Interleukin formulations for in vivo use

Abstract

Stable liquid formulation for interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-21 (IL-21) for in vivo use are provided.

Description

[0001] INTERLEUKIN FORMULATIONS FOR IN FIFO USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63 / 484,837, and U.S. Provisional Application No: 63 / 484,833 filed on February 14, 2023, which are incorporated herein by reference in their entirety.

[0004] FIELD

[0005] The disclosure relates to new pharmaceutical compositions comprising interleukins of they-chain family, such as interleukins 2, 4, 7, 9, 15, and 21.

[0006] BACKGROUND

[0007] Interleukins are molecules of medicinal interest that are difficult to stabilize in solution because of their tendencies toward adsorption, aggregation, oligomerization and oxidation. They act mainly as growth and proliferation factors for progenitors and mature cells and also have roles in lineage-specific cell differentiation.

[0008] SUMMARY

[0009] One aspect of the present invention relates to the formulation of stable aqueous solutions of recombinant interleukins of the common y - chain family by selecting excipients that, prevent them from degrading while stored at 2 - 8 °C for several months. This allow'S their manufacture for medicinal use without resorting to lengthy and expensive lyophilization procedures or extremely low storage and handling temperatures.

[0010] A second aspect is the packaging of these solutions in compatible pre-filled syringes, bags, and other containers, enabling their adoption as ready-to-use as ancillary materials for in vivo proliferation of cells or for other in vivo purposes. The incorporation of these solutions containing defined concentrations of interleukins into packaging formats suitable for direct incorporation into cell cultures enables the seamless integration of critical materials into a cell therapy manufacturing process without the need to reconstitute lyophilized proteins, titrate to achieve dose levels relevant for a given process, and mitigate the potential risk of contaminating a product due to improper aseptic technique, or other user error.

[0011] The interleukin molecules herein include variants of the present disclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-2 ammo acid sequence (SEQ ID NO: 1). These include IL-2 variants that comprise an ammo acid sequence having an N88R mutation that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type IL-2 amino acid sequence (i.e. SEQ ID NO: 1). Embodiments also include IL-2 variants that preferentially stimulate Treg cells and comprise an amino acid sequence having N88R and C125S mutations that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to the wild-type IL-2 ammo acid sequence (SEQ ID NO: 1). Embodiments also include IL-2 variants that preferentially stimulate Treg cells and comprise an ammo acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%., at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild- type IL-2 amino acid sequence (SEQ ID NO: I).

[0012] In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an ammo acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-7 amino acid sequence (SEQ ID NO: 2). These include IL-7 variants.

[0013] In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-15 ammo acid sequence (SEQ ID NO: 3). These include IL-15 variants.

[0014] In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-21 amino acid sequence (SEQ ID NO: 4). These include IL-21 vanants.

[0015] In another aspect, the liquid formulation comprising an interleukin embodied herein, is stable at temperatures ranging from 2°C to 20°C for a period of at least 12 months.

[0016] In another aspect, a liquid formulation for in vivo use comprises about 0.001 milli- international units (MIL ) to 20 MIU per ml of one or more interleukins, comprising interleukin-2 (IL- 2), interleukin-7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21). In certain embodiments, the formulation comprising IL-2 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 0.0001 to 4 mg / ml chelating agents, from about 0.01 mg / ml to about 10 mg / ml phosphates, from about 20 mg / ml to about 80 mg / ml sugar and / or sugar alcohols and from about 0.001 mg / ml to about 5 mg / ml surfactants. In certain embodiments, the formulation comprising IL-2 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 0.05 mg / ml to about 5 mg / ml phosphates, from about 30 mg / ml to about 60 mg / ml sugar and / or sugar alcohols and from about 0.01 mg / ml to about 4 mg / ml surfactants. In certain embodiments, the formulation comprises from about 0.1 milli- international units (MIU) to about 20 MIU per ml of IL-2. In certain embodiments, the formulation comprising IL-7 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 0.0001 mg / ml to about 4 mg / ml chelating agents. In certain embodiments, the formulation comprising IL-7 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 3 mg / ml to about 8 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 0.0001 mg / ml to about 0,5 mg / ml chelating agents. In certain embodiments, the formulation comprises from about 0.001 mg / ml to about 5 mg / ml of IL- 7. In certain embodiments, the formulation comprising IL-15 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 0.0001 mg / ml to about 4 mg / ml surfactants. In certain embodiments, the formulation comprising IL-15 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 3 mg / ml to about 8 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 0.0005 mg / ml to about 1 mg / ml surfactants. In certain embodiments, the formulation comprises from about 0.001 mg / ml to about 5 mg / ml of IL- 15. In certain embodiments, the formulation comprising IL-21 comprises from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 10 mg / ml to about 80 mg / ml sugar and / or sugar alcohols. In certain embodiments, the formulation comprising IL-21 comprises from about 2 mg / ml to about 9 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 30 mg / ml to about 60mg / ml sugar and / or sugar alcohols. In certain embodiments, the formulation comprises from about 0.001 mg / ml to about 5 mg / ml of IL-21. In certain embodiments, the antioxidants belong to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite. In certain embodiments, the phosphates comprise anhydrous monosodium phosphates and disodium phosphates. In certain embodiments, the monosodium phosphates and disodium phosphates comprise monosodium phosphate anhydrous, monosodium phosphate monohydrate, monosodium phosphate dihydrate, disodium phosphate anhydrous, disodium phosphate dihydrate and disodium phosphate dodecahydrate. In certain embodiments, the sugars comprise sucrose, trehalose and maltose and sugar alcohols comprise sorbitol, isomalt, xylitol, maltitol, mannitol, erythritol, and lactitol or mixtures thereof. In certain embodiments, the surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, macrogol 15 hydroxystearate, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, polyoxyethylene alkyl ethers, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, sucrose stearate, sucrose palmitate and sucrose oleate. In certain embodiments, the chelating agents comprise disodium edetate, monosodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, citric acid, tartaric acid, alanine, arginine, aspartic acid, asparagine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the acid comprises citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid and sulfuric acid. In certain embodiments, the buffer comprises sodium citrate dihydrate, potassium citrate, disodium phosphate anhydrous, disodium phosphate dihydrate, disodium phosphate dodecahydrate, monosodium phosphate anhydrous, monosodium phosphate monohydrate, monosodium phosphate dihydrate, sodium tartrate, monopotassium phosphate and dipotassium phosphate. In certain embodiments, the formulation comprises about 0.001 milli- international units (.Mil.'} to 15 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 mill!- international units (MIU) to 10 MIU per ml of IL-2. IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 9 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU ) to 8 MIU per ml of IL-2, IL-7, IL-15, or IL- 21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 7 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 6 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 5 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 4 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3.5 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 2 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 1 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation is comprised in a pharmaceutical composition or liquid formulation. In certain embodiments, the isoelectric point of IL-2, IL-7, IL- 15, or IL-21 is between 3 to 10.

[0017] In another aspect, an isolated cell comprises an expression vector encoding for interleukin- 2 (IL-2), interleukin-7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21). In certain embodiments, the cell is a bacterial cell, yeast cell, mammalian cell or cell-line. In certain embodiments, the cell is Pichia pastoris or Chinese Hamster Ovary' (CHO) cells.

[0018] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0019] Definitions

[0020] The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting of the invention. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry).

[0021] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0022] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0023] The term, “amino acid” includes the residues of the natural a-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai) in D or L. form, as well as (3-amino acids, synthetic and unnatural amino acids. Many types of amino acid residues are useful in the adipokine polypeptides, and the disclosure is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry? and Molecular Biology, CRC Press, Inc,, and the reference cited therein. Another source of a wide array of ammo acid residues is provided by the website of RSP Amino Acids LLC.

[0024] As used herein, “antioxidants” include ascorbic acid, ascorbyl palmitate, tocopherol, oxidized species such as metabisulfite, bisulfite, sulfite, etc, (these molecules act as oxygen scavengers if oxygen is the oxidizing agent involved), reducing agents (reduce the oxidized molecule to be protected, they may be preferentially oxidized, as well) and chain terminators. Methionine is preferentially oxidized and thus protects ILs from oxidation. Tins is one of the mechanisms used by antioxidants. All these are considered "antioxidants" because they are molecules that contribute to protect other molecules from oxidation either by oxygen or by other oxidizing agents via different mechanisms. In this sense methionine is an antioxidant. In summary, methionine may work as an oxygen scavenger or it may be preferentially oxidized by other oxidizing agents or may reduce the oxidized forms of ILs. In all cases it would be considered an antioxidant in chemistry and in pharmaceutical technology. This is different from the biological and medical concept of antioxidant, which mostly refers to "chain terminators", i.e. molecules that react with free radicals to stop a chain reaction in biological systems and protect cells or the food chemistry concept that mostly refers to protection against oxygen damage or auto-oxidation.

[0025] A “biological medium” as used herein, is any type of medium that is used to grow, culture and maintain organs, tissues, cells etc., in vitro. A biological medium also encompasses any biocompatible agent, any pharmaceutical excipient, pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle, tissue or organ culture media, any agent that can be administered in vivo to a subject, any agent that can be used in assays or for diluting or maintaining a biological sample, e.g. nucleic acids, peptides etc.

[0026] As used herein, the term “cell” includes prokaryotic and eukaryotic cells. In one embodiment, a cell of the invention is a bacterial cell. In another embodiment, a cell of the invention is a fungal cell, such as a yeast cell. In another embodiment, a cell of the invention is a vertebrate cell, e.g., an avian or mammalian cell. In a preferred embodiment, a cell of the invention is a murine or human cell. As used herein, the term “engineered” (as in an engineered cell) refers to a cell into which a nucleic acid molecule e.g., encoding an IL-2 protein (e.g., a spliced and / or unspliced form of IL-2) or fragments thereof, has been introduced.

[0027] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby- indicating that the defined or described item, composition, apparatus, method, process, system. etc. includes those specified elements— or, as appropriate, equivalents thereof— and that other elements can be included and still fall within the scope / definition of the defined item, composi tion, apparatus, method, process, system, etc.

[0028] “Encoding'’ refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA.) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein m a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a. gene or cDN A, can be referred to as encoding the protein or other product of that gene or cDNA.

[0029] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0030] As used herein, “expression vector” or “vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient crs-acting elements for expression: other elements for expression can be supplied by the host cell or in an m vrtro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lenti viruses. retroviruses, adenoviruses, and. adeno-associated viruses) that incorporate the recombinant polynucleotide. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. As used herein, the term “human recombinant IL-X (rhIL-X)” or simply “IL-X “(X can take the values 2, 7, 15, or 21) means a protein produced by an organism that has been transfected with the DNA sequence described below or a non-extensive modification of it.

[0031] As used herein, the term “interleukin” designates any source of interleukins, including mammalian sources such as e.g., human, mouse, rat, primate, and pig, and may be native or obtained by recombinant or synthetic techniques, including recombinant interleukin polypeptides produced by microbial hosts. The interleukins may be or comprise the native polypeptide sequence or can be an active variant of the native interleukin polypeptide. In certain embodiments, the interleukin polypeptide or active variant is derived from a human source, and includes recombinant human interleukins. Variants of the native interleukin can be fragments, analogues, and derivatives thereof. By “fragment” is intended a polypeptide comprising only a part of the intact polypeptide sequence. An “analog” designates a polypeptide comprising the native polypeptide sequence with one or more amino substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogues. “Derivatives” include any modified native interleukin polypeptide or fragment or analogue thereof, such as glycosylated, phosphorylated, fused to another polypeptide or molecule, polymerized, etc., or through chemical or enzymatic modification or addition to improve the properties of the interleukin (e.g,, stability, specificity; etc.). Active variants of a reference interleukin polypeptide generally have at least 75%, preferably at least 85%, more preferably at least 90% amino acid sequence identity to the ammo acid sequence of the reference interleukin polypeptide.

[0032] As used herein, the term “kit” refers to any delivery system for delivering materials. Inclusive of the term “kits” are kits for both research and clinical applications. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., cytokines, oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides or liposomes. The term “fragmented kit” is intended to encompass kits containing Analyte specific reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0033] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0034] The term “percent sequence identity” or having “a sequence identity” refers to the degree of identity’ between any given query sequence and a subject sequence.

[0035] The terms “pharmaceutically acceptable” (or “pharmacologically acceptable”) refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance.

[0036] The term “polynucleotide” is a chain of nucleotides, also known as a “nucleic acid”. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art and include both naturally occurring and synthetic nucleic acids.

[0037] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of ammo acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two ammo acids, and no limitation is placed on the maximum number of ammo acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The peptides provided herein for use in tire described and claimed methods and compositions can be cyclic.

[0038] As used herein, “stable” or “highly stable” refers to the biological activity of the molecule.

[0039] "Treating” or “treatment” covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a. mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting it development; and / or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain or discomfort), wherein such amelioration may or may not be directly affecting the disease (e.g., cause, transmission, expression, etc).

[0040] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the ammo acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid, sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant or can be a variant that is not known to occur naturally. Non- naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. Ali other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0041] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 is a photograph of a gel demonstrating the binding of interleukins 2, 4, 7, 9, 15, and 21, to the common gamma chain (yc) (or CD 132) receptor.

[0044] DETAILED DESCRIPTION

[0045] Embodiments are directed to highly stable solutions of recombinant non-glycosylated interleukins of the common y-chain family and the methods of producing thereof

[0046] The disclosure summarized herein includes a series of formats and formulations that enable the long-term storage of interleukin 2, 7, 15, and 21 in liquid solution. These molecules are difficult to stabilize in solution because of their tendencies toward adsorption, aggregation, oligomerization and oxidation. The formulation of these molecules in liquid solutions that enable their integrity --- as determined by their ability to retain their critical quality attributes and remain stable at 2 - 8°C for several months - facilitates their use as reagents and excipients in the manufacture of cell- based medicines applicable across a range of indications. For example, these formulations enable the delivery of these large molecules to lymphocyte cultures that form the basis of certain adoptive cell therapies without the need to reconstitute lyophilized materials. Furthermore, these molecules in the formulations described herein, when packaged in certain single-use containers that enable the direct incorporation of these molecules into a given cell therapy manufacturing process, enable the sterile integration of critical reagents, thereby simplifying and streamlining erstwhile complex unit operations, while mitigating the risk of a potential contamination.

[0047] Interleukin 2

[0048] IL-2, discovered more than 30 years ago in supernatants of activated T cells, is mainly produced by CD41 and CD81 T cells, and to a lesser extent by activated DCs, NK and NK T (NKT) cells. The IL-2 receptor (IL-2R) consists of 3 subunits: the ligand-specific a chain IL-2Ra (CD25), the b-cham IL-2Rb (CD! 22, which is also part of the IL-15R complex), and the common yc, also called IL-2Rc (CD 132). All 3 subunits are required for the assembly of the high-affinity IL-2R. On T-cell activation, IL-2Ra is rapidly induced and participates in formation of a high- affinity quaternary complex, which activates multiple signal transduction pathways. IL-2 is essential for the development of Treg cells. IL-2 also acts as a B-cell growth factor, stimulates antibody synthesis, and promotes proliferation and differentiation of NK cells to increase their cytolytic functions. Recombinant human IL-2 is used in immunotherapy for cancer and AIDS associated with HIV. Anti-IL-2Ra inhibits the immune response in patients with autoimmune diseases and prevents rejection of transplanted organs.

[0049] IL-2 (Aldesleukin) Protein Sequence, SEQ ID NO: 1:

[0050] I PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT 51 ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET

[0051] 101 TFMCEYADET ATIVEFLNRW ITFSQSIIST LT

[0052] Interleukin 7

[0053] Interleukin-7 (IL-7; RefSeq NM_000880; UniProtKB - P13232 (IL7__HUMAN)) is one of the members of the IL-2 superfamily. The IL-2 superfamily includes IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21. It binds to receptors with a common y chain subunit. In addition to a common y chain subunit, the receptor for IL-7 (IL-7R) requires an IL-7Ra chain for binding to take place. Due to the frequency of the common y chain subunit, the presence of the IL-7 receptor a chain is a better identifier for when IL-7 will actually bind to a receptor. IL-7-receptor binding results in phosphorylation of tyrosine residues on the receptor. This leads to activation of JAK1 or JAK3 depending on the cell type, which later activates many downstream signaling pathways including STAT5a / b, PB Kinase, and SRC kinases. It is well-known that IL-7 plays a critical role in the development of B-cells and T-cells.

[0054] IL-7, also known as pre-B-cell growth factor or lymphopoietin-1, is a homeostatic cytokine.40The IL-7R is present on most T cells, progenitors of B cells, and bone marrow macrophages; it consists of the IL-7Ra (CD127) chain and the common gc (CD132)- Because yc is ubiquitously expressed on lymphocytes, IL-7 responses are determined by the expression of IL- 7Ra, which is shared with thymic stromal lymphopoietin (TSLP) receptor. IL-7 signaling contributes to survival and proliferation of thymocytes and development of naive and memory B and T cells, mature T cells, and NK cells. Studies of IL-7 and IL-7Ra knockout mice have shown that IL-7 is important for homeostatic T-cell and B-cell development.41IL-7 or reagents that block IL-7 signaling might be used to treat patients with HIV-associated immunodeficiency and immunodeficiency secondary to chemotherapy, autoimmune diseases, and lymphoid malignancies.

[0055] IL- 7 Protein Sequence, SEQ ID NO: 2:

[0056] 1 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA 51 NKEGMFLFRA ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR 101 KPAALGEAQP TKSLEENKSL KEQKKLNDLC FLKRLLQEIK TC WNKILMGT 151 KEH

[0057] Interleukin- 15

[0058] Interleukin-15 (IL-15; RefSeq NM_172175, UniProt / Swiss-ProtP40933) is an important cytokine for the development, proliferation, and activation of effector NK cells and CD8° memory T cells. IL- 15 binds to the IL- 15 receptor a (IL-15Ra) and is presented in trans to the IL-2 / IL-15 receptor - common y chain (IL-15R yc) complex on effector cells. IL-15 and IL-2 share binding to the IL-15R yc, and signal through STAT3 and STATS pathways. However, IL- 2 also supports maintenance of CD4+CD254FoxP3 regulatory' T (Treg) cells and induces cell death of activated CD8+ T cells. These effects may limit the therapeutic activity of IL-2 against tumors. IL- 15 does not share these immunosuppressive activities with IL-2. Additionally, IL- 15 is the only cytokine known to provide anti-apoptotic signaling to effector CD 8+ I' cells. IL- 15, either administered alone or as a complex with the IL-15Ra, exhibits potent anti-tumor activities against well- established solid tumors in experimental animal models and, thus, has been identified as one of the most promising immunotherapeutic drugs that could potentially cure cancer.

[0059] IL- 15 is structurally homologous to IL-2 and was discovered for its ability to induce T-cell proliferation like IL-2. Many of the biological actions attributed to IL-2 can also be induced by IL- 15. The IL-15R consists of the IL-15Ra chain, the IL-2Rb chain, and the common vc. IL-15 is produced by nonimmune cells (keratmocytes and skeletal muscle cells) and immune cells (monocytes and activated CD41 T cells) in response to signals that induce innate immunity. Although IL-15 shares some functions with IL-2, such as activation of T cells, stimulation of NK- cell proliferation, and cytolytic activity, differences in their biological functions have been identified on the basis of differences observed between phenotypes of IL-2 and IL-15 knockout mice.

[0060] IL- 15 Protein Sequence, SEQ ID NO: 3:

[0061] 1 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV

[0062] 51 ISLESGDASI HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF

[0063] 101 LQSFVHIVQM FINIS

[0064] Interleukin 21

[0065] IL-21(Ensembl:ENSG00000138684, MIM:605384; AllianceGenome:HGNC:6005 is produced by T ceils, NKT cells, and the TH17 subset of CD41 T cells. The receptor for IL-21 is expressed on various cells, indicating a broad spectrum of action. IL-21 affects B~cell functions by regulating antibody isotype balance, proliferation, apoptosis, and differentiation into plasma ceils. Cytotoxic activity and proliferation of CD81 T cells, NK ceils, and NKT cells increase on stimulation with IL-21. IL-21 has been tested as an anticancer drug, and first clinical trial results are promising by slowing down tumor progression in metastatic melanoma. In contrast with its anticancer effects, IL-21 also contributes to inflammation in several disorders, as expected for a TH17-related cytokine.

[0066] IL-21 Protein Sequence, SEQ ID NO: 4:

[0067] 1 MQDRHMTRMR QLIDTVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ 51 KA QLKSANTG NNERIIN V SI KKLKRKPPST NA GRRQKHRL TCPSCDSYEK 101 KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS

[0068] EXPRESSION OF THE INTERLEUKINS

[0069] In certain embodiments the interleukins embodied here are encoded by an expression vector. The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide.

[0070] In certain embodiments, an isolated cell comprises an expression vector encoding for interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21). In certain embodiments, the isolated cell is a bacterial cell, yeast cell, mammalian cell or cell-line. In certain embodiments, the isolated cell is Pichia pastoris or Chinese Hamster Ovary (CHO) cells.

[0071] Pichia pastoris (P. pastoris) is a widely used protein expression host for the production of biopharmaceuticals and industrial enzymes. P. pastoris belongs to methylotrophic yeasts, which share a common pathway to metabolize one-carbon compounds as carbon and energy sources. The species of methylotrophic yeasts, P. pastoris (recently reclassified as Komagataella pastoris) and H. polymorpha (also named Pichiaangusta) have been widely employed and become a substantial workhorse for biotechnology including heterologous protein production.

[0072] Methylotrophic yeasts have two major features: (1) they are capable of growing to high cell densities even in unsophisticated fermentation process; (2) their high demand for methanol- oxidizing enzymes endows them with very strong and strictly regulated promoters. These features make it possible that methylotrophic yeasts can be used not only in the process development for the commercial production of feed protein (single cell protein) but also as production systems for recombinant proteins. The widely used P pastoris and H .polymorpha have different genetics in alcohol oxidases expression: P pastoris expresses AOX1 and AOX2 while H polymorpha only expresses MOX. Besides P pastoris and H .polymorpha, P. methanolica and C. hoidmii are also used as expression systems. Some points should be considered when R pastoris is used to clone and express heterologous proteins. These include the selection of host strain, the choice of promoter, transcription terminator (TT), markers combination and the application for either intracellular or secreted expression.

[0073] Host strains: The widely used commercial available strains are mainly 5 classes, wildtype strains (e.g. X-33), auxotrophic strains (e.g. GS 115, KM71), protease-deficient strains (e.g. SMD 1168), glyco-engineered strains (e.g. SuperMan5) and some other strains. Notably, engineered P pastoris has been able to secrete recombinant proteins with uniform human N- linked glycans. In natural and recombinant proteins, glycosylation is one of the most common PTMs (post-translational protein modifications), which impacts protein folding, solubility, stability, trafficking, bioavailability, immunogenicity and functional activity. The use of engineered P. pastoris broadens the applications of microbial systems in antibody expression.

[0074] Vectors: The promoters for protein expression inP pastoris include inducible promoters (A0X1, FLD1, ADH1, GUT1, etc.) and constitutive promoters (GAP, TEFL etc.). For P. pastoris, HIS4 (auxotrophic markers) and zeocin resistance (dominant markers) are the most popularly markers used. The heterologous proteins can be intracellular or secreted expression. P pastoris has the ability to secrete high titers of proteins into culture media. The prominently used secretion signals are derived from P. pastoris endogenous acid phosphatase (PHO1), X. cerevisiae a-mating factor (a -MF) and S. cerevisiae invertase (SUC2).

[0075] Polynucleotides: Nucleic acids can, for example, encode the amino acid sequence of the IL-2, IL-7, IL-15, or IL-21 with at least one or more conservative amino acid substitutions. Conservative ammo acid substitutions are known in the art, and include ammo acid, substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another ammo acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g.. Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e g., Ala, Gly, foil, fie, Leu, Met, Phe, Pro, Trp, Vai, etc.), a basic amino acid substituted for another basic amino acid (I..ys, Arg, etc ), an amino acid with a. polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Sei; Thr, Tyr, etc. ), etc.

[0076] The nucleic acids of the disclosure can, for example, encode functional variants which also include extensions of the IL-2, IL-7, IL-15, or IL-21 protein. For example, a functional variant of the IL-2, IL-7, IL-15, or IL-21 protein can include 1 , 2, 3, 4 and 5 additional amino acids from either the N -terminal or C-terminal end of IL-2, IL-7, IL-15, or IL-21 protein.

[0077] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the IL-2, IL-7, IL-15, or IL-21 protein with at least one non-conservative amino acid substitution. In this case, it is preferable for foe non-conservative amino acid substitution to not interfere with or inhibit foe biological activity of the functional variant. Preferably, foe non- conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the native IL-2, IL-7, IL-15, or IL-21 protein. The IL-2, IL-7, IL-15, or IL-21 protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e g., other amino acids, do not materially change the biological activity of the functional variant.

[0078] In some embodiments, the nucleic acids encode a mammalian IL-2, IL-7, IL-15, or IL-21 protein. In certain embodiments, the IL-2, IL-7, IL-15, or IL-21 protein can be murine, porcine, ovine, bovine, human, or combinations thereof.

[0079] In accordance with an embodiment, the present disclosure provides a composition comprising a nucleic acid encoding a IL-2, IL- 7, IL-15, or IL-21 protein, or a functional portion or fragment, or variant thereof, such as, for example, SEQ ID NOs: 1, 2, 3 or 4.

[0080] In certain embodiments, the nucleic acid sequence encodes a polypeptide comprising a sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 3 or 4.

[0081] In some embodiments, the polynucleotides provided herein encoding one or more fusion proteins include codon-optimized sequences. As used herein, the term “codon-optimized” means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by choosing different codons without altering the amino acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can increase protein expression levels (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22: 346-53) of the encoded proteins, for example, and provide other advantages. Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of A, G, C, U nucleotides, mRNA secondary structures, cis-regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285).

[0082] Any method of codon optimization can be used to codon optimize polynucleotides and nucleic acid molecules provided herein, and any variable can be altered by codon optimization. Accordingly, any combination of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method and others. The CAI method chooses a most frequently used synonymous codon for an entire protein coding sequence. As an example, the most frequently used codon for each ammo acid can be deduced from 74,218 protein-coding genes from a human genome. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon optimized.

[0083] In some embodiments, the nucleotide sequence of any region of an RNA or DNA sequence embodied herein may be codon optimized. In certain embodiments, the primary cDNA template may include reducing the occurrence or frequency of appearance of certain nucleotides in the template strand. For example, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 25% of said nucleotides in the template. In further examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 20% of said nucleotides in the template. In some examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 16% of said nucleotides in the template. The occurrence of a nucleotide in a template may be increased or reduced to a level above or below 15% and may be increased or reduced to a level above or below 12% of said nucleotides in the template.

[0084] In certain embodiments, the polynucleotides encoding the IL-2, IL-7, IL-15, or IL-21 proteins can comprise one or more chemically modified nucleotides. Examples of nucleic acid monomers include non-natural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base portion or the sugar portion. In nature, most polynucleotides comprise nucleotides that are “unmodified” or “natural” nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the T position. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates, half-life and / or expressed protein concentrations. RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and / or degradation pathways.

[0085] Examples of modified or chemically modified nucleotides include 5-hydroxycytidines, 5- alkylcytidines, 5-hydroxyalkylcytidines, 5 -carboxy cytidines, 5-formylcytidines, 5- alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2 -thiocytidines, N4-alky1 cytidines, N4- ammocytidines, N4-acetylcytidines, and N4, N4-dialkylcytidines.

[0086] Examples of modified or chemically modified nucleotides include 5-hydroxy cytidine, 5- methylcytidine, 5-hydroxymethydcytidine, 5-carboxycytidine, 5-formylcytidine, 5- methoxy cytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidme, 2-thiocytidine; N4- methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4, N4-dim ethylcytidine.

[0087] Examples of modified or chemically modified nucleotides include 5-hydroxyuridines, 5- alkyluri dines, 5-hydroxyalkyluridines, 5-carboxyundines, 5-carboxyalkylesteruridines, 5- formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thioundines, and 6- alkyluri dines.

[0088] Examples of modified or chemically modified nucleotides include 5-hydroxyundine, 5- methyluridme, 5-hydroxymethyluridine, 5-carboxyundine, 5-carboxymethylesteruridine, 5- formyl uridine, 5-methoxyuridine (also referred to herein as “SMeOU”), 5-propynyluridine, 5- bromouridine, 5-fluorouridine, 5 -iodouridine, 2-thiouridine, and 6-methyluridine.

[0089] Examples of modified or chemically-modified nucleotides include 5- metlioxycarbonylniethyl-2-tliiouridine, 5-methylaminomethyl-2-thiouridine, 5- carbamoylmethyl uridine, 5-carbamoylmethyl-2'-O-methyluridine, 1 -methyl-3-(3-amino-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5- methyldihydrouridine, 5-taurinomethyhiridine, 5-taurinomethyl-2-thiouridine, 5- (isopentenylaminomethyl)uridine, 2’-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'- O-dimethyluridine. Examples of modified or chemically-modified nucleotides include N6-methyladenosme,

[0090] 2-aminoadenosine, 3 -methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8- bromoadenosine, 2-niethylthio~N6-methyladenosine, NMsopentenyladenosine, 2-methylthio-N0- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2miethylthio-N6-(cri- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyl- adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl- adenosine, N6,N6-dimethyladenosine, N° -hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine, N6-acetyl -adenosine, 7-methyl-adenine, 2-methylthio- adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-0-methyl-adenosine, No,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyl-adenosine, 1 ,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N6-methyl-purme, 1 -thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara- adenosine, and N6-(l 9-amino-pentaoxanonadecyl)-adenosine.

[0091] Examples of modified or chemically modified nucleotides include N1-alkylguanosines, N2-alkylguanosmes, thienoguanosines, 7-deazaguanosmes, 8-oxoguanosines, 8- bromoguanosines, O6-alkylguanosines, xanthosines, inosines, and N1-alkylinosines. Examples of modified or chemically modified nucleotides include N1-methylguanosine,

[0092] N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O°-methylguanosine, xanthosine, inosine, and Nkmethylinosine. Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0093] Examples of modified and chemically modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.

[0094] Examples of modified and chemically modified nucleotide monomers include 3'-end stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidine.

[0095] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2’-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'- methoxy ethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2!-deoxy-2!-fluoro nucleotides, and 2'- O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0096] Examples of modified and chemically modified nucleotide monomers include 2',4'~ constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DN As.

[0097] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2*-O-amino nucleotides, 2!-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0098] Examples of modified and chemically modified nucleotide monomers include N6- methyladenosine nucleotides.

[0099] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2~mercapto)ethyluridine, 5- bromoundine; 8-bromoguanosine, or 7-deazaadenosine.

[0100] Examples of modified and chemically modified nucleotide monomers include 2'-O- aminopropyl substituted nucleotides. Examples of modified and chemically modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2!-R, a 2'-OR, a 2'-halogen, a 2'-SR, or a 2' -amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0101] Example of base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers may be found in nature.

[0102] Interleukin nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, rCA Proner: A Mowtta / , Dieffenbach and Dveksler, eds., Cold Spring

[0103] Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced mto a template nucleic acid.

[0104] Interleukin nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis m the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e g. , >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a. duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting m a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector, e.g. a plasmid. Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a naturally occurring portion of interleukin DNA. In some embodiments, the nucleic acid is a. synthetic polynucleotide. In some embodiments, the synthetic nucleic acid comprises a modified nucleotide. Modification of the inter-nudeoside linker (i.e., backbone) can be utilized to increase stability or pharmacodynamic properties. For example, mter-nucleoside linker modifications pre-, eni or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailabihty of the nucleic acid. Generally, a modified mter-nucleoside linker includes any linker other than other than phosphodiester (PO) liners, that covalently couples two nucleosides together. In some embodiments, the modified inter-nueleoside linker increases the nuclease resistance of the nucleic acid compared to a phosphodiester linker. For naturally occurring oligonucleotides, the mter-nucleoside linker includes phosphate groups creating a. phosphodiester bond between adjacent nucleosides. In some embodiments, the nucleic acid comprises one or more inter- nucleoside linkers modified from the natural phosphodiester. In some embodiments all of the mter-nucleoside linkers of the nucleic acid or contiguous nucleotide sequence thereof, are modified. For example, in some embodiments the inter-nudeoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nueleoside linkage.

[0105] Modifications to the ribose sugar or nucleobase can also be utilized. herein. Generally, a. modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nudeobase moiefy. In some embodiments, the nucleic acids, as described, comprise one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared to the ribose sugar moiety found m deoxyribose nucleic acid (DNA) a nd RNA. Numerous nucleosides with modifica tion of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or stability. Such modifications include those where the ribose ring structure is modified. These modifications include replacement, with a hexose ring (HNA), a bicyclic ring ha ving a biradical bridge between the C2 and C4 carbons on the ribose ring (e.g. locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids. Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non -sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids. Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2'-OH group naturally found in DMA and RNA nucleosides. Substituents may, for example be introduced at the 2', 3‘, 4?or 5' positions. Nucleosides with modified sugar moieties also include 2:modified nucleosides, such as 2:substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity. A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or OHat the 2' position (2' substituted nucleoside) or comprises a 2;linked biradicle and includes 2' substituted nucleosides and LNA (2M' biradide bridged) nucleosides. Examples of 2' substituted modified nucleosides are 2:-O-alkyl-RNA, 2;-O-metl'iyi-BNA, 2'-alkoxy-RNA, 2'-O- methoxyethyl-RNA (MOE), 2'-amino-DNA, 2’-Fluoro~RNA, and 2' -F-ANA nucleoside. By way of further example, in some embodiments, the modification m the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2?-fluoro, 2?- deoxy, and 2'~O-(2-metboxyethyl).

[0106] In some embodiments, the nucleic acid comprises one or more modified sugars. In some embodiments, the nucleic acid comprises only modified sugars In certain embodiments, the nucleic acid comprises greater than 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2f-O-methoxyethyl group. In some embodiments, the nucleic acid comprises both inter-micleoside linker modifications and nucleoside modifications.

[0107] In certain embodiments, the interleukin polypeptides are synthesized from an expression vector encoding the DNA molecule, as described m detail elsewhere herein

[0108] Polypeptides: The polypeptides embodied herein, can be modified to include one or more non-natural amino acids. As used herein, an “unnatural ammo acid,” “non-natural”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified ammo acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural ammo acids have side chain groups that distinguish them from the natural ammo acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural ammo acids, unnatural ammo acids may have an extended backbone such as beta-ammo acids. Non-limiting examples of non-natural ammo acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylaianine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcp-serme, an L-Dopa, a fluormated phenylalanine, an isopropyl-L-phenylalamne, a p-azido-L-phenylalanine, a p-acyl-L- phenylalanme, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p~ bromophenylalanine, a p-aniino-L- phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine ammo acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or ammo substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled ammo acid; a fluorescent amino acid; an ammo acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding ammo acid, a metal-containing ammo acid, a radioactive amino acid; a photocaged and / or photoisomerizable ammo acid; a biotin or biotin-analogue containing ammo acid, a glycosylated or carbohydrate modified ammo acid, a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable ammo acid, an amino acid with an elongated side chain, an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing ammo acid; a redox-active ammo acid; an a-hydroxy containing acid; an ammo thio acid containing amino acid; an a, a disubstituted ammo acid; a p- amino acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and / or one or more natural amino acids. Any one or more of SEQ ID NOs: 1 , 2, 3, or 4 can be modified to include naturally occurring and synthetic a, p, y, and 8 ammo acids. Amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycmyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, P-alanyl, p- valinyl, P-leucinyl, P-isoleucinyl, P-prolinyl, p-phenylalaninyl, P-tryptophanyl, P-methioninyl, P~ glycmyl, p-serinyl, P-threoninyi, P-cysteinyl, P-tyrosinyl, P-asparaginyl, P-glutammyl, P~ aspartoyl, p-glutaroyl, P-lysinyl, p-arginmyl or P-histidinyl. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of a, P, y, and 8 glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.

[0109] PH ASM A € 1.1 1 tc A L COMPOSITION S

[0110] Certain aspects of the instant disclosure pertain to pharmaceutical compositions of the compounds of the disclosure. The pharmaceutical compositions of the disclosure typically comprise a compound of the instant disclosure and a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable earner” includes any and ah solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, in cam oscular, topical, tmnsderma1 or oral administration Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the instant disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions. The liquid compositions of the disclosure comprise an interleukin at a concentration of 0.001 mdh- international units (MID) to 20 MIU per ml, a buffering agent, a. surfactant, acid, antioxidants, acid, sugar alcohols and an optional excipient selected from amino acids, osmolarity adjusting agents, and preservatives in water.

[0111] The surfactant is present so as to promote the solubility and the stability of the interleukin in solution. Suitable surfactants encompass, without being limited to, Q-Ceo alkylsuifate salts, certain phospholipids such as phosphati dates, cholate salts, deoxycholate salts, salts of lauroyl sarcosinate (such as sodium salt known as sarkosyl), CHAPS, CDAPSO, Triton XI 00, Triton X1 14, NP40, octyl glucoside, polyethylene glycol dodecyl ethers for instance that marketed under tradename Brij 'M35, polyethylene glycol hexadecyl ethers for instance that marketed under the tradename Brij™58, polyoxyethylene derivatives of sorbitan monolaurates such as Tween 20 and Tween 80, sorbitan esters such as sorbitan monostearate or sorbitan monolaurate, and combinations thereof

[0112] In some embodiments, the surfactant, is an anionic surfactant. Accordingly, the surfactant can be selected from C8-Ceo alkyl sulfate salts, lauroyl sarcosinate salts, cholate salts, deoxycholate salts and combinations thereof

[0113] Preferred anionic surfactants are dodecyl sulphate salts such sodium dodecyl sulphate (SDS) or lithium dodecyl sulphate. For instance the surfactant can be selected among alkali metal and alkaline-earth metal dodecyl sulphates. A preferred surfactant is SDS.

[0114] The optional excipient may be selected from preservatives, antioxidants and combinations thereof Preservatives encompass, without being limited to, benzalkonium chloride, benzoic acid, sorbic acid and salts thereof Antioxidants encompass ascorbic acid, ascorbyl palmitate, tocopherol and combinations thereof. Typically the optional excipient accounts for less than 5% by weight, preferably less than 3%, 2%, 1%, and even 0.1% by weight of the total weight of the composition.

[0115] The optional excipient may be also selected among osmolarity adjusting agents Osmolarity adj listing a gen is comprises pharmaceutically acceptable inorgani c salts such as sodium chloride and potassium chloride and organic salts such sodium or potassium organic salts, for instance potassium or sodium citrate, aspartate or acetate. The osmolarity adjusting agent is typically added in foe composition of foe invention in an amount enabling to adjust the osmolarity of the composition.

[0116] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage The composition can be formulated as a solution, micro emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for exampie, water , ethanol, polyol (for exampie, glycerol, propylene glycol, and liquid polyethyelene glycol, and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecrthm, by the maintenance of the required particle size m foe case of dispersion and by foe use of surfactants. In many cases, it will be preferable to include isotomc agents, for example, sugars, polyalcohois such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer, or in a fat pad described herein. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated deh very systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLGT Many methods for the preparation of such formulations are generally known to those skilled in the art.

[0117] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount m an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a. previously sterile-filtered solation thereof

[0118] Depending on the route of administration, the compound may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent. For example, the compound can be administered to a subject in an appropriate carrier or diluent co-adm mistered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically aeceptabie diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan, ei al., (1984) J Neuroimmunol 7:27). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and m oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0119] A therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one m which any toxic or detrimental effects of the agent are outweighed, by the therapeutically beneficial effects. In another embodiment, the active agent is formulated in the composition in a prophylactically effective amount. A '‘prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effecti ve amount will be less than the therapeutically effective amount.

[0120] The amount of active compound in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide die optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or die dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. lire specification for the dosage unit forms of die instant disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0121] METHODS OF TREATMENT lire present disclosure provides a method of treating or preventing, for example, cancer, virus infections, autoimmunity and the like. In some embodiments, the method comprises administering to a subject m need thereof, an effective amount of desired interleukin. hr certain embodiments, the compositions and methods of die present disclosure can be used in combination with one or more additional therapeutically active agents which are known to be capable of treating conditions or diseases discussed above. For example, the compositions of the present disclosure could be used in combination with one or more known therapeutically acti ve agents, to treat a neoplastic or proliferative disease such as a tumor or cancer. Non- limiting examples of other therapeutically active agents that can be readily combined nr a pharmaceutical composition with the composi tions and methods of the present disclosure are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and / or inorganic compounds including metals, salts and ions.

[0122] The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.

[0123] EXAMPLE 1: METHODS OF PRODUCING STABLE FORMULATIONS OF ILS OF THE GAMMA

[0124] FAMILY

[0125] Recombinant unglycosylated IL-2 has been approved as a medicine for cancer some decades ago under the commercial name Proleukin® and is now being investigated for medicinal use either as pharmaceutical for other indications or an ancillary material for immunotherapies for either cancer or other diseases such as graft versus host disease.

[0126] Other members of this family are being investigated for medicinal use of their recombinant versions. IL 15 is being investigated for several medicinal uses related to cancer. IL-21 is being investigated for cancer therapy.

[0127] Therefore, the possibility of formulating these interleukins into stable liquid solutions would be useful for their medicinal application. We disclose herein formulations to be used as ancillary materials for ex vivo proliferation of different lineages of leukocytes.

[0128] These interleukins are produced by fermentation of genetically modified organisms transfected with an expression vector designed to highly express the protein of the already disclosed sequences or similar sequences as defined above.

[0129] Expression vectors are commercially available and a DNA sequence coding for the aldesleukin amino-acid sequence is inserted into the vector. The cells used can be eukaryotic or prokary otic. For example, in one embodiment, the cell is a bacterial cell. In another embodiment, the cell is a fungal cell, e.g., a yeast cell. In another embodiment, the cell is a vertebrate cell, e.g., an avian or a mammalian cell. In another embodiment, the cell is a human cell. The cells of the invention can express endogenous IL-2 or fragments thereof or can be engineered to do so. For example, a cell that has been engineered to express the IL-2 or fragments thereof can be produced by introducing into the cell an expression vector encoding the protein. Among bacteria non-pathogenic strains of Escherichia coli are preferred. Escherichia coh

[0130] B serves as a research model and also for protein expression in life science laboratories and in the biotech industry . Characteristics such as protease deficiency, low acetate production at a high level of glucose, and enhanced permeability (probably due to a simple cell surface) make E. coli B a desirable host to produce genetically engineered proteins. Differences between B strains and KI 2 include the absence of flagellar component genes, the DNA cytosine methylase dem, and ompT in BL21(DE3). B strains may have an additional type II secretion system not found in KI 2. BL21 (DE3) also carries a DE3 recombinant phage harboring the T7 RNA polymerase gene that can direct high-level expression of cloned genes under the control of the T7 promoter. Typical E. coli strains used for recombinant protein expression are: BL21 (a B E. coll strain that protects target protein from Ion and ompT proteases) and their derivatives such as: Lysogenic DE3 (based on T7 polymerase), pLysS, pLysE (express T7 lysozyme reducing basal expression of target genes), Origami (allows disulfide bond formation in E. coli cytoplasm), Rosetta (enhances expression of proteins that contain codons rarely used in E. coll). Similar versions exist under the K12 E. coli genetic background. Typical plasmid vectors for high expression of recombinant proteins in E. coli are: pET series based on pBR322 origin and T7 / lac promoters; pBad with araBAD promoter and pUC origin; pGEX with tac promoter and pBR322 origin aiso. Combination of fusion tags sequences, protease cleavage sites, selection markers and strain compatibility are source for the most usual list of high expression plasmid variants. In certain embodiments, the cell is an E. coli cell. Different E. coli strains can be transfected in order to obtain optimal interleukin production.

[0131] Producing bacteria can be cultured in a suitable growth medium. For example, the medium may contain each 9 liters, 216 gr of Yeast Extract, 108 gr of Soy Peptone, 113 of gr K2HPO4, 20,8 gr KH2PO4, 36 ml of Glycerol and 4 ml of Antifoam (2% v / v). Fermentation conditions may be: Temperature: 37°C ± 0.5°C, agitation: 350 rpm ± 10 rpm, air flow: 9 L / min ± 1 L / min, pO2: set point 40%, pH between 6.95 and 7.5. After this, a feeding procedure should be followed. For example, feeding with a glucose solution 40% p / v by drip, to maintain a concentration of 0. 1 %, Once the OD600 reaches 5 to 10, an appropriated inducer such as Isopropil-P-D-1- tiogalactopiranosido (IPTG) should be added to reach an operative concentration. At this point, the feeding with Glucose may be reduced to keep a glucose concentration of about 0,01%. Fermentation can be stop usually about 18 to 24 hours after inoculation. After fermentation, bacteria can be concentrated 5 to 7 times by centrifugation or tangential filtration and processed immediately or preserved at 2-8 °C (no more than 24 hours) or preserved at -20°C (for more than 24 hours).

[0132] After culture, interleukins are inside the bacteria, predominantly in the form aggregates named inclusion bodies (lb). These lb can be isolated by disruption of bacteria (for example bysonication). For this, bacteria could be suspended in purified water. After this, the suspension could be circulated 2-4 times for the disruptor at a pressure of about 1400 bar. The lysate should be processed immediately or preserved at -20°C. The lb are separated of other components of the lysate by centrifugation or tangential filtration and washed. The lb preparation should be stored at -20°C until further processing. The lb are then suspended in a suitable buffer and submitted to a re-folding step in a regulated redox potential.

[0133] After refolding interleukins are submitted to different chromatographic and diafiltrations to get a concentrated solution of purified interleukins. Interleukins are then formulated and packaged into ready-to-use bags, pre-filled syringes, vials, ampoules or other suitable primary packaging materials for sterile solutions. Surprisingly, it was discovered that recombinant interleukins may be easily stabilized in solution using buffers of carboxylic acids at pH at least 0.3 units from their isoelectric point, isotonized with non-ionic osmolytes with the addition of methionine or other amino acids. In some cases surfactants are needed to prevent them from forming aggregates, oligomers or adsorbing onto the walls of the container.

[0134] The isoelectric point is the value of pH in which the protein does not show any net electrostatic charge. The isoelectric point of proteins may be estimated using Vector NTI 10.3.0. 2006 © Invitrogen Corporation. The results obtained for some members of the Gamma family are: It would be reasonable to expect a higher stability of these or other proteins to denaturation and aggregation, when formulated below or above their isoelectric point, but the surprising event is that all other degradation mechanisms, such as deamidations and oxidations, are minimized in these conditions as well. Methionine seems to stabilize solutions of some particularly oxidable members like IL-2, IL- 7 and IL- 15 and particularly when packaged in materials permeable to oxygen like plastic bags. Surfactants may be added to stabilize them further against aggregation, oligomerization and adsorption onto the container walls.

[0135] All interleukins seem to follow' a similar pattern regarding chemical stability. All of them tend to be very stable in liquid formulations for several months at 2 - 8 °C when formulated with buffers made up of carboxylic acids and their salts, such as citric acid / sodium citrate, acetic acid / sodium acetate, sodium tartrate / tartaric acid with pH values at least 0.2 units above or below' their isoelectric point, a non-ionic osmolyte, such as sugars like sucrose, trehalose and maltose and / or sugar alcohols such as sorbitol, isomalt, xylitol, maltitol, mannitol, erythritol, or lactitol. In some cases, methionine and disodium edetate improves stability against oxidation of some members of the family. Other suitable soluble antioxidants and / or chelating agents may be added instead of or in addition to methionine and / or sodium edetate, such as sodium metabisulfite, sodium sulfite, citric acid, citrates, tartaric acid, tartrates, and amino acids. In some embodiments surfactants from the group: polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, macrogol 15 hydroxystearate, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, polyoxyethylene alkyl ethers, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, sucrose stearate, sucrose palmitate, sucrose oleate, and others either ionic or non-ionic may be included.

[0136] EXAMPLE 2: IL-2 LIQUID FORMULATION

[0137] A solution composition for this interleukin is the following:

[0138] Anhydrous disodium phosphate 1.2 mg / m.L,

[0139] Anhydrous monosodium phosphate 0.2 mg / mL,

[0140] Mannitol 50 mg / mL,

[0141] SDS 1 mg / mL,

[0142] This composition is filled into pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume between 1 and 10 ml / bag: 1 MIU / Bag; 2 MIU / Bag; or 15 MIU / Bag

[0143] Another liquid formulation for IL-2 is the following:

[0144] Anhydrous disodium phosphate 1.2 mg / mL,

[0145] Anhydrous monosodium phosphate 0.2 mg / mL, Mannitol 50 mg / mL,

[0146] SDS 1 mg / mL,

[0147] Methionine 5 mg / inL

[0148] This composition is filled into pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume between 1 and lO ml / bag: 1 MIU / Bag; 2 MIU / Bag; or 15 MIU / Bag

[0149] The pH value of both solutions is 7.5 (more than 0.2 units above the isoelectric point of IL-2: pH 7.02)

[0150] EXAMPLE 2: IL-7 LIQUID FORMULATION

[0151] IL-7 is produced by recombinant DNA technology using a genetically engineered E. coll strain BL21 Rosetta. DE3 pLysS containing an analog of the human interleukin-7 coding region gen, inserted in a pET9a (TetR) plasmid vector at Ndel-BamHI restriction sites. It contains three disulfide bonds that are essential for its biological activity.

[0152] Genetic engineering techniques were used to modify the human IL-7 gene, and the resulting expression clone encodes a modified human interleukin-7. This modified gene has 154 codons. This recombinant form differs from native interleukin-7 in the following ways: a) IL-7 is not glycosylated because it is derived from E. coli; b) the molecule has an N-terminal Methionine.

[0153] (a) Expression System and Cloning Description Process

[0154] An inducible eukaryotic expression system for recombinant expression of hIL-7 was developed using an Escherichia coll strain as a host.

[0155] Among different E. coli BL21(DE3) strains tested, Rosetta2(DE3) pLysS strain was selected due to the fact that a high, tight and stable recombinant expression of rhIL-7 was observed. The strain supplies tRNAs for rare codons, providing for universal translation where it would otherwise be limited by the codon usage of E. coli. Furthermore, the strain carries a plasmid that encodes the T7 lysozyme gene, which is a natural inhibitor of T7 RNA polymerase that serves to repress basal expression of target genes under the control of the T7 promoter. The pET9a+expression vector was used for cloning at Ndel and BamHI restriction sites. Strain and vector combination let a tight regulation of recombinant expression based on the very well know7T7 promoter system which only is turned on when the T7 RNA polymerase is present.

[0156] Three different versions of hIL-7 genes were tested. These were synthetics hIL-7 cassettes containing the natural (wild type) gene and two modified genes versions including codon usage adaptations for E. coli expression. One of the modified genes versions (fully adapted for E. coli expression) originates the best expressing clones and one of these clones was selected for next stages of this development.

[0157] The molecular construct and gene were verified by nucleotide sequencing and protein identity was stated by SDS-PAGE (molecular size) and by Western Blot (specific antibody detection).

[0158] Table I

[0159] The pH value of this formulation is 6 ± 0.5 (more than 0.2 units below the isoelectric point of IL- 7: pl 8.72) Table 2

[0160] EXAMPLE 3 ■■■■ LIQUID FORMULATION OF IL- 15

[0161] Genetic development of recombinant clone for the Expression of IL-15.

[0162] IL-15 was produced by recombinant DM A technology using a genetically engineered E. coll strain BL21 Al containing an analog of the human interleukin- 15 coding region gen, inserted in a pET9a’ plasmid vector at Ndel-BamHI restriction sites. It contains two disulfide bridges, which the structure shows help to stabilize the conformation in regions of the loop that engage in contacts with the receptor.

[0163] Genetic engineering techniques were used to modify the human IL- 15 gene, and the resulting expression clone encodes a modified human interleukin- 15. This recombinant form differs from native interleukin- 15 in the following ways: a) IL- 15 is not glycosylated because it is derived from E. coll,' b) the molecule has an N-tenmnal Methionine.

[0164] Brief description of the expression system

[0165] An inducible eukaryotic expression system for recombinant expression of hIL-15 was developed using an Escherichia coli strain as a host. Among different tested E. colt BL21 strains, the Al strain was selected. It was due to the fact that a high, tight and stable recombinant expression of rhIL-15 was observed. The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene in the araB locus, allowing expression of T7 RNAP to be regulated by the araBAD promoter. The expression of this polymerase is induced by Arabinose. The pET9a’ expression vector was used for cloning at Ndel and BamHI restriction sites. Strain and vector combination let a tight regulation of recombinant expression based on the very well know7T7 promoter system which only is turned on when the T7 RNA polymerase is present.

[0166] Three different versions of hIL-15 genes were tested. These were synthetics hIL-15 cassettes containing: the natural (wild type) gene and two modified genes versions including codon usage adaptations for E. coll expression. One of the modified genes versions (fully adapted for E. coll expression) originates the best expressing clones and one of these clones was selected for next stages of this development. The molecular construct and gene were verified by nucleotide sequencing and protein identity was assessed by SDS-PAGE (molecular size) and by Western Blot (specific Antibody detection).

[0167] Table 3

[0168] The pH value of this formulation is 6 ± 0.5 (more than 0.2 units above the isoelectric point of IL- 15: pl 4.52)

[0169] Biological Activity Methodology

[0170] The biological activity of Interleukin-7 (IL-7) is determined through a colorimetric proliferation assay, using the TIB-239 cell line (immature mouse B lymphocytes ATCC® 1TB- 239; also known as 2E8 cells). Biological activity and Specific activity of IL-7 raw material is estimated assuming a theoretical biological activity of 1.5x108TU / nig compared against the NIBSC WHO Reference Interleukin-7 using a parallel line assay as statistical model.

[0171] EXAMPLE 4. INTERLEUKIN 21 LIQUID FORMULATION

[0172] Sodium Citrate dihydrate 4.05 g / L Citric Acid anhydrous 1.20 g / L

[0173] Mannitol 41.30 g / L

[0174] IL-21 0.30 mg / mL pH 5.0 - 5.6

[0175] The pH of this formulation is more than 0.2 units below the isoelectric point of IL-21 (pl 9.42).

[0176] Genetic development of recombinant clone for the Expression of Inter leukin-21 IL-21 is a human recombinant interleukin-21 protein with a molecular weight of approximately 15.4 kDa (Dalton). It is produced by recombinant DNA technology using a genetically engineered E. coli strain containing an analog of the human coding region for interleukin-21 gene. Genetic engineering techniques were used to modify the human IL-21 gene, and the resulting expression clone encodes human interleukin-21.

[0177] Two intramolecular disulfide bonds (between cysteine 41 and cysteine 92; and between cysteine 48 and cysteine 95) are essential for correct folded form of the protein and biologic activity.

[0178] Primary protein structure contains the following ammo acids sequence:

[0179] 1 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LP APED VEIN CEWSAFSCFQ 51 KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK

[0180] 101 KPPKEFLERF KSLI .QKMIHQ HLSSRTHGSE DS

[0181] Initial methionine (M) is needed for the recombinant protein expression in E. coli.

[0182] Human IL-21 gene source

[0183] Three nucleotide sequence synthesis (cassettes) containing the human IL-21 coding region were ordered (Genscript, NJ, USA):

[0184] Table 5

[0185] The synthetic genes do not include nucleotides coding for the signal peptide region of the hIL-21 gene. All cassettes were sent as an insert cloned into a pUC57 cloning vector (into Ndel and BamHI restriction sites). Note: All cassettes include an extra stop codon and the best option for E. coli (TA A stop codon) in the first position (\,ryas W et al., Biotechnol Prog. 2012 Mar- Apr;28(2):497-507. doi: 10.1002 / btpr,746. Epub 2011 Dec 9) that determined the presence of an extra tryptophan due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre) stop codon.

[0186] E. coll Expression Strain Generation

[0187] Once IL-21 coding regions and regulatory portions of each expression plasmid were confirmed, the WT-hIL-21 , iop-hIL-21 , and the op-hIL-21 plasmids were used for next steps. Fifteen nanograms of each supercoil expression plasmid coming from the above mentioned clones were used to transform a BL21 Al E. coll strain.

[0188] BL21-AI Genotype: F- ompT hsdSB (rB-mB-) gal dem araB::T7RNAP-tetA

[0189] The BL21-AI™ strain is an E. coli B / r strain and does not contain the Ion protease. It is also deficient in the outer membrane protease, OmpT. The lack of these proteases reduces degradation of heterologous proteins expressed in this strain. The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene in the araB locus, allowing expression of T7 RNAP to be regulated by the araBAD promoter. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline. hIL21 Protein Expression and Protein Identity Verification

[0190] Four different clones from each hIL-21 gene version (“WT”: wild type hIL-21 gene; “iop-hIL21”: first 20 codons optimized and “op-hIL21”: fully codon optimized version) were tested for protein expression. The clone “D7” was selected to make the Research Cell Bank and further development steps. See, FIG. 1.

[0191] “No induction” means bacteria without induction. The recombinant system (plasmid + E. coli strain) is and inducible genetic system that required an inducer (L-arabinose in this case) to avoid genetic repression. Non-induced bacteria are expected do not shown recombinant expression (no recombinant protein band in the SDS-PAGE). The induced bacteria should express the protein of interest (i.e. IL-21 band in the SDS-PAGE). The numbers and / or letters indicate Cion ID (i.e.

[0192] “2.2-5”; “2.2.-6”; “D7”...)

[0193] “MW” states: molecular weight marker.

[0194] SDS-PAGE 18%. Coomassie Blue stain - Reducing Condition. Induction: Overnight Induction (0.2% L- Arabinose); Clone ID: “2.2-5”; “2.2-6”; “D7” and “D8”; IL-21 STD: rhIL-21 Standard

[0195] The protein identity was verified by Western Blot analysis using a specific rabbit polyclonal antibody Anti-hIL21 (AB154767; Abeam). .Also Blast X (Search protein database using a translated nucleotide query) search results, showed 100% of identity with the hIL-21 amino acid sequence.

[0196]

[0197] Biological Activity ~ Specific activity

[0198] The biological activity of human IL-21 is determined by means of a proliferation assaybased on the B9 cell line (Mouse B cell Hybridoma) obtained from Public Health England and quantified by a colorimetric method, using MTS.

[0199] Briefly, B9 cells were washed twice with Assay Medium and a cell suspension of 2.5x105cells / mL is prepared. In parallel, serial dilutions are made for IL-21 Standard and the Samples to be analyzed. Then, 100 pL / well of each dilution are dispensed in a microplate followed by 100 pL of the cell suspension. Plates are incubated for 48 hours at 37 °C and 5% CO2.

[0200] Detection: 40 pL / well of a solution of MTS / PMS is dispensed and the microplates are incubated for 4 hours. After this time, plates are read at 490 nm.

[0201] In order to assess the biological activity and the specific activity of IL-21 raw material, the EC50 is estimated. Half maximal effective concentration (ECso ) refers to the concentration of a drug, which induces a response halfway between the baseline and maximum after a specified exposure time. The response achieved at the EC50, is considered one Unit of biological activity.

[0202] OTHER EMBODIMENTS

[0203] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0204] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure.

Claims

What is claimed is:

1. A liquid formulation for in vivo use comprising about 0.001 milli- international units (MIU) to 20 MIU per ml of one or more interleukins, comprising interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21).

2. The liquid formulation of claim 1, wherein the formulation comprising IL-2 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 0.0001 to 4 mg / ml chelating agents, from about 0.01 mg / ml to about 10 mg / ml phosphates, from about 20 mg / ml to about 80 mg / ml sugar and / or sugar alcohols and from about 0.001 mg / ml to about 5 mg / ml surfactants.

3. The liquid formulation of claim 2, wherein the formulation comprising IL-2 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 0.05 mg / ml to about 5 mg / ml phosphates, from about 30 mg / ml to about 60 mg / ml sugar and / or sugar alcohols and from about 0.01 mg / ml to about 4 mg / ml surfactants.

4. The liquid formulation of any one of claims 1-3, wherein the formulation comprises from about 0.1 milli- international units (MIU) to about 20 MIU per ml of IL-2.

5. The liquid formulation of claim 1, wherein the formulation comprising IL-7 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 0.0001 mg / ml to about 4 mg / ml chelating agents.

6. The liquid formulation of claim 5, wherein the formulation comprising IL-7 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 3 mg / ml to about 8 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 0.0001 mg / ml to about 0.5 mg / ml chelating agents.

7. The liquid formulation of any one of claims 1, 5 or 6, wherein the formulation comprises from about 0.001 mg / ml to about 5 mg / ml of IL-7.

8. The liquid formulation of claim 1, wherein the formulation comprising IL- 15 comprises from about 1 mg / ml to about 10 mg / ml antioxidants, from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 0.0001 mg / ml to about 4 mg / ml surfactants.

9. The liquid formulation of claim 8, wherein the formulation comprising IL-15 comprises from about 3 mg / ml to about 8 mg / ml antioxidants, from about 3 mg / ml to about 8 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 0.0005 mg / ml to about 1 mg / ml surfactants.

10. The liquid formulation of any one of claims 1, 8 or 9, wherein the formulation comprises from about 0.001 mg / ml to about 5 mg / ml of IL- 15.

11. The liquid formulation of claim 1, wherein the formulation comprising IL-21 comprises from about 1 mg / ml to about 10 mg / ml buffering agent, from about 0.001 mg / ml to about 5 mg / ml acid, and from about 10 mg / ml to about 80 mg / ml sugar and / or sugar alcohols.

12. The liquid formulation of claim 11, wherein the formulation comprising IL-21 comprises from about 2 mg / ml to about 9 mg / ml buffering agent, from about 0.01 mg / ml to about 3 mg / ml acid, and from about 30 mg / ml to about 60mg / ml sugar and / or sugar alcohols.

13. The liquid formulation of any one of claims 1, 11 or 12, wherein the formulation comprises from about 0,001 mg / ml to about 5 mg / ml of IL-21,14. The liquid formulation of any one of claims 2-13, wherein the antioxidants belong to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite.

15. The liquid formulation of any one of claims 2-13, wherein the phosphates comprise anhydrous monosodium phosphates and disodium phosphates.

16. The liquid formulation of claim 15, wherein the monosodium phosphates and disodium phosphates comprise monosodium phosphate anhydrous, monosodiumphosphate monohydrate, monosodium phosphate dihydrate, disodium phosphate anhydrous, disodium phosphate dihydrate and disodium phosphate dodecahydrate.

17. The liquid formulation of any one of claims 2-13, wherein the sugars comprise sucrose, trehalose and maltose and sugar alcohols comprise sorbitol, isomalt, xylitol, maltitol, mannitol, erythritol, and lactitol or mixtures thereof.

18. The liquid formulation of any one of claims 2-13, wherein the surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, macrogol 15 hydroxy stearate, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, polyoxyethylene alkyl ethers, poly oxy 1 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, sucrose stearate, sucrose palmitate and sucrose oleate.

19. The liquid formulation of any one of claims 2-13, wherein the chelating agents comprise disodium edetate, monosodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, citric acid, tartaric acid, alanine, arginine, aspartic acid, asparagine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

20. The liquid formulation of any one of claims 2-13, wherein the acid comprises citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid and sulfuric acid.

21. The liquid formulation of any one of claims 2-13, wherein the buffer comprises sodium citrate dihydrate, potassium citrate, disodium phosphate anhydrous, disodium phosphate dihydrate, disodium phosphate dodecahydrate, monosodium phosphate anhydrous, monosodium phosphate monohydrate, monosodium phosphate dihydrate, sodium tartrate, monopotassium phosphate and dipotassium phosphate.

22. The formulation of claim 1 , wherein the formulation comprises about 0.001 milli- international units (MIU) to 15 MTU per ml of IL-2, IL-7, IL-15, orIL-21 ,23. The formulation of claim 1 , wherein the formulation comprises about 0.01 nulli- international units (MIU) to 10 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

24. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 9 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

25. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 8 MIU per ml of IL-2, IL-7, IL- 15, or IL-21.

26. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 7 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

27. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 6 MIU per ml of IL-2, IL-7, IL- 15, or IL-21.

28. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 5 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

29. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 4 MIU per nil of IL-2, IL-7, IL- 15, or IL-21.

30. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 3.5 MIU per ml of IL-2, IL-7, IL- 15, or IL-21.

31. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 3 MIU per ml of IL-2, IL-7, IL- 15, or IL-21 .

32. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 2 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

33. The formulation of claim 1, wherein the formulation comprises about 0.01 milli- international units (MIU) to 1 MIU per ml of IL-2, IL-7, IL- 15, orIL-21.

34. The formulation of any one of claims 1-33, wherein the formulation is comprised in a pharmaceutical composition or liquid formulation.

35. The formulation of any one of claims I -33, wherein the isoelectric point of IL-2, IL-7, IL- 15, or IL-21 is between 3 to 10.

36. An isolated cell comprising an expression vector encoding for interleukin-2 (IL-2), interleukin- 7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21).

37. The isolated cell of claim 36, wherein the cell is a bacterial cell, yeast cell, mammalian cell or cell-line.

38. The isolated cell of claim 36, wherein the cell is Pichia pastoris or ChineseHamster Ovary (CHO) cells.

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