Method to prepare therapeutically active aldesleukin highly stable in liquid pharmaceutical compositions

Abstract

The present invention relates to a liquid pharmaceutical composition of an aldesleukin / SDS aggregate and its use in the treatment of autoimmune diseases, inflammatory diseases, and cancer. A method for preparing said composition is also disclosed.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of priority under 35 USC§119(e) to U.S. Provisional Application No. 62 / 684,288 filed June 13, 2018, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a novel pharmaceutical composition comprising an aldesleukin / SDS aggregate that is stable in solution and to the pharmaceutical use thereof. Background Technology

[0004] Interleukin 2 (IL-2) is a key cytokine that regulates the survival, proliferation, and differentiation of T cells and NK cells (1). The discovery of its biological activity led to the approval of the clinical application of IL-2 in cancer immunotherapy for metastatic renal carcinoma in 1994 and metastatic melanoma in 1998. Subsequently, IL-2 emerged as a reagent to induce in vitro expansion of T lymphocytes (tumor-infiltrating lymphocytes, TILs) extracted from a patient's tumor and then returned to the patient to prevent future cancer development (2). Recently, IL-2 has also been used for cancer treatment to expand T cells that have been transduced into antigen-specific T cell receptors (3) or transduced into chimeric antigen receptors (CAR) (4). As a result of the discovery that IL-2 has a dual function in immune responses, a new field of medical application for IL-2 has recently emerged: a) its well-known activity as an activator of inflammatory responses, and T regulatory cells (CD4 + Foxp3 +Activity as a down regulator of the immune response that induces the expansion of Treg cells (5). This seemingly paradoxical duality can be explained by the presence of two types of IL-2 receptors with high or low affinities and specific cell distributions (5). This duality of IL-2 has led to the following different therapeutic approaches: a) the use of high doses of IL-2 to stimulate an immune response, such as in the treatment of cancer, and b) the use of low doses of IL-2 to suppress an excessive or abnormal immune response, such as in the treatment of autoimmune or inflammatory diseases.

[0005] Most (if not all) current clinical applications of IL-2 are carried out by using a recombinant mutant version of the natural form IL-2 named aldesleukin (des-ala-2ser125) (PROLEUKIN™). U.S. Patent No. 4,604,377 discloses a method for preparing a pharmaceutical composition of recombinant IL-2 (containing aldesleukin) contained in a sterile and stable lyophilized formulation, wherein the recombinant IL-2 is mixed with a water-soluble carrier, such as mannitol, which provides a bulk and sufficient amount of sodium dodecyl sulfate to ensure the solubility of the recombinant IL-2 in water. The formulation is suitable for reconstitution in an aqueous injectable that is stable and highly tolerable in human patients for parenteral administration. On the other hand, a detailed description of the process for producing the PROLEUKIN™ product is disclosed more recently in European Patent EP 1,688,146 B.

[0006] One aspect of the present invention is a method for preparing a highly stable aldesleukin in a liquid pharmaceutical composition for parenteral administration to a patient requiring IL-2 immunotherapy, generally comprising the following steps: a) Escherichia coli transfected with an expression vector engineered for high expression of the aldesleukin gene ( E.coli) fermentation of the strain, b) destruction of the bacteria, c) collection of inclusion bodies containing aldesleukin aggregates, d) dissolution of aldesleukin aggregates using an SDS detergent, e) oxidation using an oxidizing compound, e.g., cupric chloride, f) ceramic hydroxyapatite chromatography, g) dilution using an organic nitrile, e.g., acetonitrile, h) C4 column HPLC chromatography, i) diafiltration, and j) filter sterilization.

[0007] A second aspect of the present invention is a composition of an aldesleukin in a pharmaceutically stable aqueous vehicle for parenteral administration to a patient requiring IL-2 immunotherapy, wherein the aldesleukin is prepared using the production method of the present invention, which ensures that the aldesleukin remains stable in the pharmaceutical liquid composition for at least one year. By using the composition of the aldesleukin of the present invention, it is possible to produce a pharmaceutical product contained in one or more pre-filled syringes, each syringe containing a suitable volume for the corresponding treatment and packaged in a box. The product does not require any manipulation prior to injection into the patient and, for example, will prevent microbial or other possible contamination. On the other hand, the stable liquid aldesleukin product of the present invention can be advantageously introduced and uniformly distributed in parenteral solutions commonly used in medicine for fluids, blood transfusions, or clinical nutrition. Recently, a treatment consisting of the drug plus aldesleukin has been developed (e.g., CEPLENE™ (histamine dihydrochloride) is administered in combination with a low dose of proleukin to maintain the first remission in patients with acute myeloid leukemia). In the case of such treatment, the stable liquid aldesleukin formulation of the present invention can serve as a basis for creating specific combination formulations or combination kits. Proleukin has also been used to induce in vitro proliferation of sensitive cells used in cell therapy procedures (5). For these procedures, the stable liquid aldesleukin of the present invention, formulated at low concentrations, can be highly advantageous for avoiding operations that may result in microbial contamination and for obtaining a rapid and uniform distribution of aldesleukin within cell cultures. However, this is only a few examples, and those skilled in the art can easily imagine other applications of the stable liquid aldesleukin formulation of the present invention.

[0008] In this specification, the IL-2 molecule comprises an IL-2 amino acid sequence (SEQ No. 1) and 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 It includes an IL-2 variant of the present disclosure comprising an amino acid sequence having 97%, at least 98%, or at least 99% sequence identity. These are the wild-type IL-2 amino acid sequence (i.e., SEQ ID NO. 1) and 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 It includes an IL-2 variant containing an amino acid sequence having an N88R mutation having 98%, or at least 99%, sequence identity.The embodiment also predominantly stimulates Treg cells and the wild-type IL-2 amino acid sequence (SEQ No. 1) and 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 It includes an IL-2 variant comprising an amino acid sequence having N88R and C125S mutations having 96%, at least 97%, or at least 98% sequence identity. The embodiment also predominantly stimulates Treg cells and the wild-type IL-2 amino acid sequence (SEQ No. 1) and 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 It includes an IL-2 variant having an amino acid sequence having 96%, at least 97%, at least 98%, or at least 99% sequence identity. Brief explanation of the drawing

[0009] Figure 1 is the amino acid sequence of recombinant human interleukin 2 (aldesleukin) (Sequence No. 1). Figure 2 compares the aldesleukin / SDS particle size range between Proleukin™ and aldesleukin produced by the method of the present invention when measured by dynamic light scattering. Figure 3 shows the particle size range of aldesleukin / SDS produced by the method of the present invention after 0, 6, or 12 months at 0-8°C. Specific details for implementing the invention

[0010] An embodiment of the present invention relates to IL-2 that is highly stable in a liquid formulation and a method for producing said stable IL-2.

[0011] definition

[0012] The technical terms used herein are for the purpose of disclosing specific embodiments and are not intended to limit the invention. Unless otherwise specifically defined, all technical and scientific terms used herein shall be deemed to have the same meaning as commonly understood by a person skilled in the art (e.g., cell culture, molecular genetics, and biochemistry).

[0013] As used herein, the singular forms “one,” “one,” and “the above” are intended to include the plural forms unless the context otherwise clearly indicates. Additionally, when the terms “comprising,” “comprising,” “having,” “having,” “together,” or variations thereof are used in the detailed description and / or claims, these terms are intended to encompass in a manner similar to the term “comprising.”

[0014] The terms “approximately” or “roughly” mean within an acceptable margin of error for a specific value as determined by a person skilled in the art, which will depend in part on the manner in which the value is measured or determined, i.e., the limitations of the measurement system. For example, “approximately” may mean within one or more standard deviations as practiced in the art. Alternatively, “approximately” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of the value or range. Alternatively, particularly in relation to biological systems or processes, the term may mean within five times the value, and within an order of magnitude of up to two times. When a specific value is disclosed in this application and claims, unless otherwise stated, the term “approximately” meaning within an acceptable margin of error for a specific value should be presumed.

[0015] As used herein, “biological medium” is any type of medium used to grow, culture, and maintain organs, tissues, cells, etc. in vitro. Biological medium also encompasses any biocompatible preparation, any pharmaceutical excipient, pharmaceutically and physiologically acceptable fluids, such as water as a vehicle, physiological saline, equilibrium salt solution, aqueous dextrose, glycerol, etc., tissue or organ culture media, any preparation that may be administered in vivo to a subject, any preparation that may be used in analytical methods, or to dilute or maintain biological samples, such as nucleic acids, peptides, etc.

[0016] As used herein, the term “cell” includes prokaryotic and eukaryotic cells. In one embodiment, the cell of the present invention is a bacterial cell. In another embodiment, the cell of the present invention is a fungal cell, such as a yeast cell. In another embodiment, the cell of the present invention is a vertebrate cell, e.g., an avian or mammalian cell. In a preferred embodiment, the cell of the present invention is a murine or human cell. As used herein, the term “manipulated” (as in “manipulated cell”) refers to a cell into which a nucleic acid molecule encoding, for example, the IL-2 protein (e.g., a spliced ​​or unspliced ​​form of IL-2) or a fragment thereof has been introduced.

[0017] As used herein, the term "cell-free composition" refers to an isolated composition that does not contain intact cells. Examples of cell-free compositions include compositions containing cell extracts and isolated proteins.

[0018] As used herein, the terms “comprising,” “comprising,” or “comprising,” and variations thereof, mean that they are comprehensive or open-ended with respect to the defined or disclosed elements, such as items, compositions, devices, methods, processes, systems, etc., and allow for additional elements, thereby indicating that the defined or disclosed items, compositions, devices, methods, processes, systems, etc., include the specified elements or, suitably, their equivalents, and that other elements are included within and still belong to the scope / definition of the defined items, compositions, devices, methods, processes, systems, etc.

[0019] The term "expression vector" refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product that can be transcribed. In some cases, when the transcription product is an mRNA molecule, it is eventually translated into a protein, polypeptide, or peptide.

[0020] As used herein, the term “human recombinant IL-2 (rhIL-2)” is a non-glycosylated protein produced by microorganisms transfected with a DNA sequence encoding human IL-2 or a non-large-scale modification thereof. In fact, the most widely used human recombinant IL-2 version in clinical practice is named Aldesleukin (Proleukin™) and is a modified version of the natural human IL-2 amino acid sequence having the following modifications: a) Aldesleukin lacks the N-terminal alanine present in natural IL-2; b) Aldesleukin has a serine substitute for cysteine ​​at amino acid position 125. The amino acid sequence of Aldesleukin is shown in Fig. 1. The composition of the Aldesleukin gene is disclosed in U.S. Patent 4518584A and an associated non-U.S. patent. The U.S. FDA has approved Aldesleukin (Proleukin™) for the treatment of adults with metastatic renal cell carcinoma and metastatic melanoma. Proleukin™ is supplied as a sterile white to yellowish-white lyophilized cake in 20 single-use vials for intravenous administration. When reconstituted with 1.2 ml of sterile water, USP for injection, each ml contains 18 million international units (1.1 mg) of Proleukin™, 50 mg of mannitol, and 0.18 mg of sodium dodecyl sulfate, and is buffered to a pH of 7.5 (range of 7.2 to 7.8) with approximately 0.17 mg of monobasic and 0.89 mg of dibasic sodium phosphate. Reconstituted or diluted Proleukin™ is stable for up to 48 hours at 2° to 25° (36° to 77°F) in a refrigerated and room temperature, as indicated in the reconstitution and dilution guide of the "Proleukin™ (Aldesleukin) Injection Label - FDA (Reference ID: 3165255)".

[0021] As used herein, the term "Interleukin 2 (IL-2)" refers to a cytokine that regulates the activity of leukocytes (primarily lymphocytes). This cytokine is part of the immune response to microbial infection and exerts its effects by binding to specific receptors present on the surface of lymphocytes. Additional information regarding IL-2 may be found in its "GeneCard" (access number GC04M123831). Other access numbers include: HGNC: 6001 Entrez Gene: 3558 Ensembl: ENSG00000109471 OMIM: 147680 UniProtKB: P60568. IL-2 proteins include the original IL-2 protein as well as variant IL-2 proteins. "Natural" or "wild-type" IL-2 sequences, as used herein, refer to human IL-2 sequences purified from natural sources or produced using recombinant technology (e.g., access number NP 000577.2). In some embodiments, the wild-type IL-2 sequence comprises a proleukin™ (aldesleukin) sequence (sequence number 1):

[0022] PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0023] As used herein, the term “kit” refers to any transport system for transporting material. The term “kit” includes kits for both research and clinical applications. In the context of reaction assays, such transport systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., cytokines, oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosed items (e.g., boxes) containing the corresponding reaction reagents and / or support materials. As used herein, the term “fragmented kit” refers to a transport system comprising two or more separate containers, each containing a portion of the entire kit components. The containers may be transported together or individually to the intended recipient. For example, the first container may contain an enzyme for use in the assay, and the second container may contain an oligonucleotide or liposome. The term “split kit” is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the U.S. Federal Food, Drug, and Cosmetic Act. In practice, any delivery system comprising two or more separate containers, each containing a portion of the entire kit components, is included in the term “split kit.” In contrast, a “combination kit” refers to a delivery system containing all components of a reaction assay within a single container (e.g., a single box housing each of the desired components). The term “kit” includes both split and combination kits.

[0024] As used in this specification and the appended claims, the term "or" is adopted to mean generally including "and / or" unless the context clearly indicates otherwise.

[0025] The phrase “pharmaceuticalally acceptable carrier” refers to a carrier for administering the therapeutic agent. Exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginate are suitable disintegrants. Binders may include starch and gelatin, and, if present, lubricants will generally be magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with a substance such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0026] As used herein, “stable” or “very stable” refers to the activity and / or nature of a molecule, e.g., IL-2, within a formulation embodied herein over an extended period when evaluated by in vitro biological activity, in vitro structural studies, and in vivo therapeutic activity. The activity of said IL-2 may be measured by any standard analytical method. Activity before storage and after a long period, e.g., at least one year of storage, may be compared. Very stable IL-2 will have the same or slightly lower activity compared to the activity of IL-2 at the time of initial formulation.

[0027] Genbank and NCBI submissions indicated by access numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. In the event of conflict, this specification, including definitions, will provide the terms. Additionally, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0028] Scope: Throughout the entire disclosure, various aspects of the invention may be provided in a scope format. It should be understood that disclosure in a scope format is for convenience and brevity only and should not be understood as an inflexible limitation on the scope of the invention. Accordingly, a scope disclosed should be deemed to specifically disclose individual numeric values ​​within that scope as well as all possible sub-scopes. For example, a scope disclosed as 1 to 6 should be deemed to specifically disclose individual numbers within said scope, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, as well as sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6. This applies regardless of the width of said scope.

[0029] Method for producing a stable IL-2 formulation

[0030] The present invention is based on a novel method for producing an aldesleukin that produces a product partially satisfying all the structural and biological characteristics of Proleukin™, but is surprisingly very stable in solution (for at least one year) when evaluated by "in vitro" biological activity, "in vitro" structural studies, and "in vivo" therapeutic activity. An exemplary method for producing an aldesleukin that is very stable in solution comprises the following steps:

[0031] a) Fermentation of an E. coli strain transfected with an expression vector engineered to high-expression of the aldesleukin gene. Bacteria are desirable microorganisms for producing IL-2, and among bacteria, E. coli strains are the most desirable. E. coli B serves as a research model in life science laboratories and the biotechnology industry, as well as for protein expression. Characteristics such as protease deficiency, low acetate production from high levels of glucose, and enhanced permeability (possibly due to a simple cell surface) make E. coli B a desirable host for the production of genetically engineered proteins. Differences between strain B and K12 include the absence of flagellar component genes, DNA cytosine methylase dcm, and ompT within BL21(DE3). Strain B may possess an additional type II secretion system not found in K12. BL21(DE3) also carries a DE3 recombinant phage containing a T7 RNA polymerase gene capable of directing high-level expression of the cloned gene under the regulation of the T7 promoter. Typical E. coli strains used for recombinant protein expression are as follows: BL21 (a B E. coli strain that protects target proteins from ion and ompT proteases) and its derivatives: lysogenic DE3 (based on T7 polymerase), pLysS, pLysE (expressing T7 lysozyme that reduces basal expression of target genes), Origami (allowing disulfide bond formation in the E. coli cytoplasm), and Rosetta (enhancing the expression of proteins containing codons rarely used in E. coli). Similar versions exist under the K12 E. coli genetic background. Typical plasmid vectors for the high expression of recombinant proteins in E. coli are as follows: the pET series based on pBR322 origin and a T7 / lac promoter; pBad with an araBAD promoter and pUC origin; and pGEX also with a tac promoter and pBR322 origin.The combination of fusion tag sequences, protease cleavage sites, screening markers, and strain compatibility is the source of the most common list of high-expression plasmid variants.

[0032] The expression vector is commercially available, and a DNA sequence encoding an aldesleukin amino acid sequence is inserted into said vector. The cell used in the analysis method may be of eukaryotic or prokaryotic origin. For example, in one embodiment, said cell is a bacterial cell. In another embodiment, said cell is a fungal cell, e.g., a yeast cell. In another embodiment, said cell is a vertebrate cell, e.g., an avian or mammalian cell. In another embodiment, said cell is a human cell. The cell of the present invention may express endogenous IL-2 or a fragment thereof, or may be engineered for this purpose. For example, a cell engineered to express IL-2 or a fragment thereof may be produced by introducing an expression vector encoding said protein into the cell.

[0033] In a given embodiment, the cell is an E. coli cell. Different E. coli strains may be transfected to obtain optimal aldesleukin production.

[0034] a) Aldesleukin-producing bacteria may be cultured in a suitable growth medium. For example, the medium may contain 9 liters, 216 grams of yeast extract, 108 grams of soybean peptone, 113 grams of K2HPO4, 20.8 grams of KH2PO4, 36 ml of glycerol, and 4 ml of antifoaming agent (2% v / v), respectively. Fermentation conditions may be as follows: temperature: 37°C ± 0.5°C, stirring: 350 rpm ± 10 rpm, air flow: 9 L / min ± 1 L / min, pO2: set point 40%, stirring cascade: 21% to 36% at a drive of 350 rpm to 440 rpm, pH 6.95 to 7.5, and injection air pressure at 2 bar before the start of oxygen flow. Subsequently, a feeding procedure follows. For example, a 40% p / v glucose solution is supplied dropwise to maintain a concentration of 0.1%. When the OD600 reaches 5 to 10, an appropriate inducer, such as isopropyl-β-D-1-thiogalactopyranoside (IPTG), must be added to reach the operating concentration. At this point, the glucose supply can be reduced to maintain a glucose concentration of approximately 0.01%. Fermentation can usually be stopped about 18 to 24 hours after inoculation. After fermentation, the bacteria are concentrated 5 to 7 times by centrifugation or tangential filtration and can be processed immediately, stored at 2–8°C (within 24 hours), or stored at -20°C (more than 24 hours).

[0035] b) After incubation, aldesleukin exists within the bacteria, predominantly in the form of aggregates designated as inclusion bodies (Ib). The Ib can be isolated by disrupting the bacteria (e.g., by ultrasound). To this end, the bacteria may be suspended in purified water at a temperature of 17 to 22°C before initiating the disruption process. Subsequently, the suspension may be circulated 2 to 3 times at a pressure of approximately 1400 bar for disruption. The lysate must be processed immediately or stored at -20°C. The Ib is separated from and washed from other components of the lysate by centrifugation or tangent filtration. The final buffer for washing the Ib may be TE (10 mL Tris-HCl, 1 mL disodium EDTA, pH 8.0). The Ib preparation must be stored at -20°C until further processing. Subsequently, the above Ib is suspended in a buffer containing 10 mL of sodium phosphate, 1% SDS, and pH 8.0, and stirred for 1 hour. Then, an oxide solution of cupric chloride is added until a final concentration of 100 μM is reached, and stirring must be continued for at least 2 hours. Afterwards, EDTA must be added until a final concentration of 100 μM is reached. The mixture must be stored at 2-8°C until used in a subsequent process.

[0036] c) The first chromatography is performed using a ceramic hydroxyapatite (Type I 80 µm Bio-Rad) column. The above aldesleukin preparation is loaded, washed with a solution of 100 ml sodium phosphate, 0.3% SDS, pH 6.5, and eluted with a solution of 250 ml sodium phosphate, 0.3%, pH 6.5.

[0037] d) A second chromatography is performed using an HPLC C4 column. The solution containing aldesleukin recovered from the first chromatography is mixed with acetonitrile (9 parts of chromatography I pool eluent and 1 part acetonitrile). Subsequently, the loaded aldesleukin is eluted using an acetonitrile gradient. The fraction containing aldesleukin is collected in a solution containing 73 ml of sodium phosphate, 0.3% SDS, and pH 7.4.

[0038] e) The final step of aldesleukin purification is volume filtration using a 5 kD cassette. Aldesleukin is equilibrated using a buffer containing 0.44 mg / mL SDS, 50 mg / mL mannitol, 1.19 mg / mL disodium phosphate, 0.26 mg / mL disodium dihydrate phosphate, and pH 7.5.

[0039] Surprisingly, the aldesleukin / SDS complex obtained by the production method of the present invention was found not to require freeze-drying as disclosed in EP 1 688 146 B1 for stabilization, as it is stable in solution for at least one year as disclosed in Examples 4 and 6.

[0040] According to the dynamic light scattering analysis shown in Examples 3 and 4, the aldesleukin / SDS aggregates obtained by the production method of the present invention have an extended size range of 4 to 18 nm with a peak at about 8 nm. Although the reconstituted Proleukin™ also exhibits a similar distribution curve, the area under the curve is clearly lower than the area under the curve of the aldesleukin / SDS particles produced by the method of the present invention. Since the mass of the protein used in both cases is the same, the results suggest that some of the Proleukin™ may remain as large aggregates upon reconstitution. In fact, it has been reported that to measure the full activity of Proleukin™, the lyophilized product must be suspended in an SDS solution to prevent aggregate formation (6).

[0041] WO 2017068031 A1 discloses a liquid pharmaceutical composition suitable for administration to a patient, in which interleukin-2 can be obtained at a concentration of 100,000 to 20,000,000 IU / mL. The composition must contain an anionic surfactant, such as sodium dodecyl sulfate (SDS), which may be present at a concentration of about 0.05 to 0.5 mg / mL to maintain the stability of interleukin-2 in solution. However, as seen in Proleukin™ products, the addition of SDS to the solution forms biologically active microaggregates with IL-2, and the microaggregation state of IL-2 is important for therapeutic efficacy (EP 1 688 146 B1). Therefore, the microaggregation state and therapeutic value of any novel IL-2 composition must be evaluated. In this regard, Examples 3 and 4 show the microaggregation state and stability, and Example 6 shows an analytical method for evaluating the therapeutic value of aldesleukin / SDS aggregates obtained by the method of the present invention.

[0042] The following examples further illustrate the invention. These examples are not intended to limit the invention in any way.

[0043] Examples

[0044] Example 1: Amino acid sequence of aldesleukin obtained by the production method of the present invention

[0045] The amino acid sequence of aldesleukin obtained by the production method of the present invention was inferred by sequencing the DNA of the aldesleukin gene present in the expression plasmid used in the method. Figure 1 shows the obtained amino acid sequence.

[0046] Example 2: Specific activity of aldesleukin / SDS aggregates obtained by the production method of the present invention

[0047] The biological activity of the aldesleukin / SDS aggregates obtained by the production method of the present invention was determined by proliferation using the HT-2 cell line assay. The inactivity of 20 independent lots was calibrated to the WHO international standard for interleukin 2 (human, rDNA-derived) NIBSC code: 86 / 500 and ranged from 16.6 to 19.5 IU / mg. The said inactivity range is similar to the inactivity disclosed for aldesleukin / SDS aggregates prepared according to EP 1 688 146 B1.

[0048] Example 3: Comparative size of SDS / proleukin aggregates obtained after reconstitution from freeze-dried commercial products and SDS / aldesleukin aggregates obtained using the method of the present invention, measured by dynamic light scattering.

[0049] Dynamic light scattering was used to determine the size and polydispersity of aldesleukin / SDS aggregates using the Zetasizer Nano ZS and Zeta Nano series software (Malvern Instruments, Germany). 100 µl of the sample was diluted in 1 ml of pure water, and measurements were taken five times at 25°C for 30 seconds with an equilibration time of 30 seconds between measurements. Particle size is expressed in nanometers as hydrodynamic diameter. Figure 2 shows the dynamic light scattering patterns for SDS / aldesleukin aggregates obtained using the method of the present invention and for Proleukin™ after reconstitution from freeze-dried commercial products. The aldesleukin / SDS aggregates obtained by the production method of the present invention exhibit an extended size range of 4 to 18 nm with a peak at approximately 8 nm. Proleukin™ also shows a similar distribution curve. However, the area under the curve is clearly lower than the area under the curve of the aldesleukin / SDS particles produced by the method of the present invention. Since the mass of the protein used in the samples analyzed from both sides was identical, the above results suggest that upon reconstruction, a portion of the Proleukin™ may remain as large aggregates that cannot be applied to research using the technology described above.

[0050] Example 4: Stability of SDS / aldesleukin products of aldesleukin obtained by the production method of the present invention

[0051] Table 1 shows the change in activity from inactivity when stored at 2–8°C. In addition to inactivity, the following characteristics were analyzed for each sample: visual observation of abnormalities, pH, volume, Western blot, SDS-PAGE (reducing and non-reducing conditions), Lowry test, endotoxin contamination (LAL test), and sterility. At all measured times, the assay appeared normal.

[0052] [Table 1]

[0053] Inactive soluble aldesleukin / SDS aggregates obtained by the method of the present invention

[0054]

[0055] In addition, Figure 3 shows that the size distribution of aldesleukin / SDS aggregates is well preserved in the liquid aldesleukin composition of the present invention after storage for 6 or 12 months at 2-8°C when measured by dynamic light scattering.

[0056] Example 5: Expansion of regulatory T cells (Treg) in human peripheral monocyte blood cells (PMBC) incubated with SDS / aldesleukin aggregates obtained by the production method of the present invention

[0057] DYNABEADS™ Regulatory CD4 Treg Cells + / CD25 + T cells were recovered from human PMBCs using a T cell kit (ThermoFisher Scientific). The ability of aldesleukin / SDS aggregates to induce Treg proliferation was evaluated using the CD3 / CD28 DYNABEADS® Human Treg Expander (ThermoFisher Scientific) protocol. 500 IU of the aldesleukin preparation was used for Treg expansion. After 7 days of expansion, Tregs were counted. Expansion was 76–125-fold for both the aldesleukin / SDS aggregates obtained using the method of the present invention and the method fully disclosed in EP1 688 146 B1. .

[0058] Example 6: Therapeutic activity of aldesleukin / SDS aggregates obtained using the production method of the present invention

[0059] The therapeutic efficacy of aldesleukin aggregates produced by the method of the present invention and aldesleukin / SDS aggregates of Proleukin™ was compared using a lung metastasis model using B16F10 tumor cells as disclosed in (7). 2×10 8-week-old female C57BL / 6 mice were injected via the tail vein. 50.5 ml of cell suspension containing B16 cells was injected intravenously. Afterward, mice were separated into three groups of 10 mice each. Mice in Group 1 received a dose of 7.0 mg / kg of Proleukin™ daily from day 3 to day 10. Mice in Group 2 received microaggregates of aldesleukin prepared by the method of the present invention daily from day 3 to day 10. Mice in Group 3 received the vehicle daily from day 3 to day 10. Mice were euthanized 16 days after tumor inoculation, and lung metastases were counted as disclosed in (7). Table 2 shows that the average number of metastases observed in the microaggregates prepared by the method of the present invention was 4.3 (range of 1-12), and in contrast, in Proleukin™ was 5.5 (range of 3-22), and these differences were not significant.

[0060] [Table 2]

[0061] Therapeutic activity of aldesleukin / SDS aggregates obtained using the production method of the present invention

[0062]

[0063] The aldesleukin produced by the method of the present invention and used in the analysis method was produced fresh. In contrast, Table 3 shows the same analysis method performed using aldesleukin produced by the method of the present invention after being stored at 2-8°C for 6 or 12 months.

[0064] [Table 3]

[0065] Therapeutic activity of aldesleukin / SDS aggregates obtained using the production method of the present invention for 6 or 12 months after storage at 2-8°C for 6 or 12 months. The result reinforces the idea that the liquid preparation of aldesleukin obtained by the method of the present invention is stable for at least 1 year.

[0066]

[0067] Sequence list

[0068] Sequence No. 1

[0069] Length: 132

[0070] protein

[0071] Biological Name: Artificial Sequence, Human Interleukin-2 Mature Des-Alanyl-1 Serine 125 Variant

[0072] Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

[0073] References

[0074] U.S. patent documents

[0075] US 4,604,377 August 5, 1986

[0076] Other patent documents

[0077] EP 1 688 146 B1 July 18, 2007

[0078] WO 2017 068 031 A1 April 27, 2017

[0079] Other public documents

[0080] 1. Malek TR. The biology of interleukin-2. Annu Rev Immunol. (2008). 26:453-5 79. DOI: 10.1146 / annurev.immunol.26.021607.090357

[0081] 2. Dudley ME et al. Adoptive Cell Transfer Therapy Following NonMyeloablative but Lymphodepleting Chemotherapy for the Treatment of Patients With Refractory Metastatic Melanoma. Journal of Clinical Oncology. (2005). 23: 2346-57. DOI: 10.1200 / JCO.2005.00.240

[0082] 3. Langerman A, Callender GG, Nishimura MI. Retroviral transduction of peptide stimulated t cells can generate dual t cell receptor-expressing (bifunctional) t cells reactive with two defined antigens. Journal of Translational Medicine. (2004). 2:4-8. DOI: 10.1186 / 1479-5876-2-42

[0083] 4. Magee MS, Snook AE. Challenges to chimeric antigen receptor (CAR)-T cell therapy for cancer. Discov Med. (2014). 18:265-71

[0084] 5. Arenas-Ramirez N, Woytschak J, Boyman O. Interleukin-2: Biology, Design and Application. Trends Immunol. (2015). 36:763-777. DOI: 10.1016 / j.it.2015.10.003

[0085] 6. Hank JA, Surfus J, Gan , Albertini M, Lindstrom M, Schiller JH, Hotton KM, Khorsand M, Sondel PM. Distinct clinical and laboratory activity of two recombinant interleukin-2 preparations. Clin Cancer Res. 1999 5:281-9

[0086] 7. Overwijk WW, Restifo NP. B16 as a Mouse Model for Human Melanoma. Curr Protoc Immunol. (2001). CHAPTER: Unit-20.1.

Claims

Claim 1 A method for producing a liquid pharmaceutical composition containing aldesleukin in a pharmaceutical aqueous vehicle, comprising the steps of: fermenting bacterial cells transfected with an expression vector encoding an aldesleukin gene; destroying said bacterial cells; collecting an inclusion body containing aldesleukin; eluting said inclusion body with sodium dodecyl sulfate (SDS) followed by oxidation using an oxidizing compound and ceramic hydroxyapatite chromatography; diluting with an organic nitrile and subjecting to high-performance liquid chromatography (HPLC) with a C4 column; and producing aldesleukin in a liquid pharmaceutical vehicle by rectifying the volume of the aldesleukin and sterilizing it; wherein the method does not include freeze-drying. Claim 2 The method of claim 1, wherein the bacterial cell is an E. coli bacterial cell. Claim 3 A method according to claim 1, wherein the aldesleukin has activity in a pharmaceutical liquid composition for at least one year when measured by an in vitro biological activity assay. Claim 4 A method according to claim 1 comprising an aldesleukin / SDS aggregate distribution in a liquid composition for at least one year in a particle size range of 4-18 nm having a peak at 8 nm when measured by dynamic light scattering. Claim 5 A method according to claim 1, wherein the aldesleukin has therapeutic activity in a liquid composition for at least one year when measured in an animal model of human cancer. Claim 6 Claim 1, wherein the aldesleukin induces proliferation of human regulatory T cells in cell culture.

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