Conjugates of il-2 moiety 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 (in particular) compositions comprising the conjugate, methods for synthesizing the conjugate, and methods for 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, consequently, cannot eliminate them, allowing cancer to progress. In patients suffering from cancer, administration of immunomodulatory proteins to the patient can help (at least in part) to restore the patient's immune system to normal and to recover 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 that 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, 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 Documents 1, 2, and 3. In other approaches, specific conjugates of IL-2 have been proposed. See, for example, Patent Documents 4, 5, 6, and 7.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Non-Patent Document

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, despite such approaches, conjugates of IL-2 are still needed. Therefore, in particular, one or more embodiments of the present invention relate to conjugates, compositions containing the conjugates, and related methods as described herein, which are novel and appear to have never been proposed in the art.

Means for Solving the Problems

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

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

[0013] In one or more embodiments of the present invention, a conjugate is provided that includes a residue of an IL-2 moiety covalently attached 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 that includes a residue of an IL-2 moiety covalently attached 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 that includes the step of subcutaneously administering to a patient a composition comprising a conjugate of a residue of IL-2 and a water-soluble polymer.

[0016] In one or more embodiments of the present invention, an isolated nucleic acid molecule is provided that encodes an IL-2 moiety, wherein the nucleic acid molecule comprises a sequence having a substantial (e.g., at least 80%) sequence identity to the sequence set forth in SEQ ID NO: 5.

[0017] In one or more embodiments of the present invention, an expression vector is provided that includes 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 is provided that includes an expression vector as provided herein (e.g., an in vitro host cell). For example, the present invention provides the following items. (Item 1) A conjugate comprising a residue of an IL-2 moiety covalently attached to a water-soluble polymer. (Item 2) The conjugate according to Item 1, wherein the IL-2 moiety covalently attached to the water-soluble polymer is covalently attached via a cleavable linkage. (Item 3) The conjugate according to item 1, wherein the IL-2 moiety covalently bound to the water-soluble polymer is covalently bound 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 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(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, and poly(acryloyl morpholine). (Item 6) The conjugate according to 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 termini with an end-cap moiety 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 bound to the amine group of the residue of the IL-2 moiety. (Item 11) The 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) The conjugate according to any one of Items 1 to 10, wherein one or more water-soluble polymers are bound to the residues of the IL-2 moiety. (Item 14) The conjugate according to any one of Items 1 to 10, wherein one water-soluble polymer is bound to the residues of the IL-2 moiety. (Item 15) A conjugate comprising residues of an IL-2 moiety covalently bound to a water-soluble polymer, wherein the water-soluble polymer is a polymer reagent having an N-hydroxysuccinimidyl group before covalent binding. (Item 16) A pharmaceutical composition comprising the conjugate according to any one of Items 1 to 15 and a pharmaceutically acceptable excipient. (Item 17) A method comprising the step of administering the pharmaceutical composition according to Item 16 to an individual. (Item 18) A method for preparing a conjugate comprising the step of contacting an IL-2 moiety with a polymer reagent under conjugate formation conditions. (Item 19) An isolated nucleic acid molecule encoding an IL-2 moiety, the nucleic acid molecule comprising a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 5. (Item 20) The nucleic acid molecule according to Item 19, which is DNA. (Item 21) An expression vector comprising the nucleic acid molecule according to Item 19. (Item 22) The expression vector according to Item 21, which is a plasmid. (Item 23) An in vitro host cell comprising the vector according to Item 22. (Item 24) A method comprising the step of placing a protein into a dialysis bag having a pore size smaller than the size of the protein to form a dialysis bag containing the protein, and the step of 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 Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] Before describing one or more embodiments of the present invention in detail, it should be understood that the present invention is not limited to specific polymers, synthetic methods, IL-2 moieties, etc., and thus can vary.

[0021] As used in this specification and the claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, when referring to "a polymer", it includes a single polymer as well as two or more of the same or different polymers, and when referring to "an optional excipient", it refers to a single optional excipient as well as two or more of the same or different optional excipients, and so on.

[0022] In the description and claims of one or more embodiments of the present invention, the following technical terms shall be used according to the following definitions.

[0023] "PEG", "polyethylene glycol", and "poly(ethylene glycol)" are synonymous when used in this specification and include any non-peptidic water-soluble poly(ethylene oxide). Typically, the PEG used in accordance with the present invention has the following structure "-(OCH2CH2) n -", where (n) is from 2 to 4000. As used in this specification, PEG also has, depending on whether the terminal oxygen is substituted, for example, during a synthetic transformation, "-CH2CH2-O(CH2CH2O) n -CH2CH2-", and "-(OCH2CH2) nAlso includes "O-". It should be noted that throughout this specification and the claims, the term "PEG" includes structures having various end groups or "end cap" groups, etc. The term "PEG" also means a polymer containing a majority, i.e., more than 50%, of the repeating sub-units of -OCH2CH2-. With respect to specific forms, PEG can take any of various molecular weights and structures or geometries such as "branched", "linear", "forked", "multifunctional", etc., which will be described in more detail below.

[0024] The terms "end-capped" and "terminally capped" are used synonymously herein and refer to the polymer having an end cap moiety at its end or terminus. Typically, although not necessarily, the end cap moiety is a hydroxy group or a C 1~20 alkoxy group, more preferably a C 1~10 alkoxy group, even more preferably a C 1~5 alkoxy group. Thus, examples of end cap moieties include alkoxy (e.g., methoxy, ethoxy, and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, etc. It should be noted that the end cap moiety may include one or more atoms of the terminal monomer in the polymer [e.g., CH3O(CH2CH2O) n - and CH3(OCH2CH2) n- the end cap portion "methoxy" in. In addition, each of the aforementioned saturated, unsaturated, substituted, and unsubstituted types is assumed. Further, 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 position of the polymer and / or the portion to which the polymer is attached (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 labels, colorimetric (e.g., dyes), metal ions, radioactive portions, and the like. Suitable detectors include photometers, films, spectrometers, and the like. The end cap group may also advantageously include a phospholipid. When the polymer has an end cap group containing a phospholipid, properties unique to the polymer and the resulting conjugate are imparted. Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholine. Specific phospholipids include, without limitation, those selected from the group consisting of dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, behenoyl phosphatidylcholine, arachidoyl phosphatidylcholine, and lecithin. The end cap group may also include a target portion so that the polymer (and everything attached thereto, such as the IL-2 portion) can be selectively localized within the desired range.

[0025] With respect to the polymers as described herein, "not naturally occurring" means a polymer that cannot be found in nature as such. However, a not naturally occurring polymer can include one or more naturally occurring monomers or monomer segments as long as the overall polymer structure is not found in nature.

[0026] The term "water-soluble" polymer, such as in "water-soluble polymer", is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer transmits at least about 75%, more preferably at least about 95%, of the light transmitted by the same solution after filtration. A water-soluble polymer 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), on a weight basis. However, most preferably, a water-soluble polymer can dissolve about 95% (by weight) in water or can dissolve completely in water.

[0027] The molecular weight associated with a water-soluble polymer, such as PEG, can be expressed as either the number average molecular weight or the weight average molecular weight. Unless otherwise indicated, whenever the molecular weight is referred to in this specification, it refers to the weight average molecular weight. The measurement of both the number average and weight average molecular weights can be performed using gel permeation chromatography or other liquid chromatography methods. Also, other methods for measuring the molecular weight value can be used, for example, determining the number average molecular weight by using end group analysis or measuring colligative properties (such as freezing point depression, boiling point elevation, or osmotic pressure), or determining the weight average molecular weight by using light scattering methods, ultracentrifugation methods, or viscosity measurement methods. The polymers of the present invention are typically polydisperse (i.e., the number average molecular weight and the 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 even more preferably less than about 1.05, and most preferably less than about 1.03.

[0028] When used in conjunction with a particular functional group, the terms "active," "reactive," or "activated" refer to a reactive functional group that readily reacts with an electrophile or nucleophile on another molecule. This is contrasted with groups that require a strong catalyst or extremely unrealistic reaction conditions to react (i.e., "non-reactive," or "inert" groups).

[0029] As used herein, the term "functional group" or any synonym thereof is intended to encompass both its protected and unprotected forms.

[0030] The terms "spacer moiety," "linkage," and "linker" are used herein to refer to a bond, atom, or collection of atoms that is optionally used to connect, for example, the terminus of a polymer segment to the IL-2 moiety or an electrophile or nucleophile of the IL-2 moiety. The spacer moiety may be stable to hydrolysis, or may contain a linkage that is physiologically hydrolysable or enzymatically degradable. Unless otherwise specifically indicated in the context, the spacer moiety may optionally be present between any two elements of a compound (e.g., a conjugate comprising a residue of an IL-2 moiety and a water-soluble polymer can be bonded either directly or indirectly via a spacer moiety).

[0031] "Alkyl" refers to a hydrocarbon chain, typically having an atomic length in the range of about 1 to 15. Such hydrocarbon chains are preferably saturated, although this is not essential, and may be branched or straight-chain, although typically straight-chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes cycloalkyl as well as cycloalkylene-containing alkyl.

[0032] "Lower alkyl" refers to an alkyl group containing 1 to 6 carbon atoms, which may be straight-chain 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 crosslinked, fused, or spirocyclized 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 an -OR group, where R is alkyl or substituted alkyl, preferably C 1~6 An -OR group that is alkyl (e.g., methoxy, ethoxy, propyloxy, etc.).

[0035] The term "substituted", such 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, e.g., cyclopropyl, cyclobutyl, etc.; halo, e.g., 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. An aryl ring may also be fused or not fused to one or more cyclic hydrocarbons, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0038] "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] "Heterocyclic" or "heterocyclic ring" means one or more rings of 5 to 12 atoms, preferably 5 to 7 atoms, which may or may not have unsaturated or aromatic characteristics and have at least one ring atom other than carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0040] "Substituted heteroaryl" is a heteroaryl having one or more non-interfering groups as substituents.

[0041] "Substituted heterocyclic" is a heterocyclic having one or more side chains formed from non-interfering substituents.

[0042] "Organic radical", as used herein, includes alkyl, substituted alkyl, aryl, and substituted aryl.

[0043] "Electrophile" and "electrophilic group" refer to an ion or an ionizable atom or atomic assembly having an electrophilic center, i.e., a center that attracts electrons, and is capable of reacting with a nucleophile.

[0044] "Nucleophile" and "nucleophilic group" refer to an ion or an ionizable atom or atomic assembly having a nucleophilic center, i.e., a center that attracts an electrophilic center, or accompanied by an electrophile.

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

[0046] "Enzymatically cleavable linkage" means a linkage that is degraded by one or more enzymes.

[0047] A "hydrolysis-stable" linkage or bond refers to a chemical bond that is substantially stable in water, i.e., does not undergo any appreciable hydrolysis over an extended period under physiological conditions, typically a covalent bond. Examples of hydrolysis-stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, etc. Generally, a hydrolysis-stable linkage exhibits a hydrolysis rate of less than about 1-2% per day under physiological conditions. For the hydrolysis rates of representative chemical bonds, many standard chemical texts can be referred to.

[0048] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that can optionally be included in the composition of the present invention and that does not cause any significant toxic adverse effects to the patient. "Pharmacologically effective amount", "physiologically effective amount", and "therapeutically effective amount" are used synonymously herein and mean the amount of the polymer-(IL-2) moiety conjugate necessary to bring about a desired level of conjugate (or the corresponding unconjugated IL-2 moiety) in the bloodstream or in the target tissue. The exact amount depends on numerous factors such as, for example, the particular IL-2 moiety, the components and physical characteristics of the therapeutic composition, the intended patient population, and the considerations of the individual patient, and one of ordinary skill in the art can readily 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 electrophilic or nucleophilic group suitable for reaction with a polymeric reagent. In addition, the term "IL-2 moiety" encompasses both the IL-2 moiety before conjugate formation and the IL-2 moiety residue after conjugate formation. As will be explained in more detail below, one of ordinary skill in the art can determine whether any given moiety has IL-2 activity. A protein comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-4, as well as any protein or polypeptide that is substantially homologous thereto, is an IL-2 moiety. 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 mutations. These terms also include analogs having 1 to 6 additional glycosylation sites, analogs having one or more additional amino acids at the carboxy-terminal end of the protein, wherein the additional amino acids include at least one glycosylation site, and analogs having an amino acid sequence that includes at least one glycosylation site. This term includes both natural and recombinantly produced moieties.

[0051] The term "substantially homologous" means that a particular subject sequence, e.g., a mutant sequence, differs from a reference sequence by only one or more substitutions, deletions, or additions, but that the net effect is that no functionally detrimental difference occurs between the reference sequence and the subject sequence. For the purposes of the present invention, sequences having a homology higher than 80% (more preferably higher than 85%, even more preferably higher than 90%, and most preferably higher than 95%), equivalent biological activity (not necessarily equivalent biological activity intensity), and equivalent expression characteristics are considered to be substantially homologous. For the purpose of determining homology, truncations of the mature sequence are to be ignored. Exemplary IL-2 moieties used herein include sequences that are substantially homologous to SEQ ID NO: 2.

[0052] The term "fragment" means any protein or polypeptide having an amino acid sequence of a part or fragment of the IL-2 moiety and having the biological activity of IL-2. Fragments include proteins or polypeptides produced by 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 suffering from, or susceptible to, a condition that can be prevented or treated by administration of an active agent (e.g., a conjugate), and includes both humans and animals.

[0054] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the description includes both the case where the circumstance occurs and the case where it does not occur.

[0055] "Substantially" means almost entirely or completely, and for example, satisfies 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, and these sequences are aligned so as to maximize the overlap between the two sequences while minimizing sequence gaps, where any protruding sequences between the two sequences are ignored. For any sequence identity described herein, preferably at least 80%, more preferably 85%, even more preferably 90%, and still even more preferably 95% sequence identity, and sequence identities of 96%, 97%, 98%, and 99% are most preferred.

[0057] The amino acid residues in the peptide 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] Looking at one or more embodiments of the present invention, there is provided a conjugate comprising a residue of an IL-2 moiety covalently bound (either directly or via a spacer moiety) to a water-soluble polymer. The conjugate of the present invention has one or more of the following characteristics.

[0059] IL-2 moiety As mentioned above, generally this conjugate comprises a residue of an IL-2 moiety covalently bound either directly or via a spacer moiety to a water-soluble polymer. As used herein, the term "IL-2 moiety" shall refer to the IL-2 moiety before conjugate formation, as well as the IL-2 moiety after binding to the non-peptidic water-soluble polymer. However, it will be understood that when the native IL-2 moiety binds to the non-peptidic water-soluble polymer, the IL-2 moiety will change slightly due to the presence of one or more covalent bonds associated with the linkage to the polymer. In many cases, this slightly changed form of the IL-2 moiety upon binding to another molecule is referred to as the "residue" of the IL-2 moiety.

[0060] The IL-2 moiety can be obtained from non-recombinant methods as well as from recombinant methods, and the present invention is not limited in this regard. In addition, the IL-2 moiety can be derived from human sources, animal sources, and plant sources.

[0061] The IL-2 moiety can be induced non-recombinantly. For example, it is possible to isolate IL-2 from biological systems and, in other ways, obtain IL-2 from the cultured medium. See, for example, the procedures described in U.S. Patent No. 4,401,756 and Pauly et al. (1984) J. Immunol Methods 75(1):73-84.

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

[0063] Any IL-2 moiety obtained by non-recombinant and recombinant techniques can be used as the IL-2 moiety in the preparation of the conjugates described herein.

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

[0065] Although there can be differences in the methods for preparing recombinant proteins, typically, recombinant methods involve the construction of nucleic acids encoding the desired polypeptide or fragment, cloning the nucleic acids 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 expression of the nucleic acids. Methods for producing and expressing recombinant polypeptides in prokaryotic and eukaryotic host cells in vitro 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 with 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 moiety (e.g., an antibody) for the epitope tag or other binding sequence in the fusion protein, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with an appropriate solution (e.g., an acid) to release the bound fusion protein. Recombinant polypeptides can also be purified by lysis of host cells, separation of the polypeptide, for example, by ion exchange chromatography, affinity binding techniques, hydrophobic interaction techniques, and then identified by MALDI or Western blot 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 moiety is not in the form of a fusion protein.

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

[0068] The IL-2 moiety can advantageously be modified to include and / or substitute one or more amino acid residues, such as lysine, cysteine and / or arginine, thereby making it easier for the polymer to bind to the atoms within the side chains of the amino acids. Examples of substitutions of the IL-2 moiety are described in U.S. Patent No. 5,206,344. In addition, the IL-2 moiety can be modified to include non-natural amino acid residues. Methods for adding amino acid residues and non-natural amino acid residues 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 advantageously be modified to include a bond of a functional group (except for 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 the 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 preferred that the IL-2 moiety is not modified to include one or more of a thiol group, the N-terminal α-carbon, a carbohydrate, an aldehyde group and a 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 Application Publication Nos. 2010 / 0036097 and 2004 / 0175337. Preferred IL-2 moieties include those having an amino acid sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 1-4, and sequences that are substantially homologous thereto. A preferred IL-2 moiety has the amino acid sequence corresponding to SEQ ID NO: 3.

[0071] In some cases, the IL-2 moiety is in the form of a "monomer" in which the single expression of the corresponding peptide is organized as an individual unit. In other cases, the IL-2 moiety is in the form of a "dimer" in which two monomeric forms of the protein are joined to each other (e.g., by a disulfide bond) (e.g., a dimer of recombinant IL-2). For example, in the context of a dimer of recombinant human IL-2, the dimer can be in the form of two monomers joined to each other by a disulfide bond formed from the Cys125 residues of each monomer.

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

[0073] Truncated forms, hybrid variants, and peptidomimetics of any of the foregoing sequences can also function as an IL-2 moiety. Any of the foregoing biologically active fragments, deletion mutants, substitution mutants, or addition mutants that maintain at least some degree of IL-2 activity can also function as an IL-2 moiety.

[0074] For any given peptide or protein moiety, it is possible to determine whether that moiety has IL-2 activity. In the art, various methods for determining IL-2 activity in vitro are described. An exemplary method is the CTTL-2 cell proliferation assay described in the following examples. An exemplary method is described in Moreau et al. (1995) Mol. Immunol. 32:1047-1056. Briefly, in a non-specific binding assay, the proposed IL-2 moiety is pre-incubated at 4°C for 1 hour in the presence of a cell line having the receptor for IL-2. Thereafter, 125 I-labeled IL-2 is incubated in that system at 4°C for 3 hours. The data are expressed as the % inhibitory ability of the proposed IL-2 moiety activity relative to wild-type IL-2. Other methodologies known in the art, including electrophysiological methods, spectrophotometric methods, chromatography, and radiometric methods, can also be used to evaluate IL-2 function.

[0075] Water-soluble polymer As previously discussed, each conjugate contains an IL-2 moiety conjugated to a water-soluble polymer. With respect to the water-soluble polymer, the water-soluble polymer is non-peptidic, non-toxic, not naturally occurring, and biocompatible. With respect to biocompatibility, a substance is considered biocompatible when the beneficial effects associated with using the substance alone or in combination with another substance (e.g., an active agent such as an IL-2 moiety) in relation to a living tissue (e.g., administration to a patient) outweigh any harmful effects when evaluated by a clinician, e.g., a physician. With respect to non-immunogenicity, a substance is considered non-immunogenic when no unwanted immune response (e.g., formation of antibodies) occurs as a result of the intended use of the substance in vivo, or, if an immune response does occur, such a response is not considered clinically significant or important in the evaluation of a clinician. It is particularly preferred that the non-peptidic water-soluble polymer be biocompatible and non-immunogenic.

[0076] Furthermore, this polymer is typically characterized as having from 2 to about 300 termini. Examples of such polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, poly(oxyethylenated polyols), poly(olefin alcohols), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol), polyphosphazenes, polyoxazolines ("POZ") (for which see International Publication No. WO 2008 / 106186), poly(N-acryloyl morpholine), and any combination of the foregoing.

[0077] The water-soluble polymer is not limited to a particular structure and may be linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched, or multi-armed (e.g., fork-shaped PEG or PEG attached to a polyol core), dendritic (or star-shaped), each of which may or may not have one or more cleavable linkages. Further, the internal structure of the water-soluble polymer may be systematized as any of a variety of repeating patterns and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers, and block terpolymers.

[0078] Typically, activated PEG and other activated water-soluble polymers (i.e., polymer reagents) are activated by suitable activating groups suitable for coupling to the desired site on the IL-2 moiety. Thus, the polymer reagent has a reactive group for reacting with the IL-2 moiety. Representative polymer reagents and methods for conjugating such polymers to an active moiety 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, J.M. Harris, Plenus Press, New York (1992), and Zalipsky (1995) Advanced Drug Reviews 16: 157-182. Exemplary activating groups suitable for coupling of the IL-2 moiety include, inter alia, hydroxyl, maleimide, ester, acetal, ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinyl ketone, thione, thiol, vinyl sulfone, hydrazine.

[0079] Preferably, the polymer reagent used in the preparation of the conjugate described herein is prepared without using phosgene. Such an approach is in contrast to the disclosure described, for example, in U.S. Patent No. 4,902,502, which specifically describes forming a chloroformate and subsequently using it to form a PEG active ester, and then reacting it with IL-2. The use of phosgene generates hydrogen chloride, which can cause chain cleavage of the polymer, thereby increasing impurities that may not be removable using prior art techniques. Thus, without wishing to be bound by theory, IL-2 partial conjugates prepared from polymer reagents formed without using phosgene provide a higher quality composition that is substantially free of high molecular weight chain degradation products. Also, in one or more embodiments, the spacer portion between the water-soluble polymer and the IL-2 moiety is not a spacer portion that includes a carbamate.

[0080] Typically, the weight average molecular weight of the water-soluble polymer in the conjugate is from about 100 Daltons to about 150,000 Daltons. However, exemplary ranges include a range of greater than 5,000 Daltons to about 100,000 Daltons, a range of about 6,000 Daltons to about 90,000 Daltons, a range of about 10,000 Daltons to about 85,000 Daltons, a range of greater than 10,000 Daltons to about 85,000 Daltons, a range of about 20,000 Daltons to about 85,000 Daltons, a range of about 53,000 Daltons to about 85,000 Daltons, a range of about 25,000 Daltons to about 120,000 Daltons, a range of about 29,000 Daltons to about 120,000 Daltons, a range of about 35,000 Daltons to about 120,000 Daltons, and a range of about 40,000 Daltons to about 120,000 Daltons for the weight average molecular weight. For any given water-soluble polymer, PEG having one or more molecular weights within these ranges is preferred.

[0081] Exemplary weight average molecular weights for the water-soluble polymer include about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branched water-soluble polymers having any of the foregoing total molecular weights (e.g., a 40,000 Dalton branched 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 moiety directly or indirectly linked to a PEG having a weight average molecular weight of less than about 6,000 Daltons.

[0082] When used as a polymer, PEG typically includes a plurality of (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" of "」. Therefore, the value of (n) is typically included in 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 monomer.

[0083] One of the polymers particularly preferred for use in the present invention is an end-capped polymer, i.e., a polymer in which at least one end is capped with a relatively inert group such as a lower C 1~6 alkoxy group (although a hydroxyl group can also be used). For example, when the polymer is PEG, it is preferred to use methoxy PEG (commonly 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 or other functional group that may optionally be chemically modified.

[0084] In one form useful in one or more embodiments of the present invention, free PEG or unbound PEG is a linear polymer having hydroxyl groups at each end: HO-CH2CH2O-(CH2CH2O) n -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 represented in a simplified form as HO-PEG-OH, where it is understood that the -PEG- symbol can represent the following structural unit: -CH2CH2O-(CH2CH2O) n -CH2CH2- Where (n) is as defined above.

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

[0087] Multi-armed or branched PEG molecules as described in U.S. Patent No. 5,932,462 can also be used as PEG polymers. For example, PEG can have the following structure:

Chemical formula

[0088] In addition, PEG can include fork-shaped PEG. An example of fork-shaped PEG is represented by the following structure:

Chemical formula

[0089] The PEG polymer can include pendant-type PEG molecules in which reactive groups such as carboxyl groups are covalently bonded along the length of the PEG rather than at the ends of the PEG chains. The pendant reactive groups can be bonded directly to the PEG or via a spacer portion such as an alkylene group.

[0090] In addition to the PEGs of the above-described forms, the polymer can also be prepared to include one or more weak or cleavable linkages in the polymer, including any of the above-described polymers. For example, the PEG can be prepared to include an ester linkage in the polymer that is susceptible to hydrolysis. As shown below, as a result of this hydrolysis, the polymer is cleaved into lower molecular weight fragments: -PEG-CO2-PEG- + H2O → -PEG-CO2H + HO-PEG-

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

[0092] The introduction of such optional features of the conjugate, i.e., one or more cleavable linkages into the polymer chain or to the IL-2 moiety, can provide additional control over the final desired pharmacological properties of the conjugate when administered. For example, a large and relatively inert conjugate (i.e., one to which is attached one or more high molecular weight PEG chains, e.g., one or more PEG chains having a molecular weight greater than about 10,000, in which case the conjugate is essentially biologically inactive) may be administered, which is released to yield a biologically active conjugate having a portion of the original PEG chain. In this way, the properties of the conjugate can be more effectively adjusted such that the biological activity of the conjugate equilibrates over time.

[0093] The water-soluble polymers related to the conjugate may also be "releasable". That is, the water-soluble polymer is released (by any of hydrolysis, enzymatic processes, catalytic processes or other methods), thereby resulting in the IL-2 moiety that is not conjugated. In some cases, the releasable polymer leaves the IL-2 moiety in vivo without leaving any fragments of the water-soluble polymer. In other cases, the releasable polymer leaves the IL-2 moiety in vivo, leaving a relatively small fragment (e.g., a succinate tag) from the water-soluble polymer. Exemplary cleavable polymers include those that bind to the IL-2 moiety via a carbonate linkage.

[0094] One of ordinary skill in the art will recognize that the foregoing discussion of non-peptidic water-soluble polymers is not exhaustive and is merely illustrative, and that any polymer material having the above qualities is contemplated. As used herein, the term "polymer reagent" generally refers to the entire molecule that may include a water-soluble polymer segment and a functional group.

[0095] As noted above, the conjugates of the present invention comprise a water-soluble polymer covalently attached to an IL-2 moiety. Typically, for any given conjugate, one to three water-soluble polymers are covalently attached to one or more moieties having IL-2 activity. However, in some cases, a conjugate may have 1, 2, 3, 4, 5, 6, 7, 8 or more water-soluble polymers individually attached to the IL-2 moiety. Any given water-soluble polymer may covalently bind to an amino acid of the IL-2 moiety or, if the IL-2 moiety is a glycoprotein (for example), to a carbohydrate of the IL-2 moiety. The attachment to the carbohydrate may be effected, for example, by metabolic functionalization using sialic acid-azide chemistry [Luchansky et al. (2004) Biochemistry 43(38):12358-12366], or by other suitable methods such as the use of glycidol to promote the introduction of aldehyde groups [Heldt et al. (2007) European Journal of Organic Chemistry 32:5429-5433].

[0096] The specific internal linkage between the moiety having IL-2 activity and the polymer depends on various factors. Such factors include, for example, the chemical nature of the specific linkage used, the specific IL-2 moiety, the available functional groups within the IL-2 moiety (either for binding to the polymer or for conversion to a suitable binding site), and the presence of other reactive functional groups within the IL-2 moiety.

[0097] The conjugate of the present invention may be a prodrug, although not essential, i.e., this means that the linkage between the polymer and the IL-2 moiety is releasable, thereby allowing the parent moiety to be released. Exemplary releasable 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 carboxyl group C-terminus of a protein, or the side chain hydroxyl group of an amino acid such as serine or threonine contained in a protein, or a similar functional group in a carbohydrate) and / or the polymer reagent using coupling methods commonly used in the art. However, most preferably, it is a releasable linkage that is readily formed by reacting a suitably activated polymer with an unmodified functional group contained in the moiety having IL-2 activity.

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

[0099] The conjugate may or may not have a measurable degree of IL-2 activity (as compared to the unconjugated IL-2 moiety). That is, the polymer-IL-2 moiety conjugate according to the present invention may have a biological activity in the range of about 0.1% to about 100% of the unmodified parent IL-2 moiety. In some cases, the polymer-IL-2 moiety conjugate may have a biological activity greater than 100% of the unmodified parent IL-2 moiety. Preferably, the conjugate having little or no IL-2 activity contains a hydrolyzable linkage connecting the moiety to the polymer, so that, regardless of the lack (or relatively lack) of activity in the conjugate, when the hydrolyzable linkage is induced to be 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 particular moiety having IL-2 activity used.

[0100] For a conjugate having a linkage stable to hydrolysis that couples a moiety having IL-2 activity to a polymer, this conjugate typically has a measurable degree of biological activity. For example, such a conjugate is typically characterized as having a biological activity that satisfies one or more of the following percentages compared to the biological activity 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 greater than 105% (when measured in a suitable model such as those well known in the art). Preferably, a conjugate having a linkage stable to hydrolysis (e.g., an amide linkage) has at least a certain degree of the biological activity of the unmodified parent moiety having IL-2 activity.

[0101] Here, exemplary conjugates according to the present invention will be described. Typically, such an IL-2 moiety is expected to (at least in part) share an amino acid sequence similar to the sequences provided in at least one of SEQ ID NOs: 1-4. Thus, although specific positions or atoms within SEQ ID NOs: 1-4 are referred to, such references are for convenience only, and one of ordinary skill in the art can readily determine the corresponding positions or atoms in other moieties having IL-2 activity. In particular, the descriptions provided herein with respect to native human IL-2 can, in many cases, be applied to any of the foregoing fragments, deletion mutants, substitution mutants, or addition mutants.

[0102] The amino group of the IL-2 moiety provides a point of attachment 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 has several lysine residues having ε-amino acids that can be utilized in the conjugate. Further, the N-terminal amine of any protein can also function as a point of attachment.

[0103] There are numerous examples of suitable polymeric reagents useful for forming a covalent linkage with an available amine of the IL-2 moiety. Specific examples are provided in Table 1 below along with the 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 polymeric reagent. Each polymeric moiety shown in Table 1 [e.g., (OCH2CH2) n or (CH2CH2O) n has a terminal “CH3” group, which 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 formation of conjugates of the polymer reagent with the amino groups of the IL-2 moiety can be achieved in various ways. In one approach, the IL-2 moiety can be conjugated with a polymer reagent functionalized with a succinimidyl derivative (or other activated ester group, and in this case, a similar approach as described for polymer reagents containing such alternative activated ester groups can be used). In this approach, the polymer having the succinimidyl derivative can bind to the IL-2 moiety in an aqueous medium at pH 7 to 9.0, but with different reaction conditions (e.g., a lower pH such as 6 to 7, or different temperatures and / or below 15 °C), as a result, the polymer can bind to different positions of the IL-2 moiety. In addition, an amide linkage can be formed by reacting a non-peptidic water-soluble polymer having an amine terminus with the IL-2 moiety having an active carboxylic acid group.

[0112] Exemplary conjugates are included in the following structures

Chemical formula

[0113] Exemplary conjugates are included in the following structure:

Chemical formula

[0114] As another method useful for conjugating the IL-2 moiety with a polymeric reagent, conjugation of a primary amine of the IL-2 moiety with a polymeric reagent functionalized with a ketone, aldehyde or its hydrated form (e.g., ketone hydrate, aldehyde hydrate) by using reductive amination is typical. In this method, the primary amine of the IL-2 moiety reacts with the carbonyl group of an aldehyde or ketone (or the hydroxyl-containing group of the corresponding hydrated aldehyde or ketone), thereby forming a Schiff base. Subsequently, the Schiff base can then be reductively converted to a stable conjugate by using a reducing agent such as sodium borohydride. In particular, when using a polymer functionalized with a ketone or α-methyl branched-chain aldehyde, and / or under specific reaction conditions (e.g., low pH), a selective reaction (e.g., at the N-terminus) is 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 included in the following structure:

Chemical formula

[0116] Exemplary conjugates of the present invention are included in the following structure:

Chemical formula

[0117] Exemplary conjugates of the present invention are included in the following structure:

Chemical formula

[0118] Other exemplary conjugates of the present invention are included in the following structure:

Chemical formula

[0119] Yet another exemplary conjugate of the present invention is encompassed by the following structure: [ka] During the ceremony: each (n) is independently an integer having a value from 2 to 4000; and IL-2 is the residue of the IL-2 moiety.

[0120] Exemplary conjugates that include a releasable linkage include those in which the IL-2 moiety is conjugated to a polymeric reagent encompassed by the following formula: [ka] During the ceremony: POLY 1 is a first water-soluble polymer; POLY 2 is a second water-soluble polymer; X 1 is the first spacer moiety; X 2 is a second spacer moiety; H α is an ionized hydrogen atom; R 1 is H or an organic radical; R 2 is H or an organic radical; (a) is either 0 or 1; (b) is either 0 or 1; R e1 is, when present, a first electron modifying group; R e2 is, when present, a second electron-modifying group; and (FG) is a functional group capable of reacting with the amino group of the active agent to form a releasable linkage such as a carbamate linkage. In this formula, polymer reagents with more definitive structures are contemplated:

Chem.

[0121] Still another exemplary polymer reagent is included in the following formula:

Chem.

Chem.

[0122] The polymer reagents providing these releasable linkages can be prepared according to the procedures described in U.S. Patent Application Publication No. 2006 / 0293499.

[0123] Exemplary conjugates formed using polymer reagents that provide releasable linkages include those of the following formula:

Chem.

[0124] Exemplary conjugates have the following structure:

Chemical formula

Chemical formula

[0125] The carboxyl group represents another functional group that can function as a point of attachment in the IL-2 moiety. Structurally, the conjugate can include the following:

Chemical formula

[0126] The C(O)-X linkage is obtained from the reaction between a polymer derivative having a terminal functional group and the carboxyl-containing IL-2 moiety. As discussed above, the specific linkage can depend on the type of functional group utilized. When the polymer is end-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-shaped polymers are used, the C(O)X moiety, particularly the X moiety, can be relatively complex and can include a longer linking structure.

[0127] Water-soluble derivatives containing a hydrazide moiety are also useful for conjugate formation in carbonyls and carboxylic acids. As long as the IL-2 moiety does not contain a carbonyl moiety or a 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 has a carbonyl moiety). For an IL-2 moiety containing a carboxylic acid, a PEG-hydrazine reagent can be covalently bonded to the IL-2 moiety in the presence of a coupling agent (e.g., DCC) [e.g., as a result of mPEG-OCH2C(O)NHNH2 + HOC(O)-(IL-2), mPEG-OCH2C(O)NHNHC(O)-IL-2 is obtained]. Specific examples of water-soluble derivatives containing a hydrazide moiety are provided in Table 2 below together with the corresponding conjugates. In addition, by reacting a water-soluble polymer derivative containing an activated ester with hydrazine (NH2-NH2) or tert-butyl carbazate [NH2NHCO2C(CH3)3], any water-soluble derivative containing an activated ester (e.g., a succinimidyl group) can be converted to contain 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. Optionally, 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 has a terminal "CH3" group, which 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 to which the water-soluble polymer binds. In particular, cysteine residues provide thiol groups when the IL-2 moiety is a protein. Next, the thiol groups of such cysteine residues can react with activation PEG specific for reaction with thiol groups, for example, N-maleimidyl polymers or other derivatives as described in U.S. Patent No. 5,739,208 and WO 01 / 62827 pamphlet. In addition, a thiol having a protecting group may be introduced into the oligosaccharide side chain of the activated glycoprotein and subsequently deprotected with a thiol-reactive water-soluble polymer.

[0130] Specific examples of the reagents are provided in Table 3 below along with the corresponding conjugates. In the table, the variable (n) represents the number of repeating monomer units, and “-S-(IL-2)” represents the IL-2 moiety residue after conjugate formation with the water-soluble polymer. Each polymer moiety shown in Table 3 [e.g., (OCH2CH2) n or (CH2CH2O) n has a terminal “CH3” group, which may be substituted with other groups (such as H and benzyl).

[0131] Regarding SEQ ID NOs: 1 and 2 corresponding to the exemplary IL-2 moieties, it can be seen that there is a cysteine residue at position 125. Thus, the exemplary thiol binding site is the cysteine located at position 125. It is preferred not to disrupt any disulfide bonds associated with a given IL-2 moiety, but it may be possible to bind polymers in one or more side chains of these cysteine residues to maintain some activity. 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, reference is made to the procedure described in WO 90 / 12874 pamphlet, and such 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 preferred not to introduce additional cysteine residues and / or thiol groups.

[0132]

Table 9

[0133]

Table 10

[0134] Regarding conjugates formed from water-soluble polymers having one or more maleimide functional groups (regardless of whether the maleimide reacts with the amine group or the thiol group of the IL-2 moiety), the corresponding water-soluble polymers of the maleamic acid type can also react with the IL-2 moiety. Under certain conditions (e.g., pH of about 7-9 and in the presence of water), the maleimide ring "opens" to form the corresponding maleamic acid. Consequently, the maleamic acid can react with the amine or thiol group of the IL-2 moiety. An exemplary maleamic acid-based reaction is schematically shown below. POLY represents a water-soluble polymer and (IL-2) represents the IL-2 moiety.

Chemical formula

[0135] Representative conjugates according to the present invention may have the following structure: POLY-L 0,1 -C(O)Z-Y-S-S-(IL-2) wherein POLY is a water-soluble polymer, L is an optional linker, Z is a heteroatom selected from the group consisting of O, NH, and S, Y is selected from the group consisting of C 2~10 alkyl, C 2~10 substituted alkyl, aryl, and substituted aryl, and (IL-2) is the IL-2 moiety. The polymer reagents that can react with the IL-2 moiety and as a result give rise to this type of conjugate are described in U.S. Patent Application Publication No. 2005 / 0014903.

[0136] As pointed out above, the exemplary conjugate of the present invention in which the water-soluble polymer is branched may have a branched water-soluble polymer having the following structure: [Chemical formula] In the formula, each (n) is an integer independently having a value of 2 to 4000.

[0137] The exemplary conjugate having a branched water-soluble polymer can be prepared using the following reagents: [Chemical formula] Thereby, a conjugate having the following structure is formed: [Chemical formula] In the formula: For each structure, each (n) is an integer independently having a value of 2 to 4000; and IL-2 is the residue of the IL-2 moiety.

[0138] Further exemplary conjugates can be formed using the following reagents: [Chemical formula] Thereby, a conjugate having the following structure is formed: [Chemical formula] In the formula: For each structure, (n) is an integer independently having a value of 2 to 4000; and IL-2 is the residue of the IL-2 moiety.

[0139] Conjugates can be formed in a variety of ways using thiol-selective polymer reagents, and the present invention is not limited in this regard. For example, the IL-2 moiety - which is in a suitable buffer (including, if necessary, a buffer containing an amine) if appropriate - is placed in an aqueous medium at a pH of about 7 to 8, and the thiol-selective polymer reagent is added in molar excess. The reaction is allowed to proceed as such for about 0.5 to 2 hours, although if it is determined that the yield of pegylation is relatively low, reaction times longer than 2 hours (e.g., 5 hours, 10 hours, 12 hours, and 24 hours) may be useful. Exemplary polymer reagents that can be used in this approach are polymer reagents having a reactive group selected from the group consisting of maleimide, sulfone (e.g., vinyl sulfone), and thiol (e.g., a functional thiol such as orthopyridinyl or "OPSS").

[0140] With respect to the 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 the polymer reagents are described in the literature.

[0141] The binding between the IL-2 moiety and the non-peptidic water-soluble polymer may be direct, in which case there are no intervening atoms between the IL-2 moiety and the polymer, or the binding may be indirect, in which case one or more atoms are located between the IL-2 moiety and the polymer. With respect to the indirect binding, a "spacer moiety" functions as a linker between the residue of the IL-2 moiety and the water-soluble polymer. Examples of one or more atoms constituting the spacer moiety include one or more of a carbon atom, a nitrogen atom, a sulfur atom, an oxygen atom, and combinations thereof. The spacer moiety can include an amide, secondary amine, carbamate, thioether, and / or disulfide group. Non-limiting examples of specific spacer moieties include -O-, -S-, -S-S-, -C(O)-, -C(O)-NH-, -NH-C(O)-NH-, -O-C(O)-NH-, -C(S)-, -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, -CH2-CH2-CH2-O-, -O-CH2-CH2-CH2-CH2-, -CH2-O-CH2-CH2-CH2-, -CH2-CH2-O-CH2-CH2-, -CH2-CH2-CH2-O-CH2-, -CH2-CH2-CH2-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-CH2-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)-CH2-CH2-, -CH2-CH2-NH-C(O)-CH2-CH2-, -C(O)-NH-CH2-, -C(O)-NH-CH2-CH2-, -O-C(O)-NH-CH2-, -O-C(O)-NH-CH2-CH2-, -NH-CH2-, -NH-CH2-CH2-, -CH2-NH-CH2-, -CH2-CH2-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-, -O-C(O)-NH-[CH2], h -(OCH2CH2) j -, a divalent cycloalkyl group, -O-, -S-, an amino acid, -N(R 6 )-, and those selected from the group consisting of any two or more combinations of the foregoing, wherein R 6 is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) is from 0 to 6, and (j) is from 0 to 20. Other specific spacer moieties have the following structures: -C(O)-NH-(CH2) 1~6 -NH-C(O)-, -NH-C(O)-NH-(CH2) 1~6 -NH-C(O)-, and -O-C(O)-NH-(CH2) 1~6 -NH-C(O)-, wherein the value of the subscript after each methylene indicates the number of methylenes contained in the structure, for example, (CH2)1~6 means that its structure can contain 1, 2, 3, 4, 5 or 6 methylenes. In addition, any of the above spacer moieties can further contain an ethylene oxide oligomer chain containing 1 to 20 ethylene oxide monomer units [i.e., -(CH2CH2O) 1~20 . That is, the ethylene oxide oligomer chain can be present before or after the spacer moiety and, in some cases, between any two atoms of the spacer moiety containing two or more atoms. Also, if the oligomer chain is adjacent to the polymer segment and is merely an extension of the polymer segment, it is not considered part of the spacer moiety.

[0142] Composition The conjugate is typically part of a composition. Generally, the composition contains a plurality of conjugates and, although not essential, preferably each conjugate contains the same IL-2 moiety (i.e., there is only one type of IL-2 moiety present throughout the composition). In addition, the composition can contain a plurality of conjugates, where any given conjugate contains 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 present throughout the composition). However, optimally, substantially all of the conjugates in the composition (e.g., 85% or more of the plurality of conjugates in the composition) each contain the same IL-2 moiety.

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

[0144] Regarding the conjugate in the composition, the composition satisfies one or more of the following characteristics, namely, at least about 85% of the conjugate in the composition has 1 to 4 polymers bound to the IL-2 moiety; at least about 85% of the conjugate in the composition has 1 to 3 polymers bound to the IL-2 moiety; at least about 85% of the conjugate in the composition has 1 to 2 polymers bound to the IL-2 moiety; at least about 85% of the conjugate in the composition has 1 polymer bound to the IL-2 moiety; at least about 95% of the conjugate in the composition has 1 to 5 polymers bound to the IL-2 moiety; at least about 95% of the conjugate in the composition has 1 to 4 polymers bound to the IL-2 moiety; at least about 95% of the conjugate in the composition has 1 to 3 polymers bound to the IL-2 moiety; at least about 95% of the conjugate in the composition has 1 to 2 polymers bound to the IL-2 moiety; at least about 95% of the conjugate in the composition has 1 polymer bound to the IL-2 moiety; at least about 99% of the conjugate in the composition has 1 to 5 polymers bound to the IL-2 moiety; at least about 99% of the conjugate in the composition has 1 to 4 polymers bound to the IL-2 moiety; at least about 99% of the conjugate in the composition has 1 to 3 polymers bound to the IL-2 moiety; at least about 99% of the conjugate in the composition has 1 to 2 polymers bound to the IL-2 moiety; and at least about 99% of the conjugate in the composition has 1 polymer bound to the IL-2 moiety. For example, when referring to the range of polymers as "x to y polymers", it is intended that the number of polymers is x to y, including the endpoints (i.e., for example, "1 to 3 polymers" is intended to mean 1 polymer, 2 polymers, and 3 polymers, and "1 to 2 polymers" is intended to mean 1 polymer and 2 polymers, etc.).

[0145] In one or more embodiments, the composition comprising the conjugate preferably does not contain or is substantially free of albumin. Also preferably, the composition does not contain or is substantially free of proteins that do not have IL-2 activity. Thus, preferably, the composition is 85%, more preferably 95%, most preferably 99% free of albumin. Additionally, preferably, the composition is 85%, more preferably 95%, most preferably 99% free of any protein that does not have IL-2 activity. To the extent albumin is present in the composition, the exemplary compositions of the invention are substantially free of conjugates comprising a poly(ethylene glycol) polymer that links residues of the IL-2 moiety to albumin.

[0146] PROLEUKIN® brand of Aldesleukin (Prometheus (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 advantageously may not require SDS and do not contain (or substantially do not contain) SDS as well as commonly used surfactants (such as Tween 20 and Tween 80). As a result, 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 do not contain or substantially do not contain surfactants such as SDS, Tween 20, and Tween 80 (e.g., less than about 20%, more preferably less than about 15%, even more preferably less than about 10%, even still more preferably less than about 9%, even still more preferably less than about 8%, even still more preferably less than about 7%, even still more preferably less than about 6%, even still more preferably less than about 5%, even still more preferably less than about 4%, even still more preferably less than about 3%, even still more preferably less than about 2%, even still more preferably less than about 1%, even still more preferably less than about 0.5%, and most preferably less than 0.001%). In addition, the compositions and conjugates of the present invention can be prepared without performing the step of removing surfactants such as SDS, Tween 20, and Tween 80 (e.g., by ultrafiltration). Furthermore, the compositions and conjugates of the present invention can be prepared without performing the step of removing surfactants (e.g., by ultrafiltration).

[0147] Control of the desired number of polymers for 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 means can be used to reduce or remove unwanted conjugates (e.g., conjugates having four or more polymers attached).

[0148] For example, a polymer-IL-2 partial conjugate can be purified to obtain / isolate different conjugates. Specifically, the resulting mixture can be purified to obtain PEG in an average range of 1, 2, 3, 4, 5 or more per IL-2 moiety, typically 1, 2 or 3 PEGs per IL-2 moiety. The purification strategy for the final conjugate reaction mixture can 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. That is, using gel filtration chromatography, based on the difference in their molecular weights (which essentially corresponds to the average molecular weight of the water-soluble polymer moiety), species with different polymer-to-IL-2 moiety ratios (e.g., 1mer, 2mer, 3mer, etc., where "1mer" indicates 1 polymer per IL-2 moiety, "2mer" indicates 2 polymers per IL-2 moiety, etc.) are fractionated. For example, in an exemplary reaction where a 35,000 Dalton protein is randomly conjugated to a polymer reagent with a molecular weight of approximately 20,000 Daltons, the resulting reaction mixture can include unmodified protein (molecular weight approximately 35,000 Daltons), monopegylated protein (molecular weight approximately 55,000 Daltons), dipegylated protein (molecular weight approximately 75,000 Daltons), and so on.

[0150] By using this method, it is possible to separate PEGs having different molecular weights 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, it is possible to separate a mixture of PEG 1mer, 2mer, 3mer, etc. from each other, but each of the recovered conjugate compositions may 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 choice of a particular column may depend on the desired fractionation range desired. Elution is generally performed using a suitable buffer such as phosphate, acetate, etc. The collected fractions can be analyzed by various 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. Using either approach, 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 the polymer-IL-2 partial conjugate and the non-conjugated IL-2 moiety.

[0154] Optionally, the composition of the 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., alditols, 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, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin.

[0157] Excipients may also 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 antibacterial agent to prevent or inhibit the growth of microorganisms. Non-limiting examples of antibacterial agents suitable for one or more embodiments of the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0159] Antioxidants may likewise be present in the composition. The use of antioxidants prevents oxidation and thereby prevents the degradation of conjugates or other components of the preparation. Antioxidants suitable for use in one or more embodiments of the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0160] Surfactants may be present as excipients. Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80", and pluronics such as F68 and F88 (both available from BASF, Mount Olive, New Jersey); sorbitan esters; phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamine (preferably not in liposomal form), fatty acids, and lipids such as fatty acid esters; steroids such as cholesterol; and IL-2lating agents such as EDTA, zinc, and other such suitable cations.

[0161] An acid or a base may be present as an excipient in the composition. 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. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

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

[0163] The amount of conjugate in the composition (i.e., the conjugate formed between the active agent and the polymeric reagent) can vary depending on various factors, but optimally, when the composition is stored in a unit dose container (e.g., a vial), it can be a therapeutically effective dose. Additionally, the pharmaceutical preparation may be contained in a syringe. A therapeutically effective dose can be determined experimentally by administering the conjugate repeatedly while increasing the amount thereof in order to determine which amount results in a clinically desirable endpoint.

[0164] The amount of any individual excipient in the composition can vary depending on the activity of the excipient and the specific needs of the composition. Typically, the determination of the optimal amount for any individual excipient is made through routine experimentation, i.e., by preparing compositions containing various amounts of the excipient (ranging from low to high amounts) and examining stability and other parameters, and then determining at which point an 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, more preferably about 15% to about 95% by weight of the excipient, and most preferably the concentration is less than 30% by weight.

[0166] These aforementioned pharmaceutical excipients, together 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, A.H., “Handbook of Pharmaceutical Excipients”, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0167] The composition includes all types of formulations, particularly those suitable for injection, such as powders that can be reconstituted or proliquids and liquids. Examples of diluents suitable for reconstitution of solid compositions prior to injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, Ringer's solution, normal saline, sterile water, deionized water, and combinations thereof. For liquid pharmaceutical compositions, solutions and suspensions are contemplated.

[0168] The composition of one or more embodiments of the present invention is typically administered by injection, although not necessarily, and thus is generally a liquid solution or suspension immediately prior to administration. The pharmaceutical preparation can also take other forms such as syrups, creams, ointments, tablets, powders, etc. Other methods of administration include intralung, rectal, transdermal, transmucosal, oral, intrathecal, intratumoral, peritumoral, intraperitoneal, subcutaneous, intraarterial, etc.

[0169] The present invention also provides a method of administering a conjugate as provided herein to a patient suffering from a condition responsive to treatment with the conjugate. This method includes administering to the patient, generally by injection, a therapeutically effective amount of the conjugate (preferably provided as part of a pharmaceutical composition). As described above, the conjugate can be injected (e.g., intramuscularly, subcutaneously and parenterally). Types of formulations suitable for parenteral administration include, in particular, ready-to-inject solutions, dry powders that are miscible with a solvent prior to use, ready-to-inject suspensions, insoluble dry compositions that are miscible with a vehicle prior to use, and emulsions and liquid concentrates that are diluted prior to administration.

[0170] The method of administering the conjugate (preferably provided as part of a pharmaceutical composition) can optionally be carried out so as to localize the conjugate to a particular area. For example, liquids, gels and solid formulations containing the conjugate can be surgically implanted into the affected area (such as within the tumor, in the vicinity of the tumor, within the area of inflammation, and in the vicinity of the area of inflammation, etc.). Conveniently, organs and tissues can 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 administering the conjugate can be treated. Those skilled in the art 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 can 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 reduced and / or eliminated.

[0173] Any given conjugate of unit dosage (here also 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] The present invention has been described in connection with its preferred specific embodiments, but 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 present invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains.

[0175] All papers, works, patents, and other publications referred to in this specification are hereby incorporated by reference in their entirety.

Example

[0176] In the practice of the present invention, unless otherwise indicated, conventional techniques such as organic synthesis, biochemistry, protein purification, etc. are used, which are within the scope of the art. Such methods are described in detail in the literature. See, for example, J. March, "Advanced Organic Chemistry: Reactions Mechanisms and Structure", 4th Edition (New York: Wiley-Interscience, 1992), supra.

[0177] In the following examples, efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations must be taken into account. Unless otherwise indicated, temperatures are in degrees Celsius and pressures are at or near sea level. Each of the following examples is considered to be a teaching for those skilled in the art to perform one or more of the embodiments described in this specification.

[0178] For use in this example, an aqueous solution ("stock solution") containing recombinant IL-2 ("rIL-2") corresponding to the amino acid sequence of SEQ ID NO: 3, which is a mature protein sequence, 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 as described by the manufacturer, loaded onto the gels, and electrophoresis was performed.

[0180] Cation exchange chromatography An SP-HP Sepharose (GE Healthcare) cation exchange column with a bed volume of approximately 100 ml was prepared using standard methods. 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 flow rate of the column 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 Since the human IL-2 cDNA sequence may not be optimally expressed in prokaryotes such as Escherichia coli (E. coli) because of the large differences in codon usage frequency among organisms, the gene was completely synthesized using PCR techniques instead of introducing multiple point mutations into existing human-derived cDNA sequences to maximize E. coli codon usage frequency.

[0183] The method of synthesizing genes from overlapping primers was essentially a slight modification of a combination of two methods. The 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 divided into forward and reverse oligonucleotides, with few exceptions, less than 35 bp in length, and there were no gaps between the oligonucleotides. Each oligonucleotide was such that two adjacent ones overlapped the opposing strand by at least 10 nucleotides at the 3' end and at least 15 nucleotides at the 5' end. Partial gene fragments were constructed using double asymmetric PCR and combined using overlap extension PCR to construct the entire gene. Next, as described by Young et al., the T7 endonuclease I selection step was used to remove mismatched duplexes. See Young et al. (2004) Nucleic Acids Research 32(7):e59. Restriction enzyme sites were included at the gene termini, and the final gene fragment was cloned into a commercially available expression vector for Escherichia coli (E. coli). DNA sequence analysis was used to confirm the sequence obtained as shown in Figure 1 and SEQ ID NO:5.

[0184] Using this approach, in this amino acid sequence, amino acid position 1 (alanine) is excluded when compared to the native mature human sequence, and a C→S amino acid mutation is included at amino acid position 125 for the sequence as shown. The first amino acid of this sequence is the methionine of direct bacterial expression (without the encoded signal peptide). However, during expression, the first methionine is removed by the host methionine aminopeptidase.

[0185] The gene was cloned into one of the pET (T7) expression vectors. Protein expression was carried out in the E. coli strain BL21(DE3), and one of the strains was typically used for the T7 expression system. This expression system is commercially available, and the method of expression is available from EMD Biosciences, Merck KGaA, Darmstadt, Germany. The use of this system was based on a research license from Brookhaven National Laboratory. The protein was expressed as inclusion bodies in E. coli by 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, the cells were harvested by centrifugation. To further homogenize, the cell mass pellet was stored at -80 °C. The frozen cell mass pellet was resuspended in cell wash 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 at 4 - 15 °C with a Microfluidizer (M-110P from Microfluidics, Newton, Massachusetts, USA) for one pass. The homogenate was diluted 2-fold with cell wash buffer (50 mM Tris, 5 mM EDTA, pH 8.0) and centrifuged at 13860 × g for 60 minutes. The supernatant was discarded. The inclusion body pellet was successively washed 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, 1M Washing was performed in three steps using a buffer of NaCl and pH 8.0. After washing, a crude IL-2 inclusion body was obtained.

[0187] The crude IL-2 inclusion body was dissolved in a buffer of 6M guanidine, 100 mM Tris, and pH 8. EDTA was added to a final concentration of 2 mM. Next, dithiothreitol (DTT) was 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 lower the guanidine concentration to 4.8. After centrifugation at 13,860×g for 1 hour, the resulting gel-like pellet was discarded. By adding water, the guanidine concentration of the supernatant was further reduced to 3.5 M. 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 13,860×g for 1 hour. The resulting pellet was suspended in a buffer of 3.5 M guanidine, 20 mM acetate, 5 mM DTT, and pH 5 and centrifuged at 13,860×g for 1 hour. This washing step was repeated once more.

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

[0189] Another embodiment of the present invention relates to an improved method for causing a protein to adopt a tertiary structure. In this regard, previous methods often rely on serial dilution, which is often harsh on the protein. Thus, an improved technique that enables protein folding under milder conditions is provided, the method comprising the steps of placing the expressed protein (e.g., an IL-2 moiety such as IL-2 prepared according to this example) into a dialysis bag having 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, e.g., 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 (e.g., buffers and water) lacking (or substantially lacking) guanidine, urea, lithium perchlorate, 2-mercaptoethanol, dithiothreitol, and surfactants. 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 bag was placed into a reservoir containing a buffer of 4.8 M guanidine, 0.1 M Tris, pH 8. After equilibration for 3 hours, the guanidine concentration in the reservoir was gradually decreased to 2 M 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 to remove the precipitate. The supernatant was concentrated using a Pellicon XL TFF membrane system (Millipore Corporation, USA).

[0191] The 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 running buffer was 2 M guanidine, 20 mM Tris, pH 8, and the flow rate was 25 mL / min. The fractions under the IL-2 monomer peak were pooled. It should be noted that other suitable purification methods such as ion exchange chromatography and hydrophobic interaction chromatography (HIC chromatography) can also be used.

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

[0193] Example 2 PEGylation of rIL-2 with mPEG2-C2-fmoc-20K-NHS [Chemical Structure] mPEG2-C2-fomc-20K-N-hydroxysuccinimide derivative, 20 kDa (「mPEG2-C2-fmoc-20K-NHS」) mPEG2-C2-fmoc-20K-NHS, which had been 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 reach 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 a final concentration of 20 mM, and conjugate formation was allowed to proceed for 30 minutes, providing the [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 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, purification by column chromatography was performed.

[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). From the purity analysis of [mPEG2-C2-fmoc-20K]-[rIL-2] by reverse phase HPLC, a purity of 100% purified conjugate was detected at 280 nm. See Figure 2.2. The purity was greater than 95% when determined by 4-12% NuPage bis-tris SDS-PAGE gel (gel not shown) with Coomassie blue staining of 20 μg of purified [mPEG2-C2-fmoc-20K]-[rIL-2]. The apparently large, >200 kDa molecular weight conjugate was thought to result in a low mobility of the conjugate through the gel due to high PEG hydration and as a result a relatively large hydrodynamic radius. From these tests, the formation of three conjugates was confirmed: 4mer, 3mer, 2mer, and 1mer, i.e., for the 4mer four “[mPEG2-C2-fmoc-20K]” are attached to a single “[rIL-2]”, for the 3mer three “[mPEG2-C2-fmoc-20K]” are attached to a single “[rIL-2]”, for the 2mer two “[mPEG2-C2-fmoc-20K]” are attached to a single [rIL-2], and for the 1mer one “[mPEG2-C2-fmoc-20K]” is attached to a single [rIL-2] of [mPEG2-C2-fmoc-20K]-[rIL-2].

[0195] By detecting the species change by reverse-phase HPLC, the release property of [mPEG2-C2-fmoc-20K]-[rIL-2] that releases rIL-2 was shown. Briefly, the 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 tested for the disappearance of the [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate and the presence of the released rIL-2. The appearance of rIL-2 reached a plateau about 10 hours after incubation and gradually decreased, presumably due to precipitation. The data are provided in Figure 2.3.

[0196] Example 3 Pegylation of rIL-2 with mPEG2-CAC-fmoc-20K-NHS

Chemical Structure

[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, and the lines correspond to absorbance at various wavelengths. From the purity analysis of [mPEG2-CAC-fmoc-20K]-[rIL-2] by reverse phase HPLC analysis, a purity of 98.5% of the purified conjugate was detected at 280 nm. The peak at 19.6 minutes corresponds to unreacted mPEG2-CAC-fmoc-20K-NHS (which constitutes <0.1%). See Figure 3.2. The purity was >95% when determined by 4-12% NuPage Bis-Tris SDS-PAGE gel (gel not shown) with Coomassie blue staining of 20 μg of purified [mPEG2-CAC-fmoc-20K]-[rIL-2]. The apparently large conjugate with a molecular weight >200 kDa was thought to be the result of low mobility of the conjugate through the gel due to high PEG hydration. The molecular weight of the purified [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate was also determined by MALDI-TOF spectrometry. As can be seen in Figure 3.3, the main peak at 79.6 kDa is within the predicted range of the molecular weight of the 3mer [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate. The peak at 100.8 kDa is within the predicted range of the molecular weight of the 4mer [mPEG2-CAC-fmoc-20K]-[rIL-2]. Peaks at MW 40 kDa and 58.7 kDa may correspond to the divalent 3mer IL-2 conjugate and 4mer IL-2 conjugate, respectively.

[0198] Example 4 Pegylation of rIL-2 with branched mPEG-N-hydroxysuccinimidyl derivative, 20 kDa [Chemical Structure] mPEG2-ru-20K-N-hydroxysuccinimidyl derivative, 20 kDa (「mPEG2-ru-20K-NHS」) mPEG2-ru-20K-NHS, which had been 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 reach 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 a final concentration of 20 mM, and when conjugate formation was allowed to proceed for 30, [mPEG2-ru-20K]-[rIL-2] conjugate was provided. After 30 minutes, quenching was achieved by adding 1 M glycine (pH 6.0) to the reaction mixture 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, it 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, and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm). From the purity analysis of [mPEG2-ru-20K]-[rIL-2] by reverse phase HPLC analysis, a purity of 100% of the purified conjugate was detected at 280 nm. See Figure 4.2. The purity was greater than 95% when determined by 4-12% NuPage bis-tris SDS-PAGE gel (gel not shown) with Coomassie blue staining of 20 μg of purified [mPEG2-ru-20K]-[rIL-2]. The apparently large conjugate with a molecular weight greater than 200 kDa was the result of low mobility of the conjugate through the gel due to high PEG hydration.

[0200] Example 5 Branched mPEG-N-hydroxysuccinimidyl derivative, pegylation of rIL-2 with 40 kDa

Chemical formula

[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. From the purity analysis of [mPEG2-ru-40K]-[rIL-2] by reverse phase HPLC, a purity of 100% purified conjugate was detected at 280 nm. The purity was greater than 95% when determined by 4-12% NuPage bis-tris SDS-PAGE gel (gel not shown) with Coomassie blue staining of 20 μg of purified [mPEG2-ru-40K]-[rIL-2]. The apparently large conjugate with a molecular weight greater than 200 kDa (presumably 3mer type of [mPEG2-ru-40K]-[rIL-2]) was a result of low mobility of the conjugate through the gel due to high PEG hydration. Unreacted mPEG2-ru-40K-NHS was eluted first through the column, followed by the [mPEG2-ru-40K]-[rIL-2] conjugate.

[0202] Example 6 Branched mPEG-N-hydroxysuccinimidyl derivative, Pegylation of rIL-2 with 4 kDa

Chemical formula

[0203] A typical cation exchange chromatography purification profile of [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 elution fractions. As shown in Figure 6, the fractions containing the mixture of 3mer / 2mer [mPEG2-ru-4K]-[rIL2], and the fractions containing the mixture of 2mer / 1mer [mPEG2-ru-4K]-[rIL2] were pooled separately.

[0204] Example 7 Pegylation of rIL-2 with Linear mPEG-Butyraldehyde Derivative, 30 kDa [Chemical Structure] Linear mPEG-Butyraldehyde Derivative, 30 kDa (「mPEG-ButyrALD」) After adding all reaction components and buffer, design the pegylation reaction such that the final rIL-2 concentration is 2.5 mg / ml. Warm the mPEG-ButyrALD, 30 kDa, stored at -20 °C under argon to ambient temperature. Weigh out an amount of PEG reagent equal to 10 - 50 molar equivalents of the rIL-2 to be pegylated and dissolve it in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. Add the 12% PEG reagent solution to an aliquot of the stock rIL-2 solution and stir for 15 - 30 minutes. Next, add the reducing agent sodium cyanoborohydride (NaCNBH3) in a 10 - 100 molar excess relative to the PEG reagent and ensure coupling via secondary amine linkages by stirring the reaction mixture at room temperature for 5 - 18 hours, thereby forming a conjugate solution.

[0205] It has been found that when the aldehyde group of mPEG-ButyrALD is reduced by a reducing reagent such as sodium cyanoborohydride, it reacts with the primary amine associated with rIL-2 and covalently binds to it via a secondary amine. The selectivity for one or more amines to bind to the polymer can be adjusted by adjusting the pH of the conjugate formation conditions. Relatively low pH conditions (e.g., pH of about 5.5) can induce conjugate formation at the N-terminus. At relatively neutral pH conditions (e.g., about 7.5 and slightly above), covalent bonding occurs more frequently at other positions (i.e., at 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 the conjugate is formed, and thus the ability to achieve the desired positional isomers can be enhanced.

[0206] Using this same approach, other conjugates are prepared using mPEG-BuryrALD with other weight-average molecular weights.

[0207] Example 8 Pegylation of rIL-2 with branched mPEG-butylaldehyde derivative, 40 kDa [Chemical] Branched-chain mPEG-butylaldehyde derivative, 40 kDa ("mPEG2-ButyrALD") After adding all reaction components and buffer, design the pegylation reaction so that the final rIL-2 concentration is 2.5 mg / ml. Warm the mPEG2-ButyrALD, 40 kDa, stored at -20 °C under argon to ambient temperature. Weigh out an amount of the PEG reagent equal to 10 - 50 molar equivalents of the rIL-2 to be pegylated and dissolve it in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. Add the 12% PEG reagent solution to an aliquot of the stock rIL-2 solution and stir for 15 - 30 minutes. Next, add the reducing agent sodium cyanoborohydride (NaCNBH3) in a 10 - 100 molar excess relative to the PEG reagent and stir the reaction mixture 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 reagent such as sodium cyanoborohydride, it reacts with the primary amine related to rIL-2 and covalently binds to it via a secondary amine.

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

[0210] Example 9 Pegylation of rIL-2 with linear mPEG-succinimidyl α-methylbutanoate derivative, 30 kDa [Chemical] Linear mPEG-succinimidyl α-methylbutanoate derivative, 30 kDa ("mPEG-SMB") After adding all the reaction components and buffer, design the pegylation reaction so that the final rIL-2 concentration is 2.5 mg / ml. Warm mPEG-SMB, 30 kDa, which was stored at -20 °C under argon, to ambient temperature. Weigh out an amount of the PEG reagent equal to 10 - 50 molar equivalents of the rIL-2 to be pegylated and dissolve it in 20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12% reagent solution. Add the 12% PEG reagent solution to an aliquot of the stock rIL-2 solution and stir at room temperature for 5 - 18 hours to obtain a conjugate solution. The conjugate solution is quenched with lysine solution (pH 7.5) such that the final lysine molar concentration is 10 - 100 times the PEG reagent molar concentration.

[0211] The mPEG-SMB derivative has been found to provide a sterically hindered active NHS ester that reacts selectively with lysine and terminal amines.

[0212] Using this same procedure, other conjugates are prepared using mPEG-SMB having other weight average molecular weights.

[0213] Example 10 Pegylation of rIL-2 with mPEG-PIP, 20 kDa The basic structure of the polymer reagent is provided below:

Chemical Structure

[0214] It has been found that when the ketone group of mPEG-PIP is reduced by a reducing reagent such as sodium cyanoborohydride, it reacts with the primary amine associated with rIL-2 and covalently bonds to it via a secondary amine.

[0215] Using this same approach, other conjugates are prepared using mPEG-PIP having other weight average molecular weights.

[0216] Example 11 Activity of exemplary (rIL-2)-PEG conjugates The activities of Aldesleukin (control), [mPEG2-C2-fmoc-20K]-[rIL-2] of Example 2, [mPEG2-CAC-fmoc-20K]-[rIL-2] of Example 3, and [mPEG2-ru-20K]-[rIL-2] of Example 4 were evaluated in a cell proliferation assay using CTLL-2 cells.

[0217] CTLL-2 cells (mouse cytotoxic T lymphocyte cell line) were maintained in complete RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, and 10% IL-2 culture supplement (containing T-STIM™, ConA (Concanavalin A)) at 37 °C in a 5% CO2 atmosphere. The cells were cultured in suspension until they reached a cell density of 2 - 3×10 5 cells / mL and then split. For the activity assay, 3 - 4 days after the last split, the cells were washed three times with Dulbecco's phosphate buffered saline. The cells were then resuspended at a cell density of approximately 2×10 5 cells / mL in supplemented medium without T-STIM™ and seeded at 90 μL / well into a 96-well microplate with a white wall and clear bottom. The experiment was also carried out using supplemented medium (without T-STIM™) adjusted to pH 6.7 - 7 to minimize the release of conjugates during incubation. Then, 10 μL of a 10-fold concentrated test compound diluted in supplemented medium without T-STIM™ was added. The cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 hours. After 24 hours of incubation, 100 μL of Promega's CellTiter-Glo® reagent was added to each well. The plate was mixed on 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. 5 cells / mL in the suspension until they reached a cell density of 2 - 3×10 5 cells / mL and then split.

[0218] For the activity assay, 3 - 4 days after the last split, the cells were washed three times with Dulbecco's phosphate buffered saline. The cells were then resuspended at a cell density of approximately 2×10 5 cells / mL in supplemented medium without T-STIM™ and seeded at 90 μL / well into a 96-well microplate with a white wall and clear bottom. The experiment was also carried out using supplemented medium (without T-STIM™) adjusted to pH 6.7 - 7 to minimize the release of conjugates during incubation. Then, 10 μL of a 10-fold concentrated test compound diluted in supplemented medium without T-STIM™ was added. The cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 hours. After 24 hours of incubation, 100 μL of Promega's CellTiter-Glo® reagent was added to each well. The plate was mixed on 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. 5 cells / mL in the suspension until they reached a cell density of 2 - 3×10 5 cells / mL and then split.

[0219] For the releasable conjugates of [mPEG2-C2-fmoc-20K]-[rIL-2] of Example 2 and [mPEG2-CAC-fmoc-20K]-[rIL-2] of Example 3, the activities of both the released IL-2 and the unreleased conjugate were 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 conjugate, the sample was diluted from the storage buffer to the supplemented medium approximately 1 hour before the assay. To test the activity of the released IL-2, the releasable conjugates {i.e., the [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate of Example 2 and the [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate of 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 the start of the assay.

[0220] Using GraphPad's Prism 5.01 software, the EC of cell proliferation was determined by non-linear regression analysis of the dose-response curve 50 value (the concentration of the test compound required to show 50% of the maximum response) was obtained.

[0221] The activity of Aldesleukin and conjugates was measured using a cell proliferation assay. A summary of the results is shown in Table 4. All test substances induced the growth of CTLL-2 cells in a dose-dependent manner. Since the releasable conjugate was pre-incubated under conditions that forced protein release, Aldesleukin was also pre-incubated as a control to test the stability of the protein itself under forced release treatment conditions. As shown in Table 4, Aldesleukin remained stable after pre-incubation under release conditions (8 hours at 37°C, pH 9), and the relative potency against Aldesleukin stored under recommended conditions was shown. After pre-incubating [mPEG2-C2-fmoc-20K]-[rIL-2] of Example 2 and [mPEG2-CAC-fmoc-20K]-[rIL-2] of Example 3 under conditions that caused the release of IL-2, as shown in Figure 8, the activity was restored; the IL-2 released from these conjugates showed relative potency against the control Aldesleukin, while a portion of the non-released conjugate had lower potency compared to Aldesleukin. The stable 3mer [mPEG2-ru-20K]-[rIL-2] conjugate showed the lowest potency (Figure 7), which was 0.04% of Aldesleukin, while the 1mer [mPEG2-ru-20K]-[rIL-2] showed potency equivalent 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] of Example 2, [mPEG2-CAC-fmoc-20K]-[rIL-2] of Example 3, and [mPEG2-ru-20K]-[rIL-2] of Example 4 were evaluated by ELISA after a single injection in mice.

[0224] The concentration of aldosterone was measured by a heterogeneous sandwich ELISA. Briefly, a 96-well microtiter plate was coated with a mouse monoclonal antibody against IL-2 and blocked. Samples and standards were prepared in native plasma and subsequently diluted to 10% plasma with a buffer containing a biotinylated rabbit polyclonal antibody against IL-2, followed by incubation on the assay plate. Streptavidin-horseradish peroxidase and subsequently the colorimetric substrate 3,3’,5,5’-tetramethylbenzidine (TMB) were used to detect IL-2. A stop solution was added, and the absorbance was read at 450 nm with background subtraction at 650 nm. A standard curve was generated by a weighted four-parameter algorithm, and the sample concentration was determined by interpolation on the standard curve. The lower limit of quantification was 0.05 ng / mL.

[0225] The concentration of pooled 1mer / 2mer [mPEG2-ru-20K]-[rIL-2] was measured by a homogeneous HTRF® assay (Cisbio US, Bedford MA). The reaction mixture (15 μL, europium cryptate-conjugated 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. Samples and standards (5 μL) diluted in native plasma were added, and the plate was incubated. The plate was read on a fluorescence reader at 615 and 665 nm, and the delta F was calculated. A standard curve was generated by a weighted five-parameter algorithm, and the sample concentration was determined by interpolation on the standard curve. The lower limit of quantification was 0.5 ng / mL.

[0226] The 3mer [mPEG2-C2-fmoc-20K]-[rIL-2] and 3mer [mPEG2-CAC-fmoc-20K]-[rIL-2] were measured in the total IL-2 assay. Since the [mPEG2-C2-fmoc-20K]-[rIL-2] and [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugates are releasable conjugates and different molecular species may be present in the sample, individual quantification is difficult; therefore, the total IL-2 level was measured. The samples and standard stock were prepared in native plasma, diluted 1:1 with release buffer (100 mM HEPES / 100 mM Tris-HCL, pH 9), and incubated at 37 °C for 30 - 36 hours to force the release of the polymeric component of the conjugate from the conjugate. After incubation, 25% by volume of 0.1 M acetic acid was added to neutralize the high pH. The released IL-2 was measured by ELISA as 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). Heparinized plasma samples were collected at the 10-minute time point and at 1, 6, 24, 48, 72, 96, 120, 168, and 336 hours time points. The geometric mean concentration was calculated from 3 mice for each time point. As shown in Figure 9, aldesleukin had a short half-life and was not detectable after 6 hours (<0.05 ng / mL), while the conjugate had a long half-life and was still detectable at the 336-hour time point.

[0228] Example 13 Lung metastatic melanoma efficacy test For the evaluation of the efficacy of compounds intended to have IL-2 activity, a metastatic melanoma lung model is widely used and developed in C57BL / 6 mice. In this model, first, B16F10 melanoma cells are intravenously administered to the mice, thereby generating pulmonary nodules of various numbers and sizes. The number of pulmonary nodules and the total surface area of their lesions vary depending on the transplanted cell concentration. Next, the test compound of interest is administered to the treatment group of mice, and another group of mice is left untreated as a control. The efficacy of the test compound can be determined as the reduction rate of the number and size of pulmonary nodules and the total lesion area for each lung between the treatment group and the untreated group.

[0229] In this test, 100,000 B16F10 cells (passage P8 or less) were transplanted by tail vein injection. On the 3rd day after cell transplantation, the test compound of interest (or vehicle) was administered according to the administration by either the IP (intraperitoneal) route or the IV (intravenous) route as shown in Table 5.

[0230]

Table 12

[0231] On the 14th day after cell transplantation, the mice were sacrificed, and at the same time, the lungs were excised and fixed in formaldehyde-containing Bouin's (Bowen) solution for 1 to 2 days. The lungs (fixed in Bouin's (Bowen) solution) were examined with a stereomicroscope, and the number and size of the lesions for each lung were determined.

[0232] As shown in Figure 10, on the 14th day after cell transplantation, the mice were sacrificed, and the excised lungs were fixed in Bouin's (Bowen) solution. Aldesleukin (Prometheus For each of Laboratories Inc., San Diego CA), the IL-2 moiety 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], tumor nodules and their sizes were counted.

[0233] Example 14 Subcutaneous B16F10 Melanoma Efficacy Test For the evaluation of the efficacy of compounds intended to have IL-2 activity, a very robust subcutaneous melanoma model in syngeneic mice, i.e., C57BL / 6 mice, has been used. Briefly, 1 million B16F10 cells were subcutaneously transplanted into the dorsal region of each 5 - 6-week-old C57BL / 6 mouse. After the tumors grew to a palpable size, i.e., up to 70 - 120 square mm, they were randomized and assigned to the groups as shown in Table 6. The mice were administered the test compound, i.e., Aldesleukin (Prometheus Laboratories Inc., San Diego CA), rIL-2-polymer conjugate or vehicle, at various dose concentrations and dosing regimens. Body weight and tumor volume were measured every other day. The endpoint of this test is the earlier of when the median tumor volume of a given group reaches 1500 square mm or 45 days.

[0234] [Table 13]

[0235] The dose-response curves of tumor growth inhibition after administering Aldesleukin (Prometheus Laboratories Inc.) and rIL-2-polymer conjugates in various dosing schemes are provided in FIGS. 11A and 11B. These results show evidence that the efficacy of the single-dose of the rIL-2-polymer conjugates tested is superior to that of Aldesleukin (Prometheus Laboratories Inc.) administered at 3 mg / kg twice daily for 5 days.

[0236] FIG. 11A shows the tumor growth inhibition period until the median tumor volume reaches 1500 mm 3 It was found that the tumor growth delay (TGD) from the tumor progression curve was 4.6 days and 6.2 days, respectively, for the pooled 3mer / 4mer [mPEG2-CAC-fmoc-20K]-[rIL-2] at 2 mg / kg and 4 mg / kg dose concentrations. For the pooled 3mer / 4mer [mPEG2-C2-fmoc-20K]-[rIL-2], it was found that the TGD was 6.4 days and 7.6 days at 2 mg / kg and 4 mg / kg dose concentrations, respectively.

[0237] FIG. 11B shows the tumor growth inhibition period until the median tumor volume reaches 1500 mm 3 It was found that the TGD from the tumor progression curve was 3.6 days and 4.6 days, respectively, for the pooled 1mer / 2mer [mPEG2-C2-fmoc-20K]-[rIL-2] at 6 mg / kg and 8 mg / kg dose concentrations. For the pooled 1mer / 2mer [mPEG2-ru-20K]-[rIL-2], it was found that the TGD was 3.8 at 2 mg / kg, while the 4 mg / kg dose concentration was found to be essentially toxic. It was found that the TGD from the tumor progression curve was 2.2 days and 3.6 days, respectively, for the pooled 1mer / 2mer [mPEG2-CAC-fmoc-20K]-[rIL-2] at 2 mg / kg and 4 mg / kg dose concentrations.

[0238] Briefly stated, in both the pulmonary metastasis model (Example 13) and the subcutaneous mouse melanoma model (Example 14), the rIL-2 polymer conjugate achieved efficacy with substantially less frequent dosing and less total protein mass when compared to Aldesleukin (Prometheus Laboratories Inc.).

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【Claim 1】 The composition or method described in the specification.

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