Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5418—IL-7
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5443—IL-15
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/55—IL-2
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2302—Interleukin-2 (IL-2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2307—Interleukin-7 (IL-7)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2315—Interleukin-15 (IL-15)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2321—Interleukin-21 (IL-21)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/84—Pichia

Definitions

• the disclosure relates to new pharmaceutical compositions comprising interleukins of the y-chain family, such as interleukins 2, 4, 7, 9, 15, and 21.
• 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.
• One aspect of the present invention relates to the formulation of stable aqueous solutions of recombinant interleukins of the common y - chain family by selecting excipients that prevent them from degrading while stored at 2 --- 8 °C for several months, while avoiding the use of human- or animal-derived carrier proteins such as bovine serum albumin or human serum albumin.
• human- or animal-derived carrier proteins such as bovine serum albumin or human serum albumin.
• 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 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 ammo acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-2 amino acid sequence (SEQ ID NO: 1).
• 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%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the wild-type IL-2 amino acid sequence (i.e, SEQ ID NO: 1 ).
• Embodiments also include IL-2 variants that preferentially stimulate Treg cells and comprise an ammo 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%, 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 wildtype IL-2 amino acid sequence (SEQ ID NO: 1).
• 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-7 variants.
• 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).
• SEQ ID NO: 3 amino acid sequence that has at least 60%
• the interleukin molecules herein include variants of the present disclosure comprising an ammo acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the IL-21 amino acid sequence (SEQ ID NO: 4).
• SEQ ID NO: 4 amino acid sequence
• a liquid formulation for ex vivo use comprises about 0.001 milliinternational units (MIL) to 20 MIL 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).
• MIL milliinternational units
• 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.
• 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 (.MU.') to about 20 MIU per ml of IL-2.
• the formulation comprising IL-7 comprises from about 1 mg/ml to about 10 mg/ml antioxidants, from about 1 mg/ml to about 10 mg/ml buffering agent, from about 0,001 mg/ml to about 5 mg/ml acid, and from about 0.0001 mg/ml to about 4 mg/ml chelating agents.
• the formulation comprising IL- 7 comprises from about 3 mg/ml to about 8 mg/ml antioxidants, from about 3 mg/ml to about 8 mg/ml buffering agent, from about 0.01 mg/ml to about 3 mg/ml acid, and from about 0.0001 mg/ml to about 0,5 mg/ml chelating agents.
• the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL- 7.
• 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.
• the formulation comprising IL-15 comprises from about 3 mg/ml to about 8 mg/ml antioxidants, from about 3 mg/ml to about 8 mg/ml buffering agent, from about 0.01 mg/ml to about 3 mg/ml acid, and from about 0.0005 mg/ml to about 1 mg/ml surfactants.
• the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL- 15.
• the formulation comprising IL-21 comprises from about 1 mg/ml to about 10 mg/ml buffering agent, from about 0.001 mg/ml to about 5 mg/ml acid, and from about 10 mg/ml to about 80 mg/ml sugar and/or sugar alcohols.
• the formulation comprising IL-21 comprises from about 2 mg/ml to about 9 mg/ml buffering agent, from about 0.01 mg/ml to about 3 mg/ml acid, and from about 30 mg/ml to about 60mg/ml sugar and/or sugar alcohols. In certain embodiments, the formulation comprises from about 0.001 mg/ml to about 5 mg/ml of IL-21.
• the antioxidants belong to the group formed by sodium metabisulfite, sodium sulfite, potassium metabisulfite and potassium sulfite.
• the phosphates comprise anhydrous monosodium phosphates and disodium phosphates.
• 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.
• the sugars comprise sucrose, trehalose and maltose and sugar alcohols comprise sorbitol, isomait, xylitol, maltitol, mannitol, erythritol, and lactitol or mixtures thereof.
• 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.
• the chelating agents comprise disodium edetate, monosodium edetate, trisodium edetate, tetrasodium edetate, sodium citrate, citric acid, tartaric acid, alanine, argmine, aspartic acid, asparagine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
• the acid comprises citric acid, tartaric acid, phosphoric acid, hydrochloric acid, nitric acid and sulfuric acid.
• 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.
• 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, IL-15, or IL-21.
• 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.
• MIU milli- international units
• the formulation comprises about 0.01 milli- international units (MIU) to 5 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 4 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3.5 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 3 MIU per ml of IL-2, IL-7, IL-15, or IL-21.
• MIU milli- international units
• the formulation comprises about 0.01 milli- international units (MIU) to 2 MIU per ml of IL-2, IL-7, IL- 15, or IL-21. In certain embodiments, the formulation comprises about 0.01 milli- international units (MIU) to 1 MIU per ml of IL-2, IL-7, IL-15, or IL-21. In certain embodiments, the formulation is comprised in a pharmaceutical composition or liquid formulation. In certain embodiments, the isoelectric point of IL-2, IL-7, IL-15, or IL-21 is between 3 to 10.
• 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).
• the cell is a bacterial cell, yeast cell, mammalian cell or cell-line.
• the cell isPichia pastoris or Chinese Hamster Ovary (CHO) cells.
• compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
• 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.
• amino acid includes the residues of the natural a-aniino 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.
• a-aniino acids e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Lys, He, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai
• Many types of amino acid residues are useful in the adipokine polypeptides, and the disclosure is not limited to natural, genetically-encoded ammo acids.
• ammo acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc,, and the reference cited therein. Another source of a wide array of ammo acid residues is provided by the website of RSP Amino Acids LLC.
• 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 molecules that contribute to protect other molecules from oxidation either by oxygen or by other oxidizing agents via different mechanisms.
• methionine In tins sense methionine is an antioxidant.
• 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.
• a cell of the invention includes prokaryotic and eukaryotic cells.
• a cell of the invention is a bacterial cell.
• a cell of the invention is a fungal cell, such as a yeast cell.
• a cell of the invention is a vertebrate cell, e.g., an avian or mammalian cell.
• a cell of the invention is a murine or human cell.
• engineered 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.
• 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.
• 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.
• Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a ceil or other biological system.
• a polynucleotide such as a gene, a cDNA, or an mRNA
• Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
• expression is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
• expression vector ' 1 or “vector ' 1 refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
• An expression vector comprises sufficient cw-acting elements for expression; other elements for expression can be supplied by the host cell or in an wtro expression system.
• Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained m liposomes) and viruses (e.g., ientiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
• vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
• 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.
• viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
• the term human recombinant IL-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.
• 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 deliver ⁇ ' of reaction reagents (e.g., cytokines, oligonucleotides, enzymes, etc.
• kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
• fragment kit refers to delivery systems comprising two or more separate containers that each contains a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides or liposomes.
• 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 subportion of the total kit components are included in the term “fragmented kit.”
• 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).
• kit includes both fragmented and combined kits.
• sequence identity refers to the degree of identity between any given query' sequence and a subject sequence.
• pharmaceutically acceptable refers 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.
• pharmaceutically acceptable carrier 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.
• 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.
• peptide refers to a compound comprised of ammo acid residues covalently linked by peptide bonds.
• a protein or peptide must contain at least two ammo acids, and no limitation is placed on the maximum number of ammo acids that can comprise a protein's or peptide's sequence.
• Polypeptides include any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds.
• 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 and claimed methods and compositions can be cyclic.
• stable or “highly stable” refers to the biological activity of the molecule.
• transfected or “transformed” or “transduced” means to a process by which 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 ammo acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
• a variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant or can be a variant that is not known to occur naturally. Non- naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.
• 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.
• description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
• 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 (ye) (or CD 132) receptor.
• Embodiments are directed to highly stable solutions of recombinant non-glycosylated interleukins 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 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 cellbased medicines applicable across a range of indications.
• 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.
• 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 réellewhile complex unit operations, while mitigating the risk of a potential contamination.
• IL-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-chain 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.
• IL-2Ra 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:
• Interleukin 7 Interleukin-7 (IL-7; RefSeq NM 000880; UniProtKB - P13232 (IL7 HUMAN)) is one of the members of the IL-2 superfamily.
• the IL-2 superfamily includes IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21. It binds to receptors with a common y chain subunit.
• the receptor for IL-7 (IL-7R) requires an IL-7Roc chain for binding to take place. Due to the frequency of the common y chain subunit, the presence of the IL-7 receptor a chain is a better identifier for when IL-7 will actually bind to a receptor.
• IL-7-receptor binding results in phosphorylation of tyrosine residues on the receptor. This leads to activation of JAK1 or JAK3 depending on the cell type, which later activates many downstream signaling pathways including STAT5a/b, PB Kinase, and SRC kinases. It is well-known that IL-7 plays a critical role in the development of B-cells and T-cells.
• 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- 7Ra, which is shared with thymic stromal lymphopoietin (TSLP) receptor.
• TSLP thymic stromal lymphopoietin
• 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.
• 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-l 5Ra) and is presented in trans to the IL-2/IL-15 receptor - common y chain (IL-15Ryc) complex on effector cells.
• IL- 15 and IL-2 share binding to the IL-15R yc, and signal through STAT3 and STATS pathways.
• IL- 2 also supports maintenance of CD4 + CD25 + FoxP3 + regulatory I' (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.
• 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-21 is produced by T cells, NKT cells, and the Till 7 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 with its anticancer effects, IL-21 also contributes to inflammation in several disorders, as expected for a TH17-reiated cytokine.
• the interleukins embodied here are encoded by an expression vector.
• 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.
• 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).
• the isolated cell is a bacterial cell, yeast cell, mammalian cell or cell-line.
• the isolated cell is Pichia pastoris or Chinese Hamster Ovary (CHO) cells.
• Pichia pastoris is a widely used protein expression host for the production of biopharmaceuticals and industrial enzymes.
• P pastoris belongs to methylotrophic yeasts, which share a common pathway to metabolize one-carbon compounds as carbon and energy sources.
• the species of methylotrophic yeasts, P pastoris (recently reclassified as Komagataella pastoris) and H. polymorpha (also named Pichiaangusta) have been widely employed and become a substantial workhorse for biotechnology including heterologous protein production.
• 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 methanoloxidizing enzymes endows them with very strong and strictly regulated promoters. These features make it possible that methylotrophic yeasts can be used not only in the process development for the commercial production of feed protein (single cell protein) but also as production systems for recombinant proteins.
• the widely used P. pastoris and H. polymorpha have different genetics in alcohol oxidases expression: P pastoris expresses AOX1 and AOX2 while H polymorpha only expresses MOX. Besides P pastoris and H. polymorpha, P methanolica and C. boidinii are also used as expression systems.
• P pastoris is used to clone and express heterologous proteins. These include the selection of host strain, the choice of promoter, transcription terminator (TT), markers combination and the application for either intracellular or secreted expression.
• TT transcription terminator
• Host strains The widely used commercial available strains are mainly 5 classes, wildtype strains (e.g. X-33), auxotrophic strains (e.g. GS 115, KM71), protease-deficient strains (e.g. SMD 1168), glyco-engineered strains (e.g. SuperManS) and some other strains.
• engineered P pastoris has been able to secrete recombinant proteins with uniform human N- linked glycans.
• glycosylation is one of the most common PTMs (post-translational protein modifications), which impacts protein folding, solubility, stability; trafficking, bioavailability, immunogenicity and functional activity;
• the use of engineered P. pastoris broadens the applications of microbial systems in antibody expression.
• the promoters for protein expression in P. pastoris include inducible promoters (A0X1, FLD1, ADH1, GUT1, etc.) and constitutive promoters (GAP, TEF1, etc.).
• HIS4 auxotrophic markers
• zeocin resistance zeocin resistance
• the heterologous proteins can be intracellular or secreted expression.
• P. pastoris has the ability to secrete high titers of proteins into culture media.
• the prominently used secretion signals are derived from P. pastoris endogenous acid phosphatase (PHO1), 8. cerevisiae a-mating factor (a -MF) and X. cerevisiae invertase (SUC2).
• Nucleic acids can, for example, encode the amino acid sequence of the IL-2, IL-7, IL-15, or IL-21 with at least one or more conservative amino acid substitutions.
• Conservative ammo acid substitutions are known in the art, and include ammo acid, substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid that has the same chemical or physical properties.
• the conservative amino acid substitution can be an acidic ammo acid, substituted for another acidic amino acid (e.g., Asp or Glu), amino acid with a nonpolar side chain substituted for another ammo acid with a nonpolar side chain (e.g., Ala, Gly, Vai, lie.
• the nucleic acids of the disclosure can, for example, encode fenctional variants which also include extensions of the IL-2, IL-7, IL-15, or IL-21 protein.
• 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 -terminal or C-terminal end of IL-2, IL-7, IL-15, or IL-21 protein.
• the functional variants can comprise the amino acid sequence of the IL-2, IL-7, IL-15, or IL-21 protein with at least one nomconservative amino acid substitution.
• the non-conservative amino acid substitution it is preferable for the non-conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant.
• the nonconservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the native IL-2, IL-7, IL-15, or IL-21 protein.
• the IL-2, IL-7, IL-15, or IL-21 protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the functional variant, e.g., other amino acids, do not materially change the biological activity of the functional varian t.
• the nucleic acids encode a mammalian IL-2, IL-7, IL-15, or IL-21 protein.
• the IL-2, IL-7, IL- 15, or IL-21 protein can be murine, porcine, ovine, bovine, human, or combinations thereof.
• the present disclosure provides a composition
• 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.
• the nucleic acid sequence encodes a polypeptide comprising a sequence of at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 3 or 4.
• the polynucleotides provided herein encoding one or more interleukin proteins include codon-optimized sequences.
• codon- optimized means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by choosing different codons without altering the ammo acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can increase protein expression levels (Gustafsson et al., Codon bias and heterologous protein expression.
• Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of A, G, C, U nucleotides, mRNA secondary structures, cis-regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos et al.. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285).
• CAI codon adaptation index
• 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.
• CAI high codon adaptation index
• the CAI method chooses a most frequently used synonymous codon for an entire protein coding sequence. As an example, the most frequently used codon for each 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.
• the nucleotide sequence of any region of an RNA or DNA sequence embodied herein may be codon optimized.
• 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 m a template may be increased or reduced to a level above or below 25% of said nucleotides m the template. In further examples, the occurrence of a nucleotide in a template may be increased or reduced to a level above or below 20% of said nucleotides in the template.
• 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.
• the polynucleotides encoding the IL-2, IL-7, IL- 15, or IL-21 proteins can comprise one or more chemically modified nucleotides.
• nucleic acid monomers include non-natural, modified, and chemically modified nucleotides, including any such nucleotides known in the art.
• Nucleotides can be artificially modified at either the base portion or the sugar portion.
• 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).
• 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.
• modified or chemically modified nucleotides include 5-hydroxycytidmes, 5- alkylcytidines, 5-hydroxyalkylcytidines, 5 -carboxy cytidines, 5-formylcytidmes, 5- alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N 4 -alkylcytidines, N 4 - aminocytidines, N 4 -acetylcytidines, and N 4 , N 4 -dialkyIcytidines.
• modified or chemically modified nucleotides include 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidme, 5-formylcytidine, 5- m ethoxy cytidine, 5-propynyl cytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine; N 4 - methylcytidine, N 4 -aminocytidine, N 4 -acetylcytidine, and N 4 , N 4 -dimethylcytidine.
• modified or chemically modified nucleotides include 5-hydroxyuridines, 5- al kyl uridines, 5-hydroxyalky 1 uridines, 5 -carboxyurid ines, 5 -carboxyalkylesteruridines, 5 - formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thiouridines, and 6- alkylundmes.
• modified or chemically modified nucleotides include 5-hydroxyuridine, 5- methyl uridine, 5-hydroxymethyluridme, 5-carboxyuridine, 5-carboxymethylesteruridine, 5- formyl uridine, 5-methoxyuridine (also referred to herein as “SMeOU”), 5-propynyluridine, 5- bromouridine, 5-fluorouridme, 5-iodouridine, 2-thiouridine, and 6-methyluridine.
• modified or chemically-modified nucleotides include 5- methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5- carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, l-methyl-3-(3-amino-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5- niethyldihydrouridme, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5- (isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine, and 3,2'- O-dimethyluridine.
• 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, NMsopentenyladenosine, 2-methylthio-N 6 - isopentenyladenosine, N 6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cz,s- hydroxyisopentenyl)adenosine, N 6 -glycinylcarbamoyladenosine, N 6 -threonylcarbamoyladenosine, N 6 -methyl-N 6 -threonylcarbamoyl-adenosine, 2-methylthio-N
• modified or chemically modified nucleotides include N ! -alkylguanosines, N z -alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8- bromoguanosines, O 6 -alkylguanosines, xanthosines, inosines, and N 1 -alkylinosines.
• modified or chemically modified nucleotides include N 1 -methylguanosine, N 2 -methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O°-methy1guanosine, xanthosine, inosine, and N 1 -methylinosine.
• nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.
• modified and chemically modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-()-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.
• nucleotides known in the art for example, 2'-O-methyl ribonucleotides, 2'-()-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides
• modified and chemically modified nucleotide monomers include 3'-end stabilized nucleotides, 3'-glyceryl nucleotides, 3’-inverted abasic nucleotides, and 3'-inverted thymidine.
• 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.
• the modified monomer is a locked nucleic acid nucleotide (LNA).
• modified and chemically modified nucleotide monomers include 2',4'- constrained 2'-O-niethoxy ethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs.
• modified and chemically modified nucleotide monomers include 2' -amino nucleotides, 2’-O-aniino nucleotides, 2 ! -C-allyl nucleotides, and 2'-O-allyl nucleotides.
• modified and chemically modified nucleotide monomers include N6- methyladenosine nucleotides.
• 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.
• modified and chemically modified nucleotide monomers include 2'-O- aminopropyl substituted nucleotides.
• modified and chemically modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'-halogen, a 2 ! -SR, or a 2' -amino, where R can be H, alkyl, alkenyl, or alkynyl.
• Example of base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers may be found in nature.
• Interleukin nucleic acid molecules can be produced by standard techniques. For example, PCR techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RM A. Various PCR methods are described in, for example, JCmmr; ,4 Lofmmftwy AAmrmZ, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995.
• sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
• Various PCR strategies also are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.
• Interleukin nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphorarmdite technology) or as a series of oligonucleotides.
• one or more pairs of long oligonucleotides e.g., >50-100 nucleotides
• each pair containing a short segment of complementarity e.g., about 15 nucleotides
• DM A polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector, e g. a plasmid.
• Isolated nucleic acids of the disclosure also can be obtained by mutagenesis of, e.g., a. naturally occurring portion of interleukin DM A.
• the nucleic acid is a synthetic polynucleotide.
• the synthetic nucleic acid comprises a modified nucleotide.
• Modification of the inter-nucleoside linker can be utilized to increase stability or pharmacodynamic properties.
• inter-nucleoside tinker modifications prevent or reduce degradation by cellular nucleases, thus increasing the pharmacokinetics and bioavailability of the nucleic acid.
• a modified inter-nucleoside linker includes any linker other than other than phospbodiester (PO) liners, that covalently couples two nucleosides together.
• PO phospbodiester
• the modified inter-nucleoside linker increases the nuclease resistance of the nucleic acid compared to a phospbodiester linker.
• the inter-nucleoside linker includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
• the nucleic acid comprises one or more inter- nucleoside linkers modified from the natural phosphodi ester. In some embodimen ts all of the inter-nucleoside linkers of the nucleic acid or contiguous nucleotide sequence thereof, are modified.
• the inter-nucleoside linkage comprises Sulphur (S), such as a phosphorothioate inter-nucleoside linkage.
• a modified nucleoside includes the introduction of one or more modifications of the sugar moiety or the nuc-leobase moiety.
• the nucleic acids, as described comprise one or more nucleosides comprising a modified sugar moiety, wherein the modified sugar moiety is a modification of the sugar moiety when compared, to the ribose sugar moiety found in deoxyribose nucleic acid (DNA) and RN A.
• DNA deoxyribose nucleic acid
• RN A deoxyribose nucleic acid
• Numerous nucleosides with modification of the ribose sugar moiety can be utilized, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or stability.
• Such modifications include those where the ribose ring structure is modified. These modifications include replacement with a hexose ring (HNA), a bicyclic ring ha ving a. biradical bridge between the C2 and C4 carbons on the ribose ring (e.g. locked nucleic acids (LNA)), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA)
• HNA hexose ring
• LNA locked nucleic acids
• UPA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
• Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids or tricyclic nucleic acids. Modified nucleosides also include nucleosides where the sugar moiety is replaced with a.
• Non-sugar moiety for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
• Sugar modifications also include modifications made by altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2' -OH group naturally found in DNA and RNA. nucleosides. Substituents may, for example be introduced at the 2', 3‘, 4' or 5‘ positions.
• Nucleosides with modified sugar moieties also include 2’ modified nucleosides, such as 2' substituted nucleosides.
• a 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or tin i at the 2' position (2' substituted nucleoside) or comprises a 2' linked biradicle and includes 2' substituted nucleosides and LNA(2'-4 ! biradicle bridged) nucleosides.
• 2 f substituted modified nucleosides are 2 : -O-alkyl-RNA, 2 ; -O-metliyl-RNA, 2'-alkoxy-RNA, 2 ! -O- methoxyethyl-RNA (MOE), 2 f --ammo-DNA, 2'-Fluoro-RNA, and 2 : -F--ANA nucleoside.
• the modification m the ribose group comprises a modification at the 2' position of the ribose group.
• the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2 ? -fh.ioro, 2'- deoxy, and 2 ; --O-(2-methoxyethyi).
• the nucleic acid comprises one or more modified sugars. In some embodiments, the nucleic acid comprises only modified sugars. In certain embodiments, the nucleic acid comprises greater than 1.0%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a ti'-O-metboxyethyl group In some embodiments, the nucleic acid comprises both in ter-nucleoside linker modifications and nucleoside modifications.
• the interleukin polypeptides are synthesized frtsn an expression vector encoding the DNA molecule, as described in detail elsewhere herein.
• polypeptides can be modified to include one or more non-natural amino acids.
• an “unnatural ammo acid,” “non-natural”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified ammo acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids.
• Unnatural ammo acids have side chain groups that distinguish them from the natural ammo acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural ammo acids may have an extended backbone such as beta-ammo acids.
• Non-limiting examples of iron-natural ammo acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosme, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcp-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L- phenylalamne, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine
• one or more amino acids of the helicase are substituted with one or more unnatural amino acids and/or one or more natural amino acids.
• Any one or more of SEQ ID NOs: 1, 2, 3, or 4 can be modified to include naturally occurring and synthetic a, p, -y, and 5 ammo acids.
• the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutamyl, lysinyl, argininyl, histidinyl, p- alanyl, P-valinyl, P-leucinyl, P-isoleucmyl, P-prolinyl, P-phenylalaninyl, P-tryptophanyl, P ⁇ methioninyl, P-glycinyl, P-serinyl, P-threoninyl, P-cysteinyl, P-tyrosinyl, P ⁇
• amino acid 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.
• compositions of the compounds of the disclosure typically comprise a compound of the instant disclosure and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable earner includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media, or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the instant disclosure is contemplated Supplementary active compounds can also be incorporated into the compositions.
• the liquid compositions of the disclosure comprise an interleukin ai a concentration of 0.001 rnilli- international units (MIU) to 200 MIU, a buffering agent, a surfactant, acid, antioxidants, acid, sugar alcohols and an optional excipient selected from amino acids, osmolarity adjusting agents, and preservatives in water.
• MIU rnilli- international units
• the surfactant is present so as to promote the solubility and the stability of the interleukin in solution.
• Suitable surfactants encompass, without being limited to, Cs-Czo alkylsulfate salts, certain phospholipids such as phosphati dates, chelate salts, deoxycholate salts, salts of lauroyl sarcosinate (such as sodium salt known as sarkosyl), CHAPS, CHAPSO, Triton XI 00, Triton XI 14, NP40, octyl glucoside, polyethylene glycol dodecyl ethers for instance that marketed under tra dename BrijTM35, polyethylene glycol hexadecyl ethers for instance that marketed under the tradename BrijTM58, polyoxyethylene derivatives of sorbitan monolaurates such as Tween 20 and Tween 80, sorbitan esters such as sorbitan monostearate or sorbitan monolaurate,
• the surfactant is an anionic surfactant. Accordingly, the surfactant can be selected from C8-C20 alky Isulfate salts, lauroyl sarcosinate salts, cholate salts, deoxycholate salts and combinations thereof
• Preferred anionic surfactants are dodecyl sulphate salts such sodium dodecyl sulphate (SOS) or lithium dodecyl sulphate.
• SOS sodium dodecyl sulphate
• the surfactant can be selected among alkali metal and alkaline-earth metal dodecyl sulphates.
• a preferred surfactant is SDS.
• the optional excipient may be selected from preservatives, antioxidants and combinations thereof
• Preservatives encompass, without being limited to, benzalkonium chloride, benzoic acid, sorbic acid and salts thereof
• Antioxidants encompass ascorbic acid, ascorbyl palmitate, tocopherol and combinations thereof
• the optional excipient accounts for less than 5% by weight, preferably less than 3%, 2%, 1%, and even 0. 1% by weight of the total weight of the composition.
• the optional excipient may be also selected among osmolarity adjusting agents.
• Osmolarity adjusting agents comprises pharmaceutically acceptable inorganic salts such as sodium chloride and potassium chloride and organic salts such sodium or potassium organic salts, for instance potassium or sodium citrate, aspartate or acetate.
• the osmolarity adjusting agent is typically added in the composition of the invention in an amount enabling to adjust the osmolarity of the composition.
• 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.
• IL 15 is being investigated for several medicinal uses related to cancer.
• IL-21 is being investigated for cancer therapy.
• 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 ammo-acid sequence is inserted into the vector.
• the cells used can be eukaryotic or prokaryotic.
• the cell is a bacterial cell.
• the ceil is a fungal cell, e.g., a yeast cell.
• the cell is a vertebrate ceil, e.g., an avian or a mammalian cell.
• 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.
• 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.
• Escherichia coli B selves as a research model and also for protein expression in life science laboratories and in the biotech industry. Characteristics such as protease deficiency, iow acetate production at a high level of glucose, and enhanced permeability (probably due to a simple cell surface) make E. coli B a desirable host to produce genetically engineered proteins. Differences between B strains and KI 2 include the absence of flagellar component genes, the DM A cytosine methylase dem, and ompT in BL21(DE3). B strains may have an additional type II secretion system not found in KI 2.
• BL21(DE3) also carries a DE3 recombinant phage harboring the T7 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. coll strain that protects target protein from Ion and ompT proteases) and their derivatives such as: Lysogenic DEB (based on T7 polymerase), pLysS, pLysE (express T7 lysozyme reducing basal expression of target genes), Origami (allows disulfide bond formation in E.
• coli cytoplasm cytoplasm
• Rosetta Enhances expression of proteins that contain codons rarely used in E. coli.
• 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.
• 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.
• the medium may contain each 9 liters, 216 gr of Yeast Extract, 108 gr of Soy Peptone, 113 of gr K2HPO4, 20,8 gr KH2PO4, 36 ml of Glycerol and 4 ml of Antifoam (2% v/v).
• Fermentation conditions may be: Temperature: 37°C ⁇ 0,5°C, agitation: 350 rpm ⁇ 10 rpm, air flow: 9 L/min ⁇ 1 L/min, pO2: set point 40%, pH between 6.95 and 7.5. After this, a feeding procedure should be followed.
• a glucose solution 40% p/v by drip, to maintain a concentration of 0.1 %.
• an appropriated inducer such as Isopropil-P-D-1 - tiogalactopiranosido (IPTG) should be added to reach an operative concentration.
• IPTG Isopropil-P-D-1 - tiogalactopiranosido
• 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.
• bacteria can be concentrated 5 to 7 times by centrifugation or tangential filtration and processed immediately or preserved at 2-8 °C (no more than 24 hours) or preserved at -20°C (for more than 24 hours).
• inclusion bodies lb
• lb inclusion bodies
• bacteria could be suspended in purified water. After this, the suspension could be circulated 2-4 times for the disruptor at a pressure of about 1400 bar.
• the lysate should be processed immediately or preserved at -20°C.
• the lb are separated of other components of the lysate by centrifugation or tangential filtration and washed.
• the lb preparation should be stored at -20°C until further processing.
• the lb are then suspended in a suitable buffer and submitted to a re-folding step in a regulated redox potential.
• interleukins After refolding interleukins are submitted to different chromatographic and diafiltrations to get a concentrated solution of purified interleukins. Interleukins are then formulated and packaged into ready-to-use bags, pre-filled syringes, vials, ampoules or other suitable primary packaging materials for sterile solutions. Surprisingly, it was discovered that recombinant interleukins may be easily stabilized in solution using buffers of carboxylic acids at pH at least 0.3 units from their isoelectric point, isotonized with non-ionic osmolytes with the addition of methionine or other amino acids. In some cases surfactants are needed to prevent them from forming aggregates, oligomers or adsorbing onto the walls of the container.
• 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.
• methionine and disodium edetate improves stability against oxidation of some members of the family.
• 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.
• a solution composition for this interleukin is the following:
• composition is filled into pharmaceutically acceptable plastic bags containing the following amoun ts 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:
• 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)
• IL-7 is produced by recombinant DNA technology using a genetically engineered E. colt strain BL21 Rosetta. DE3 pLysS containing an analog of the human interleukin-7 coding region gen, inserted in a pET9a (TetR) plasmid vector at Ndel-BamHI restriction sites. It contains three disulfide bonds that are essential for its biological activity.
• IL-7 is not glycosylated because it is derived from E. coir
• molecule has an N-termmal Methionine
• An inducible eukaryotic expression system for recombinant expression of hIL-7 was developed using an Escherichia, coli strain as a host.
• Escherichia, coli strain as a host.
• Rosetta2(DE3) pLysS strain was selected due to the fact that a high, tight and stable recombinant expression of rliIL-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.
• 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 cassettes containing the natural (wild type) gene and two modified genes versions including codon usage adaptations for E. coli expression.
• One of the modified genes versions (fully adapted for E. coli expression) originates the best expressing clones and one of these clones was selected for next stages of this development.
• the molecular construct and gene were verified by nucleotide sequencing and protein identity’ was stated by SDS-PAGE (molecular size) and by’ Western Blot (specific antibody’ detection).
• IL- 15 was produced by recombinant DNA technology using a genetically engineered E. coll strain BL21 Al containing an analog of the human interleukin- 15 coding region gen, inserted in a pET9a plasmid vector at Ndel-BamHI restriction sites. It contains two disulfide bridges, which the structure shows help to stabilize the conformation in regions of the loop that engage in contacts with the receptor.
• An inducible eukaryotic expression system for recombinant expression of hIL-15 was developed using an Escherichia, coli strain as a host.
• the Al strain was selected. It was due to the fact that a high, tight and stable recombinant expression of rhIL-15 was observed.
• the strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7 RNAP) gene in the araB locus, allowing expression of T7 RNAP to be regulated by the araB AD promoter. The expression of this polymerase is induced by Arabinose.
• T7 RNAP T7 RNA polymerase
• 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.
• hIL-15 genes Three different versions of hIL-15 genes were tested. These were synthetics hIL-15 cassettes containing: the natural (wild type) gene and two modified genes versions including codon usage adaptations for E. 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 by Western Blot (specific Antibody detection).
• the pH value of this formulation is 6 ⁇ 0.5 (more than 0.2 u: its above the isoelectric point of IL- 15: pl 4.52) Biological Activity Methodology
• Interleukin-7 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 1.5x10 8 lU/mg compared against the NIBSC WHO Reference Interleukin-7 using a parallel line assay as statistical model.
• the pH of this formulation is more than 0.2 units below the isoelectric point of IL-21 (pl 9.42).
• IL-21 is a human recombinant interleukin- 21 protein with a molecular weight of approximately 15.4 kDa (Dalton). It is produced by recombinant DN A technology using a genetically engineered E. coll 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.
• Primary' protein structure contains the following amino acids sequence:
• the synthetic genes do not include nucleotides coding for the signal peptide region of the hIL-21 gene. All cassettes were sent as an insert cloned into a pUC57 cloning vector (into Ndel and BamHI restriction sites). Note: All cassettes include an extra stop codon and the best option for E. coli (TAA stop codon) in the first position (Vyas W et al., Biolechnol Prog. 2012 Mar- Apr;28(2):497-507. doi: 10.1002/btpr.746. Epub 2011 Dec 9) that determined the presence of an extra tryptophan due to a stop codon wobble effect, which could be eliminated by replacing TGA (opal) stop codon with TAA (ochre) stop codon.
• the BL21-AITM 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.
• T7 RNAP T7 RNA polymerase
• the presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline.
• No induction means bacteria without induction.
• the recombinant system (plasmid +E. coli strain) is and inducible genetic system that required an inducer (L-arabinose in this case) to avoid genetic repression.
• Non-induced bacteria are expected do not shown recombinant expression (no recombinant protein band in the SDS-PAGE).
• the induced bacteria should express the protein of interest (i.e. IL-21 band in the SDS-PAGE).
• the numbers and/or letters indicate Cion ID (i.e.
• 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 biological activity of human IL-21 is determined by means of a proliferation assaybased on the B9 cell line (Mouse B cell Hybridoma) obtained from Public Health England and quantified by a colorimetric method, using MTS.
• B9 cell line Mae B cell Hybridoma
• B9 cells were washed twice with Assay Medium and a cell suspension of 2.5x10 5 cells/mL is prepared.
• serial dilutions are made for IL-21 Standard and the Samples to be analyzed.
• 100 pL/well of each dilution are dispensed in a microplate followed by 100 ,uL of the cell suspension. Plates are incubated for 48 hours at 37 °C and 5% CO?.
• 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.
• ECso Half maximal effective concentration 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 ECso is considered one Unit of biological activity.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Wood Science & Technology (AREA)
• Biophysics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Toxicology (AREA)
• Gastroenterology & Hepatology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Mycology (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Cell Biology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=92420743

Family Applications (1)

Country Status (4)

Family Cites Families (4)

• 2024
  • 2024-02-14 IL IL322076A patent/IL322076A/en unknown
  • 2024-02-14 AU AU2024221181A patent/AU2024221181A1/en active Pending
  • 2024-02-14 EP EP24757632.5A patent/EP4665381A1/de active Pending
  • 2024-02-14 WO PCT/US2024/015832 patent/WO2024173576A1/en not_active Ceased

Also Published As

Similar Documents

Legal Events