Interleukin preparations for in vivo use

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 484,837, filed February 14, 2023, and U.S. Provisional Application No. 63 / 484,833, filed February 14, 2023, which applications are incorporated herein by reference in their entireties.

[0002] The present disclosure relates to novel pharmaceutical compositions comprising interleukins of the gamma chain family, such as interleukins 2, 4, 7, 9, 15, and 21. [Background technology]

[0003] Interleukins are molecules of medical interest that are difficult to stabilize in solution due to their tendency to adsorption, aggregation, oligomerization, and oxidation. They act primarily as growth and proliferation factors for progenitor and mature cells and also have a role in lineage-specific cell differentiation. Summary of the Invention

[0004] One aspect of the present invention relates to the formulation of stable aqueous solutions of recombinant interleukins of the common gamma chain family by selecting excipients that prevent degradation during storage at 2-8° C. for several months, allowing for pharmaceutical production without resorting to time-consuming and costly lyophilization procedures or extremely low storage and handling temperatures.

[0005] A second aspect is the packaging of these solutions into compatible pre-filled syringes, bags, and other containers, allowing for their adoption as supplements for in vivo cell growth or as ready-to-use materials for other in vivo purposes. Incorporation of these solutions containing defined concentrations of interleukins into a packaging format suitable for direct incorporation into cell culture allows for the seamless integration of critical materials into cell therapy manufacturing processes, eliminating the need for lyophilized protein reconstitution, titration to achieve specific process-related dose levels, and mitigating the potential risk of product contamination due to improper aseptic technique or other user error.

[0006] Interleukin molecules herein include variants of the present disclosure comprising an amino 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 amino acid sequence of IL-2 (SEQ ID NO:1). These include IL-2 variants having an N88R mutation and comprising an amino 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 (i.e., SEQ ID NO:1).Also included in the embodiment are IL-2 antibodies that preferentially stimulate Treg cells and have the N88R and C125S mutations and are 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%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 1 Also included are IL-2 variants comprising an amino acid sequence having 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 IL-2 variant. Embodiments also include IL-2 variants that preferentially stimulate Treg cells and comprise an amino 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: 1).

[0007] In another embodiment, the interleukin molecules herein include variants of the present disclosure comprising an amino 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 amino acid sequence of IL-7 (SEQ ID NO:2). These include IL-7 variants.

[0008] In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an amino 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 amino acid sequence of IL-15 (SEQ ID NO:3). These include IL-15 variants.

[0009] In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an amino 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 amino acid sequence of IL-21 (SEQ ID NO: 4). These include IL-21 variants.

[0010] In another embodiment, a liquid formulation comprising an interleukin embodied herein is stable for at least 12 months at a temperature range of 2°C to 20°C.

[0011] In another aspect, a liquid formulation for in vivo use comprises about 0.001 milli-international units (MIU) to 20 MIU per ml of one or more interleukins, including interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), or interleukin-21 (IL-21). In a specific embodiment, the IL-2-containing formulation comprises about 1 mg / ml to about 10 mg / ml of antioxidant, about 0.0001 to 4 mg / ml of chelating agent, about 0.01 mg / ml to about 10 mg / ml of phosphate, about 20 mg / ml to about 80 mg / ml of sugar and / or sugar alcohol, and about 0.001 mg / ml to about 5 mg / ml of surfactant. In certain embodiments, a formulation comprising IL-2 comprises about 3 mg / ml to about 8 mg / ml of antioxidant, about 0.05 mg / ml to about 5 mg / ml of phosphate, about 30 mg / ml to about 60 mg / ml of sugar and / or sugar alcohol, and about 0.01 mg / ml to about 4 mg / ml of surfactant. In certain embodiments, the formulation comprises about 0.1 milli-international units (MIU) to about 20 MIU of IL-2 per ml. In certain embodiments, a formulation comprising IL-7 comprises about 1 mg / ml to about 10 mg / ml of antioxidant, about 1 mg / ml to about 10 mg / ml of buffer, about 0.001 mg / ml to about 5 mg / ml of acid, and about 0.0001 mg / ml to about 4 mg / ml of chelating agent. In certain embodiments, a formulation comprising IL-7 comprises about 3 mg / ml to about 8 mg / ml of antioxidant, about 3 mg / ml to about 8 mg / ml of buffer, about 0.01 mg / ml to about 3 mg / ml of acid, and about 0.0001 mg / ml to about 0.5 mg / ml of chelating agent. In certain embodiments, a formulation comprises about 0.001 mg / ml to about 5 mg / ml of IL-7. In certain embodiments, a formulation comprising IL-15 comprises about 1 mg / ml to about 10 mg / ml of antioxidant, about 1 mg / ml to about 10 mg / ml of buffer, about 0.001 mg / ml to about 5 mg / ml of acid, and about 0.0001 mg / ml to about 4 mg / ml of surfactant.In certain embodiments, a formulation comprising IL-15 comprises about 3 mg / ml to about 8 mg / ml of antioxidant, about 3 mg / ml to about 8 mg / ml of buffer, about 0.01 mg / ml to about 3 mg / ml of acid, and about 0.0005 mg / ml to about 1 mg / ml of surfactant. In certain embodiments, a formulation comprises about 0.001 mg / ml to about 5 mg / ml of IL-15. In certain embodiments, a formulation comprising IL-21 comprises about 1 mg / ml to about 10 mg / ml of buffer, about 0.001 mg / ml to about 5 mg / ml of acid, and about 10 mg / ml to about 80 mg / ml of sugar and / or sugar alcohol. In certain embodiments, a formulation comprising IL-21 comprises about 2 mg / ml to about 9 mg / ml of buffer, about 0.01 mg / ml to about 3 mg / ml of acid, and about 30 mg / ml to about 60 mg / ml of sugar and / or sugar alcohol. In certain embodiments, the formulation comprises about 0.001 mg / ml to about 5 mg / ml of IL-21. In certain embodiments, the antioxidant belongs to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite, and potassium sulfite. In certain embodiments, the phosphate salt comprises anhydrous monosodium phosphate and disodium phosphate. In certain embodiments, the monosodium phosphate and disodium phosphate comprise anhydrous monosodium phosphate, monosodium phosphate monohydrate, monosodium phosphate dihydrate, anhydrous disodium phosphate, disodium phosphate dihydrate, and disodium phosphate dodecahydrate. In certain embodiments, the sugar comprises sucrose, trehalose, and maltose, and the sugar alcohol comprises sorbitol, isomalt, xylitol, maltitol, mannitol, erythritol, and lactitol, or a mixture thereof.In certain embodiments, surfactants include 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 agent includes edetate disodium, edetate monosodium, edetate trisodium, edetate tetrasodium, 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 includes citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid, and sulfuric acid. In certain embodiments, the buffer includes 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 (MIU) to 15 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 10 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 9 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 8 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 7 MIU of IL-2, IL-7, IL-15, or IL-21 per ml.In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 6 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 5 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 4 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 3.5 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation comprises about 0.01 milli-international units (MIU) to 3 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation contains about 0.01 milli-international units (MIU) to 2 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation contains about 0.01 milli-international units (MIU) to 1 MIU of IL-2, IL-7, IL-15, or IL-21 per ml. In certain embodiments, the formulation is in the form of 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 and 10.

[0012] In another aspect, the isolated cell comprises an expression vector encoding 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, a yeast cell, a mammalian cell, or a cell line. In certain embodiments, the cell is a Pichia pastoris or a Chinese hamster ovary (CHO) cell.

[0013] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0014] definition The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the invention. Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of cell culture, molecular genetics, and biochemistry).

[0015] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0016] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, as practiced in the art. Alternatively, "about" can mean within 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 five-fold and two-fold of a value. When a particular value is described in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for the particular value.

[0017] The term "amino acid" includes residues in the D or L form of natural α-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), as well as β-amino acids, synthetic, and unnatural amino acids. Many amino acid residues are useful in adipokine polypeptides, and the present 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 references cited therein. Another source of a wide variety of amino acid residues is provided by the RSP Amino Acids LLC website.

[0018] As used herein, "antioxidants" include oxidizing species such as ascorbic acid, ascorbyl palmitate, tocopherol, metabisulfite, bisulfite, and sulfite (these molecules act as oxygen scavengers when the oxidizing agent involved is oxygen), reducing agents (which reduce oxidized molecules to be protected and can also be preferentially oxidized), and chain terminators. Methionine is preferentially oxidized, thus protecting ILs from oxidation. This is one of the mechanisms used by antioxidants. These are all considered "antioxidants" because they are molecules that contribute to protecting other molecules from oxidation by oxygen or other oxidants through various mechanisms. In this sense, methionine is an antioxidant. In summary, methionine can act as an oxygen scavenger, be preferentially oxidized by other oxidants, or reduce the oxidized forms of ILs. In all cases, it would be considered an antioxidant in chemical and formulation technology. This differs from the biological and medical concept of antioxidants, which refers primarily to "chain terminators", i.e. molecules that react with free radicals to stop chain reactions in biological systems and protect cells, or the food chemistry concept, which refers primarily to protection against oxygen damage and autooxidation.

[0019] As used herein, a "biological medium" is any type of medium used to grow, culture, or maintain organs, tissues, cells, etc. in vitro. Biological medium also includes any biocompatible agent, any pharmaceutical excipient, any pharmaceutically and physiologically acceptable fluid such as water, saline, balanced salt solution, aqueous dextrose, glycerol, etc. as a carrier, tissue or organ culture medium, any agent that can be administered in vivo to a subject, any agent that can be used in an assay or that can be used to dilute or maintain biological samples such as nucleic acids, peptides, etc.

[0020] 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, such as 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" (e.g., engineered cell) refers to a cell into which a nucleic acid molecule, such as an IL-2 protein (e.g., spliced ​​and / or unspliced ​​forms of IL-2) or a fragment thereof, has been introduced.

[0021] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, are intended to be inclusive or open-ended, allowing for additional elements with respect to a defined or described element of an item, composition, apparatus, method, process, system, etc., thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those identified elements (or their equivalents, as appropriate), and that other elements may be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0022] "Encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to function in biological processes as a template for the synthesis of other polymers and macromolecules having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and refers to the biological property resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0023] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0024] As used herein, "expression vector" or "vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (e.g., naked or liposomal), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, and viruses. Thus, the term includes autonomously replicating plasmids or viruses. The term is also intended to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0025] As used herein, the term "human recombinant IL-X (rhIL-X)" or simply "IL-X" (where X can have a value of 2, 7, 15, or 21) refers to the protein produced by an organism transfected with the DNA sequence set forth below, or non-extensive modifications thereof.

[0026] As used herein, the term "interleukin" refers to interleukins of any source, including mammalian sources such as human, mouse, rat, primate, and porcine, and may be naturally occurring or obtained by recombinant or synthetic techniques, including recombinant interleukin polypeptides produced by microbial hosts. Interleukins may be or include naturally occurring polypeptide sequences or may be active variants of naturally occurring interleukin polypeptides. In certain embodiments, the interleukin polypeptide or active variant is of human origin and includes recombinant human interleukins. Variants of naturally occurring interleukins may be fragments, analogs, and derivatives. By "fragment" is intended a polypeptide comprising only a portion of the complete polypeptide sequence. "Analog" refers to a polypeptide comprising a naturally occurring polypeptide sequence with one or more amino acid substitutions, insertions, or deletions. Muteins and pseudopeptides are specific examples of analogs. "Derivatives" include any native interleukin polypeptide or fragment or analog thereof that has been modified, such as by glycosylation, phosphorylation, fusion to another polypeptide or molecule, polymerization, or 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%, and more preferably at least 90% amino acid sequence identity to the amino acid sequence of the reference interleukin polypeptide.

[0027] As used herein, the term "kit" refers to any delivery system for delivering materials. The term "kit" includes both research and clinical kits. 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 appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit may include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term "split kit" refers to a delivery system containing two or more separate containers, each containing a portion 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 "split kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system containing two or more separate containers, each containing a portion of the total kit components, is encompassed by the term "split kit." In contrast, a "combined kit" refers to a delivery system containing all components of a reaction assay in a single container (e.g., a single box containing each desired component). The term "kit" encompasses both split kits and combined kits.

[0028] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the context clearly dictates otherwise.

[0029] The terms "percent sequence identity" or "has sequence identity" refer to the degree of identity between any given query sequence and subject sequence.

[0030] The term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like, that can be used as a vehicle for a pharmaceutically acceptable substance.

[0031] The term "polynucleotide" is a chain of nucleotides, also known as a "nucleic acid." As used herein, polynucleotide includes all nucleic acid sequences obtained by any method available in the art, including, but not limited to, both naturally occurring and synthetic nucleic acids.

[0032] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. The peptides provided herein for use in the methods and compositions described and claimed may be cyclic.

[0033] As used herein, "stable" or "very stable" refers to the biological activity of a molecule.

[0034] "Treating" or "treatment" refers to the treatment of a disease state in a mammal, and includes (a) preventing the disease state from occurring in the mammal, particularly when such a mammal is susceptible to but has not yet been diagnosed with the disease state, (b) inhibiting the disease state, e.g., halting its progression, and / or (c) alleviating the disease state, e.g., causing regression of the disease state until a desired endpoint is reached. Treatment also includes ameliorating symptoms of the disease (e.g., reducing pain or discomfort), which may or may not directly affect the disease (e.g., cause, propagation, manifestation, etc.).

[0035] As used herein, a "variant" is a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence but retains essential properties of the reference molecule. The sequence changes in a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are usually limited or conservative, resulting in close overall similarity and, in many regions, identity between the reference peptide and the variant. The variant and reference peptide may differ in amino acid sequence by any combination of one or more amino acid substitutions, additions, or deletions. Nucleic acid or peptide variants may be naturally occurring, such as allelic variants, or variants known not to occur in nature. Non-naturally occurring nucleic acid and peptide variants may be generated by mutagenesis techniques or direct synthesis.

[0036] The Genbank and NCBI submissions indicated by the accession numbers cited herein are incorporated herein by reference.All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference.In case of discrepancy, the present specification, including definitions, shall prevail.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0037] Ranges: Throughout this disclosure, various aspects of the invention may 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 not only each individual numerical value within that range but also all possible subranges. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed each individual number within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the broadness of the range. [Brief explanation of the drawings]

[0038] [Figure 1] 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 (γc) (or CD132) receptor. DETAILED DESCRIPTION OF THE INVENTION

[0039] Embodiments relate to highly stable solutions of recombinant non-glycosylated interleukins of the common gamma chain family and methods for their production.

[0040] The disclosure summarized herein includes a series of formats and formulations that enable long-term storage of interleukins 2, 7, 15, and 21 in liquid solution. These molecules are difficult to stabilize in solution due to their tendency to adsorption, aggregation, oligomerization, and oxidation. Formulating these molecules into liquid solutions that preserve their integrity (determined by their ability to retain their critical quality attributes and remain stable for several months at 2-8°C) facilitates their use as reagents and excipients in the manufacture of cell-based medicines applicable to a variety of indications. For example, these formulations enable the delivery of these macromolecules to lymphocyte cultures that form the basis of certain adoptive cell therapies without the need to reconstitute lyophilized material. Furthermore, the molecules in the formulations described herein, when packaged in specific single-use containers, allow for the direct incorporation of these molecules into a given cell therapy manufacturing process, thereby enabling aseptic integration of critical reagents, simplifying and streamlining previously complex unit operations while reducing the risk of potential contamination.

[0041] Interleukin 2 IL-2 was discovered in the supernatant of activated T cells over 30 years ago and is produced primarily by CD41 and CD81 T cells, and to a lesser extent by activated DCs, NK, and NKT cells. The IL-2 receptor (IL-2R) consists of three subunits: the ligand-specific α chain IL-2Rα (CD25), the β chain IL-2Rβ (CD122) (also part of the IL-15R complex), and the common γc, also known as IL-2Rγ (CD132). All three subunits are required for assembly of the high-affinity IL-2R. Upon T cell activation, IL-2Rα is rapidly induced and participates in the formation of a high-affinity tetrameric complex, which activates multiple signaling 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 the proliferation and differentiation of NK cells, enhancing their cytolytic function. Recombinant human IL-2 is used in immunotherapy for cancer and HIV-related AIDS. Anti-IL-2Rα suppresses immune responses in patients with autoimmune diseases and prevents rejection of transplanted organs.

[0042] Sequence of IL-2 (aldesleukin) protein, SEQ ID NO:1: 1 PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT 51 ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET 101 TFMCEYADET ATIVEFLNRW ITFSQSIIST LT

[0043] Interleukin 7 Interleukin-7 (IL-7; RefSeq NM_000880; UniProtKB-Pl3232(IL7_HUMAN)) is a member of the IL-2 superfamily, which includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. It binds to receptors that share a common gamma chain subunit. In addition to the common gamma chain subunit, the IL-7 receptor (IL-7R) requires the IL-7R α chain for binding to occur. Due to the frequency of the common gamma chain subunit, the presence of the IL-7R α chain is a better indicator of when IL-7 actually binds to the receptor. IL-7 receptor binding results in the phosphorylation of tyrosine residues on the receptor. This leads to the activation of JAK1 or JAK3, depending on the cell type, which subsequently activates many downstream signaling pathways, including STAT5a / b, PB kinase, and SRC kinase. It is well known that IL-7 plays an important role in B and T cell development.

[0044] IL-7, also known as pre-B cell growth factor or lymphopoietin-1, is a homeostatic cytokine 40IL-7R is present on most T cells, B cell precursors, and bone marrow macrophages and is composed of the IL-7Rα (CD127) chain and the common γc (CD132) chain. Because γc is ubiquitously expressed on lymphocytes, IL-7 responses are determined by the expression of IL-7Rα, which is shared with the thymic stromal lymphopoietin (TSLP) receptor. IL-7 signaling contributes to thymocyte survival and proliferation, as well as the development of naive and memory B and T cells, mature T cells, and NK cells. Studies of IL-7 and IL-7Rα knockout mice indicate that IL-7 is important for homeostatic T and B cell development. 41 Reagents that block IL-7 or IL-7 signaling could be used to treat patients with HIV-associated immunodeficiency and immunodeficiency secondary to chemotherapy, autoimmune diseases, and lymphoid malignancies.

[0045] Sequence of IL-7 protein, SEQ ID NO:2: 1 MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA 51 NKEGMFLFRA ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR 101 KPAALGEAQP TKSLEENKSL KEQKKLNDLC FLKRLLQEIK TCWNKILMGT 151 KEH

[0046] Interleukin-15 Interleukin-15 (IL-15; RefSeq NM_l72175, UniProt / Swiss-Prot P40933) stimulates effector NK cells and CD8 +IL-15 is a cytokine important for the development, proliferation, and activation of memory T cells. IL-15 binds to the IL-15 receptor α (IL-15Rα) and is trans-presented to the IL-2 / IL-15 receptor-common γ chain (IL-15Rγc) complex on effector cells. IL-15 and IL-2 share binding to IL-15Rγc and signal through the STAT3 and STAT5 pathways. However, IL-2 also binds to CD4 + CD25 + FoxP3 + Supports the maintenance of regulatory T (Treg) cells and activates CD8 + Induce T cell death. These effects may limit the therapeutic activity of IL-2 against tumors. IL-15 does not share these immunosuppressive activities with IL-2. In addition, IL-15 inhibits the effector CD8 + IL-15 is the only cytokine known to provide antiapoptotic signaling to T cells. Administered alone or in complex with IL-15Rα, IL-15 exhibits potent antitumor activity against well-established solid tumors in experimental animal models, and has therefore been identified as one of the most promising immunotherapeutic agents that can potentially cure cancer.

[0047] IL-15 is structurally homologous to IL-2 and was discovered by its ability to induce T cell proliferation, like IL-2. Many of the biological effects attributed to IL-2 can also be induced by IL-15. IL-15R consists of the IL-15Rα chain, the IL-2Rβ chain, and the common γc chain. IL-15 activates non-immune cells (keratinocytes and skeletal muscle cells) and immune cells (monocytes and activated CD4) in response to signals that induce innate immunity. + IL-15 is produced by T cells. IL-15 shares several functions with IL-2, such as T cell activation, stimulation of NK cell proliferation, and cytolytic activity, but differences in their biological functions have been identified based on the differences observed between the phenotypes of IL-2 and IL-15 knockout mice.

[0048] Sequence of IL-15 protein, SEQ ID NO:3: 1 MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV 51 ISLESGDASI HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF 101 LQSFVHIVQM FINTS

[0049] Interleukin 21 IL-21 (Ensembl: ENSG00000138684, MIM: 605384; AllianceGenome: HGNC: 6005) is produced by the TH17 subset of T cells, NKT cells, and CD41 T cells. IL-21 receptors are expressed on a variety of cells, indicating a broad spectrum of action. IL-21 influences B cell function by regulating antibody isotype balance, proliferation, apoptosis, and differentiation into plasma cells. The cytotoxic activity and proliferation of CD81 T cells, NK cells, and NKT cells are increased upon stimulation with IL-21. IL-21 is being tested as an anticancer agent, and initial clinical trial results are promising by delaying tumor progression in metastatic melanoma. In contrast to its anticancer effects, IL-21 also contributes to inflammation in several diseases, as expected of a TH17-related cytokine.

[0050] Sequence of IL-21 protein, SEQ ID NO:4: 1 MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ 51 KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK 101 KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS

[0051] Interleukin expression In certain embodiments, the interleukins embodied herein are encoded by expression vectors. The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least part of a gene product that can be transcribed. In some cases, when the transcription product is an mRNA molecule, it is then translated into a protein, polypeptide, or peptide.

[0052] In certain embodiments, the isolated cell comprises an expression vector encoding 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, a yeast cell, a mammalian cell, or a cell line. In certain embodiments, the isolated cell is a Pichia pastoris or a Chinese hamster ovary (CHO) cell.

[0053] P. pastoris is a widely used protein expression host for the production of biopharmaceuticals and industrial enzymes. P. pastoris belongs to the methylotrophic yeasts that share a common pathway for metabolizing one-carbon compounds as carbon and energy sources. The methylotrophic yeast species P. pastoris (recently reclassified as Komagataella pastoris) and H. polymorpha (also known as Pichia angusta) have been widely adopted and have become a virtual mainstay of biotechnology, including heterologous protein production.

[0054] Methylotrophic yeasts have two key characteristics: (1) they can grow to high cell densities even in unsophisticated fermentation processes; (2) their high requirement for methanol oxidase endows them with very strong and tightly regulated promoters. These characteristics enable methylotrophic yeasts to be used not only in process development for the commercial production of feed proteins (single-cell proteins) but also as production systems for recombinant proteins. The widely used P. pastoris and H. polymorpha have different genetics for alcohol oxidase expression: P. pastoris expresses AOX1 and AOX2, whereas H. polymorpha expresses only MOX. In addition to P. pastoris and H. polymorpha, P. methanolica and C. boidinii are also used as expression systems.

[0055] Several points should be considered when using P. pastoris for cloning and expression of heterologous proteins. These include the choice of host strain, the choice of promoter, transcription terminator (TT), marker combination, and the application of either intracellular or secretory expression.

[0056] Host Strains: Widely used commercially available strains fall into five main classes: wild-type strains (e.g., X-33), auxotrophic strains (e.g., GS115, KM71), protease-deficient strains (e.g., SMD1168), glycoengineered strains (e.g., SuperMan5), and several other strains. Notably, engineered P. pastoris can secrete recombinant proteins with uniform human N-linked glycans. Glycosylation is one of the most common post-translational protein modifications (PTMs) in native and recombinant proteins, affecting protein folding, solubility, stability, transport, bioavailability, immunogenicity, and functional activity. The use of engineered P. pastoris broadens the application of antibody expression in microbial systems.

[0057] Vectors: Promoters for protein expression in P. pastoris include inducible promoters (such as AOX1, FLD1, ADH1, and GUT1) and constitutive promoters (such as GAP and TEF1). For P. pastoris, HIS4 (an auxotrophic marker) and Zeocin resistance (a dominant marker) are the most commonly used markers. Heterologous proteins can be expressed intracellularly or secreted. P. pastoris has the ability to secrete high titers of proteins into the culture medium. The most commonly used secretion signals are derived from the endogenous acid phosphatase (PHO1) of P. pastoris, the alpha-mating factor (α-MF) of S. cerevisiae, and the invertase (SUC2) of S. cerevisiae.

[0058] Polynucleotides: Nucleic acids can encode, for example, the amino acid sequence of IL-2, IL-7, IL-15, or IL-21 with at least one or more conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which an amino acid with particular physical and / or chemical properties is replaced with another amino acid with the same chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, etc.), a basic amino acid for another basic amino acid (e.g., Lys, Arg), an amino acid with a polar side chain for another amino acid with a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0059] The nucleic acids of the disclosure can encode functional variants that also include extensions of, for example, IL-2, IL-7, IL-15, or IL-21 proteins. For example, functional variants of IL-2, IL-7, IL-15, or IL-21 proteins can include 1, 2, 3, 4, and 5 additional amino acids from either the N-terminus or C-terminus of the IL-2, IL-7, IL-15, or IL-21 protein.

[0060] Alternatively, or in addition, a functional variant can comprise the amino acid sequence of an IL-2, IL-7, IL-15, or IL-21 protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not prevent or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the native IL-2, IL-7, IL-15, or IL-21 protein. An IL-2, IL-7, IL-15, or IL-21 protein can consist essentially of the specific amino acid sequence described herein, such that other components of the functional variant, e.g., other amino acids, do not substantially alter the biological activity of the functional variant.

[0061] In some embodiments, the nucleic acid encodes 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 a combination thereof.

[0062] According to one embodiment, the present disclosure provides a composition comprising a nucleic acid encoding an IL-2, IL-7, IL-15, or IL-21 protein, or a functional part or fragment thereof, or a variant thereof, such as SEQ ID NO: 1, 2, 3, or 4.

[0063] In certain embodiments, the nucleic acid sequence encodes a polypeptide comprising a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, 2, 3, or 4.

[0064] In some embodiments, a polynucleotide encoding one or more fusion proteins provided herein comprises a codon-optimized sequence. As used herein, the term "codon-optimized" means that a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by selecting different codons without changing the amino acid sequence of the encoded protein. Thus, codon optimization generally refers to replacing codons with synonymous codons to optimize protein expression while maintaining the same amino acid sequence of the translated protein. Codon optimization of a sequence can, for example, increase the protein expression level of the encoded protein (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22:346-53) and can provide other benefits. For example, codon usage preference as measured by the codon adaptability index (CAI), the presence or frequency of A, G, C, U nucleotides, mRNA secondary structure, cis-regulatory sequences, GC content, and other variables can be correlated with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285).

[0065] Any codon optimization method can be used to codon-optimize the polynucleotides and nucleic acid molecules provided herein, and any variable can be changed by codon optimization. Therefore, any combination of codon optimization methods can be used. Exemplary methods include the high codon fitness index (CAI) method. The CAI method selects the most frequently used synonymous codons for the entire protein-coding sequence. As an example, the most frequently used codons for each amino acid can be estimated from 74,218 protein-coding genes from the human genome. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon-optimized.

[0066] In some embodiments, the nucleotide sequence of any region of the RNA or DNA sequence embodied herein may be codon-optimized. In certain embodiments, the primary cDNA template may include a reduced occurrence or frequency of a particular nucleotide in the template strand. For example, the occurrence of a nucleotide in the template may be increased or decreased to a level greater than or less than 25% of the nucleotide in the template. In a further example, the occurrence of a nucleotide in the template may be increased or decreased to a level greater than or less than 20% of the nucleotide in the template. In some examples, the occurrence of a nucleotide in the template may be increased or decreased to a level greater than or less than 16% of the nucleotide in the template. The occurrence of a nucleotide in the template may be increased or decreased to a level greater than or less than 15% of the nucleotide in the template, or greater than or less than 12%.

[0067] In certain embodiments, polynucleotides encoding IL-2, IL-7, IL-15, or IL-21 proteins may contain one or more chemically modified nucleotides. Examples of nucleic acid monomers include unnatural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides may be artificially modified at either the base or sugar moiety. In nature, most polynucleotides contain "unmodified" or "natural" nucleotides, including the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). These bases are typically anchored at the 1' position to ribose or deoxyribose. The use of RNA polynucleotides containing chemically modified nucleotides has been shown to improve RNA expression, expression rate, half-life, and / or expressed protein concentration. RNA polynucleotides containing chemically modified nucleotides have also been useful for optimizing protein localization, thereby avoiding adverse biological responses, such as immune responses and / or degradation pathways.

[0068] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N 4 -Alkylcytidine, N 4 -aminocytidine, N 4 -acetylcytidine, and N 4 ,N 4 -Contains dialkylcytidine.

[0069] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; 4 -methylcytidine, N 4-aminocytidine, N 4 -acetylcytidine, and N 4 ,N 4 -Contains dimethylcytidine.

[0070] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine, and 6-alkyluridine.

[0071] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylester uridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "SMeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

[0072] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'-O-dimethyluridine.

[0073] Examples of modified or chemically modified nucleotides include N 6-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, N 6 -methyl-N 6 -Threonylcarbamoyladenosine, 2-methylthio-N 6 -Threonylcarbamoyladenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyladenosine, 2-methylthio-N 6 -hydroxynorvalylcarbamoyl adenosine, N 6 -acetyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxy-adenine, α-thioadenosine, 2'-O-methyladenosine, N 6 ,2'-O-dimethyladenosine,N 6 ,N 6 ,2'-O-trimethyladenosine, 1,2'-O-dimethyladenosine, 2'-O-ribosyladenosine, 2-amino-N 6 -methylpurine, 1-thioadenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N 6 -(19-amino-pentaoxanonadecyl)-adenosine.

[0074] Examples of modified or chemically modified nucleotides include N 1 -Alkylguanosine, N 2-Alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O 6 -Alkylguanosine, xanthosine, inosine, and N 1 -Alkyl inosines are included.

[0075] Examples of modified or chemically modified nucleotides include N 1 -methylguanosine, N 2 -methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O 6 -methylguanosine, xanthosine, inosine, and N 1 -Methylinosine is included.

[0076] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0077] Examples of modified and chemically modified nucleotide monomers include any such nucleotide known in the art, e.g., 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic monomer residues.

[0078] Examples of modified and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides, and 3'-inverted thymidines.

[0079] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid (LNA) nucleotides, 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methylthioethyl, 2'-deoxy-2'-fluoronucleotides, and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid (LNA) nucleotide.

[0080] Examples of modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0081] 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.

[0082] Examples of modified and chemically modified nucleotide monomers include N 6 -Methyl adenosine nucleotides are included.

[0083] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0084] Examples of modified and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0085] Examples of modifications and chemically modified nucleotide monomers include replacing the 2'-OH group of the nucleotide with 2'-R, 2'-OR, 2'-halogen, 2'-SR, or 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0086] The examples of base modifications described above can be combined with additional modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers can be found in nature.

[0087] Interleukin nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain isolated nucleic acids containing the nucleotide sequences described herein, including nucleotide sequences encoding the polypeptides described herein. PCR can be used to amplify specific sequences from DNA and RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described, for example, in "PCR Primer: A Laboratory Manual," Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of or beyond the region of interest is used to design oligonucleotide primers whose sequences are identical or similar to those of the opposite strand of the template to be amplified. Various PCR strategies are also available, which allow site-specific nucleotide sequence modifications to be introduced into the template nucleic acid.

[0088] Interleukin nucleic acids can also be chemically synthesized as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using the phosphoramidite method) or as a series of oligonucleotides. For example, one or more long oligonucleotide pairs (e.g., >50-100 nucleotides) containing the desired sequence can be synthesized, each pair containing a short complementary segment (e.g., about 15 nucleotides) such that a double-stranded sequence forms when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single double-stranded nucleic acid molecule for each oligonucleotide pair, which can then be ligated into a vector, e.g., a plasmid. Isolated nucleic acids of the present disclosure can also be obtained, for example, by mutagenesis of a portion of naturally occurring interleukin DNA.

[0089] In some embodiments, the nucleic acid is a synthetic polynucleotide. In some embodiments, the synthetic nucleic acid comprises modified nucleotides. Modifications of the internucleoside linker (i.e., backbone) can be utilized to increase stability or pharmacodynamic properties. For example, internucleoside linker modifications prevent or reduce degradation by cellular nucleases, thereby increasing the pharmacokinetics and bioavailability of the nucleic acid. Generally, modified internucleoside linkers include any linker other than a phosphodiester (PO) linker that covalently links two nucleosides. In some embodiments, modified internucleoside linkers increase the nuclease resistance of the nucleic acid compared to phosphodiester linkers. In the case of naturally occurring oligonucleotides, the internucleoside linker contains a phosphate group that creates a phosphodiester bond between adjacent nucleosides. In some embodiments, the nucleic acid comprises one or more internucleoside linkers modified from natural phosphodiester. In some embodiments, all internucleoside linkers of the nucleic acid or its contiguous nucleotide sequence are modified. For example, in some embodiments, the internucleoside linkages include sulfur (S), such as phosphorothioate internucleoside linkages.

[0090] Modifications to the ribose sugar or nucleobase may also be utilized herein. Generally, modified nucleosides involve the introduction of one or more modifications to the sugar or nucleobase moieties. In some embodiments, as described, nucleic acids include one or more nucleosides containing a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety compared to the ribose sugar moiety found in deoxyribonucleic acid (DNA) and RNA. Numerous nucleosides with modifications to the ribose sugar moiety may be utilized, primarily for the purpose of improving oligonucleotide properties, such as affinity and / or stability. Such modifications include those in which the ribose ring structure is modified. These modifications include substitution with a hexose ring (HNA), a bicyclic ring having a diradical bridge between the C2 and C4 carbons of 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 or tricyclic nucleic acids. Modified nucleosides also include nucleosides in which a sugar moiety is replaced with a non-sugar moiety, such as in peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0091] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or the 2'-OH group naturally found in DNA and RNA nucleosides. The substituent can be introduced, for example, at the 2', 3', 4', or 5' position. Nucleosides with modified sugar moieties also include 2'-modified nucleosides, such as 2'-substituted nucleosides. Indeed, much focus has been placed on the development of 2'-substituted nucleosides, and many 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as improved nucleoside tolerance and affinity. 2'-sugar-modified nucleosides are nucleosides with a substituent other than H or -OH at the 2' position (2'-substituted nucleosides) or contain 2'-linked diradicals, including 2'-substituted nucleosides and LNA (2'-4' diradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. By way of further example, in some embodiments, the modification in 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-methoxyethyl).

[0092] 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 more than 10%, more than 25%, more than 50%, more than 75%, or more than 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the nucleic acid comprises both an internucleoside linker modification and a nucleoside modification.

[0093] In certain embodiments, the interleukin polypeptides are synthesized from expression vectors encoding DNA molecules, as described in detail elsewhere herein.

[0094] Polypeptide: Polypeptides embodied herein can be modified to include one or more unnatural amino acids. As used herein, "unnatural amino acid," "unnatural," "modified amino acid," or "chemically modified amino acid" refers to an amino acid other than the 20 genetically encoded α-amino acids, a modified amino acid, or an amino acid analog. Unnatural amino acids have side chain groups that distinguish them from natural amino acids, but unnatural amino acids can be naturally occurring compounds other than the 20 proteinogenic α-amino acids. In addition to side chain groups that distinguish them from natural amino acids, unnatural amino acids can have elongated backbones, such as β-amino acids.

[0095] Non-limiting examples of unnatural amino acids include selenocysteine, pyrrolidine, homocysteine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, and the like. Phosphorus, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, non-natural analogs of tyrosine amino acid; non-natural analogs of glutamine amino acid; non-natural analogs of phenylalanine amino acid; non-natural analogs of serine amino acid; non-natural analogs of threonine amino acid; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, selenium amino acids that interact covalently or non-covalently with another molecule; metal-binding amino acids, metal-containing amino acids, radioactive amino acids; photocaged and / or photoisomerizable amino acids; biotin or biotin analog-containing amino acids, glycosylated or carbohydrate-modified amino acids, keto-containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy atom-substituted amino acids; chemically cleavable or photocleavable amino acids, amino acids with extended side chains, amino acids containing toxic groups, sugar-substituted amino acids, such as sugar-substituted serine; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothioacid-containing amino acids; α,α-disubstituted amino acids; β-amino acids; and cyclic amino acids other than proline.In one 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.

[0096] Any one or more of SEQ ID NOs: 1, 2, 3, or 4 may be modified to include naturally occurring and synthetic alpha, beta, gamma, and delta amino acids, namely, those found in proteins, namely, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl, or β-histidinyl. Where the term amino acid is used, it is considered to be a specific and independent disclosure of each of the alpha, beta, gamma, and delta esters of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine, in the D- and L-configuration.

[0097] Pharmaceutical Composition A particular aspect of the present disclosure relates to pharmaceutical compositions of the compounds of the present disclosure. Pharmaceutical compositions of the present disclosure typically contain a compound of the present disclosure and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The type of carrier can be selected based on the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal, or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0098] The liquid compositions of the present disclosure comprise an interleukin at a concentration of 0.001 milli-international units (MIU) to 20 MIU per ml, a buffer, a surfactant, an acid, an antioxidant, an acid, a sugar alcohol, and optional excipients selected from amino acids, osmolality adjusters, and preservatives in water.

[0099] The surfactant is present to promote the solubility and stability of the interleukin in solution. Suitable surfactants include C8-C 20These include, but are not limited to, alkyl sulfates, certain phospholipids such as phosphatidates, cholate, deoxycholate, salts of lauroyl sarcosine (such as the sodium salt known as sarcosyl), CHAPS, CHAPSO, Triton X100, Triton X114, NP40, octyl glucoside, polyethylene glycol dodecyl ether (e.g., that commercially available under the trade name Brij™ 35), polyethylene glycol hexadecyl ether (e.g., that commercially available under the trade name Brij™ 58), polyoxyethylene derivatives of sorbitan monolaurate (e.g., Tween 20 and Tween 80), sorbitan esters (e.g., sorbitan monostearate or sorbitan monolaurate), and combinations thereof.

[0100] In some embodiments, the surfactant is an anionic surfactant. 20 It may be selected from alkyl sulfates, lauroyl sarcosinates, cholates, deoxycholates and combinations thereof.

[0101] Preferred anionic surfactants are dodecyl sulfates, such as sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate. For example, surfactants can be selected from alkali metal and alkaline earth metal dodecyl sulfates. Preferred surfactants are SDS.

[0102] Optional excipients can be selected from preservatives, antioxidants, and combinations thereof. Preservatives include, but are not limited to, benzalkonium chloride, benzoic acid, sorbic acid, and their salts. Antioxidants include ascorbic acid, ascorbyl palmitate, tocopherol, and combinations thereof. Typically, optional excipients account for less than 5% by weight, preferably less than 3%, 2%, 1%, or even less than 0.1% by weight, based on the total weight of the composition.

[0103] Optional excipients can also be selected from osmolality adjusters.Osmolality adjusters include pharmaceutically acceptable inorganic salts such as sodium chloride and potassium chloride, and organic salts of sodium or potassium, such as potassium or sodium citrate, aspartate or acetate.Osmolality adjusters are typically added to the compositions of the present invention in an amount that allows them to adjust the osmolality of the composition.

[0104] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin in the composition. Furthermore, the compounds can be administered in sustained-release formulations, such as in compositions with slow-release polymers or in fat pads as described herein. The active compounds can be prepared with carriers that protect the compound against rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-polyglycolic acid copolymers (PLG). Many methods for the preparation of such formulations are generally known to those skilled in the art.

[0105] Sterile injectable solution can be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the above-mentioned components as needed, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other components required from the above-listed list.For the preparation of sterile injectable solution, the specific preparation method is vacuum drying and freeze-drying, which produces a powder of the active compound plus any additional desired components from the solution that has been previously sterile-filtered.

[0106] Depending on the route of administration, the compound may be coated with a material to protect it from the action of enzymes, acids, and other natural conditions that may inactivate the agent. For example, the compound can be administered to a subject in a suitable carrier or diluent co-administered with enzyme inhibitors, or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP), and trasylol. Liposomes include water-in-oil-in-water emulsions and conventional liposomes (Strejan, et al., (1984) J. Neuroimmunol 7:27). Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may contain preservatives to prevent the growth of microorganisms.

[0107] The therapeutically effective amount of an active agent can vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the agent to induce a desired response in the individual. Dosage regimens can be adjusted to provide an optimal therapeutic response. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the agent are outweighed by the therapeutically beneficial effects. In another embodiment, the active agent is formulated in a prophylactically effective amount in the composition. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve the desired preventive result. Typically, because a prophylactic dose is used in subjects before or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0108] The amount of active compound in the composition may vary depending on factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit refers to a physically discrete unit suitable as a unitary dosage for the mammalian subject being treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications for dosage unit forms of the present disclosure are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology for compounding such active compounds to treat individual sensitivities.

[0109] Treatment method The present disclosure provides methods for treating or preventing, for example, cancer, viral infection, autoimmunity, etc. In some embodiments, the methods comprise administering to a subject in need thereof an effective amount of a desired interleukin.

[0110] In certain embodiments, the compositions and methods of the present disclosure can be used in combination with one or more additional therapeutically active agents known to treat the conditions or diseases discussed above. For example, the compositions of the present disclosure can be used in combination with one or more known therapeutically active agents to treat neoplastic or proliferative diseases such as tumors or cancer. Non-limiting examples of other therapeutically active agents that can be easily combined with the compositions and methods of the present disclosure into pharmaceutical compositions include 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.

[0111] The following examples further illustrate the present invention and are not intended to limit the invention in any way. [Example]

[0112] Example 1: Method for producing stable formulations of ILs of the gamma family Recombinant non-glycosylated IL-2 was approved several decades ago as a cancer treatment under the trade name Proleukin® and is currently being investigated for pharmaceutical use as a drug for other indications or as an adjunct to immunotherapy for other diseases such as cancer or graft-versus-host disease.

[0113] Other members of this family are being investigated for their recombinant versions as medicines. IL-15 is being investigated for several cancer-related medicines. IL-21 is being investigated for cancer treatment.

[0114] Therefore, the possibility of formulating these interleukins into stable liquid solutions would be useful for their pharmaceutical application. We disclose herein a formulation to be used as a support for the ex vivo expansion of different lineages of leukocytes.

[0115] These interleukins are produced by fermentation of genetically modified organisms transfected with expression vectors designed to highly express proteins of the sequences already disclosed or similar sequences as defined above.

[0116] Expression vectors are commercially available, and a DNA sequence encoding the amino acid sequence of aldesleukin is inserted into the vector. The cells used can be eukaryotic or prokaryotic. For example, in one embodiment, the cells are bacterial cells. In another embodiment, the cells are fungal cells, such as yeast cells. In another embodiment, the cells are vertebrate cells, such as avian or mammalian cells. In another embodiment, the cells are human cells. The cells of the present invention can express, or can be engineered to express, endogenous IL-2 or a fragment thereof. For example, cells engineered to express IL-2 or a fragment thereof can be produced by introducing an expression vector encoding the protein into the cells.

[0117] Among bacteria, nonpathogenic strains of Escherichia coli are preferred. E. coli B serves as a research model and for protein expression in life science laboratories and the biotechnology industry. Characteristics such as protease deficiency, low acetate production at high levels of glucose, and enhanced permeability (presumably due to a simple cell surface) make E. coli B a desirable host for producing engineered proteins. Differences between B and K12 strains include the absence of flagellar component genes, DNA cytosine methylase dcm, and ompT in BL21(DE3). B strains may possess an additional type II secretion system not found in K12. BL21(DE3) also harbors the DE3 recombinant phage, which carries the T7 RNA polymerase gene, which can direct high-level expression of cloned genes under the control of a T7 promoter. E. coli strains typically used for recombinant protein expression include BL21 (a B E. coli strain that protects target proteins from lon and ompT proteases) and its derivatives, such as lysogenic DE3 (based on T7 polymerase), pLysS, pLysE (expressing T7 lysozyme, which reduces basal expression of target genes), Origami (allowing disulfide bond formation within the E. coli cytoplasm), and Rosetta (enhancing expression of proteins containing codons rarely used in E. coli). Similar versions exist in the K12 E. coli genetic background. Typical plasmid vectors for high-level expression of recombinant proteins in E. coli include the pET series, based on the pBR322 replication origin and T7 / lac promoter; pBad, which has the araBAD promoter and pUC replication origin; and pGEX, which also has the tac promoter and pBR322 replication origin. Combinations of fusion tag sequences, protease cleavage sites, selectable markers, and strain compatibility provide the basis for a list of the most common high-expression plasmid variants. In a particular embodiment, the cells are E. coli cells. Different E. coli strains can be transfected to obtain optimal interleukin production.

[0118] The production bacteria can be cultured in an appropriate growth medium. For example, for every 9 liters of medium, 216 g of yeast extract, 108 g of soy peptone, 113 g of KHPO, 20.8 g of KHPO, 36 ml of glycerol, and 4 ml of antifoam (2% v / v) can be included. Fermentation conditions can be: temperature: 37°C ± 0.5°C, agitation: 350 rpm ± 10 rpm, air flow: 9 L / min ± 1 L / min, pO set point: 40%, and pH between 6.95 and 7.5. After this, a feeding procedure should be followed. For example, a 40% p / v glucose solution can be fed dropwise to maintain a concentration of 0.1%. Once the OD600 reaches 5 to 10, an appropriate inducer, such as isopropyl-β-D-1-thiogalactopyranoside (IPTG), should be added to reach the working concentration. At this point, the glucose feed may be reduced to maintain a glucose concentration of approximately 0.01%. Fermentation can usually be stopped approximately 18 to 24 hours after inoculation. After fermentation, the bacteria are concentrated 5 to 7 times by centrifugation or tangential flow filtration and either processed immediately, stored at 2-8°C (within 24 hours), or stored at -20°C (for more than 24 hours).

[0119] After incubation, interleukins are present inside the bacteria, primarily as aggregates called inclusion bodies (Ib). These Ib can be isolated by disrupting the bacteria (e.g., by sonication). To do this, the bacteria can be suspended in purified water. The suspension can then be cycled 2-4 times through a disruptor under a pressure of approximately 1400 bar. The lysate can be processed immediately or stored at -20°C. Ib is separated from other components of the lysate by centrifugation or tangential flow filtration and washed. The Ib preparation can be stored at -20°C until further processing. Ib is then suspended in an appropriate buffer and subjected to a refolding step at an adjusted redox potential.

[0120] After refolding, interleukin is subjected to different chromatography and diafiltration to obtain a concentrated solution of purified interleukin.Then, interleukin is formulated and packaged in a ready-to-use bag, pre-filled syringe, vial, ampule or other suitable primary packaging material for sterile solution.Surprisingly, it has been discovered that recombinant interleukin can be easily stabilized in solution by using a carboxylic acid buffer with a pH at least 0.3 units away from its isoelectric point, adjusting tonicity with a non-ionic osmotic agent, and adding methionine or other amino acids.In some cases, surfactants are required to prevent them from forming aggregates, oligomers, or adsorbing to the wall of the container.

[0121] The isoelectric point is the pH value at which a protein exhibits no net electrostatic charge. The isoelectric point of a protein can be estimated using Vector NTI 10.3.0 (2006 (C) Invitrogen Corporation). The results obtained for several members of the gamma family are as follows:

[0122] [Table A]

[0123] While it would be reasonable to expect that these and other proteins would be more stable against denaturation and aggregation when formulated below or above their isoelectric points, what is surprising is that all other degradation mechanisms, such as deamidation and oxidation, are also minimized under these conditions. Methionine appears to stabilize solutions of some particularly oxidation-prone members, such as IL-2, IL-7, and IL-15, especially when packaged in oxygen-permeable materials such as plastic bags. Surfactants can be added to further stabilize them against aggregation, oligomerization, and adsorption to container walls.

[0124] All interleukins appear to follow a similar pattern with regard to chemical stability. All of them tend to be very stable in liquid formulations for several months at 2-8°C when formulated in buffers consisting of carboxylic acids and their salts, e.g., citric acid / sodium citrate, acetic acid / sodium acetate, and sodium tartrate / tartaric acid, at pH values ​​at least 0.2 units above or below their isoelectric points, with nonionic osmolality adjusters such as sugars, such as 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 improve the oxidative stability of some members of the family. Other suitable soluble antioxidants and / or chelating agents, such as sodium metabisulfite, sodium sulfite, citric acid, citrate salts, tartaric acid, tartrate salts, and amino acids, can be added instead of, or in addition to, methionine and / or sodium edetate. In some embodiments, surfactants from the group of 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, ionic or non-ionic, may be included.

[0125] [Example 2] IL-2 liquid preparation The solution composition for this interleukin is as follows: anhydrous disodium phosphate 1.2 mg / mL, anhydrous monosodium phosphate 0.2 mg / mL, mannitol 50 mg / mL, SDS 1mg / mL.

[0126] The composition is packaged in pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume of 1-10 ml per bag: 1 MIU / bag; 2 MIU / bag; or 15 MIU / bag.

[0127] Another liquid formulation for IL-2 is as follows: anhydrous disodium phosphate 1.2 mg / mL, anhydrous monosodium phosphate 0.2 mg / mL, mannitol 50 mg / mL, SDS 1mg / mL, Methionine 5mg / mL.

[0128] The composition is packaged in pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume of 1-10 ml per bag: 1 MIU / bag; 2 MIU / bag; or 15 MIU / bag.

[0129] The pH value of both solutions is 7.5 (more than 0.2 units higher than the isoelectric point of IL-2: pH 7.02).

[0130] [Example 2] IL-7 liquid formulation IL-7 is produced by recombinant DNA technology using the genetically engineered Escherichia coli strain BL21 Rosetta DE3 pLysS, which contains an analog of the human interleukin-7 coding region gene inserted into the pET9a(TetR) plasmid vector at the NdeI-BamHI restriction enzyme sites, which contains three disulfide bonds essential for its biological activity.

[0131] The human IL-7 gene was modified using genetic engineering techniques, and the resulting expression clone encodes a modified human interleukin-7. The modified gene contains 154 codons. This recombinant form differs from native interleukin-7 in the following ways: a) IL-7 is derived from E. coli and is therefore not glycosylated; b) the molecule contains an N-terminal methionine.

[0132] (a) Expression system and cloning process description An inducible eukaryotic expression system for the recombinant expression of hIL-7 was developed using an Escherichia coli strain as the host.

[0133] Among the different E. coli BL21(DE3) strains tested, we selected the Rosetta2(DE3)pLysS strain due to the fact that high, robust, and stable recombinant expression of rhIL-7 was observed. This strain supplies tRNAs for rare codons, enabling universal translation that would otherwise be limited by E. coli's codon usage. Furthermore, this strain harbors a plasmid encoding the T7 lysozyme gene, a natural inhibitor of T7 RNA polymerase, which helps suppress basal expression of target genes under the control of the T7 promoter.

[0134] pET9a for cloning at NdeI and BamHI restriction enzyme sites + An expression vector was used, and the strain and vector combination allowed for tight regulation of recombinant expression based on the well-known T7 promoter system, which is only turned on in the presence of T7 RNA polymerase.

[0135] Three different versions of the hIL-7 gene were tested. These were synthetic hIL-7 cassettes containing the native (wild-type) gene and two modified gene versions, including codon usage optimization for E. coli expression. One of the modified gene versions (fully optimized for E. coli expression) yielded the best-expressing clones, and one of these clones was selected for the next stage of this development.

[0136] The molecular constructs and genes were verified by sequencing, and the protein identities were confirmed by SDS-PAGE (molecular size) and Western blot (specific antibody detection).

[0137] [Table 1]

[0138] [Table 2]

[0139] [Example 3] Liquid formulation of IL-15 Genetic development of recombinant clones for IL-15 expression IL-15 was expressed as pET9a + It was produced by recombinant DNA technology using the genetically engineered Escherichia coli strain BL21 A1, which contains an analog of the human interleukin-15 coding region gene inserted into the NdeI-BamHI restriction enzyme sites of a plasmid vector. It contains two disulfide bridges, a structure that has been shown to help stabilize the conformation of the loop region involved in contact with the receptor.

[0140] The human IL-15 gene was modified using genetic engineering techniques, 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 derived from E. coli and is therefore not glycosylated; b) the molecule contains an N-terminal methionine.

[0141] Brief description of the expression system An inducible eukaryotic expression system for recombinant expression of hIL-15 was developed using an Escherichia coli strain as a host. Among the different E. coli BL21 strains tested, strain A1 was chosen due to the fact that high, robust, and stable recombinant expression of rhIL-15 was observed. This strain harbors a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene at the araB locus, allowing expression of T7 RNAP to be regulated by the araBAD promoter. Expression of this polymerase is induced by arabinose.

[0142] pET9a for cloning at NdeI and BamHI restriction enzyme sites +An expression vector was used, and the strain and vector combination allowed for tight regulation of recombinant expression based on the well-known T7 promoter system, which is only turned on in the presence of T7 RNA polymerase.

[0143] Three different versions of the hIL-15 gene were tested. These were synthetic hIL-15 cassettes containing the native (wild-type) gene and two modified gene versions, including codon usage optimization for E. coli expression. One of the modified gene versions (fully adapted for E. coli expression) yielded the best-expressing clones, and one of these clones was selected for the next stage of development. The molecular construct and gene were verified by sequencing, and the protein identity was assessed by SDS-PAGE (molecular size) and Western blot (specific antibody detection).

[0144] [Table 3]

[0145] [Table 4]

[0146] Biological Activity Methodology The biological activity of interleukin-7 (IL-7) is determined through a colorimetric proliferation assay using the TIB-239 cell line (immature murine B lymphocyte ATCC® TIB-239; also known as 2E8 cells). The biological activity and specific activity of the IL-7 source was compared to the NIBSC WHO reference interleukin-7 at 1.5 x 10 using a parallel line assay as the statistical model. 8 Estimated assuming a theoretical biological activity of IU / mg.

[0147] [Example 4] Interleukin-21 liquid formulation Sodium citrate dihydrate 4.05g / L Anhydrous citric acid 1.20g / L Mannitol 41.30g / L IL-21 0.30 mg / mL pH 5.0~5.6

[0148] The pH of this formulation is more than 0.2 units below the isoelectric point of IL-21 (pI 9.42).

[0149] Genetic development of recombinant clones for interleukin-21 expression IL-21 is a human recombinant interleukin-21 protein with a molecular weight of approximately 15.4 kDa (daltons). It is produced by recombinant DNA technology using a genetically engineered strain of Escherichia coli containing an analog of the coding region of the human interleukin-21 gene. Genetic engineering techniques have been used to modify the human IL-21 gene, and the resulting expression clone encodes human interleukin-21.

[0150] Two intramolecular disulfide bonds (between cysteine ​​41 and cysteine ​​92, and between cysteine ​​48 and cysteine ​​95) are essential for the protein's correctly folded conformation and biological activity.

[0151] The primary structure of the protein comprises the following amino acid sequence: 1 M QDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ 51 KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK 101 KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DS

[0152] For recombinant protein expression in E. coli, the initiating methionine ( M ) is required.

[0153] Human IL-21 gene source Three nucleotide sequence composites (cassettes) containing the human IL-21 coding region were ordered (Genscript, New Jersey, USA):

[0154] [Table 5]

[0155] The synthetic gene does not contain the nucleotides encoding the signal peptide region of the hIL-21 gene. All cassettes were delivered as inserts cloned into the pUC57 cloning vector (NdeI and BamHI restriction enzyme sites). Note: All cassettes contain an extra stop codon (TAA stop codon), which is the best option for E. coli, at the first position, revealing the presence of an extra tryptophan due to the stop codon's wobble effect. However, this wobble effect can be eliminated by replacing the TGA (opal) stop codon with a TAA (ochre) stop codon (Vyas VV et al., Biotechnol Prog. 2012 Mar-Apr;28(2):497-507. doi:10.1002 / btpr.746. Epub 2011 Dec 9).

[0156] Generation of E. coli expression strains After confirming the IL-21 coding region and regulatory portion of each expression plasmid, the WT-hIL-21, iop-hIL-21, and op-hIL-21 plasmids were used in the next step. Fifteen nanograms of each supercoiled expression plasmid obtained from the above clones was used to transform the BL21 A1 E. coli strain.

[0157] BL21-AI genotype: F-ompT hsdSB(rB-mB-)gal dcm araB::T7RNAP-tetA The BL21-AI™ strain is an E. coli B / r strain that lacks the lon protease. It also lacks the outer membrane protease, OmpT. The lack of these proteases reduces the degradation of heterologous proteins expressed in this strain. This strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene at 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, allowing the identity of the strain to be verified using tetracycline.

[0158] Expression of hIL21 protein and verification of protein identity 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. Clone "D7" was selected for research cell bank and further development steps. See Figure 1.

[0159] "No induction" means bacteria without induction. The recombinant system (plasmid + E. coli strain) is an inducible gene system that requires an inducer (in this case, L-arabinose) to avoid gene repression. Uninduced bacteria are expected to show no recombinant expression (no recombinant protein band on SDS-PAGE). Induced bacteria should express the protein of interest (i.e., IL-21 band on SDS-PAGE). Numbers and / or letters indicate clone ID (i.e., "2.2-5", "2.2.-6", "D7"...).

[0160] "MW" indicates molecular weight markers.

[0161] SDS-PAGE 18%. Coomassie blue staining - reducing conditions. Induction: Overnight induction (0.2% L-arabinose); Clone IDs: "2.2-5", "2.2-6", "D7" and "D8"; IL-21 STD: rhIL-21 standard

[0162] Protein identity was verified by Western blot analysis using the specific rabbit polyclonal antibody Anti-hIL21 (AB154767; Abcam), and Blast X (which searches protein databases using translated nucleotide queries) search results showed 100% identity with the hIL-21 amino acid sequence.

[0163] [Table B]

[0164] Biological activity - specific activity The biological activity of human IL-21 is determined by a proliferation assay based on the B9 cell line (a murine B cell hybridoma) obtained from Public Health England and quantified by a colorimetric method using MTS.

[0165] Briefly, B9 cells were washed twice with assay medium and 2.5 × 10 5 A cell suspension of 1000 cells / mL was prepared. In parallel, serial dilutions of the IL-21 standard and the samples to be analyzed were performed. Then, 100 μL of each dilution was dispensed per well into a microplate, followed by 100 μL of the cell suspension. The plate was incubated at 37°C and 5% CO2 for 48 hours.

[0166] Detection: 40 μL / well of MTS / PMS solution is dispensed and the microplate is incubated for 4 hours, after which time the plate is read at 490 nm.

[0167] To evaluate the biological activity and specific activity of IL-21 materials, EC 50 is estimated. The median effective concentration (EC 50 ) refers to the drug concentration that induces a response halfway between baseline and maximum after a specific exposure time.50 The response achieved by is considered one unit of biological activity.

[0168] Other embodiments From the foregoing description, it will be apparent that variations and modifications may be made to adapt the disclosure described herein to various uses and conditions. Such embodiments also fall within the scope of the following claims.

[0169] All citations to sequences, patents, and publications herein are incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Applicants do not, by citation of various references in this document, admit that any particular reference is "prior art" to their disclosure.

Claims

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, including interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-15 (IL-15), or interleukin-21 (IL-21).

2. 10. The liquid formulation of claim 1, wherein the formulation comprising IL-2 comprises from about 1 mg / ml to about 10 mg / ml of antioxidant, from about 0.0001 to 4 mg / ml of chelating agent, from about 0.01 mg / ml to about 10 mg / ml of phosphate, from about 20 mg / ml to about 80 mg / ml of sugar and / or sugar alcohol, and from about 0.001 mg / ml to about 5 mg / ml of surfactant.

3. 3. The liquid formulation of claim 2, wherein the formulation comprising IL-2 comprises about 3 mg / ml to about 8 mg / ml of antioxidant, about 0.05 mg / ml to about 5 mg / ml of phosphate, about 30 mg / ml to about 60 mg / ml of sugar and / or sugar alcohol, and about 0.01 mg / ml to about 4 mg / ml of surfactant.

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

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

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

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

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

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

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

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

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

13. 13. The liquid formulation of any one of claims 1, 11 or 12, comprising about 0.001 mg / ml to about 5 mg / ml of IL-21.

14. The liquid formulation according to any one of claims 2 to 13, wherein the antioxidant belongs to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite.

15. 14. The liquid formulation of any one of claims 2 to 13, wherein the phosphate salts include anhydrous monosodium phosphate and disodium phosphate.

16. 16. The liquid formulation of claim 15, wherein the mono- and disodium phosphate comprises mono-sodium phosphate anhydrous, mono-sodium phosphate monohydrate, mono-sodium phosphate dihydrate, disodium phosphate anhydrous, disodium phosphate dihydrate, and disodium phosphate dodecahydrate.

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

18. 14. The liquid formulation of any one of claims 2 to 13, wherein the surfactant comprises 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 ether, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, sucrose stearate, sucrose palmitate, and sucrose oleate.

19. 14. The liquid formulation of any one of claims 2 to 13, wherein the chelating agent comprises edetate disodium, edetate monosodium, edetate trisodium, edetate tetrasodium, 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 to 13, wherein the acid comprises citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid and sulfuric acid.

21. 14. The liquid formulation of any one of claims 2 to 13, wherein the buffering agent 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. 10. The formulation of claim 1, comprising about 0.001 milli-international units (MIU) to 15 MIU of IL-2, IL-7, IL-15, or IL-21 per ml.

23. 10. The formulation of claim 1, comprising about 0.01 milli-international units (MIU) to 10 MIU of IL-2, IL-7, IL-15, or IL-21 per ml.

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

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

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

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

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

29. 10. The formulation of claim 1, comprising about 0.01 milli-international units (MIU) to 4 MIU of IL-2, IL-7, IL-15, or IL-21 per ml.

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

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

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

33. 10. The formulation of claim 1, comprising about 0.01 milli-international units (MIU) to 1 MIU of IL-2, IL-7, IL-15, or IL-21 per ml.

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

35. 34. The formulation of any one of claims 1 to 33, wherein the isoelectric point of IL-2, IL-7, IL-15, or IL-21 is between 3 and 10.

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

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

38. 37. The isolated cell of claim 36, wherein the cell is a Pichia pastoris or a Chinese hamster ovary (CHO) cell.

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