3-25 functional groups; from 3-15 functional groups; from 3 to 10 functional groups.
  • the number of functional groups can be selected from the group consisting of 3, 4, 5, 6, 7, 8, 9 and 10 functional groups within the polymer.
  • IL-1 1 moiety refers to a moiety having human
  • the IL-1 1 moiety will also have at least one electrophilic group or
  • IL-11 moiety encompasses both the IL-11 moiety prior to conjugation as well as the IL-1 1 moiety residue following conjugation. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has IL-11 activity. Proteins comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1 through 24 is an IL-1 1 moiety, as well as any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-1 1 moiety” includes such proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations.
  • analogs having from 1 to 6 additional glycosylation sites analogs having at least one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site.
  • the term includes both natural and
  • substantially homologous means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more
  • IL-1 1 moieties for use herein include those sequences that are substantially homologous SEQ ID NO: 2.
  • fragment means any protein or polypeptide having the amino acid sequence of a portion or fragment of an IL-1 1 moiety, and which has the biological activity of IL-1 1. Fragments include proteins or polypeptides produced by proteolytic degradation of an IL-1 1 moiety as well as proteins or polypeptides produced by chemical synthesis by methods routine in the art.
  • the term "patient,” refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of an active agent (e.g., conjugate), and includes both humans and animals.
  • an active agent e.g., conjugate
  • substantially means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater of the condition.
  • Amino acid residues in peptides are abbreviated as follows: Phenylalanine is
  • Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
  • Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or ; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
  • a conjugate comprising a residue of an IL-11 moiety covalently attached (either directly or through a spacer moiety) to a water-soluble polymer.
  • the conjugates of the invention will have one or more of the following features.
  • the conjugate generically comprises a residue of an IL-1 1 moiety covalently attached, either directly or through a spacer moiety, to a water-soluble polymer.
  • IL-1 1 moiety shall refer to the IL-11 moiety prior to conjugation as well as to the IL-1 1 moiety following attachment to a nonpeptidic,
  • IL-1 1 moiety when the original IL-1 1 moiety is attached to a nonpeptidic, water-soluble polymer, the IL-11 moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer(s). Often, this slightly altered form of the IL-11 moiety attached to another molecule is referred to a "residue" of the IL-1 1 moiety.
  • the IL-1 1 moiety can be derived from non-recombinant methods and from recombinant methods and the invention is not limited in this regard.
  • the IL-1 1 moiety can be derived from human sources, animal sources, and plant sources.
  • the IL-1 1 moiety can be derived non-recombinantly. For example, it is possible to isolate IL-11 from biological systems and otherwise obtain IL-1 1 from cultured media.
  • the IL-1 1 moiety can be derived from recombinant methods. See, for example, U.S. Patent No. 5,371 , 193, U.S. Patent Application Publication No. 2009/0191 148 and Paul et al. (1990) Proc. Natl. Acad. Sci. USA 87:7512-7516.
  • the IL-1 1 moiety can be expressed in bacterial [e.g., E. coli, see, for example,
  • recombinant-based methods for preparing proteins typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as a Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment.
  • a host cell e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as a Chinese hamster ovary cell or baby hamster kidney cell.
  • nucleic acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or added in- frame with the coding sequence, thereby producing a fusion protein comprised of the desired polypeptide and a polypeptide suited for binding.
  • Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein.
  • binding moieties e.g., antibodies
  • the recombinant polypeptide can also be purified by lysing the host cells, separating the polypeptide, e.g., by ion-exchange chromatography, affinity binding approaches, hydrophobic interaction approaches, and thereafter identify by MALDI or western blot, and collecting the polypeptide.
  • separating the polypeptide e.g., by ion-exchange chromatography, affinity binding approaches, hydrophobic interaction approaches, and thereafter identify by MALDI or western blot, and collecting the polypeptide.
  • IL-1 1 moiety can be unglycosylated or glycosylated and either may be used. That is, the IL-1 1 moiety can be unglycosylated or the IL-11 moiety can be glycosylated. In one or more embodiments of the invention, the IL-1 1 moiety is unglycosylated.
  • the IL-11 moiety can advantageously be modified to include and/or substitute one or more amino acid residues such as, for example, lysine, cysteine and/or arginine, in order to provide facile attachment of the polymer to an atom within the side chain of the amino acid.
  • substitution of an IL-1 1 moiety is described in U.S. Patent No. 5,206,344.
  • the IL-1 1 moiety can be modified to include a non-naturally occurring amino acid residue.
  • Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art. Reference is made to J. March, Advanced Organic IL-1 lmistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).
  • the IL-1 1 moiety can advantageously be modified to include attachment of a functional group (other than through addition of a functional
  • the IL-11 moiety can be modified to include a thiol group.
  • the IL-1 1 moiety can be modified to include an N-terminal alpha carbon.
  • the IL-1 1 moiety can be modified to include one or more carbohydrate moieties.
  • the IL-1 1 moiety can be modified to include an aldehyde group.
  • the IL-1 1 moiety can be modified to include a ketone group. In some embodiments of the invention, it is preferred that the IL-11 moiety is not modified to include one or more of a thiol group, an N-terminal alpha carbon, carbohydrate, adehyde group and ketone group.
  • IL-1 1 moieties are described in the literature and in, for example,
  • IL-11 moieties include those having an amino acid sequence comprising sequences selected from the group consisting of SEQ ID NOs: 1 through 24, and sequences substantially homologous thereto.
  • a preferred IL-11 moiety has the amino acid sequence corresponding to SEQ ID NO: 2.
  • the IL-1 1 moiety will be in a "monomer” form, wherein a single expression of the corresponding peptide is organized into a discrete unit.
  • the IL-11 moiety will be in the form of a "dimer” (e.g., a dimer of recombinant IL- 1 1) wherein two monomer forms of the protein are associated to each other or other
  • precursor forms IL-1 1 can be used as the IL-11 moiety.
  • An exemplary precursor form of IL-1 1 has the sequence of SEQ ID NO: 1 ,
  • Truncated versions, hybrid variants, and peptide mimetics of any of the foregoing sequences can also serve as the IL-11 moiety.
  • Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of IL-1 1 activity can also serve as an IL-1 1 moiety.
  • a Ba/F3 cell line (DSMZ, Germany) stably expressing IL-1 1 receptors, gpl 30 and the IL-1 1 receptor a chain (IL-1 IR), can be prepared by transduction of Ba/F3 cells with two retroviral vectors, MIN-IL-1 IR and MIH-gpl30, expressing the IL-1 1 receptor a chain (NCBI NM 004512.3) and gpl30 (NCBI NM 602184), respectively.
  • the retroviral plasmid pMIN-IL-11 R can be prepared by the insertion of the
  • IL-1 IR gene into the pMIN vector (Yu et al (2003) Gene Therapy 10:706-71 1).
  • the pMIH-gpl30 can be prepared by the substitution of the neomycin resistance gene of the pMIN with a hygromycin resistance gene, followed by gpl30 gene insertion.
  • pMIN-IL-1 IR can be transfected into HEK293T cells with pVM-GP and pVM-AE, expressing gag-pol and amphotropic envelope, respectively as shown by Yu et al. (2003) Gene Therapy 10:706-71 1).
  • the retroviral vector MIH-gpl30 can be generated by the same procedure as MIN-IL-1 IR, except pMIH-gpl 30 is transfected instead of pMIN-IL-1 IR.
  • the transfected cells can then be grown for two days in DMEM media containing 10% fetal bovine serum. After cultivation, the cell-free virus can be prepared by filtering the culture supernatant through a 0.45 ⁇ filter.
  • Ba F3 cells can be transduced with the retroviral vector MIN-IL-1 IR and selected in the presence of 2 mg/ml G418.
  • IL-1 1 receptor a chain The expression of IL-1 1 receptor a chain can be confirmed by flow cytometry using FITC-anti-IL-1 IR (Thermo Scientific, Rockford, IL, USA). Then, the cells stably expressing IL-1 IR can then be transduced with the retroviral vector MIH-gpl30 and selected in the presence of 2 mg/ml G418 and 0.5 mg/ml hygromycin. The expression of both IL-1 1R and gpl 30 can be confirmed by flow cytometry using FITC- anti-IL-1 1R and PE-anti-hgpl30 (BD Biosciences, San Jose, CA, USA), respectively.
  • the single clones expressing both IL11R and gpl30 can be obtained by limited dilution in the presence of 2 mg/ml G418 and 0.5 mg/ml hygromycin.
  • the production of IL-1 1 receptor a chain and gpl 30 mRNA from the cells was confirmed by RT-PCR using the following primer pairs.
  • gpl 30 SEQ ID NO: 27: 5'-ACGCGTATGTTGACGTTGCAGACT-S '
  • each conjugate comprises an IL-1 1 moiety attached to a water-soluble polymer.
  • the water-soluble polymer is nonpeptidic, nontoxic, non-naturally occurring and biocompatible.
  • biocompatibility a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as an IL-1 1 moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician.
  • non-immunogenicity a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. It is particularly preferred that the nonpeptidic water-soluble polymer is biocompatible and non-immunogenic.
  • the polymer is typically characterized as having from 2 to about 300 termini.
  • poly(alkylene glycols) such as polyethylene glycol (“PEG”), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
  • PEG polyethylene glycol
  • PPG poly(propylene glycol)
  • copolymers of ethylene glycol and propylene glycol and the like poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(
  • POZ polyoxazolines
  • poly(N-acryloylmorpholine) poly(N-acryloylmorpholine), and combinations of any of the foregoing.
  • the water-soluble polymer is not limited to a particular structure and can be linear (for example, an end capped, e.g., alkoxy, PEG or a bifunctional PEG), branched or multi-armed (e.g., individual PEGs attached to the hydroxyl groups of a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages.
  • the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
  • activated PEG and other activated water-soluble polymers are activated with a suitable activating group appropriate for coupling to a desired site on the IL-11 moiety.
  • a polymeric reagent will possess a reactive group for reaction with the IL-1 1 moiety.
  • Representative polymeric reagents and methods for conjugating these polymers to an active moiety are known in the art and further described in Zalipsky, S., et al., "Use of Functionalized Poly (Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M.
  • exemplary activating groups suitable for coupling to an IL-11 moiety include hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, hydrazine, among others.
  • the weight-average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons.
  • Exemplary ranges include weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons.
  • PEGs having a molecular weight in one or more of these ranges are preferred.
  • Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1 ,000 Daltons, about 1 ,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Dal
  • Branched versions of the water-soluble polymer e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers
  • the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Daltons.
  • PEGs When used as the polymer, PEGs will typically comprise a number of
  • (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900.
  • n the number of repeating units
  • One particularly preferred polymer for use in the invention is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower Ci -6 alkoxy group, although a hydroxyl group can also be used.
  • a relatively inert group such as a lower Ci -6 alkoxy group
  • mPEG methoxy-PEG
  • mPEG methoxy-PEG
  • free or unbound PEG is a linear polymer terminated at each end with hydroxyl groups:
  • PEG useful in one or more embodiments of the present invention is methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group.
  • mPEG The structure of mPEG is given below.
  • Multi-armed or branched PEG molecules such as those described in U.S.
  • Patent No. 5,932,462 can also be used as the PEG polymer.
  • PEG can have the structure:
  • poly a and poly b are PEG P°ty b Q
  • backbones (either the same or different), such as methoxy poly(ethylene glycol);
  • Pv is a nonreactive moiety, such as H, methyl or a PEG backbone
  • the branched PEG polymer is methoxy poly(ethylene glycol) disubstituted lysine.
  • the reactive ester functional group of the disubstituted lysine may be further modified to form a functional group suitable for reaction with the target group within the IL-1 1 moiety.
  • the PEG can comprise a forked PEG.
  • An example of a forked PEG is a forked PEG.
  • PEG is represented by the following structure:
  • X is a spacer moiety of one or more atoms and each Z is an activated terminal group linked to CH by a chain of atoms of defined length.
  • International Patent Application Publication WO 99/45964 discloses various forked PEG structures capable of use in one or more embodiments of the present invention.
  • the chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof.
  • the PEG polymer may comprise a pendant PEG molecule having reactive groups, such as carboxyl, covalently attached along the length of the PEG rather than at the end of the PEG chain.
  • the pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.
  • the polymer can also be prepared with one or more weak or degradable linkages in the polymer, including any of the above-described polymers.
  • PEG can be prepared with ester linkages in the polymer that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight:
  • hydrolytically degradable linkages useful as a degradable linkage within a polymer backbone and/or as a degradable linkage to an IL-1 1 moiety, include: carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al. (1997) Polymer Preprints 38 .
  • phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between a formate and an alcohol; amide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of another PEG chain; urethane linkages formed from reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group,
  • Such optional features of the conjugate may provide for additional control over the final desired pharmacological properties of the conjugate upon administration.
  • a large and relatively inert conjugate i.e., having one or more high molecular weight PEG chains attached thereto, for example, one or more PEG chains having a molecular weight greater than about 10,000, wherein the conjugate possesses essentially no bioactivity
  • a large and relatively inert conjugate may be administered, which is hydrolyzed to generate a bioactive conjugate possessing a portion of the original PEG chain.
  • the properties of the conjugate can be more effectively tailored to balance the bioactivity of the conjugate over time.
  • the water-soluble polymer associated with the conjugate can also be any water-soluble polymer associated with the conjugate.
  • releasable That is, the water-soluble polymer releases (either through hydrolysis, enzymatic processes, catalytic processes or otherwise), thereby resulting in the unconjugated IL-11 moiety.
  • releasable polymers detach from the IL-1 1 moiety in vivo without leaving any fragment of the water-soluble polymer.
  • releasable polymers detach from the IL-11 moiety in vivo leaving a relatively small fragment (e.g., a succinate tag) from the water-soluble polymer.
  • An exemplary cleavable polymer includes one that attaches to the IL-1 1 moiety via a carbonate linkage.
  • polymeric reagent generally refers to an entire molecule, which can comprise a water-soluble polymer segment and a functional group.
  • a conjugate of the invention comprises a water-soluble polymer covalently attached to an IL-1 1 moiety.
  • a water-soluble polymer covalently attached to an IL-1 1 moiety.
  • the conjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers individually attached to an IL-11 moiety.
  • Any given water-soluble polymer may be covalently attached to either an amino acid of the IL-1 1 moiety, or, when the IL-1 1 moiety is (for example) a glycoprotein, to a carbohydrate of the IL-1 1 moiety. Attachment to a carbohydrate may be carried out, e.g., using metabolic functionalization employing sialic acid-azide chemistry [Luchansky et al. (2004)
  • the particular linkage within the moiety having IL- 11 activity and the polymer depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular IL-1 1 moiety, the available functional groups within the IL-1 1 moiety (either for attachment to a polymer or conversion to a suitable attachment site), the presence of additional reactive functional groups within the IL-11 moiety, and the like.
  • the conjugates of the invention can be, although not necessarily, prodrugs, meaning that the linkage between the polymer and the IL-1 1 moiety is hydrolytically releasable to allow release of the parent moiety.
  • exemplary releasable linkages include carboxylate ester, phosphate ester, thiol ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.
  • linkages can be readily prepared by appropriate modification of either the IL-1 1 moiety (e.g., the carboxyl group C terminus of the protein, or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein, or a similar functionality within the carbohydrate) and/or the polymeric reagent using coupling methods commonly employed in the art.
  • the IL-1 1 moiety e.g., the carboxyl group C terminus of the protein, or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein, or a similar functionality within the carbohydrate
  • hydrolyzable linkages that are readily formed by reaction of a suitably activated polymer with a non-modified functional group contained within the moiety having IL-11 activity.
  • a hydrolytically stable linkage such as an amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide) linkage can also be employed as the linkage for coupling the IL-1 1 moiety.
  • a preferred hydrolytically stable linkage is an amide.
  • a water-soluble polymer bearing an activated ester can be reacted with an amine group on the IL-1 1 moiety to thereby result in an amide linkage.
  • the conjugates may or may not possess a measurable degree of IL-1 1 activity. That is to say, a polymer- IL-11 moiety conjugate in accordance with the invention will possesses anywhere from about 0.1% to about 100% of the bioactivity of the unmodified parent IL-1 1 moiety. In some instances, the polymer-IL-1 1 moiety conjugates may have greater than 100% bioactivity of the unmodified parent IL-1 1 moiety.
  • conjugates possessing little or no IL-11 activity contain a hydrolyzable linkage connecting the polymer to the moiety, so that regardless of the lack (or relative lack) of activity in the conjugate, the active parent molecule (or a derivative thereof) is released upon aqueous-induced cleavage of the hydrolyzable linkage.
  • Such activity may be determined using a suitable in-vivo or in-vitro model, depending upon the known activity of the particular moiety having IL-1 1 activity employed.
  • conjugates possessing a hydrolytically stable linkage that couples the moiety having IL-11 activity to the polymer will typically possess a measurable degree of bioactivity.
  • such conjugates are typically characterized as having a bioactivity satisfying one or more of the following percentages relative to that of the unconjugated IL-1 1 moiety: at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%), at least about 97%>, at least about 100%, and more than 105%> (when measured in a suitable model, such as those well known in the art).
  • conjugates having a hydrolytically stable linkage e.g., an amide linkage
  • such an IL-1 1 moiety is expected to share (at least in part) a similar amino acid sequence as the sequence provided in at least one of SEQ ID NOs: 1 through 24 and 29.
  • a similar amino acid sequence as the sequence provided in at least one of SEQ ID NOs: 1 through 24 and 29.
  • SEQ ID NOs: 1 through 24 and 29 such a reference is for convenience only and one having ordinary skill in the art will be able to readily determine the corresponding location or atom in other moieties having IL-11 activity.
  • the description provided herein for native human IL-1 1 is often applicable to fragments, deletion variants, substitution variants or addition variants of any of the foregoing.
  • IL-1 1 moiety and the water-soluble polymer.
  • amino acid sequence provided in SEQ ID NOs: 1 through 24 and 29 it is evident that there are several lysine residues, each lysine residue having an ⁇ -amino acid that may be available for conjugation.
  • the N-terminal amine of any protein can also serve as a point of attachment.
  • Conjugation of a polymeric reagent to an amino group of an IL-1 1 moiety can be accomplished by a variety of techniques.
  • an IL-1 1 moiety can be conjugated to a polymeric reagent functionalized with a succinimidyl derivative (or other activated ester group, wherein approaches similar to those described for these alternative activated ester group-containing polymeric reagents can be used).
  • the polymer bearing a succinimidyl derivative can be attached to the IL-11 moiety in an aqueous media at a pH of 7 to 9.0, although using different reaction conditions (e.g., a lower pH such as 6 to 7, or different temperatures and/or less than 15 °C) can result in the attachment of the polymer to a different location on the IL-1 1 moiety.
  • an amide linkage can be formed by reacting an amine-terminated nonpeptidic, water-soluble polymer with an IL- 11 moiety bearing an activating a carboxylic acid group.
  • (n) is an integer having a value of from 2 to 4000;
  • X is a spacer moiety
  • R 1 is an organic radical
  • IL-1 1 is a residue of an IL-11 moiety.
  • Typical of another approach useful for conjugating the IL-1 1 moiety to a polymeric reagent is use of reductive amination to conjugate a primary amine of an IL-11 moiety with a polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate, aldehyde hydrate).
  • a polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate, aldehyde hydrate).
  • the primary amine from the IL-11 moiety reacts with the carbonyl group of the aldehyde or ketone (or the corresponding hydroxyl-containing group of a hydrated aldehyde or ketone), thereby forming a Schiff base.
  • the Schiff base in turn, can then be reductively converted to a stable conjugate through use of a reducing agent such as sodium borohydride.
  • a reducing agent such as sodium borohydride.
  • Selective reactions e.g., at the N-terminus
  • exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form include those wherein the water-soluble polymer is encompassed within the following structure:
  • each (n) is independently an integer having a value of from 2 to 4000.
  • each (n) is independently an integer having a value of from 2 to 4000;
  • X is spacer moiety
  • (b) is an integer having a value 2 through 6;
  • (c) is an integer having a value 2 through 6;
  • R 2 in each occurrence, is independently H or lower alkyl
  • IL-1 1 is a residue of an IL-11 moiety.
  • each (n) is independently an integer having a value of from 2 to 4000;
  • IL-1 1 is a residue of an IL-1 1 moiety.
  • each (n) is independently an integer having a value of from 2 to 4000;
  • X when present, is a spacer moiety comprised of one or more atoms
  • (b') is zero or an integer having a value of one through ten;
  • (c) is an integer having a value of one through ten
  • R 2 in each occurrence, is independently H or an organic radical
  • R 3 in each occurrence, is independently H or an organic radical
  • IL-1 1 is a residue of an IL-1 1 moiety.
  • each (n) is independently an integer having a value of from 2 to 4000;
  • IL-1 1 is a residue of IL-11 moiety.
  • exemplary conjugates that include a releasable linkage include those in which an IL-1 1 moiety are conjugated to a polymeric reagent encompassed within the following formula:
  • POLY 1 is a first water-soluble polymer
  • POLY 2 is a second water-soluble polymer
  • X 1 is a first spacer moiety
  • X 2 is a second spacer moiety
  • H a is an ionizable hydrogen atom
  • PJ is H or an organic radical
  • R 2 is H or an organic radical
  • R el when present, is a first electron altering group
  • R e2 when present, is a second electron altering group
  • FG is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage.
  • a releasable linkage such as a carbamate linkage.
  • each of POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 , H a and (FG) is as previously defined in this paragraph, and R el is a first electron altering group; and R e2 is a second electron altering group.
  • (n) is independently an integer from 4 to 1500.
  • exemplary conjugates formed using releasable linkage-providing polymeric reagents include those of the following formulae:
  • POLY 1 is a first water-soluble polymer
  • POLY 2 is a second water-soluble polymer
  • X 1 is a first spacer moiety
  • X 2 is a second spacer moiety
  • H a is an ionizable hydrogen atom
  • R 1 is H or an organic radical
  • R 2 is H or an organic radical
  • R el when present, is a first electron altering group
  • R e2 when present, is a second electron altering group
  • Y 1 is O or S
  • Y 2 is O or S
  • IL-1 1 is a residue of an IL-11 moiety.
  • (n) is independently an integer from 4 to 1500, and (IL-1 1) is a residue of an IL-11 moiety.
  • Carboxyl groups represent another functional group that can serve as a point of attachment on the IL-1 1 moiety. Structurally, the conjugate will comprise the following:
  • IL-1 1 and the adjacent carbonyl group corresponds to the carboxyl -containing IL-1 1 moiety
  • X is a linkage, preferably a heteroatom selected from O, N(H), and S
  • POLY is a water-soluble polymer such as PEG, optionally terminating in an end-capping moiety.
  • the C(0)-X linkage results from the reaction between a polymeric derivative bearing a terminal functional group and a carboxyl-containing IL-1 1 moiety.
  • the specific linkage will depend on the type of functional group utilized. If the polymer is end-functionalized or "activated" with a hydroxyl group, the resulting linkage will be a carboxylic acid ester and X will be O. If the polymer backbone is functionalized with a thiol group, the resulting linkage will be a thioester and X will be S.
  • the C(0)X moiety, and in particular the X moiety may be relatively more complex and may include a longer linkage structure.
  • Water-soluble derivatives containing a hydrazide moiety are also useful for conjugation at a carbonyl and carboxylic acid.
  • the IL-11 moiety does not contain a carbonyl moiety or a carboxylic acid, one can be added using techniques known to one of ordinary skill in the art.
  • a carbonyl moiety can be introduced by reducing a carboxylic acid (e.g., the C-terminal carboxylic acid) and/or by providing glycosylated or glycated (wherein the added sugars have a carbonyl moiety) versions of the IL-11 moiety.
  • a PEG-hydrazine reagent can, in the presence of a coupling agent (e.g., DCC), covalently attach to the IL-1 1 moiety [e.g., mPEG-OCH 2 C(0)NHNH 2 + HOC(0)-(IL-l 1) results in mPEG-OCH 2 C(0)NHNHC(0)-IL- 11].
  • a coupling agent e.g., DCC
  • covalently attach to the IL-1 1 moiety e.g., mPEG-OCH 2 C(0)NHNH 2 + HOC(0)-(IL-l 1) results in mPEG-OCH 2 C(0)NHNHC(0)-IL- 11.
  • any water-soluble derivative containing an activated ester e.g., a succinimidyl group
  • a hydrazide moiety by reacting the water-soluble polymer derivative containing the activated ester with hydrazine (NH 2 -NH 2 ) or tert-butyl carbazate
  • the variable (n) represents the number of repeating monomeric units and "-C(0)-(IL-1 1)" represents the residue of the IL-1 1 moiety following conjugation to the polymeric reagent.
  • the hydrazone linkage can be reduced using a suitable reducing agent. While each polymeric portion [e.g., (OCH 2 CH 2 ) n or (CH 2 CH 2 0) n ] presented in Table 2 terminates in a "CH 3 " group, other groups (such as H and benzyl) can be substituted therefor.
  • Thiol groups contained within the IL-1 1 moiety can serve as effective sites of attachment for the water-soluble polymer.
  • cysteine residues provide thiol groups when the IL-11 moiety is a protein.
  • the thiol groups in such cysteine residues can then be reacted with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl polymer or other derivative as described in U.S. Patent No. 5,739,208 and in WO 01/62827.
  • a protected thiol may be incorporated into an oligosaccharide side chain of an activated glycoprotein, followed by deprotection with a thiol-reactive
  • WO 90/12874 for adding cysteine residues, wherein such procedure can be adapted for an IL-1 1 moiety.
  • conventional genetic engineering processes can also be used to introduce a cysteine residue into the IL-1 1 moiety. In some embodiments, however, it is preferred not to introduce an additional cysteine residue and/or thiol group.
  • the corresponding maleamic acid form(s) of the water-soluble polymer can also react with the IL-1 1 moiety. Under certain conditions (e.g., a pH of about 7-9 and in the presence of water), the maleimide ring will "open" to form the corresponding maleamic acid.
  • the maleamic acid in turn, can react with an amine or thiol group of an IL-1 1 moiety.
  • Exemplary maleamic acid-based reactions are schematically shown below.
  • POLY represents the water-soluble polymer
  • (IL-11) represents the IL-11 moiety.
  • a representative conjugate in accordance with the invention can have the following structure:
  • Polymeric reagents that can be reacted with an IL-1 1 moiety and result in this type of conjugate are described in U.S. Patent Application Publication No. 2005/0014903.
  • exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form will have the branched form of the
  • water-soluble polymer comprise the following structure:
  • each (n) is independently an integer having a value of from 2 to 4000;
  • IL-1 1 is a residue of IL-1 1 moiety.
  • An additional exemplary conjugate can be formed using a reagent:
  • (n) is independently an integer having a value of from 2 to 4000;
  • IL-11 is a residue of IL-11 moiety.
  • Conjugates can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • the IL-11 moiety can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • the IL-11 moiety can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • the IL-11 moiety can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • the IL-11 moiety can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • the IL-11 moiety can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard.
  • ⁇ optionally in a suitable buffer (including amine-containing buffers, if desired) ⁇ is placed in an aqueous media at a pH of about 7-8 and the thiol-selective polymeric reagent is added at a molar excess.
  • the reaction is allowed to proceed for about 0.5 to 2 hours, although reaction times of greater than 2 hours (e.g., 5 hours, 10 hours, 12 hours, and 24 hours) can be useful if PEGylation yields are determined to be relatively low.
  • Exemplary polymeric reagents that can be used in this approach are polymeric reagents bearing a reactive group selected from the group consisting of maleimide, sulfone (e.g., vinyl sulfone), and thiol (e.g., functionalized thiols such as an ortho pyridinyl or "OPSS").
  • a reactive group selected from the group consisting of maleimide, sulfone (e.g., vinyl sulfone), and thiol (e.g., functionalized thiols such as an ortho pyridinyl or "OPSS").
  • polymeric reagents those described here and elsewhere can be purchased from commercial sources or prepared from commercially available starting materials.
  • methods for preparing the polymeric reagents are described in the literature.
  • the attachment between the IL-1 1 moiety and the non-peptidic, water-soluble polymer can be direct, wherein no intervening atoms are located between the IL-11 moiety and the polymer, or indirect, wherein one or more atoms are located between the IL-11 moiety and the polymer.
  • a "spacer moiety" serves as a linker between the residue of the IL-11 moiety and the water-soluble polymer.
  • the one or more atoms making up the spacer moiety can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • the spacer moiety can comprise an amide, secondary amine, carbamate, thioether, and/or disulfide group.
  • specific spacer moieties include those selected from the group consisting of -0-, -S-, -S-S-, -C(O)-, -C(0)-NH-, -NH-C(0)-NH-, -0-C(0)-NH-, -C(S)-, -C3 ⁇ 4-, -CH 2 -CH 2 -,
  • R 6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is zero to six, and (j) is zero to 20.
  • R 6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is zero to six, and (j) is zero to 20.
  • Other specific spacer moieties have the following structures:
  • any of the above spacer moieties may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., - (CH 2 CH 2 0)i -2 o].
  • the ethylene oxide oligomer chain can occur before or after the spacer moiety, and optionally in between any two atoms of a spacer moiety comprised of two or more atoms. Also, the oligomer chain would not be considered part of the spacer moiety if the oligomer is adjacent to a polymer segment and merely represent an extension of the polymer segment. For clarity, amino acids belonging to the IL-1 1 moiety are not considered part of the spacer moiety.
  • the conjugates are typically part of a composition.
  • the composition comprises a plurality of conjugates, preferably although not necessarily, each conjugate is comprised of the same IL-11 moiety (i.e., within the entire composition, only one type of IL-1 1 moiety is found).
  • the composition can comprise a plurality of conjugates wherein any given conjugate is comprised of a moiety selected from the group consisting of two or more different IL-1 1 moieties (i.e., within the entire composition, two or more different IL-1 1 moieties are found).
  • substantially all conjugates in the composition are each comprised of the same IL-1 1 moiety.
  • composition can comprise a single conjugate species (e.g., a
  • compositions can also comprise other conjugates having four, five, six, seven, eight or more polymers attached to any given moiety having IL-11 activity.
  • compositions comprises a plurality of conjugates, each conjugate comprising one water-soluble polymer covalently attached to one IL-1 1 moiety, as well as compositions comprising two, three, four, five, six, seven, eight, or more
  • water-soluble polymers covalently attached to one IL-1 1 moiety.
  • the composition will satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have from one to four polymers attached to the IL-11 moiety; at least about 85% of the conjugates in the composition will have from one to three polymers attached to the IL-1 1 moiety; at least about 85% of the conjugates in the composition will have from one to two polymers attached to the IL-1 1 moiety; at least about 85% of the conjugates in the composition will have one polymer attached to the IL-1 1 moiety; at least about 95% of the conjugates in the composition will have from one to five polymers attached to the IL-11 moiety; at least about 95% of the conjugates in the composition will have from one to four polymers attached to the IL-11 moiety; at least about 95% of the conjugates in the composition will have from one to three polymers attached to the IL-1 1 moiety; at least about 95% of the conjugates in the composition will have from one to two polymers attached to the IL
  • the conjugate-containing composition is free or substantially free of albumin. It is also preferred that the composition is free or substantially free of proteins that do not have IL-1 1 activity. Thus, it is preferred that the composition is 85% > , more preferably 95%, and most preferably 99% free of albumin. Additionally, it is preferred that the composition is 85%, more preferably 95%, and most preferably 99% free of any protein that does not have IL-11 activity. To the extent that albumin is present in the composition, exemplary compositions of the invention are substantially free of conjugates comprising a poly(ethylene glycol) polymer linking a residue of an IL-1 1 moiety to albumin.
  • Control of the desired number of polymers for any given moiety can be achieved by selecting the proper polymeric reagent, the ratio of polymeric reagent to the IL-1 1 moiety, temperature, pH conditions, and other aspects of the conjugation reaction.
  • reduction or elimination of the undesired conjugates e.g., those conjugates having four or more attachedpolymers
  • the polymer- IL-11 moiety conjugates can be purified to obtain/isolate different conjugated species.
  • the product mixture can be purified to obtain an average of anywhere from one, two, three, four, five or more PEGs per IL-11 moiety, typically one, two or three PEGs per IL-11 moiety.
  • the strategy for purification of the final conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular IL-11 moiety, the desired dosing regimen, and the residual activity and in vivo properties of the individual conjugate(s).
  • conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography. That is to say, gel filtration chromatography is used to fractionate differently numbered polymer-to-IL-1 1 moiety ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1 polymer to IL-1 1 moiety, “2-mer” indicates two polymers to IL-11 moiety, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble polymer portion).
  • gel filtration chromatography is used to fractionate differently numbered polymer-to-IL-1 1 moiety ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1 polymer to IL-1 1 moiety, "2-mer” indicates two polymers to IL-11 moiety, and so on) on the basis of their differing molecular weights (where the
  • the resulting reaction mixture may contain unmodified protein (having a molecular weight of about 35,000 Daltons), monoPEGylated protein (having a molecular weight of about 55,000 Daltons), diPEGylated protein (having a molecular weight of about 75,000 Daltons), and so forth.
  • compositions are preferably substantially free of proteins that do not have a reverse phase-high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a CI 8 column or C3 column, available commercially from companies such as Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a SepharoseTM ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-active agent isomers having the same molecular weight (i.e., positional isoforms).
  • RP-HPLC reverse phase-high performance liquid chromatography
  • compositions preferably are substantially free of all other noncovalently attached water-soluble polymers. In some circumstances, however, the composition can contain a mixture of polymer- IL-1 1 moiety conjugates and unconjugated IL- 1 1 moiety.
  • composition of the invention further comprises a
  • the pharmaceutically acceptable excipient can be added to a conjugate to form a composition.
  • Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient.
  • Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose,
  • maltodextrins dextrans, starches, and the like
  • alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and the like.
  • the excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
  • an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
  • the composition can also include an antimicrobial agent for preventing or deterring microbial growth.
  • antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
  • An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
  • a surfactant can be present as an excipient.
  • exemplary surfactants include: polysorbates, such as “Tween 20” and “Tween 80,” and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and IL-1 Hating agents, such as EDTA, zinc and other such suitable cations.
  • Acids or bases can be present as an excipient in the composition.
  • acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.
  • Suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
  • amino acids can be present as an excipient in the compositions described herein.
  • exemplary amino acids in this regard include arginine, lysine and glycine.
  • the amount of the conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial).
  • a unit dose container e.g., a vial
  • the pharmaceutical preparation can be housed in a syringe.
  • a therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.
  • the amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine
  • compositions containing vaiying amounts of the excipient ranging from low to high
  • stability examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.
  • the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
  • compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • compositions of one or more embodiments of the present invention are typically, although not necessarily, administered via injection and are therefore generally liquid solutions or suspensions immediately prior to administration.
  • the pharmaceutical preparation can also take other forms such as syrups, creams, ointments, tablets, powders, and the like.
  • Other modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, intratumorally, peritumorally, intraperitonally, subcutaneous, intra-arterial, and so forth.
  • the invention also provides a method for administering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with conjugate.
  • the method comprises administering to a patient, generally via injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition).
  • the conjugates can be injected (e.g., intramuscularly, subcutaneously and parenterally).
  • suitable formulation types for parenteral administration include ready-for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.
  • the method of administering the conjugate can optionally be conducted so as to localize the conjugate to a specific area.
  • the liquid, gel and solid formulations comprising the conjugate could be surgically implanted in a diseased area (such as in a tumor, near a tumor, in an inflamed area, and near an inflamed area).
  • a diseased area such as in a tumor, near a tumor, in an inflamed area, and near an inflamed area.
  • organs and tissue can also be imaged in order to ensure the desired location is better exposed to the conjugate.
  • the method of administering may be used to treat any condition that can be remedied or prevented by administration of the conjugate.
  • Those of ordinary skill in the art appreciate which conditions a specific conjugate can effectively treat.
  • the conjugates can be used either alone or in combination with other pharmacotherapy to treat patients suffering from a condition.
  • Exemplary conditions include, without limitation:
  • TH2-mediated disorders e.g., asthma, congestive obstruction pulmonary disorder, rhinitis, allergies, and atopic dermatitis
  • the conjugate can be administered to the patient prior to, simultaneously with, or after administration of another active agent.
  • the actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered.
  • Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature. Generally, a therapeutically effective amount will range from about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10 mg day to 50 mg/day.
  • a given dose can be periodically administered up until, for example, symptoms of the condition being treated lessen and/or are eliminated entirely.
  • the unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • the specific dosing schedule will be known by those of ordinary skill in the art or can be determined
  • Exemplary dosing schedules include, without limitation, administration once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
  • Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared samples were loaded onto a NuPAGE Novex precast gel and run for approximately thirty minutes.
  • Agilent 1 100 HPLC system (Agilent). Samples were analyzed using a Shodex protein W- 804 column (300 x 8 mm, Phenomenex), and a mobile phase consisting of sodium phosphate and sodium sulfate, pH 7. The flow rate for the column was 0.5 ml/min. Eluted protein and PEG-protein conjugates were detected using UV at 280nm and 220nm.
  • RP-HPLC Reversed phase high-performance liquid chromatography
  • Agilent 1 100 HPLC system Agilent 1 100 HPLC system
  • Samples were analyzed using a Zorbax 300SB-C3 column (3.5 ⁇ particle size, 150 mm x 3.0 mm, Agilent), and mobile phases consisting of 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B).
  • the flow rate for the column was 0.3 ml/min.
  • the protein and PEG-protein conjugates were Eluted with a linear gradient over 20 minutes, and were detected using UV at 280nm.
  • mPEG2-NHS Branched mPEG-N-Hydroxysuccinimide Derivative, 40kDa,
  • mPEG2-NHS (40kDa) stored at -20 °C under argon, was warmed to ambient temperature. A five-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-11 solution) of the warmed mPEG2-NHS (40kDa) was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (2.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well.
  • the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 hours first, and then at 4°C overnight. The reaction was quenched with acetic acid to lower the pH to 4.0.
  • the conjugate solution was characterized by SDS-PAGE and RP-HPLC.
  • Figure 1 shows the chromatogram following the RP-HPLC analysis of the conjugate solution.
  • the PEGylation reaction yielded 64% 1-mer (mono-conjugate or one PEG attached to each IL-11 molecule) and 21 % 2-mer (di-conjugate or two PEGs attached to each IL-1 1 molecule).
  • a cation-exchange chromatography method using SP Sepharose High Performance column along with sodium acetate and sodium phosphate buffers was also used to purify the conjugates.
  • mPEG2-NHS Branched mPEG-N-Hydroxysuccinimide Derivative, 20kDa,
  • mPEG2-NHS (20kDa) stored at -20°C under argon, was warmed to ambient temperature. A three-fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-11 solution) of the warmed mPEG2-NHS (20kDa) was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.5 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 to 3 hours. The reaction was quenched with acetic acid to lower the pH to 4.0.
  • the conjugate solution was characterized by SDS-PAGE and HPLC. A cation-exchange chromatography method using SP Sepharose High Performance column along with sodium phosphate buffers was also used to purify the conjugates.
  • mPEG-ButyrALD Linear mPEG-Butyraldehyde Derivative, 20kDa
  • mP EG-Butyr ALD 20kDa, stored at -20 °C under argon, was warmed to ambient temperature.
  • An eighteen- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1) of the warmed mPEG-ButryALD was dissolved in Milli-Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (1.5 mg/ml in sodium acetate and sodium phosphate buffer, pH 5.8) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for thirty minutes.
  • a reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH 3 .
  • the reaction solution was well mixed and was kept at 4°C overnight.
  • the aldehyde group of mPEG-ButyrALD can react with the primary amines associated with IL-11 and covalently bond to them via secondary amine upon reduction by sodium cyanoborohydride.
  • Figure 2 shows the SEC-HPLC chromatogram of the conjugate solution. The PEGylation reaction yielded 40% 1 -mer (one PEG attached to IL-11 or mono- conjugate).
  • mPEG2-ru-ButyrALD 40kDa
  • mPEG2-ru-ButyrALD 40kDa
  • Millis H 2 0 a 10% reagent solution
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (1.9 mg/ml in sodium acetate buffer, pH 5.3) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for ten minutes.
  • a reducing agent, sodium cyanoborohydride was then added to make 10 raM NaCNBH 3 .
  • the reaction solution was well mixed and was kept at 4°C overnight.
  • the aldehyde group of mPEG2-ButyrALD (40kDa) can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. Because the PEGylation reaction was carried at pH 5, attachment of the PEG derivative to IL-1 1 was more selective to the N-terminal.
  • Figure 3 shows the SEC-HPLC chromatogram of the conjugate solution. The PEGylation reaction yielded 72% mono-conjugate, 21% di-conjugate and 1% higher species. A cation-exchange chromatography method using SP Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugates.
  • Figure 4 shows the separation profile of a reaction mixture.
  • mPEG2-ru-ButyrALD 20kDa, stored at -20 °C under argon, was warmed to ambient temperature.
  • a twenty-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1) of the warmed mPEG2-ButryALD (20kDa) was dissolved in Milli - Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.4 mg/ml in 50mM sodium phosphate buffer, lOOmM sodium chloride, 5% sorbital, pH 6) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for 15 minutes.
  • a reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH 3 .
  • the reaction solution was well mixed and was kept at room temperature for 15minutes, and then at 4°C overnight.
  • the conjugate solution was characterized by SDS-PAGE and HPLC.
  • the aldehyde group of mPEG2-ButyrALD (20kDa) can react with the primary amines associated with IL-11 and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. Because the PEGylation reaction was carried at pH 5, attachment of the PEG derivative to IL-1 1 was more selective to the N-terminal. A cation-exchange chromatography method using SP Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugates.
  • RotoMix Slow Speed Lab Rotator
  • the reaction was quenched by the addition of IM acetic acid to lower the pH to 5.
  • the conjugate solution was characterized by SDS-PAGE ( Figure 5). Based on the gel result, the reaction yielded 50% mono-conjugate and 36% di-conjugate.
  • the PEGs Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
  • PEG2-FMOC-NHS having other weight average molecular weights.
  • a three- fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed C2-PEG2-FMOC-NHS(40kDa) was dissolved in 2mM HC1 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-11 solution (3.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well.
  • the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for two hours.
  • the reaction was quenched by the addition of IM acetic acid to lower the pH to 5.
  • the conjugate solution was characterized by SEC-HPLC ( Figure 6).
  • the PEGylation reaction yielded 95% mono-conjugate.
  • a cation-exchange chromatography method using SP Sepharose High Performance column along with sodium acetate and sodium phosphate buffers was also used to purify the conjugates.
  • the PEGs Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-11 conjugates under physiological condition.
  • CG-PEG2-FMOC-NHS 40kDa, stored at -20 °C under argon, was warmed to ambient temperature.
  • a three- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-11 solution) of the warmed CG-PEG2-FMOC-NHS was dissolved in 2mM HC1 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (3.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well.
  • the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 hr first, and then at 4°C overnight.
  • the reaction was quenched by the addition of 1M acetic acid to lower the pH to 5.
  • the conjugate solution was characterized by SDS-PAGE ( Figure 5). Based on the gel result, the reaction yielded 55% mono-conjugate and 30% di-conjugate.
  • the PEGs Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
  • mPEG-HP-ALD 20kDa, stored at -20°C under argon, was warmed to ambient temperature.
  • a twenty to thirty- fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-HP-ALD was dissolved in Milli-Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for thirty minutes.
  • the pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques.
  • the reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C.
  • the reaction was quenched with 0.1M HCl to pH 6.
  • the conjugate solution was characterized by HPLC and SDS-
  • the aldehyde group of mPEG-HP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent.
  • Figure 7 shows the SEC-HPLC chromatogram of a conjugate solution. The PEGylation reaction yielded 29% 1 -mer (mono-conjugate, or one PEG attached to one IL-1 1 molecule).
  • ALD having other weight average molecular weights.
  • mPEG-MAHP-ALD 20kDa, stored at -20°C under argon, was warmed to ambient temperature.
  • a five to ten-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-MAHP-ALD was dissolved in Milli-Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.5 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for thirty minutes.
  • the pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques.
  • the reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C.
  • the reaction was quenched with 0.1M HC1 to pH 6.
  • the conjugate solution was characterized by HPLC
  • the aldehyde group of mPEG-MAHP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent.
  • Figure 8 shows the SEC-HPLC chromatogram of a conjugate solution.
  • MAHP-ALD having other weight average molecular weights.
  • mPEG-PipHP-ALD 20kDa, stored at -20°C under argon, was warmed to ambient temperature.
  • a twenty- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-PipHP-ALD was dissolved in Milli-Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the reaction solution was kept at room temperature on a RotoMix for thirty minutes.
  • the pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques.
  • the reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C.
  • the reaction was quenched with 0.1M HC1 to pH6.
  • the conjugate solution was characterized by
  • the aldehyde group of mPEG-PipHP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent.
  • Figure 9 shows the SEC-HPLC chromatogram of a conjugate solution. The PEGylation reaction yielded 49% 1-mer (mono-conjugate, or one PEG attached to one IL-11 molecule).
  • PipHP-ALD having other weight average molecular weights.
  • mPEG-ALD Linear mPEG-Propionaldehyde Derivative, 20kDa
  • a ten to twenty- fold excess (relative to the amount of IL-1 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-ALD was dissolved in MiUi-Q H 2 0 to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-11 solution (4.4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the pH of the solution was adjusted to pH 9 using conventional techniques.
  • the reaction solution was kept at room temperature on a RotoMix for thirty minutes.
  • a reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH 3 .
  • the reaction solution was well mixed and was kept at room temperature or 4°C overnight.
  • the aldehyde group of mPEG-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine upon reduction by sodium cyanoborohydride.
  • Figure 10 shows the SEC-HP LC chromatogram of the conjugate solution. The PEGylation reaction yielded 49% 1-mer (one PEG attached to IL-1 1 or mono- conjugate).
  • a four- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1 solution) of the warmed C2-PEG2-FMOC-NHS(20kDa) was dissolved in 2mM HCl to form a 10% reagent solution.
  • the 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (5.2 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well.
  • the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for one hour.
  • the reaction was quenched by the addition of 0.1 M hydrochloric acid to lower the pH to 6.
  • the conjugate solution was characterized by SEC-HPLC.
  • the PEGylation reaction yielded 60-70% mono-conjugate.
  • a cation-exchange chromatography method using CM Sepharose High Performance column along with sodium phosphate buffers was also used to purify the conjugates (Figure 1 1).
  • the PEGs Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
  • mPEG-SMB mPEG-Succinimidyl a-Methylbutanoate Derivative, 30kDa
  • PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rIL-11 concentration is 2.5 mg/ml.
  • a quantity of the PEG reagent equal to 10 - 50 mol equivalents of the rIL-11 to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution.
  • the 12% PEG reagent solution is added to the aliquot of stock rIL-11 solution and stirred for 5 - 18 hours at room temperature thereby resulting in a conjugate solution.
  • the conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration.
  • the mPEG-SMB derivative is found to provide a sterically hindered active
  • NHS ester which selectively reacts with lysine and terminal amines.
  • the basic structure of the polymeric reagent is provided below:
  • PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rIL-1 1 concentration is 2.5 mg/ml.
  • a quantity of the PEG reagent equal to 10 - 50 mol equivalents of the rIL-1 1 to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution.
  • the 12% PEG reagent solution is added to the aliquot of stock rIL-11 solution and stirred for 15 - 30 minutes.
  • a reducing agent sodium cyanoborohydride (NaCNBH )
  • NaCNBH sodium cyanoborohydride
  • the conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration.
  • ketone group of mPEG-PIP is found to react with the primary amines associated with rIL-1 1 and covalently bond to them via a secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride.
  • PEG-IL-11 conjugates were tested for IL-1 1 activity in a bioassay. Briefly, 3 to 4 days prior to assay, enhanced IL-1 1 responsive cells (prepared by obtaining commercially available IL-1 1 -sensitive cells from R&D Systems, Minneapolis MN and subcloning these cells to obtain a more highly IL-1 1 responsive cell line) were seeded at 5xl0 4 cells/mL in complete growth medium (RPMI-1640 supplemented with 10% heat-inactivated FBS and 50 ⁇ 2-mercaptoethanol) containing 10 ng/mL IL-1 1 (R&D systems).
  • complete growth medium RPMI-1640 supplemented with 10% heat-inactivated FBS and 50 ⁇ 2-mercaptoethanol
  • EC50 values were calculated by non-linear regression curve fitting (GraphPad). To simplify comparisons between conjugates, all concentrations refer to the mass of the IL-11 moiety, rather than the entire conjugate. Calculated EC50S for stable conjugates are shown in Table 4, below, and representative dose response curves in Figure 12.
  • mice Four hours later, the mice were injected i.p. with 2 mg/kg of E. coli LPS (Sigma) or vehicle (PBS). Two hours later, blood was collected by cardiac puncture and serum separated by centrifugation and stored at -80° C until ready for analysis by ELISA. LPS-induced plasma TNFa was measured using a mouse TNFa-specific ELISA (Pierce ThermoScientific) according to the manufacturer's instructions.
  • the IL-1 1 and PEGylated-IL- 11 conjugates suppressed production of murine TNFa in response to LPS. Though not statistically significant, the data suggests PEGylated-IL- 1 1 conjugates may be more effective than IL-1 1 itself in this inflammation model. There was little apparent difference between the mono conjugate of Example 1 1 in its "native" state or following forced-release of IL-1 1 from the conjugate prior to administration. This is consistent with the rapid release of this conjugate under

Abstract

Conjugates of an IL-1 1 moiety and one or more nonpeptidic, water-soluble polymers are provided. Typically, the nonpeptidic, water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, and methods of administering compositions to an individual.

Description

CONJUGATES OF AN IL-11 MOIETY AND A POLYMER
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35 U.S.C. §1 19(e) to U.S.
Provisional Patent Application No. 61/515,1 13 filed August 4, 201 1 , the disclosure of which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] Among other things, one or more embodiments of the present invention relate generally to the field of conjugates comprising an IL-1 1 moiety (i.e., a moiety having at least some activity similar to human IL-1 1) and a water-soluble, non-peptidic polymer. In addition, the invention relates to (among other things) the fields of compositions comprising conjugates, methods for synthesizing conjugates, and methods of administering a
composition.
BACKGROUND OF THE INVENTION
[0003] Interleukin 1 1 ("IL-1 1 ," sometimes referred to as adipogenesis inhibitory factor) is a cytokine that interacts with a variety of hematopoietic and non-hematopoietic cell types. For example, IL-11 has been shown to increase the number of platelets in blood.
Gordon et al. (1996) Blood 87:3615-3624. In addition, it has been suggested that IL-11 acts to promote epithelial growth of damaged epithelial tissues located in the gastrointestinal tract. See Schwertschlag et al. (1999) Leukemia 13: 1307-1315. Also, IL-1 1 has been shown to have immunomodulatory effects on both macrophages and T cells, which result in reduced inflammatory responses. Trepicchio et al. (1999) J. Clin. Invest. 104(1 1): 1527-1537. Thus, IL-1 1 exhibits a myriad of effects, some of which have been proposed as being clinically useful.
[0004] Oprelvekin is a form of human recombinant IL-11 ("rhIL-11 ") marketed under the NEUMEGA brand (Wyeth Pharmaceuticals Inc., Philadelphia PA) and is indicated for the prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in adult patients with nonmyeloid malignancies who are at high risk of severe thrombocytopenia. Following administration, rhIL-11 is rapidly cleared. Takagi et al. (1995) J. Pharmacol. Exp. Ther. 275:537-543. As a result, administration of oprelvekin typically occurs once daily via a single subcutaneous injection in the abdomen, thigh, hip or upper arm. Daily injections are often inconvenient and potentially painful, which is suboptimal.
[0005] Attempts at improving the pharmacokinetic profile have been proposed. For example, certain conjugates of IL-11 have been suggested. See, for example, Takagi et al. (2007) J. Control. Release 119:271-278, WO 2010/024557 and U.S. Patent Application Publication No. 2009/0281281.
[0006] Notwithstanding these conjugates, however, there remains a need for conjugates of IL-1 1 having improved characteristics and profiles. Among other things, one or more embodiments of the present invention is therefore directed to such conjugates as well as compositions comprising the conjugates and related methods as described herein, which are believed to be new and completely unsuggested by the art.
SUMMARY OF THE INVENTION
[0007] Accordingly, in one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-11 moiety covalently attached to a water-soluble polymer.
[0008] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein the residue of the IL-11 moiety is covalently attached to the water-soluble polymer via a releasable linkage.
[0009] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-11 moiety covalently attached to a water-soluble polymer, wherein the IL-11 moiety is free of cysteine residues. [0010] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-1 1 moiety covalently attached to a branched water-soluble polymer, wherein the branched water-soluble polymer lacks a lysine residue,
[0011] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein an amine of the IL-11 moiety is covalently attached to the water-soluble polymer via a linkage other than an amide linkage.
[0012] In one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein an amine of the IL-11 moiety is covalently attached to the water-soluble
Dolvmer vi a a" arnine linknpe
[0013] In one or more embodiments of the invention, a composition is provided, the composition comprising a conjugate as described herein along with a pharmaceutically acceptable excipient.
[0014] In one or more embodiments of the invention, a method for delivering a conjugate is provided, the method comprising the step of subcutaneously administering to the patient a composition comprised of a conjugate of a residue of an IL-1 1 moiety and a water-soluble polymer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 is a representation of a chromatogram following reverse phase HPLC analysis of [mPEG2-ru-NHS, 40kDa]-[rIL-l 1] conjugate soution, as further described in Example 1.
[0016] Figure 2 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-ButyrALD, 20kDa]-[rIL-l 1] conjugate solution, as further described in Example 2.
[0017] Figure 3 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG2-ru-ButyrALD, 40kDa]-[rIL-l 1] conjugate solution, as further described in Example 3. [0018] Figure 4 is a representation of a typical chromatogram following cation exchange chromatography of [mPEG-ButyrALD, 40kDa]-[rIL-l l] conjugates, as further described in Example 3.
[0019] Figure 5 is a representation of the results following SDS-PAGE analysis of the reaction mixture prepared in accordance with the procedures set forth in Example 4 and Example 6.
[0020] Figure 6 is a representation of a chromatogram following SEC-HPLC analysis of [C2-PEG2-FMOC-NHS, 40kDa]-[rIL-l 1] conjugate solution, as further described in Example 5.
[0021] Figure 7 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-HP-ALD]-[rIL-l 1] conjugate solution, as further described in Example 7.
[0022] Figure 8 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-MAHP-ALD]-[rIL-l 1 ] conjugate solution, as further described in Example 8,
[0023] Figure 9 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-PipHP-ALD]-[rIL-l 1] conjugate solution, as further described in Example 9.
[0024] Figure 10 is a representation of a chromatogram following SEC-HPLC analysis of [mPEG-ALD20K]-[rIL-l l] conjugate solution, as further described in Example 10.
[0025] Figure 11 is a representation of a typical chromatogram following cation exchange chromatography of [C2-PEG2-FMOC-20 ]-[rIL-l 1] conjugates, as further described in Example 11.
[0026] Figure 12 depicts non-linear regression curves of stable conjugates tested in the IL-1 1 bioassay as further described in Example 14.
[0027] Figure 13 depicts non-linear regression curves of releasable conjugates tested in the IL-1 1 bioassay as further described in Example 14.
[0028] Figure 14 is a plot of the TNFoc production in response to acute LPS challenge in mice pretreated with a test article, as further described in Example 5. DETAILED DESCRIPTION OF THE INVENTION
[0029] Before describing one or more embodiments of the present invention in detail, it is to be understood that this invention is not limited to the particular polymers, synthetic techniques, IL-11 moieties, and the like, as such may vary.
[0030] It must be noted that, as used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers, reference to "an optional excipient" refers to a single optional excipient as well as two or more of the same or different optional excipients, and the like.
[0031] In describing and claiming one or more embodiments of the present invention, the following terminology will be used in accordance with the definitions described below.
[0032] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as used herein, are interchangeable and encompass any nonpeptidic, water-soluble poly(ethylene oxide).
Typically, PEGs for use in accordance with the invention comprise the following structure "-(OCH2CH2)n-M where (n) is 2 to 4000. As used herein, PEG also includes
"-CH2CH2-0(CH2CH20)n-CH2CH2-" and M-(OCH2CH2)nO-," depending upon whether or not the terminal oxygens have been displaced, e.g., during a synthetic transformation.
Throughout the specification and claims, it should be remembered that the term "PEG" includes structures having various terminal or "end capping" groups and so forth. The term "PEG" also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH2CH2- repeating subunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as "branched," "linear," "forked," "multifunctional," and the like, to be described in greater detail below.
[0033] The terms "end-capped" and "terminally capped" are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.
Typically, although not necessarily, the end-capping moiety comprises a hydroxy or Ci_2o alkoxy group, more preferably a Cj.io alkoxy group, and still more preferably a Ci-5 alkoxy group. Thus, examples of end-capping moieties include alkoxy (e.g. , methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. It must be remembered that the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH30(CH2CH20)n- and CH3(OCH2CH2)n-]. In addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned. Moreover, the end-capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, and the like. Suitable detectors include photometers, films,
spectrometers, and the like. The end-capping group can also advantageously comprise a phospholipid. When the polymer has an end-capping group comprising a phospholipid, unique properties are imparted to the polymer and the resulting conjugate. Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines. Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine,
dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin. The end-capping group may also include a targeting moiety, such that the polymer - as well as anything, e.g., an IL-1 1 moiety, attached thereto ~ can preferentially localize in an area of interest.
[0034] "Non-naturally occurring" with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature. A non-naturally occurring polymer may, however, contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.
[0035] The term "water soluble" as in a "water-soluble polymer" is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35%) (by weight) soluble in water, more preferably at least about 50%> (by weight) soluble in water, still more preferably about 70%> (by weight) soluble in water, and still more preferably about 85%) (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water. [0036] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight average molecular weight. The polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.
[0037] The terms "active," "reactive" or "activated" when used in conjunction with a particular functional group, refer to a functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive" or "inert" group).
[0038] As used herein, the term "functional group" or any synonym thereof is meant to encompass protected forms thereof as well as unprotected forms.
[0039] The terms "spacer moiety," "linkage" and "linker" are used herein to refer to a bond or an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymeric reagent and an IL-1 1 moiety or an electrophile or nucleophile of an IL-11 moiety. The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of an IL-1 1 moiety and a water-soluble polymer can be attached directly or indirectly through a spacer moiety). [0040] "Alkyl" refers to a hydrocarbon chain, typically ranging from about 1 to 15 atoms in length. Such hydrocarbon chains are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 3-methylpentyl, and the like.
[0041] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, i'-butyl, and /-butyl.
[0042] "Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms. "Cycloalkylene" refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.
[0043] "Alkoxy" refers to an -OR group, wherein R is alkyl or substituted alkyl, preferably C1-6 alkyl (e.g., methoxy, ethoxy, propyloxy, and so forth).
[0044] The term "substituted" as in, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl, C3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like. "Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).
[0045] "Noninterfering substituents" are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.
[0046] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms.
Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.
[0047] "Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings. [0048] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.
[0049] "Substituted heteroaryl" is heteroaryl having one or more noninterfering groups as substituents.
[0050] "Substituted heterocycle" is a heterocycle having one or more side chains formed from noninterfering substituents.
[0051] An "organic radical" as used herein shall include akyl, substituted alkyl, aryl, and substituted aryl.
[0052] "Electrophile" and "electrophilic group" refer to an ion or atom or collection of atoms, which may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.
[0053] "Nucleophile" and "nucleophilic group" refers to an ion or atom or collection of atoms, which may be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center or an electrophile.
[0054] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. A "releasable linkage" is a covalent linkage that cleaves under physiological conditions at a rate that is clinically useful and includes, for example and without limitation, hydrolyzable bonds.
[0055] An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.
[0056] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, which is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1-2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
[0057] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
[0058] "Pharmacologically effective amount," "physiologically effective amount," and
"therapeutically effective amount" are used interchangeably herein to mean the amount of a polymer-(lL-l 1) moiety conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated IL-11 moiety) in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g., the particular IL-1 1 moiety, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one of ordinary skill in the art, based upon the information provided herein.
[0059] "Multi-functional" means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different. Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, and can contain, for example, a number satisfying one or more of the following ranges: from 3-50 functional groups; from 3-25 functional groups; from 3-15 functional groups; from 3 to 10 functional groups. For example, the number of functional groups can be selected from the group consisting of 3, 4, 5, 6, 7, 8, 9 and 10 functional groups within the polymer.
[0060] The term "IL-1 1 moiety," as used herein, refers to a moiety having human
IL-1 1 activity. The IL-1 1 moiety will also have at least one electrophilic group or
nucleophilic group suitable for reaction with a polymeric reagent. In addition, the term "IL-11 moiety" encompasses both the IL-11 moiety prior to conjugation as well as the IL-1 1 moiety residue following conjugation. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has IL-11 activity. Proteins comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1 through 24 is an IL-1 1 moiety, as well as any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-1 1 moiety" includes such proteins modified deliberately, as for example, by site directed mutagenesis or accidentally through mutations. These terms also include analogs having from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy terminal end of the protein wherein the additional amino acid(s) includes at least one glycosylation site, and analogs having an amino acid sequence which includes at least one glycosylation site. The term includes both natural and
recombinantly produced moieties.
[0061] The term "substantially homologous" means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more
substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. For purposes of the present invention, sequences having greater than 95 percent homology, equivalent biological activity (although not necessarily equivalent strength of biological activity), and equivalent expression characteristics are considered substantially homologous. For purposes of determining homology, truncation of the mature sequence should be disregarded. Exemplary IL-1 1 moieties for use herein include those sequences that are substantially homologous SEQ ID NO: 2.
[0062] The term "fragment" means any protein or polypeptide having the amino acid sequence of a portion or fragment of an IL-1 1 moiety, and which has the biological activity of IL-1 1. Fragments include proteins or polypeptides produced by proteolytic degradation of an IL-1 1 moiety as well as proteins or polypeptides produced by chemical synthesis by methods routine in the art.
[0063] The term "patient," refers to a living organism suffering from or prone to a condition that can be prevented or treated by administration of an active agent (e.g., conjugate), and includes both humans and animals.
[0064] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.
[0065] "Substantially" means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80% or greater, 90% or greater, and 95% or greater of the condition. [0066] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is
Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;
Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or ; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is Gly or G.
[0067] Turning to one or more embodiments of the invention, a conjugate is provided, the conjugate comprising a residue of an IL-11 moiety covalently attached (either directly or through a spacer moiety) to a water-soluble polymer. The conjugates of the invention will have one or more of the following features.
[0068] The IL-11 Moiety
[0069] As previously stated, the conjugate generically comprises a residue of an IL-1 1 moiety covalently attached, either directly or through a spacer moiety, to a water-soluble polymer. As used herein, the term "IL-1 1 moiety" shall refer to the IL-11 moiety prior to conjugation as well as to the IL-1 1 moiety following attachment to a nonpeptidic,
water-soluble polymer. It will be understood, however, that when the original IL-1 1 moiety is attached to a nonpeptidic, water-soluble polymer, the IL-11 moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer(s). Often, this slightly altered form of the IL-11 moiety attached to another molecule is referred to a "residue" of the IL-1 1 moiety.
[0070] The IL-1 1 moiety can be derived from non-recombinant methods and from recombinant methods and the invention is not limited in this regard. In addition, the IL-1 1 moiety can be derived from human sources, animal sources, and plant sources.
[0071] The IL-1 1 moiety can be derived non-recombinantly. For example, it is possible to isolate IL-11 from biological systems and otherwise obtain IL-1 1 from cultured media.
[0072] The IL-1 1 moiety can be derived from recombinant methods. See, for example, U.S. Patent No. 5,371 , 193, U.S. Patent Application Publication No. 2009/0191 148 and Paul et al. (1990) Proc. Natl. Acad. Sci. USA 87:7512-7516.
[0073] The IL-1 1 moiety can be expressed in bacterial [e.g., E. coli, see, for example,
Fischer et al. (1995) Biotechnol. Appl. Biotechnol. 21_(3):295-31 1], mammalian [see, for example, Kronman et al. (1 92) Gene 121:295-304], yeast [e.g., Pichia pastoris, see, for example, Morel et al. (1997) Biochem. J. 328(1): 121-129], and plant [see, for example, Mor et al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression.
[0074] Although recombinant-based methods for preparing proteins can differ, recombinant methods typically involve constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria, yeast, transgenic animal cell, or mammalian cell such as a Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art.
[0075] To facilitate identification and purification of the recombinant polypeptide, nucleic acid sequences that encode for an epitope tag or other affinity binding sequence can be inserted or added in- frame with the coding sequence, thereby producing a fusion protein comprised of the desired polypeptide and a polypeptide suited for binding. Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein. The recombinant polypeptide can also be purified by lysing the host cells, separating the polypeptide, e.g., by ion-exchange chromatography, affinity binding approaches, hydrophobic interaction approaches, and thereafter identify by MALDI or western blot, and collecting the polypeptide. These and other methods for identifying and purifying recombinant polypeptides are known to those of ordinary skill in the art. In one or more embodiments of the invention, however, the IL-11 moiety is not in the form of a fusion protein.
[0076] Depending on the system used to express proteins having IL-1 1 activity, the
IL-1 1 moiety can be unglycosylated or glycosylated and either may be used. That is, the IL-1 1 moiety can be unglycosylated or the IL-11 moiety can be glycosylated. In one or more embodiments of the invention, the IL-1 1 moiety is unglycosylated. [0077] The IL-11 moiety can advantageously be modified to include and/or substitute one or more amino acid residues such as, for example, lysine, cysteine and/or arginine, in order to provide facile attachment of the polymer to an atom within the side chain of the amino acid. An example of substitution of an IL-1 1 moiety is described in U.S. Patent No. 5,206,344. In addition, the IL-1 1 moiety can be modified to include a non-naturally occurring amino acid residue. Techniques for adding amino acid residues and non-naturally occurring amino acid residues are well known to those of ordinary skill in the art. Reference is made to J. March, Advanced Organic IL-1 lmistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).
[0078] In addition, the IL-1 1 moiety can advantageously be modified to include attachment of a functional group (other than through addition of a functional
group-containing amino acid residue). For example, the IL-11 moiety can be modified to include a thiol group. In addition, the IL-1 1 moiety can be modified to include an N-terminal alpha carbon. In addition, the IL-1 1 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-1 1 moiety can be modified to include an aldehyde group. In addition, the IL-1 1 moiety can be modified to include a ketone group. In some embodiments of the invention, it is preferred that the IL-11 moiety is not modified to include one or more of a thiol group, an N-terminal alpha carbon, carbohydrate, adehyde group and ketone group.
[0079] Exemplary IL-1 1 moieties are described in the literature and in, for example,
U.S. Patent No. 5,371,193, U.S. Patent Application Publication No. 2009/0191 148 and Paul et al. (1990) Proc. Natl, Acad. Sci. USA 87:7512-7516. Preferred IL-11 moieties include those having an amino acid sequence comprising sequences selected from the group consisting of SEQ ID NOs: 1 through 24, and sequences substantially homologous thereto. A preferred IL-11 moiety has the amino acid sequence corresponding to SEQ ID NO: 2.
[0080] In some instances, the IL-1 1 moiety will be in a "monomer" form, wherein a single expression of the corresponding peptide is organized into a discrete unit. In other instances, the IL-11 moiety will be in the form of a "dimer" (e.g., a dimer of recombinant IL- 1 1) wherein two monomer forms of the protein are associated to each other or other
"multimer." [0081] In addition, precursor forms IL-1 1 can be used as the IL-11 moiety. An exemplary precursor form of IL-1 1 has the sequence of SEQ ID NO: 1 ,
[0082] Truncated versions, hybrid variants, and peptide mimetics of any of the foregoing sequences can also serve as the IL-11 moiety. Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of IL-1 1 activity can also serve as an IL-1 1 moiety.
[0083] For any given peptide or protein moiety, it is possible to determine whether that moiety has IL-1 1 activity. Various methods for determining the in vitro IL-11 activity are described in the art. An exemplary approach involves an in vitro cell proliferation assay using Ba/F3 cells expressing gpl30 and IL-1 1 receptor a chain, similar to the method described by Lebeau et al. (1997) FEB S Letters 407: 141-147.
[0084] Briefly, a Ba/F3 cell line (DSMZ, Germany) stably expressing IL-1 1 receptors, gpl 30 and the IL-1 1 receptor a chain (IL-1 IR), can be prepared by transduction of Ba/F3 cells with two retroviral vectors, MIN-IL-1 IR and MIH-gpl30, expressing the IL-1 1 receptor a chain (NCBI NM 004512.3) and gpl30 (NCBI NM 602184), respectively.
[0085] The retroviral plasmid pMIN-IL-11 R can be prepared by the insertion of the
IL-1 IR gene into the pMIN vector (Yu et al (2003) Gene Therapy 10:706-71 1). The pMIH-gpl30 can be prepared by the substitution of the neomycin resistance gene of the pMIN with a hygromycin resistance gene, followed by gpl30 gene insertion. To produce the retroviral vector MIN-IL-1 IR, pMIN-IL-1 IR can be transfected into HEK293T cells with pVM-GP and pVM-AE, expressing gag-pol and amphotropic envelope, respectively as shown by Yu et al. (2003) Gene Therapy 10:706-71 1). The retroviral vector MIH-gpl30 can be generated by the same procedure as MIN-IL-1 IR, except pMIH-gpl 30 is transfected instead of pMIN-IL-1 IR. The transfected cells can then be grown for two days in DMEM media containing 10% fetal bovine serum. After cultivation, the cell-free virus can be prepared by filtering the culture supernatant through a 0.45 μιη filter. To produce Ba/F3 cells expressing IL-1 1 receptor a chain, Ba F3 cells can be transduced with the retroviral vector MIN-IL-1 IR and selected in the presence of 2 mg/ml G418. The expression of IL-1 1 receptor a chain can be confirmed by flow cytometry using FITC-anti-IL-1 IR (Thermo Scientific, Rockford, IL, USA). Then, the cells stably expressing IL-1 IR can then be transduced with the retroviral vector MIH-gpl30 and selected in the presence of 2 mg/ml G418 and 0.5 mg/ml hygromycin. The expression of both IL-1 1R and gpl 30 can be confirmed by flow cytometry using FITC- anti-IL-1 1R and PE-anti-hgpl30 (BD Biosciences, San Jose, CA, USA), respectively. The single clones expressing both IL11R and gpl30 can be obtained by limited dilution in the presence of 2 mg/ml G418 and 0.5 mg/ml hygromycin. The production of IL-1 1 receptor a chain and gpl 30 mRNA from the cells was confirmed by RT-PCR using the following primer pairs.
[0086] For the IL-1 1 receptor a chain: SEQ ID NO: 25:
5'-CGACGCGTATGAGCAGCAGCTGCTCAGGG-S' (forward) SEQ ID NO: 26: 5'- G AAG ATCTCTAC AGGTTTGG AGCTCCTGG- S ' (reverse).
[0087] For gpl 30: SEQ ID NO: 27: 5'-ACGCGTATGTTGACGTTGCAGACT-S '
(forward) SEQ ID NO: 28: 5'-GGATCCTCACTGAGGCATGTAGCC-S' (reverse). In vitro biological activity assay of compounds of interest can be diluted as necessary and placed in 96- well plates.
[0088] Thereafter, one hundred microliters of Ba/F3 cells (3 X 104 cells/ml) can be added to the diluted samples and grown for 72 hours at 37°C, 5% C02 in 96-well plates. At the end of cultivation, cells can be treated with XTT agent (Cell proliferation kit II, Roche, Indianapolis, IN, USA) for 4 hours at 37°C, 5% C02. The optical densities of samples at 492 nm can be measured with a microplate reader (VERSA max, Molecular Devices, Sunnyvale, CA, USA). The optical density at 690 nm can be subtracted from each sample to remove the scattering signal of the cells.
[0089] Other methodologies known in the art can also be used to assess IL-11 function, including electrometry, spectrophotometry, chromatography, and radiometric methodologies.
[0090] The Water-Soluble Polymer
[0091] As previously discussed, each conjugate comprises an IL-1 1 moiety attached to a water-soluble polymer. With respect to the water-soluble polymer, the water-soluble polymer is nonpeptidic, nontoxic, non-naturally occurring and biocompatible. With respect to biocompatibility, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as an IL-1 1 moiety) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician. With respect to non-immunogenicity, a substance is considered non-immunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g., the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. It is particularly preferred that the nonpeptidic water-soluble polymer is biocompatible and non-immunogenic.
[0092] Further, the polymer is typically characterized as having from 2 to about 300 termini. Examples of such polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene,
polyoxazolines ("POZ") (which are described in WO 2008/106186),
poly(N-acryloylmorpholine), and combinations of any of the foregoing.
[0093] The water-soluble polymer is not limited to a particular structure and can be linear (for example, an end capped, e.g., alkoxy, PEG or a bifunctional PEG), branched or multi-armed (e.g., individual PEGs attached to the hydroxyl groups of a polyol core), a dendritic (or star) architecture, each with or without one or more degradable linkages.
Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeat patterns and can be selected from the group consisting of homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
[0094] Typically, activated PEG and other activated water-soluble polymers (i.e., polymeric reagents) are activated with a suitable activating group appropriate for coupling to a desired site on the IL-11 moiety. Thus, a polymeric reagent will possess a reactive group for reaction with the IL-1 1 moiety. Representative polymeric reagents and methods for conjugating these polymers to an active moiety are known in the art and further described in Zalipsky, S., et al., "Use of Functionalized Poly (Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press, New York (1992), and in Zalipsky (1995) Advanced Drug Reviews 16: 157-182. Exemplary activating groups suitable for coupling to an IL-11 moiety include hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, hydrazine, among others. [0095] Typically, the weight-average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons. Exemplary ranges, however, include weight-average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of from about 6,000 Daltons to about 90,000 Daltons, in the range of from about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of from about 20,000 Daltons to about 85,000 Daltons, in the range of from about 53,000 Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to about 120,000 Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons, in the range of from about 35,000 Daltons to about 120,000 Daltons, and in the range of from about 40,000 Daltons to about 120,000 Daltons. For any given water-soluble polymer, PEGs having a molecular weight in one or more of these ranges are preferred.
[0096] Exemplary weight-average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1 ,000 Daltons, about 1 ,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branched versions of the water-soluble polymer (e.g., a branched 40,000 Dalton water-soluble polymer comprised of two 20,000 Dalton polymers) having a total molecular weight of any of the foregoing can also be used. In one or more embodiments, the conjugate will not have any PEG moieties attached, either directly or indirectly, with a PEG having a weight average molecular weight of less than about 6,000 Daltons.
[0097] When used as the polymer, PEGs will typically comprise a number of
(OCH2CH2) monomers [or (CH2CH20) monomers, depending on how the PEG is defined]. As used throughout the description, the number of repeating units is identified by the subscript "n" in "(OCH2CH2)n " Thus, the value of (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., "n") by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.
[0098] One particularly preferred polymer for use in the invention is an end-capped polymer, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower Ci-6 alkoxy group, although a hydroxyl group can also be used. When the polymer is PEG, for example, it is preferred to use a methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG wherein one terminus of the polymer is a methoxy (-OCH3) group, while the other terminus is a hydroxyl or other functional group that can be optionally chemically modified.
[0099] In one form useful in one or more embodiments of the present invention, free or unbound PEG is a linear polymer terminated at each end with hydroxyl groups:
HO-CH2CH20-(CH2CH20)n-CH2CH2-OH, wherein (n) typically ranges from zero to about 4,000.
[0100] The above polymer, alpha-, omega-dihydroxylpoly( ethylene glycol), can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol can represent the following structural unit:
-CH2CH20-(CH2CH20)n-CH2CH2-, wherein (n) is as defined as above.
[0101] Another type of PEG useful in one or more embodiments of the present invention is methoxy-PEG-OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group. The structure of mPEG is given below.
CH30-CH2CH20-(CH2CH20)n-CH2CH2-OH wherein (n) is as described above.
[0102] Multi-armed or branched PEG molecules, such as those described in U.S.
Patent No. 5,932,462, can also be used as the PEG polymer. For example, PEG can have the structure:
poly a— P
wherein: R" ^
polya and polyb are PEG P°tyb Q
backbones (either the same or different), such as methoxy poly(ethylene glycol);
Pv" is a nonreactive moiety, such as H, methyl or a PEG backbone; and
P and Q are nonreactive linkages. In a preferred embodiment, the branched PEG polymer is methoxy poly(ethylene glycol) disubstituted lysine. Depending on the specific IL-1 1 moiety used, the reactive ester functional group of the disubstituted lysine may be further modified to form a functional group suitable for reaction with the target group within the IL-1 1 moiety.
[0103] In addition, the PEG can comprise a forked PEG. An example of a forked
PEG is represented by the following structure:
Z
/
PEG-X-CH
\
Z
wherein: X is a spacer moiety of one or more atoms and each Z is an activated terminal group linked to CH by a chain of atoms of defined length. International Patent Application Publication WO 99/45964 discloses various forked PEG structures capable of use in one or more embodiments of the present invention. The chain of atoms linking the Z functional groups to the branching carbon atom serve as a tethering group and may comprise, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof.
[0104] The PEG polymer may comprise a pendant PEG molecule having reactive groups, such as carboxyl, covalently attached along the length of the PEG rather than at the end of the PEG chain. The pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.
[0105] In addition to the above-described forms of PEG, the polymer can also be prepared with one or more weak or degradable linkages in the polymer, including any of the above-described polymers. For example, PEG can be prepared with ester linkages in the polymer that are subject to hydrolysis. As shown below, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight:
-PEG-CO2-PEG- + H20 >- -PEG-CO2H + HO-PEG-
[0106] Other hydrolytically degradable linkages, useful as a degradable linkage within a polymer backbone and/or as a degradable linkage to an IL-1 1 moiety, include: carbonate linkages; imine linkages resulting, for example, from reaction of an amine and an aldehyde (see, e.g., Ouchi et al. (1997) Polymer Preprints 38.(1): 582-3); phosphate ester linkages formed, for example, by reacting an alcohol with a phosphate group; hydrazone linkages which are typically formed by reaction of a hydrazide and an aldehyde; acetal linkages that are typically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between a formate and an alcohol; amide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of another PEG chain; urethane linkages formed from reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol; peptide linkages formed by an amine group, e.g., at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by, for example, a phosphoramidite group, e.g., at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0107] Such optional features of the conjugate, e.g., the introduction of one or more degradable linkages into the polymer chain, may provide for additional control over the final desired pharmacological properties of the conjugate upon administration. For example, a large and relatively inert conjugate (i.e., having one or more high molecular weight PEG chains attached thereto, for example, one or more PEG chains having a molecular weight greater than about 10,000, wherein the conjugate possesses essentially no bioactivity) may be administered, which is hydrolyzed to generate a bioactive conjugate possessing a portion of the original PEG chain. In this way, the properties of the conjugate can be more effectively tailored to balance the bioactivity of the conjugate over time.
[0108] The water-soluble polymer associated with the conjugate can also be
"releasable." That is, the water-soluble polymer releases (either through hydrolysis, enzymatic processes, catalytic processes or otherwise), thereby resulting in the unconjugated IL-11 moiety. In some instances, releasable polymers detach from the IL-1 1 moiety in vivo without leaving any fragment of the water-soluble polymer. In other instances, releasable polymers detach from the IL-11 moiety in vivo leaving a relatively small fragment (e.g., a succinate tag) from the water-soluble polymer. An exemplary cleavable polymer includes one that attaches to the IL-1 1 moiety via a carbonate linkage.
[0109] Those of ordinary skill in the art will recognize that the foregoing discussion concerning nonpeptidic, water-soluble polymers is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated. As used herein, the term "polymeric reagent" generally refers to an entire molecule, which can comprise a water-soluble polymer segment and a functional group.
[0110] As described above, a conjugate of the invention comprises a water-soluble polymer covalently attached to an IL-1 1 moiety. Typically, for any given conjugate, there will be one to three water-soluble polymers covalently attached to one or more moieties having IL-1 1 activity. In some instances, however, the conjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers individually attached to an IL-11 moiety. Any given water-soluble polymer may be covalently attached to either an amino acid of the IL-1 1 moiety, or, when the IL-1 1 moiety is (for example) a glycoprotein, to a carbohydrate of the IL-1 1 moiety. Attachment to a carbohydrate may be carried out, e.g., using metabolic functionalization employing sialic acid-azide chemistry [Luchansky et al. (2004)
Biochemistry 43(38): 12358-12366] or other suitable approaches such as the use of glycidol to facilitate the introduction of aldehyde groups [Heldt et al. (2007) European Journal of Organic Chemistry 32:5429-5433].
[0111] The particular linkage within the moiety having IL- 11 activity and the polymer depends on a number of factors. Such factors include, for example, the particular linkage chemistry employed, the particular IL-1 1 moiety, the available functional groups within the IL-1 1 moiety (either for attachment to a polymer or conversion to a suitable attachment site), the presence of additional reactive functional groups within the IL-11 moiety, and the like.
[0112] The conjugates of the invention can be, although not necessarily, prodrugs, meaning that the linkage between the polymer and the IL-1 1 moiety is hydrolytically releasable to allow release of the parent moiety. Exemplary releasable linkages include carboxylate ester, phosphate ester, thiol ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides. Such linkages can be readily prepared by appropriate modification of either the IL-1 1 moiety (e.g., the carboxyl group C terminus of the protein, or a side chain hydroxyl group of an amino acid such as serine or threonine contained within the protein, or a similar functionality within the carbohydrate) and/or the polymeric reagent using coupling methods commonly employed in the art. Most preferred, however, are hydrolyzable linkages that are readily formed by reaction of a suitably activated polymer with a non-modified functional group contained within the moiety having IL-11 activity.
[0113] Alternatively, a hydrolytically stable linkage, such as an amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide) linkage can also be employed as the linkage for coupling the IL-1 1 moiety.
Again, a preferred hydrolytically stable linkage is an amide. In one approach, a water-soluble polymer bearing an activated ester can be reacted with an amine group on the IL-1 1 moiety to thereby result in an amide linkage.
[0114] The conjugates (as opposed to an unconjugated IL-1 1 moiety) may or may not possess a measurable degree of IL-1 1 activity. That is to say, a polymer- IL-11 moiety conjugate in accordance with the invention will possesses anywhere from about 0.1% to about 100% of the bioactivity of the unmodified parent IL-1 1 moiety. In some instances, the polymer-IL-1 1 moiety conjugates may have greater than 100% bioactivity of the unmodified parent IL-1 1 moiety. Preferably, conjugates possessing little or no IL-11 activity contain a hydrolyzable linkage connecting the polymer to the moiety, so that regardless of the lack (or relative lack) of activity in the conjugate, the active parent molecule (or a derivative thereof) is released upon aqueous-induced cleavage of the hydrolyzable linkage. Such activity may be determined using a suitable in-vivo or in-vitro model, depending upon the known activity of the particular moiety having IL-1 1 activity employed.
[0115] For conjugates possessing a hydrolytically stable linkage that couples the moiety having IL-11 activity to the polymer, the conjugate will typically possess a measurable degree of bioactivity. For instance, such conjugates are typically characterized as having a bioactivity satisfying one or more of the following percentages relative to that of the unconjugated IL-1 1 moiety: at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 80%>, at least about 85%, at least about 90%, at least about 95%), at least about 97%>, at least about 100%, and more than 105%> (when measured in a suitable model, such as those well known in the art). Preferably, conjugates having a hydrolytically stable linkage (e.g., an amide linkage) will possess at least some degree of the bioactivity of the unmodified parent moiety having IL-11 activity.
[0116] Exemplary conjugates in accordance with the invention will now be described.
Typically, such an IL-1 1 moiety is expected to share (at least in part) a similar amino acid sequence as the sequence provided in at least one of SEQ ID NOs: 1 through 24 and 29. Thus, while reference will be made to specific locations or atoms within SEQ ID NOs: 1 through 24 and 29, such a reference is for convenience only and one having ordinary skill in the art will be able to readily determine the corresponding location or atom in other moieties having IL-11 activity. In particular, the description provided herein for native human IL-1 1 is often applicable to fragments, deletion variants, substitution variants or addition variants of any of the foregoing.
[0117] Amino groups on IL-1 1 moieties provide a point of attachment between the
IL-1 1 moiety and the water-soluble polymer. Using the amino acid sequence provided in SEQ ID NOs: 1 through 24 and 29, it is evident that there are several lysine residues, each lysine residue having an ε-amino acid that may be available for conjugation. Further, the N-terminal amine of any protein can also serve as a point of attachment.
[0118] There are a number of examples of suitable polymeric reagents useful for forming covalent linkages with available amines of an IL-11 moiety. Specific examples, along with the corresponding conjugate, are provided in Table 1 , below. In the table, the variable (n) represents the number of repeating monomelic units and "-NH-(IL-1 1)" represents the residue of the IL-1 1 moiety following conjugation to the polymeric reagent. While each polymeric portion [e.g., (OCH2CH2)n or (CH2CH20)n] presented in Table 1 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor.
Table 1
Amine-Selective Pol meric Rea ents and the IL-11 Moiet Con u ate Formed Therefrom
Figure imgf000026_0001
Figure imgf000027_0001
mPEG-Benzotriazole Carbonate Reagents Carbamate Linkage
Figure imgf000028_0001
mPEG-Succinimidyl Reagents
Figure imgf000029_0001
Reagents
Figure imgf000030_0001
to a secon ary car on
Figure imgf000031_0001
Figure imgf000032_0001
[0119] Conjugation of a polymeric reagent to an amino group of an IL-1 1 moiety can be accomplished by a variety of techniques. In one approach, an IL-1 1 moiety can be conjugated to a polymeric reagent functionalized with a succinimidyl derivative (or other activated ester group, wherein approaches similar to those described for these alternative activated ester group-containing polymeric reagents can be used). In this approach, the polymer bearing a succinimidyl derivative can be attached to the IL-11 moiety in an aqueous media at a pH of 7 to 9.0, although using different reaction conditions (e.g., a lower pH such as 6 to 7, or different temperatures and/or less than 15 °C) can result in the attachment of the polymer to a different location on the IL-1 1 moiety. In addition, an amide linkage can be formed by reacting an amine-terminated nonpeptidic, water-soluble polymer with an IL- 11 moiety bearing an activating a carboxylic acid group.
[0120] Exemplary conjugates are encompassed within the following structure
O II
H3CO-(CH2CH20)n-X-CH-C-NH-(IL-11)
R1
wherein:
(n) is an integer having a value of from 2 to 4000;
X is a spacer moiety;
R1 is an organic radical; and
IL-1 1 is a residue of an IL-11 moiety.
[0121] Exemplary conjugates are encompassed by the following structure:
O II
H3CO-(CH2CH20)n-CH2-CH-C-NH-(IL-1 1 )
CH3 wherein (n) an integer having a value of from 2 to 4000 and IL-1 1 is a residue of an IL-1 1 moiety.
[0122] Typical of another approach useful for conjugating the IL-1 1 moiety to a polymeric reagent is use of reductive amination to conjugate a primary amine of an IL-11 moiety with a polymeric reagent functionalized with a ketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate, aldehyde hydrate). In this approach, the primary amine from the IL-11 moiety reacts with the carbonyl group of the aldehyde or ketone (or the corresponding hydroxyl-containing group of a hydrated aldehyde or ketone), thereby forming a Schiff base. The Schiff base, in turn, can then be reductively converted to a stable conjugate through use of a reducing agent such as sodium borohydride. Selective reactions (e.g., at the N-terminus) are possible, particularly with a polymer functionalized with a ketone or an alpha-methyl branched aldehyde and/or under specific reaction conditions (e.g., reduced pH). [0123] Exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form include those wherein the water-soluble polymer is encompassed within the following structure:
Figure imgf000034_0001
wherein each (n) is independently an integer having a value of from 2 to 4000.
[0124] Exemplary conjugates of the invention are encompassed within the following structure:
O
H3CO-(CH2CH20)n-CH2CH2-NH-C-O n 2
O HO-X-(CH2CH20)b C-kNH-(IL-1 1 )
II
H3CO-(CH2CH20)n-CH2CH2-NH-C-0-J H wherein:
each (n) is independently an integer having a value of from 2 to 4000;
X is spacer moiety;
(b) is an integer having a value 2 through 6;
(c) is an integer having a value 2 through 6;
R2, in each occurrence, is independently H or lower alkyl; and
IL-1 1 is a residue of an IL-11 moiety.
[0125] Exemplary conjugates of the invention are encompassed within the following structure:
Figure imgf000034_0002
wherein:
each (n) is independently an integer having a value of from 2 to 4000; and
IL-1 1 is a residue of an IL-1 1 moiety.
[0126] Other exemplary conjugates of the invention are encompassed within following structure: H3CO-(CH2CH20)n-CH2CH2-NH-C R2 O
I
O ■0-(X)a-(CH2CH20)b.- C-j-C-NH-(IL-11)
H3CO-(CH2CH20)n-CH2CH2-NH-C-0- R: -•c
wherein:
each (n) is independently an integer having a value of from 2 to 4000;
(a) is either zero or one;
X, when present, is a spacer moiety comprised of one or more atoms;
(b') is zero or an integer having a value of one through ten;
(c) is an integer having a value of one through ten;
R2, in each occurrence, is independently H or an organic radical;
R3, in each occurrence, is independently H or an organic radical; and
IL-1 1 is a residue of an IL-1 1 moiety.
[0127] Still further exemplary conjugates of the invention are encompassed within the following structure:
O
H3CO-(CH2CH20)n-CH2CH2-NH-C-0-i O
O kO-CH2CH2CH2C-NH-(IL-11)
II
H3CO-(CH2CH20)n-CH2CH2-NH-C-0- wherein:
each (n) is independently an integer having a value of from 2 to 4000; and
IL-1 1 is a residue of IL-11 moiety.
[0128] Exemplary conjugates that include a releasable linkage include those in which an IL-1 1 moiety are conjugated to a polymeric reagent encompassed within the following formula:
Figure imgf000035_0001
POLY1 is a first water-soluble polymer;
POLY2 is a second water-soluble polymer;
X1 is a first spacer moiety;
X2 is a second spacer moiety;
Ha is an ionizable hydrogen atom;
PJ is H or an organic radical;
R2 is H or an organic radical;
(a) is either zero or one;
(b) is either zero or one;
Rel, when present, is a first electron altering group;
Re2, when present, is a second electron altering group; and
(FG) is a functional group capable of reacting with an amino group of an active agent to form a releasable linkage, such as a carbamate linkage. Within this formula, polymeric reagents having the more defined structure are contemplated:
Figure imgf000036_0001
wherein each of POLY1, POLY2, X1, X2, R1, R2, Ha and (FG) is as previously defined in this paragraph, and Rel is a first electron altering group; and Re2 is a second electron altering group.
[0129] Still further exemplary polymeric reagents fall within the following formulae:
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000037_0002
CH30-(CH2CH20)n-CH2CH2-0
Figure imgf000037_0003
Figure imgf000037_0004
Figure imgf000038_0001
wherein, for each structure and in each instance, (n) is independently an integer from 4 to 1500.
[0130] These releasable linkage-providing polymeric reagents can be prepared in accordance with the procedures set forth in U.S. Patent Application Publication No.
2006/0293499.
[0131] Exemplary conjugates formed using releasable linkage-providing polymeric reagents include those of the following formulae:
Figure imgf000038_0002
wherein:
POLY1 is a first water-soluble polymer;
POLY2 is a second water-soluble polymer;
X1 is a first spacer moiety;
X2 is a second spacer moiety;
Ha is an ionizable hydrogen atom;
R1 is H or an organic radical;
R2 is H or an organic radical;
(a) is either zero or one;
(b) is either zero or one;
Rel, when present, is a first electron altering group;
Re2, when present, is a second electron altering group;
Y1 is O or S; Y2 is O or S; and
(IL-1 1) is a residue of an IL-11 moiety.
[0132] Exemplary conjugates have the following structure:
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000039_0004
Figure imgf000040_0001
Figure imgf000040_0002
wherein, for each structure and in each instance, (n) is independently an integer from 4 to 1500, and (IL-1 1) is a residue of an IL-11 moiety.
[0133] Carboxyl groups represent another functional group that can serve as a point of attachment on the IL-1 1 moiety. Structurally, the conjugate will comprise the following:
O
(IL-l l)-C-X-POLY
where (IL-1 1) and the adjacent carbonyl group corresponds to the carboxyl -containing IL-1 1 moiety, X is a linkage, preferably a heteroatom selected from O, N(H), and S, and POLY is a water-soluble polymer such as PEG, optionally terminating in an end-capping moiety.
[0134] The C(0)-X linkage results from the reaction between a polymeric derivative bearing a terminal functional group and a carboxyl-containing IL-1 1 moiety. As discussed above, the specific linkage will depend on the type of functional group utilized. If the polymer is end-functionalized or "activated" with a hydroxyl group, the resulting linkage will be a carboxylic acid ester and X will be O. If the polymer backbone is functionalized with a thiol group, the resulting linkage will be a thioester and X will be S. When certain multi-arm, branched or forked polymers are employed, the C(0)X moiety, and in particular the X moiety, may be relatively more complex and may include a longer linkage structure. [0135] Water-soluble derivatives containing a hydrazide moiety are also useful for conjugation at a carbonyl and carboxylic acid. To the extent that the IL-11 moiety does not contain a carbonyl moiety or a carboxylic acid, one can be added using techniques known to one of ordinary skill in the art. For example, a carbonyl moiety can be introduced by reducing a carboxylic acid (e.g., the C-terminal carboxylic acid) and/or by providing glycosylated or glycated (wherein the added sugars have a carbonyl moiety) versions of the IL-11 moiety. With respect to IL-11 moieties containing a carboxylic acid, a PEG-hydrazine reagent can, in the presence of a coupling agent (e.g., DCC), covalently attach to the IL-1 1 moiety [e.g., mPEG-OCH2C(0)NHNH2 + HOC(0)-(IL-l 1) results in mPEG-OCH2C(0)NHNHC(0)-IL- 11]. Specific examples of water-soluble derivatives containing a hydrazide moiety, along with the corresponding conjugates, are provided in Table 2, below. In addition, any water-soluble derivative containing an activated ester (e.g., a succinimidyl group) can be converted to contain a hydrazide moiety by reacting the water-soluble polymer derivative containing the activated ester with hydrazine (NH2-NH2) or tert-butyl carbazate
[NH2NHC02C(CH3)3] . In the table, the variable (n) represents the number of repeating monomeric units and "-C(0)-(IL-1 1)" represents the residue of the IL-1 1 moiety following conjugation to the polymeric reagent. Optionally, the hydrazone linkage can be reduced using a suitable reducing agent. While each polymeric portion [e.g., (OCH2CH2)n or (CH2CH20)n] presented in Table 2 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor.
Table 2
Carbox l-S ecific Pol meric Rea ents and the IL-1 1 Moiet Con u ate Formed Therefrom
Figure imgf000041_0001
Polymeric Reagent Corresponding Conjugate
0
II 0
H3CO-(CH2CH20)nCH2CH2-NH- O NH- NH2
H3CO-(CH2CH20)nCH2CH2- N H- C - NH- NH-C(OH IL-11 ) mPEG-Hydrazine Reagents
Hydrazone Linkage
0 0
II H
H3CCHCH2CH20)nCH2CH2- NH- NH- C- NH- NH2 H3CO-(CH2CH20)nCH2CH2-N-NH-C II-NH-NH-C(0)-(IL-11 )
Hydrazone Linkage
mPEG-Hydrazine Reagents
S S II
H3CO-(CH2CH20)nCH2CH2-NH-C-NH-NH2 H3CO-(CH2CH20)nCH2CH2 - NH- C - NH- NH-C(OH IL-1 1 ) mPEG-Hydrazine Reagents
Hydrazone Linkage
S s
II H
H3CO-(CH2CH20)nCH2CH2- NH- NH- C~ NH- NH2 II
Η30Ο-(0Η20Η20)π0Η2α-Ι2-Ν— NH-C-NH-NH-C()-(IL-11)
Hydrazone Linkage
mPEG-Hydrazine Reagents o o
H3Ca(CH2CH20)nCH2CH2-NH-C II-NH-NH-C II-NH-NH2 II II
H3Ca(CH2CH20)nCH2CH2-NH-C- NH-NH-C- NH- NH-C(OXIL.-11) mPEG-Hydrazine Reagents
Hydrazone Linkage
0 0
H3C )-(CH2CH20)nCH2CH2-0— C - NH- NHj H3CCKCH2CH20)nCH2CH2--0-C-NH-NH-C(OHIL-11 ) mPEG-Hydrazine Reagents Hydrazone Linkage
0
II 0
H3CO-{CH2CH20)nCH2-C-NH-NH2 o
H3CO-(CH2CH20)nCH2-C-NH - NH-C -(IL-1 1 ) mPEG-Hydrazine Reagents
C(0)NHNHC(0) Linkage
[0136] Thiol groups contained within the IL-1 1 moiety can serve as effective sites of attachment for the water-soluble polymer. In particular, cysteine residues provide thiol groups when the IL-11 moiety is a protein. The thiol groups in such cysteine residues can then be reacted with an activated PEG that is specific for reaction with thiol groups, e.g., an N-maleimidyl polymer or other derivative as described in U.S. Patent No. 5,739,208 and in WO 01/62827. In addition, a protected thiol may be incorporated into an oligosaccharide side chain of an activated glycoprotein, followed by deprotection with a thiol-reactive
water-soluble polymer.
[0137] Specific examples of reagents, along with the corresponding conjugate, are provided in Table 3, below. In the table, the variable (n) represents the number of repeating monomeric units and "-S-(IL-11)" represents the IL-11 moiety residue following conjugation to the water-soluble polymer. While each polymeric portion [e.g., (OCH2CH2)n or
(CH2CH20)n] presented in Table 3 terminates in a "CH3" group, other groups (such as H and benzyl) can be substituted therefor.
[0138] With respect to SEQ ID NOs: 1 and 2 corresponding to exemplary IL-1 1 moieties, it can be seen that there is a cysteine residue at position 125. Thus, an exemplary thiol attachment sites is the cysteine located at position 125. Although it is preferred not to disrupt any disulfide bonds associated with a given IL-1 1 moiety, it may be possible to attach a polymer within the side chain of one or more of these cysteine residues and retain a degree of activity. In addition, it is possible to add a cysteine residue to the IL-1 1 moiety using conventional synthetic techniques. See, for example, the procedure described in
WO 90/12874 for adding cysteine residues, wherein such procedure can be adapted for an IL-1 1 moiety. In addition, conventional genetic engineering processes can also be used to introduce a cysteine residue into the IL-1 1 moiety. In some embodiments, however, it is preferred not to introduce an additional cysteine residue and/or thiol group.
Table 3
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
[0139] With respect to conjugates formed from water-soluble polymers bearing one or more maleimide functional groups (regardless of whether the maleimide reacts with an amine or thiol group on the IL-1 1 moiety), the corresponding maleamic acid form(s) of the water-soluble polymer can also react with the IL-1 1 moiety. Under certain conditions (e.g., a pH of about 7-9 and in the presence of water), the maleimide ring will "open" to form the corresponding maleamic acid. The maleamic acid, in turn, can react with an amine or thiol group of an IL-1 1 moiety. Exemplary maleamic acid-based reactions are schematically shown below. POLY represents the water-soluble polymer, and (IL-11) represents the IL-11 moiety.
Figure imgf000046_0001
[0140] A representative conjugate in accordance with the invention can have the following structure:
POLY-Lo,i-C(0)Z-Y-S-S-(IL-l 1) wherein POLY is a water-soluble polymer, L is an optional linker, Z is a heteroatom selected from the group consisting of O, NH, and S, and Y is selected from the group consisting of C2-io alkyl, C2-10 substituted alkyl, aryl, and substituted aryl, and (IL-11) is an IL-1 1 moiety. Polymeric reagents that can be reacted with an IL-1 1 moiety and result in this type of conjugate are described in U.S. Patent Application Publication No. 2005/0014903.
[0141] As previously indicated, exemplary conjugates of the invention wherein the water-soluble polymer is in a branched form, will have the branched form of the
water-soluble polymer comprise the following structure:
I I
H3CO-(CH2CH20)n— CH2CH2-NH
O
II
H3CO-(CH2CH20)n-CH2CH2-NH -c-o- wherein each (n) is independently an integer having a value of from 2 to 4000.
[0142] Exemplary conjugates having a water-soluble polymer in branched form are prepared using the following reagent:
Figure imgf000047_0001
thereby forming a conjugate having the following structure:
Figure imgf000047_0002
wherein:
(for each structure) each (n) is independently an integer having a value of from 2 to 4000; and
IL-1 1 is a residue of IL-1 1 moiety.
[0143] An additional exemplary conjugate can be formed using a reagent:
Figure imgf000047_0003
thereby forming a conjugate having the following structure:
Figure imgf000047_0004
wherein:
(for each structure) (n) is independently an integer having a value of from 2 to 4000; and
IL-11 is a residue of IL-11 moiety.
[0144] Conjugates can be formed using thiol-selective polymeric reagents in a number of ways and the invention is not limited in this regard. For example, the IL-11 moiety
~ optionally in a suitable buffer (including amine-containing buffers, if desired) ~ is placed in an aqueous media at a pH of about 7-8 and the thiol-selective polymeric reagent is added at a molar excess. The reaction is allowed to proceed for about 0.5 to 2 hours, although reaction times of greater than 2 hours (e.g., 5 hours, 10 hours, 12 hours, and 24 hours) can be useful if PEGylation yields are determined to be relatively low. Exemplary polymeric reagents that can be used in this approach are polymeric reagents bearing a reactive group selected from the group consisting of maleimide, sulfone (e.g., vinyl sulfone), and thiol (e.g., functionalized thiols such as an ortho pyridinyl or "OPSS").
[0145] With respect to polymeric reagents, those described here and elsewhere can be purchased from commercial sources or prepared from commercially available starting materials. In addition, methods for preparing the polymeric reagents are described in the literature.
[0146] The attachment between the IL-1 1 moiety and the non-peptidic, water-soluble polymer can be direct, wherein no intervening atoms are located between the IL-11 moiety and the polymer, or indirect, wherein one or more atoms are located between the IL-11 moiety and the polymer. With respect to the indirect attachment, a "spacer moiety" serves as a linker between the residue of the IL-11 moiety and the water-soluble polymer. The one or more atoms making up the spacer moiety can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. The spacer moiety can comprise an amide, secondary amine, carbamate, thioether, and/or disulfide group. Nonlimiting examples of specific spacer moieties include those selected from the group consisting of -0-, -S-, -S-S-, -C(O)-, -C(0)-NH-, -NH-C(0)-NH-, -0-C(0)-NH-, -C(S)-, -C¾-, -CH2-CH2-,
-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -0-CH2-, -CH2-0-, -0-CH2-CH2-, -CH2-0-CH2-, -CH2-CH2-0-, -0-CH2-CH2-CH2-, -CH2-0-CH2-CH2-, -CH2-CH2-0-CH2-,
-CH2-CH2-CH2-0-, -0-CH2-CH2-CH2-CH2-, -CH2-0-CH2-CH2-CH2-,
-CH2-CH2-0-CH2-CH2-, -CH2-CH2-CH2-0-CH2-, -CH2-CH2-CH2-CH2-0-, -C(0)-NH-CH2-, -C(0)-NH-CH2-CH2-, -CH2-C(0)-NH-CH2-, -CH2-CH2-C(0)-NH-,
-C(0)-NH-CH2-CH2-CH2-, -CH2-C(0)-NH-CH2-CH2-, -CH2-CH2-C(0)-NH-CH2-,
-CH2-CH2-CH2-C(0)-NH-, -C(0)-NH-CH2-CH2-CH2-CH2-, -CH2-C(0)-NH-CH2-CH2-CH2-, -CH2-CH2-C(0)-NH-CH2-CH2-, -CH2-CH2-CH2-C(0)-NH-CH2-,
-CH2-CH2-CH2-C(0)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(0)-NH-, -C(0)-0-CH2-, -CH2-C(0)-0-CH2-, -CH2-CH2-C(0)-0-CH2-, -C(0)-0-CH2-CH2-, -NH-C(0)-CH2-, -CH2-NH-C(0)-CH2-, -CH2-CH2-NH-C(0)-CH2-, -NH-C(0)-CH2-CH2-, -CH2-NH-C(0)-CH2-CH2-, -CH2-CH2-NH-C(0)-CH2-CH2-, -C(0)-NH-CH2-, -C(0)-NH-CH2-CH2-, -0-C(0)-NH-CH2-, -0-C(0)-NH-CH2-CH2-, -NH-C¾-,
-NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-NH-CH2-, -C(0)-CH2-, -C(0)-CH2-CH2-, -CH2-C(0)-CH2-, -CH2-CH2-C(0)-CH2-, -CH2-CH2-C(0)-CH2-CH2-, -CH2-CH2-C(0)-, -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)-, -CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)-CH2-,
-CH2-CH2-CH2-C(0)-NH-CH2-CH2-NH-C(0)-CH2-CH2-,
-0-C(0)-NH-[CH2]h-(OCH2CH2)j-, bivalent cycloalkyl group, -0-, -S-, an amino acid, -N(R6)-, and combinations of two or more of any of the foregoing, wherein R6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is zero to six, and (j) is zero to 20. Other specific spacer moieties have the following structures:
-C(0)-NH-(CH2)i_6-NH-C(0)-, -NH-C(0)-NH-(CH2),.6-NH-C(0)-, and -0-C(0)-NH-(CH2)i_ 6-NH-C(0)-, wherein the subscript values following each methylene indicate the number of methylenes contained in the structure, e.g., (CH2)i-6 means that the structure can contain 1 , 2, 3, 4, 5 or 6 methylenes. Additionally, any of the above spacer moieties may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., - (CH2CH20)i-2o]. That is, the ethylene oxide oligomer chain can occur before or after the spacer moiety, and optionally in between any two atoms of a spacer moiety comprised of two or more atoms. Also, the oligomer chain would not be considered part of the spacer moiety if the oligomer is adjacent to a polymer segment and merely represent an extension of the polymer segment. For clarity, amino acids belonging to the IL-1 1 moiety are not considered part of the spacer moiety.
[0147] Compositions
[0148] The conjugates are typically part of a composition. Generally, the composition comprises a plurality of conjugates, preferably although not necessarily, each conjugate is comprised of the same IL-11 moiety (i.e., within the entire composition, only one type of IL-1 1 moiety is found). In addition, the composition can comprise a plurality of conjugates wherein any given conjugate is comprised of a moiety selected from the group consisting of two or more different IL-1 1 moieties (i.e., within the entire composition, two or more different IL-1 1 moieties are found). Optimally, however, substantially all conjugates in the composition (e.g., 85% or more of the plurality of conjugates in the composition) are each comprised of the same IL-1 1 moiety.
[0149] The composition can comprise a single conjugate species (e.g., a
monoPEGylated conjugate wherein the single polymer is attached at the same location for substantially all conjugates in the composition) or a mixture of conjugate species (e.g., a mixture of monoPEGylated conjugates where attachment of the polymer occurs at different sites and/or a mixture monoPEGylated, diPEGylated and triPEGylated conjugates). The compositions can also comprise other conjugates having four, five, six, seven, eight or more polymers attached to any given moiety having IL-11 activity. In addition, the invention includes instances wherein the composition comprises a plurality of conjugates, each conjugate comprising one water-soluble polymer covalently attached to one IL-1 1 moiety, as well as compositions comprising two, three, four, five, six, seven, eight, or more
water-soluble polymers covalently attached to one IL-1 1 moiety.
[0150] With respect to the conjugates in the composition, the composition will satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have from one to four polymers attached to the IL-11 moiety; at least about 85% of the conjugates in the composition will have from one to three polymers attached to the IL-1 1 moiety; at least about 85% of the conjugates in the composition will have from one to two polymers attached to the IL-1 1 moiety; at least about 85% of the conjugates in the composition will have one polymer attached to the IL-1 1 moiety; at least about 95% of the conjugates in the composition will have from one to five polymers attached to the IL-11 moiety; at least about 95% of the conjugates in the composition will have from one to four polymers attached to the IL-11 moiety; at least about 95% of the conjugates in the composition will have from one to three polymers attached to the IL-1 1 moiety; at least about 95% of the conjugates in the composition will have from one to two polymers attached to the IL-1 1 moiety; at least about 95% of the conjugates in the composition will have one polymer attached to the IL-11 moiety; at least about 99%) of the conjugates in the composition will have from one to five polymers attached to the IL-11 moiety; at least about 99% of the conjugates in the composition will have from one to four polymers attached to the IL-1 1 moiety; at least about 99% of the conjugates in the composition will have from one to three polymers attached to the IL-1 1 moiety; at least about 99% of the conjugates in the composition will have from one to two polymers attached to the IL-1 1 moiety; and at least about 99% of the conjugates in the composition will have one polymer attached to the IL-11 moiety. It is understood that a reference to a range of polymers, e.g., "from x to y polymers," contemplates a number of polymers x to y inclusive (that is, for example, "from one to three polymers" contemplates one polymer, two polymers and three polymers, "from one to two polymers" contemplates one polymer and two polymers, and so forth).
[0151] In one or more embodiments, it is preferred that the conjugate-containing composition is free or substantially free of albumin. It is also preferred that the composition is free or substantially free of proteins that do not have IL-1 1 activity. Thus, it is preferred that the composition is 85%>, more preferably 95%, and most preferably 99% free of albumin. Additionally, it is preferred that the composition is 85%, more preferably 95%, and most preferably 99% free of any protein that does not have IL-11 activity. To the extent that albumin is present in the composition, exemplary compositions of the invention are substantially free of conjugates comprising a poly(ethylene glycol) polymer linking a residue of an IL-1 1 moiety to albumin.
[0152] Control of the desired number of polymers for any given moiety can be achieved by selecting the proper polymeric reagent, the ratio of polymeric reagent to the IL-1 1 moiety, temperature, pH conditions, and other aspects of the conjugation reaction. In addition, reduction or elimination of the undesired conjugates (e.g., those conjugates having four or more attachedpolymers) can be achieved through purification means.
[0153] For example, the polymer- IL-11 moiety conjugates can be purified to obtain/isolate different conjugated species. Specifically, the product mixture can be purified to obtain an average of anywhere from one, two, three, four, five or more PEGs per IL-11 moiety, typically one, two or three PEGs per IL-11 moiety. The strategy for purification of the final conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular IL-11 moiety, the desired dosing regimen, and the residual activity and in vivo properties of the individual conjugate(s).
[0154] If desired, conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography. That is to say, gel filtration chromatography is used to fractionate differently numbered polymer-to-IL-1 1 moiety ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein "1-mer" indicates 1 polymer to IL-1 1 moiety, "2-mer" indicates two polymers to IL-11 moiety, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble polymer portion). For example, in an exemplary reaction where a 35,000 Dalton protein is randomly conjugated to a polymeric reagent having a molecular weight of about 20,000 Daltons, the resulting reaction mixture may contain unmodified protein (having a molecular weight of about 35,000 Daltons), monoPEGylated protein (having a molecular weight of about 55,000 Daltons), diPEGylated protein (having a molecular weight of about 75,000 Daltons), and so forth.
[0155] While this approach can be used to separate PEG and other polymer-IL-11 moiety conjugates having different molecular weights, this approach is generally ineffective for separating positional isoforms having different polymer attachment sites within the IL-11 moiety. For example, gel filtration chromatography can be used to separate from each other mixtures of PEG 1 -mers, 2-mers, 3-mers, and so forth, although each of the recovered conjugate compositions may contain PEG(s) attached to different reactive groups (e.g., lysine residues) within the IL-1 1 moiety.
[0156] Gel filtration columns suitable for carrying out this type of separation include
Superdex™ and Sephadex™ columns available from Amersham Biosciences (Piscataway, NJ). Selection of a particular column will depend upon the desired fractionation range desired. Elution is generally carried out using a suitable buffer, such as phosphate, acetate, or the like. The collected fractions may be analyzed by a number of different methods, for example, (i) absorbance at 280 nm for protein content, (ii) dye-based protein analysis using bovine serum albumin (BSA) as a standard, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. Biochem, 107:60-63), (iv) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), followed by staining with barium iodide, and (v) high performance liquid chromatography (HPLC).
[0157] Separation of positional isoforms is carried out by reverse phase
chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) using a suitable column (e.g., a CI 8 column or C3 column, available commercially from companies such as Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a Sepharose™ ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-active agent isomers having the same molecular weight (i.e., positional isoforms). [0158] The compositions are preferably substantially free of proteins that do not have
IL-1 1 activity. In addition, the compositions preferably are substantially free of all other noncovalently attached water-soluble polymers. In some circumstances, however, the composition can contain a mixture of polymer- IL-1 1 moiety conjugates and unconjugated IL- 1 1 moiety.
[0159] Optionally, the composition of the invention further comprises a
pharmaceutically acceptable excipient. If desired, the pharmaceutically acceptable excipient can be added to a conjugate to form a composition.
[0160] Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.
[0161] A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, and the like.
[0162] The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
[0163] The composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.
[0164] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.
[0165] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and IL-1 Hating agents, such as EDTA, zinc and other such suitable cations.
[0166] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.
[0167] One or more amino acids can be present as an excipient in the compositions described herein. Exemplary amino acids in this regard include arginine, lysine and glycine.
[0168] The amount of the conjugate (i.e., the conjugate formed between the active agent and the polymeric reagent) in the composition will vary depending on a number of factors, but will optimally be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial). In addition, the pharmaceutical preparation can be housed in a syringe. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the conjugate in order to determine which amount produces a clinically desired endpoint.
[0169] The amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine
experimentation, i.e., by preparing compositions containing vaiying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.
[0170] Generally, however, the excipient will be present in the composition in an amount of about 1% to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred.
[0171] These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician's Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D,C, 2000.
[0172] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned.
[0173] The compositions of one or more embodiments of the present invention are typically, although not necessarily, administered via injection and are therefore generally liquid solutions or suspensions immediately prior to administration. The pharmaceutical preparation can also take other forms such as syrups, creams, ointments, tablets, powders, and the like. Other modes of administration are also included, such as pulmonary, rectal, transdermal, transmucosal, oral, intrathecal, intratumorally, peritumorally, intraperitonally, subcutaneous, intra-arterial, and so forth.
[0174] The invention also provides a method for administering a conjugate as provided herein to a patient suffering from a condition that is responsive to treatment with conjugate. The method comprises administering to a patient, generally via injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition). As previously described, the conjugates can be injected (e.g., intramuscularly, subcutaneously and parenterally). Suitable formulation types for parenteral administration include ready-for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.
[0175] The method of administering the conjugate (preferably provides as part of a pharmaceutical composition) can optionally be conducted so as to localize the conjugate to a specific area. For example, the liquid, gel and solid formulations comprising the conjugate could be surgically implanted in a diseased area (such as in a tumor, near a tumor, in an inflamed area, and near an inflamed area). Conveniently, organs and tissue can also be imaged in order to ensure the desired location is better exposed to the conjugate.
[0176] The method of administering may be used to treat any condition that can be remedied or prevented by administration of the conjugate. Those of ordinary skill in the art appreciate which conditions a specific conjugate can effectively treat. For example, the conjugates can be used either alone or in combination with other pharmacotherapy to treat patients suffering from a condition. Exemplary conditions include, without limitation:
prevention of severe thrombocytopenia and the reduction of the need for platelet transfusions following myelosuppressive chemotherapy in adult patients with nonmyeloid malignancies who are at high risk of severe thrombocytopenia; TH2-mediated disorders (e.g., asthma, congestive obstruction pulmonary disorder, rhinitis, allergies, and atopic dermatitis);
psoriasis; antibiotic-induced diarrhea; inflammatory bowel disease; liver inflammation in hepatitis C patients; Crohn's disease; mucositis; fertility modulation; gingivitis; and spondyloarthropies. Advantageously, the conjugate can be administered to the patient prior to, simultaneously with, or after administration of another active agent.
[0177] The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and/or are described in the pertinent reference texts and literature. Generally, a therapeutically effective amount will range from about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10 mg day to 50 mg/day. A given dose can be periodically administered up until, for example, symptoms of the condition being treated lessen and/or are eliminated entirely. [0178] The unit dosage of any given conjugate (again, preferably provided as part of a pharmaceutical preparation) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined
experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
[0179] It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
[0180] All articles, books, patents and other publications referenced herein are hereby incorporated by reference in their entireties.
EXPERIMENTAL
[0181] The practice of the invention will employ, unless otherwise indicated, conventional techniques of organic synthesis, biochemistry, protein purification and the like, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992), supra.
[0182] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.) but some experimental error and deviation should be taken into account. Unless indicated otherwise, temperature is in degrees C and pressure is at or near atmospheric pressure at sea level. Each of the following examples is considered to be instructive to one of ordinary skill in the art for carrying out one or more of the embodiments described herein.
[0183] An aqueous solution ("stock solution") comprising recombinant IL-1 1 ("rlL-
11 ") corresponding to the amino acid sequence of SEQ ID NO: 3 for use in the examples. [0184] SDS-PAGE Analysis
[0185] Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Invitrogen gel electrophoresis system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared samples were loaded onto a NuPAGE Novex precast gel and run for approximately thirty minutes.
[0186] SEC-HPLC Analysis
[0187] Size exclusion chromatography (SEC-HPLC) analysis was performed on an
Agilent 1 100 HPLC system (Agilent). Samples were analyzed using a Shodex protein W- 804 column (300 x 8 mm, Phenomenex), and a mobile phase consisting of sodium phosphate and sodium sulfate, pH 7. The flow rate for the column was 0.5 ml/min. Eluted protein and PEG-protein conjugates were detected using UV at 280nm and 220nm.
[0188] RP-HPLC Analysis
[0189] Reversed phase high-performance liquid chromatography (RP-HPLC) was performed on an Agilent 1 100 HPLC system (Agilent). Samples were analyzed using a Zorbax 300SB-C3 column (3.5 μηι particle size, 150 mm x 3.0 mm, Agilent), and mobile phases consisting of 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B). The flow rate for the column was 0.3 ml/min. The protein and PEG-protein conjugates were Eluted with a linear gradient over 20 minutes, and were detected using UV at 280nm.
Example 1
PEGylation of IL-11 with Branched mPEG-N-Hydroxysuccinimide Derivative, 40kDa
Figure imgf000058_0001
Branched mPEG-N-Hydroxysuccinimide Derivative, 40kDa, ("mPEG2-NHS")
[0190] mPEG2-NHS (40kDa), stored at -20 °C under argon, was warmed to ambient temperature. A five-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-11 solution) of the warmed mPEG2-NHS (40kDa) was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (2.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well. To allow for coupling of the mPEG2-NHS (40kDa) to IL-1 1 via an amide linkage, the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 hours first, and then at 4°C overnight. The reaction was quenched with acetic acid to lower the pH to 4.0. The conjugate solution was characterized by SDS-PAGE and RP-HPLC. Figure 1 shows the chromatogram following the RP-HPLC analysis of the conjugate solution. The PEGylation reaction yielded 64% 1-mer (mono-conjugate or one PEG attached to each IL-11 molecule) and 21 % 2-mer (di-conjugate or two PEGs attached to each IL-1 1 molecule). A cation-exchange chromatography method using SP Sepharose High Performance column along with sodium acetate and sodium phosphate buffers was also used to purify the conjugates.
Example la
PEGylation of IL-11 with Branched mPEG-N-Hydroxysuccinimide Derivative, 20kDa
Figure imgf000059_0001
Branched mPEG-N-Hydroxysuccinimide Derivative, 20kDa, ("mPEG2-NHS")
[0191] mPEG2-NHS (20kDa), stored at -20°C under argon, was warmed to ambient temperature. A three-fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-11 solution) of the warmed mPEG2-NHS (20kDa) was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.5 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. To allow for coupling of the mPEG2-NHS (20kDa) to IL-1 1 via an amide linkage, the reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 to 3 hours. The reaction was quenched with acetic acid to lower the pH to 4.0. The conjugate solution was characterized by SDS-PAGE and HPLC. A cation-exchange chromatography method using SP Sepharose High Performance column along with sodium phosphate buffers was also used to purify the conjugates. Example 2
PEGylation of IL-11 with Linear mPEG-Butyraldehyde Derivative, 20kDa
Figure imgf000060_0001
Linear mPEG-Butyraldehyde Derivative, 20kDa ("mPEG-ButyrALD")
[0192] mP EG-Butyr ALD , 20kDa, stored at -20 °C under argon, was warmed to ambient temperature. An eighteen- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1) of the warmed mPEG-ButryALD was dissolved in Milli-Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (1.5 mg/ml in sodium acetate and sodium phosphate buffer, pH 5.8) and mixed well. The reaction solution was kept at room temperature on a RotoMix for thirty minutes. A reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH3. The reaction solution was well mixed and was kept at 4°C overnight.
[0193] The aldehyde group of mPEG-ButyrALD can react with the primary amines associated with IL-11 and covalently bond to them via secondary amine upon reduction by sodium cyanoborohydride. Figure 2 shows the SEC-HPLC chromatogram of the conjugate solution. The PEGylation reaction yielded 40% 1 -mer (one PEG attached to IL-11 or mono- conjugate).
[0194] Using this same approach, other conjugates can be prepared using
mPEG-ButyrALD having other weight average molecular weights.
Example 3
PEGylation of IL-11 with Branched mPEG-Butyraldehyde Derivative, 40kDa
Figure imgf000060_0002
Branched mPEG-Butyraldehyde Derivative, 40kDa ("mPEG2-ru-ButyrALD") [0195] mPEG2-ru-ButyrALD, 40kDa, stored at -20 °C under argon, was warmed to ambient temperature. A ten- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1) of the warmed mPEG2-ButryALD (40kDa) was dissolved in Millis H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (1.9 mg/ml in sodium acetate buffer, pH 5.3) and mixed well. The reaction solution was kept at room temperature on a RotoMix for ten minutes. A reducing agent, sodium cyanoborohydride was then added to make 10 raM NaCNBH3. The reaction solution was well mixed and was kept at 4°C overnight.
[0196] The aldehyde group of mPEG2-ButyrALD (40kDa) can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. Because the PEGylation reaction was carried at pH 5, attachment of the PEG derivative to IL-1 1 was more selective to the N-terminal. Figure 3 shows the SEC-HPLC chromatogram of the conjugate solution. The PEGylation reaction yielded 72% mono-conjugate, 21% di-conjugate and 1% higher species. A cation-exchange chromatography method using SP Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugates. Figure 4 shows the separation profile of a reaction mixture.
[0197] Using this same approach, other conjugates can be prepared using
mPEG2-ButyrALD having other weight average molecular weights.
Example 3a
PEGylation of IL-11 with Branched mPEG-Butyraldehyde Derivative, 20kDa
O
H3c -OCH2CH2)-NH-C-0— I O
O 0CH2CH2CH2-C-NH-(-CH2CH20 -CH2CH2CH2CH0
H3C-(-OCH2CH2)-NH-C-0- Branched mPEG-Butyraldehyde Derivative, 20kDa ("mPEG2-ru-ButyrALD")
[0198] mPEG2-ru-ButyrALD, 20kDa, stored at -20 °C under argon, was warmed to ambient temperature. A twenty-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1) of the warmed mPEG2-ButryALD (20kDa) was dissolved in Milli - Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.4 mg/ml in 50mM sodium phosphate buffer, lOOmM sodium chloride, 5% sorbital, pH 6) and mixed well. The reaction solution was kept at room temperature on a RotoMix for 15 minutes. A reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH3. The reaction solution was well mixed and was kept at room temperature for 15minutes, and then at 4°C overnight. The conjugate solution was characterized by SDS-PAGE and HPLC.
[0199] The aldehyde group of mPEG2-ButyrALD (20kDa) can react with the primary amines associated with IL-11 and covalently bond to them via secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride. Because the PEGylation reaction was carried at pH 5, attachment of the PEG derivative to IL-1 1 was more selective to the N-terminal. A cation-exchange chromatography method using SP Sepharose High Performance column and sodium phosphate buffers was also developed to purify the conjugates.
Example 4
PEGylation of IL-11 with 9-Hydroxymethyl-4-(mPEG(20,000)-carboxyamide)-7-(3- (mPEG(20,000))carbamoyI-propyI)-fluorene-N-hydroxysuccinimidyl Carbonate, 40kDa
Figure imgf000062_0001
Branched mPEG-FMOC-N-Hydroxysuccinimide Derivative, 40kDa, ("CAC-PEG2-FMOC-
NHS")
[0200] CAC-PEG2-FMOC-NHS, 40kDa, stored at -20 °C under argon, was warmed to ambient temperature. A three- fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed CAC-PEG2-FMOC-NHS was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (3.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well. The reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 hr first, and then at 4°C overnight. The reaction was quenched by the addition of IM acetic acid to lower the pH to 5. The conjugate solution was characterized by SDS-PAGE (Figure 5). Based on the gel result, the reaction yielded 50% mono-conjugate and 36% di-conjugate.
[0201] Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
[0202] Using this same approach, other conjugates can be prepared using CAC-
PEG2-FMOC-NHS having other weight average molecular weights.
Example 5
PEGylation of IL-11 with 9-Hydroxymethyl-2,7-(bis-mPEG(20,000)-carboxyamide)- fluorene-N-hydroxysuccinimidyl Carbonate, 40kDa
Figure imgf000063_0001
Branched mPEG-FMOC-N-Hydroxysuccinimide Derivative, 40kDa,
("C2-PEG2-FMOC-NHS(40kDa)")
[0203] C2-PEG2-FMOC-NHS(40kDa), 40kDa, stored at -20 °C under argon, was warmed to ambient temperature. A three- fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed C2-PEG2-FMOC-NHS(40kDa) was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-11 solution (3.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well. The reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for two hours. The reaction was quenched by the addition of IM acetic acid to lower the pH to 5. The conjugate solution was characterized by SEC-HPLC (Figure 6). [0204] The PEGylation reaction yielded 95% mono-conjugate. A cation-exchange chromatography method using SP Sepharose High Performance column along with sodium acetate and sodium phosphate buffers was also used to purify the conjugates.
[0205] Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-11 conjugates under physiological condition.
[0206] Using this same approach, other conjugates can be prepared using C2-PEG2-
FMOC-NHS(40kDa) having other weight average molecular weights.
Example 6
PEGylation of IL-11 with 9-Hydroxymethyl-4-(mPEG(20,000)-carboxyamide)-7- (mPEG(20,000) amdioglutaric amide)fluorene-N-hydroxysuccinimidyl Carbonate,
40kDa
Figure imgf000064_0001
Branched mPEG-FMOC-N-Hydroxysuccinimide Derivative, 40kDa, ("CG-PEG2-FMOC-
NHS")
[0207] CG-PEG2-FMOC-NHS, 40kDa, stored at -20 °C under argon, was warmed to ambient temperature. A three- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-11 solution) of the warmed CG-PEG2-FMOC-NHS was dissolved in 2mM HC1 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (3.4 mg/ml in sodium phosphate buffer, pH 7.3) and mixed well. The reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for 2 hr first, and then at 4°C overnight. The reaction was quenched by the addition of 1M acetic acid to lower the pH to 5. The conjugate solution was characterized by SDS-PAGE (Figure 5). Based on the gel result, the reaction yielded 55% mono-conjugate and 30% di-conjugate.
[0208] Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
[0209] Using this same approach, other conjugates can be prepared using CG-PEG2-
FMOC-NHS having other weight average molecular weights.
Example 7
PEGylation of IL-11 with Linear mPEG-2-Hydroxy-pentanal Aldehyde Derivative,
20kDa
Figure imgf000065_0001
mPEG-2-hydroxy-pentanal Aldehyde Derivative, 20kDa, ("mPEG-HP-ALD-20K")
[0210] mPEG-HP-ALD, 20kDa, stored at -20°C under argon, was warmed to ambient temperature. A twenty to thirty- fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-HP-ALD was dissolved in Milli-Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. The reaction solution was kept at room temperature on a RotoMix for thirty minutes. The pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques. The reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C. The reaction was quenched with 0.1M HCl to pH 6. The conjugate solution was characterized by HPLC and SDS-PAGE.
[0211] The aldehyde group of mPEG-HP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent. Figure 7 shows the SEC-HPLC chromatogram of a conjugate solution. The PEGylation reaction yielded 29% 1 -mer (mono-conjugate, or one PEG attached to one IL-1 1 molecule). [0212] A cation-exchange chromatography method using SP Sepharose High
Performance column along with sodium phosphate buffers was also used to purify the conjugates.
[0213] Using this same approach, other conjugates can be prepared using mPEG-HP-
ALD having other weight average molecular weights.
Example 8
PEGylation of IL-11 with Linear mPEG-2-Hydroxy-methylamino-pentanal Aldehyde
Derivative, 20kDa
I OH
mPEG-2-Hydroxy-methylamino-pentanal Aldehyde Derivative, 20kDa,
("mPEG-MAHP-ALD-20K")
[0214] mPEG-MAHP-ALD, 20kDa, stored at -20°C under argon, was warmed to ambient temperature. A five to ten-fold excess (relative to the amount of IL-11 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-MAHP-ALD was dissolved in Milli-Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4.5 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. The reaction solution was kept at room temperature on a RotoMix for thirty minutes. The pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques. The reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C. The reaction was quenched with 0.1M HC1 to pH 6. The conjugate solution was characterized by HPLC and SDS-PAGE.
[0215] The aldehyde group of mPEG-MAHP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent. Figure 8 shows the SEC-HPLC chromatogram of a conjugate solution. The
PEGylation reaction yielded 49% 1-mer (mono-conjugate, or one PEG attached to one IL-11 molecule). [0216] A cation-exchange chromatography method using SP Sepharose High
Performance column along with sodium phosphate buffers was also used to purify the conjugates.
[0217] Using this same approach, other conjugates can be prepared using mPEG-
MAHP-ALD having other weight average molecular weights.
Example 9
PEGylation of IL-11 with Linear mPEG-2-hydroxy-piperazine-pentanal Aldehyde
Derivative, 20kDa
Figure imgf000067_0001
mPEG-2-hydroxy-piperazine-pentanal aldehyde Derivative, 20kDa, ("mPEG-PipHP-ALD-20K")
[0218] mPEG-PipHP-ALD, 20kDa, stored at -20°C under argon, was warmed to ambient temperature. A twenty- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-PipHP-ALD was dissolved in Milli-Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. The reaction solution was kept at room temperature on a RotoMix for thirty minutes. The pH of the reaction mixture was determined and adjusted to pH 9 using conventional techniques. The reaction solution was then placed on a RotoMix overnight to facilitate conjugation at room temperature, or at 4°C. The reaction was quenched with 0.1M HC1 to pH6. The conjugate solution was characterized by HPLC and SDS-PAGE.
[0219] The aldehyde group of mPEG-PipHP-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine without reducing agent. Figure 9 shows the SEC-HPLC chromatogram of a conjugate solution. The PEGylation reaction yielded 49% 1-mer (mono-conjugate, or one PEG attached to one IL-11 molecule). [0220] A cation-exchange chromatography method using SP Sepharose High
Performance column along with sodium phosphate buffers was also used to purify the conjugates.
[0221] Using this same approach, other conjugates can be prepared using mPEG-
PipHP-ALD having other weight average molecular weights.
Example 10
PEGylation of IL-11 with Linear mPEG-Propionaldehyde Derivative, 20kDa
o
II
H3C-(OCH2CH2)n-0-CH2CH2-CH
Linear mPEG-Propionaldehyde Derivative, 20kDa ("mPEG-ALD")
[0222] mPEG-ALD, 20kDa, stored at -20 °C under argon, was warmed to ambient temperature. A ten to twenty- fold excess (relative to the amount of IL-1 in a measured aliquot of the stock IL-1 1 solution) of the warmed mPEG-ALD was dissolved in MiUi-Q H20 to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-11 solution (4.4 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. The pH of the solution was adjusted to pH 9 using conventional techniques. The reaction solution was kept at room temperature on a RotoMix for thirty minutes. A reducing agent, sodium cyanoborohydride was then added to make 10 mM NaCNBH3. The reaction solution was well mixed and was kept at room temperature or 4°C overnight.
[0223] The aldehyde group of mPEG-ALD can react with the primary amines associated with IL-1 1 and covalently bond to them via secondary amine upon reduction by sodium cyanoborohydride. Figure 10 shows the SEC-HP LC chromatogram of the conjugate solution. The PEGylation reaction yielded 49% 1-mer (one PEG attached to IL-1 1 or mono- conjugate).
[0224] Using this same approach, other conjugates can be prepared using mPEG-ALD having other weight average molecular weights. Example 11
PEGylation of IL-11 with 9-hydroxymethyl-2,7-di[M-PEG(10,000)-carboxamido] fiuorene-N-Hydroxysuccinimide Derivative, 20kDa
Figure imgf000069_0001
Branched mPEG-FMOC-N-Hydroxysuccinimide Derivative, 20kDa,
C*C2-PEG2-FMOC-NHS(20kDa)")
[0225] C2-PEG2-FMOC-NHS(20kDa), 20kDa, stored at -20°C under argon, was warmed to ambient temperature. A four- fold excess (relative to the amount of IL-1 1 in a measured aliquot of the stock IL-1 1 solution) of the warmed C2-PEG2-FMOC-NHS(20kDa) was dissolved in 2mM HCl to form a 10% reagent solution. The 10% reagent solution was quickly added to the aliquot of stock IL-1 1 solution (5.2 mg/ml in PBS buffer with 5% sorbital, pH 7.3) and mixed well. The reaction solution was placed on a Slow Speed Lab Rotator (RotoMix) at room temperature for one hour. The reaction was quenched by the addition of 0.1 M hydrochloric acid to lower the pH to 6. The conjugate solution was characterized by SEC-HPLC. The PEGylation reaction yielded 60-70% mono-conjugate. A cation-exchange chromatography method using CM Sepharose High Performance column along with sodium phosphate buffers was also used to purify the conjugates (Figure 1 1).
[0226] Due to the degradable linkage in the PEG structure, the PEGs are releasable from the PEG-IL-1 1 conjugates under physiological condition.
[0227] Using this same approach, other conjugates can be prepared using C2-PEG2-
FMOC-NHS(20kDa) having other weight average molecular weights. Example 12
PEGvIation of rIL-11 with
Linear mPEG-Succinimidyl a-Methylbutanoate Derivative, 30kDa
Figure imgf000070_0001
mPEG-Succinimidyl a-Methylbutanoate Derivative, 30kDa ("mPEG-SMB")
[0228] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rIL-11 concentration is 2.5 mg/ml. mPEG-SMB, 30kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10 - 50 mol equivalents of the rIL-11 to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rIL-11 solution and stirred for 5 - 18 hours at room temperature thereby resulting in a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration.
[0229] The mPEG-SMB derivative is found to provide a sterically hindered active
NHS ester, which selectively reacts with lysine and terminal amines.
[0230] Using this same approach, other conjugates are prepared using mPEG-SMB having other weight average molecular weights.
Example 13
PEGvIation of rIL-11 with mPEG-PIP, 20kDa
The basic structure of the polymeric reagent is provided below:
Figure imgf000070_0002
(hydrated form) [0232] PEGylation reactions are designed such that after addition of all the reaction components and buffers, the final rIL-1 1 concentration is 2.5 mg/ml. mPEG-PIP, 20kDa, stored at -20 °C under argon, is warmed to ambient temperature. A quantity of the PEG reagent equal to 10 - 50 mol equivalents of the rIL-1 1 to be PEGylated is weighed out and dissolved in 20mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to the aliquot of stock rIL-11 solution and stirred for 15 - 30 minutes. A reducing agent, sodium cyanoborohydride (NaCNBH ), is then added at 10 - 100 molar excess relative to the PEG reagent and the reaction stirred for 5 - 18 hours at room temperature to ensure coupling via a secondary amine linkage (to a secondary carbon) to thereby form a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration.
[0233] The ketone group of mPEG-PIP is found to react with the primary amines associated with rIL-1 1 and covalently bond to them via a secondary amine upon reduction by a reducing reagent such as sodium cyanoborohydride.
[0234] Using this same approach, other conjugates are prepared using mPEG-PIP having other weight average molecular weights.
Example 14
IL-11 Bioassay of IL-11 and PEGylated IL-11 conjugates
[0235] PEG-IL-11 conjugates were tested for IL-1 1 activity in a bioassay. Briefly, 3 to 4 days prior to assay, enhanced IL-1 1 responsive cells (prepared by obtaining commercially available IL-1 1 -sensitive cells from R&D Systems, Minneapolis MN and subcloning these cells to obtain a more highly IL-1 1 responsive cell line) were seeded at 5xl04 cells/mL in complete growth medium (RPMI-1640 supplemented with 10% heat-inactivated FBS and 50 μΜ 2-mercaptoethanol) containing 10 ng/mL IL-1 1 (R&D systems). On assay day, cells were counted using a ViCell counter (Beckman Coulter) and centrifuged in a tabletop centrifuge at 1200 rpm for five minutes. The supernatant was discarded and the cells were washed three times by resuspension in 30 mL of IX Dulbecco's phosphate-buffered saline (D-PBS; Life Technologies) and centriiugation at 1200 rpm for five minutes. After the final wash, the cells were resuspended in complete growth medium without IL- 1 1, counted again, and diluted to a concentration of l l 06 cells/mL. In between washes, serial dilutions of the PEG-IL-11 conjugates were performed. To start the bioassay, 50 ΐ, of diluted cells were added into white, clear-bottom 96-well assay plates (Perkin Elmer VisiPlate), followed by 50 μΐ. of diluted IL-1 1 conjugates. The assay plates were then incubated for 24 hours in a 37°C, 5% C02 water-jacketed incubator. After 24 hours of incubation, the assay plates were removed from the incubator, 100 of Cell TiterGlo reagent (Promega) was added into each well, and the plates incubated at room temperature for ten minutes. The assay plates were read on a TopCount plate reader (Perkin Elmer) to quantify luminescent signal. EC50 values were calculated by non-linear regression curve fitting (GraphPad). To simplify comparisons between conjugates, all concentrations refer to the mass of the IL-11 moiety, rather than the entire conjugate. Calculated EC50S for stable conjugates are shown in Table 4, below, and representative dose response curves in Figure 12.
Table 4
Figure imgf000072_0001
[0236] Forced-release of releasable PEG conjugates. Releasable PEG conjugates were force-released in 200 mM Tris, pH 9.0, for 24 hours at 37°C. Released conjugates were then immediately tested for bioactivity as described in the bioassay method. Calculated EC50S for releasable conjugates are shown in Table 5, below, and representative dose response curves in Figure 13. Table 5
Summar of EC50 Values of Releasable Con u ates in the IL-1 1 Bioassa
Figure imgf000073_0001
Example 15
In vivo anti-inflammatory activity of PEGylated-IL-11 conjugates
[0237] The in vivo anti-inflammatory activity of IL- 1 1 and PEGylated-IL- 1 1 conjugates was demonstrated using an LPS-induced TNFa-expression model as described by Trepicchio et al (J Immunol 157:3627, 1996). Briefly, male C57BL/6 mice were pretreated (i.p.) with PBS, IL-11 (100 g/kg), or PEGylated-IL-11 conjugates (mono conjugates of Example 8, mono conjugates of Example 7 or mono conjugates of Example 11) (100 or 500 μ kg). The releasable conjugate of Example 1 1 was assayed following forced-release (see Example 14, above), as well as in its native state. Four hours later, the mice were injected i.p. with 2 mg/kg of E. coli LPS (Sigma) or vehicle (PBS). Two hours later, blood was collected by cardiac puncture and serum separated by centrifugation and stored at -80° C until ready for analysis by ELISA. LPS-induced plasma TNFa was measured using a mouse TNFa-specific ELISA (Pierce ThermoScientific) according to the manufacturer's instructions.
[0238] As shown in Figure 14, the IL-1 1 and PEGylated-IL- 11 conjugates suppressed production of murine TNFa in response to LPS. Though not statistically significant, the data suggests PEGylated-IL- 1 1 conjugates may be more effective than IL-1 1 itself in this inflammation model. There was little apparent difference between the mono conjugate of Example 1 1 in its "native" state or following forced-release of IL-1 1 from the conjugate prior to administration. This is consistent with the rapid release of this conjugate under
physiological conditions in plasma.

Claims

wnat is ciaimea is:
1. A conjugate comprising a residue of an IL-11 moiety covalently attached to a water-soluble polymer.
2. The conjugate of claim 1 , wherein the IL-1 1 moiety covalently attached to the water-soluble polymer is covalently attached via a releaseable linkage.
3. The conjugate of claim 1 , wherein the IL-1 1 moiety covalently attached to the water-soluble polymer is covalently attached via a stable linkage.
4. The conjugate of any one of claims 1, 2 and 3, wherein the water-soluble polymer is a branched water-soluble polymer.
5. The conjugate of any one of claims 1, 2, 3 and 4, wherein the water-soluble polymer is a polymer selected from the group consisting of poly(alkylene oxide), poly( inyl pyrrolidone), poly( vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).
6. The conjugate of claim 5, wherein the water-soluble polymer is a poly(alkyIene oxide).
7. The conjugate of claim 6, wherein the poly(alkylene oxide) is a polyethylene glycol).
8. The conjugate of claim 7, wherein the poly( ethylene glycol) is terminally capped with an end-capping moiety selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy and substituted aryloxy.
9. The conjugate of any one of claims 1 , 2, 3, 4, 5, 6 and 7, wherein the water-soluble polymer has a weight- average molecular weight in a range of from about 500 Daltons to about 100,000 Daltons.
78
10. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the conjugate is covalently attached at an amine group of the residue of the IL-1 1 moiety.
1 1. The conjugate of any one of claims 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one, two, three or four water-soluble polymers are attached to the residue of the IL-1 1 moiety.
12. The conjugate of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one, two or three water-soluble polymers are attached to the residue of the IL-1 1 moiety.
13. The conjugate of any one of claims 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one or water-soluble polymers are attached to the residue of the IL-11 moiety.
14. The conjugate of any one of claims 1 , 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein one water-soluble polymer is attached to the residue of the IL-1 1 moiety.
15. A conjugate comprising a residue of an IL-1 1 moiety covalently attached to a water-soluble polymer, wherein the water-soluble polymer, prior to being covalently attached, is a polymeric reagent bearing an N-hydroxysuccinimidyl group.
16. A pharmaceutical composition comprising a conjugate of any one of claims 1 through 15 and a pharmaceutically acceptable excipient.
17. A method comprising administering to an individual a pharmaceutical composition of claim 16.
18. A method for making a conjugate comprising contacting, under conjugation conditions, an IL-1 1 moiety with a polymeric reagent.
- 79 -
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