Conjugate of IL-2 portion and polymer

Abstract

To provide conjugates of an IL-2 moiety and a polymer.SOLUTION: Provided is a conjugate of an IL-2 moiety and one or more nonpeptidic, water-soluble polymers. Typically, the nonpeptidic water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are a composition comprising the conjugate, a method for preparing the conjugate, a method for administering the composition to an individual, as well as a nucleic acid sequence, an expression system, a host cell and a method for preparing the IL moiety. In one or more embodiments of the invention, a conjugate comprising a residue of an IL-2 moiety covalently attached to a water-soluble polymer, where the residue of the IL-2 moiety is covalently attached to the water-soluble polymer via a releasable linkage.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 61 / 413,236, filed on November 12, 2010, under 35 U.S.C. § 119(e), the disclosure of which is hereby incorporated by reference in its entirety.

[0002] In particular, one or more embodiments of the present invention generally relate to conjugates comprising an IL - 2 moiety (i.e., a moiety having at least some activity similar to human IL - 2) and a polymer. In addition, the present invention relates to compositions (especially those) comprising the conjugate, methods of synthesizing the conjugate, and methods of administering the composition.

Background Art

[0003] In healthy humans, the immune system can distinguish between normal cells and cancerous cells. When a given cell is identified as cancerous, the immune system typically eliminates it. Thus, when the immune system is broken or overwhelmed, the impaired immune system cannot distinguish cancer cells and, thus, cannot eliminate them, allowing cancer to progress. In patients suffering from cancer, administration of immunomodulatory proteins to the patient can help to (at least in part) normalize the patient's immune system and restore the person's ability to eliminate cancer cells. In this way, the progression of cancer can be slowed or even eliminated.

[0004] One such immunomodulatory protein used in the treatment of patients suffering from certain cancers is interleukin - 2. Interleukin - 2 (IL - 2) is a naturally occurring cytokine and has activity both as a stimulator of natural killer cells (NK cells) and as an inducer of T - cell proliferation. In its non - glycosylated form, IL - 2 has a molecular weight of approximately 15,300 daltons (however, IL - 2 exists in various glycosylated forms in vivo).

[0005] Aldesleukin, a commercially available non-glycosylated recombinant human IL-2 product (available from Prometheus Laboratories Inc., San Diego, CA under the PROLEUKIN® brand of des-alanyl-1, serine-125 human interleukin-2), is approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for administration to patients suffering from or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer and bladder cancer.

[0006] However, even at recommended doses, aldesleukin can cause severe side effects including capillary leak syndrome (CLS) and neutrophil dysfunction. Considering the potential for such severe side effects and the recommended treatment cycle of 14 doses by intravenous infusion over 15 minutes every 8 hours, the administration of aldesleukin is carried out in a clinical setting. Furthermore, commercially available formulations of aldesleukin contain the presence of sodium dodecyl sulfate, a substance thought to be necessary for maintaining optimal activity due to the stability of the three-dimensional structure. See Non-Patent Document 1.

[0007] Attempts have been made to address concerns about the toxicity of IL-2. In one approach, formulation methods have been attempted. See, for example, Patent Document 1, Patent Document 2 and Patent Document 3. In another approach, specific conjugates of IL-2 have been proposed. See, for example, Patent Document 4, Patent Document 5, Patent Document 6 and Patent Document 7.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

[0009] [Non-Patent Document 1] Arakawa et al. Int.J.Peptide Protein Res.(1994)43:583-587 [Overview of the project] [Problems that the invention aims to solve]

[0010] However, despite such approaches, there is still a need for IL-2 conjugates. Therefore, in particular, one or more embodiments of the present invention relating to conjugates, compositions containing conjugates, and related methods as described herein appear to be novel and never before proposed in the art. [Means for solving the problem]

[0011] Accordingly, in one or more embodiments of the present invention, a conjugate is provided which comprises a residue of an IL-2 moiety covalently bonded to a water-soluble polymer.

[0012] In one or more embodiments of the present invention, a conjugate is provided comprising a residue of an IL-2 moiety covalently bonded to a water-soluble polymer, wherein the residue of the IL-2 moiety covalently bonded to the water-soluble polymer via a releaseable linkage.

[0013] In one or more embodiments of the present invention, a conjugate is provided which comprises a residue of an IL-2 moiety covalently bonded to a water-soluble polymer, wherein the IL-2 moiety is a precursor IL-2 moiety.

[0014] In one or more embodiments of the present invention, a conjugate is provided which comprises a residue of an IL-2 moiety covalently bonded to a water-soluble polymer, wherein the IL-2 moiety is a non-precursor IL-2 moiety.

[0015] In one or more embodiments of the present invention, a method for delivering a conjugate is provided, comprising the step of subcutaneously administering a composition comprising a conjugate of an IL-2 residue and a water-soluble polymer to a patient.

[0016] In one or more embodiments of the present invention, an isolated nucleic acid molecule is provided, which encodes an IL-2 moiety, wherein the nucleic acid molecule comprises a sequence having substantial (e.g., at least 80%) sequence identity with the sequence shown in Sequence ID No. 5.

[0017] In one or more embodiments of the present invention, an expression vector is provided which comprises a nucleic acid molecule provided herein (e.g., an in vitro expression vector).

[0018] In one or more embodiments of the present invention, a host cell (e.g., an in vitro host cell) is provided, which comprises an expression vector as provided herein. For example, the present invention provides the following items: (Item 1) A conjugate containing a residue of the IL-2 moiety covalently bonded to a water-soluble polymer. (Item 2) The conjugate according to item 1, wherein the IL-2 portion covalently bonded to the water-soluble polymer is covalently bonded via a releaseable linkage. (Item 3) The conjugate according to item 1, wherein the IL-2 portion covalently bonded to the water-soluble polymer is covalently bonded via a stable linkage. (Item 4) The conjugate according to any one of items 1 to 3, wherein the water-soluble polymer is a branched-chain water-soluble polymer. (Item 5) The conjugate according to any one of items 1 to 4, wherein the water-soluble polymer is a polymer selected from the group consisting of poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine). (Item 6) The conjugate described in item 5, wherein the water-soluble polymer is poly(alkylene oxide). (Item 7) The conjugate according to item 6, wherein the poly(alkylene oxide) is poly(ethylene glycol). (Item 8) The conjugate according to item 7, wherein the poly(ethylene glycol) is capped at the ends with end cap portions selected from the group consisting of hydroxy, alkoxy, substituted alkoxy, alkenoxy, substituted alkenoxy, alkynoxy, substituted alkynoxy, aryloxy, and substituted aryloxy. (Item 9) The conjugate according to any one of items 1 to 7, wherein the water-soluble polymer has a weight-average molecular weight in the range of about 500 daltons to about 100,000 daltons. (Item 10) The conjugate according to any one of items 1 to 10, wherein the conjugate is covalently bonded to the amine group of the residue of the IL-2 portion. (Item 11) A conjugate according to any one of items 1 to 10, wherein one, two, three, or four water-soluble polymers are bound to the residue of the IL-2 moiety. (Item 12) The conjugate according to any one of items 1 to 10, wherein one, two, or three water-soluble polymers are bound to the residue of the IL-2 moiety. (Item 13) A conjugate according to any one of items 1 to 10, wherein one or a water-soluble polymer is bound to the residue of the IL-2 moiety. (Item 14) A conjugate according to any one of items 1 to 10, wherein one water-soluble polymer is bound to a residue of the IL-2 moiety. (Item 15) A conjugate comprising a residue of an IL-2 moiety covalently bonded to a water-soluble polymer, wherein the water-soluble polymer is a polymer reagent having an N-hydroxysuccinimidyl group before covalent bonding. (Item 16) A pharmaceutical composition comprising a conjugate described in any one of items 1 to 15 and a pharmaceutically acceptable excipient. (Item 17) A method comprising the step of administering a pharmaceutical composition described in item 16 to an individual. (Item 18) A method for preparing a conjugate, comprising the step of contacting the IL-2 moiety with a polymer reagent under conjugate formation conditions. (Item 19) An isolated nucleic acid molecule encoding the IL-2 portion, comprising a sequence having at least 95% sequence identity with the sequence shown in Sequence ID No. 5. (Item 20) DNA, the nucleic acid molecule described in item 19. (Item 21) An expression vector containing the nucleic acid molecule described in item 19. (Item 22) A plasmid, an expression vector as described in item 21. (Item 23) In vitro host cells containing the vector described in item 22. (Item 24) A method comprising the steps of: placing a protein into a dialysis bag having a pore diameter smaller than the size of the protein to form a dialysis bag containing the protein; and subjecting the dialysis bag containing the protein to a protein denaturant-free solution. (Item 25) A composition comprising an IL-2 moiety, 5-15 mM sodium acetate, and 2-7% trehalose. [Brief explanation of the drawing]

[0019] [Figure 1] The DNA sequence of the gene described in Example 1 and the resulting amino acid sequence are provided. [Figure 2-1] This figure shows a typical chromatogram after cation exchange chromatography of [mPEG2-C2-fmoc-20K]-[rIL-2] prepared according to the procedure described in Example 2. [Figure 2-2] As further described in Example 2, this is a chromatogram of [mPEG2-C2-fmoc-20K]-[rIL-2] after reverse-phase HPLC analysis. [Figure 2-3] This is a plot of the emission profiles of various conjugates prepared according to the procedure described in Example 2. [Figure 3-1] This figure shows a typical chromatogram after cation exchange chromatography of [mPEG2-CAC-fmoc-20K]-[rIL-2] prepared according to the procedure described in Example 3. [Figure 3-2] As further described in Example 3, this is a chromatogram of [mPEG2-CAC-fmoc-20K]-[rIL-2] after reverse-phase HPLC analysis. [Figure 3-3] This figure shows the results after MALDI-TOF analysis of various conjugates prepared according to the procedure described in Example 3. [Figure 4-1] This figure shows a typical chromatogram after cation exchange chromatography of [mPEG2-ru-20K]-[rIL-2] prepared according to the procedure described in Example 4. [Figure 4-2] As further described in Example 4, this is a chromatogram of [mPEG2-ru-20K]-[rIL-2] after reverse-phase HPLC analysis. [Figure 5]As further described in Example 5, this is a diagram of the chromatogram after cation exchange chromatography of [mPEG2-ru-40K]-[rIL-2]. [Figure 6] As further described in Example 6, this is a figure of the chromatogram after cation exchange chromatography of [mPEG2-ru-4K]-[rIL-2]. [Figure 7] As further described in Example 11, a plot of CTLL-2 cell proliferation in response to aldesleukin and stable [mPEG2-ru-20K]-[rIL-2] is shown. The data points are the mean values ​​from one experiment measured in triplicates. Error bars represent the standard error of the mean. [Figure 8] As further described in Example 11, plots of CTLL-2 cell proliferation in response to aldesleukin, released and unreleased [mPEG2-C2-fmoc-20K]-[rIL-2] and [mPEG2-CAC-fmoc-20K]-[rIL-2] are shown. Data points are the mean values ​​from one experiment measured in triplicates. Error bars represent the standard error of the mean. [Figure 9] As further described in Example 12, the concentration-time curves after a single injection in mice are plotted. [Figure 10] As further described in Example 13, plots of the total lesion area (mm2) for several test compounds are shown. [Figure 11A] Figures 11A and 11B are plots showing the tumor growth inhibition duration curves of the test substance under various administration schemes, as further described in Example 14. [Figure 11B] Figures 11A and 11B are plots showing the tumor growth inhibition duration curves of the test substance under various administration schemes, as further described in Example 14. [Modes for carrying out the invention]

[0020] Before describing in detail one or more embodiments of the present invention, it should be understood that the invention is not limited to any particular polymer, synthesis method, IL-2 moiety, etc., and therefore may vary.

[0021] It should be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context specifically indicates otherwise. For example, "a polymer" includes a single polymer as well as two or more identical or different polymers, and "an optional excipient" refers to a single optional excipient as well as two or more identical or different optional excipients.

[0022] In the description of one or more embodiments of the present invention and in the claims, the following technical terms shall be used in accordance with their definitions below.

[0023] "PEG," "polyethylene glycol," and "poly(ethylene glycol)" are synonymous as used herein and encompass any non-peptide water-soluble poly(ethylene oxide). Typically, PEG used in accordance with the present invention has the following structure "-(OCH2CH2) n - (wherein (n) is 2 to 4000) is included. When used herein, PEG also includes "-CH2CH2-O(CH2CH2O)" depending on whether the terminal oxygen was substituted, for example, during the synthetic conversion. n -CH2CH2-" and "-(OCH2CH2) nThis also includes "O-". Throughout this specification and the claims, it should be noted that the term "PEG" includes structures having various terminal groups or "end-cap" groups, etc. The term "PEG" also means polymers containing a majority, i.e., more than 50%, of -OCH2CH2- repeating subunits. In terms of specific forms, PEG may have various molecular weights, as well as any of the structures or geometric shapes such as "branched," "linear," "fork-shaped," and "polyfunctional," which are described in further detail below.

[0024] The terms “end-capped” and “terminus capped” are used synonymously herein and refer to the state in which the end or endpoint of a polymer has an end-capped portion. Typically, although not required, the end-capped portion consists of a hydroxyl group or C 1~20 Alkoxy group, more preferably C 1~10 Alkoxy group, more preferably C 1~5 It contains an alkoxy group. Therefore, examples of end cap portions include alkoxy (e.g., methoxy, ethoxy, and benzyloxy), as well as aryl, heteroaryl, cyclo, and heterocyclo groups. It should be noted that the end cap portion may contain one or more atoms of the terminal monomers in the polymer [e.g., CH3O(CH2CH2O)]. n - and CH3(OCH2CH2) n- The end cap portion in the [methoxy]. In addition, saturated, unsaturated, substituted, and unsubstituted forms of each of the above are conceivable. Furthermore, the end cap group may be a silane. The end cap group may also advantageously include a detectable label. When the polymer has an end cap group containing a detectable label, the amount or location of the polymer and / or the portion to which the polymer is bound (e.g., an active agent) can be determined using a suitable detector. Such labels include, without limitation, fluorescent agents, chemiluminescent agents, portions used for enzyme labeling, colorimetric (e.g., dyes), metal ions, and radioactive portions. Suitable detectors include photometers, films, and spectrometers. The end cap group may also advantageously include a phospholipid. When the polymer has an end cap group containing a phospholipid, the polymer and the resulting conjugate are given specific properties. 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 cap group may also include a target moiety so that the polymer (and anything that binds to it, e.g., the IL-2 moiety) can be selectively localized to the desired range.

[0025] With respect to polymers as described herein, "not naturally occurring" means a polymer that is not found in nature in its original form. However, a polymer not naturally occurring may include one or more naturally occurring monomers or monomer segments, as long as the polymer structure as a whole is not found in nature.

[0026] The term "water-soluble polymer," as found in "water-soluble polymer," refers to any polymer that is soluble in water at room temperature. Typically, water-soluble polymers transmit at least about 75%, more preferably at least about 95%, of the light transmitted by the same solution after filtration. Water-soluble polymers can preferably dissolve in water at least about 35% (by weight), more preferably at least about 50% (by weight), even more preferably about 70% (by weight), and even more preferably about 85% (by weight). However, most preferably, water-soluble polymers can dissolve in water at about 95% (by weight), or can dissolve completely in water.

[0027] The molecular weight of water-soluble polymers, such as PEG, can be expressed as either a number-average molecular weight or a weight-average molecular weight. Unless otherwise specified, all instances of "molecular weight" in this specification refer to the weight-average molecular weight. Both number-average and weight-average molecular weights can be measured using gel permeation chromatography or other liquid chromatography methods. Alternatively, the number-average molecular weight may be determined using other methods for measuring molecular weight, such as end-group analysis or by measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure), or the weight-average molecular weight may be determined using light scattering, ultracentrifugation, or viscometric methods. The polymers of the present invention are typically polydisperse (i.e., the number-average molecular weight and weight-average molecular weight of the polymer are not equal) and preferably have a low polydispersity value of less than about 1.2, more preferably less than about 1.15, even more preferably less than about 1.10, still more preferably less than about 1.05, and most preferably less than about 1.03.

[0028] The terms “active,” “reactive,” or “activated” refer to reactive functional groups that readily react with electrophiles or nucleophiles on other molecules, when used in conjunction with specific functional groups. This is in contrast to groups that require a strong catalyst or extremely impractical reaction conditions to react (i.e., “unreactive” or “inactive” groups).

[0029] As used herein, the term “functional group” or any of its synonyms is intended to encompass both its protected and unprotected forms.

[0030] The terms “spacer portion,” “linking,” and “linker” are used herein to refer to bonds or atoms or atomic assemblies used, for example, to optionally link the ends of a polymer segment to an IL-2 moiety or an interconnection portion of an IL-2 moiety with an electrophile or nucleophile. Spacer portions may be stable to hydrolysis, or they may include links that are physiologically hydrolyzable or enzymatically digestible. Unless otherwise specifically indicated in the context, spacer portions may optionally be present between any two elements of a compound (for example, the provided conjugates, comprising residues of an IL-2 moiety and a water-soluble polymer, may be linked directly or indirectly via spacer portions).

[0031] "Alkyl" refers to a hydrocarbon chain, typically with an atomic length ranging from about 1 to 15 atoms. Such hydrocarbon chains are preferably saturated, though not essential, and may be branched or linear, although typically linear is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, and 3-methylpentyl. As used herein, "alkyl" includes cycloalkyl and cycloalkylene-containing alkyl groups.

[0032] "Lower alkyl" refers to alkyl groups containing 1 to 6 carbon atoms, which may be linear or branched, as exemplified by methyl, ethyl, n-butyl, i-butyl, and t-butyl.

[0033] "Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain, including cross-linked, condensed, or spiro-cyclized compounds, preferably composed of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms. "Cycloalkylene" refers to a cycloalkyl group inserted into an alkyl chain by bonding a chain with any two carbons in a cyclic ring system.

[0034] "Alkoxy" is a -OR group, where R is alkyl or substituted alkyl, preferably C 1~6 -OR groups that are alkyl (e.g., methoxy, ethoxy, propyloxy, etc.).

[0035] The term "substituted", as in "substituted alkyl", refers to a moiety (e.g., an alkyl group) substituted with one or more non-interfering substituents, including but not limited to alkyl, C 3~8 Cycloalkyl, such as cyclopropyl, cyclobutyl, etc.; halo, such as fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; etc. "Substituted aryl" is aryl having one or more non-interfering groups as substituents. For substitution on a phenyl ring, the substituents may be in any orientation (i.e., ortho, meta, or para).

[0036] "Non-interfering substituents" are groups that, when present in a molecule, typically do not have reactivity with other functional groups contained in that molecule.

[0037] "Aryl" means one or more aromatic rings, each having 5 or 6 central carbon atoms. Aryl includes multiple aryl rings that may be fused, as in naphthyl, or unfused, as in biphenyl. The aryl rings may also be fused or unfused with one or more cyclic hydrocarbons, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0038] A "heteroaryl" is an aryl group containing 1 to 4 heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. The heteroaryl ring may also be fused with one or more cyclic hydrocarbons, heterocyclic rings, aryl rings, or heteroaryl rings.

[0039] A "heterocyclic ring" or "heterocyclic formula" refers to one or more rings of 5 to 12 atoms, preferably 5 to 7 atoms, which may or may not have unsaturated or aromatic properties, and which have at least one ring atom other than carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0040] A "substituted heteroaryl" is a heteroaryl molecule that has one or more non-interfering groups as substituents.

[0041] A "substituted heterocycle" is a heterocycle having one or more side chains formed from non-interfering substituents.

[0042] As used herein, "organic radical" includes akyl, substituted alkyl, aryl, and substituted aryl.

[0043] "Electrophile" and "electrophile group" refer to ions or atoms or sets of atoms that have an electrophilic center, i.e., a center that attracts electrons, and are capable of reacting with a nucleophile.

[0044] "Nucleophile" and "nucleophile group" refer to an ion or an ionizable atom or group of atoms that has a nucleophilic center, i.e., a center that withdraws from an electrophile, or that is accompanied by an electrophile.

[0045] A "physiologically cleavable," "hydrolyzable," or "degradable" bond is one that reacts with water under physiological conditions (i.e., is hydrolyzed). Whether a bond is easily hydrolyzed in water can depend not only on the general type of linkage connecting the two central atoms, but also on the substituents bonded to those central atoms. Suitable links that are unstable to or easily hydrolyzed include, but are not limited to, carboxylic acid esters, phosphate esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, and oligonucleotides.

[0046] "Enzyme-degradable linkage" refers to a linkage that can be broken down by one or more enzymes.

[0047] A "hydrolysis-stable" linkage or bond refers to a chemical bond that is substantially stable in water, that is, one that does not undergo any noticeable degree of hydrolysis over long periods under physiological conditions, typically a covalent bond. Examples of hydrolysis-stable links include, but are not limited to, carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, and urethanes. Generally, a hydrolysis-stable linkage exhibits a hydrolysis rate of less than approximately 1-2% per day under physiological conditions. For typical hydrolysis rates of chemical bonds, refer to many standard chemistry textbooks.

[0048] "Pharmacologically acceptable excipients or carriers" means excipients that may be included in the compositions of the present invention and that do not cause any significant toxic adverse effects to patients. "Pharmacologically effective amount," "physiologically effective amount," and "therapeutic effective amount" are used synonymously herein and mean the amount of polymer-(IL-2) moiety conjugate required to bring the desired level of conjugate (or the corresponding unconjugated IL-2 moiety) into the bloodstream or target tissue. The exact amount depends on a number of factors, such as the specific IL-2 moiety, the components and physical properties of the therapeutic composition, the target patient population, and individual patient considerations, and a person skilled in the art can easily determine it based on the information provided herein.

[0049] "Polyfunctional" means a polymer containing three or more functional groups, where the functional groups may be the same or different. The polyfunctional polymer reagents of the present invention typically contain about 3 to 100 functional groups, or 3 to 50 functional groups, or 3 to 25 functional groups, or 3 to 15 functional groups, or 3 to 10 functional groups within the polymer backbone, or contain 3, 4, 5, 6, 7, 8, 9, or 10 functional groups.

[0050] As used herein, the term "IL-2 moiety" refers to a moiety having human IL-2 activity. The IL-2 moiety may also have at least one electrophile or nucleophile suitable for reaction with polymer reagents. In addition, the term "IL-2 moiety" encompasses both the IL-2 moiety before conjugate formation and the IL-2 moiety residues after conjugate formation. As will be further described below, those skilled in the art can determine whether any given moiety has IL-2 activity. An IL-2 moiety is a protein containing an amino acid sequence corresponding to any one of SEQ ID NOs: 1-4, and any protein or polypeptide substantially homologous thereto. As used herein, the term "IL-2 moiety" includes such proteins that have been intentionally modified, for example, by site-directed mutagenesis, or accidentally modified by mutation. These terms also include analogs having 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxyl terminus of the protein, which includes at least one glycosylation site, and analogs having an amino acid sequence containing at least one glycosylation site. These terms include both native and recombinantly produced parts.

[0051] The term "substantially homologous" means that a particular target sequence, such as a mutant sequence, differs from the reference sequence by only one or more substitutions, deletions, or additions, but the net effect is that no functionally detrimental difference occurs between the reference sequence and the target sequence. For the purposes of the present invention, sequences having homology of higher than 80 percent (more preferably higher than 85 percent, even more preferably higher than 90 percent, and most preferably higher than 95 percent), equivalent biological activity (not essential, but equivalent biological activity intensity), and equivalent expression characteristics are considered substantially homologous. For the purpose of determining homology, truncation of mature sequences is disregarded. The exemplary IL-2 moieties used herein include a substantially homologous sequence that is sequence number 2.

[0052] The term "fragment" means any protein or polypeptide having the amino acid sequence of a part of or a fragment of the IL-2 moiety, and possessing the biological activity of IL-2. Fragments include proteins or polypeptides produced by the proteolysis of the IL-2 moiety, as well as proteins or polypeptides produced by chemical synthesis by routine methods in the art.

[0053] The term "patient" refers to an organism that is suffering from or is susceptible to a condition that can be prevented or treated by the administration of an active drug (e.g., a conjugate), and includes both humans and animals.

[0054] "Optional" or "depending on circumstances" means that the following circumstances may or may not occur, and therefore the description includes both the cases in which the circumstances occur and the cases in which they do not.

[0055] "Substantially" means almost entirely or completely, for example, satisfying one or more of the following: more than 50%, 51% or more, 75% or more, 80% or more, 90% or more, and 95% or more of the condition.

[0056] As used herein, “sequence identity” is determined by comparing the sequence of a reference DNA sequence with the corresponding portion of another DNA sequence, where these sequences are aligned such that the overlap between the two sequences is maximized while minimizing sequence gaps, and any protruding sequences between the two sequences are ignored. With respect to any sequence identity described herein, preferably it is at least 80%, more preferably 85%, even more preferably 90%, and still more preferably 95%, with 96%, 97%, 98%, and 99% sequence identity being most preferred.

[0057] The amino acid residues in peptides are abbreviated as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile 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 Gln or Q; asparagine is Asn or N; lysine is Lys or K; 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.

[0058] In one or more embodiments of the present invention, a conjugate is provided comprising a residue of an IL-2 moiety covalently bonded (directly or via a spacer portion) to a water-soluble polymer. The conjugate of the present invention has one or more of the following features:

[0059] IL-2 part As mentioned above, this conjugate generally contains residues of the IL-2 moiety covalently bonded directly to the water-soluble polymer or via a spacer moiety. As used herein, the term "IL-2 moiety" refers to the IL-2 moiety before conjugate formation, as well as the IL-2 moiety after bonding with a non-peptide water-soluble polymer. However, it will be understood that when the original IL-2 moiety bonds with a non-peptide water-soluble polymer, the IL-2 moiety is slightly altered due to the presence of one or more covalent bonds associated with the bonding with the polymer. In many cases, this slightly altered form of the IL-2 moiety, resulting from bonding with another molecule, is referred to as a "residue" of the IL-2 moiety.

[0060] The IL-2 portion can be obtained from both non-recombinant and recombinant methods, and the present invention is not limited to this. In addition, the IL-2 portion can be derived from human sources, animal sources, and plant sources.

[0061] The IL-2 moiety can be non-recombinantly induced. For example, IL-2 can be isolated from biological systems and obtained from culture media by other means. See, for example, U.S. Patent No. 4,401,756 and the procedures described in Pauly et al. (1984) J. Immunol Methods 75(1):73-84.

[0062] The IL-2 portion can be derived from a recombinant method. See, for example, U.S. Patent No. 5,614,185, the disclosures and examples provided herein.

[0063] Any IL-2 portion obtained by non-recombinant or recombinant methods can be used as the IL-2 portion in the preparation of the conjugate described herein.

[0064] The IL-2 moiety can be expressed in bacterial (e.g., Escher coli, see Fischer et al. (1995) Biotechnol.Appl.BioIL-2m.21(3):295-311) expression systems, mammalian (e.g., see Kronman et al. (1992)Gene 121:295-304) expression systems, yeast (e.g., Pichia pastoris, see Morel et al. (1997) Biochem.J.328(1):121-129) expression systems, and plant (e.g., see Mor et al. (2001) Biotechnol.Bioeng.75(3):259-266) expression systems. Expression can occur either by exogenous expression (if the host cell naturally contains the desired genetic code) or endogenous expression.

[0065] While there may be differences in methods for preparing recombinant proteins, typically, recombinant methods involve constructing nucleic acids encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming host cells (e.g., plants, bacteria, yeast, transgenic animal cells, or mammalian cells such as Chinese hamster ovary cells or baby hamster kidney cells), and producing the desired polypeptide or fragment by nucleic acid expression. Methods for producing and expressing recombinant polypeptides in vitro in prokaryotic and eukaryotic host cells are known to those skilled in the art.

[0066] To facilitate the identification and purification of recombinant polypeptides, a nucleic acid sequence encoding an epitope tag or other affinity-binding sequence can be inserted or added in-frame to the coding sequence, thereby producing a fusion protein containing the desired polypeptide and a polypeptide suitable for binding. Identification and purification of the fusion protein can be performed by first passing a mixture containing the fusion protein through an affinity column having a binding portion (e.g., an antibody) to the epitope tag or other binding sequence in the fusion protein, thereby binding the fusion protein to the column. The fusion protein can then be recovered by washing the column with a suitable solution (e.g., an acid) to release the bound fusion protein. Recombinant polypeptides can also be purified by lysis of host cells, separation of polypeptides by, for example, ion-exchange chromatography, affinity-binding techniques, hydrophobic interaction techniques, and subsequently identified by MALDI or Western blotting, and the polypeptide recovered. These and other methods for identifying and purifying recombinant polypeptides are known to those skilled in the art. However, in one or more embodiments of the present invention, the IL-2 portion is not in the form of a fusion protein.

[0067] Depending on the system used for expressing the IL-2 activity-containing protein, the IL-2 moiety may or may not be glycosylated, and either can be used. That is, the IL-2 moiety may or may not be glycosylated. In one or more embodiments of the present invention, the IL-2 moiety is not glycosylated.

[0068] The IL-2 moiety can be advantageously modified to include and / or substitute one or more amino acid residues, such as lysine, cysteine, and / or arginine, thereby facilitating the polymer's bonding to atoms in the side chains of those amino acids. Examples of IL-2 moiety substitutions are described in U.S. Patent No. 5,206,344. In addition, the IL-2 moiety can be modified to include amino acid residues that do not exist naturally. Methods for adding amino acid residues and those that do not exist naturally are known to those skilled in the art. See J. March, Advanced Organic IL-2mistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley-Interscience, 1992).

[0069] In addition, the IL-2 moiety can be advantageously modified to include the attachment of a functional group (excluding the addition of an amino acid residue containing a functional group). For example, the IL-2 moiety can be modified to include a thiol group. In addition, the IL-2 moiety can be modified to include an N-terminal α-carbon. In addition, the IL-2 moiety can be modified to include one or more carbohydrate moieties. In addition, the IL-2 moiety can be modified to include an aldehyde group. In addition, the IL-2 moiety can be modified to include a ketone group. In some embodiments of the present invention, it is preferable that the IL-2 moiety is not modified to include one or more of the thiol group, N-terminal α-carbon, carbohydrate, aldehyde group, and ketone group.

[0070] Exemplary IL-2 moieties are described in the literature and, for example, in U.S. Patent Nos. 5,116,943, 5,153,310, 5,635,597, 7,101,965, and 7,567,215, and U.S. Patent Publication Nos. 2010 / 0036097 and 2004 / 0175337. Preferred IL-2 moieties include those having an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 4, and sequences substantially homologous thereto. A preferred IL-2 moiety has an amino acid sequence corresponding to SEQ ID NO. 3.

[0071] In some cases, the IL-2 moiety is in the form of a "monomer," where the single expression of the corresponding peptide is systematized as a distinct unit. In other cases, the IL-2 moiety is in the form of a "dimer," where two monomeric proteins are linked to each other (e.g., by a disulfide bond) (e.g., a recombinant IL-2 dimer). For example, in relation to recombinant human IL-2 dimers, the dimer can be in the form of two monomers linked to each other by a disulfide bond formed from the Cys125 residue of each monomer.

[0072] In addition, a precursor form of IL-2 can be used as the IL-2 portion. An exemplary precursor form of IL-2 has the sequence shown in SEQ ID NO: 1.

[0073] Truncates, hybrid mutants, and peptide mimes of any of the aforementioned sequences can also function as IL-2 moieties. Any of the aforementioned biologically active fragments, deletion mutants, substitution mutants, or addition mutants that maintain at least some degree of IL-2 activity can also function as IL-2 moieties.

[0074] It is possible to determine whether any given peptide or protein moiety possesses IL-2 activity. Various methods for determining in vitro IL-2 activity have been described in the art. An exemplary method is the CTTL-2 cell proliferation assay described in the following examples. The exemplary method is described in Moreau et al. (1995) Mol.Immunol. 32:1047-1056. Briefly, in the nonspecific binding assay, the proposed IL-2 moiety is pre-incubated at 4°C for 1 hour in the presence of a cell lineage possessing the IL-2 receptor. Subsequently, 125 I-labeled IL-2 is incubated in the system at 4°C for 3 hours. The data are expressed as the percentage inhibitory activity of the proposed IL-2 partial activity compared to wild-type IL-2. Other methodologies known in the art, including electrometric, spectrometric, chromatographic, and radiometric methods, can also be used to evaluate IL-2 function.

[0075] Water-soluble polymers As discussed earlier, each conjugate contains an IL-2 moiety bound to a water-soluble polymer. Regarding the water-soluble polymer, it is non-peptidic, non-toxic, not naturally occurring, and biocompatible. Regarding biocompatibility, a substance is considered biocompatible if, when used alone or in combination with another substance (e.g., an active agent such as the IL-2 moiety) in relation to living tissue (e.g., administration to a patient), the beneficial effects outweigh any adverse effects as assessed by a clinician, e.g., a physician. Regarding non-immunogenicity, a substance is considered non-immunogenic if its intended use in vivo does not result in undesirable immune responses (e.g., antibody formation), or if immune responses do occur, such responses are not considered clinically significant or important as assessed by a clinician. It is particularly preferable that non-peptidic water-soluble polymers are both biocompatible and non-immunogenic.

[0076] Furthermore, these polymers are typically characterized by having 2 to approximately 300 terminals. Examples of such polymers include, but are not limited to, polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), poly(alkylene glycols) such as copolymers of ethylene glycol and propylene glycol, poly(oxyethylene-polymerized polyols), poly(olefin alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazene, polyoxazoline ("POZ") (as described in International Publication No. 2008 / 106186), poly(N-acryloylmorpholine), and any combination of the above.

[0077] Water-soluble polymers are not limited to a specific structure and may be linear (e.g., end-capped, e.g., alkoxyPEG or bifunctional PEG), branched, or multi-armed (e.g., fork-type PEG or PEG bonded to a polyol core), or tree-like (or star-shaped), each having one or more degradable links, or not having any. Furthermore, the internal structure of water-soluble polymers may be systematized as any of various repeating patterns, and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating tricopolymers, random tricopolymers, and block tricopolymers.

[0078] Typically, activated PEG and other activated water-soluble polymers (i.e., polymer reagents) are activated by suitable activating groups that are suitable for coupling with a desired site on the IL-2 moiety. Thus, the polymer reagents have reactive groups for reacting with the IL-2 moiety. Representative polymer reagents and methods for conjugating such polymers with active moieties are known in the art and are 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,” JM Harris, Plenus Press, New York (1992), and Zalipsky (1995), Advanced Drug Reviews 16: pp. 157-182. Examples of suitable activating groups for coupling the IL-2 moiety include, in particular, hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thion, thiol, vinyl sulfone, and hydrazine.

[0079] Preferably, the polymer reagents used in the preparation of the conjugates described herein are prepared without the use of phosgene. Such a method is in contrast to, for example, the disclosure described in U.S. Patent No. 4,902,502, which specifically describes forming a chloroformate, then using it to form a PEG-active ester, and then reacting it with IL-2. The use of phosgene can generate hydrogen chloride, which can lead to polymer chain cleavage and increase impurities, which may not be removable by prior art. Therefore, although we do not wish to be constrained by theory, IL-2 partial conjugates prepared from polymer reagents formed without the use of phosgene provide a higher quality composition that is substantially free of polymer chain degradation products. Also, in one or more embodiments, the spacer portion between the water-soluble polymer and the IL-2 portion is not a spacer portion containing a carbamate.

[0080] Typically, the weight-average molecular weight of the water-soluble polymer in the conjugate is approximately 100 daltons to 150,000 daltons. However, exemplary ranges include weight-average molecular weights in the following ranges: over 5,000 daltons to approximately 100,000 daltons, approximately 6,000 daltons to approximately 90,000 daltons, approximately 10,000 daltons to approximately 85,000 daltons, over 10,000 daltons to approximately 85,000 daltons, approximately 20,000 daltons to approximately 85,000 daltons, approximately 53,000 daltons to approximately 85,000 daltons, approximately 25,000 daltons to approximately 120,000 daltons, approximately 29,000 daltons to approximately 120,000 daltons, approximately 35,000 daltons to approximately 120,000 daltons, and approximately 40,000 daltons to approximately 120,000 daltons. For any given water-soluble polymer, PEG having one or more molecular weights within these ranges is preferred.

[0081] Examples of weight-average molecular weights for WL polymers include approximately 100 daltons, 200 daltons, 300 daltons, 400 daltons, 500 daltons, 600 daltons, 700 daltons, 750 daltons, 800 daltons, 900 daltons, 1,000 daltons, 1,500 daltons, 2,000 daltons, 2,200 daltons, 2,500 daltons, 3,000 daltons, 4,000 daltons, 4,400 daltons, 4,500 daltons, 5,000 daltons, 5,500 daltons, 6,000 daltons, 7,000 daltons, and 7,500 daltons. Examples include 00 Daltons, approximately 8,000 Daltons, approximately 9,000 Daltons, approximately 10,000 Daltons, approximately 11,000 Daltons, approximately 12,000 Daltons, approximately 13,000 Daltons, approximately 14,000 Daltons, approximately 15,000 Daltons, approximately 20,000 Daltons, approximately 22,500 Daltons, approximately 25,000 Daltons, approximately 30,000 Daltons, approximately 35,000 Daltons, approximately 40,000 Daltons, approximately 45,000 Daltons, approximately 50,000 Daltons, approximately 55,000 Daltons, approximately 60,000 Daltons, approximately 65,000 Daltons, approximately 70,000 Daltons, and approximately 75,000 Daltons. Branched-chain water-soluble polymers having any of the aforementioned total molecular weights (for example, a 40,000-dalton branched-chain water-soluble polymer containing two 20,000-dalton polymers) can also be used. In one or more embodiments, the conjugate does not have a PEG portion directly or indirectly bound to PEG having a weight-average molecular weight of less than about 6,000 daltons.

[0082] When used as a polymer, PEG typically contains multiple (OCH2CH2) monomers [or (CH2CH2O) monomers, depending on how PEG is defined]. For use throughout this description, the number of repeating units is "(OCH2CH2) nIt is specified by the subscript "n". Thus, the value of (n) typically falls within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer with a known molecular weight, 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 monomers.

[0083] One polymer particularly preferred for use in the present invention is an end-capped polymer, i.e., at least one end is lower C 1~6 This polymer is capped with a relatively inert group such as an alkoxy group (however, a hydroxyl group can also be used). For example, when the polymer is PEG, it is preferable to use methoxyPEG (generally referred to as mPEG), which is a linear PEG in which one end of the polymer is a methoxy(-OCH3) group and the other end is a hydroxyl group or other functional group, which may be chemically modified in some cases.

[0084] In one or more embodiments useful of the present invention, free PEG or unbound PEG is a linear polymer having each end terminated with a hydroxyl group: HO-CH2CH2O-(CH2CH2O) n The formula is -CH2CH2-OH, where (n) is typically in the range of 0 to about 4,000.

[0085] The above polymer, α-, ω-dihydroxyl poly(ethylene glycol), can be expressed in a simplified form as HO-PEG-OH, where the -PEG- symbol represents the following structural units: -CH2CH2O-(CH2CH2O) n -CH2CH2- In the formula, (n) is as defined above.

[0086] Another type of PEG useful in one or more embodiments of the present invention is methoxyPEG-OH, or simply mPEG, which has a relatively inert methoxy group at one end and a hydroxyl group at the other. The structure of mPEG is as follows. CH3O-CH2CH2O-(CH2CH2O) n -CH2CH2-OH In the formula, (n) is as described above.

[0087] Multi-armed or branched PEG molecules, such as those described in U.S. Patent No. 5,932,462, can also be used as PEG polymers. For example, PEG may have the following structure: [ka] During the ceremony: poly a and poly b These are PEG skeletons such as methoxypoly(ethylene glycol) (they are either the same or different); R'' is a non-reactive part such as H, methyl, or PEG skeleton; and P and Q are non-reactive links. In a preferred embodiment, the branched PEG polymer is methoxypoly(ethylene glycol) disubstituted lysine. Depending on the specific IL-2 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-2 moiety.

[0088] In addition, PEGs can include forked PEGs. An example of a forked PEG is represented by the following structure: [ka] In the formula, X is a spacer portion of one or more atoms, and each Z is an active end group linked to CH by an atomic chain of a fixed length. International Publication No. 99 / 45964 discloses various fork-type PEG structures that can be used in one or more embodiments of the present invention. The atomic chain linking the Z functional group to the branched carbon atoms acts as a tether group and may include, for example, alkyl chains, ether chains, ester chains, amide chains and combinations thereof.

[0089] PEG polymers may include pendant-type PEG molecules in which reactive groups such as carboxyls are covalently bonded along the length of the PEG chain, rather than at the ends of the PEG chain. The pendant-type reactive groups can be bonded directly to the PEG or via spacer groups such as alkylene groups.

[0090] In addition to the forms of PEG described above, polymers may also be prepared to include one or more weak or degradable links within the polymer, including any of the polymers described above. For example, PEG may be prepared to include hydrolyzable ester links within the polymer. As a result of this hydrolysis, the polymer is cleaved into fragments with lower molecular weights, as shown below: -PEG-CO2-PEG-+H2O→-PEG-CO2H+HO-PEG-

[0091] Other hydrolytically degradable links useful as degradable links within the polymer backbone and / or as degradable links with the IL-2 moiety include carbonate links; for example, imine links obtained from the reaction of an amine and an aldehyde (see, e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); for example, phosphate ester links formed by the reaction of an alcohol with a phosphate group; typically hydrazone links formed by the reaction of a hydrazide and an aldehyde; typically acetal links formed by the reaction between an aldehyde and an alcohol; for example, orthoester links formed by the reaction between a formate and an alcohol; for example, amide links formed by an amine group at the end of a polymer such as PEG and a carboxyl group of another PEG chain; for example, urethane links formed by the reaction of PEG having a terminal isocyanate group with PEG alcohol; peptide links formed by an amine group at the end of a polymer such as PEG and a carboxyl group of a peptide; and, for example, oligonucleotide links formed by a phosphoramidite group at the end of a polymer and a 5' hydroxyl group of an oligonucleotide.

[0092] Such optional features of the conjugate, namely the introduction of one or more degradable links into the polymer chain or to the IL-2 moiety, can provide further control over the final desired pharmacological properties of the conjugate when administered. For example, a large, relatively inactive conjugate (i.e., one or more high molecular weight PEG chains, e.g., one or more PEG chains with a molecular weight greater than about 10,000, bound thereto, in which case the conjugate is essentially bioactive) may be administered, which is released to produce a bioactive conjugate containing part of the original PEG chain. In this way, the properties of the conjugate can be more effectively tuned so that the bioactivity of the conjugate equilibrium over time.

[0093] The water-soluble polymer associated with the conjugate may also be “releaseable.” That is, the water-soluble polymer is released (by hydrolysis, enzymatic processes, catalytic processes, or other means), thereby yielding an unconjugated IL-2 moiety. In some cases, the releaseable polymer detaches from the IL-2 moiety in vivo without leaving any fragments of the water-soluble polymer. In other cases, the releaseable polymer detaches from the IL-2 moiety in vivo, leaving relatively small fragments from the water-soluble polymer (e.g., succinic acid tags). Exemplary cleavable polymers include those that bond to the IL-2 moiety via carbonate linkages.

[0094] Those skilled in the art will recognize that the above-mentioned considerations regarding non-peptide water-soluble polymers are not exhaustive but merely illustrative, and that any polymer material possessing the above qualities is intended. As used herein, the term “polymer reagent” generally refers to an entire molecule that may contain water-soluble polymer segments and functional groups.

[0095] As described above, the conjugate of the present invention comprises a water-soluble polymer covalently bonded to the IL-2 moiety. Typically, for any given conjugate, 1 to 3 water-soluble polymers are covalently bonded to one or more moieties having IL-2 activity. However, in some cases, the conjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers individually bonded to the IL-2 moiety. Any given water-soluble polymer may be covalently bonded to the amino acids of the IL-2 moiety, or, if the IL-2 moiety is (for example) a glycoprotein, to the carbohydrates of the IL-2 moiety. The binding to carbohydrates may be carried out using other preferred methods, such as metabolic functionalization using the sialic acid-azide chemical reaction [Luchansky et al. (2004) Biochemistry 43(38):12358-12366], or by promoting the introduction of aldehyde groups using glycidol [Heldt et al. (2007) European Journal of Organic Chemistry 32:5429-5433].

[0096] The specific internal linkage between the IL-2 active moiety and the polymer depends on a variety of factors. These factors include, for example, the chemical properties of the specific linkage used, the specific IL-2 moiety, the available functional groups within the IL-2 moiety (whether for bonding to the polymer or for conversion to a suitable bonding site), and the presence of other reactive functional groups within the IL-2 moiety.

[0097] The conjugate of the present invention may, although not essential, be a prodrug, meaning that the linkage between the polymer and the IL-2 moiety is releaseable, thereby releasing the parent moiety. Exemplary releaseable linkages include carboxylic acid esters, phosphate esters, thiol esters, anhydrides, acetals, ketals, acyloxyalkyl ethers, imines, orthoesters, peptides, and oligonucleotides. Such linkages can be readily prepared by appropriately modifying either the IL-2 moiety (e.g., the C-terminus of a carboxyl group of a protein, or a hydroxyl side chain of an amino acid such as serine or threonine contained in a protein, or a similar functional group in a carbohydrate) and / or a polymer reagent using coupling methods commonly used in the art. However, most preferably, the releaseable linkage is readily formed by reacting a suitably activated polymer with an unmodified functional group contained in the IL-2 active moiety.

[0098] Alternatively, hydrolysis-stable linkages, such as amide, urethane (also known as carbamate), amine, thioether (also known as sulfide), or urea (also known as carbamide), can also be used as coupling links for the IL-2 moiety. Furthermore, amides are preferred as hydrolysis-stable linkages. In one method, a water-soluble polymer having an activated ester can be reacted with the amine group of the IL-2 moiety to generate an amide linkage.

[0099] The conjugate (as opposed to the unconjugated IL-2 moiety) may or may not have measurable levels of IL-2 activity. That is, the polymer-IL-2 moiety conjugate according to the present invention may have bioactivity ranging from about 0.1% to about 100% of that of the unmodified parent IL-2 moiety. In some cases, the polymer-IL-2 moiety conjugate may have bioactivity greater than 100% of that of the unmodified parent IL-2 moiety. Preferably, a conjugate with little or no IL-2 activity includes hydrolyzable links connecting the polymer to the moiety, and therefore, regardless of the lack (or relative lack) of activity in the conjugate, when the hydrolyzable links are cleaved by water, the active parent molecule (or its derivative) is released. Such activity can be measured using a suitable in vivo or in vitro model, depending on the known activity of the specific IL-2 activity moiety used.

[0100] For conjugates having a hydrolysis-stable linkage that couples an IL-2 active moiety with a polymer, the conjugate typically exhibits measurable bioactivity. For example, such a conjugate is typically characterized by having bioactivity that satisfies one or more of the following percentages compared to the bioactivity of the unconjugated IL-2 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 over 105% (when measured using a suitable model, such as those well known in the art). Preferably, a conjugate having a hydrolysis-stable linkage (e.g., an amide linkage) has at least some degree of bioactivity of the unmodified parent IL-2 active moiety.

[0101] Herein, an exemplary conjugate according to the present invention is described. Typically, such an IL-2 moiety is expected to share (at least in part) an amino acid sequence similar to the sequence provided in at least one of SEQ ID NOs: 1-4. Thus, although certain positions or atoms in SEQ ID NOs: 1-4 are referenced, such references are for convenience only, and those skilled in the art can easily determine the corresponding positions or atoms in other moieties that have IL-2 activity. In particular, the description provided herein with respect to natural human IL-2 can often be applied to any of the aforementioned fragments, deletion mutants, substitution mutants, or addition mutants.

[0102] The amino group of the IL-2 moiety provides a binding site between the IL-2 moiety and the water-soluble polymer. Using the amino acid sequences provided in SEQ ID NOs: 1-2, it is clear that each contains several lysine residues with ε-amino acids that can be used as conjugates. Furthermore, the N-terminal amine of any of these proteins can also function as a binding site.

[0103] There are many suitable polymer reagents useful for forming covalent bonds between the IL-2 moiety and available amines. Specific examples are provided in Table 1 below, along with their corresponding conjugates. In the table, the variable (n) represents the number of repeating monomer units, and "-NH-(IL-2)" represents the residue of the IL-2 moiety after conjugate formation with the polymer reagent. Each polymer moiety shown in Table 1 [e.g., (OCH2CH2)] n or (CH2CH2O) n The term ] has a "CH3" group at its terminus, but this may be substituted with other groups (such as H and benzyl).

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] [Table 4]

[0108] [Table 5]

[0109] [Table 6]

[0110] [Table 7]

[0111] The conjugate formation of polymer reagents with the amino group of the IL-2 moiety can be achieved by various methods. In one method, the IL-2 moiety can be conjugated with a polymer reagent functionalized with a succinimidyl derivative (or other activated ester groups; in this case, a method similar to those described for polymer reagents containing such alternative activated ester groups can be used). In this method, the polymer having the succinimidyl derivative can conjugate with the IL-2 moiety in an aqueous medium at pH 7-9.0, but by using different reaction conditions (e.g., lower pH such as 6-7, or different temperatures and / or below 15°C), the polymer may conjugate to different positions on the IL-2 moiety. In addition, amide linkages can be formed by reacting a non-peptide water-soluble polymer having an amine terminus with an IL-2 moiety having an activated carboxylic acid group.

[0112] An exemplary conjugate is included in the following structure: [ka] During the ceremony: (n) is an integer with values ​​between 2 and 4000; X is the spacer part; R 1 is an organic radical; and IL-2 is the residue in the IL-2 portion.

[0113] An exemplary conjugate is included in the following structure: [ka] In the formula, (n) is an integer with a value between 2 and 4000, and IL-2 is the residue of the IL-2 portion.

[0114] Another useful method for conjugating the IL-2 moiety with polymer reagents is typically the reductive amination of the primary amine in the IL-2 moiety with a polymer reagent functionalized with a ketone, aldehyde, or their hydrate form (e.g., ketone hydrate, aldehyde hydrate). In this method, the primary amine in the IL-2 moiety reacts with the carbonyl group of the aldehyde or ketone (or the corresponding hydroxyl-containing group of the hydrated aldehyde or ketone) to form a Schiff base. Subsequently, the Schiff base can be reductively converted to a stable conjugate using a reducing agent such as sodium borohydride. In particular, when using polymers functionalized with ketones or α-methyl branched aldehydes, and / or under specific reaction conditions (e.g., low pH), selective reactions (e.g., at the N-terminus) are possible.

[0115] Exemplary conjugates of the present invention in which the water-soluble polymer is branched include those in which the water-soluble polymer is incorporated into the following structure: [ka] In the formula, each (n) is an integer that independently has a value between 2 and 4000.

[0116] An exemplary conjugate of the present invention is encompassed in the following structure: [ka] During the ceremony: Each (n) is an independent integer with a value between 2 and 4000; X is the spacer part; (b) is an integer with a value between 2 and 6; (c) is an integer with a value between 2 and 6; R 2 Each present is independently H or a lower alkyl; and IL-2 is the residue in the IL-2 portion.

[0117] An exemplary conjugate of the present invention is encompassed in the following structure: [ka] During the ceremony: Each (n) is an integer with a value between 2 and 4000; and IL-2 is the residue in the IL-2 portion.

[0118] Other exemplary conjugates of the present invention are included in the following structures: [ka] During the ceremony: Each (n) is an independent integer with a value between 2 and 4000; (a) is either 0 or 1; X is a spacer portion containing one or more atoms when present; (b') is an integer with a value of 0 or between 1 and 10; (c) is an integer with a value between 1 and 10; R 2 Each of them is independently either H or an organic radical; R 3 Each present is independently H or an organic radical; and IL-2 is the residue in the IL-2 portion.

[0119] Yet another exemplary conjugate of the present invention is encompassed in the following structure: [ka] During the ceremony: Each (n) is an integer with a value between 2 and 4000; and IL-2 is the residue in the IL-2 portion.

[0120] Exemplary conjugates containing releaseable links include those in which the IL-2 moiety is conjugated with a polymer reagent encompassed by the following formula: [ka] During the ceremony: POLY 1 is the first water-soluble polymer; POLY 2 It is a second water-soluble polymer; X 1 This is the first spacer portion; X 2 This is the second spacer portion; H α is an ionized hydrogen atom; R 1 is either H or an organic radical; R 2 is either H or an organic radical; (a) is either 0 or 1; (b) is either 0 or 1; R e1 When present, it is the first electronically modified group; R e2 It is, when present, a second electronically modified group; and (FG) is a functional group capable of reacting with the amino group of the active agent to form releaseable links such as carbamate links. A polymer reagent with a more definitive structure is envisioned in this formula: [ka] In the formula, POLY 1 , POLY 2 , X 1 , X 2 , R 1 , R 2 H α Each of (FG) is as defined above, and R e1 is the first electronically modified group; and R e2 This is the second electronically modified group.

[0121] Another example polymer reagent is included in the following formula: [ka] [ka] In the formula, for each structure and in each example, (n) is an integer between 4 and 1500, independently.

[0122] These polymer reagents providing the releaseable linkages can be prepared according to the procedure described in U.S. Patent Application Publication No. 2006 / 0293499.

[0123] Exemplary conjugates formed using polymer reagents that provide releaseable linkages include those of the following formulas: [ka] During the ceremony: POLY 1 is the first water-soluble polymer; POLY2 It is a second water-soluble polymer; X 1 This is the first spacer portion; X 2 This is the second spacer portion; H α is an ionized hydrogen atom; R 1 is either H or an organic radical; R 2 is either H or an organic radical; (a) is either 0 or 1; (b) is either 0 or 1; R e1 When present, it is the first electronically modified group; R e2 When present, it is a second electronic modifier; Y 1 is either O or S; Y 2 is O or S; and (IL-2) is the residue in the IL-2 portion.

[0124] An exemplary conjugate has the following structure: [ka] [ka] In the formula, for each structure and in each example, (n) is an integer between 4 and 1500, and (IL-2) is the residue of the IL-2 moiety.

[0125] The carboxyl group represents another functional group that can function as a bonding site in the IL-2 moiety. Structurally, the conjugate may include: [ka] In the formula, (IL-2) and the adjacent carbonyl group correspond to the carboxyl-containing IL-2 moiety, X is a linking chain, preferably a heteroatom selected from O, N(H), and S, and POLY is a water-soluble polymer such as PEG, which may have end cap portions at its ends.

[0126] C(O)-X linkages are obtained from the reaction between a polymer derivative having terminal functional groups and a carboxyl-containing IL-2 moiety. As discussed above, the specific linkage may depend on the type of functional group used. When the polymer is terminally functionalized or "activated" with a hydroxyl group, the resulting linkage is a carboxylic acid ester, and X is O. When the polymer backbone is functionalized with a thiol group, the resulting linkage is a thioester, and X is S. When certain multi-armed, branched, or fork-type polymers are used, the C(O)X moiety, particularly the X moiety, can be relatively complex and may contain longer linkage structures.

[0127] Water-soluble derivatives containing a hydrazide moiety are also useful for conjugate formation in carbonyl and carboxylic acids. As long as the IL-2 moiety does not contain a carbonyl moiety or carboxylic acid, it can be added using methods known to those skilled in the art. For example, a carbonyl moiety can be introduced by reducing a carboxylic acid (e.g., a C-terminal carboxylic acid) and / or by providing an IL-2 moiety in a glycosylated or saccharified form (in this case, the added sugar having a carbonyl moiety). Regarding IL-2 moieties containing carboxylic acids, PEG-hydrazine reagents can be covalently bonded to the IL-2 moiety in the presence of a coupling agent (e.g., DCC) [for example, mPEG-OCH2C(O)NHNH2+HOC(O)-(IL-2) yields mPEG-OCH2C(O)NHNHC(O)-IL-2]. Specific examples of water-soluble derivatives containing a hydrazide moiety, along with their corresponding conjugates, are provided in Table 2 below. In addition, by reacting a water-soluble polymer derivative containing an activated ester with hydrazine (NH2-NH2) or tert-butyl carbadate [NH2NHCO2C(CH3)3], any water-soluble derivative containing an activated ester (e.g., succinimidyl group) can be converted to include a hydrazide moiety. In the table, the variable (n) represents the number of repeating monomer units, and "-C(O)-(IL-2)" represents the residue of the IL-2 moiety after conjugate formation with the polymer reagent. If necessary, the hydrazone linkage may be reduced using a suitable reducing agent. Each polymer moiety shown in Table 2 [e.g., (OCH2CH2)] n or (CH2CH2O) n The term ] has a "CH3" group at its terminus, but this may be substituted with other groups (such as H and benzyl).

[0128] [Table 8]

[0129] The thiol groups contained in the IL-2 moiety can function as effective sites for binding to water-soluble polymers. In particular, cysteine ​​residues provide thiol groups when the IL-2 moiety is a protein. The thiol groups of such cysteine ​​residues can then react with activated PEGs specific to reactions with thiol groups, such as N-maleimidyl polymer or other derivatives as described in U.S. Patent No. 5,739,208 and International Publication No. 01 / 62827. In addition, thiols with protecting groups may be introduced into the oligosaccharide side chains of activated glycoproteins, followed by deprotection with thiol-reactive water-soluble polymers.

[0130] Specific examples of reagents, along with their corresponding conjugates, are provided in Table 3 below. In the table, the variable (n) represents the number of repeating monomer units, and "-S-(IL-2)" represents the IL-2 partial residue after conjugate formation with the water-soluble polymer. Each polymer part shown in Table 3 [e.g., (OCH2CH2)] n or (CH2CH2O) n The term ] has a "CH3" group at its terminus, but this may be substituted with other groups (such as H and benzyl).

[0131] Regarding Sequence IDs 1 and 2, which correspond to the exemplary IL-2 moiety, it can be seen that there is a cysteine ​​residue at position 125. Therefore, the exemplary thiol-binding site is the cysteine ​​located at position 125. It is preferable not to cleave any disulfide bonds associated with a given IL-2 moiety, but it may be possible to maintain some degree of activity by binding polymers in one or more side chains of these cysteine ​​residues. In addition, it is possible to add cysteine ​​residues to the IL-2 moiety using conventional synthetic techniques. For example, for the addition of cysteine ​​residues, please refer to the procedure described in International Publication No. 90 / 12874, and such a procedure can be adapted to the IL-2 moiety. In addition, cysteine ​​residues can also be introduced into the IL-2 moiety using conventional genetic engineering methods. However, in some embodiments, it is preferable not to introduce additional cysteine ​​residues and / or thiol groups.

[0132] [Table 9]

[0133] [Table 10]

[0134] With respect to conjugates formed from water-soluble polymers having one or more maleimide functional groups (whether the maleimide reacts with the amine group or the thiol group of the IL-2 moiety), the corresponding one or more maleamic acid-type water-soluble polymers can also react with the IL-2 moiety. Under certain conditions (e.g., pH approximately 7-9 and in the presence of water), the maleimide ring "opens" to form the corresponding maleamic acid. Subsequently, the maleamic acid can react with the amine group or thiol group of the IL-2 moiety. Exemplary maleamic acid-based reactions are schematically shown below. POLY represents the water-soluble polymer, and (IL-2) represents the IL-2 moiety. [ka]

[0135] A typical conjugate according to the present invention may have the following structure: POLY-L 0,1 -C(O)ZYSS-(IL-2) In the formula, 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 C 2~10 Alkyl, C 2~10 A polymer reagent is selected from the group consisting of substituted alkyls, aryls, and substituted aryls, where (IL-2) is the IL-2 moiety. A polymer reagent that can react with the IL-2 moiety and result in this type of conjugate is described in U.S. Patent Application Publication No. 2005 / 0014903.

[0136] As previously noted, the exemplary conjugate of the present invention, in which the water-soluble polymer is branched, may have a branched water-soluble polymer comprising the following structure: [ka] In the formula, each (n) is an integer that independently has a value between 2 and 4000.

[0137] Exemplary conjugates having branched-chain water-soluble polymers can be prepared using the following reagents: [ka] This results in the formation of a conjugate having the following structure: [ka] During the ceremony: (For each structure) Each (n) is an integer that independently has values ​​from 2 to 4000; and IL-2 is the residue in the IL-2 portion.

[0138] Further exemplary conjugates can be formed using the following reagents: [ka] This results in the formation of a conjugate having the following structure: [ka] During the ceremony: (For each structure) (n) is an integer that independently has values ​​from 2 to 4000; and IL-2 is the residue in the IL-2 portion.

[0139] The conjugate can be formed in various ways using thiol-selective polymer reagents, and the present invention is not limited thereto. For example, an IL-2 portion—in a preferably suitable buffer (including, if necessary, a buffer containing an amine)—is placed in an aqueous medium at a pH of approximately 7–8, and a thiol-selective polymer reagent is added in molar excess. The reaction is then allowed to proceed for approximately 0.5–2 hours, however, if the yield of pegylation is deemed relatively low, longer reaction times (e.g., 5, 10, 12, and 24 hours) may be useful. Exemplary polymer reagents that can be used in this method are polymer reagents having reactive groups selected from the group consisting of maleimides, sulfones (e.g., vinyl sulfones), and thiols (e.g., orthopyridinyl or functional thiols such as "OPSS").

[0140] With regard to polymer reagents, those described herein and elsewhere can be purchased from commercial suppliers or prepared from commercially available starting materials. In addition, methods for preparing polymer reagents are described in the literature.

[0141] The bond between the IL-2 moiety and the non-peptide water-soluble polymer may be direct, in which case there are no intervening atoms between the IL-2 moiety and the polymer; or the bond may be indirect, in which case one or more atoms are located between the IL-2 moiety and the polymer. With regard to indirect bonding, a "spacer moiety" functions as a linker between the residues of the IL-2 moiety and the water-soluble polymer. The one or more atoms constituting the spacer moiety may include one or more carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. The spacer moiety may include amides, secondary amines, carbamates, thioethers, and / or disulfide groups. Non-specific examples of spacer parts include -O-, -S-, -SS-, -C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -OC(O)-NH-, -C(S)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH 2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH 2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-C H2-CH2-O-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -CH2-C(O)-NH-CH2-, -CH2-CH2-C(O)-NH-, -C(O)- NH-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-, -C( O)-NH-CH2-CH2-CH2-CH2-, -CH2-C(O)-NH-CH2-CH2-CH2-, -CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-C H2-C(O)-NH-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-, -CH2-CH2-CH2-CH2-C(O)-NH-, -C(O)-O-CH2-,-CH2-C(O)-O-CH2-, -CH2-CH2-C(O)-O-CH2-, -C(O)-O-CH2-CH2-, -NH-C(O)-CH2-, - CH2-NH-C(O)-CH2-, -CH2-CH2-NH-C(O)-CH2-, -NH-C(O)-CH2-CH2-, -CH2-NH-C(O)-C H2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -OC(O)-NH-CH2-, -OC(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH 2-NH-CH2-, -C(O)-CH2-, -C(O)-CH2-CH2-, -CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-, -CH2-CH2-C(O)-CH2-CH2-, -CH2-CH2-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-, -CH2-CH2-CH2-C(O)-NH-CH2-CH2-NH-C(O)-CH2-CH2-, -OC(O)-NH-[CH2], h -(OCH2CH2) j -, divalent cycloalkyl group, -O-, -S-, amino acid, -N(R) 6 )-, and selected from the group consisting of any two or more combinations of the above, where R 6 (h) 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, where (h) is 0 to 6 and (j) is 0 to 20. Another specific spacer portion has the following structure: -C(O)-NH-(CH2) 1~6 -NH-C(O)-, -NH-C(O)-NH-(CH2) 1~6 -NH-C(O)- and -OC(O)-NH-(CH2) 1~6 -NH-C(O)-, where the subscript after each methylene group indicates the number of methylene groups contained in the structure, for example, (CH2)1~6 This means that the structure may contain 1, 2, 3, 4, 5, or 6 methylene groups. In addition, any of the above spacer portions may contain 1 to 20 ethylene oxide monomer units [i.e., -(CH2CH2O)] 1~20 The material may further contain an ethylene oxide oligomer chain containing ]. That is, the ethylene oxide oligomer chain can be located before or after the spacer portion, and possibly between any two atoms of the spacer portion containing two or more atoms. Furthermore, the oligomer chain is not considered part of the spacer portion if the oligomer is adjacent to the polymer segment and merely corresponds to an extension of the polymer segment.

[0142] composition A conjugate is typically part of a composition. Generally, a composition contains multiple conjugates, and although not required, preferably each conjugate contains the same IL-2 moiety (i.e., there is only one type of IL-2 moiety in the entire composition). In addition, a composition may contain multiple conjugates, any given conjugate containing a moiety selected from the group consisting of two or more different IL-2 moieties (i.e., there are two or more different IL-2 moieties in the entire composition). However, optimally, substantially all conjugates in a composition (e.g., 85% or more of the multiple conjugates in the composition) each contain the same IL-2 moiety.

[0143] The composition may comprise a single conjugate species (e.g., a monopegated conjugate in which a single polymer is bonded at the same position for substantially all conjugates in the composition) or a mixture of conjugate species (e.g., a mixture of monopegated conjugates in which polymer bonding occurs at different sites, and / or a mixture of monopegated, dipegated, and tripeggated conjugates). The composition may also comprise other conjugates in which 4, 5, 6, 7, 8 or more polymers are bonded to any given moiety having IL-2 activity. In addition, the present invention also comprises compositions comprising a plurality of conjugates, each conjugate comprising one water-soluble polymer covalently bonded to one IL-2 moiety, as well as compositions comprising 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers covalently bonded to one IL-2 moiety.

[0144] With respect to the conjugates in the composition, the composition satisfies one or more of the following properties: at least about 85% of the conjugates in the composition have 1 to 4 polymers bonded to the IL-2 moiety; at least about 85% of the conjugates in the composition have 1 to 3 polymers bonded to the IL-2 moiety; at least about 85% of the conjugates in the composition have 1 to 2 polymers bonded to the IL-2 moiety; at least about 85% of the conjugates in the composition have 1 polymer bonded to the IL-2 moiety; at least about 95% of the conjugates in the composition have 1 to 5 polymers bonded to the IL-2 moiety; at least about 95% of the conjugates in the composition have 1 to 4 polymers bonded to the IL-2 moiety; at least about 95% of the conjugates in the composition have I Having 1 to 3 polymers bonded to the L-2 moiety; at least about 95% of the conjugates in the composition have 1 to 2 polymers bonded to the IL-2 moiety; at least about 95% of the conjugates in the composition have 1 polymer bonded to the IL-2 moiety; at least about 99% of the conjugates in the composition have 1 to 5 polymers bonded to the IL-2 moiety; at least about 99% of the conjugates in the composition have 1 to 4 polymers bonded to the IL-2 moiety; at least about 99% of the conjugates in the composition have 1 to 3 polymers bonded to the IL-2 moiety; at least about 99% of the conjugates in the composition have 1 to 2 polymers bonded to the IL-2 moiety; and at least about 99% of the conjugates in the composition have 1 polymer bonded to the IL-2 moiety. For example, when referring to a range of polymers as "x to y polymers," it is understood that the number of polymers is intended to be x to y, including the endpoints (i.e., for example, "1 to 3 polymers" refers to 1 polymer, 2 polymers, and 3 polymers, and "1 to 2 polymers" refers to 1 polymer and 2 polymers, etc.).

[0145] In one or more embodiments, the composition containing the conjugate is preferably free of albumin or substantially free of albumin. It is also preferable that the composition is free of or substantially free of proteins that do not have IL-2 activity. Therefore, it is preferable that the composition is 85%, more preferably 95%, and most preferably 99% albumin-free. In addition, it is preferable that the composition is 85%, more preferably 95%, and most preferably 99% free of any proteins that do not have IL-2 activity. To the extent that albumin is present in the composition, the exemplary compositions of the present invention are substantially free of conjugates containing a poly(ethylene glycol) polymer linking the IL-2 moiety residue to albumin.

[0146] Prometheus (registered trademark) brand of Aldesleukin Available from Laboratories Inc., San Diego, CA, IL-2 is provided in combination with sodium dodecyl sulfate ("SDS"). In contrast, the compositions of the present invention may advantageously be SDS-free and may be free of (or substantially free of) SDS and surfactants in general (e.g., Tween 20 and Tween 80). Consequently, the compositions and conjugates of the present invention can be prepared without performing the step of adding SDS, Tween 20, and Tween 80. In addition, the compositions and conjugates of the present invention can be prepared without performing the step of adding surfactants or other excipients. Furthermore, the compositions of the present invention are free from or substantially free from surfactants such as SDS, Tween 20, and Tween 80 (for example, less than about 20%, more preferably less than about 15%, even more preferably less than about 10%, even more preferably less than about 9%, even more preferably less than about 8%, even more preferably less than about 7%, even more preferably less than about 6%, even more preferably less than about 5%, even more preferably less than about 4%, even more preferably less than about 3%, even more preferably less than about 2%, even more preferably less than about 1%, even more preferably less than about 0.5%, with less than 0.001% being the most preferred). In addition, the compositions and conjugates of the present invention can be prepared without performing a step to remove surfactants such as SDS, Tween 20, and Tween 80 (for example, by ultrafiltration). Furthermore, the compositions and conjugates of the present invention can be prepared without performing a step to remove surfactants (for example, by ultrafiltration).

[0147] Controlling the desired number of polymers relative to any given moiety can be achieved by selecting appropriate polymer reagents, the ratio of the polymer reagents to the IL-2 moiety, temperature, pH conditions, and other aspects of the conjugate formation reaction. In addition, purification methods can be used to reduce or remove undesirable conjugates (e.g., conjugates with four or more polymers bonded together).

[0148] For example, polymer-IL-2 partial conjugates can be purified to obtain / isolate different conjugate species. Specifically, the resulting mixture can be purified to obtain an average of 1, 2, 3, 4, 5 or more PEGs per IL-2 moiety, typically 1, 2, or 3 PEGs per IL-2 moiety. The purification strategy for the final conjugate reaction mixture may depend on various factors, including, for example, the molecular weight of the polymer reagent used, the specific IL-2 moiety, the desired dosing regimen, the individual residue activity of one or more conjugates, and the in vivo properties.

[0149] If necessary, conjugates with different molecular weights can be isolated using gel filtration chromatography and / or ion exchange chromatography. Specifically, using gel filtration chromatography, conjugates with different polymer-to-IL-2 moiety ratios (e.g., 1mer, 2mer, 3mer, etc., where "1mer" represents 1 polymer to IL-2 moiety, "2mer" represents 2 polymer to IL-2 moieties, etc.) can be fractionated based on their molecular weight differences (this difference essentially corresponds to the average molecular weight of the water-soluble polymer). For example, in an exemplary reaction in which a 35,000 dalton protein is randomly conjugated with a polymer reagent of approximately 20,000 daltons, the resulting reaction mixture may contain unmodified protein (molecular weight approximately 35,000 daltons), monopegylated protein (molecular weight approximately 55,000 daltons), dipegylated protein (molecular weight approximately 75,000 daltons), etc.

[0150] By using this method, PEGs with different molecular weights can be separated from other polymer-IL-2 partial conjugates, but generally this method is not useful for separating positional isoforms with different polymer binding sites in the IL-2 moiety. For example, using gel filtration chromatography, a mixture of PEG 1mer, 2mer, 3mer, etc. can be separated from each other, but each of the recovered conjugate compositions can contain one or more PEGs bound to different reactive groups (e.g., lysine residues) in the IL-2 moiety.

[0151] Suitable gel filtration columns for performing this type of separation include Superdex™ columns and Sephadex™ columns available from Amersham Biosciences (Piscataway, NJ). The selection of a particular column may depend on the desired fractionation range. Elution is generally performed using a suitable buffer such as phosphate, acetate, etc. The collected fractions can be analyzed by a variety of different methods such as, 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 test 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).

[0152] The separation of positional isomers is carried out by reverse-phase chromatography using reverse-phase high-performance liquid chromatography (RP-HPLC) with a suitable column (e.g., a C18 column or a C3 column commercially available 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. By either method, isomers of the polymer-active agent having the same molecular weight (i.e., positional isomers) can be separated.

[0153] The composition preferably substantially does not contain proteins having no IL-2 activity. In addition, the composition preferably substantially does not contain any other non-covalently bound water-soluble polymers. However, under certain circumstances, the composition may contain a mixture of polymer-IL-2 partial conjugates and non-conjugated IL-2 moieties.

[0154] Optionally, the composition of the present invention further comprises a pharmaceutically acceptable excipient. If necessary, the pharmaceutically acceptable excipient can be added to the conjugate to form the composition.

[0155] Exemplary excipients include, without limitation, those selected from the group consisting of carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, amino acids, and combinations thereof.

[0156] Carbohydrates such as sugars, derivatized sugars, e.g., algitols, aldonic acids, esterified sugars, and / or sugar polymers may be present as excipients. Specific carbohydrate excipients include, for example: monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosylsorbitol, myo-inositol, and cyclodextrin.

[0157] Other examples of excipients include inorganic salts or buffers such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and combinations thereof.

[0158] The composition may also include an antimicrobial agent for preventing or inhibiting the growth of microorganisms. Non-limiting 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, phenylmercury nitrate, thimersol, and combinations thereof.

[0159] Antioxidants may also be present in the composition. The use of antioxidants prevents oxidation, thereby preventing 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 sulfoxylate formaldehyde, sodium metabisulfite, and combinations thereof.

[0160] A surfactant may be present as an excipient. Examples of surfactants include polysorbates such as "Tween 20" and "Tween 80," as well as pluronic sorbitan esters such as F68 and F88 (both available from BASF, Mount Olive, New Jersey), phospholipids such as lecithin and other phosphatidylcholines, lipids such as phosphatidylethanolamine (preferably not in liposomal form), fatty acids, and fatty acid esters, steroids such as cholesterol, and IL-2 rating agents such as EDTA, zinc, and other suitable cations.

[0161] Acids or bases may be present in the composition as excipients. Non-limiting examples of acids that can be used include 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 examples of bases, without limitation, include 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 fumarate, and combinations thereof.

[0162] One or more amino acids may be present as excipients in the compositions described herein. Exemplary amino acids in this regard include arginine, lysine, and glycine.

[0163] The amount of conjugate in the composition (i.e., the conjugate formed between the active agent and the polymer reagent) may vary depending on various factors, but optimally, it may be a therapeutically effective dose when the composition is stored in a unit dose container (e.g., a vial). In addition, the pharmaceutical preparation may be contained in a syringe. The therapeutically effective dose can be experimentally determined by repeated administrations with gradually increasing amounts of conjugate to determine which amount produces the clinically desirable endpoint.

[0164] The amount of any individual excipient in a composition may vary depending on the activity of the excipient and the specific needs of the composition. Typically, the determination of the optimal amount of any individual excipient is done through routine experiments, i.e., by preparing compositions containing various amounts of the excipient (ranging from low to high) and examining their stability and other parameters, and then determining at what point the optimal effect is obtained without significant side effects.

[0165] However, generally, the excipient is present in the composition in an amount of about 1% to about 99% by weight, preferably about 5% to about 98% by weight, and more preferably about 15% to about 95% by weight, with a concentration of less than 30% by weight being most preferable.

[0166] These aforementioned pharmaceutical excipients, along with other excipients, are described in "Remington: The Science & Practice of Pharmacy," 19th edition, Williams & Williams, (1995), "Physician's Desk Reference," 52nd edition, Medical Economics, Montvale, NJ (1998), and Kibbe, AH, "Handbook of "Pharmaceutical Excipients", 3rd edition, American This information is from the Pharmaceutical Association, Washington, DC, 2000.

[0167] The compositions encompass all types of formulations, particularly those suitable for injection, such as reconstituteable powders or parent liquids and liquids. Examples of suitable diluents for pre-injection reconstitution of solid compositions include bacteriostatic water for injection, 5% dextrose in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. Liquid pharmaceutical compositions are envisioned as solutions and suspensions.

[0168] The compositions of one or more embodiments of the present invention are, though not essential, typically administered by injection and are therefore generally liquid solutions or suspensions immediately before administration. Pharmaceutical preparations may also take other forms, such as syrups, creams, ointments, tablets, and powders. Other methods of administration include intrapulmonary, rectal, percutaneous, transmucosal, oral, intrathecal, intratumoral, peritumoral, intraperitoneal, subcutaneous, and intraarterial administration.

[0169] The present invention also provides a method for administering a conjugate, as provided herein, to a patient suffering from a condition responsive to treatment with the conjugate. This method involves administering to the patient a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition), generally by injection. As previously stated, the conjugate can be administered by injection (e.g., intramuscular, subcutaneous, and parenterally). Suitable formulation types for parenteral administration include, in particular, solutions for immediate injection, dry powders to be miscible with a solvent before use, suspensions for immediate injection, insoluble dry compositions to be miscible with a medium before use, and emulsions and liquid concentrates to be diluted before administration.

[0170] Methods of administering the conjugate (preferably provided as part of a pharmaceutical composition) may be carried out to localize the conjugate to a specific area. For example, liquid, gel, and solid formulations containing the conjugate may be surgically implanted in the affected area (e.g., within a tumor, near a tumor, within an inflamed area, and near an inflamed area). Conveniently, organs and tissues may also be imaged to ensure that the desired location is better exposed to the conjugate.

[0171] Using such an administration method, any pathological condition that can be cured or prevented by the administration of the conjugate can be treated. Those skilled in the art will understand which pathological conditions a specific conjugate can effectively treat. For example, the conjugate can be used alone or in combination with other drug therapies to treat patients suffering from a disease selected from the group consisting of renal cell carcinoma, metastatic melanoma, hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile idiopathic arthritis, atopic dermatitis, breast cancer, and bladder cancer. Advantageously, the conjugate can be administered to the patient before, simultaneously with, or after the administration of another active agent.

[0172] The actual dosage administered may vary depending on the age, weight, and general condition of the subject, as well as the severity of the pathological condition to be treated, the judgment of the medical practitioner, and the conjugate being administered. Therapeutically effective amounts are known to those skilled in the art and / or are described in relevant reference texts and literature. Generally, therapeutically effective amounts can range from about 0.001 mg to 100 mg, preferably dosages of 0.01 mg / day to 75 mg / day, more preferably dosages of 0.10 mg / day to 50 mg / day. A given dosage can be administered periodically, for example, until the symptoms of organophosphate poisoning are alleviated and / or eliminated completely.

[0173] Any given conjugate of a unit dosage (again, preferably provided as part of a pharmaceutical preparation) can be administered according to various dosing schedules depending on the judgment of the clinician, the needs of the patient, etc. Specific dosing schedules are well known to those skilled in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, once daily, three times a week, twice a week, once a week, twice a month, once a month, and any combination thereof. When a clinical endpoint is achieved, dosing of the composition is discontinued.

[0174] Although the present invention has been described in relation to its preferred specific embodiments, it should be understood that the foregoing description and the following examples are for illustrative purposes only and are not intended to 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 relating to the invention.

[0175] All papers, works, patents, and other publications referenced herein are incorporated herein by reference in their entirety. [Examples]

[0176] Unless otherwise specified, the implementation of this invention utilizes prior arts such as organic synthesis, biochemistry, and protein purification, which are within the scope of the said art. Such methods are described in detail in the literature. For example, see J. March, "Advanced Organic Chemistry: Reactions Mechanisms and Structure," 4th edition (New York: Wiley-Interscience, 1992), mentioned above.

[0177] In the following embodiments, efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be taken into consideration. Unless otherwise specified, temperatures are in Celsius, and pressures are at or near sea pressure. Each of the following examples is to be considered a teaching for those skilled in the art to carry out one or more embodiments described herein.

[0178] For use in this example, an aqueous solution ("stock solution") containing recombinant IL-2 ("rIL-2") corresponding to the amino acid sequence of the mature protein sequence SEQ ID NO: 3 was obtained from Myoderm (Norristown PA) or prepared according to Example 1. The concentration of the stock solution varied between 1 and 100 mg / mL.

[0179] SDS-PAGE analysis Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Invitrogen NuPAGE system and Novex 4-10% bis-tris precast gels (Invitrogen, Carlsbad, CA). Samples were prepared according to the manufacturer's instructions, loaded onto the gel, and electrophoresis was performed.

[0180] Cation exchange chromatography A cation exchange column (SP-HP Sepharose, GE Healthcare) with a bed volume of approximately 100 ml was prepared using a standard method. The column was connected to a GE Healthcare (Chalfont St. Giles, UK) AKTA Explorer 100, and the prepared PEG-rIL-2 conjugate was purified. Details of the purification process are described below.

[0181] RP-HPLC analysis Reverse-phase chromatography (RP-HPLC) analysis was performed using an Agilent (Santa Clara, CA) 1100 HPLC system. Samples were analyzed using a Silverton (Japan) Intrada WP-RP column (3 μm particle size, 2.1 × 150 mm). The column flow rate was 0.5 ml / min. The mobile phases were 0.09% TFA in water (solvent A) and 0.04% TFA in acetonitrile (solvent B).

[0182] Example 1 Cloning of the IL-2 gene and expression of rIL-2 Because the codon usage frequency of human IL-2 cDNA sequences varies greatly from organism to organism, they may not be optimally expressed in prokaryotes such as Escherichia coli (E. coli). Instead of maximizing E. coli codon usage frequency by making numerous point mutations in an existing human-derived cDNA sequence, the gene was completely synthesized using PCR techniques.

[0183] The method for synthesizing genes from overlap primers was essentially a slightly modified combination of two methods. Basic considerations of each individual method are provided in Young et al. (2004) Nucleic Acids Research 32(7):e59 and Devlin et al. (1988) Gene 65:13-22. Briefly, the DNA sequence was split into forward and reverse oligonucleotides of less than 35 bp, with some exceptions, and there were no gaps between the oligonucleotides. Each oligonucleotide had two adjacent strands overlapping by at least 10 nucleotides at the 3' end and at least 15 nucleotides at the 5' end. Partial fragments of the gene were constructed using double asymmetric PCR, and these were combined using overlap extension PCR to construct the entire gene. Then, mismatched double helixes were removed using a T7 endonuclease I selection step, as described by Young et al. See Young et al. (2004) Nucleic Acids Research 32(7):e59. The final gene fragment, including restriction enzyme sites at the gene ends, was cloned into a commercially available expression vector for Escherichia coli (E. coli). The obtained sequence was confirmed using DNA sequencing analysis, as shown in Figure 1 and Sequence ID No. 5.

[0184] Using this method, this amino acid sequence omits the amino acid at position 1 (alanine) compared to the natural mature human sequence, and includes a C→S amino acid mutation at position 125 for the sequence as shown. The first amino acid in this sequence is methionine, which is directly expressed in bacteria (there is no encoded signal peptide). However, during expression, the initial methionine is removed by the host methionine aminopeptidase.

[0185] The gene was cloned into one of the pET(T7) expression vectors. The protein was expressed in *E. coli* strain BL21(DE3), and one of the strains was typically used as the T7 expression system. This expression system is commercially available, and the expression method is available from EMD Biosciences, Merck KGaA, Darmstadt, Germany. Use of this system was performed under a research license from Brookhaven National Laboratory. The protein was expressed as inclusion bodies in *E. coli* via the vector. Typical formulations used for expression can be found in the literature and in *Protein Production by Auto-Induction in High-Density Shaking Cultures*, by F. William Studier, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (December 20, 2007).

[0186] After fermentation, cells were recovered by centrifugation. The cell mass pellet was stored at -80°C for further homogenization. The frozen cell mass pellet was resuspended in cell washing buffer (50 mM Tris, 5 mM EDTA, pH 8.0) to a concentration of 10% (W / V) and centrifuged at 13860 × g for 30 minutes. The supernatant was discarded. The washed pellet was resuspended in homogenization buffer (50 mM Tris, 5 mM EDTA, 1 mM PMSF, pH 8.0) and homogenized in a microfluidizer (M-110P from Microfluidics, Newton, Massachusetts, USA) at 4-15°C per pass. The homogenate was diluted 2-fold with cell washing buffer (50 mM Tris, 5 mM EDTA, pH 8.0) and centrifuged at 13860 × g for 60 minutes. The supernatant was discarded. The inclusion pellets are sequentially treated with 50 mM Tris, 5 mM EDTA, 2% Triton X-100, pH 8.0; 50 mM Tris, 5 mM EDTA, 1% sodium deoxycholate, pH 8.0; and 50 mM Tris, 5 mM EDTA, 1 M The samples were washed in three steps using a NaCl buffer solution at pH 8.0. After washing, crude IL-2 inclusion bodies were obtained.

[0187] Crude IL-2 inclusions were dissolved in a buffer of 6 M guanidine, 100 mM Tris, pH 8. EDTA was added to a final concentration of 2 mM. Dithiothreitol (DTT) was then added to a final concentration of 50 mM. The mixture was incubated at 50°C for 30 minutes. After reduction, water was added to the mixture to reduce the guanidine concentration to 4.8. After centrifugation at 13860 × g for 1 hour, the resulting gel-like pellet was discarded. The guanidine concentration of the supernatant was further reduced to 3.5 M by adding water. The pH was adjusted to 5 by titration with 100% acetic acid. The mixture was incubated at room temperature for 60 minutes and centrifuged at 13860 × g for 1 hour. The resulting pellet was suspended in a buffer of 3.5 M guanidine, 20 mM acetate, 5 mM DTT, pH 5, and centrifuged at 13860 × g for 1 hour. This washing step was repeated once more.

[0188] A clean, reduced IL-2 inclusion was dissolved in a buffer solution of 6M guanidine, 100mM Tris, and pH 8. 100mM CuCl2 stock was added to obtain the final Cu content. 2+ The concentration was set to 0.1 mM. The mixture was incubated overnight at 4°C.

[0189] Another embodiment of the present invention relates to an improved method for causing a protein to adopt a tertiary structure. In this regard, conventional methods often rely on serial dilution, which is often harsh on the protein. Therefore, an improved method is provided that allows protein folding under milder conditions, and this method comprises the steps of placing an expressed protein (e.g., an IL-2 moiety such as IL-2 prepared according to this embodiment) into a dialysis bag with a pore size smaller than the size of the expressed protein, and adding a protein denaturant-free solution (e.g., water) (preferably over several hours, for example, over 6 hours, more preferably over 10 hours, and even more preferably over 15 hours). Those skilled in the art will recognize exemplary protein denaturant-free solutions, such as solutions lacking (or substantially lacking) guanidine, urea, lithium perchlorate, 2-mercaptoethanol, dithiothreitol, and surfactants (e.g., buffer and water). Thus, in this method, the expressed IL-2 solution was placed into a dialysis bag (molecular weight pore size of 3.5 kilodaltons). The dialysis bags were placed in a reservoir containing 4.8M guanidine, 0.1M Tris, and pH 8 buffer. After equilibration for 3 hours, the guanidine concentration in the reservoir was gradually reduced to 2M over 15 hours by pumping water into the reservoir. The entire refolding process was completed at 4°C. The refolded IL-2 was confirmed by SEC-HPLC.

[0190] The refolded IL-2 was centrifuged at 13860 × g for 60 minutes, and the precipitate was removed. The supernatant was concentrated using a Pellicon XL TFF membrane system (Millipore Corporation, USA).

[0191] Refolded and concentrated IL-2 was loaded onto a BPG column (GE Healthcare Bio-Sciences AB, Uppsala Sweden) packed with Sephacryl S-100 HR resin. The liquid buffer was 2M guanidine, 20mM Tris, pH 8, and flow rate was 25 mL / min. The fraction below the IL-2 monomer peak was pooled. It should be noted that other suitable purification methods, such as ion exchange chromatography and hydrophobic interaction chromatography (HIC chromatography), may also be used.

[0192] Using a Pellicon XL TFF membrane system (Millipore Corporation, USA), the IL-2 monomer fraction pool was concentrated to approximately 1–2 mg / mL at 4°C and an operating pressure of 30–40 psi. The concentrated IL-2 monomer solution was dialyzed to the final formulation buffer (10 mM sodium acetate, 5% trehalose, pH 4.5), and the guanidine concentration was reduced to less than 0.1 mM by changing the formulation buffer several times (usually 4–5 times). The formulated IL-2 solution was sterilized by passing it through a 0.22 μm filter and stored at -80°C for subsequent use.

[0193] Example 2 Pegging of rIL-2 using mPEG2-C2-fmoc-20K-NHS [ka] mPEG2-C2-fomc-20K-N-hydroxysuccinimide derivative, 20kDa ("mPEG2-C2-fmoc-20K-NHS") mPEG2-C2-fmoc-20K-NHS, stored at -80°C under argon, was warmed to ambient temperature under nitrogen purge. A stock solution of mPEG2-C2-fmoc-20K-NHS (200 mG / mL) was prepared in 2 mM HCl, and mPEG2-C2-fmoc-20K-NHS was added to rIL-2 in an amount sufficient to achieve a molar ratio of mPEG2-C2-fmoc-20K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 in the mixture was 0.5 mG / mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture to bring the final concentration to 20 mM, and conjugate formation was allowed to proceed for 30 minutes, providing a [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate. After 30 minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture, bringing the final concentration to 100 mM. The quenched reaction mixture was then diluted with H2O to a conductivity of less than 0.5 mS / cm (25°C). After adjusting the pH to 4.0 using glacial acetic acid, the mixture was purified by column chromatography.

[0194] A typical cation exchange chromatography purification profile of [mPEG2-C2-fmoc-20K]-[rIL-2] is provided in Figure 2.1. [mPEG2-C2-fmoc-20K]-[rIL-2] and unreacted PEG are shown, and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm). Purity analysis of [mPEG2-C2-fmoc-20K]-[rIL-2] by reverse-phase HPLC revealed a purity of 100% for the purified conjugate at 280 nm. See Figure 2.2. Purity was ≥95% when determined by 4-12% NuPage bis-Tris SDS-PAGE gel (gel not shown) with Coomassie blue staining using 20 μg of purified mPEG2-C2-fmoc-20K]-[rIL-2]. The apparently large conjugates with molecular weights exceeding 200 kDa were thought to have low mobility through the gel due to a high degree of PEG hydration, resulting in a relatively large hydrodynamic half. From these tests, the formation of three conjugates was confirmed: 4mer, 3mer, 2mer, and 1mer. Specifically, for 4mer, four "[mPEG2-C2-fmoc-20K]" molecules bound to a single "[rIL-2]" molecule; for 3mer, three "[mPEG2-C2-fmoc-20K]" molecules bound to a single "[rIL-2]" molecule; for 2mer, two "[mPEG2-C2-fmoc-20K]" molecules bound to a single [rIL-2] molecule; and for 1mer, one "[mPEG2-C2-fmoc-20K]" molecule bound to a single [rIL-2] molecule, resulting in [mPEG2-C2-fmoc-20K]-[rIL-2]".

[0195] The release-capable nature of [mPEG2-C2-fmoc-20K]-[rIL-2] was demonstrated by detecting species changes using reverse-phase HPLC. In short, purified [mPEG2-C2-fmoc-20K]-[rIL-2] was incubated in 100 mM NaHCO3 solution at pH 9.0 and 37°C for several hours. Aliquots of this system were taken at regular intervals, and tests were conducted to detect the disappearance of the [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate and the presence of released rIL-2. The appearance of rIL-2 plateaued approximately 10 hours after incubation and gradually decreased, likely due to precipitation. The data are provided in Figure 2.3.

[0196] Example 3 Pegging of rIL-2 using mPEG2-CAC-fmoc-20K-NHS [ka] mPEG2-CAC-fmoc-20K-N-hydroxysuccinimide derivative, 20kDa ("mPEG2-CAC-fmoc-20K-NHS") mPEG2-CAC-fmoc-20K-NHS, stored at -80°C under argon, was warmed to ambient temperature under nitrogen purge. A stock solution of mPEG2-CAC-fmoc-20K-NHS (200 mG / mL) was prepared in 2 mM HCl, and mPEG2-CAC-fmoc-20K-NHS was added to rIL-2 in an amount sufficient to achieve a molar ratio of mPEG2-CAC-fmoc-20K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 in the mixture was 0.5 mG / mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture to bring the final concentration to 20 mM, and conjugate formation was allowed to proceed for 30 minutes, providing a [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate. After 30 minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture, bringing the final concentration to 100 mM. The quenched reaction mixture was then diluted with H2O to a conductivity of less than 0.5 mS / cm (25°C). After adjusting the pH to 4.0 using glacial acetic acid, the mixture was purified by column chromatography.

[0197] A typical cation exchange chromatography purification profile of [mPEG2-CAC-fmoc-20K]-[rIL-2] is provided in Figure 3.1. [mPEG2-CAC-fmoc-20K]-[rIL-2] is shown, with lines corresponding to absorbance at various wavelengths. Purity analysis of [mPEG2-CAC-fmoc-20K]-[rIL-2] by reverse-phase HPLC revealed a purity of 98.5% of the purified conjugate at 280 nm. The peak at 19.6 min corresponds to unreacted mPEG2-CAC-fmoc-20K-NHS (which constitutes <0.1%). See Figure 3.2. The purity was determined to be over 95% when measured on a 4-12% NuPage bis-tris SDS-PAGE gel (gel not shown) with Coomassie blue staining using 20 μg of purified [mPEG2-CAC-fmoc-20K]-[rIL-2]. The seemingly large molecular weight of the conjugate, exceeding 200 kDa, was thought to be a result of low conjugate mobility through the gel due to the high degree of PEG hydration. The molecular weight of the purified [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate was also determined by MALDI-TOF spectroscopy. As can be seen in Figure 3.3, the main peak at 79.6 kDa is within the predicted molecular weight range for the 3-mer [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate. The 100.8 kDa peak falls within the predicted molecular weight range for 4mer[mPEG2-CAC-fmoc-20K]-[rIL-2]. The MW peaks at 40 kDa and 58.7 kDa may correspond to the divalent 3mer IL-2 conjugate and the 4mer IL-2 conjugate, respectively.

[0198] Example 4 Pegylation of rIL-2 by branched-chain mPEG-N-hydroxysuccinimidyl derivative at 20kDa [ka] mPEG2-ru-20K-N-hydroxysuccinimidyl derivative, 20kDa ("mPEG2-ru-20K-NHS") mPEG2-ru-20K-NHS, stored at -80°C under argon, was warmed to ambient temperature under nitrogen purge. A stock solution of mPEG2-ru-20K-NHS (200 mG / mL) was prepared in 2 mM HCl, and mPEG2-ru-20K-NHS was added to rIL-2 in an amount sufficient to achieve a molar ratio of mPEG2-ru-20K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 in the mixture was 0.5 mG / mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture to bring the final concentration to 20 mM, and conjugate formation was allowed to proceed for 30 minutes, providing a [mPEG2-ru-20K]-[rIL-2] conjugate. After 30 minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture, bringing the final concentration to 100 mM. The quenched reaction mixture was then diluted with H2O to a conductivity of less than 0.5 mS / cm (25°C). After adjusting the pH to 4.0 using glacial acetic acid, the mixture was purified by column chromatography.

[0199] A typical cation exchange chromatography purification profile of [mPEG2-ru-20K]-[rIL-2] is provided in Figure 4.1. [mPEG2-ru-20K]-[rIL-2] and unreacted mPEG2-ru-20K-NHS are shown, with lines corresponding to absorbance at various wavelengths (e.g., 280 nm and 225 nm). Purity analysis of [mPEG2-ru-20K]-[rIL-2] by reverse-phase HPLC revealed a purity of 100% for the purified conjugate at 280 nm. See Figure 4.2. Purity was determined to be over 95% when 20 μg of purified [mPEG2-ru-20K]-[rIL-2] was used on a 4–12% NuPage bis-Tris SDS-PAGE gel (gel not shown) with Coomassie blue staining. The conjugates with apparently large molecular weights exceeding 200 kDa exhibited low mobility through the gel, likely due to their high PEG hydration level.

[0200] Example 5 Pegylation of rIL-2 by branched-chain mPEG-N-hydroxysuccinimidyl derivative at 40kDa [ka] mPEG2-ru-40K-N-hydroxysuccinimidyl derivative, 40kDa ("mPEG2-ru-40K-NHS") mPEG2-ru-40K-NHS, stored at -80°C under argon, was warmed to ambient temperature under nitrogen purge. A stock solution of mPEG2-ru-40K-NHS (200 mG / mL) was prepared in 2 mM HCl, and mPEG2-ru-40K-NHS was added to rIL-2 in an amount sufficient to achieve a molar ratio of mPEG2-ru-40K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 in the mixture was 0.5 mG / mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture to bring the final concentration to 20 mM, and conjugate formation was allowed to proceed for 30 minutes, providing the [mPEG2-ru-40K]-[rIL-2] conjugate. After 30 minutes, 1 M HCl was added to the reaction mixture. Quenching was achieved by adding glycine (pH 4.0) to a final concentration of 100 mM. The quenched reaction mixture was then diluted with H2O to a conductivity of less than 0.5 mS / cm (25°C). After adjusting the pH to 4.0 using glacial acetic acid, the mixture was purified by column chromatography.

[0201] A typical cation exchange chromatography purification profile of [mPEG2-ru-40K]-[rIL-2] is provided in Figure 5. [mPEG2-ru-40K]-[rIL-2] and unreacted PEG are shown, and the line corresponds to the absorbance at 280 nm. Purity analysis of [mPEG2-ru-40K]-[rIL-2] by reverse-phase HPLC detected a purity of 100% of the purified conjugate at 280 nm. The purity was greater than 95% when determined by 20 μg of purified [mPEG2-ru-40K]-[rIL-2] on a 4-12% NuPage bis-Tris SDS-PAGE gel (gel not shown) with Coomassie blue staining. The conjugate, which appeared large and had a molecular weight exceeding 200 kDa (likely a 3-mer type [mPEG2-ru-40K]-[rIL-2]), exhibited low mobility through the gel due to its high degree of PEG hydration. Through the column, unreacted mPEG2-ru-40K-NHS was eluted first, followed by the elution of the [mPEG2-ru-40K]-[rIL-2] conjugate.

[0202] Example 6 Pegylation of rIL-2 by branched-chain mPEG-N-hydroxysuccinimidyl derivative at 4kDa [ka] mPEG2-ru-20K-N-hydroxysuccinimidyl derivative, 4kDa ("mPEG2-ru-4K-NHS") mPEG2-ru-4K-NHS, stored at -80°C under argon, was warmed to ambient temperature under nitrogen purge. A stock solution of mPEG2-ru-4K-NHS (200 mG / mL) was prepared in 2 mM HCl, and mPEG2-ru-4K-NHS was added to rIL-2 in an amount sufficient to achieve a molar ratio of mPEG2-ru-4K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 in the mixture was 0.5 mG / mL (0.035 mM) after solubilization with 0.015% SDS. Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture to bring the final concentration to 100 mM, and conjugate formation was allowed to proceed for 30 minutes, providing a [mPEG2-ru-4K]-[rIL-2] conjugate. After 30 minutes, quenching was achieved by adding 1 M glycine (pH 4.0) to the reaction mixture, bringing the final concentration to 100 mM. The quenched reaction mixture was then diluted with H2O to a conductivity of less than 0.5 mS / cm (25°C). After adjusting the pH to 4.0 using glacial acetic acid, the mixture was purified by column chromatography.

[0203] A typical cation exchange chromatography purification profile for [mPEG2-ru-4K]-[rIL-2] is provided in Figure 6. The eluted [mPEG2-ru-4K]-[rIL-2] conjugate showed a mixture of 3mer, 2mer, and 1mer [mPEG2-ru-4K]-[rIL-2] conjugates in the eluted fraction. As shown in Figure 6, the fractions containing the 3mer / 2mer [mPEG2-ru-4K]-[rIL2] mixture and the fraction containing the 2mer / 1mer [mPEG2-ru-4K]-[rIL2] mixture were pooled separately.

[0204] Example 7 Pegmentation of rIL-2 with linear mPEG-butyraldehyde derivatives at 30 kDa [ka] Linear mPEG-butyraldehyde derivative, 30 kDa ("mPEG-ButyrALD") The pegylation reaction is designed so that the final rIL-2 concentration is 2.5 mg / ml after adding all the reaction components and buffers. mPEG-ButyrALD, 30 kDa, stored under argon at -20°C, is warmed to ambient temperature. A quantity of PEG reagent equal to 10-50 mol equivalents of the rIL-2 to be pegylated is weighed and dissolved in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to an aliquot of the stock rIL-2 solution and stirred for 15-30 minutes. Next, sodium borohydride cyanohydride (NaCNBH3), a reducing agent, is added in an excess of 10-100 mol relative to the PEG reagent, and the reaction mixture is stirred at room temperature for 5-18 hours to ensure coupling via secondary amine linkage, thereby forming a conjugate solution.

[0205] It is known that the aldehyde group of mPEG-ButyrALD, when reduced with a reducing agent such as sodium borocyanohydride, reacts with the primary amine associated with rIL-2 and covalently bonds to it via a secondary amine. The selectivity with which one or more amines bond to the polymer can be adjusted by adjusting the pH of the conjugate formation conditions. Relatively low pH conditions (e.g., pH around 5.5) can induce conjugate formation at the N-terminus. At relatively neutral pH conditions (e.g., around 7.5 and slightly above), covalent bonding occurs more frequently at other positions (i.e., on the amine side chains of lysine residues contained in the protein). Adjusting the pH of the conjugate formation conditions allows for some control over where conjugates occur, and thus can increase the ability to realize desired positional isomers.

[0206] Using this same method, other conjugates can be prepared using mPEG-BuryrALD with different weight-average molecular weights.

[0207] Example 8 Pegmentation of rIL-2 with branched-chain mPEG-butylaldehyde derivative and 40kDa [ka] Branched-chain mPEG-butyraldehyde derivative, 40 kDa ("mPEG2-ButyrALD") The pegylation reaction is designed so that the final rIL-2 concentration is 2.5 mg / ml after adding all the reaction components and buffers. mPEG2-ButyrALD, 40 kDa, stored under argon at -20°C, is warmed to ambient temperature. A quantity of PEG reagent equal to 10-50 mol equivalents of the rIL-2 to be pegylated is weighed and dissolved in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to an aliquot of the stock rIL-2 solution and stirred for 15-30 minutes. Next, sodium borohydride cyanohydride (NaCNBH3), a reducing agent, is added in an excess of 10-100 mol relative to the PEG reagent, and the reaction mixture is stirred at room temperature for 5-18 hours to ensure coupling via secondary amine linkage, thereby forming a conjugate solution.

[0208] It has been found that when the aldehyde group of mPEG2-ButyrALD is reduced with a reducing agent such as sodium borocyanohydride, it reacts with the primary amine associated with rIL-2 and forms a covalent bond with it via a secondary amine.

[0209] Using this same method, other conjugates can be prepared using mPEG2-BuryrALD with other weight-average molecular weights.

[0210] Example 9 pegylation of rIL-2 with linear mPEG-succinimidyl α-methylbutanoate derivative at 30 kDa [ka] Linear mPEG-succinimidyl α-methylbutanoate derivative, 30 kDa ("mPEG-SMB") The pegylation reaction is designed so that the final rIL-2 concentration is 2.5 mg / ml after adding all the reaction components and buffers. mPEG-SMB, 30 kDa, stored under argon at -20°C, is warmed to ambient temperature. The amount of PEG reagent equivalent to 10 to 50 molar equivalents of the rIL-2 to be pegylated is weighed and dissolved in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to an aliquot of the stock rIL-2 solution and stirred at room temperature for 5 to 18 hours to obtain a conjugate solution. The conjugate solution is quenched with lysine solution (pH 7.5) so that the final lysine molar concentration is 10 to 100 times the PEG reagent molar concentration.

[0211] mPEG-SMB derivatives have been shown to provide sterically hindrance active NHS esters that selectively react with lysine and terminal amines.

[0212] Using this same method, other conjugates can be prepared using mPEG-SMB with other weight-average molecular weights.

[0213] Example 10 Pegging rIL-2 using mPEG-PIP and 20kDa The basic structure of polymer reagents is provided below: [ka] After adding all the reaction components and buffers, the pegylation reaction is designed so that the final rIL-2 concentration is 2.5 mg / ml. mPEG-PIP, 20 kDa, stored under argon at -20°C, is warmed to ambient temperature. An amount of PEG reagent equal to 10-50 mol equivalents of the rIL-2 to be pegylated is weighed and dissolved in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. The 12% PEG reagent solution is added to an aliquot of the stock rIL-2 solution and stirred for 15-30 minutes. Next, sodium borohydride cyanohydride (NaCNBH3), a reducing agent, is added in an excess of 10-100 mol relative to the PEG reagent, and the reaction mixture is stirred at room temperature for 5-18 hours to ensure coupling (with secondary carbon) via secondary amine linkage, thereby forming a conjugate solution. The conjugate solution is quenched with a lysine solution (pH 7.5) so that the final lysine molar concentration is 10 to 100 times that of the PEG reagent molar concentration.

[0214] It is known that when the ketone group of mPEG-PIP is reduced with a reducing agent such as sodium borocyanohydride, it reacts with a primary amine associated with rIL-2 and forms a covalent bond with it via a secondary amine.

[0215] Using this same method, other conjugates can be prepared using mPEG-PIPs with different weight-average molecular weights.

[0216] Example 11 Activity of exemplary (rIL-2)-PEG conjugate The activity of aldesleukin (control), [mPEG2-C2-fmoc-20K]-[rIL-2] from Example 2, [mPEG2-CAC-fmoc-20K]-[rIL-2] from Example 3, and [mPEG2-ru-20K]-[rIL-2] from Example 4 was evaluated using a cell proliferation assay with CTLL-2 cells.

[0217] Complete RPMI was performed by supplementing CTLL-2 cells (mouse cytotoxic T lymphocyte cell line) with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, and 10% IL-2 culture additive (T-STIM®, containing ConA (concanavalin A)). Cells were maintained in 1640 medium at 37°C under a 5% CO2 atmosphere. 5 After culturing in suspension until a cell density of cells / mL was reached, the cells were divided.

[0218] For the activity assay, cells were washed three times with Dulbecco's phosphate-buffered saline 3-4 days after the last division. The cells were then placed in supplemental medium without T-STIM®, approximately 2 × 10⁶ cells. 5 The cells were resuspended at a cell density of cells / mL and seeded at 90 μl / well in a 96-well microplate with white walls and a clear bottom. The experiment was also conducted using supplemental medium (T-STIM®-free) adjusted to pH 6.7–7 to minimize conjugate release during incubation. Next, 10 μl of a 10-fold concentration of the test compound was added to the T-STIM®-free supplemental medium. The cells were incubated at 37°C for 24 hours under a 5% CO2 atmosphere. After 24 hours of incubation, 100 μL of Promega CellTiter-Glo® reagent was added to each well. The plate was mixed in an orbital shaker for 2 minutes and then incubated at room temperature for 10 minutes. Luminescence was then recorded using a Perkin Elmer TopCount® instrument with an integration time of 1 second / well.

[0219] For the [mPEG2-C2-fmoc-20K]-[rIL-2]-releasing conjugate of Example 2 and the [mPEG2-CAC-fmoc-20K]-[rIL-2]-releasing conjugate of Example 3, the activity of both the released IL-2 and the unreleased conjugate was tested. The test compounds were stored under acidic conditions (10 mM sodium acetate buffer, pH 4) to stabilize conjugate formation. To test the activity of the conjugates, the samples were diluted in supplemental medium from the storage buffer approximately one hour before the assay. To test the activity of released IL-2, the releaseable conjugates {i.e., the [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate from Example 2 and the [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate from Example 3} were diluted 10-fold in 100 mM (final concentration) sodium bicarbonate buffer, pH 9, and pre-incubated at 37°C for 8 hours before starting the assay.

[0220] Using GraphPad's Prism 5.01 software, we performed nonlinear regression analysis of dose-response curves to determine the EC of cell proliferation. 50 The value (the concentration of the test compound required to show 50% of the maximum reaction) was obtained.

[0221] The activity of aldethleukin and its conjugate was measured using a cell proliferation assay. A summary of the results is shown in Table 4. All test substances induced CTLL-2 cell growth in a dose-dependent manner. Since the releaseable conjugate was pre-incubated under conditions that forced protein release, aldethleukin was also pre-incubated as a control to test the stability of the protein itself under forced release conditions. As shown in Table 4, aldethleukin remained stable after pre-incubation under release conditions (37°C for 8 hours, pH 9), demonstrating relative potency to aldethleukin stored under recommended conditions. After pre-incubating [mPEG2-C2-fmoc-20K]-[rIL-2] from Example 2 and [mPEG2-CAC-fmoc-20K]-[rIL-2] from Example 3 under conditions that induce IL-2 release, activity was restored as shown in Figure 8; the IL-2 released from these conjugates showed relative potency to control aldesleukin, while some of the unreleased conjugates showed lower potency compared to aldesleukin. The stable 3mer[mPEG2-ru-20K]-[rIL-2] conjugate showed the lowest potency (Figure 7), at 0.04% of aldesleukin, while 1mer[mPEG2-ru-20K]-[rIL-2] showed comparable potency to aldesleukin, considering the known assay standard deviation.

[0222] [Table 11]

[0223] Example 12 Pharmacokinetics of Exemplary (rIL-2)-PEG Conjugates The pharmacokinetic profiles of aldesleukin (control), [mPEG2-C2-fmoc-20K]-[rIL-2] from Example 2, [mPEG2-CAC-fmoc-20K]-[rIL-2] from Example 3, and [mPEG2-ru-20K]-[rIL-2] from Example 4 were evaluated by ELISA after a single injection in mice.

[0224] Aldesleukin concentrations were measured using a heterogeneous sandwich ELISA. In short, 96-well microtiter plates were coated with mouse monoclonal antibody against IL-2 and blocked. Samples and standards were prepared in stock plasma, then diluted to 10% plasma with a buffer containing biotinylated rabbit polyclonal antibody against IL-2, and incubated in assay plates. IL-2 was detected using streptavidin-horseradish peroxidase, followed by the colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB). Stop solution was added, absorbance was read at 450 nm, and background subtraction was performed at 650 nm. A standard curve was constructed using a weighted four-parameter algorithm, and sample concentrations were determined by interpolation to the standard curve. The limit of quantification was 0.05 ng / mL.

[0225] The pooled 1mer / 2mer [mPEG2-ru-20K]-[rIL-2] concentrations were measured using a homogeneous HTRF® assay (Cisbio US, Bedford MA). The reaction mixture (15 μL, containing europium chromate conjugate mouse monoclonal antibody against IL-2, streptavidin-d2, and biotinylated rabbit monoclonal antibody against PEG) was added to a white, low-volume 384-well microtiter plate. Diluted samples and standards (5 μL) were added to the stock plasma, and the plate was incubated. The plate was read at 615 and 665 nm using a fluorescence reader, and the delta-F was calculated. A standard curve was created using a weighted 5-parameter algorithm, and the sample concentration was determined by interpolation to the standard curve. The limit of quantification was 0.5 ng / mL.

[0226] In the overall IL-2 assay, 3mer[mPEG2-C2-fmoc-20K]-[rIL-2] and 3mer[mPEG2-CAC-fmoc-20K]-[rIL-2] were measured. Since the [mPEG2-C2-fmoc-20K]-[rIL-2] and [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugates are releaseable conjugates, different molecular species may be present in the sample, making individual quantification difficult; therefore, the total IL-2 level was measured. Samples and standard stocks were prepared in stock plasma, diluted 1:1 with release buffer (100mM HEPES / 100mM Tris-HCl, pH 9), and incubated at 37°C for 30-36 hours to force the release of polymer-containing components from the conjugates. After incubation, 25% volume of 0.1M acetic acid was added to neutralize the high pH. The released IL-2 was measured by the ELISA method described above.

[0227] Figure 9 shows the concentration-time plot of the test substance in C57BL / 6 mice after a single intramuscular injection (1 mg / kg). Sodium heparinized plasma samples were collected at 10 minutes and at 1 hour, 6 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, and 336 hours. Geometric mean concentrations were calculated from three mice at each time point. As shown in Figure 9, aldesleukin had a short half-life and was undetectable after 6 hours (<0.05 ng / mL), while the conjugate had a longer half-life and was still detectable at 336 hours.

[0228] Example 13 Efficacy trial for metastatic melanoma of the lung A metastatic melanoma lung model is widely used to evaluate the efficacy of compounds intended to possess IL-2 activity, and this model is developed using C57BL / 6 mice. In this model, mice are first intravenously administered B16F10 melanoma cells, which induce the development of lung nodules of varying numbers and sizes. The number of lung nodules and the total surface area of ​​their lesions differ depending on the concentration of transplanted cells. Next, a treatment group of mice is administered the test compound of interest, while another group of mice is kept untreated as a control. The efficacy of the test compound can be determined as the percentage reduction in the number and size of lung nodules and the total lesion area in each lung between the treatment group and the untreated group.

[0229] In this study, 100,000 B16F10 cells (passaged to P8 or less) were transplanted via tail vein injection. On the third day after cell transplantation, the target test compound (or vehicle) was administered via either the IP (intraperitoneal) or IV (intravenous) route as shown in Table 5.

[0230] [Table 12]

[0231] Fourteen days after cell transplantation, mice were sacrificed, and their lungs were simultaneously removed and fixed in formaldehyde-containing Bowen's solution for 1-2 days. The lungs (fixed in Bowen's solution) were examined under a stereomicroscope, and the number and size of lesions in each lung were determined.

[0232] As shown in Figure 10, 14 days after cell transplantation, a mouse was sacrificed, and its excised lung was fixed in Bowen's solution. Tumor nodules and their sizes were counted for each of the following: the IL-2 portion of Example 1, pooled 3mer / 4mer[mPEG2-CAC-fmoc-20K]-[rIL-2], pooled 3mer / 4mer[mPEG2-ru-20K]-[rIL-2], and pooled 1mer / 2mer[mPEG2-ru-20K]-[rIL-2].

[0233] Example 14 Subcutaneous B16F10 melanoma efficacy trial To evaluate the efficacy of compounds intended to possess IL-2 activity, a highly robust subcutaneous melanoma model in syngeneic mice, namely C57BL / 6 mice, was used. Briefly, 1 million B16F10 cells were subcutaneously transplanted into the dorsal region of each 5-6 week old C57BL / 6 mouse. After allowing the tumors to grow to a palpable size, i.e., 70-120 square mm, they were randomized and assigned to groups as shown in Table 6. The mice were administered the test compounds, namely aldesleukin (Prometheus Laboratories Inc., San Diego, CA), rIL-2 polymer conjugate, or vehicle, at various dose concentrations and dose regimes. Body weight and tumor volume were measured every other day. The endpoint of this study was when the median tumor volume in a given group reached 1500 square mm or 45 days, whichever came first.

[0234] [Table 13]

[0235] Dose-response curves for tumor growth inhibition after administration of aldesleukine (Prometheus Laboratories Inc.) and rIL-2 polymer conjugate under various administration schemes are provided in Figures 11A and 11B. These results provide evidence that the efficacy of the single-dose rIL-2 polymer conjugate tested was superior to that of aldesleukine (Prometheus Laboratories Inc.) administered at 3 mg / kg twice daily for 5 days.

[0236] Figure 11A shows that after a single dose of rIL-2 polymer conjugate, the median tumor volume was 1500 mm². 3 This shows the duration of tumor growth suppression until the tumor progression curve is reached. The tumor growth delay (TGD) from the tumor progression curve was found to be 4.6 days and 6.2 days for pooled 3mer / 4mer[mPEG2-CAC-fmoc-20K]-[rIL-2] at dose concentrations of 2 mg / kg and 4 mg / kg, respectively. For pooled 3mer / 4mer[mPEG2-C2-fmoc-20K]-[rIL-2], the TGD was found to be 6.4 days and 7.6 days for dose concentrations of 2 mg / kg and 4 mg / kg, respectively.

[0237] Figure 11B shows that after a single dose of rIL-2 polymer conjugate, the median tumor volume was 1500 mm². 3 This indicates the duration of tumor growth suppression until the tumor reaches a certain stage. The TGD from the tumor progression curve was found to be 3.6 days and 4.6 days for pooled 1mer / 2mer[mPEG2-C2-fmoc-20K]-[rIL-2] at dose concentrations of 6 mg / kg and 8 mg / kg, respectively. For pooled 1mer / 2mer[mPEG2-ru-20K]-[rIL-2], the TGD was found to be 3.8 at 2 mg / kg, while the 4 mg / kg dose concentration was found to be inherently toxic. The TGD from the tumor progression curve was found to be 2.2 days and 3.6 days for pooled 1mer / 2mer[mPEG2-CAC-fmoc-20K]-[rIL-2] at dose concentrations of 2 mg / kg and 4 mg / kg, respectively.

[0238] In short, in both the lung lesion metastasis model (Example 13) and the subcutaneous mouse melanoma model (Example 14), efficacy was achieved with rIL-2 polymer conjugates at substantially lower administration frequencies and with lower total protein levels compared to aldezleukin (Prometheus Laboratories Inc.).

number

Claims

1. A conjugate comprising an interleukin-2 (IL-2) moiety covalently bonded to a branched poly(ethylene glycol) polymer via a stable amide linkage, wherein the branched poly(ethylene glycol) polymer is bonded to the amino group of the IL-2 moiety via the stable amide linkage, the IL-2 moiety has the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3, the branched poly(ethylene glycol) polymer has two poly(ethylene glycol) polymer chains, and the branched poly(ethylene glycol) polymer comprises the following structure: 【Chemistry 51】 In the formula, each (n) is an integer that independently has values ​​between 2 and 4000, forming a conjugate.

2. The conjugate according to claim 1, wherein the branched poly(ethylene glycol) polymer is capped at the ends with methoxy groups.

3. The conjugate according to claim 1, wherein one, two, three, or four branched poly(ethylene glycol) polymers are bonded to the IL-2 portion.

4. The conjugate according to claim 1, wherein one, two, or three branched poly(ethylene glycol) polymers are bonded to the IL-2 portion.

5. The following structure is included: 【Chemistry 52】 The conjugate according to claim 1, wherein each (n) is an integer having a value between 2 and 4000, and (IL-2) is the IL-2 portion.

6. The conjugate according to claim 1, wherein the branched poly(ethylene glycol) polymer has a weight-average molecular weight in the range of 500 daltons to 100,000 daltons.

7. The conjugate according to claim 6, wherein the branched poly(ethylene glycol) polymer has a weight-average molecular weight selected from 20,000 daltons and 40,000 daltons.

8. The conjugate according to claim 6, wherein the IL-2 portion has the amino acid sequence of SEQ ID NO:

3.

9. A pharmaceutical composition for use in the treatment of cancer, comprising a conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition for use according to claim 9, wherein the cancer is selected from renal cell carcinoma, metastatic melanoma, acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, breast cancer, and bladder cancer.

11. A pharmaceutical composition for use according to claim 9, which is in a form suitable for injection.

12. The conjugate contained in the composition comprises the following structure: 【Chemistry 53】 The pharmaceutical composition for use according to claim 9, wherein each (n) is an integer having a value between 2 and 4000, and (IL-2) is the IL-2 portion.

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