IL322076A - Interleukin formulations for ex vivo use - Google Patents

Description

Atty Docket: 348356.01402 INTERLEUKIN FORMULATIONS FOREX FIFO USE 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: 63/484,841, and U.S. Provisional Patent Application No. 63/484,839, filed on February 14, 2023, which is incorporated herein by reference in its entirety.

FIELD The disclosure relates to new pharmaceutical compositions comprising interleukins of the y-chain family, such as interleukins 2, 4, 7, 9, 15, and 21.

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

SUMMARY One aspect of the present invention relates to the formulation of stable aqueous solutions of recombinant interleukins of the common v - chain family by selecting excipients that prevent them from degrading while stored at 2 --- 8 °C for several months, while avoiding the use of human- or animal-derived carrier proteins such as bovine serum albumin or human serum albumin. This allows the manufacture of recombinant interleukins for further manufacturing or medicinal use without resorting to lengthy and expensive lyophilization procedures or extremely low' storage and handling temperatures. Moreover, the ability to stabilize recombinant interleukins without the use of human- or animal-derived carrier proteins improves their safety profile and mitigates risk in further manufacturing or medicinal applications.

A second aspect is the packaging of these solutions in compatible pre-filled syringes, bags, and other containers, enabling their adoption as ready-to-use as ancillary materials for ex- vivo proliferation of cells or for other ex-vivo purposes. The incorporation of these solutions Atty Docket: 348356.01402 containing defined concentrations of interleukins into packaging formats suitable for direct incorporation into cell cultures enables the seamless integration of critical materials into a cell therapy manufacturing process without the need to reconstitute lyophilized proteins, titrate to achieve dose levels relevant for a given process, and mitigate the potential risk of contaminating a product due to improper aseptic technique, or other user error.

The interleukin molecules herein include variants of the present disclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-2 amino acid sequence (SEQ ID NO: 1). These include IL-2 variants that comprise an amino acid sequence having an N88R mutation that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 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 2.0 least 99% sequence identity to the wild-type IL-2 amino acid sequence (i.e. SEQ ID NO: 1).Embodiments also include IL,-2 variants that preferentially stimulate Treg cells and comprise an amino acid sequence having N88R and C125S mutations that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to the wild-type IL-2 amino acid sequence (SEQ ID NO: 1). Embodiments also include IL-2 variants that preferentially stimulate Treg cells and comprise an ammo acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%,2 Atty Docket: 348356.01402 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).

In another aspect, the interleukin molecules herein include variants of the present disclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%,at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-7 amino acid sequence (SEQ ID NO: 2). These include IL-15 variants.

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

In another aspect, the interleukin molecules herein include variants of the presentdisclosure comprising an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at Atty Docket: 348356.01402 least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-21 amino acid sequence (SEQ ID NO: 4). These include IL-variants.

In another aspect, a liquid formulation for ex vivo use comprises about 0.001 milli-international units (MIU) to 20 MIU per ml of one or more interleukins, comprising interleukin-2 (IL- 2), interleukin-7 (IL-7), interleukin- 15 (IL-15), or interleukin -21 (IL-21). In certain embodiments, the formulation comprising IL-2 comprises from about 1 mg/ml to about 10 mg/ml antioxidants, from about 0.0001 to 4 mg/ml chelating agents, from about 0.01 mg/ml to about 10 mg/ml phosphates, from about 20 mg/ml to about 80 mg/ml sugar and/or sugar alcohols and from about 0.001 mg/ml to about 5 mg/ml surfactants. In certain embodiments, the formulation comprising IL-2 comprises from about 3 mg/ml to about 8 mg/ml antioxidants, from about 0.05 mg/ml to about 5 mg/ml phosphates, from about 30 mg/ml to about 60 mg/ml sugar and/or sugar alcohols and. from about 0.01 mg/ml to about 4 mg/ml surfactants. In certain embodiments, the formulation comprises from about 0.1 milli- international units (AHU) to about 20 MIU per ml of IL-2. In certain embodiments, the formulation comprising IL-7 comprises from about 1 mg/ml to about mg/ml antioxidants, from about 1 mg/ml to about 10 mg/ml buffering agent, from about 0.0mg/ml to about 5 mg/ml acid, and from about 0.0001 mg/ml to about 4 mg/ml chelating agents. In certain embodiments, the formulation comprising IL-7 comprises from about 3 mg/ml to about 20 mg/ml antioxidants, from about 3 mg/ml to about 8 mg/ml buffering agent, from about 0.01 mg/ml to about 3 mg/ml acid, and from about 0.0001 mg/ml to about 0.5 mg/ml chelating agents. In certain embodiments, the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL- 7. In certain embodiments, the formulation comprising IL-15 comprises from about 1 mg/ml to about 10 mg/ml antioxidants, from about 1 mg/ml to about 10 mg/ml buffering agent, from about 0.001 mg/ml to about 5 mg/ml acid, and from about 0.0001 mg/ml to about 4 mg/ml surfactants.In certain embodiments, the formulation comprising IL-15 comprises from about 3 mg/ml to about mg/ml antioxidants, from about 3 mg/ml to about 8 mg/ml buffering agent, from about 0.mg/ml to about 3 mg/ml acid, and from about 0.0005 mg/ml to about 1 mg/ml surfactants. In certain embodiments, the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL- 15. In certain embodiments, the formulation comprising IL-21 comprises from about 1 mg/ml to4 Atty Docket: 348356.01402 about 10 mg/ml buffering agent, from about 0.001 mg/ml to about 5 mg/ml acid, and from about mg/ml to about 80 mg/ml sugar and/or sugar alcohols. In certain embodiments, the formulation comprising IL-21 comprises from about 2 mg/ml to about 9 mg/ml buffering agent, from about 0.01 mg/ml to about 3 mg/ml acid, and from about 30 mg/ml to about 60mg/ml sugar and/or sugar alcohols. In certain embodiments, the formulation comprises from about 0.001 mg/ml to about 5mg/ml of IL-21. In certain embodiments, the antioxidants belong to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite. In certain embodiments, the phosphates comprise anhydrous monosodium phosphates and disodium phosphates. In certain embodiments, the monosodium phosphates and disodium phosphates comprise monosodium phosphate anhydrous, monosodium phosphate monohydrate, monosodium phosphate dihydrate, disodium phosphate anhydrous, disodium phosphate dihydrate and disodium phosphate dodecahydrate. In certain embodiments, the sugars comprise sucrose, trehalose and maltose and sugar alcohols comprise sorbitol, isomalt, xylitol, maltitol, mannitol, erythritol, and lactitol or mixtures thereof. In certain embodiments, the surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, macrogol 15hydroxystearate, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407, polyoxyethylene alkyl ethers, poly oxy 1 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, sucrose stearate, sucrose palmitate and sucrose oleate. In certain embodiments, the chelating agents comprise disodium edetate, monosodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, citric acid, tartaric acid, alanine, arginine, aspartic acid, asparagine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In certain embodiments, the acid comprises citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid and sulfuric acid. In certain embodiments, the buffer comprises sodium citrate dihydrate, potassium citrate, disodium phosphate anhydrous, disodium phosphate dihydrate, disodium phosphate dodecahydrate, monosodium phosphate anhydrous, monosodium phosphate monohydrate, monosodium phosphate dihydrate, sodium tartrate, monopotassium phosphate and dipotassium phosphate. In certain embodiments, the formulation comprises about 0.001 milli- international units (MIU) to 15 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 10 MIU per ml of IL-2,IL-7, Atty Docket: 348356.01402 IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 9 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 8 MIU per ml of IL-2, IL-7, IL-15, or IL- 21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 7 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 6 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 4 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3.5MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 1 MIU per ml of II.,-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation is comprised in a pharmaceutical composition or liquid formulation. In certain embodiments, the isoelectric point of IL-2, IL-7, IL-15, or IL-21 is between to 10.

In another aspect, an isolated cell comprises an expression vector encoding for interleukin- 2.0 2 (IL-2), interleukin- 7 (IL-7), interleukin- 15 (IL-15), or interleukin-21 (IL-21). In certainembodiments, the cell is a. bacterial cell, yeast cell, mammalian cell or cell-line. In certain embodiments, the cell sPichiapastoris or Chinese Hamster Ovary (CHO) cells.

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

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

Atty Docket: 348356.01402 As used herein, the singular forms "a ", "an " and "the " are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including ", "includes ", "having ", "has ", "with ", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising. " The term "about " or "approximately " means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about " can mean within 1 or more than 1 standard deviation, per the practice in the art.Alternatively, "about " can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value or range. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about " meaning within an acceptable error range for the particular value should be assumed.

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

As used herein, "antioxidants " include ascorbic acid, ascorbyl palmitate, tocopherol, oxidized species such as metabisulfite, bisulfite, sulfite, etc. (these molecules act as oxygen scavengers if oxygen is the oxidizing agent involved), reducing agents (reduce the oxidized molecule to be protected, they may be preferentially oxidized, as well) and chain terminators. Methionine is preferentially oxidized and thus protects ILs from oxidation. This is one of the mechanisms used by antioxidants. All these are considered "antioxidants" because they are 7 Atty Docket: 348356.01402 molecules that contribute to protect other molecules from oxidation either by oxygen or by other oxidizing agents via different mechanisms. In this sense methionine is an antioxidant. In summary, methionine may work as an oxygen scavenger or it may be preferentially oxidized by other oxidizing agents or may reduce the oxidized forms of ILs. In all cases it would be considered an antioxidant in chemistry and in pharmaceutical technology. This is different from the biological and medical concept of antioxidant, which mostly refers to "chain terminators", i.e. molecules that react with free radicals to stop a chain reaction in biological systems and protect cells or the food chemistry concept that mostly refers to protection against oxygen damage or auto-oxidation.

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

As used herein, the term "cell " includes prokaryotic and eukaryotic cells. In one embodiment, a cell of the invention is a bacterial cell. In another embodiment, a cell of the invention is a fungal cell, such as a yeast cell. In another embodiment, a cell of the invention is a vertebrate cell, e.g., an avian or mammalian cell. In a preferred embodiment, a cell of the invention 2.0 is a murine or human cell. As used herein, the term "engineered " (as in an engineered cell) refers to a. cell into which a nucleic acid molecule e.g., encoding an IL-2 protein (e.g., a spliced and/or unspliced form of IL-2) or fragments thereof, has been introduced.

As used herein, the terms "comprising, " "comprise " or "comprised, " and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements —or, as appropriate, equivalents thereof —and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.8 Atty Docket: 348356.01402 Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

As used herein, the term human recombinant IL-X (rhIL-X) or simply IL-X (X can take the values 2, 4, 7, 9, 15, 18 or 21) means a protein produced by an organism that has been transfected with the DNA sequence described below or a non-extensive modification of it.

Atty Docket: 348356.01402 As used herein, the term "kit " refers to any delivery system for delivering materials. Inclusive of the term "kits " are kits for both research and clinical applications. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery ׳ of reaction reagents (e.g., cytokines, oligonucleotides, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials. As used herein, the term "fragmented kit " refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intendedrecipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides or liposomes. The term "fragmented kit " is intended to encompass kits containing Analyte specific reagents (ASRs) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a subportionof the total kit components are included in the term "fragmented kit. " In contrast, a "combined kit " refers to a delivery system containing all of the components of a reaction assay in a single container (e.g,, in a single box housing each of the desired components). The term "kit " includes both fragmented and combined kits.

As used in this specification and the appended claims, the term "or " is generally employed 2.0 in its sense including "and/or " unless the content clearly dictates otherwise.

The term "percent sequence identity " or having "a. sequence identity " refers to the degree of identity between any given query' sequence and a subject sequence.

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

Atty Docket: 348356.01402 The term "polynucleotide " is a chain of nucleotides, also known as a "nucleic acid ". As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art and include both naturally occurring and synthetic nucleic acids.

The terms "peptide, " "polypeptide, " and "protein " are used interchangeably, and refer to acompound comprised of ammo acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides " include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs,fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The peptides provided herein for use in the described&, y ) .־ AtU BakkAR/Gss 1A ، lit 1.*.$) C As used herein, "stable " or "highly stable " refers to the biological activity of the molecule.

The term "transfected " or "transformed " or "transduced " means to a process by which2.0 exogenous nucleic acid is transferred or introduced into the host cell. A "transfected " or "transformed " or "transduced " cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The transfected/transformed/transduced cell includes the primary subject cell and its progeny.

"Variant " as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the 11 Atty Docket: 348356.01402 reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant or can be a variant that is not known to occur naturally. Non- naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed, all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1,2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the 2.0 breadth of the range.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph of a. gel demonstrating the binding of interleukins 2, 4, 7, 9, 15, and 21, to the common gamma. chain (yc) (or CD 13 2) receptor.

DETAILED DESCRIPTION Embodiments are directed to highly stable solutions of recombinant non-glycosylatedinterleukins of the common y-chain family and the methods of producing thereof.

The disclosure summarized herein includes a series of formats and formulations that enable the long-term storage of interleukin 2, 7, 15, and 21 in liquid solution. These molecules are difficult Atty Docket: 348356.01402 to stabilize in solution because of their tendencies toward adsorption, aggregation, oligomerization and oxidation. The formulation of these molecules in liquid solutions that enable their integrity - as determined by their ability to retain their critical quality attributes and remain stable at 2 --- 8°C for several months -- facilitates their use as reagents and excipients in the manufacture of cell- based medicines applicable across a range of indications. For example, these formulations enable the delivery of these large molecules to lymphocyte cultures that form the basis of certain adoptive cell therapies without the need to reconstitute lyophilized materials. Furthermore, these molecules in the formulations described herein, when packaged in certain single-use containers that enable the direct incorporation of these molecules into a given cell therapy manufacturing process, enable the sterile integration of critical reagents, thereby simplifying and streamlining erstwhile complex unit operations, while mitigating the risk of a potential contamination.

Interleukin 2 IL-2, discovered more than 30 years ago in supernatants of activated T cells, is mainly produced by CD41 and CD81 T cells, and to a lesser extent by activated. DCs, NK and NK T (NKT) cells. The IL-2 receptor (IL-2R) consists of 3 subunits: the ligand-specific a. chain IL-2Ra (CD25), the P-cham IL-2RP (CD122, which is also part of the IL-15R complex), and the common yc, also called IL-2R% (CD 132). All 3 subunits are required for the assembly of the high-affinity IL-2R. On T-cell activation, IL-2Ra is rapidly induced and participates in formation of a. high- affinity quaternary complex, winch activates multiple signal transduction pathways. IL-2 is essential for the development of Treg cells. IL-2 also acts as a B-cell growth factor, stimulates antibody synthesis, and promotes proliferation and differentiation of NK cells to increase their cytolytic functions. Recombinant human IL-2 is used in immunotherapy for cancer and AIDS associated with HIV. Anti-IL-2Ra inhibits the immune response in patients with autoimmune diseases and prevents rejection of transplanted organs.

IL-2 (,Aldesleukin) Protein Sequence, SEQ ID NO: 1: I PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKATELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET101 TFMCEYADET ATIVEFLNRW ITFSQSIIST LTInterleukin 7 Atty Docket: 348356.01402 Interleukin-7 (IL-7; RefSeq NM000880; UniProtKB - P13232 (IL7 HUMAN)) is one of the members of the IL-2 superfamily. The IL-2 superfamily includes IL-2, IL-4, IL-7, IL-9, IL- and IL-21. It binds to receptors with a common y chain subunit. In addition to a common y chain subunit, the receptor for IL-7 (IL-7R) requires an IL-7Ra chain for binding to takeplace. Due to the frequency of the common y chain subunit, the presence of the IL-7 receptor a chain is a. better identifier for when IL-7 will actually bind to a. receptor. IL-7-receptor binding results in phosphorylation of tyrosine residues on the receptor. This leads to activation of JAKI or JAK3 depending on the cell type, which later activates many downstream signaling pathways including STAT5a/b, PB Kinase, and SRC kinases. It is well-known that IL-7 playsa critical role in the development of B-cells and T-cells.

IL-7, also known as pre-B-cell growth factor or lymphopoietin-1 , is a homeostatic cytokine. 40 The IL-7R is present on most T cells, progenitors of B cells, and bone marrow macrophages; it consists of the IL-7Ra (CD 127) chain and the common yc (CD 132)- Because yc is ubiquitously expressed on lymphocytes, IL-7 responses are determined by the expression of IL- TRa, which is shared with thymic stromal lymphopoietin (TSLP) receptor. IL-7 signaling contributes to survival and proliferation of thymocytes and development of naive and memory B and T cells, mature T cells, and NK cells. Studies of IL-7 and IL-7Ra knockout mice have shown that IL-7 is important for homeostatic T-cell and B-cell development. 41 IL-7 or reagents that block IL-7 signaling might be used to treat patients with HIV-associated immunodeficiency and immunodeficiency secondary' to chemotherapy, autoimmune diseases, and lymphoid malignancies.

IL-7 Protein Sequence, SEQ ID NO: 2: 2.5 1101151 MDCDIEGKDG KQYESVLM.VS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL KEQKKLNDLC FLKRLLQE1K TCWNKILMGT Interieukm-15 Interleukin-15 (IL-15; RefSeq NMJ72175, UniProt/Swiss-ProtP40933) is an important cytokine for the development, proliferation, and activation of effector NK cells and CD8+ memory T cells. IL-15 binds to the IL-15 receptor a (IL-15Ra) and is presented in trans to the IL-2/IL- Atty Docket: 348356.01402 receptor - common ■y chain (IL-15Ryc) complex on effector cells. IL-15 and IL-2 share binding to the IL-15Ryc, and signal through STATS and STATS pathways. However, IL- 2 also supports maintenance of CD4+ CD25+ FoxP3 + regulatory T (Treg) cells and induces ceil death of activated CD8+ T cells. These effects may limit the therapeutic activity of IL-2 against tumors. IL-15 does not share these immunosuppressive activities with IL-2. .Additionally, IL-15 is the only cytokine known to provide anti-apoptotic signaling to effector CD8+ T cells. IL-15, either administered alone or as a complex with the IL-15Ra, exhibits potent anti-tumor activities against well- established solid tumors in experimental animal models and, thus, has been identified as one of the most promising immunotherapeutic drugs that could, potentially cure cancer.

IL-15 is structurally homologous to IL-2 and was discovered for its ability' to induce T-cell proliferation like IL-2. Many of the biological actions attributed to IL-2 can also be induced by IL- 15. The IL-15R consists of the IL-15Ra chain, the IL-2Rp chain, and the common yc. IL-15 is produced by nonimmune cells (keratinocytes and skeletal muscle cells) and immune cells (monocytes and activated CD41 T cells) in response to signals that induce innate immunity. Although IL-15 shares some functions with IL-2, such as activation of T cells, stimulation of NK- cell proliferation, and cytolytic activity, differences in their biological functions have been identified on the basis of differences observed between phenotypes of IL-2 and IL-15 knockout mice.

IL-15 Protein Sequence, SEQ ID NO: 3:1MNWNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV 51ISLESGDASI HDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF101LQ SFVHIVQM FINTSInterleukin 21 IL-21 is produced by T cells, NKT cells, and the TH17 subset of CD41 T cells. The receptor for IL-21 is expressed on various cells, indicating a broad spectrum of action. IL-21 affects B-cell functions by regulating antibody isotype balance, proliferation, apoptosis, and differentiation into plasma cells. Cytotoxic activity and proliferation of CD81 T cells, NK cells, and NKT cells increase on stimulation with IL-21. IL-21 has been tested as an anticancer drug, and first clinical trial results are promising by slowing down tumor progression in metastatic melanoma. In contrast Atty Docket: 348356.01402 with its anticancer effects, IL-21 also contributes to inflammation in several disorders, as expected for a TH17-related cytokine.

IL-21 Protein Sequence, SEQ ID NO: 4:1MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ51KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK101 KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DSExpression of the Interleukins In certain embodiments the interleukins embodied here are encoded by an expression vector. The term "expression vector " refers to a. vector containing a. nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, when the transcription product is an mRNA molecule, this is in turn translated into a protein, polypeptide, or peptide.

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

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

Methylotrophic yeasts have two major features: (1) they are capable of growing to high cell densities even in unsophisticated fermentation process; (2) their high demand for methanol- oxidizing enzymes endows them with very strong and strictly regulated promoters. These features make it possible that methylotrophic yeasts can be used not only in the process development for the commercial production of feed protein (single cell protein) but also as production systems for recombinant proteins. The widely used. P. pastoris and H. polymorpha Atty Docket: 348356.01402 have different genetics in alcohol oxidases expression; P. pastoris expresses AOXI and AOXwhile H polymorpha only expresses MOX. Besides P pastoris and H. polymorpha, P methanolica and C. boidinii are also used as expression systems.

Some points should be considered when P pastoris is used to clone and expressheterologous proteins. These include the selection of host strain, the choice of promoter, transcription terminator (TT), markers combination and the application for either intracellular or secreted expression.

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

Vectors: The promoters for protein expression in P. pastoris include inducible promoters (AOXI, FLD1, ADH1, GUT1, etc.) and constitutive promoters (GAP, TEF1, etc.). For P.pastoris, HIS4 (auxotrophic markers) and zeocin resistance (dominant markers) are the most popularly markers used. The heterologous proteins can be intracellular or secreted expression. P.2.0 pastoris has the ability to secrete high titers of proteins into culture media. The prominently used secretion signals are derived from P. pastoris endogenous acid phosphatase (PHOI), 8. cerevisiae a-mating factor (a -MF) and & cerevisiae invertase (SUC2).

Polynucleotides: Nucleic acids can, for example, encode the amino acid sequence of the IL-2, IL-7, IL-15, or IL-21 with at least one or more conservative ammo acid substitutions.

Atty Docket: 348356.01402 The nucleic acids of the disclosure can, for example, encode functional variants whichalso include extensions of the IL-2, IL-7, IL-15, or IL-21 protein. For example, a functional variant of the IL-2, IL-7, IL-15, or IL-21 protein can include 1, 2, 3, 4 and 5 additional ammo acids from either the N-termmai or C-termmal end of IL-2, IL-7, IL-15, or IL-21 protein.

In some embodiments, the nucleic acids encode a mammalian IL-2, IL-7, IL-15, or IL-20 protein. In certain embodiments, the IL-2, IL-7, IL-15, or IL-21 protein can be murine, porcine, ovine, bovine, human, or combinations thereof.

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

In certain embodiments, the nucleic acid sequence encodes a polypeptide comprising asequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 3 or 4, Atty Docket: 348356.01402 In some embodiments, the polynucleotides provided herein encoding one or more interleukin proteins include codon-optimized sequences. As used herein, the term "codon- optimized " means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, orcoding sequence by choosing different codons without altering the amino acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can increase protein expression levels (Gustafsson etal., Codon bias and heterologous protein expression. 2004,Trends Biotechnol 22: 346-53) of the encoded proteins, for example, and provide other advantages. Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of A, G, C, U nucleotides, mRNA secondary structures, cis-regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructingartificial DNA segments. 2006, BMC Bioinformatics 7:285).

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

In some embodiments, the nucleotide sequence of any region of an RNA or DNAsequence embodied herein may be codon optimized. In certain embodiments, the primary cDNA template may include reducing the occurrence or frequency of appearance of certain nucleotides in the template strand. For example, the occurrence of a nucleotide in a template may be increased or reduced to a. level above or below 25% of said nucleotides in the template. In further examples, the occurrence of a. nucleotide in a template may be increased or reduced to a level Atty Docket: 348356.01402 above or below 20% of said nucleotides in the template. In some examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 16% of said nucleotides in the template. The occurrence of a nucleotide in a template may be increased or reduced to a level above or below 15% and may be increased or reduced to a level above or below 12% of said nucleotides in the template.

In certain embodiments, the polynucleotides encoding the IL-2, IL-7, IL-15, or IL-proteins can comprise one or more chemically modified nucleotides. Examples of nucleic acid monomers include non-natural, modified, and chemically modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the baseportion or the sugar portion. In nature, most polynucleotides comprise nucleotides that are "unmodified " or "natural " nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the 1' position. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates,half-life and/or expressed protein concentrations. RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and/or degradation pathways.

Examples of modified or chemically modified nucleotides include 5-hydroxycytidines, 5- alkylcytidines, 5-h y dr oxya iky !cytidines, 5-carboxy cytidines, 5-formylcytidines, 5-2.0 alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N4-alkylcytidines, N4- aminocytidines, N4-acetyIcytidines, and N4, N4-dialk.ylcytidines.

Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5- methoxy cytidine, 5-propynyl cytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4, N4-dimethyIcytidine.

Examples of modified or chemically modified nucleotides include 5-hydroxyuridines, 5- alkyluridines, 5-hydroxyalkyl uridines, 5-carboxyuridines, 5-carboxyalkylesteruridines, 5- formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thiouridines, and 6- alkyluridines.20 Atty Docket: 348356.01402 Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5- methyluridine, 5-hydroxymethyluridme, 5-carboxyuridine, 5-carboxymethylesteruridine, 5- formyl uridine, 5-methoxyuridine (also referred to herein as "SMeOU"), 5-propynyluridine, 5- bromouridine, 5-fluoroundine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

Examples of modified or chemically-modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-th1ouridine, 5- carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridme, l-methyl-3 -(3-ammo-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenoundine, 5-carboxymethyluridine, 5- methyldihydrouridine, 5-taurmomethy !uridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudoundine, 2-thio-2'O-methyluridine, and 3,2'- O-dimethyluridine.

Examples of modified or chemically-modified nucleotides include N6-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8- bromoadenosine, 2-methylth1o-N 6-methyladenos1ne, N״-isopentenyladenosine, 2-methylthio-N 6-isopentenyladenosine, N6-(c1s-hydroxyisopentenyl)adenosine, 2-methylthio-N 6-(cx ؟- hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyl- adenosine, N6-methyl-N 6-threonylcarbamoyl-adenosine, 2-methylthio-N ״-threonylcarbamoyl- adenosine, N6,N6-dimethyladenosine, N6־hydroxynorvalylcarbamoyladenosine, 2-methylthio-N 6- hydroxynorvalylcarbamoyl-adenosine, N^-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2!-0-methyl-adenosine, N6,2'-O-dimethyl- adenosine, N6,N6,2'-O-trimethyI-adenos1ne, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-am1no-N 6-methyl-purine, 1 -thio-adenosine, 2-F-ara-adenosine, 2'-F-adenosine, 2!-OH-ara- adenosine, and N6-(! 9-amino-pentaoxanonadecy])-adenosine.

Examples of modified or chemically modified nucleotides include N1 -alkylguanosines,Nz-alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8- bromoguanosines, O6-alkylguanosines, xanthosines, inosines, and N1-alkylinosines.

Examples of modified or chemically modified nucleotides include N1 -methylguanosine, N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, ()0-methylguanosine, xanthosine, inosine, and N1-methylinosine.21 Atty Docket: 348356.01402 Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

Examples of modified and chemically modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-0-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.

Examples of modified and chemically modified nucleotide monomers include 3'-end stabilized nucleotides, 3'-glyceryl nucleotides, 3!-inverted abasic nucleotides, and 3'-1nverted thymidine.

Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'- methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2!-deoxy-2 !-fluoro nucleotides, and 2'- O-methyl nucleotides. In an exemplar} ׳ embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

Examples of modified and chemically modified nucleotide monomers include 2',4'- constrained 2’-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs.

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.

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

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

Examples of modified and chemically modified nucleotide monomers include 2'-O- aminopropyl substituted nucleotides. ־ליל Atty Docket: 348356.01402 Examples of modified and chemically modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2!-K a 2'-OR, a ?'-halogen, a 2؛-SR, or a 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

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

Atty Docket: 348356.01402 Atty Docket: 348356.01402 embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar Polypeptides: The polypeptides embodied herein, can be modified to include one ormore non-natural amino acids. As used herein, an "unnatural ammo acid, " "non-natural ", "modified amino acid " or "chemically modified amino acid " refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural ammo acids have side chain groups that distinguish them from the natural Atty Docket: 348356.01402 ammo acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural ammo acids may have an extended backbone such as beta-amino acids.

Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine,homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthy!)alanine, a 3-methyl-phenylalanine, an 0-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GIcNAcp-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L- phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, aphosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L- phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid: an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl,ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a. photoactivatable cross-linker; a spin-labeled amino acid; a. fluorescent amino acid; an ammo acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding ammo.0 acid; a metal-containing ammo acid; a radioactive amino acid; a. photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid, an amino acid with an elongated side chain, an amino acidcontaining a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like, a carbon-linked sugar-containing ammo acid; a. redox-active amino acid, an a-hydroxy containing acid; an amino thio acid containing amino acid; an a,a disubstituted ammo acid; a. p- ammo acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or moreunnatural amino acids and/or one or more natural amino acids.26 Atty Docket: 348356.01402 Any one or more of SEQ ID NOs: 1, 2, 3, or 4 can be modified to include naturally occurring and synthetic a, P, ׳y, and 5 ammo acids. Amino acids found in proteins, i.e. gly cine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine andhistidine. Alternatively, the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucmyl, prolinyl, phenylalaninyl, tryptophanyl, methionmyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, p- alanyl, P-valinyL P-leucinyl, P-isoleucinyl, P-prolinyl, P-phenylalaninyl, P-tryptophanyl, P״ methioninyl, P-glycinyl, P-serinyl, P-threoninyl, P-cysteinyl, P-tyrosinyl, P-asparaginyl, P־glutaminyl, P-aspartoyl, P-glutaroyl, P-lysinyl, P-argininyl or P-histidinyl. When the term amino acid is used, it is considered to be a specific and independent disclosure of each of the esters of a, P, y, and 5 glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L-configurations.

Pharmaceutical Compositions Atty Docket: 348356.01402 Atty Docket: 348356.01402 The following examples further illustrate the invention. These examples are not intended to limit the invention in any manner.

EXAMPLES Example 1: Methods of Producing Stable Formulations of ILs of the Gamma Family Recombinant unglycosylated IL-2 has been approved as a medicine for cancer some decades ago under the commercial name Proleukin® and is now being investigated for medicinal use either as pharmaceutical for other indications or an ancillary material for immunotherapies for either cancer or other diseases such as graft versus host disease.

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

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

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

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

Among bacteria non-pathogenic strains of Escherichia coli are preferred. Escherichia coli B serves as a research model and also for protein expression in life science laboratories and. in the 70 Atty Docket: 348356.01402 biotech industry. Characteristics such as protease deficiency, low acetate production at a high level of glucose, and enhanced permeability (probably due to a simple cell surface) make E. coli B a desirable host to produce genetically engineered proteins. Differences between B strains and Kinclude the absence of flagellar component genes, the DMA cytosine methylase dem, and ompT in BL21(DE3). B strains may have an additional type II secretion system not found in K12.BL2I(DE3) also carries a DE3 recombinant phage harboring the T7 RM A polymerase gene that can direct high-level expression of cloned genes under the control of the T7 promoter. Typical E. coli strains used for recombinant protein expression are: BL21 (a B E. coli strain that protects target protein from ion and ompT proteases) and their derivatives such as: Lysogenic DES (based on T7 polymerase), pLysS, pLysE (express T7 lysozyme reducing basal expression of target genes),Origami (allows disulfide bond formation in E. coli cytoplasm), Rosetta (enhances expression of proteins that contain codons rarely used in E. coli). Similar versions exist under the K12 E. coli genetic background. Typical plasmid vectors for high expression of recombinant proteins in E. coli are: pET series based on pBR322 origin and T7/lac promoters; pBad with araBAD promoter and pUC origin; pGEX with tac promoter and pBR322 origin also. Combination of fusion tags sequences, protease cleavage sites, selection markers and strain compatibility are source for the most usual list of high expression plasmid variants. In certain embodiments, the cell is an E. coli cell. Different E. coli strains can be transfected in order to obtain optimal interleukin production.

Producing bacteria can be cultured in a suitable growth medium. For example, the medium may contain each 9 liters, 216 gr of Yeast Extract, 108 gr of Soy Peptone, 113 of gr K2HPO4, 20,gr KH2PO4, 36 ml of Glycerol and 4 ml of Antifoam (2% v/v). Fermentation conditions may be: Temperature: 37OC ± 0.5o C, agitation: 350 rpm ± 10 rpm, air flow: 9 L/min ± l L/min, pO2: set point 40%, pH between 6.95 and 7.5. After this, a. feeding procedure should be followed. For example, feeding with a glucose solution 40% p/v by drip, to maintain a. concentration of 0.1 %. Once the OD600 reaches 5 to 10, an appropriated inducer such as Isoprop1l-P ־D-1 - tiogalaetopiranosido (IPTG) should be added to reach an operative concentration. At this point, the feeding with Glucose may be reduced to keep a glucose concentration of about 0.01%. Fermentation can be stop usually about 18 to 24 hours after inoculation. After fermentation, bacteria, can be concentrated 5 to 7 times by centrifugation or tangential filtration and processed Atty Docket: 348356.01402 immediately or preserved at 2-8 °C (no more than 24 hours) or preserved at -20°C (for more than hours).

After culture, interleukins are inside the bacteria, predominantly in the form aggregates named inclusion bodies (1b). These Ib can be isolated by disruption of bacteria (for example by sonication). For this, bacteria could be suspended in purified water. After this, the suspension could be circulated 2-4 times for the disruptor at a pressure of about 1400 bar. The lysate should be processed immediately or preserved at -20°C. The Ib are separated of other components of the lysate by centrifugation or tangential filtration and washed. The Ib preparation should be stored at -20°C until further processing. The Ib are then suspended in a suitable buffer and submitted to a re-folding step in a regulated redox potential.

After refolding interleukins are submitted to different chromatographic and diafiltrations to get a concentrated solution of purified interleukins. Interleukins are then formulated and packaged into ready-to-use bags, pre-filled syringes, vials, ampoules or other suitable primary packaging materials for sterile solutions. Surprisingly, it was discovered that recombinant interleukins may be easily stabilized in solution using buffers of carboxylic acids at pH at least 0.units from their isoelectric point, isotonized with non-ionic osmolytes with the addition of methionine or other amino acids. In some cases surfactants are needed, to prevent them from forming aggregates, oligomers or adsorbing onto the walls of the container.

The isoelectric point is the value of pH in which the protein does not show' any net electrostatic charge. The isoelectric point of proteins may be estimated using Vector NTI 10.3.0. 2006 © Invitrogen Corporation. The results obtained for some members of the Gamma. family are: Interleukin Isoelectric point (pH) IL-2 7.02 IL-7 8.72 IL- 15 4.52 Atty Docket: 348356.01402 IL-21 9.42 It would be reasonable to expect a higher stability of these or other proteins to denaturation and aggregation, when formulated below or above their isoelectric point, but the surprising event is that all other degradation mechanisms, such as deamidations and oxidations, are minimized in these conditions as well. Methionine seems to stabilize solutions of some particularly oxidable members like IL-2, IL-7 and IL-15 and particularly when packaged in materials permeable to oxygen like plastic bags. Surfactants may be added to stabilize them further against aggregation, oligomerization and adsorption onto the container walls.

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

Example 2: IL-2 Liquid Formulation A solution composition for this interleukin is the following:Anhydrous disodium phosphate 1.2 mg/mL,Anhydrous monosodium phosphate 0,2 mg/mL, Mannitol 50 mg/mL, Atty Docket: 348356.01402 SDS 1 mg/mL, This composition is filled into pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume between 1 and 10 ml/bag: 1 MIU/'Bag; 2 MIU/Bag; or 15 MIU/Bag Another liquid formulation for IL-2 is the following: Anhydrous disodium phosphate 1.2 mg/mL,Anhydrous monosodium phosphate 0.2 mg/mL, Mannitol 50 mg/mL, SDS 1 mg/mL, Methionine 5 mg/mL This composition is filled into pharmaceutically acceptable plastic bags containing the following amounts of IL-2 in a volume between 1 and 10 ml/bag: 1 MIU/'Bag; 2 MIU/Bag; or 15 MIU/Bag The pH value of both solutions is 7.5 (more than 0.2 units above the isoelectric point of IL-2: pH 7.02) Example 2: IL-7 Liquid Formulation IL-7 is produced by recombinant DNA technology using a genetically engineered E. coli strain BL21 Rosetta. DE3 pLysS containing an analog of the human interleukin-7 coding region gen, inserted in a pET9a (TetR) plasmid vector at Ndel-BamTII restriction sites. It contains three disulfide bonds that are essential for its biological activity.

Genetic engineering techniques were used to modify the human TL-7 gene, and the resulting expression clone encodes a modified human interleukin-7. This modified gene has 1codons. This recombinant form differs from native interleukin-7 in the following ways: a) IL-7 is not glycosylated because it is derived from E. coli; b) the molecule has an N-termmal Methionine.

Expression System and Cloning Description Process An inducible eukaryotic expression system for recombinant expression of hIL-7 was developed using an Escherichia, coli strain as a host.

Atty Docket: 348356.01402 Among different E. coli BL21(DE3) strains tested, Rosetta2(DE3 ) pLysS strain was selected due to the fact that a high, tight and stable recombinant expression of rhIL-7 was observed. The strain supplies tRNAs for rare codons, providing for universal translation where it would otherwise be limited by the codon usage of E. coli. Furthermore, the strain carries a plasmid that encodes the T7 lysozyme gene, which is a natural inhibitor of T7 RNA polymerase that serves to repress basal expression of target genes under the control of the T7 promoter.

The pET9a + expression vector was used for cloning at Ndel and BamHI restriction sites. Strain and vector combination let a tight regulation of recombinant expression based on the very ׳־ well know' T7 promoter system which only is turned on when the T7 RNA polymerase is present.

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

The molecular construct and gene w׳ere verified by nucleotide sequencing and protein identity' was stated by SDS-PAGE (molecular size) and by Western Blot (specific antibody detection).

Table 1 ComponentAmount Unit IL-70.01 mg Sodium citrate dihydratemg Citric acid anhydrous0.56 mg Methioninemg Disodium Edetate0.0146 mg Water for Injectionq.s. 1 mL The pH value of this formulation is 6 ± 0.5 (more than 0.2 units below' the isoelectric point of IL-7: pl 8.72) Atty Docket: 348356.01402 Table 2 Time (months)Biological activity (MIU/mL) Chromatographic purity (% peak area) 01.8499.1 11.4799.3 31.4899.4 61.699.3 91.499.2 Example 3 - Liquid Formulation of IL-15 Genetic development of recombinant clone for th e Expression of JL-15.

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

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

Brief description of the expression system An inducible eukaryotic expression system for recombinant expression of hIL-1 5 was developed using an Escherichia, coli strain as a host. Among different tested E. coll BL21 strains, Atty Docket: 348356.01402 the AI strain was selected. It was due to the fact that a high, tight and stable recombinant expression of rhIL-15 was observed. The strain carries a chromosomal insertion of a cassette containing the T7 RINA polymerase (T7 RNAP) gene in the araB locus, allowing expression of T7 RNAP to be regulated by the araB AD promoter. The expression of this polymerase is induced by Arabinose.

The pET9a + expression vector was used for cloning at Ndel and BamHI restriction sites. Strain and vector combination let a tight regulation of recombinant expression based on the very well know T7 promoter system which only is turned on when the T7 RNA polymerase is present.

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

Table 3 Component Amount Unit Interleukin 15 0.01 mg Sodium CitrateDihydratemg Citric Acidanhydrous0.56 mg Methionine 5 mg Polysorbate 80 0.005 % Atty Docket: 348356.01402 Water forq.s. 1 mLinjectionThe pH value of this formulation is 6 ± 0.5 (more than 0.2 units above the isoelectric point of IL-15: pl 4.52) Table 4 Time (months)Biological activity(MIU/mL) 0 0.10 1 0.12 3 0.11 6 0.10 9 0.09 12 0.10 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 mouse B lymphocytes ATCC® TIB- 239; also known as 2E8 cells). Biological activity and Specific activity of IL-7 raw material is estimated assuming a theoretical biological activity of 1.5x1 08 lU/mg compared against the MIBSC WHO Reference Interleukin- 7 using a parallel line assay as statistical model.

Example 4. INTERLEUKIN 21 Liquid FORMULATION Sodium Citrate dihydrate 4.05 g/L Citric Acid anhydrous Mannitol 1.20 g/L 41.30 g/L37 Atty Docket: 348356.01402 IL-21 pH 0.30 mg/mL .0 - 5.6 The pH of this formulation is more than 0.2 units below the isoelectric point of IL-21 (pi 9.42).

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

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

Primary' protein structure contains the following amino acids sequence: 1. MQDRHMERMR QLIDTVDQLK NYVNDLVPEF LP APED ATTN CEWSAFSCFQ 51 KAQLKSANTG NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK101 KPPKEFLERF KSLLQKMIHQ HLSSRTHGSE DSInitial methionine (M) is needed for the recombinant protein expression in E. coli.Human IL-21 gene source Three nucleotide sequence synthesis (cassettes) containing the human IL-21 codingregion were ordered (Genscript, NJ, USA): Table 5 NAME ML-21 SEQ. DESC RIPTION Wild type version of hlL-21 gene coding region iop-hIL-21The first 20 codons (60 nucleotides) of the hlL-21 gene were optimized forE. coli expression 0p-hIL-21Fully optimized version for E. coli expression of the ML-coding region gene Atty Docket: 348356.01402 The synthetic genes do not include nucleotides coding for the signal peptide region of the hIL-21 gene. All cassettes were sent as an insert cloned into a pUC57 cloning vector (into Ndel and BamHI restriction sites). Note: 7X11 cassettes include an extra stop codon and the best option for E. coli (TAA stop codon) in the first position (Vyas W et al., BiolechnolProg. 2012 Mar- Apr;28(2):497-507. doi: 10.1002/btpr.746. Epub 2011 Dec 9) that determined the presence of an extra tryptophan due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre) stop codon.

E. coli Expression Strain GenerationOnce IL-21 coding regions and regulatory ׳־ portions of each expression plasmid were confirmed, the WT-hIL-21 , iop-hIL-21 , and the op-hIL-21 plasmids were used for next steps. Fifteen nanograms of each supercoil expression plasmid coming from the above mentioned clones were used to transform a BL21 Al E. coli strain.

BL21-AI Genotype: F- ompT hsdSB (rB-mB-) gal dem araB::T7RN/XP-tetA The BL21-AI™ strain is an E. coli B/r strain and does not contain the ion protease. It is also deficient in the outer membrane protease, OmpT. The lack of these proteases reduces degradation of heterologous proteins expressed in this strain. The strain carries a. chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene in the araB locus, allowing expression of T7 RNAP to be regulated by the araB AD promoter. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline. hIL21 Protein Expression and Protein Identity Verification Four different clones from each hIL-21 gene version ("WT": wild type hIL-21 gene; "iop-hIL21 ": first 20 codons optimized and "0p-hIL21 ": fully codon optimized version) were tested for protein expression. The clone "D7" was selected to make the Research Cell Bank and further development steps. See, FIG. 1.

"No induction " means bacteria without induction. The recombinant system (plasmid +E. coli strain) is and inducible genetic system that required an inducer (L-arabinose in this case) to avoid Atty Docket: 348356.01402 genetic repression. Non-induced bacteria are expected do not shown recombinant expression (no recombinant protein band in the SDS-PAGE). The induced bacteria should express the protein of interest (i.e. IL-21 band in the SDS-PAGE). The numbers and/or letters indicate Cion ID (i.e."2.2-5"; "2.2.-6";"D7"...) "MW" states: molecular weight marker.SDS-PAGE 18%. Coomassie Blue stain - Reducing Condition.Induction: Overnight Induction (0.2% L-Arabinose); Clone ID: "2.2-5"; "2.2-6"; "D7" and "D8"; IL-21 STD: rhIL-21 Standard The protein identity was verified by Western Blot analysis using a specific rabbitpolyclonal antibody Ant1-hIL21 (AB 154767; Abeam). Also Blast X (Search protein database using a translated nucleotide query) search results, showed 100% of identity with the hIL-amino acid sequence. ( b،s1n )؛؛ Specifications zero) iiiiiiliiiifi 4236 Pontx. SDS״Page (non،reduvUtg fffffffffffffff^^Not more than 3° a of total Not more than 3% of total impuritiesNot more tlian 3% of total impuritiesNot more ,Jian 3% of total impmitiesNotmore than 3% of total impuritiesNot more tlian 3% of total impuritiesNotmore tlian 3% of total impurities HPLC)Not mere than 5% of total0.62 % total impurities 0,67 % total impurities 0,44 % total impurities 0,82 0/’e total impurities 0,25 % total impurities 4,67/0 total impurities 1 rng/ml 0.98 rng/ml 0.99 mg/ml 0 95 mg/rnl 1.05 rng/ml 0.94 rng/ml Biological Actix ity _ L51־ ml34.100 U/ml 49,246.3 U/ml 33,502.5 U/ml 28,307.5 U/ml 22,4.58.7 U/ml 71,212.05 U/ml EC50: 29.34 ng/ml EC50: 19.9 ng/ml EC50: 29.55 ng/rnl EC50 33.56 ng/rnl EC50: 42.3 ng/ml EC50: 13.2 ng/mlffffffffffff^ff^J 00 U/mg 50,251 3 U/mg 33,840.9 U/rng 29,797.4 U/mg 23,640.7 U/mg 75,757 5 U/mg(*) As there is no international reference standard of IL-21, EC50 ״was adopted as a measure of biological activity. This parameter has a larger variance than the usual values of biological activity determined by a parallel-line regression. See text for further information.

Atty Docket: 348356.01402 Biological Activity - Specific activityThe biological activity of human IL-21 is determined by means of a proliferation assay based on the B9 cell line (Mouse B cell Hybridoma) obtained from Public Health England and quantified by a colorimetric method, using MTS.

Briefly, B9 cells were washed twice with Assay Medium and a cell suspension of 2.5x1 05cells/mL is prepared. In parallel, serial dilutions are made for IL-21 Standard and the Samples to be analyzed. Then, 100 pL/well of each dilution are dispensed in a microplate followed by 1،uL of the cell suspension. Plates are incubated for 48 hours at 37 °C and 5% CO2.

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

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

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

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

Claims (38)

Atty Docket: 348356.01402 What is claimed is:

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

2. The liquid formulation of claim I, wherein the formulation comprising IL-2 comprises from about 1 mg/ml to about 10 mg/ml antioxidants, from about 0.0001 to 4 mg/ml chelating agents, from about 0,01 mg/ml to about 10 mg/ml phosphates, from about 20 mg/ml to about 80 mg/ml sugar and/or sugar alcohols and from about 0.001 mg/ml to about 5 mg/ml surfactants.

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

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

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

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

7. The liquid formulation of any one of claims 1, 5 or 6, wherein the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL-7.43 Atty Docket: 348356.01402

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

9. The liquid formulation of claim 8, wherein the formulation comprising IL-15 comprises from about 3 mg/ml to about 8 mg/ml antioxidants, from about mg/ml to about 8 mg/ml buffering agent, from about 0.01 mg/ml to about mg/ml acid, and from about 0,0005 mg/ml to about 1 mg/ml surfactants.

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

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

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

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

14. The liquid formulation of any one of claims 2-13, wherein the antioxidants belong to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite.

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

16. The liquid formulation of claim 15, wherein the monosodium phosphates and disodium phosphates comprise monosodium phosphate anhydrous, monosodium 44 Atty Docket: 348356.01402 phosphate monohydrate, monosodium phosphate dihydrate, disodium phosphate anhydrous, disodium phosphate dihydrate and disodium phosphate dodecahydrate.

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

18. The liquid formulation of any one of claims 2-13, wherein the surfactants comprise polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, macrogol 15 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.

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

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

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

22. The formulation of claim 1, wherein the formulation comprises about Atty Docket: 348356.01402 0.001 milli- international units (MIU) to 15 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

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

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

25. The formulation of claim 1, wherein the formulation comprises about0.01 milli- international units (MIU) to 8 MILT per ml of IL-2, IL-7, IL-15, or IL-21.

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

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

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

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

30. The formulation of claim 1, wherein the formulation comprises about0.01 milli- international units (MIU) to 3.5 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

31. The formulation of claim 1, wherein the formulation comprises about Atty Docket: 348356.01402 0.01 milli- international units (MIU) to 3 MIU per ml of IL-2, IL-7, IL-15, or IL-21.

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

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

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

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

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

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

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