Pharmaceutical compositions and pharmaceutical products of heterodimer human interleukin-15 (hetIL-15).

Abstract

The present disclosure relates to stable pharmaceutical compositions comprising heterodimeric complexes of IL-15 and IL-15Rα, and to pharmaceutical products comprising such compositions. The disclosure also relates to the use of these compositions (e.g., as part of a kit with instructions for use) and pharmaceutical products for the treatment of lymphopenia, cancer, or infectious diseases.

Description

[Technical Field]

[0001] This disclosure relates to pharmaceutical compositions of heterodimer human interleukin-15 (IL-15 / IL-15Rα) complexes, pharmaceutical products containing such pharmaceutical compositions, and the use of such pharmaceutical compositions. In particular, this disclosure relates to stable liquid and solid pharmaceutical formulations containing, for example, the heterodimer IL-15 / IL-15Rα complex disclosed herein.

[0002] Sequence List This application includes a sequence listing, which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the above ASCII document, created on March 31, 2021, is named PAT058681-SL.txt and is 19,095 bytes in size. [Background technology]

[0003] The cytokine interleukin-15 (IL-15) is a member of the alpha-4 helix bundle family of lymphokines produced by many cells in the body. IL-15 plays a crucial role in modulating the activity of both the innate and adaptive immune systems, such as maintaining the memory T cell response to invading pathogens, inhibiting apoptosis, activating dendritic cells, and inducing natural killer (NK) cell proliferation and cytotoxic activity.

[0004] The IL-15 receptor consists of three polypeptides shared by multiple cytokine receptors: type-specific IL-15 receptor alpha ("IL-15Rα"), IL-2 / IL-15 receptor beta (or CD122) ("β"), and common gamma chain (or CD132) ("γ"). IL-15Rα is thought to be expressed by a wide range of cell types, but not necessarily simultaneously with β and γ. IL-15 signaling has been shown to occur via heterodimer complexes of IL-15Rα, β, and γ; via heterodimer complexes of β and γ; or via a subunit found on mast cells, IL-15RX.

[0005] IL-15 specifically binds to IL-15Rα with high affinity via the "sushi domain" in exon 2 of the receptor's extracellular domain. After trans-endosomal recycling and re-transport to the cell surface, these IL-15 complexes acquire the property of activating bystander cells expressing the IL-15Rβγ low-affinity receptor complex, inducing IL-15-mediated signaling via the Jak / Stat pathway. Wild-type soluble IL-15Rα ("sIL-15Rα") has been observed to be cleaved at a cleavage site in the extracellular domain immediately distal to the receptor's transmembrane domain.

[0006] Based on its multifaceted role in the immune system, various therapeutic approaches designed to modulate IL-15-mediated function have been explored. Recent reports suggest that IL-15 maintains its immune-enhancing function when complexed with sIL-15Rα, or the sushi domain. Recombinant IL-15 and IL-15 / IL-15Rα complexes have been shown to promote memory CD8 T cells and NK cells to varying degrees and improve tumor rejection in various preclinical models. Furthermore, tumor targeting of IL-15 or IL-15 / IL-15Rα complex-containing constructs in mouse models resulted in improved antitumor responses in both immunocompetent animals transplanted with syngeneic tumors or in T and B cell-deficient SCID mice (containing NK cells) injected with human tumor cell lines.

[0007] Therapeutic proteins are typically formulated either as an aqueous form ready for parenteral administration or as a lyophilized product to be reconstituted with a suitable diluent before administration.

[0008] Therapeutic proteins in lyophilized materials remain stable over long periods and can be reconstituted to obtain a solution of the active ingredient. The reconstituted solution should preferably have low levels of protein aggregation for delivery to the patient.

[0009] Pharmaceutical compositions have short shelf lives, and formulated proteins may lose biological activity due to chemical and physical instability during storage. Pharmaceutical products containing proteins are highly susceptible to physical and chemical degradation, and the critical stability of proteins in liquid compositions often hinders long-term storage at room temperature or refrigerated conditions, although lyophilized products are generally more stable. Physical and chemical reactions (aggregation [covalent and non-covalent], deamidation, oxidation, clipping, isomerization, denaturation) can occur in solution, potentially leading to increased levels of degradation products and / or loss of biological activity.

[0010] Compositions containing proteins or protein complexes, such as the IL-15 / IL-15Rα complex, need to provide sufficient physical and chemical stability of the protein or protein complex to ensure that the dosage and product safety requirements are met when the molecule is administered to a patient during transport and handling. Specifically, acceptable compositions containing proteins or protein complexes, such as the IL-15 / IL-15Rα complex, must have enhanced stability and minimize protein degradation, particularly protein aggregation, to avoid serious immunogenic reactions and retain biologically active molecules. Furthermore, the composition must also have an acceptable osmotic pressure and pH value for subcutaneous application and be low viscosity as a prerequisite for manufacturing (formulation, filtration, filling) and injectability. However, a long-recognized problem in pharmaceutical formulations of protein-based therapeutics is stability and aggregation, where protein molecules can clump together, forming opaque, insoluble substances or precipitates that can clog syringes or pumps, or cause undesirable reactions after administration, making them unsafe for patients. [Overview of the project] [Means for solving the problem]

[0011] For example, a long-term stable pharmaceutical composition comprising the heterodimer IL-15 / IL-15Rα complex disclosed herein is provided herein. Such a pharmaceutical composition may be a solid composition or a liquid composition.

[0012] In one embodiment, a liquid pharmaceutical composition is disclosed herein, comprising, for example, the heterodimer IL-15 / IL-15Rα complex disclosed herein, about 0.0001% to about 1% (w / v) of a surfactant, optionally further comprising about 1 mM to about 100 mM of a buffer providing a pH in the range of about 4.5 to about 8.5, and optionally further comprising about 1 mM to about 500 mM of at least one stabilizer and its subcombinations. The liquid composition is a ready-to-use liquid composition, rather than being reconstituted from a lyophilized product.

[0013] In one embodiment, a liquid pharmaceutical composition is disclosed herein, for example, comprising the heterodimer IL-15 / IL-15Rα complex disclosed herein, without surfactants, optionally further comprising about 1 mM to about 100 mM of a buffering agent providing a pH in the range of about 4.5 to about 8.5, and optionally further comprising about 1 mM to about 500 mM of at least one stabilizer and its subcombinations. The liquid composition is a ready-to-use liquid composition, rather than being reconstituted from a lyophilized product.

[0014] Also disclosed herein is a pharmaceutical product comprising a container and a liquid pharmaceutical composition disposed within the container, wherein the composition comprises, for example, a heterodimer IL-15 / IL-15Rα complex disclosed herein in a concentration of about 0.1 mg / mL to about 50 mg / mL or about 0.1 mg / mL to about 10 mg / mL, optionally comprising about 0.0001% to about 1% (w / v) of a surfactant, optionally further comprising about 1 mM to about 100 mM of a buffer providing a pH in the range of about 4.5 to about 8.5, optionally further comprising about 1 mM to about 500 mM of at least one stabilizer and its sub-combinations, wherein the liquid pharmaceutical composition is not reconstituted from a lyophilized product.

[0015] In another aspect, solid pharmaceutical compositions are disclosed herein that comprise a heterodimeric IL-15 / IL-15Rα complex, a buffer of about 1 mM to about 100 mM that provides a pH in the range of about 6.5 to about 8.5, at least one stabilizer of about 1 mM to about 500 mM, and sub-combinations thereof.

[0016] Also disclosed herein are pharmaceutical solid products comprising a container and a pharmaceutical composition disposed within the container, wherein the composition comprises, for example, a heterodimeric IL-15 / IL-15Rα complex of about 0.1 mg / mL to about 50 mg / mL or about 0.1 mg / mL to about 10 mg / mL as disclosed herein, a buffer of about 1 mM to 100 mM that provides a pH in the range of about 6.5 to about 8.5, at least one stabilizer of about 1 mM to about 500 mM, and sub-combinations thereof.

[0017] The disclosure also relates to the use of these pharmaceutical compositions for the treatment of lymphopenia, cancer, or infectious diseases, and kits containing these pharmaceutical products and compositions.

[0018] Further compositions, products, methods, regimens, uses, and kits are provided in the following description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1] [FIGS. 1A-C] Total aggregates of formulations F1 - F3 after storage at A) 2 - 8°C for 6 months (24 weeks), B) 25°C for 3 months (12 weeks), and C) 40°C for 1.5 months (6 weeks). [Figure 2] [FIGS. 2A-C] Total degradation products by SEC for formulations F1 - F3 after storage at A) 2 - 8°C for 6 months (24 weeks), B) 25°C for 3 months (12 weeks), and C) 40°C for 1.5 months (6 weeks). [Figure 3-1] [FIGS. 3A-B] Total charge variants for formulations F1 - F3 after storage at A) 2 - 8°C for 6 months (24 weeks) and B) 25°C for 3 months (12 weeks). [Figure 3-2]Same as above. [Figure 4-1] [Figure 4A-D] Purity of A) IL-15 receptor α (IL-15Ra), B) major species of IL-15, C) high molecular weight species of IL-15 (HMW), and D) aglycosylated IL-15 in preparations F1-F3 after storage at 2-8°C for 6 months (24 weeks), as determined by CE-SDS. [Figure 4-2] Same as above. [Figure 5] [Figure 5A-C] Purity of formulations F1-F3 by RP-HPLC after storage at 2-8°C for 6 months (24 weeks), B) 25°C for 3 months (12 weeks), and C) 40°C for 1.5 months (6 weeks). [Figure 6] [Figure 6A-B] Number of invisible particles (SVPs) evaluated by PAMAS in formulations F1-F3 after storage at 2-8°C for 6 months (24 weeks), A) size greater than 2 μm and B) size greater than 10 μm. [Figure 7] [Figure 7] Turbidity of formulations F1-F3 after storage at 2-8°C for 6 months (24 weeks) (NTU = Turbidity Units of Turbidimetric Meter). [Figure 8] [Figure 8A-B] The results of mechanical stress on F2 and F3 are subjected to five freeze / thaw cycles or overnight shaking. Shown are A) total aggregates and B) total fragments as assessed by SEC. [Figure 9-1] [Figure 9A-E] The number of invisible particles (SVPs) A) larger than 2 μm and B) larger than 10 μm, as assessed by PAMAS, in formulations containing acetate in the presence of polysorbate 20 or poloxamer 188 at pH 4.7-5.5 after storage at 2-8°C for 5 months (SVP > 2 μm) and 4 months (SVP > 10 μm), respectively. The number of invisible particles (SVPs) C) larger than 2 μm, D) larger than 5 μm, and E) larger than 10 μm, as assessed by PAMAS, for all formulations after storage at 2-8°C for up to 12 months. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 10][Figure 10] Purity of all formulations by RP-HPLC after storage at 40°C for up to 3 months. [Modes for carrying out the invention]

[0020] The present invention relates to pharmaceutical formulations comprising, for example, the heterodimer IL-15 / IL-15Rα complex disclosed herein. The pharmaceutical formulations may be in solid (e.g., lyophilized) or liquid form.

[0021] To make this disclosure more easily understandable, certain terms are defined throughout the following and detailed descriptions. Unless otherwise defined, all scientific and technical terms used in connection with this disclosure have the same meaning as those generally understood by those skilled in the art. All publications, patent applications, patents, scientific publications, and other references referenced herein are incorporated herein by reference in their entirety. In the event of any conflict between a cited document and the disclosure herein, the specification shall prevail. Throughout the text of this application, in the event of any conflict between the text of the specification (e.g., Table 1) and the sequence listings, the text of the specification shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the scope of the invention. All methods described herein may be performed in any suitable order unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any examples or exemplary words (e.g., "etc.") provided herein is solely for the purpose of further illustrating the invention and does not imply any limitation to the scope of the invention as otherwise asserted.

[0022] Details of one or more aspects and embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings and the claims.

[0023] definition As used herein and in these claims, unless otherwise explicitly indicated, the singular forms "a," "an," and "the" refer to one or more (e.g., at least one) grammatical objects of the article. For example, the term "cell" includes multiple cells, including mixtures thereof.

[0024] Throughout this specification and the following claims, unless the context requires otherwise, the terms “comprise,” and variations such as “comprises” and “compriing,” are used herein in their unrestricted and uncompromising sense. As used herein, the term “comprising” encompasses “including” and “consisting of,” for example, a composition “comprising” X may consist solely of X or may include some additional elements (e.g., X + Y).

[0025] As used herein, "consisting of" excludes any element, step, or component not specified in the aspects, embodiments, and / or claims. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the fundamental novel features of the aspects, embodiments, and / or claims.

[0026] In each example herein, the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced by any one of the other two terms.

[0027] In this specification, the term "or" is used to mean the term "and / or" unless the context explicitly states otherwise, and is used interchangeably with it.

[0028] All numerical expressions, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximate values ​​that vary in increments of (+) or (-)0.1. While not always explicitly stated, it should be understood that all numerical expressions are preceded by the term “about.” The terms “about” and “approximately” used herein in relation to reference numbers and their grammatical equivalents may include the number itself and values ​​within a range of plus or minus 10% from that number. For example, the quantity “about 10” includes 10 and any quantity between 9 and 11. For example, the term “about” in relation to a reference number may also include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. In some cases, a numerical value disclosed collectively may be “about” that number, even if the terms “about” or “approximately” are not specifically mentioned. Furthermore, although not always explicitly stated, the reagents described herein are merely illustrative, and it should be understood that equivalents of such reagents are known in the art.

[0029] The compositions, methods, and uses described herein include polypeptides and nucleic acids having the specified sequence, or a sequence substantially identical or similar thereto, for example, a sequence that is at least about 85%, at least about 90%, at least about 95%, or more identical to the specified sequence. With respect to amino acid sequences, the term “substantially identical” is used herein to mean a first amino acid sequence that is i) identical to the second amino acid sequence, or ii) contains a sufficient or minimum number of amino acid residues that are conservative substitutions of amino acid residues aligned in the second amino acid sequence, such that the first and second amino acid sequences may have a common structural domain and / or common functional activity. For example, an amino acid sequence containing a common structural domain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the reference sequence, for example, the sequence provided herein.

[0030] With respect to nucleotide sequences, the term “substantially identical” is used herein to mean a first nucleic acid sequence containing a sufficient or minimum number of nucleotides identical to the nucleotides aligned in the second nucleic acid sequence, such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or common functional polypeptide activity. For example, a nucleotide sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with a reference sequence, e.g., a sequence provided herein.

[0031] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as the wild-type sequence, or is encoded by substantially the same nucleotide sequence, and may possess one or more of the activities of the wild-type sequence.

[0032] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Preferably, the length of the reference sequence aligned for comparison purposes is at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the length of the reference sequence. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then those molecules are identical at that position (as used herein, "identity" of amino acids or nucleic acids is equivalent to "homology" of amino acids or nucleic acids).

[0033] The percentage of identity between two arrays is a function of the number of gaps that must be introduced for optimal alignment of the two arrays, and the number of identical positions shared by those arrays, taking into account the length of each gap.

[0034] The comparison of sequences between two sequences and the determination of their identity percentage can be achieved using mathematical algorithms. For example, the identity percentage between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm, incorporated into the GAP program in the GCG software package (available from NCBI), using a Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. Preferably, the identity percentage between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap stretching penalty of 4, and a frameshift gap penalty of 5.

[0035] The percentage of identity between two amino acid or nucleotide sequences can be determined using the algorithm by E. Meyers and W. Miller ((1989) CABIOS, 4:11-17), incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

[0036] The protein sequences described herein can be used as "query sequences" to perform searches against public databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul, et al. (1990) J.Mol.Biol.215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the protein molecule of the present invention. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used (available from NBCI).

[0037] It is understood that the molecules described herein may have additional conservative or non-essential amino acid substitutions that do not substantially affect their function.

[0038] The term “amino acid” is intended to encompass all molecules that may be present in polymers of wild-type amino acids, whether natural or synthetic, including both amino- and acid-functional forms. Exemplary amino acids include wild-type amino acids; their analogs, derivatives, and homologues; amino acid analogues with variant side chains; and all stereoisomers of any of the above. As used herein, the term “amino acid” includes both D- and L-optical isomers and peptide mimetic compounds.

[0039] A "conservative amino acid substitution" is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0040] The terms “polypeptide,” “peptide,” and “protein” (when single-chain) are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have undergone modifications, such as disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other operation, such as conjugation with labeled components. Polypeptides can be isolated from natural sources, produced by recombinant techniques from eukaryotic or prokaryotic hosts, or are products of synthetic procedures.

[0041] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” “polynucleotide sequence,” and “polynucleotide” are used interchangeably. These refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides can be single-stranded or double-stranded, and if single-stranded, they can be coding or non-coding (antisense) strands. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The nucleotide sequence may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, for example, by conjugation with labeling components. Nucleic acids may be recombinant polynucleotides, or genomic, cDNA, or semi-synthetic or synthetic polynucleotides linked with other polynucleotides in configurations not naturally occurring or found in nature.

[0042] The term "glycosylation" refers to the bonding of a polysaccharide to a polypeptide. Preferably, the polysaccharide consists of 2 to 12 monosaccharides linked together by glycosidic bonds. The glycoprotein may contain O-linked sugar moieties and / or N-linked sugar moieties. The structure and number of sugar moieties bonded to a particular glycosylation site may be variable. Such sugar moieties may be, for example, N-acetylglucosamine, N-acetylgalactosamine, mannose, galactose, glucose, fucose, xylose, glucuronic acid, iduronic acid, and / or sialic acid.

[0043] The term "N-linked glycosylation" refers to the binding of an amino acid chain of a polysaccharide to an asparagine residue.

[0044] The term "O-linked glycosylation" refers to the binding of the amino acid chain of the sugar portion to a serine or threonine residue.

[0045] The terms "sugar properties" or "glycosylation properties" are used to describe the glycanity of glycosylated polypeptides. These properties include the glycosylation site, or the occupancy of the glycosylation site, or the identity, structure, composition or amount of the glycan and / or non-sugar portion of the polypeptide, or the identity and amount of a particular glycoform.

[0046] As used herein, the term “glycan” can be a monomer or polymer of sugar residues, such as at least three sugars, and can be linear or branched (e.g., having α1,3 arms and α1,6 arms). “Glycan” may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'-sulfo-N-acetylglucosamine, etc.). The term “glycan” includes homopolymers and heteropolymers of sugar residues. The term “glycan” also encompasses the glycan component of complex carbohydrates (e.g., glycoproteins, glycolipids, proteoglycans, etc.). The term also encompasses free glycans, including glycans that have been cleaved from or otherwise released from complex carbohydrates.

[0047] As used herein, the term “glycoprotein” refers to a protein containing a peptide backbone covalently linked to one or more sugar moieties (i.e., glycans). The sugar moieties may be monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. The sugar moieties may consist of a single unbranched chain of sugar residues, or one or more branched chains. Glycoproteins may contain O-linked sugar moieties and / or N-linked sugar moieties. The polysaccharides are linked via either the OH group of serine or threonine (O-glycosylated polypeptide) or the amide group (NH2) of asparagine (N-glycosylated polypeptide). Glycoproteins may be homologous to host cells, or preferably heterologous, i.e., exogenous, to the host cells expressing them (e.g., human proteins produced by CHO cells).

[0048] As used herein, the term "complex carbohydrate" encompasses all molecules in which at least one sugar moiety is covalently bonded to at least one other moiety. Specifically, the term includes all biomolecules having covalently bonded sugar moieties, such as N-linked glycoproteins, O-linked glycoproteins, glycolipids, and proteoglycans.

[0049] As used herein, the term “glycosylation pattern” refers to a set of glycan structures present on a particular sample. For example, a particular complex carbohydrate (e.g., glycoprotein) or a set of complex carbohydrates (e.g., a set of glycoproteins) will have a glycosylation pattern. In some embodiments, the glycosylation pattern of cell surface glycans is referred to. A glycosylation pattern can be characterized by, for example, glycan identity, the amount (absolute or relative) of individual glycans or a particular type of glycan, the degree of occupancy of glycosylation sites, or a combination of such parameters.

[0050] As used herein, in the context of receptor (e.g., native IL-15Rα or IL-15 receptor βγ) and ligand (e.g., native IL-15) interactions, the terms “specifically bind,” “specifically recognize,” and similar terms refer to specific binding or association between the ligand and the receptor. Preferably, the ligand has a higher affinity for the receptor than other molecules. In a specific embodiment, the ligand is native IL-15 and the native receptor is IL-15Rα. In another specific embodiment, the ligand is a native IL-15 / IL-15Rα complex and the native receptor is a βγ receptor complex. In a further embodiment, the IL-15 / IL-15Rα complex binds to the βγ receptor complex and activates IL-15-mediated signaling. Ligands that specifically bind to receptors can be identified, for example, by immunoassays, surface plasmon resonance, e.g., BIAcore, or other techniques known to those skilled in the art.

[0051] When used herein, in the context of compounds or drugs (including protein-based drugs such as polypeptides) that can be obtained from natural sources, e.g., cells, the terms “purified” and “isolated” mean a compound or drug that is substantially free of contaminants from natural sources, e.g., soil particles, minerals, chemicals, and / or not limited to those of environmental origin, but also including cellular materials present in cells, such as cell fragments, cell wall materials, membranes, organelles, bulk nucleic acids, carbohydrates, proteins, and / or lipids. The phrase “substantially free of natural source materials” means a compound or drug preparation isolated from the material from which the preparation is isolated (e.g., cellular components of cells). Thus, isolated compounds or drugs include compound or drug preparations having about 30%>, 20%>, 10%>, 5%, 2%, or less than 1% (by dry weight) of cellular material and / or contaminants.

[0052] IL-15 As used herein, the terms "IL-15" and "interleukin-15" refer to wild-type IL-15 or IL-15 derivatives. As used herein with respect to proteins or polypeptides, the terms "wild-type IL-15" and "wild-type interleukin-15" refer to any mammalian interleukin-15 amino acid sequence, e.g., immature or precursor and mature forms. Non-limiting examples of GeneBank accession numbers for amino acid sequences of wild-type mammalian interleukin-15 in various species include: NP_000576 (human, immature form), CAA62616 (human, immature form), NP_001009207 (domestic cat (Felis catus), immature form), AAB94536 (Norway rat (Rattus norvegicus), immature form), AAB41697 (Norway rat (Rattus norvegicus), immature form), NP_032383 (house mouse (Mus musculus), immature form), AAR19080 (dog), AAB60398 (rhesus macaque (Macaca mulatta), immature form), AAI00964 (human, immature form), AAH23698 (house mouse (Mus Examples include musculus, immature form, and AAH18149 (human). Table 1 shows the amino acid sequences of immature / precursor forms of human IL-15, including the long-chain signal peptide (underlined) provided in SEQ ID NO: 1 and mature human wild-type IL-15 (italicized). In some embodiments, wild-type IL-15 is the immature or precursor form of mammalian IL-15. In other embodiments, IL-15 is the mature form of mammalian IL-15. In specific embodiments, wild-type IL-15 is the precursor form of human IL-15. In yet another embodiment, wild-type IL-15 is the mature form of human IL-15. In one embodiment, the wild-type IL-15 protein / polypeptide is isolated or purified.

[0053] In certain embodiments, mature human IL-15 is [ka] It contains the amino acid sequence.

[0054] The terms “IL-15 derivative” and “interleukin-15 derivative” as used herein with respect to proteins or polypeptides mean: (a) a polypeptide that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the wild-type mammalian IL-15 polypeptide; (b) a polypeptide containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions, and / or substitutions) to the wild-type mammalian IL-15 polypeptide; and / or (c) a fragment of the wild-type mammalian IL-15 polypeptide. Examples of IL-15 derivatives include polypeptides containing the amino acid sequence of the mature form of the mammalian IL-15 polypeptide and heterologous signal peptide amino acid sequences. In one embodiment, the IL-15 derivative is a derivative of the wild-type human IL-15 polypeptide. In another embodiment, the IL-15 derivative is a derivative of an immature or precursor form of the human IL-15 polypeptide. In another embodiment, the IL-15 derivative is a derivative of a mature form of the human IL-15 polypeptide. In another embodiment, the IL-15 derivative is, for example, IL-15N72D as described in Zhu et al., (2009), J.Immunol. 183:3598 or U.S. Patent No. 8,163,879. In another embodiment, the IL-15 derivative is an IL-15 variant as described in U.S. Patent No. 8,163,879. In one embodiment, the IL-15 derivative is isolated or purified.

[0055] Preferably, the IL-15 derivative retains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the function of the wild-type mammalian IL-15 polypeptide that binds to the IL-15Rα polypeptide as measured by assays known in the art, such as ELISA, SPR (e.g., BIAcore®), or co-immunoprecipitation. Preferably, the IL-15 derivative retains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the function of the wild-type mammalian IL-15 polypeptide that induces IL-15-mediated signaling as measured by assays known in the art, such as electrophoretic mobility shift assays, ELISA, or other immunoassays. Preferably, the IL-15 derivative binds to IL-15Rα and / or IL-15Rβγ as evaluated by ligand / receptor binding assays known in the art, for example. The identity percentage can be determined as described above using any method known to those skilled in the art.

[0056] IL-15Rα As used herein, the terms “IL-15Rα” and “interleukin-15 receptor alpha” refer to wild-type IL-15Rα, IL-15Rα derivatives, or wild-type IL-15Rα and IL-15Rα derivatives. As used herein with respect to proteins or polypeptides, the terms “wild-type IL-15Rα” and “wild-type interleukin-15 receptor alpha” refer to any mammalian interleukin-15 receptor alpha ("IL-15Rα") amino acid sequence, e.g., immature or precursor and mature forms and isoforms. Non-limiting examples of GeneBank accession numbers for amino acid sequences of various wild-type mammalian IL-15Rα include NP_002180 (human), ABK41438 (rhesus monkey (Macaca mulatta)), NP_032384 (house mouse (Mus musculus)), Q60819 (house mouse (Mus musculus)), and CAI41082 (human). Table 1 shows the amino acid sequence of the immature form of full-length human IL-15Rα, including the signal peptide (underlined) and mature human wild-type IL-15Rα (italic) provided in SEQ ID NO: 3. Table 1 also shows the amino acid sequence of the immature form of soluble human IL-15Rα, including the signal peptide (underlined) and mature human soluble IL-15Rα (italic) provided in SEQ ID NO: 4. In some embodiments, wild-type IL-15Rα is the immature form of the mammalian IL-15Rα polypeptide. In other embodiments, wild-type IL-15Rα is the mature form of the mammalian IL-15Rα polypeptide. In some embodiments, wild-type IL-15Rα is the soluble form of the mammalian IL-15Rα polypeptide. In other embodiments, wild-type IL-15Rα is the full-length form of the mammalian IL-15Rα polypeptide. In specific embodiments, wild-type IL-15Rα is the immature form of the human IL-15Rα polypeptide. In another embodiment, wild-type IL-15Rα is the mature form of the human IL-15Rα polypeptide. In some embodiments, wild-type IL-15Rα is the soluble form of the human IL-15Rα polypeptide. In other embodiments, wild-type IL-15Rα is the full-length form of the human IL-15Rα polypeptide.In one embodiment, the wild-type IL-15Rα protein or polypeptide is isolated or purified.

[0057] In certain embodiments, the soluble form of human IL-15Rα is [ka] It contains the amino acid sequence.

[0058] The terms “IL-15Rα derivative” and “interleukin-15 receptor alpha derivative” as used herein with respect to proteins or polypeptides mean: (a) polypeptides that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to wild-type mammalian IL-15 polypeptide; (b) polypeptides containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions, and / or substitutions) to wild-type mammalian IL-15Rα polypeptide; (c) fragments of wild-type mammalian IL-15Rα polypeptide; and / or (d) the specified IL-15Rα derivatives as described herein. Examples of IL-15Rα derivatives include polypeptides containing the amino acid sequence of the mature form of mammalian IL-15Rα polypeptide and heterologous signal peptide amino acid sequences. In one embodiment, the IL-15Rα derivative is a derivative of the wild-type human IL-15Rα polypeptide. In one embodiment, the IL-15Rα derivative is a derivative of an immature form of the human IL-15 polypeptide. In one embodiment, the IL-15Rα derivative is a derivative of a mature form of the human IL-15 polypeptide. In one embodiment, the IL-15Rα derivative is a soluble form of the mammalian IL-15Rα polypeptide. In other words, in some embodiments, the IL-15Rα derivative is a soluble form of mammalian IL-15Rα, and these soluble forms are not naturally occurring. Another example of an IL-15Rα derivative is a truncated soluble form of human IL-15Rα. In one embodiment, the IL-15Rα derivative is purified or isolated.

[0059] Preferably, the IL-15Rα derivative retains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the function of the wild-type mammalian IL-15Rα polypeptide that binds to the IL-15 polypeptide as measured by assays known in the art, such as ELISA, SPR (BIAcore®), and co-immunoprecipitation. In one embodiment, the IL-15Rα derivative retains at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the function of the wild-type mammalian IL-15Rα polypeptide that induces IL-15-mediated signaling as measured by assays known in the art, such as electrophoretic mobility shift assays, ELISA, and other immunoassays. In one embodiment, the IL-15Rα derivative binds to IL-15 as evaluated by methods known in the art, such as ELISA.

[0060] Soluble forms of human IL-15Rα are provided herein. Specified IL-15Rα derivatives, which are truncated soluble forms of human IL-15Rα, are also provided herein. These specified IL-15Rα derivatives and soluble forms of human IL-15Rα are based in part on the identification of the proteolytic cleavage site of human IL-15Rα. Soluble forms of IL-15Rα characterized based on the glycosylation of IL-15Rα are further provided herein.

[0061] Proteolytic cleavage of human IL-15Rα occurs between Gly170 and His171, shown in bold and underlined in the provided amino acid sequence of the immature form of wild-type full-length human IL-15Rα: [ka] (Sequence ID 3 in Table 1)

[0062] Accordingly, a soluble form of human IL-15Rα (e.g., a purified soluble form of human IL-15Rα) is provided herein, wherein the amino acid sequence of the soluble form of human IL-15Rα terminates at the site of proteolytic cleavage of wild-type membrane-bound human IL-15Rα. In one embodiment, a soluble form of human IL-15Rα (e.g., a purified soluble form of human IL-15Rα) is provided herein, wherein the amino acid sequence of the soluble form of human IL-15Rα terminates at PQG (Sequence ID 11 in Table 1) (where G is Gly170). In one embodiment, a soluble form of human IL-15Rα (e.g., a purified soluble form of human IL-15Rα) having the amino acid sequence shown in Sequence ID 4 in Table 1 is provided herein. In one embodiment, an IL-15Rα derivative (e.g., a purified and / or soluble form of the IL-15Rα derivative) is provided herein (i) identical to SEQ ID NO: 4 in Table 1 by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%; and (ii) is a polypeptide ending with the amino acid sequence PQG (SEQ ID NO: 11 in Table 1). In one embodiment, a soluble form of human IL-15Rα (e.g., a purified and / or soluble form of human IL-15Rα) having the amino acid sequence of SEQ ID NO: 5 in Table 1 is provided herein. In some embodiments, IL-15Rα derivatives (e.g., purified and / or soluble forms of IL-15Rα derivatives) are provided herein, which are polypeptides identical to at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO: 5 in Table 1, and optionally, the amino acid sequence of the soluble form of the IL-15Rα derivative terminates at PQG (SEQ ID NO: 11 in Table 1).

[0063] In one embodiment, the following IL-15Rα derivatives of human IL-15Rα are provided herein, which are soluble and consist of: (a) the last amino acid at the C-terminus of the IL-15Rα derivative being the amino acid residue PQGHSDTT (SEQ ID NO: 6 in Table 1); (b) the last amino acid at the C-terminus of the IL-15Rα derivative being the amino acid residue PQGHSDT (SEQ ID NO: 7 in Table 1); (c) the last amino acid at the C-terminus of the IL-15Rα derivative being the amino acid residue PQGHSD (SEQ ID NO: 8 in Table 1); (d) the last amino acid at the C-terminus of the IL-15Rα derivative being the amino acid residue PQGHS (SEQ ID NO: 9 in Table 1); or (e) the last amino acid at the C-terminus of the IL-15Rα derivative being the amino acid residue PQGH (SEQ ID NO: 10 in Table 1). In one embodiment, the amino acid sequences of these IL-15Rα derivatives are at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 12 in Table 1. In one embodiment, these IL-15Rα derivatives are purified.

[0064] Furthermore, glycosylated forms of IL-15Rα (for example, purified glycosylated forms of IL-15Rα) are also provided herein, wherein the glycosylation of IL-15Rα accounts for at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or 20%~25%, 20%~30%, 25%~30%, 25%~35%, 30%~35%, 30%~40%, 35%~40%, 35%~45%, 40%~50%, 45%~50%, 20%~40%, or 25%~50% of the mass (molecular weight) of IL-15Rα, as evaluated by techniques known to those skilled in the art. The proportion of IL-15Rα (e.g., purified IL-15Rα) by glycosylation can be determined, for example, by gel electrophoresis and quantitative density measurement of the gel, and by comparing the average mass (molecular weight) of the glycosylated form of IL-15Rα (e.g., purified glycosylated form of IL-15Rα) with that of the unglycosylated form of IL-15Rα (e.g., purified unglycosylated form of IL-15Rα). In one embodiment, the average mass (molecular weight) of IL-15Rα (e.g., purified IL-15Rα) is determined using MALDI-TOF MS spectroscopy on a Voyager De-Pro equipped with a CovalX HM-1 high-mass detector using sinapic acid as the matrix, and the proportion of the mass accounted for by glycosylation is determined by comparing the mass of the glycosylated form of IL-15Rα (e.g., purified glycosylated form of IL-15Rα) with the mass of the unglycosylated form of IL-15Rα (e.g., purified unglycosylated form of IL-15Rα).

[0065] Furthermore, glycosylated forms of IL-15Rα are also provided herein, in which IL-15Rα is glycosylated (N- or O-glycosylated) at a certain amino acid residue. In one embodiment, the glycosylation site is as follows: (i) amino acid sequence in IL-15Rα [ka] (ii) O-glycosylation of threonine at position 5 of (Sequence ID 13 in Table 1); (ii) Amino acid sequence in IL-15Rα [ka] (iii) O-glycosylation of serine at position 7 of (Sequence ID 13 in Table 1); (iii) Amino acid sequence in IL-15Rα [ka] (Sequence ID 14 in Table 1) Amino acid sequence on serine at position 8 or in IL-15Rα [ka] (Sequence ID 15 in Table 1) N-glycosylation on the serine at position 8; (iv) Amino acid sequence in IL-15Rα ITCPPPMSVEHADIWVKSYSLYSRERYICNS (Sequence ID 15 in Table 1) N-glycosylation on Ser18; (v) Amino acid sequence in IL-15Rα [ka] (Sequence ID 15 in Table 1) N-glycosylation of serine at position 20; (vi) Amino acid sequence in IL-15Rα [ka] N-glycosylation of serine at position 23 of (Sequence ID 15 in Table 1); and / or amino acid sequence in (vii)IL-15Rα [ka] Human IL-15Rα is provided herein in which 1, 2, 3, 4, 5, 6, 7, or all of the N-glycosylations on the serine at position 31 of (SEQ ID NO: 15 in Table 1) are glycosylated. In one embodiment, glycosylated IL-15Rα is wild-type human IL-15Rα. In one embodiment, glycosylated IL-15Rα is an IL-15Rα derivative of human IL-15Rα. In one embodiment, glycosylated IL-15Rα is wild-type soluble human IL-15Rα, for example, SEQ ID NO: 4 or 5 in Table 1. In one embodiment, glycosylated IL-15Rα is an IL-15Rα derivative that is a soluble form of human IL-15Rα. In one embodiment, glycosylated IL-15Rα is purified or isolated.

[0066] IL-15 / IL-15Rα complex As used herein, the terms “IL-15 / IL-15Rα complex,” “IL-15 / IL-15Rα heterocomplex,” or “hetIL-15” refer to a complex comprising IL-15 and IL-15Rα covalently or noncovalently bound to each other. In a preferred embodiment, IL-15Rα has a relatively high affinity for IL-15, for example, measured by techniques known in the art, such as KinExA assay, plasma surface resonance (e.g., BIAcore® assay), with a K of 10–50 pM. D It has the following characteristics. In one embodiment, the IL-15 / IL-15Rα complex induces IL-15-mediated signaling as measured by assays well known in the art, such as electrophoretic mobility shift assays, ELISA, and other immunoassays. In one embodiment, the IL-15 / IL-15Rα complex retains the ability to specifically bind to the βγ chain. In one embodiment, the IL-15 / IL-15Rα complex is isolated from cells.

[0067] Provided herein is a complex comprising IL-15 covalently or noncovalently bound to the interleukin-15 receptor alpha ("IL-15Rα") (also referred herein as the "IL-15 / IL-15Rα complex" or "IL-15 / IL-15Rα heterocomplex"), which binds to the βγ subunit of the IL-15 receptor and induces IL-15 signaling (e.g., Jak / Stat signaling) to enhance IL-15-mediated immune function. The IL-15 / IL-15Rα complex can bind to the βγ receptor complex.

[0068] The IL-15 / IL-15Rα complex can be composed of wild-type IL-15 or an IL-15 derivative and wild-type IL-15Rα or an IL-15Rα derivative. In one embodiment, the IL-15 / IL-15Rα complex includes wild-type IL-15 or an IL-15 derivative and the above-mentioned IL-15Rα.

[0069] In one embodiment, the IL-15 / IL-15Rα complex comprises human IL-15 complexed with a soluble form of human IL-15Rα. The complex may contain IL-15 covalently or non-covalently bound to the soluble form of IL-15Rα. In a preferred embodiment, human IL-15 is non-covalently bound to the soluble form of IL-15Rα. In a particularly preferred embodiment, the IL-15 / IL-15Rα complex comprises human IL-15, including SEQ ID NO: 2, non-covalently bound to a soluble form of human IL-15Rα, including SEQ ID NO: 5.

[0070] In one embodiment, the IL-15 / IL-15Rα complex comprises wild-type IL-15 or an IL-15Rα derivative and soluble IL-15Rα (e.g., wild-type soluble human IL-15Rα). In one embodiment, the IL-15 / IL-15Rα complex is composed of an IL-15 derivative and an IL-15Rα derivative. In one embodiment, the IL-15 / IL-15Rα complex is composed of wild-type IL-15 and an IL-15Rα derivative. In one embodiment, the IL-15Rα derivative is a soluble form of IL-15Rα. Specific examples of the soluble form of IL-15Rα are those described above. In one embodiment, the soluble form of IL-15Rα lacks the transmembrane domain of wild-type IL-15Rα and, optionally, the intracellular domain of wild-type IL-15Rα. In one embodiment, the IL-15Rα derivative is the extracellular domain of wild-type IL-15Rα or a fragment thereof. In one embodiment, the IL-15Rα derivative is the sushi domain or a fragment of the extracellular domain containing exon 2 of wild-type IL-15Rα. In one embodiment, the IL-15Rα derivative comprises the sushi domain or a fragment of the extracellular domain containing exon 2 of wild-type IL-15Rα and at least one amino acid encoded by exon 3. In one embodiment, the IL-15Rα derivative comprises the sushi domain or a fragment of the extracellular domain containing exon 2 of wild-type IL-15Rα and the IL-15Rα hinge region or a fragment thereof. In one embodiment, IL-15Rα comprises the amino acid sequence of Sequence ID No. 5 in Table 1.

[0071] In one embodiment, the IL-15Rα derivative contains a mutation in the extracellular domain cleavage site that inhibits cleavage by an endogenous protease that cleaves wild-type IL-15Rα. In one embodiment, the extracellular domain cleavage site of IL-15Rα is replaced with a cleavage site that is recognized and cleaved by a heterologous known protease.

[0072] In one embodiment, IL-15 is encoded by a nucleic acid sequence optimized to enhance IL-15 expression using, for example, the methods described in International Publication No. 2007 / 084342 and International Publication No. 2010 / 020047; and U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498.

[0073] In one embodiment, an IL-15 / IL-15Rα complex is provided herein, comprising human IL-15Rα glycosylated at 1, 2, 3, 4, 5, 6, 7, or all of the above glycosylation sites, with reference to SEQ ID NOs. 13, 14, and 15 in Table 1. In one embodiment, glycosylated IL-15Rα is wild-type human IL-15Rα. In one embodiment, glycosylated IL-15Rα is an IL-15Rα derivative of human IL-15Rα. In one embodiment, glycosylated IL-15Rα is wild-type soluble human IL-15Rα, for example, SEQ ID NOs. 4 or 5 in Table 1. In one embodiment, glycosylated IL-15Rα is an IL-15Rα derivative that is a soluble form of human IL-15Rα. In one embodiment, the IL-15 / IL-15Rα complex is purified or isolated.

[0074] In addition to IL-15 and IL-15Rα, the IL-15 / IL-15Rα complex may contain heterologous molecules. In some embodiments, heterologous molecules increase protein stability. Non-limiting examples of such molecules include polyethylene glycol (PEG), which increases the in vivo half-life of IL-15 or IL-15Rα, the Fc domain or fragment thereof of IgG immunoglobulin, or albumin. In some embodiments, IL-15Rα is conjugated / fused to the Fc domain or fragment thereof of an immunoglobulin (e.g., IgG1). In specific embodiments, the IL-15RαFc fusion protein contains the amino acid sequence of SEQ ID NO: 16 or 17 in Table 1. In another embodiment, the IL-15RαFc fusion protein is the IL-15Rα / Fc fusion protein described in Han et al., (2011), Cytokine 56:804-810, U.S. Patent No. 8,507,222, or U.S. Patent No. 8,124,084. In these IL-15 / IL-15Rα complexes containing heterologous molecules, the heterologous molecules can be conjugated to IL-15 and / or IL-15Rα. In one embodiment, the heterologous molecule is conjugated to IL-15Rα. In another embodiment, the heterologous molecule is conjugated to IL-15. In yet another embodiment, the heterologous molecule is bound to IL-15Rα and to IL-15.

[0075] [Table 1]

[0076] [Table 2]

[0077] [Table 3]

[0078] The components of the IL-15 / IL-15Rα complex can be directly fused using either non-covalent or covalent bonds (e.g., by combining amino acid sequences via peptide bonds) and / or combined using one or more linkers. Suitable linkers for the preparation of the IL-15 / IL-15Rα complex include peptides, alkyl groups, chemically substituted alkyl groups, polymers, or any other covalent or non-covalent chemicals that can be bound together to two or more components. Polymer linkers include any polymer known in the art, e.g., polyethylene glycol (PEG). In some embodiments, the linker is a peptide with an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. In one embodiment, the linker is long enough to preserve the ability of IL-15 to bind to IL-15Rα. In other embodiments, the linker is long enough to conserve the ability of the IL-15 / IL-15Rα complex to bind to the βγ receptor complex and act as an agonist mediating IL-15 signaling.

[0079] In some embodiments, the IL-15 / IL-15Rα complex is pre-coupled before use in the methods described herein (e.g., before contacting cells with the IL-15 / IL-15Rα complex or before administering the IL-15 / IL-15Rα complex to a subject). In other embodiments, the IL-15 / IL-15Rα complex is not pre-coupled before use in the methods described herein.

[0080] In specific embodiments, the IL-15 / IL-15Rα complex enhances or induces immune function in a subject by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% compared to immune function in a subject not administered with the IL-15 / IL-15Rα complex, using assays known in the art, such as ELISPOT, ELISA, and cell proliferation assays. In one embodiment, the immune function is cytokine release (e.g., interferon-gamma, IL-2, IL-5, IL-10, IL-12, or transforming growth factor (TGF)-beta). In some embodiments, IL-15-mediated immune function is NK cell proliferation, which can be assayed, for example, by flow cytometry to detect the number of cells expressing an NK cell marker (e.g., CD56). In some embodiments, IL-15-mediated immune function is antibody production, which can be assayed, for example, by ELISA. In some embodiments, IL-15-mediated immune function is effector function, which can be assayed, for example, by cytotoxicity assays or other assays known in the art.

[0081] In specific embodiments, examples of immune function enhanced by the IL-15 / IL-15Rα complex include lymphocyte proliferation / expansion (e.g., increased lymphocyte count), inhibition of lymphocyte apoptosis, activation of dendritic cells (or antigen-presenting cells), and / or antigen presentation. In certain embodiments, the immune function enhanced by the IL-15 / IL-15Rα complex is CD4 + T cells (e.g., Th1 and Th2 helper T cells), CD8 +T cells (e.g., cytotoxic T lymphocytes, alpha / beta T cells, and gamma / delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, dendritic cells (immature or mature), antigen-presenting cells, macrophages, mast cells, natural killer T cells (NKT cells), tumor-resident T cells, CD122 + This involves the proliferation / expansion or activation of T cells or natural killer cells (NK cells). In one embodiment, the IL-15 / IL-15Rα complex increases the proliferation / expansion or number of lymphocyte progenitor cells. In some embodiments, the IL-15 / IL-15Rα complex is CD4 + T cells (e.g., Th1 and Th2 helper T cells), CD8 + T cells (e.g., cytotoxic T lymphocytes, alpha / beta T cells, and gamma / delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, dendritic cells (immature or mature), antigen-presenting cells, macrophages, mast cells, natural killer T cells (NKT cells), tumor-resident T cells, CD122 + The number of T cells or natural killer cells (NK cells) is increased by approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, or more compared to the negative control (for example, the number of cells that are not treated with, cultured with, or in contact with the IL-15 / IL-15Rα complex described herein).

[0082] In specific embodiments, the IL-15 / IL-15Rα complex increases the expression of IL-2 in whole blood activated by Staphylococcus enterotoxin B (SEB). For example, the IL-15 / IL-15Rα complex increases IL-2 expression by at least approximately 2-fold, 3-fold, 4-fold, or 5-fold compared to the expression of IL-2 when SEB is used alone.

[0083] In the present invention, the terms “subject” and “patient” include any human or non-human animal. The term “non-human animal” includes all vertebrates, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, mammals and non-mammals. In preferred embodiments, the subject is a human patient. The terms “subject” and “patient” are used interchangeably herein.

[0084] The term "pharmaceutical preparation" or "pharmaceutical composition" means a preparation containing, for example, the heterodimer IL-15 / IL-15Rα complex described herein in a form that enables the biological activity of the complex, and which does not contain additional components that are unacceptably toxic to the subject to which the preparation is administered.

[0085] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that, within the bounds of sound medical judgment, is suitable for contact with a subject, such as mammalian or human tissue, without excessive toxicity, irritation, allergic response, and other problematic complications commensurate with a reasonable benefit / risk ratio, and does not impede the efficacy of the biological activity of the active ingredient.

[0086] For example, with respect to compounds such as the IL-15 / IL-15Rα complex or other drugs, the term "administer" is used to refer to the delivery of that compound to a patient via any route.

[0087] As used herein, “therapeutic dose” means the amount of, for example, the IL-15 / IL-15Rα complex disclosed herein, which, in a single or multiple dose to a patient (such as a human), is effective in treating, preventing, preventing the onset of, curing, delaying, reducing the severity of, or improving at least one symptom of a disorder or recurrent disorder, or in extending the patient’s survival beyond what would be expected in the absence of such treatment. When applied to an individual active ingredient administered alone (for example, the IL-15 / IL-15Rα complex disclosed herein), the term means that ingredient alone. When applied to a combination, the term means the combined amount of active ingredients that produce a therapeutic effect, whether administered together, sequentially, or simultaneously.

[0088] The terms "combination" or "in combination with" do not mean that the treatments or therapeutic agents must be administered simultaneously and / or formulated together for delivery, although their delivery methods are within the scope of those described herein. The therapeutic agents in a combination may be administered simultaneously with, before, or after one or more other additional treatments or therapeutic agents. The therapeutic agents or treatment protocols may be administered in any order. Generally, each agent is administered in a dose and / or according to a time schedule determined for that agent. It is further recognized that the additional therapeutic agents used in this combination may be administered together in a single composition or separately in different compositions. Generally, it is expected that the additional therapeutic agents used in a combination will be used at levels that do not exceed those used individually. In some embodiments, the levels used in a combination are lower than those used individually.

[0089] The terms “to treat,” “treatment,” and “to treat” refer to a reduction or improvement in the progression, severity, and / or duration of a disorder, e.g., proliferative disorder, or improvement of one or more symptoms (preferably one or more identifiable symptoms) of a disorder resulting from the administration of one or more treatments. For example, the terms “to treat,” “treatment,” and “to treat” refer to improvement in at least one measurable physical parameter of proliferative disorder that is not necessarily identifiable by the patient, e.g., tumor growth. Preferably, the terms “to treat,” “treatment,” and “to treat” refer to inhibition of the progression of a proliferative disorder, e.g., physically by stabilizing an identifiable symptom, physiologically by stabilizing a physical parameter, or both. Preferably, the terms “to treat,” “treatment,” and “to treat” refer to a reduction or stabilization of tumor size or the number of cancer cells.

[0090] The terms “disease” and “disorder” are used interchangeably to refer to pathological conditions, particularly pathological conditions. In certain embodiments, the terms “disease” and “disorder” are used interchangeably to refer to diseases affected by IL-15 signaling and / or diseases affected by enhancement of immune effector responses.

[0091] As used herein, the terms “therapies” and “therapy” may refer to any protocol, method, composition, formulation, and / or agent that can be used in the prevention, treatment, management, or improvement of diseases, such as cancer, infectious diseases, lymphopenia, and immunodeficiency, or symptoms associated therewith. In certain embodiments, the terms “therapies” and “therapy” may refer to biotherapies, supportive therapies, and / or other therapies that are useful in the treatment, management, prevention, or improvement of diseases or symptoms associated therewith that are known to those skilled in the art.

[0092] The terms "anti-cancer effect" or "anti-tumor effect" refer to biological effects that can be manifested by various means, including, but are not limited to, a reduction in tumor volume, a reduction in the number of cancer cells or tumor cells, a reduction in the number of metastases, an extension of life expectancy, a reduction in the proliferation of cancer cells or tumor cells, a reduction in the survival of cancer or tumor cells, or an improvement in various physiological symptoms associated with cancerous conditions.

[0093] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described herein include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain tumor, lymphoma, leukemia, and lung cancer. The terms "tumor" and "cancer" are used interchangeably herein, and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. As used in the present invention, the terms "cancer" or "tumor" include pre-malignant and malignant cancers and tumors.

[0094] As used herein, the terms “immune effector,” “effector function,” or “response” refer to the function or response of, for example, immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response refers to the properties of T cells or NK cells that promote the killing of target cells or the inhibition of their growth or proliferation. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector function or response. Other examples of T cell effector functions include cytolytic activity or helper activity, for example, cytokine secretion.

[0095] The phrase “means for administration” is used to indicate any device available for systemically administering a drug to a patient, including, but not limited to, pre-filled syringes, vials and syringes, injection pens, autoinjectors, intravenous (IV) infusions and bags, pumps, patch pumps, etc. Using such items, a patient can self-administer the drug (i.e., administer the drug on their behalf) or a physician can administer the drug. In some embodiments of the methods, kits and uses of this disclosure, for example, the IL-15 / IL-15Rα complex disclosed herein is delivered to the patient via an IV route. In some embodiments of the methods, kits and uses of this disclosure, for example, the IL-15 / IL-15Rα complex disclosed herein is delivered to the patient via a subcutaneous (SC) route.

[0096] Where the dose of the IL-15 / IL-15Rα complex is referenced herein, the dose is based on the mass of single-stranded IL-15. The single-stranded IL-15 equivalent is calculated from (i) the mass of the IL-15 / IL-15Rα complex determined by amino acid analysis in a specific preparation experimentally determined by RP-HPLC or amino acid analysis, and (ii) the ratio of IL-15 to IL-15Rα (e.g., soluble IL-15Rα).

[0097] The term “pharmaceutical product” means a container (e.g., a pen, syringe, bag, pump, etc.) containing a pharmaceutical composition placed inside the container. “Container” means any means for holding a liquid or solid pharmaceutical composition, e.g., a pen, syringe, vial, autoinjector, patch, etc. for storing, transporting, and maintaining the composition of the Disclosure. Examples of pharmaceutically acceptable containers for use as part of the disclosed pharmaceutical include syringes (e.g., available from Beckton Dickinson, Nuova Ompi, et al.), stoppered vials, cartridges, autoinjectors, patch pumps, and injection pens.

[0098] A “stable” composition is one in which the proteins or protein complexes contained therein, for example, those disclosed herein, essentially retain their stability (e.g., physical stability and / or chemical stability and / or biological activity) after storage. Various analytical techniques for measuring protein stability are available in the art and are outlined in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected time. A “stable liquid pharmaceutical composition” or “stable solid pharmaceutical composition” is a pharmaceutical composition in which there are no significant physical, chemical, and / or biological changes in the protein, such as the IL-15 / IL-15Rα complex disclosed herein, when stored at refrigerated temperatures (approximately 2°C to 8°C) for at least approximately 6 months, at least approximately 12 months, at least approximately 2 years, or at least approximately 3 years; or at room temperature (approximately 20°C to 25°C) for at least approximately 3 months, at least approximately 6 months, and at least approximately 1 year; or under stress conditions (approximately 40°C) for at least approximately 1 month, at least approximately 3 months, and at least approximately 6 months. Various stability criteria can be used, for example, less than 10% or less of the protein is degraded, less than 5% (measured by, for example, SEC purity, RP-HPLC purity, charge heterogeneity by AEX, CE-SDS purity (non-reduced), etc.). Alternatively, stability can be indicated if the solution remains clear to slightly milky by visual analysis or turbidimetric analysis. Alternatively, stability can be demonstrated if the concentration, pH, and osmolality of the composition have a variation of no more than + / -10% over a given period, for example, at least about 3 months, at least about 6 months, and at least about 1 year. Alternatively, stability can be demonstrated using the biological activity described herein.Alternatively, stability may be indicated if less than 1%, preferably less than 0.5%, of aggregates are formed over a given period, for example, at least 1 month, at least 3 months, at least 6 months, or at least 12 months (as measured by AP-SEC, DP-SEC, etc.). Alternatively, stability may be indicated if, after storage at 2-8°C for 6 months, the formation of degradation products (as measured by RP-HPLC (total impurities)) is < about 10%, < about 15%, < about 10%, or < about 5%.

[0099] For example, if a protein, e.g., IL-15 and / or IL-15Rα (e.g., disclosed herein), or a protein complex, e.g., IL-15 / IL-15Rα complex (e.g., disclosed herein), does not show a significant increase in aggregation, precipitation, and / or denaturation when measured by visual inspection of color and / or transparency (turbidity), or by UV light scattering, size exclusion chromatography (SEC), SDS-PAGE, dynamic light scattering (DLS), and / or other methods known in the art, then the protein, e.g., IL-15 and / or IL-15Rα complex (e.g., disclosed herein), maintains its physical stability in a pharmaceutical composition. In addition, the protein conformation should not be significantly altered when evaluated by, for example, fluorescence spectroscopy (to determine the tertiary structure), circular dichroism spectroscopy (to determine the secondary and tertiary structures), and / or FTIR spectroscopy (to determine the secondary structure).

[0100] If it does not exhibit significant chemical modification, the protein, e.g., IL-15 and / or IL-15Rα (e.g., disclosed herein), or the protein complex, e.g., IL-15 / IL-15Rα complex (e.g., disclosed herein), retains its chemical stability in the pharmaceutical composition. Chemical stability can be assessed by detecting and / or quantifying chemically modified forms of the protein, e.g., IL-15 and / or IL-15Rα (e.g., disclosed herein), or the protein complex, e.g., IL-15 / IL-15Rα complex (e.g., disclosed herein). Degradation processes that often alter the chemical structure of proteins include hydrolysis or clipping (e.g., evaluated by methods such as size exclusion chromatography [SEC], SDS-PAGE, and / or MALDI-TOF MS), oxidation (e.g., evaluated by methods such as mass spectrometry or peptide mapping in combination with MALDI-TOF MS), deamide (e.g., evaluated by methods such as cation exchange chromatography (CEX), capillary isoelectric focusing, peptide mapping, and isoaspartic acid measurement), and isomerization (e.g., evaluated by measurements such as isoaspartic acid content, peptide mapping, or other methods known in the art).

[0101] If the biological activity of a protein, such as IL-15 and / or IL-15Rα (as disclosed herein, for example), or a protein complex, such as the IL-15 / IL-15Rα complex (as disclosed herein, for example), at a given time point is within a predetermined range of the biological activity shown at the time the pharmaceutical composition is prepared, then the protein, such as IL-15 and / or IL-15Rα (as disclosed herein, for example), or the protein complex, such as the IL-15 / IL-15Rα complex (as disclosed herein, for example), retains its biological activity in the pharmaceutical composition. The biological activity of proteins, such as IL-15 and / or IL-15Rα, or protein complexes, such as the IL-15 / IL-15Rα complex, can be determined, for example, by cytokine release assays (e.g., interferon-gamma, IL-2, IL-5, IL-10, IL-12, or transforming growth factor (TGF)-beta), NK cell proliferation assays determined by flow cytometry to detect the number of cells expressing NK cell markers (e.g., CD56), antibody production assays which can be determined by ELISA, or effector function by cytotoxicity assays. The biological activity of proteins, such as IL-15 and / or IL-15Rα, or protein complexes, such as the IL-15 / IL-15Rα complex, can be determined, for example, by assaying the activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells. Activity can be expressed relative to the "original activity" by comparing the activity of a sample containing the proteins described herein, e.g., IL-15 and / or IL-15Rα, or protein complexes, e.g., IL-15 / IL-15Rα complexes, after storage, and by comparing the above sample to a reference sample.

[0102] As used herein, “RP-HPLC purity” refers to the percentage of IL-15-related peaks and IL-15Rα-related peaks obtained by RP-HPLC, which can be used to assess the stability of proteins, such as IL-15 and IL-15Rα as disclosed herein. RP-HPLC is used to separate proteins, such as IL-15 and IL-15Rα and their variants, by their hydrophobicity. Other peaks obtained by RP-HPLC may contain fragmented, isomerized, and oxidized species of IL-15, IL-15Rα, and / or the IL-15 / IL-15Rα complex disclosed herein.

[0103] As used herein, “charge heterogeneity by AEX” refers to the percentage of basic or acidic variants in AEX and can be used to evaluate the stability of IL-15, IL-15Rα, and / or the IL-15 / IL-15Rα complex disclosed herein. AEX is used to evaluate the charge heterogeneity of the IL-15 / IL-15Rα complex disclosed herein, for example, by measuring the percentage of acidic and basic variants.

[0104] As used herein, “Purity by SEC” refers to the percentage of monomers in SEC and can be used to evaluate the stability of IL-15, IL-15Rα, and / or the IL-15 / IL-15Rα complexes disclosed herein. SEC is used to separate the isolated monomers disclosed herein from aggregates and fragments by their size under non-denaturing conditions. The sum of peaks eluting before the main peak is reported as the percentage of aggregated products (AP-SEC), and the sum of peaks eluting after the main peak is reported as the percentage of decomposition products (DP-SEC).

[0105] In the present invention, "purity by CE-SDS" refers to the percentage of intact IL-15Rα, intact IL-15, IL-15 high molecular weight (HMW) species, and aglycosylated IL-15 in CE-SDS, and can be used, for example, to evaluate the stability of the IL-15 / IL-15Rα complex disclosed herein. CE-SDS is used to separate by-products and degradation products from the IL-15 / IL-15Rα complex disclosed herein, for example, due to their molecular size under non-reducing conditions. The sum of the peaks separated from the identified IL-15Rα and IL-15 related peaks is reported as a percentage of impurities.

[0106] As used herein, the term “liquid pharmaceutical composition” refers to an aqueous composition that is not reconstituted from a lyophilized product and contains at least, for example, the IL-15 / IL-15Rα complex disclosed herein and at least one additional excipient (e.g., a surfactant or buffer). The liquid pharmaceutical composition may also contain additional excipients (e.g., stabilizers) and additional active ingredients. This type of formulation is also called a “ready-to-use” formulation.

[0107] As used herein, the term “lyophilized” refers to a dry (e.g., freeze-dried) pharmaceutical composition with little to no water content. Cryopreservation techniques for proteins are known in the art; see, for example, Rey & May (2004) Freeze-Drying / Lyophilization of Pharmaceutical & Biological Products ISBN 0824748689. Because reconstituted lyophilized materials tend to have a limited shelf life, they are typically reconstituted to give aqueous compositions for immediate use (e.g., within 1-10 days).

[0108] As used herein, the term “solid pharmaceutical composition” refers to a pharmaceutical composition that is reconstituted from a lyophilized product prior to administration and contains, for example, the IL-15 / IL-15Rα complex disclosed herein, at least one buffer, at least one stabilizer, and at least one tonic modifier. The solid pharmaceutical composition may also contain additional excipients and additional active ingredients.

[0109] As used herein, the term “buffer” refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical composition. Buffers may be present in the liquid or solid (e.g., lyophilized) formulations of the present invention. Suitable buffers for use with the pharmaceutical compositions of this disclosure include, but are not limited to, gluconate buffers, histidine buffers, citrate buffers, phosphate [e.g., sodium and / or potassium] buffers, succinate [e.g., sodium] buffers, acetate buffers [e.g., sodium or potassium], Tris buffers, glycine, arginine, and combinations thereof. Buffers are generally used at concentrations of about 1 mM to about 100 mM, about 10 mM to about 50 mM, about 15 mM to about 30 mM, and about 20 mM to about 30 mM. Regardless of the buffer used, the pH can be adjusted to the desired value, for example, in the range of about 4.5 to about 8.5, using acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, and citric acid, sodium hydroxide, and potassium hydroxide.

[0110] Stabilizers help prevent the oxidation and aggregation of proteins in pharmaceutical compositions. Various analytical methods can be used to evaluate the stability of a given composition. For example, RP-HPLC can be used to assay the level of oxidation products (pre-main peak) in the liquid and / or solid pharmaceutical compositions disclosed herein, and SEC can be used to assay the level of aggregation in the liquid and / or solid pharmaceutical compositions disclosed herein. Suitable stabilizers for use in the liquid and / or solid pharmaceutical compositions of this disclosure include ionic and nonionic stabilizers, and examples of stabilizers include sugars (e.g., monosaccharides, disaccharides, trisaccharides, and oligosaccharides), amino acids (e.g., glycine, arginine), sugar alcohols / polyols (e.g., mannitol, sorbitol, xylitol, dextran, glycerol, arabitol, propylene glycol, polyethylene glycol), and cyclodextrins (e.g., hydroxypropyl-β-cyclodextrin, sulfobutylethyl-β-cyclodextrin). Examples of stabilizers include, but are not limited to, clodextrin, β-cyclodextrin, polyethylene glycol (e.g., PEG3000, PEG3350, PEG4000, PEG6000), albumin (e.g., human serum albumin (HSA), bovine serum albumin (BSA)), salts (e.g., sodium chloride, magnesium chloride, calcium chloride), chelating agents (e.g., EDTA), and antioxidants (e.g., sodium ascorbate, cysteine, sodium bisulfate, sodium citrate, methionine, benzyl alcohol). Two or more stabilizers selected from the same or different groups may be present in the liquid and / or solid pharmaceutical composition.

[0111] The term "bulking agent" includes agents that can provide additional structure to freeze-dried products (e.g., providing a pharmaceutically acceptable cake). Commonly used bulking agents include mannitol, glycine, lactose, and sucrose. In addition to providing a pharmaceutically acceptable cake, bulking agents typically impart useful qualities to solid compositions, such as altering the decay temperature, providing freeze-thaw protection, and further enhancing protein stability over long-term storage. These agents may also function as tonic modifiers and / or stabilizers.

[0112] The term "cryoprotectant" generally refers to agents that stabilize proteins or protein derivatives against freeze-induced stress. They typically also provide protection during primary and secondary drying and long-term storage of products. Examples of such cryoprotectants include polymers such as dextran and polyethylene glycol; sugars such as sucrose, glucose, trehalose, and lactose; surfactants such as polysorbates; and amino acids such as glycine, arginine, and serine.

[0113] The term "lyphoprotectant" includes agents that provide stability to proteins by providing a possibly amorphous, glassy matrix during drying or "dehydration" processes (primary and secondary drying cycles) and by binding to proteins or protein complexes, for example, as disclosed herein, via hydrogen bonding, for example, by exchanging water molecules removed during the drying process. This maintains the protein structure, minimizes protein degradation during the freeze-drying cycle, and improves the long-term stability of the protein or protein derivative. Examples include polyols or sugars such as sucrose and trehalose.

[0114] "Reconstitution time" is the time required to rehydrate a solid formulation with a liquid to provide, for example, a clear, particle-free solution.

[0115] The term "isotonic" means that the preparation of interest has essentially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of approximately 270–328 mOsm. Slightly hypotonic osmotic pressures are approximately 250–269 mOsm, and slightly hypertonic osmotic pressures are approximately 328–350 mOsm. Osmotic pressure is measured, for example, using a vapor pressure or ice-freezing osmometer.

[0116] Examples of tonic modifiers useful in the formulations of the present invention include salts such as NaCl, KCl, MgCl2, and CaCl2, which are used to control osmotic pressure. In addition, cryoprotectants / lioprotectants and / or bulking agents such as sucrose, mannitol, and glycine, as well as others, may function as tonic modifiers.

[0117] Pharmaceutical composition This disclosure relates to stable liquid and solid pharmaceutical compositions comprising, for example, a buffer, a surfactant, and a stabilizer, comprising at least one IL-15 / IL-15Rα complex as disclosed herein and described above. In some embodiments, the pharmaceutical composition comprises at least two additional excipients, such as a buffer and a stabilizer. In some embodiments, the pharmaceutical composition comprises a buffer, at least one stabilizer, and a surfactant.

[0118] The object of the present invention is to provide a pharmaceutical composition that is stable after storage and after delivery, comprising, for example, at least one IL-15 / IL-15Rα complex disclosed herein. A stable composition is one in which at least one IL-15 / IL-15Rα complex, for example, disclosed herein, retains its physical and / or chemical stability, and / or retains its biological activity after storage. For example, the pharmaceutical composition should exhibit a shelf life of more than about 6 months, more than about 12 months, more than about 18 months, more than about 24 months, or more than about 36 months according to lyophilization and storage in the case of a liquid formulation. The stability of the pharmaceutical composition can be measured using a bioactivity assay.

[0119] Generally, pharmaceutical compositions are formulated with excipients suitable for the intended route of administration (for example, oral compositions generally include inert diluents or food carriers). Examples of routes of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., ingested or inhaled), transdermal (topical), transmucosal, and rectal. The compositions of this disclosure are suitable for parenteral administration, such as intravenous, intramuscular, intraperitoneal, or subcutaneous injection; they are particularly suitable for subcutaneous injection.

[0120] For example, the viscosity of a pharmaceutical composition containing at least one IL-15 / IL-15Rα complex disclosed herein can be controlled for subcutaneous or intravenous administration. Viscosity may be affected by protein concentration and pH. For example, increasing protein concentration may increase viscosity. Increasing pH may decrease the viscosity of the IL-15 / IL-15Rα complex composition. In some compositions, sodium chloride is added to lower the viscosity of the formulation. Additional components that may affect the viscosity of the IL-15 / IL-15Rα complex composition are amino acids such as histidine and arginine.

[0121] Pharmaceutical compositions may be liquids or solids. Liquid formulations are aqueous solutions or suspensions prepared in a suitable aqueous solvent such as water or an aqueous / organic mixture such as a water-alcohol mixture. Liquid formulations may be stored at room temperature, refrigerated (e.g., 2-8°C) or frozen (e.g., -20°C or -70°C).

[0122] Solid formulations can be prepared by any preferred method, for example, by adding a lioprotectant to form a cake or powder. In one embodiment, the solid formulation is prepared by drying the liquid formulation described herein, for example, by freeze-drying or spray-drying. When the formulation is a solid formulation, the formulation may have a water content of about 5% or less, about 4.5% or less, about 4% or less, about 3.5% or less, about 3% or less, about 2.5% or less, about 2% or less, about 1.5% or less, or about 1% or less, or is substantially anhydrous. The solid formulation can be dissolved, i.e., reconstituted, in a preferred medium or solvent to make a liquid suitable for administration. Preferred solvents for reconstituting the solid formulation include water, isotonic saline, buffers, such as phosphate-buffered saline, Ringer's (lactate or dextrose) solution, minimal essential medium, alcohol / aqueous solution, and dextrose solution. Depending on the amount of solvent, a therapeutic protein concentration higher, the same as, or lower than the concentration before drying may be obtained.

[0123] In some embodiments, the pharmaceutical compositions disclosed herein maintain a purity of at least about 75%, about 80%, about 85%, about 90%, or about 95% by RP-HPLC after storage at about 2°C to about 8°C for at least about 6 months, at least about 12 months, or at least about 24 months; a purity of at least about 75%, at least about 80%, at least about 85%, or at least about 90% by RP-HPLC after storage at about 25°C for at least 6 months or at least about 12 months; and / or maintain a purity of at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% by RP-HPLC after storage at about 40°C for at least about 6 months. In some embodiments, the pharmaceutical compositions disclosed herein maintain a purity of at least about 85% by RP-HPLC after storage at about 2 to about 8°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of at least about 90% by RP-HPLC after storage at about 2°C to about 8°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of about 95% by RP-HPLC after storage at about 2°C to about 8°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of about 85% by RP-HPLC after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the present disclosure maintain a purity of about 90% by RP-HPLC after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months.In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of approximately 95% by RP-HPLC after storage at approximately 25°C for at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months. In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of approximately 85% by RP-HPLC after storage at approximately 40°C for at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months. In some embodiments, the pharmaceutical compositions of the Disclosure maintain a purity of approximately 90% by RP-HPLC after storage at approximately 40°C for at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months. In some embodiments, the pharmaceutical compositions of the present disclosure maintain a purity of approximately 95% by RP-HPLC after storage at approximately 40°C for at least approximately 1 month, at least approximately 2 months, at least approximately 3 months, at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months.

[0124] In some embodiments, the pharmaceutical compositions disclosed herein maintain, as assessed by AEX, approximately 25% basic variants and approximately 75% acidic variants after storage at approximately 2°C to approximately 8°C for at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months; approximately 25% basic variants and approximately 75% acidic variants after storage at approximately 20°C to approximately 25°C for at least approximately 6 months or at least approximately 12 months; and / or maintain approximately 25% basic variants and approximately 75% acidic variants after storage at approximately 40°C for at least approximately 6 months, as assessed by AEX.

[0125] In some embodiments, the pharmaceutical compositions of the present disclosure contain less than 200 >2 μm particles by PAMAS after storage at about 2°C to about 8°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months, and / or less than 20 >10 μm particles by PAMAS after storage at about 2°C to about 8°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the present disclosure contain less than 200 >2 μm particles by PAMAS after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months, and / or less than 20 >10 μm particles by PAMAS after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months. In some embodiments, the pharmaceutical compositions of the present disclosure contain less than 200 >2 μm particles by PAMAS after storage at about 40°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months, and / or less than 20 >10 μm particles by PAMAS after storage at about 40°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 12 months, or at least about 24 months.

[0126] The stability of the pharmaceutical composition can be measured using a bioactivity assay. Preferably, the bioactivity after storage is about 70% to 125% of the original activity. Bioactivity can be evaluated by assaying the activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells.

[0127] In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at approximately 2°C to approximately 8°C for at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months, with activity at approximately 70% to approximately 125% of the original activity. In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at approximately 2°C to approximately 8°C for at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months, with activity at approximately 80% to approximately 125% of the original activity.

[0128] In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 12 months, with activity at about 70% to about 125% of the original activity. In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 12 months, with activity at about 80% to about 125% of the original activity.

[0129] In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 40°C for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks (about 1 month), at least about 2 months, or at least about 3 months, with activity at about 70% to about 125% of the original activity. In some embodiments, the liquid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 40°C for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks (about 1 month), at least about 2 months, or at least about 3 months, with activity at about 80% to about 125% of the original activity.

[0130] In some embodiments, the solid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at approximately 2°C to approximately 8°C for at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months, with activity at approximately 70% to approximately 125% of the original activity. In some embodiments, the solid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at approximately 2°C to approximately 8°C for at least approximately 6 months, at least approximately 12 months, or at least approximately 24 months, with activity at approximately 80% to approximately 125% of the original activity.

[0131] In some embodiments, the solid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 12 months, with activity at about 70% to about 125% of the original activity. In some embodiments, the solid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 25°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least about 12 months, with activity at about 80% to about 125% of the original activity.

[0132] In some embodiments, the solid pharmaceutical compositions disclosed herein maintain biological activity, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 40°C for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks (about 1 month), at least about 2 months, or at least about 3 months, with activity at about 70% to about 125% of the original activity. In some embodiments, the solid pharmaceutical compositions disclosed herein maintain stability, as assessed by activation of the IL-15 receptor on U2OS IL2Rβ / IL2Rγ cells, after storage at about 40°C for at least about 2 weeks, at least about 3 weeks, at least about 4 weeks (about 1 month), at least about 2 months, or at least about 3 months, with activity at about 80% to about 125% of the original activity.

[0133] IL-15 / IL-15Rα concentration The IL-15 / IL-15Rα complex used in the pharmaceutical compositions of this disclosure (for example, as disclosed herein) is described herein. In one embodiment, the IL-15 / IL-15Rα complex comprises IL-15 comprising SEQ ID NO: 2 and IL-15Rα comprising SEQ ID NO: 5. In another embodiment, the IL-15 / IL-15Rα complex comprises IL-15 comprising SEQ ID NO: 5 and IL-15Rα comprising SEQ ID NO: 5.

[0134] In some embodiments, the concentration of the IL-15 / IL-15Rα protein complex is about 0.1 mg / mL to about 50 mg / mL in the pharmaceutical composition. In one embodiment, the concentration of the IL-15 / IL-15Rα protein complex is about 0.1 mg / mL to about 20 mg / mL in the pharmaceutical composition. The concentration of the IL-15 / IL-15Rα protein complex is preferably about 0.1 mg / mL to about 20 mg / mL, and most preferably about 0.1 mg / mL to about 10 mg / mL. Non-limiting examples include approximately 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.

[0135] The liquid and / or solid pharmaceutical compositions of this disclosure may contain one or more stabilizers, and nonionic stabilizers are preferred. Suitable nonionic stabilizers include polyols or sugars such as monosaccharides, disaccharides, or trisaccharides, for example, sucrose, trehalose, raffinose, maltose, sorbitol, or mannitol. The sugar may be a sugar alcohol or an amino sugar. In a preferred embodiment, the nonionic stabilizer is a polyol or a sugar. In a preferred embodiment, the nonionic stabilizer is sucrose, trehalose, glycerol, mannitol, or sorbitol. In another preferred embodiment, the nonionic stabilizer is sucrose. The concentration of the nonionic stabilizer is approximately 50 mM to 500 mM, for example, approximately 120 mM to 350 mM, for example, approximately 175 mM to 350 mM, for example, approximately 180 mM to 300 mM, for example, approximately 200 mM to 300 mM, for example, approximately 220 mM to 300 mM, for example, approximately 250 mM to 270 mM, approximately 175 mM, approximately 180 mM, approximately 185 mM, approximately 190 mM, approximately 195 mM, approximately 200 mM, approximately 205 mM, approximately 210 mM, approximately 215 mM, approximately 220 mM, approximately 225 mM, approximately 230 mM, approximately 235 mM, approximately 240 mM, approximately 2 It may also be 45mM, approximately 250mM, approximately 255mM, approximately 260mM, approximately 265mM, approximately 270mM, approximately 275mM, approximately 280mM, approximately 285mM, approximately 290mM, approximately 295mM, approximately 300mM, approximately 310mM, approximately 320mM, approximately 330mM, approximately 340mM, approximately 350mM, approximately 360mM, approximately 370mM, approximately 380mM, approximately 390mM, approximately 400mM, approximately 410mM, approximately 420mM, approximately 430mM, approximately 440mM, approximately 450mM, approximately 460mM, approximately 470mM, approximately 480mM, approximately 490mM, or approximately 500mM. In a preferred embodiment, the concentration of the nonionic stabilizer in the stable liquid pharmaceutical composition is about 120 mM to about 350 mM. In another preferred embodiment, the concentration of the nonionic stabilizer in the stable liquid pharmaceutical composition is about 180 mM to about 300 mM. In yet another preferred embodiment, the nonionic stabilizer is sucrose, trehalose, glycerol, mannitol, or sorbitol, and the concentration of the nonionic stabilizer is about 120 mM to about 350 mM.In yet another preferred embodiment, the nonionic stabilizer is sucrose, trehalose, glycerol, mannitol, or sorbitol, and the concentration of the nonionic stabilizer is about 180 mM to about 300 mM. In yet another preferred embodiment, the nonionic stabilizer is mannitol, and the concentration of mannitol is about 120 mM to about 350 mM. In yet another preferred embodiment, the nonionic stabilizer is mannitol, and the concentration of mannitol is about 180 mM to about 300 mM. In yet another embodiment, the nonionic stabilizer is mannitol, and the concentration of mannitol is about 260 mM. In yet another preferred embodiment, the nonionic stabilizer is sucrose, and the concentration of sucrose is about 120 mM to about 350 mM. In yet another preferred embodiment, the nonionic stabilizer is sucrose, and the concentration of sucrose is about 180 mM to about 300 mM. In yet another embodiment, the nonionic stabilizer is sucrose, and the concentration of sucrose is approximately 260 mM.

[0136] Other excipients The liquid and / or solid pharmaceutical compositions provided herein may contain further excipients, e.g., additional buffers, salts (e.g., sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride), additional stabilizers, tonic modifiers (e.g., salts and amino acids [e.g., proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine, and histidine]), glycerol, albumin, alcohols, preservatives, additional surfactants, antioxidants, and the like. The liquid and / or solid pharmaceutical compositions may also contain one or more isotonic agents. The term “isotonic agent” refers to a pharmaceutically acceptable excipient used to adjust the tonicity of the liquid and / or solid pharmaceutical composition. The liquid and / or solid pharmaceutical compositions may be hypotonic, isotonic, or hypertonic. Isotonicity generally relates to the osmotic pressure of a solution, typically to the osmotic pressure of human serum (approximately 250–350 mOsmol / kg). The liquid and / or solid pharmaceutical compositions described herein may be hypotonic, isotonic, or hypertonic, but are preferably isotonic. An isotonic formulation means a solution that has the same tonicity as another comparable solution, such as physiological saline or serum. Suitable isotonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerin, and any component from the group of amino acids or sugars, particularly glucose. Isotonic agents are generally used in amounts of approximately 0.1 mM to approximately 500 mM.

[0137] Among stabilizers and isotonic agents, there is a group of compounds that can function in both ways; that is, they can be both stabilizers and isotonic agents simultaneously. Examples can be found in the group of sugars, amino acids, polyols, cyclodextrins, polyethylene glycols, and salts. An example of a sugar that can be both a stabilizer and an isotonic agent simultaneously is sucrose. A complete discussion of such additional pharmaceutical components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0138] Liquid pharmaceutical composition For example, a stable liquid pharmaceutical composition is disclosed herein, comprising a heterodimer IL-15 / IL-15Rα complex as described herein, about 0.0001% to about 1% (w / v) of a surfactant, optionally further comprising about 1 mM to about 100 mM of a buffering agent providing a pH in the range of about 4.5 to about 8.5, and optionally further comprising about 1 mM to about 500 mM of at least one stabilizer as described above. Preferred heterodimer IL-15 / IL-15Rα complexes that may be included in the stable liquid pharmaceutical composition are described in detail herein. Particularly preferred is an IL-15 / IL-15Rα complex comprising IL-15 including SEQ ID NO: 2 and IL-15Rα including SEQ ID NO: 5 as disclosed herein.

[0139] Suitable surfactants for use with the stable liquid pharmaceutical compositions of this disclosure include, but are not limited to, nonionic surfactants, ionic surfactants, zwitterionic surfactants, and combinations thereof. Typical surfactants used include sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerin fatty acid esters (e.g., glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate), polyglycerin fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), and polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate). Polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glyceryl monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g.,Examples of surfactants include, but are not limited to, polyoxyethylene stearamide, C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (e.g., sodium polyoxyethylene lauryl sulfate) and C1-C18 alkyl sulfosuccinate esters (e.g., sodium lauryl sulfosuccinate) having an average of 2-4 moles of ethylene oxide units added, as well as natural surfactants such as lecithin, glycerophospholipids, and sphingophospholipids (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids. The composition may contain one or more of these surfactants. Preferred surfactants are poloxamers (e.g., poloxamer 188, poloxamer 407, poloxamer 403, poloxamer 402, poloxamer 181, poloxamer 401, poloxamer 185, and poloxamer 338) or polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, 40, 60, or 80). Polysorbate 20 (Tween 20) (e.g., in concentrations of about 0.01% to about 0.1% (w / v), e.g., about 0.01% to about 0.04% (w / v), e.g., about 0.01%, about 0.02%, about 0.04%, about 0.06%, about 0.08%, and about 0.1%) is useful. Polysorbate 80 (Tween 80) (for example, in concentrations of about 0.01% to about 0.1% (w / v), for example, about 0.01% to about 0.04% (w / v), for example, about 0.01%, about 0.02%, about 0.04%, about 0.06%, about 0.08%, and about 0.1%) is useful. Poloxamer 188 (for example, in concentrations of about 0.01% to about 1% (w / v), for example, about 0.1% to about 0.5% (w / v), for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, and about 1%) is particularly useful. In one embodiment, the stable liquid pharmaceutical composition contains about 0.2% (w / v) of poloxamer 188. In alternative embodiments, the surfactant contained in the stable liquid pharmaceutical composition is a polysorbate, preferably polysorbate 20 or polysorbate 80, and more preferably polysorbate 20. Preferably, the polysorbate isThe concentration is approximately 0.01% to 0.1% (w / v), preferably approximately 0.02% to 0.05% (w / v), and preferably approximately 0.04% (w / v).

[0140] In one embodiment, the concentration of the heterodimer IL-15 / IL-15Rα complex contained in the stable liquid pharmaceutical composition is in the range of about 0.1 mg / mL to about 50 mg / mL, more preferably about 0.1 mg / mL to about 10 mg / mL, and most preferably about 1 mg / mL.

[0141] In certain embodiments, the buffering agent included in the stable liquid pharmaceutical composition is an acetate buffer, succinate buffer, citrate buffer, or histidine buffer. Particularly preferred is an L-histidine / HCl buffer (i.e., L-histidine as a buffer). Acetate buffers, particularly sodium acetate buffers, have been evaluated as beneficial in liquid pharmaceutical compositions with respect to decomposition products by SEC, AEX, and aggregation products by RP-HPLC. In one embodiment, the stable liquid composition contains about 10 mM to about 50 mM, for example, about 10 mM, for example, about 15 mM, for example, about 20 mM, for example, about 25 mM, for example, about 30 mM, for example, about 35 mM, for example, about 40 mM, for example, about 45 mM, for example, about 50 mM of a sodium acetate buffer. In another embodiment, the stable liquid composition contains about 15 mM to about 30 mM of a sodium acetate buffer. In another embodiment, the stable liquid composition contains about 20 mM to about 30 mM of sodium acetate buffer. In another embodiment, the stable liquid composition contains about 10 mM to about 30 mM of sodium acetate buffer. In one embodiment, the stable liquid composition contains about 10 mM to about 50 mM, for example, about 10 mM, for example, about 15 mM, for example, about 20 mM, for example, about 25 mM, for example, about 30 mM, for example, about 35 mM, for example, about 40 mM, for example, about 45 mM, for example, about 50 mM of histidine buffer. In another embodiment, the stable liquid composition contains about 15 mM to about 30 mM of histidine buffer. In another embodiment, the stable liquid composition contains about 20 mM to about 30 mM of histidine buffer. In another embodiment, the stable liquid composition contains about 10 mM to about 30 mM of histidine buffer.

[0142] In one embodiment, the pH of the stable liquid pharmaceutical composition is in the range of about 4.5 to about 8.5, for example, about 4.5 to about 7.5, for example, about 4.5 to about 6.5, for example, about 4.5 to about 5.5, for example, about 4.7 to about 5.5, for example, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, about 6.2, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, and about 7.5. In a preferred embodiment, the pH of the stable liquid composition is in the range of about 4.5 to about 5.5. Overall testing has shown that the ideal compositional pH of the liquid pharmaceutical compositions of this disclosure is about 5.0. Therefore, in one embodiment, the pH of the stable liquid composition is about 5.0. In one embodiment, the stable liquid composition contains about 10 mM to about 50 mM of sodium acetate buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 8.5. In yet another embodiment, the stable liquid pharmaceutical composition contains about 20 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 8.5. In yet another embodiment, the stable liquid composition contains about 10 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 50 mM of sodium acetate buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 20 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 30 mM of sodium acetate buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 50 mM of sodium acetate buffer at a pH of about 5.0. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM of sodium acetate buffer at a pH of about 5.0. In another embodiment, the stable liquid pharmaceutical composition contains a sodium acetate buffer at a pH of about 5.0 and about 20 mM to about 30 mM.In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 30 mM of sodium acetate buffer at a pH of about 5.0. In another embodiment, the stable liquid pharmaceutical composition contains about 20 mM of sodium acetate buffer at a pH of about 5.0. In one embodiment, the stable liquid composition contains about 10 mM to about 50 mM of histidine buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM of histidine buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid pharmaceutical composition contains about 20 mM to about 30 mM of histidine buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid composition contains about 10 mM to about 30 mM of histidine buffer at a pH of about 4.5 to about 8.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 50 mM histidine buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM histidine buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 20 mM to about 30 mM histidine buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 30 mM histidine buffer at a pH of about 4.5 to about 5.5. In another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 50 mM histidine buffer at a pH of about 5.0. In another embodiment, the stable liquid pharmaceutical composition contains about 15 mM to about 30 mM histidine buffer at a pH of about 5.0. In another embodiment, the stable liquid pharmaceutical composition contains about 20 mM to about 30 mM histidine buffer at a pH of about 5.0. In yet another embodiment, the stable liquid pharmaceutical composition contains about 10 mM to about 30 mM histidine buffer at a pH of about 5.0. In yet another embodiment, the stable liquid pharmaceutical composition contains about 20 mM histidine buffer at a pH of about 5.0.

[0143] In a particular embodiment, a stable liquid pharmaceutical composition is: a. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, wherein the pH of the composition is approximately 4.7. b. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, wherein the pH of the composition is approximately 4.7. c. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, wherein the pH of the composition is approximately 5. d. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, wherein the pH of the composition is approximately 5. e. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, with a pH of approximately 5.5. f. The composition comprises approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, and the pH of the composition is approximately 5.5. The composition comprises approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM sodium acetate, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, with a pH of approximately 4.7. The composition contains approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM histidine, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, and the pH of the composition is approximately 4.7. i. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM histidine, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, wherein the pH of the composition is approximately 5. j. A composition comprising approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM histidine, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, wherein the pH of the composition is approximately 5. k. The composition comprises approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM histidine, approximately 260 mM sucrose, and approximately 0.2% poloxamer 188, and the pH of the composition is approximately 5.5, or 1. The composition contains approximately 1 mg / mL of the IL-15 / IL-15Ra complex described herein, approximately 20 mM histidine, approximately 260 mM sucrose, and approximately 0.04% polysorbate 20, with a pH of approximately 5.5.

[0144] Solid pharmaceutical composition Furthermore, a solid pharmaceutical composition is disclosed herein, comprising a buffering agent of about 10 mM to about 50 mM, which contains the heterodimer IL-15 / IL-15Rα complex described herein and provides a pH in the range of about 6.5 to about 8.5; at least one stabilizer of about 1 mM to about 500 mM, described above; and at least one isotonic agent of about 0.1 mM to about 50 mM, described above.

[0145] The solid formulations of the present invention are generally prepared by drying a liquid formulation. Any suitable drying method, such as freeze-drying or spray-drying, can be used. In one embodiment, a lioprotectant is added to the formulation before freeze-drying. Freeze-drying typically involves freezing the liquid formulation in a container used for storage, transport, and distribution of the formulation (e.g., vials, syringes (single or dual-chamber syringes, etc.), or cartridges (e.g., single or dual-chamber syringes, etc.)) (see, for example, Gatlin and Nail in Protein Purification Process Engineering, ed. Roger G. Harrison, Marcel Dekker Inc., 317-367 (1994)). Once the formulation is frozen, the atmospheric pressure is reduced and the temperature is adjusted so that the frozen solvent can be removed, for example, by sublimation. This step of the freeze-drying process is sometimes called primary drying. If desired, the temperature can then be increased to remove any solvent still bound to the dried formulation by evaporation. This step of the freeze-drying process is sometimes called secondary drying. Once the formulation reaches the desired degree of dryness, the drying process is terminated and the container is sealed. The final solid formulation is sometimes called a “lyophilized formulation” or “cake.” The lyophilization process can be carried out using any suitable equipment. Suitable lyophilization equipment is available from numerous private suppliers (e.g., SP Scientific, Stone Ridge, NY).

[0146] Various suitable apparatuses can be used to dry liquid formulations to produce solid (e.g., lyophilized) formulations. Generally, lyophilized formulations are prepared by those skilled in the art using a sealed chamber containing shelves on which vials of the liquid formulation to be dried are placed. The temperature of the shelves, as well as the cooling and heating rates, can be controlled, as can the pressure within the chamber. It will be understood that the various process parameters discussed herein refer to processes performed using this type of apparatus. Those skilled in the art can readily adapt the parameters described herein to other types of drying apparatuses, if desired.

[0147] Suitable temperatures and vacuum levels for primary and secondary drying can be easily determined by those skilled in the art. Generally, the formulation is frozen at a temperature of about -30°C or lower, such as -40°C or -50°C. The cooling rate can affect the amount and size of ice crystals in the matrix. Primary drying is generally carried out at a temperature about 10°C, 20°C, 30°C, 40°C, or 50°C higher than the freezing temperature.

[0148] After freeze-drying, vials, syringes, or cartridges can be sealed, for example, by stoppering under vacuum. Alternatively, a gas, such as dry air or nitrogen, can be introduced into the container before sealing. If oxidation is a concern, the gas introduced into the freeze-drying chamber may include a gas that delays or prevents oxidation of the freeze-dried product. The gas may be a non-oxygenating gas, such as nitrogen, or an inert gas, such as helium, neon, argon, krypton, or xenon.

[0149] Preferred heterodimer IL-15 / IL-15Rα complexes that may be included in solid pharmaceutical compositions are described in detail herein. An IL-15 / IL-15Rα complex comprising IL-15 comprising SEQ ID NO: 2 as disclosed herein and IL-15Rα comprising SEQ ID NO: 5 is particularly preferred.

[0150] In one embodiment, the concentration of the heterodimer IL-15 / IL-15Rα complex contained in the solid pharmaceutical composition is in the range of about 0.1 mg / mL to about 50 mg / mL, more preferably about 0.1 mg / mL to about 10 mg / mL, and most preferably about 0.1 mg / mL to about 0.5 mg / mL.

[0151] In certain embodiments, the buffering agent included in the solid pharmaceutical composition is a phosphate buffering agent, an acetate buffering agent, a succinate buffering agent, a citrate buffering agent, or a histidine buffering agent. Particularly preferred is a Na / K phosphate buffering agent. In one embodiment, the solid composition contains about 10 mM to about 50 mM, for example, about 10 mM, for example, about 15 mM, for example, about 20 mM, for example, about 25 mM, for example, about 30 mM, for example, about 35 mM, for example, about 40 mM, for example, about 45 mM, for example, about 50 mM of Na / K phosphate buffering agent. In another embodiment, the solid composition contains about 15 mM to about 30 mM of Na / K phosphate buffering agent. In another embodiment, the solid composition contains about 20 mM to about 30 mM of Na / K phosphate buffering agent. In yet another embodiment, the solid composition contains about 10 mM to about 30 mM of Na / K phosphate buffering agent.

[0152] In one embodiment, the pH of the solid pharmaceutical composition is in the range of about 6.5 to about 8.5, e.g., about 6.5 to about 8, e.g., about 6.5 to about 7.5, e.g., about 6.8 to about 7.5, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, and about 8.5. In a preferred embodiment, the pH of the solid pharmaceutical composition is in the range of about 6.5 to about 7.5. Overall testing has shown that the ideal composition pH of the solid pharmaceutical composition of this disclosure is about 7.3. Therefore, in one embodiment, the pH of the solid pharmaceutical composition is about 7.3. In one embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 8.5 and about 1 mM to about 50 mM. In another embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 8.5 and about 1 mM to about 30 mM. In yet another embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 8.5 and about 1 mM to about 10 mM. In yet another embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 7.5 and about 1 mM to about 50 mM. In yet another embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 7.5 and about 1 mM to about 30 mM. In yet another embodiment, the solid pharmaceutical composition contains a Na / K phosphate buffer at a pH of about 6.5 to about 7.5 and about 1 mM to about 10 mM. In another embodiment, the solid pharmaceutical composition contains about 1 mM to about 50 mM of Na / K phosphate buffer at a pH of about 7.3. In another embodiment, the solid pharmaceutical composition contains about 1 mM to about 30 mM of Na / K phosphate buffer at a pH of about 7.3. In another embodiment, the solid pharmaceutical composition contains about 1 mM to about 10 mM of Na / K phosphate buffer at a pH of about 7.3. In another embodiment, the solid pharmaceutical composition contains about 1 mM to about 5 mM of Na / K phosphate buffer at a pH of about 7.3. In another embodiment, the solid pharmaceutical composition contains about 1.35 mM of Na / K phosphate buffer at a pH of about 7.3.

[0153] The solid pharmaceutical composition also contains at least one stabilizer in a concentration of about 1 mM to about 500 mM. In a preferred embodiment, the solid pharmaceutical composition contains at least two stabilizers in a concentration of about 1 mM to about 500 mM. Preferred stabilizers are, for example, those described above. In one embodiment, the solid pharmaceutical composition contains about 1 mM to about 500 mM sucrose and about 1 mM to about 500 mM mannitol. In another embodiment, the solid pharmaceutical composition contains about 5 mM to about 50 mM sucrose and about 100 mM to about 300 mM mannitol. In yet another embodiment, the solid pharmaceutical composition contains about 30 mM sucrose and about 220 mM mannitol.

[0154] The solid pharmaceutical composition also contains at least one isotonic agent in a concentration of about 0.1 mM to about 50 mM. In a preferred embodiment, the solid pharmaceutical composition contains at least two isotonic agents in a concentration of about 0.1 mM to about 50 mM. Preferred isotonic agents are, for example, those described above. In one embodiment, the solid pharmaceutical composition contains about 0.1 mM to about 50 mM KCl and about 0.1 mM to about 50 mM NaCl. In another embodiment, the solid pharmaceutical composition contains about 0.1 mM to about 1 mM KCl and about 10 mM to about 50 mM NaCl. In yet another embodiment, the solid pharmaceutical composition contains about 0.375 mM KCl and about 20 mM NaCl.

[0155] In one embodiment, the solid pharmaceutical composition contains approximately 0.24 mg / mL of IL-15 / IL-15Rα complex, approximately 30 mM sucrose, approximately 220 mM mannitol, approximately 0.375 mM KCl, approximately 20 mM NaCl, and approximately 1.35 mM Na / K phosphate buffer at pH 7.3.

[0156] manufactured goods In another embodiment, a manufacturing article containing a pharmaceutical formulation disclosed herein and providing instructions for its use is provided herein. The manufacturing article includes a container. Suitable containers include, for example, bottles, vials (e.g., double-chamber vials, vials for liquid formulations with or without needles, vials for solid formulations with or without needles and reconstituted solutions), syringes (e.g., double-chamber syringes, pre-filled syringes (e.g., for use with autoinjector devices), autoinjectors), cartridges, pens, and test tubes. Containers can be formed from a variety of materials such as glass, metal, or plastic. The container holds the formulation, and labels on or attached to the container may indicate the method of use. In another embodiment, the formulation may be prepared for self-administration and / or may include instructions for self-administration. In one embodiment, the container holding the formulation may be a single-use vial. In another embodiment, the container holding the formulation may be a multi-use vial that allows repeated administration of the formulation, for example, using two or more portions of a reconstituted formulation. The manufactured articles may further include other materials desirable from a commercial and user perspective, such as other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use, as described in the previous section.

[0157] In one embodiment, a manufactured article is provided comprising a container and a liquid pharmaceutical composition disposed within the container, wherein the composition comprises a heterodimer IL-15 / IL-15Rα complex (e.g., about 0.1 mg / mL to about 50 mg / mL or about 0.1 to about 10 mg / mL); about 0.0001% to about 1% (w / v) of a surfactant, optionally further comprising about 1 mM to about 100 mM of a buffering agent providing a pH in the range of about 4.5 to about 8.5, optionally further comprising about 1 mM to about 500 mM of at least one stabilizer, wherein the liquid pharmaceutical composition is not reconstituted from a lyophilized product.

[0158] In another embodiment, a manufactured article is provided comprising a container and a solid pharmaceutical composition disposed within the container, wherein the composition comprises: a heterodimer IL-15 / IL-15Rα complex (e.g., about 0.1 mg / mL to about 50 mg / mL or about 0.1 mg / mL to about 10 mg / mL); about 10 mM to about 50 mM of a buffering agent providing a pH in the range of about 6.5 to about 8.5; about 1 mM to about 500 mM of at least one stabilizer; and about 0.1 mM to about 50 mM of at least one isotonic agent. In one embodiment, the composition is lyophilized and stored in a single-dose container. The container can be stored at about 2 to 8°C or 25°C until administered to a subject requiring it.

[0159] In some embodiments, a liquid or solid pharmaceutical composition contains a sufficient amount of heterodimer IL-15 / IL-15Rα complex to enable the delivery of at least about 0.1 to about 10 μg / kg of the heterodimer IL-15 / IL-15Rα complex (e.g., as disclosed herein) per unit dose. In some embodiments, a liquid or solid pharmaceutical product contains a sufficient amount of heterodimer IL-15 / IL-15Rα complex to enable the delivery of at least about 0.1 μg / kg, about 0.25 μg / kg, about 0.5 μg / kg, about 1 μg / kg, about 2 μg / kg, or about 5 μg / kg of the heterodimer IL-15 / IL-15Rα complex (e.g., as disclosed herein) per unit dose. In some embodiments, the liquid or solid pharmaceutical product is formulated in a dose that enables subcutaneous delivery of the heterodimer IL-15 / IL-15Rα complex (e.g., as disclosed herein) at a dose of about 0.1 μg / kg to about 10 μg / kg per unit dose. In some embodiments, the liquid or solid pharmaceutical composition is formulated in a dose that enables intravenous delivery of the heterodimer IL-15 / IL-15Rα complex (e.g., as disclosed herein) at a dose of about 0.1 μg / kg to about 10 μg / kg per unit dose.

[0160] Kits containing pharmaceutical products and compositions This disclosure also encompasses kits for treating patients. Broadly speaking, such kits include at least one of the disclosed pharmaceutical products or liquid or solid compositions and instructions for use. The instructions disclose appropriate techniques for providing the pharmaceutical composition to a patient as part of a drug administration regimen. These kits may also contain additional agents for delivery (i.e., concurrent or sequential [pre- or post-]) therapy in combination with the encapsulated pharmaceutical composition.

[0161] A kit for the treatment of a patient in need thereof is disclosed herein, the kit comprising: a) a container; b) a liquid pharmaceutical composition disposed within the container, i) a heterodimer IL-15 / IL-15Rα complex (e.g., about 0.1 mg / mL to about 50 mg / mL or about 0.1 mg / mL to about 10 mg / mL); and about 0.0001% to about 1% (w / v) of a surfactant, further optionally comprising about 1 mM to about 100 mM of a buffer providing a pH in the range of about 4.5 to about 8.5, and further optionally comprising about 1 mM to about 500 mM of at least one stabilizer, and not reconstituted from a lyophilized product; and c) instructions for use for administering the liquid pharmaceutical composition to a patient. In some embodiments, the container is a pen, a pre-filled syringe, an autoinjector, or a vial. In one embodiment, the container is a syringe. The syringe may be contained in an autoinjector. In another embodiment, the container is an auto-injector containing a liquid formulation as described herein.

[0162] A kit for the treatment of a patient in need thereof is disclosed herein, comprising: a) a container; b) a solid pharmaceutical composition disposed within the container, wherein the composition comprises: i) a heterodimer IL-15 / IL-15Rα complex (e.g., about 0.1 mg / mL to about 0.5 mg / mL); about 10 mM to about 50 mM of a buffer providing a pH in the range of about 6.5 to about 8.5; about 1 mM to about 500 mM of at least one stabilizer; and about 0.1 mM to about 50 mM of at least one isotonic agent; and c) instructions for administering the liquid pharmaceutical composition to a patient. In some embodiments, the container is a pen, a pre-filled syringe, an autoinjector, or a vial. In one embodiment, the container is a syringe. The syringe may be contained in an autoinjector. In another embodiment, the container is an autoinjector containing the solid formulation described herein.

[0163] Methods for using pharmaceutical products and compositions The pharmaceutical compositions of this disclosure are used to treat patients who would benefit from treatment with the IL-15 / IL-15Rα complex disclosed herein, for example. Of course, the appropriate dosage will vary depending on, for example, the specific IL-15 / IL-15Rα complex used, the host, the mode of administration, the nature and severity of the condition being treated, and the nature of any previous treatments the patient has received. Ultimately, the healthcare provider in charge will determine the amount of IL-15 / IL-15Rα complex to treat an individual patient.

[0164] For example, a therapeutic method is provided herein, comprising administering a therapeutically effective amount of the IL-15 / IL-15Rα complex disclosed herein to a patient in need, for example, by subcutaneous injection, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0165] Furthermore, provided herein are pharmaceutical compositions, for example, for use in the treatment of patients in need thereof, which include administering to a patient, for example, a therapeutically effective amount of the IL-15 / IL-15Rα complex disclosed herein, for example, by subcutaneous injection, and the IL-15 / IL-15Rα complex is provided as part of the pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0166] Furthermore, for example, the use of the IL-15 / IL-15Rα complex disclosed herein for the manufacture of therapeutic agents for patients in need thereof, as described herein, is provided herein, for example, including administering a therapeutically effective amount of the IL-15 / IL-15Rα complex disclosed herein to a patient, for example by subcutaneous injection, and the IL-15 / IL-15Rα complex is provided as part of the pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0167] In one embodiment, a method for improving IL-15-mediated immune function is provided herein, comprising administering the IL-15 / IL-15Rα complex disclosed herein to a subject in a prescribed dose regimen, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. Since improving IL-15-mediated immune function is beneficial for the prevention, treatment, and / or management of certain disorders, a method for preventing, treating, and / or managing such disorders is also provided herein, comprising administering the IL-15 / IL-15Rα complex disclosed herein to a subject in need thereof, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. Non-limiting examples of disorders for which improving IL-15-mediated immune function is beneficial include cancer, lymphopenia, immunodeficiency, infectious diseases, and wounds.

[0168] In one embodiment, a method is provided herein for preventing, treating, and / or managing a disorder in a subject in which improved IL-15-mediated immune function is beneficial for the prevention, treatment, and / or management of such disorder, comprising administering the same dose of the IL-15 / IL-15Rα complex disclosed herein to the subject over the duration of a therapeutic cycle, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In one embodiment, the dose is in the range of 0.1 μg / kg and 0.5 μg / kg. In one embodiment, the dose is in the range of 0.25 μg / kg and 1 μg / kg. In a specific embodiment, the dose is in the range of 0.5 μg / kg and 2 μg / kg. In another embodiment, the dose is 1 μg / kg to 4 μg / kg. In another embodiment, the dose is 2 μg / kg to 8 μg / kg. In another embodiment, the doses are 0.1 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, and 8 μg / kg. In a specific embodiment, the dose is 1 μg / kg. In one embodiment, the dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 2-4, 2-5, 2-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times. In some embodiments, the dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more over periods of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days, or 1-3, 1-4, 2-4, 2-5, 1-5, 2-6, 3-6, 4-6, or 6-8 times. In specific embodiments, each dose is administered at least 1, 2, 3, 4, 5, 6 times or more over periods of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days. In other specific embodiments, each dose is administered at least once, and the subject is administered weekly over a period of 3 weeks.

[0169] In another embodiment, the present invention provides a method in which, in preventing, treating, and / or managing a disorder in a subject, improved IL-15-mediated immune function is beneficial for preventing, treating, and / or managing such a disorder, for example, by administering the IL-15 / IL-15Rα complex disclosed herein to a subject at least 1, 2, 4, or 6 times in an administration cycle prior to a non-administration period in an administration regime, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In a specific embodiment, for example, the IL-15 / IL-15Rα complex disclosed herein is administered once a week for three weeks, with no administration in the fourth week, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. This administration cycle is then repeated.

[0170] In alternative embodiments, methods are provided herein for preventing, treating, and / or managing disorders in subjects in which enhanced IL-15-mediated immune function is beneficial for preventing, treating, and / or managing such disorders, comprising (a) administering at least one initial low dose of, for example, the IL-15 / IL-15Rα complex disclosed herein to the subject; and (b) administering successively increasing doses of, for example, the IL-15 / IL-15Rα complex disclosed herein to the subject over the duration of a therapeutic cycle, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition described herein or as a solid pharmaceutical composition. In specific embodiments, methods for preventing, treating, and / or controlling cancer in a subject are provided herein, comprising (a) administering an initial dose of, for example, the IL-15 / IL-15Rα complex disclosed herein over the duration of a treatment cycle; and (b) administering successively increasing doses of the IL-15 / IL-15Rα complex over the duration of a treatment cycle, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In specific embodiments, the initial dose is in the range of 0.1 μg / kg and 0.5 μg / kg. In specific embodiments, the initial dose is in the range of 0.25 μg / kg and 1 μg / kg. In another embodiment, the initial dose is in the range of 0.5 μg / kg and 2 μg / kg. In specific embodiments, the initial dose is 1 μg / kg to 4 μg / kg. In another embodiment, the initial dose is 2 μg / kg and 8 μg / kg. In another embodiment, the initial dose is approximately 0.25 μg / kg. In another embodiment, the initial dose is approximately 0.5 μg / kg. In another embodiment, the initial dose is approximately 1 μg / kg. In another embodiment, the initial doses are 0.1 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, and 8 μg / kg.In one embodiment, the initial dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 2-4, 2-5, 2-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times. In some embodiments, the initial dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 2-4, 2-5, 1-5, 2-6, 3-6, 4-6, or 6-8 times over a period of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days. In one embodiment, each successively increasing dose is 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times higher than the previous dose, or 1.2-2, 2-3, 2-4, 1-5, 2-6, 3-4, 3-6, or 4-6 times higher than the previous dose, or 2 times higher than the previous dose. In some embodiments, each successively increasing dose is 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or 200% higher than the previous dose. In specific embodiments, each dose is administered at least one, two, three, four, five, six or more times over periods of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days. In another specific embodiment, each dose is administered at least once, and the subject is administered the dose three times a week for a period of two weeks (e.g., Monday, Wednesday, and Friday).

[0171] In one embodiment, the subjects are the following adverse events, e.g., grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade 3 or 4 leukocytosis (white blood cell count (WBC) > 100,000 mmHg). 3) Monitoring is performed for a decrease in grade 3 or 4 WBC, absolute lymphocyte count (ALC) and / or absolute neutrophil count (ANC), lymphocyte increase, and organ dysfunction (e.g., liver or kidney dysfunction). In certain embodiments, the dose is not increased and the subject experiences an adverse event, e.g., grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade 3 or 4 leukocytosis (white blood cell (WBC) > 100,000 mm 3 ) If a subject experiences a decrease in grade 3 or 4 WBC, absolute lymphocyte count (ALC) and / or absolute neutrophil count (ANC), lymphocyte increase, and organ dysfunction (e.g., liver or kidney dysfunction), the dose may remain the same and can be stopped or reduced. According to these embodiments, the dose of, for example, the IL-15 / IL-15Rα complex disclosed herein administered to a subject can be reduced until the adverse event decreases or disappears, or can remain the same, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, e.g., as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0172] In another embodiment, a method is provided herein in which enhancement of IL-15-mediated immune function is beneficial in preventing, treating, and / or managing a disorder in a subject, the method comprising administering the IL-15 / IL-15Rα complex disclosed herein to a human subject in a dose regimen in which the dose is increased by 2 to 3 times in subsequent cycles, starting with a first cycle containing an initial dose of 0.25 μg / kg to 4 μg / kg, and the dose is increased by 2 to 3 times compared to the previous dose, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. Each dose is administered at least once, twice, four times, or six times before increasing the dose to the next level, and the concentration of free IL-15 in a sample obtained from the subject (e.g., a plasma sample) is monitored for a certain period after administration of a certain dose of the IL-15 / IL-15Rα complex disclosed herein (e.g., approximately 24 to 48 hours, approximately 24 to 36 hours, approximately 24 to 72 hours, approximately 48 to 72 hours, approximately 36 to 48 hours, or approximately 48 to 60 hours after administration of a certain dose of the IL-15 / IL-15Rα complex, and before administration of another dose of the IL-15 / IL-15Rα complex) before increasing the dose to the next level, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0173] In another embodiment, a method is provided herein in which, in preventing, treating, and / or managing a disorder in a subject, enhancement of IL-15-mediated immune function is beneficial for preventing, treating, and / or managing such a disorder, and the method comprises, for example, administering the IL-15 / IL-15Rα complex disclosed herein to a subject in the following sequential dose regimens: (i) 0.25 μg / kg; (ii) 0.5 μg / kg; (iii) 1 μg / kg; (iv) 2 μg / kg; (v) 4 μg / kg; and (vi) 8 μg / kg, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In certain embodiments, for example, the IL-15 / IL-15Rα complex disclosed herein is administered to a subject in a dose regimen of the following sequential doses: (i) 1 μg / kg; (ii) 2 μg / kg; (iii) 4 μg / kg; and (iv) 8 μg / kg, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. Each dose is administered at least 1, 2, 4, or 6 times in an administration cycle before increasing the dose to the next level, and the concentration of free IL-15 in a sample obtained from a subject (e.g., a plasma sample) is monitored for a certain period after administration of a certain dose of the IL-15 / IL-15Rα complex disclosed herein (e.g., approximately 24 to 48 hours, approximately 24 to 36 hours, approximately 24 to 72 hours, approximately 48 to 72 hours, approximately 36 to 48 hours, or approximately 48 to 60 hours after administration of a certain dose of the IL-15 / IL-15Rα complex), and before administration of another dose of the IL-15 / IL-15Rα complex before increasing the dose to the next level, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0174] In another embodiment, a method for preventing, treating, and / or managing cancer in a subject is provided herein, for example, comprising administering the IL-15 / IL-15Rα complex disclosed herein to the subject in a dose regimen of the following sequential doses: (i) 1 μg / kg; (ii) 2 μg / kg; (iii) 4 μg / kg; and (iv) 8 μg / kg, wherein each dose is administered at least 1, 2, 4, or 6 times in an administration cycle before increasing the dose to the next level, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In specific embodiments, the method includes administering the IL-15 / IL-15Rα complex disclosed herein to a subject using a cyclic dosing regimen, which comprises (a) subcutaneous administration of the IL-15 / IL-15Rα complex to the subject at a dose of 0.1 to 10 μg / kg every 1, 2, or 3 days over a first period of 1 to 3 weeks; and (b) subcutaneous administration of the IL-15 / IL-15Rα complex to the subject at a dose of 0.1 to 10 μg / kg every 1, 2, or 3 days over a third period of 1 to 3 weeks, after a second period of 1 to 2 months in which the IL-15 / IL-15Rα complex is not administered to the subject, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0175] In certain embodiments, the subjects are human subjects. In some embodiments, the dose in a treatment cycle is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 1-5, 2-4, 2-5, 1-6, 2-6, 1-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times. In some embodiments, the dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 1-5, 2-4, 2-5, 2-6, 1-6, 3-6, 4-6, or 6-8 times over a period of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days. In one embodiment, each dose is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per administration cycle, or 1-3, 1-4, 1-5, 2-4, 2-5, 1-6, 2-6, 1-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times. In a specific embodiment, each dose is administered at least 1, 2, 3, 4, 5, 6 times or more over a period of 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14-21 days, or 1-3, 1-4, 1-5, 2-4, 2-5, 1-6, 2-6, 1-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times.

[0176] In another specific embodiment, the subject is administered a dose three times a week (e.g., Monday, Wednesday, and Friday). In one embodiment, the subject is monitored for the following adverse events, e.g., grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade 3 or 4 leukocytosis (white blood cells (WBC) > 100,000 mm3), grade 3 or 4 decrease in WBC, absolute lymphocyte count (ALC) and / or absolute neutrophil count (ANC), lymphocytosis, and organ dysfunction (e.g., hepatic or renal dysfunction). In some embodiments, the dose may remain the same, discontinue, or reduce if the subject experiences adverse events, such as grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade 3 or leukocytosis (white blood cells (WBC) > 100,000 mm3), grade 3 or 4 decrease in WBC, absolute lymphocyte count (ALC) and / or absolute neutrophil count (ANC), lymphocytosis, and organ dysfunction (e.g., hepatic or renal dysfunction), without increasing the dose. According to these embodiments, the dose of the IL-15 / IL-15Rα complex administered to the subject, for example, as described herein, may be reduced or remain the same until the adverse events decrease or disappear.

[0177] In specific embodiments, each dose is administered once a week for three weeks, according to the method described herein. In specific embodiments, each dose is administered once, three times a week for two weeks, according to the method described herein. In specific embodiments, each dose is administered once, three times a week for two, three, or four weeks, according to the method described herein. In specific embodiments, each dose is administered once, six times a week for two, three, or four weeks, according to the method described herein. In specific embodiments, each dose is administered once every other day for two, three, or four weeks, according to the method described herein. In specific embodiments, each dose is administered once daily for two, three, or four weeks, according to the method described herein.

[0178] In certain embodiments, for example, the IL-15 / IL-15Rα complex disclosed herein is administered subcutaneously to a subject by the method described herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In some embodiments, for example, the IL-15 / IL-15Rα complex disclosed herein is administered intravenously or intramuscularly to a subject by the method described herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In certain embodiments, for example, the IL-15 / IL-15Rα complex disclosed herein is administered intratumorally to a subject by the method described herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In some embodiments, for example, the IL-15 / IL-15Rα complex disclosed herein is administered topically to a site in a subject (e.g., a site of infection) by the method described herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0179] In one embodiment, the sample obtained from the subject by the method described herein is a blood sample. In a specific embodiment, the sample is a plasma sample. The basal plasma level of IL-15 is approximately 1 pg / ml in humans, approximately 8-10 pg / ml in monkeys (e.g., macaques), and approximately 12 pg / ml in rodents (e.g., mice). Samples can be obtained from the subject using techniques known to those skilled in the art.

[0180] Plasma levels of IL-15 can be assessed using standard techniques known to those skilled in the art. For example, plasma can be obtained from blood samples taken from a subject, and the level of IL-15 in the plasma can be measured by ELISA.

[0181] In specific embodiments, examples of immune function enhanced by the method described herein include lymphocyte proliferation / expansion (e.g., increase in lymphocyte count), inhibition of lymphocyte apoptosis, activation of dendritic cells (or antigen-presenting cells), and antigen presentation. In certain embodiments, the immune function enhanced by the method described herein is CD4 + T cells (e.g., Th1 and Th2 helper T cells), CD8 + T cells (e.g., cytotoxic T lymphocytes, alpha / beta T cells, and gamma / delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, dendritic cells (immature or mature), antigen-presenting cells, macrophages, mast cells, natural killer T cells (NKT cells), tumor-resident T cells, CD122 + This involves the proliferation / increase in the number of T cells or natural killer (NK) cells, or their activation. In one embodiment, the method described herein involves the proliferation / increase in the number of lymphocyte progenitor cells or an improvement thereof. In some embodiments, the method described herein involves CD4 + T cells (e.g., Th1 and Th2 helper T cells), CD8 + T cells (e.g., cytotoxic T lymphocytes, alpha / beta T cells, and gamma / delta T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, dendritic cells (immature or mature), antigen-presenting cells, macrophages, mast cells, natural killer T cells (NKT cells), tumor-resident T cells, CD122 + The number of T cells or natural killer cells (NK cells) is increased by approximately 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, or more compared to a negative control.

[0182] In specific embodiments, the methods described herein use assays well known in the art, such as ELISPOT, ELISA, and cell proliferation assays, to improve or induce immune function in a subject by at least 0.2, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or at least 10 times compared to a subject that has not been administered the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In specific embodiments, the methods described herein, using assays well known in the art, such as ELISPOT, ELISA, and cell proliferation assays, improve or induce immune function in a subject by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% compared to immune function in a subject that has not been administered, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In specific embodiments, the immune function is cytokine release (e.g., interferon-gamma, IL-2, IL-5, IL-10, IL-12, or transforming growth factor (TGF)-beta). In one embodiment, IL-15-mediated immune function is NK cell proliferation, which can be detected, for example, by assaying with flow cytometry to determine the number of cells expressing an NK cell marker (e.g., CD56). In one embodiment, IL-15-mediated immune function is CD8 +In another embodiment, IL-15-mediated immunization is antibody production, which can be assayed, for example, by flow assay. In some embodiments, IL-15-mediated immunization is effector function, which can be assayed, for example, by cytotoxicity assay or other assays well known in the art. The effect of one or more administrations of a combination of one or more IL-15 / IL-15Rα complexes and anti-PD-1 antibody molecules on peripheral blood lymphocyte counts can be monitored / evaluated using standard techniques known to those skilled in the art. Peripheral blood lymphocyte counts in mammals can be determined, for example, by obtaining a peripheral blood sample from the mammal, separating lymphocytes from other components of the peripheral blood, e.g., plasma, using, for example, Ficoll-Hypaque (Pharmacia) gradient centrifugation, and counting the lymphocytes using trypan blue. Peripheral blood T cell counts in mammals can be determined by, for example, separating lymphocytes from other components of peripheral blood, such as plasma, using methods such as Ficoll-Hypaque (Pharmacia) gradient centrifugation, labeling the T cells with antibodies conjugated to T cell antigens, such as FITC or phycoerythrin directed towards CD3, CD4, and CD8, and counting the number of T cells by FACS. Furthermore, specific subsets of T cells (e.g., CD2) can be identified. + CD4 + CD8 + CD4 + RO + CD8 + RO + CD4 + RA + , or CD8 + RA + The effect on ) or NK cells can be determined using standard techniques known to those skilled in the art, such as FACS.

[0183] Combination therapy Other therapies that can be used in combination with the IL-15 / IL-15Rα complex disclosed herein are also provided. In one embodiment, a method for preventing, treating, and / or controlling cancer is provided herein, comprising administering an effective amount of the IL-15 / IL-15Rα complex disclosed herein and at least one additional therapeutic agent, wherein the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0184] In one embodiment, at least one additional therapeutic agent is an anti-PD-1 antibody.

[0185] In preferred embodiments, the anti-PD-1 antibody is pembrolizumab, nivolumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, or tripalimab.

[0186] In a particularly preferred embodiment, the anti-PD-1 antibody is spartalizumab.

[0187] In specific embodiments, administration of a combination of the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein to a subject by the method described herein achieves one, two, three or more of the following results: (1) reduction of tumor or neoplasm growth; (2) reduction of tumor formation; (3) eradication, removal or control of primary, local and / or metastatic cancer; (4) reduction of metastatic spread; (5) reduction of mortality; (6) increase of survival rate; (7) increase of survival time; (8) increase of remission patients; (9) decrease of hospitalization rate; (10) decrease of hospitalization period; and (11) maintenance of tumor size such that the tumor size does not increase by more than 10%, or more than 8%, or more than 6%, or more than 4%; preferably, the tumor size does not increase by more than 2%. The IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0188] In specific embodiments, administration of the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein to a subject having cancer by the method described herein inhibits or reduces tumor growth by at least 2 times, preferably at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, or at least 10 times compared to tumor growth in a subject having cancer treated with a negative control, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In another embodiment, administration of the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein to a subject having cancer by the method described herein inhibits or reduces tumor growth by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 8%, at least 85%, at least 90%, or at least 95% compared to a negative control or a subject having cancer administered the IL-15 / IL-15Rα complex or the anti-PD-1 antibody molecule disclosed herein as a single agent, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0189] Examples of cancerous disorders include, but are not limited to, solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas (e.g., adenocarcinoma and squamous cell carcinoma), such as those affecting the liver, lungs, breasts, lymph nodes, gastrointestinal tract (e.g., colon), urogenital tract (e.g., renal cells, urothelial cells), prostate, and pharynx. Examples of adenocarcinomas include malignant tumors such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Examples of squamous cell carcinomas include malignant tumors in the lungs, esophagus, skin, head and neck region, oral cavity, anus, and neck. In one embodiment, the cancer is melanoma, such as advanced melanoma. Metastatic lesions of the above cancers can also be treated or prevented using the methods and compositions of the present invention.

[0190] For example, exemplary cancers whose growth can be inhibited using the combination of IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules disclosed herein include cancers that are typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory adenocarcinoma of the prostate), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Furthermore, refractory or recurrent malignancies can be treated using the combination therapies described herein.

[0191] Other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, kidney cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, for example, acute myeloid leukemia. Examples include chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in infants, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, kidney or ureteral cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, vertebral axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancers, such as those induced by asbestos (e.g., mesothelioma), and combinations of the above cancers.

[0192] In specific embodiments, the cancer is melanoma, kidney cancer, colon cancer, or prostate cancer. In one embodiment, the cancer is melanoma. In another embodiment, the cancer is metastatic. In yet another embodiment, the cancer is metastatic melanoma. In yet another embodiment, the subject has been previously treated with an immune checkpoint inhibitor (CPI), such as anti-PD-1 and / or anti-PD-L1 and / or anti-CTLA-4, and has responded and is progressing.

[0193] The combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein can be administered together with one or more other therapeutic agents, such as anticancer agents, cytokines, or antihormone agents, to treat and / or manage cancer, and the IL-15 / IL-15Rα complex is provided as part of the pharmaceutical compositions described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. Non-limiting exemplary anticancer agents are described below.

[0194] In one embodiment, a method is provided herein for preventing, treating and / or managing a disorder in a subject, such as a hyperproliferative state or disorder (e.g., cancer) in the subject, the method comprising administering the IL-15 / IL-15Rα complex and an anti-PD-1 antibody molecule disclosed herein to the subject, the IL-15 / IL-15Rα complex being provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In some embodiments, the anti-PD-1 antibody molecule is administered by injection (e.g., subcutaneously or intravenously) in doses of about 200 mg to 500 mg, for example, about 250 mg to 450 mg, about 300 mg to 400 mg, about 250 mg to 350 mg, about 350 mg to 450 mg, or about 300 mg or about 400 mg (e.g., a uniform dose). The administration schedule (e.g., a uniform administration schedule) may vary, for example, from once a week to once every two, three, four, five, or six weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose of approximately 300 mg to 400 mg once every three weeks or once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose starting from approximately 300 mg once every three weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose starting from approximately 400 mg once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose starting from approximately 300 mg once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is administered at a dose starting from approximately 400 mg once every three weeks.

[0195] As described herein, for example, the IL-15 / IL-15Rα complex disclosed herein may be administered to a subject in a pharmaceutical composition, for example, in a liquid pharmaceutical composition disclosed herein, or in a solid pharmaceutical composition disclosed herein. In one embodiment, for example, the IL-15 / IL-15Rα complex disclosed herein is administered to a subject in a liquid pharmaceutical composition. In another embodiment, for example, the IL-15 / IL-15Rα complex disclosed herein is administered to a subject in a solid pharmaceutical composition. In a specific embodiment, for example, the IL-15 / IL-15Rα complex disclosed herein is administered in combination with one or more other therapeutic agents, for example, an anti-PD-1 antibody molecule, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition described herein or as a solid pharmaceutical composition. Combination therapy includes, for example, simultaneous and sequential administration of the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein, wherein the IL-15 / IL-15Rα complex is provided as part of the pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. When used herein, for example, the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein are said to be administered simultaneously if they are administered to a patient on the same day, for example, at the same time, or at intervals of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours. In contrast, for example, the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein are said to be administered sequentially if they are administered to a patient on different days, for example, the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein may be administered at intervals of 1 day, 2 days, or 3 days. In the methods and uses described herein, for example, the administration of the IL-15 / IL-15Rα complex disclosed herein may precede or follow the administration of an anti-PD-1 antibody molecule. When administered simultaneously, for example, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein may be in the same pharmaceutical composition or in different pharmaceutical compositions.

[0196] For example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein can also be administered in conjunction with radiotherapy, including, for example, the use of X-rays, gamma rays, and other radiation sources, to destroy cancer cells. In specific embodiments, radiotherapy is administered as external beam radiation or remote therapy (radiation directed from a distance source). In other embodiments, radiotherapy is administered as medical therapy or close-range radiotherapy, where the radioactive source is positioned inside the body near the cancer cells or tumor mass. For example, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein can also be administered in combination with chemotherapy. In one embodiment, for example, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein can be administered before, during, or after radiotherapy or chemotherapy by the methods or uses described herein. In one embodiment, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein can be administered before, during, or after surgery.

[0197] In some embodiments, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is administered to a subject with or diagnosed with cancer, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein. In other embodiments, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is administered to a subject prone to or suspected of developing cancer, and the IL-15 / IL-15Rα complex is provided as part of a pharmaceutical composition described herein, for example, as a liquid pharmaceutical composition or a solid pharmaceutical composition described herein.

[0198] In one embodiment, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is administered to subjects aged 0–6 months, 6–12 months, 1–5 years, 5–10 years, 10–15 years, 15–20 years, 20–25 years, 25–30 years, 30–35 years, 35–40 years, 40–45 years, 45–50 years, 50–55 years, 55–60 years, 60–65 years, 65–70 years, 70–75 years, 75–80 years, 80–85 years, 85–90 years, 90–95 years, or 95–100 years. In another embodiment, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is administered to adult humans. In some embodiments, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein, for example, is administered to subjects who are undergoing, scheduled to undergo, or have undergone surgery, chemotherapy, and / or radiotherapy. In some embodiments, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein, for example, is administered to refractory patients. In one embodiment, a refractory patient is a patient who is refractory to standard anticancer therapy. In one embodiment, a patient with cancer is refractory to treatment if the cancer is not significantly eradicated and / or the symptoms are not significantly alleviated. The determination of whether a patient is refractory can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment, using “refractory” in the sense recognized in the art in such a background. In various embodiments, a patient with cancer is refractory if the cancerous tumor does not decrease or increases.

[0199] Other methods and uses described herein are used to treat patients exposed to specific toxins or pathogens. Accordingly, in another embodiment, a method is provided for treating an infectious disease in a subject, comprising administering to the subject a combination disclosed herein, for example, a combination comprising, for example, the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein, thereby treating the subject for an infectious disease.

[0200] In the treatment of infections (e.g., acute and / or chronic), administration of combinations of IL-15 / IL-15Rα complexes and anti-PD-1 antibody molecules, for example, as disclosed herein, can be combined with conventional treatments in addition to stimulating innate host immune defenses against infection, or instead. Innate host immune defenses against infection include, but are not limited to, inflammation, fever, antibody-mediated host defense, T lymphocyte-mediated host defense, e.g., lymphokine secretion and cytotoxic T cells (especially during viral infections), complement-mediated lysis and opsonization (facilitation of phagocytosis), and phagocytosis. The ability of anti-PD-1 antibody molecules to reactivate dysfunctional T cells is useful for treating chronic infections, particularly those in which cell-mediated immunity is crucial for complete recovery.

[0201] Antibody-mediated PD-1 blockade can stimulate immune responses to pathogens, toxins, and autoantigens by acting as an adjuvant to IL-15 / IL-15Rα complex administration, or in combination with IL-15 / IL-15Rα complex and / or vaccines. Examples of pathogens for which this therapeutic approach is particularly useful include those for which no effective vaccine currently exists, or for which conventional vaccines are not entirely effective. These include, but are not limited to, human immunodeficiency virus (HIV), hepatitis viruses (A, B, and C), influenza virus, herpes simplex virus, Giardia, Malaria, Leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. Immune system stimulation by blocking the IL-15 / IL-15Rα complex and PD-1 is particularly useful against established infections caused by agents that present modified antigens throughout the course of infection, such as HIV. These novel epitopes are recognized as exogenous substances at the time of treatment and therefore induce a robust T cell response that is not attenuated by negative signaling mediated by PD-1, for example.

[0202] Other therapeutic agents that can be used in combination with the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules disclosed herein, for example, for the prevention, treatment and / or management of diseases such as cancer, infectious diseases, lymphopenia, immunodeficiency and wounds include, but are not limited to, small molecules, synthetic drugs, peptides (including cyclic peptides), polypeptides, proteins, nucleic acids (e.g., DNA and RNA nucleotides, for example, antisense nucleotide sequences, triple helices, RNAi, and nucleotide sequences encoding biologically active proteins, polypeptides, or peptides), antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules.Specific examples of such treatments, though not limited to them, include immunomodulators (e.g., interferon), anti-inflammatory drugs (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone, glucocorticoids, steroids, and nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors)), analgesics, leukotriene antagonists (e.g., montelukast, methylxanthine, zafirlukast, and xyluterin), beta-2 agonists (e.g., albuterol, biterol, fenoterol, isoetalie, metaprotereol) Examples include (e.g., pirbuterol, salbutamol, terbutalineformoterol, salmeterol, and salbutamolterbutaline), anticholinergic agents (e.g., ipratropium bromide and oxytropium bromide), sulfasalazine, penicillamine, dapsone, antihistamines, antimalarial agents (e.g., hydroxychloroquine), antiviral agents (e.g., nucleoside analogs (e.g., zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and AZT), and antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, erythromycin, penicillin, mitramycin, and anthramycin).

[0203] Any therapeutic method known, used, or currently used for the prevention, management, and / or treatment of diseases affected by IL-15 function / signaling and / or immune checkpoint modulation can be used in combination with, for example, the combination therapy of the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein. For information regarding therapies (e.g., prophylactic or therapeutic agents) used or currently used for the prevention, treatment, and / or management of diseases or disorders, such as cancer, infectious diseases, lymphopenia, immunodeficiency, and wounds, see, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; The Merck Manual of Diagnosis and Therapy, Berkow, MD et al. (eds.), 17th Ed., Merck Sharp & Dohme Research Laboratories, Rahway, NJ, 1999; Cecil Textbook of Medicine, 20th Ed., Bennett and Plum (eds.), WBSaunders, Philadelphia, 1996, and Physicians' Desk Reference (66th ed. 2012).

[0204] For example, non-limiting examples of one or more other therapies that can be used in addition to the combination therapy of the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein include immunomodulators, for example, chemotherapeutic agents and non-chemotherapeutic immunomodulators. Non-limiting examples of chemotherapeutic agents include methotrexate, cyclosporine A, leflunomide, cisplatin, ifosfamide, taxanes, for example, taxol and paclitaxol, topoisomerase I inhibitors (e.g., CPT11, topotecan, 9AC, and GG211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, cytochalasin B, and gram Examples include cidin D, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin homologs, as well as cyclophosphamide.

[0205] biological activity For example, the IL-15 / IL-15Rα complex and / or anti-PD-1 antibody molecules disclosed herein increase an immune response that may be, for example, an antibody response (humoral response) or a cellular immune response, such as cytokine secretion (e.g., interferon-gamma), helper activity, or cytotoxicity. In one embodiment, the increase in the immune response is an increase in cytokine secretion, antibody production, effector function, T cell proliferation, and / or NK cell proliferation. Various assays for measuring such activity are well known in the art, and include enzyme-linked immunosorbent assays (ELISA; see, e.g., Section 2.1 of Current Protocols in Immunology, Coligan et al. (eds.), John Wiley and Sons, Inc. 1997), "tetramer staining" assays for identifying antigen-specific T cells (see Altman et al., (1996), Science 274:94-96), mixed lymphocyte target culture assays (see, e.g., Palladino et al., (1987), Cancer Res. 47:5074-5079), and the ELISPOT assay, which can be used to measure cytokine release in vitro (see, e.g., Scheibenbogen et al., (1997), Int. J. Cancer 71:932-936).

[0206] For example, the immune response induced or enhanced by the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, or 12-fold enhanced or increased compared to the immune response induced by the IL-15 / IL-15Rα complex or the anti-PD-1 antibody molecule disclosed herein, administered by any method known in the art and a negative control, or by single-agent administration. In some embodiments, the immune response induced by the combination of the IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule disclosed herein, for example, is improved by at least 0.5 to 2 times, at least 2 to 5 times, at least 5 to 10 times, at least 10 to 50 times, at least 50 to 100 times, at least 100 to 200 times, at least 200 to 300 times, at least 300 to 400 times, or at least 400 to 500 times compared to the immune response induced by a negative control assayed by any method known in the art. In some embodiments, the assay used to evaluate the immune response measures the level of antibody production, cytokine production, or cytotoxicity. In some embodiments, the assay used to measure the immune response is an enzyme-linked immunosorbent assay (ELISA) to determine antibody or cytokine levels, an ELISPOT assay to determine cytokine release, or a [ 51 This is a Cr release assay.

[0207] In specific embodiments, for example, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein increases the expression of IL-2 in whole blood activated by Staphylococcus enterotoxin B (SEB). For example, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein increases IL-2 expression by at least about 2-fold, about 3-fold, about 4-fold, or about 5-fold compared to the expression of IL-2 when the IL-15 / IL-15Rα complex, the anti-PD-1 antibody molecule, or an isotype control (e.g., IgG4) is used alone.

[0208] In one embodiment, for example, the proliferation or viability of cancer cells contacted with the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule disclosed herein is inhibited or reduced by at least about 2 times, preferably at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 7 times, or at least about 10 times compared to the proliferation of cancer cells contacted with the IL-15 / IL-15Rα complex or the anti-PD-1 antibody molecule disclosed herein as a negative control or single agent. Alternatively, cell viability is measured by an assay that measures lactate dehydrogenase (LDH), a stable cytoplasmic enzyme released during cell lysis, or during cell lysis [ 51 This can be measured by the release of Cr. In another embodiment, the proliferation of cancer cells contacted with a combination of the IL-15 / IL-15Rα complex and an anti-PD-1 antibody molecule is measured using assays well known in the art, such as CSFE, BrdU, or cell proliferation assays using radioactive thymidine uptake, to be inhibited or reduced by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to cancer cells contacted with, for example, the IL-15 / IL-15Rα complex or anti-PD-1 antibody molecule disclosed herein, as a negative control or single agent.

[0209] Cancer cell lines capable of performing such assays are known to those skilled in the art. Necrosis, apoptosis, and proliferation assays can also be performed on primary cells, for example, tissue explants.

[0210] Details of one or more embodiments of this disclosure are described in the accompanying description above. Furthermore, it should be understood that each embodiment can be combined with one or more other embodiments, insofar as such combinations are consistent with the description of the embodiment. Furthermore, it should be understood that the embodiments provided above include all embodiments, including such embodiments arising from combinations of embodiments. Any methods and materials similar to or equivalent to those described herein can be used in carrying out or testing this disclosure, but preferred methods and materials are described below. Other features, purposes, and advantages of this disclosure will become apparent from the description and claims. [Examples]

[0211] The following embodiments are provided to better illustrate preferred embodiments of the Disclosure. Those skilled in the art will recognize a number of modifications and changes that can be performed without altering the spirit or scope of the Disclosure. Such modifications and changes are included within the scope of the Disclosure. These embodiments should not be construed as limiting the scope of the Disclosed Matter as defined by the appended claims.

[0212] Example 1 The objective of this study was to evaluate the stability of liquid pharmaceutical compositions containing heterodimer IL-15 / IL-15Rα complexes, which include IL-15 (containing SEQ ID NO: 2) and IL-15Rα (containing SEQ ID NO: 5), in a direct comparison of three different formulations for long-term storage at 2–8°C. Furthermore, stability under stress (40°C) and accelerated (25°C) conditions was evaluated, and freeze-thaw and shaking stress were applied to compositions filled in 1.2 mL vials.

[0213] Materials and equipment The heterodimer IL-15 / IL-15Rα, each possessing SEQ ID NO: 2 and SEQ ID NO: 5, was provided at a concentration of 10 mg / mL in 5 mM histidine at pH 6.5. A total of three formulations were evaluated during this study, as detailed in Table 2.

[0214] [Table 4]

[0215] Preparation of the heterodimer IL-15 / IL-15Rα complex The heterodimer IL-15 / IL-15Rα complex in 5 mM histidine at pH 6.5 was thawed in a water bath at 30°C ± 5°C until completely thawed.

[0216] To obtain an acetate-based formulation, the heterodimer IL-15 / IL-15Rα complex was subjected to buffer exchange: the complex was diluted to 120 mL of stock solution E6 (20 mM acetate buffer, pH 5.0) at a concentration of 1 mg / mL, and then divided into a total of eight 15 mL spin columns (MWCO 10 kDa). The spin columns were centrifuged at 4500 rpm at 10°C for 10 minutes. The filtrate was removed, and the columns were resuspended by filling them with 15 mL of concentrate. This process was repeated for a total of six buffer exchange cycles. After the final cycle of centrifugation, filtrate removal, refilling, and resuspension, the concentration step was initiated. The spin columns were centrifuged at 4500 rpm for 10 minutes. After each centrifugation, the removed volume was replaced with protein solution from the other spin columns, thereby reducing the number of spin columns from eight to one, and the target volume of concentrated active pharmaceutical ingredient was obtained. After concentrating the complex, the concentrated substance was transferred to a Nalgene vial and its concentration was determined by nanodropping. To simplify handling during preparation, the concentrated solution was diluted to a concentration of 10 ± 0.5 mg / mL with each buffer.

[0217] Mixing and filtration Histidine-based formulations were prepared by adding the appropriate stock solution to achieve the final composition detailed in Figure 2. Preparation was performed directly in Nalgene vials with a minimum volume of 60 mL. The following amounts of stock solution were added to various formulations as needed:

number

[0218] The volume was then adjusted to 50 mL, the pH was measured, and adjusted using 1 M NaOH (acetic acid buffer) and 1 M HCl (histidine buffer). Changes in density were ignored. The amount added was recorded, and the solution was finally filled to 55 mL with Milli-Q water. The formulation was sterile filtered through a 0.22 μm PVDF filter under laminar flow and dispensed into 6R vials, each filled to 1.2 mL. The vials were crimped, labeled accordingly, and stored according to the stability plan described below. At each pull point, all samples were analyzed according to the analytical plan outlined below.

[0219] Stability research and analysis The samples were subjected to stability studies at the time points and storage conditions described in Table 3, and analyzed as detailed in Table 4.

[0220] [Table 5]

[0221] [Table 6]

[0222] Purity by SEC This study is based on size exclusion chromatography (SEC) with UV detection. Variants of the heterodimer IL-15 / IL-15Rα complex of different sizes (e.g., low molecular weight and high molecular weight variants and related substances) are separated by SEC under the native conditions of a suitable column. The purity of the main peak, as well as the amount of aggregates and fragments, was determined as a percentage of the total area obtained in each chromatogram of the sample.

[0223] Purity measured by CE-SDS Capillary electrophoresis SDS (CE-SDS) separates proteins according to size using an electric field by adding a hydrophilic sieving polymer to the separation buffer. The sample was injected into the inlet side of the capillary, and separation was performed along the long portion of the capillary from the inlet to the detector. Detection was performed using UV light.

[0224] Purification by RP-HPLC The heterodimer IL-15 / IL-15Rα complex product-related substances were separated by reverse-phase HPLC (RP-HPLC). Depending on the hydrophobicity, the proteins could be eluted separately from the hydrophobic matrix by applying different organic solvent concentrations. For separation on a C8 column, a gradient with increasing amounts of acetonitrile was used. Protein elution was monitored by UV absorption at a wavelength of 215 nm.

[0225] Charge variant by AEX Using anion exchange chromatography (AEX), charge-based variants of the heterodimeric IL-15 / IL-15Rα complex were separated after removal of N and O-linked glycans by enzymatic digestion. During chromatographic separation, the protein, which is negatively charged overall, was retained by the positively charged functional groups of the stationary phase. By applying a gradient while increasing the salt concentration, weakly bound variants (positively charged) eluted first, followed by progressively negatively charged variants. Elution was monitored by UV absorption at 210 nm.

[0226] result All formulations showed good stability, and no significant differences were observed among the tested formulations in terms of total aggregates (see Figure 1), total degradation products by SEC (see Figure 2), changes in charge variants (see Figure 3), and purity by CE-SDS (see Figure 4). However, when analyzed by RP-HPLC, F3 showed superior stability to accelerated and stress temperatures, in contrast to F2 and F1 (see Figure 5). When stored at 2–8°C, F1 and F2 showed a significant increase in invisible particles >2 μm as well as >10 μm particles, but no invisible particles were observed in F3 (see Figure 6). These findings were supported by increased turbidity in F1 and F2, as shown in Figure 7.

[0227] Mechanical stress test Mechanical stress tests were performed on F2 and F3 by freeze / thaw (F / T) stress and overnight shaking. For the purpose of testing, 1.2 mL was filled into glass vials and subjected to a total of five freeze-thaw cycles by repeatedly deep freezing the vials in a -80°C freezer and then thawing them at room temperature. For shaking, the vials were placed horizontally on a shaker and shaken overnight under normal light. All samples were analyzed by SEC. The results are shown in Figure 8. Both formulations showed good mechanical stability; that is, there was no change in aggregates or fragments after F / T or shaking stress.

[0228] Example 2 The objective of this study was to evaluate the stability of liquid pharmaceutical compositions containing heterodimer IL-15 / IL-15Rα complexes, which include IL-15 (containing SEQ ID NO: 2) and IL-15Rα (containing SEQ ID NO: 5), using a fully factorial design to identify factors contributing to stability. Twelve different formulations were tested (see Table 5).

[0229] [Table 7]

[0230] The purity and particle formation by RP-HPLC were evaluated for all the above formulations. A decrease in purity by RP-HPLC was observed for all the formulations containing polysorbate 20 stored under stress conditions at 40°C. In contrast, for the formulations containing poloxamer 188 at 40°C, only a slight decrease in purity was observed by RP-HPLC. This observation was less prominent in the formulations containing acetate and more prominent in the formulations containing histidine (see Figure 10). The formation of subvisible particles (SVP) with sizes >2 μm (see Figure 9A) and >10 μm (see Figure 9B) in the presence of surfactants stored at 2 - 8°C was slightly more prominent in the compositions containing polysorbate 20, but subvisible particles were observed in the presence of poloxamer 188. This increase in particles was less prominent in the formulations containing histidine than in those containing acetate (see Figures 9C, 9D, and 9E), but in both cases, it remained within the acceptable range. The present disclosure provides, for example, the following: [Item 1] A liquid pharmaceutical composition comprising an IL-15 / IL-15Rα complex and a surfactant in an amount of about 0.0001% to about 1% (w / v). [Item 2] The composition according to item 1, further comprising a buffer in an amount of about 1 mM to about 100 mM that provides a pH in the range of about 4.5 to about 8.5. [Item 3] The composition according to item 2, further comprising at least one stabilizer in an amount of about 1 mM to about 500 mM. [Item 4] The composition according to any one of items 1 to 3, wherein the surfactant is a poloxamer. [Item 5] The composition according to item 4, wherein the poloxamer is poloxamer 188. [Item 6] The composition according to item 4 or 5, wherein the poloxamer is present at a concentration of about 0.05% to about 0.5% (w / v). [Item 7] The composition according to any one of items 1 to 3, wherein the surfactant is a polysorbate. [Item 8] The composition according to item 7, wherein the polysorbate is polysorbate 20 or polysorbate 80. [Item 9] The composition according to item 7 or 8, wherein the polysorbate is polysorbate 20. [Item 10] The composition according to item 7 or 8, wherein the polysorbate is polysorbate 80. [Item 11] The composition according to any one of items 7 to 10, wherein the surfactant is present at a concentration of about 0.01% to about 0.1% (w / v). [Item 12] The composition according to any one of items 2 to 11, wherein the buffer is an acetate buffer, a succinate buffer, a citrate buffer, or a histidine buffer. [Item 13] The composition according to any one of items 2 to 12, wherein the buffer is an acetate buffer. [Item 14] The composition according to item 13, wherein the acetate buffer is a Na-acetate buffer. [Item 15] The composition according to any one of items 2 to 14, wherein the buffer is at a concentration of about 10 mM to about 50 mM. [Item 16] The composition according to any one of items 2 to 15, wherein the buffer is at a concentration of about 15 mM to about 30 mM. [Item 17] The composition according to any one of items 2 to 16, wherein the buffer is at a concentration of about 20 mM. [Item 18] The composition according to any one of items 2 to 17, wherein the buffer provides a pH of about 4.7 to about 5.5. [Item 19] The composition according to any one of items 3 to 18, wherein the at least one stabilizer is a polyol or a sugar. [Item 20] The composition according to any one of items 3 to 19, wherein the at least one stabilizer is a sugar which is sucrose. [Item 21] A composition according to any one of claims 3 to 20, wherein at least one stabilizer is present at a concentration of about 100 mM to about 350 mM. [Section 22] The composition according to any one of claims 3 to 21, wherein at least one stabilizer is present at a concentration of about 220 mM to about 300 mM. [Section 23] The composition according to any one of claims 3 to 22, wherein at least one stabilizer is present at a concentration of about 260 mM. [Section 24] The composition according to any one of claims 1 to 23, wherein the concentration of the IL-15 / IL-15Rα complex is approximately 0.1 mg / mL to approximately 50 mg / mL. [Section 25] The composition according to any one of claims 1 to 24, wherein the concentration of the IL-15 / IL-15Rα complex is approximately 0.1 mg / mL to approximately 10 mg / mL. [Section 26] A composition according to any one of claims 1 to 25, comprising approximately 1 mg / mL of IL-15 / IL-15Ra complex, approximately 0.2% of poloxamer 188, approximately 260 mM of sucrose, and approximately 20 mM of sodium acetate, with a pH of approximately 5.0. [Section 27] A solid pharmaceutical composition comprising an IL-15 / IL-15Rα complex, a buffering agent in a concentration of approximately 10 mM to 50 mM that provides a pH in the range of approximately 6.5 to 8.5, at least one stabilizer in a concentration of approximately 1 mM to 500 mM, and at least one isotonic agent in a concentration of approximately 0.1 mM to 50 mM. [Section 28] The composition according to claim 27, wherein the buffer is a phosphate buffer, an acetate buffer, a succinate buffer, a citrate buffer, or a histidine buffer. [Section 29] The composition according to item 27 or 28, wherein the buffer is a Na / K phosphate buffer. [Section 30] A composition according to any one of claims 27 to 29, comprising approximately 1 mM to approximately 50 mM of a buffering agent. [Section 31] The composition according to any one of items 27 to 30, containing a buffer of about 1 mM to about 5 mM. [Item 32] The composition according to any one of items 27 to 31, wherein the pH of the composition is about 6.5 to about 7.5. [Item 33] The composition according to any one of items 27 to 32, wherein the pH of the composition is about 7.3. [Item 34] The composition according to any one of items 27 to 33, further containing at least two stabilizers of about 1 mM to about 500 mM. [Item 35] The composition according to any one of items 27 to 34, containing sucrose of about 1 mM to about 500 mM and mannitol of about 1 mM to about 500 mM. [Item 36] The composition according to any one of items 27 to 35, containing sucrose of about 5 mM to about 50 mM and mannitol of about 100 mM to about 300 mM. [Item 37] The composition according to any one of items 27 to 36, containing sucrose of about 30 mM and mannitol of about 220 mM. [Item 38] The composition according to any one of items 27 to 37, further containing at least two tonicity agents of about 0.1 mM to about 50 mM. [Item 39] The composition according to any one of items 27 to 38, containing KCl of about 0.1 mM to about 50 mM and NaCl of about 0.1 mM to about 50 mM. [Item 40] The composition according to any one of items 27 to 39, containing KCl of about 0.1 mM to about 1 mM and NaCl of about 10 mM to about 50 mM. [Item 41] The composition according to any one of items 27 to 40, containing KCl of about 0.375 mM and NaCl of about 20 mM. [Item 42] The composition according to any one of items 27 to 41, containing an IL-15 / IL-15Rα complex of about 0.1 mg / mL to about 50 mg / mL. [Item 43] A composition according to any one of claims 27 to 42, comprising approximately 0.1 mg / mL to approximately 10 mg / mL of IL-15 / IL-15Rα complex. [Section 44] A composition according to any one of claims 27 to 43, comprising approximately 0.1 mg / mL to approximately 0.5 mg / mL of IL-15 / IL-15Rα complex. [Section 45] The composition according to any one of claims 27 to 44, wherein the composition is freeze-dried. [Section 46] A composition according to any one of claims 27 to 45, comprising approximately 0.24 mg / mL of IL-15 / IL-15Rα complex, approximately 30 mM sucrose, approximately 220 mM mannitol, approximately 0.375 mM potassium chloride, approximately 20 mM NaCl, and approximately 1.35 mM Na / K phosphate buffer at pH 7.3. [Section 47] The composition according to any one of claims 1 to 46, wherein the IL-15 / IL-15Rα complex comprises IL-15 including SEQ ID NO: 2. [Section 48] The composition according to any one of claims 1 to 47, wherein the IL-15 / IL-15Rα complex comprises IL-15Rα containing SEQ ID NO: 5. [Section 49] The composition according to any one of claims 1 to 48, wherein the IL-15 / IL-15Rα complex comprises IL-15 containing SEQ ID NO: 2 and IL-15Rα containing SEQ ID NO: 5. [Section 50] A composition according to any one of items 1 to 49, for use in the treatment of cancer, lymphopenia, immunodeficiency, infectious diseases, and / or wounds. [Section 51] The composition described in item 50 for use in the treatment of cancer. [Section 52] The aforementioned cancers include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, kidney cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, for example, acute myeloid leukemia, chronic myeloid leukemia, acute phosphorus The composition according to item 50 or 51, which includes pablastic leukemia, chronic lymphocytic leukemia, solid tumors in infants, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, vertebral axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancer, such as those induced by asbestos (e.g., mesothelioma), and combinations of the said cancers. [Section 53] The composition according to any one of claims 50 to 52, wherein the cancer is melanoma, kidney cancer, colorectal cancer, or prostate cancer. [Section 54] The composition according to any one of claims 50 to 53, wherein the cancer is melanoma. [Section 55] A composition according to any one of items 50 to 54, wherein the cancer is metastatic. [Section 56] A dosage form comprising a pharmaceutical composition as described in any one of items 1 to 49. [Section 57] A vial containing a pharmaceutical composition as described in any one of items 1 to 49. [Section 58] A syringe containing a pharmaceutical composition as described in any one of items 1 to 49. [Section 59] An autoinjector including the syringe described in item 58. [Section 60] An autoinjector comprising a composition described in any one of items 1 to 49. [Section 61] The liquid composition is a. After storage at 2-8°C for 24 weeks, the total amount of aggregates is approximately 0.1% to approximately 0.25%. b. After storage at 25°C for 12 weeks, the total amount of aggregates is approximately 0.15% to approximately 0.35%. c. After storage at 40°C for 6 weeks, the total amount of aggregates is approximately 0.3% to 0.6%. d. After 24 weeks of storage at 2-8°C, the total amount of fragments was approximately 0.25% to 0.75%. e. After 12 weeks of storage at 25°C, the total amount of fragments is approximately 1.5% to 2.0%. At 40°C, after 6 weeks of storage, the total amount of fragments was approximately 2.5% to 3.5%. g. After 24 weeks of storage at 2-8°C, the total amount of basic variants is approximately 42.5% to 45%. After 24 weeks of storage at 2-8°C, the total amount of acidic variants is approximately 55% to 57.5%. i. After 12 weeks of storage at 25°C, the total amount of basic variants is approximately 38% to 42%. j. After 12 weeks of storage at 25°C, the acidic variant was approximately 55% to 60%. After storage at 2-8°C for 24 weeks, IL-15Rα levels were approximately 65% ​​to 67% according to CE-SDS. After 24 weeks of storage at 2-8°C, IL-15 levels were approximately 17% to 19% according to CE-SDS. After 24 weeks of storage at m.2~8°C, IL-15 HMW was found to be approximately 7% to 8% according to CE-SDS. n.2 After storage at 8°C for 24 weeks, CE-SDS showed that approximately 5% to 6% of aglycosylated IL-15 was present. After storage at 2-8°C for 24 weeks, RP-HPLC determined that the total amount of impurities was approximately 2% to 4%. After storage at 25°C for 12 weeks, RP-HPLC determined that the total amount of impurities was approximately 3% to 5%. q. After storage at 40°C for 6 weeks, RP-HPLC determines whether the total amount of impurities is less than 5%. After storage at 2-8°C for 24 weeks, PAMAS was used to determine if there were virtually no SVPs >2μm. After storage at 2-8°C for 24 weeks, PAMAS tests showed that there were virtually no SVPs >10 μm. After storage at t.2~8℃ for 24 weeks, the turbidity should be less than 1.0 NTU, u. After being subjected to 5 freeze / thaw cycles or overnight shaking, the total amount of aggregates assessed by SEC is less than 0.25%, or The composition according to any one of claims 1 to 26, wherein, after being subjected to 5 freeze / thaw cycles or overnight shaking, the total amount of fragments evaluated by SEC remains less than 0.35%.

Claims

1. A liquid pharmaceutical composition comprising an IL-15 / IL-15Rα complex, approximately 0.2% to approximately 0.3% (w / v) of surfactant, approximately 1 mM to approximately 100 mM of buffering agent, and approximately 220 mM to approximately 300 mM of stabilizer, (i) The surfactant is poloxamer 188, (ii) The buffer is an acetate (Na-acetate) buffer, and (iii) The stabilizer is sucrose, Here, the IL-15 / IL-15Rα complex comprises: an IL-15 polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 2, and an IL-15Rα polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:

5. A liquid pharmaceutical composition wherein the buffer provides a pH in the range of 4.7 to 5.

5.

2. The aforementioned buffering agent: (a) a concentration of approximately 10 mM to approximately 50 mM; and / or (b) Provides a pH of approximately 4.7 or approximately 5.0 The composition according to claim 1.

3. The composition according to claim 1 or 2, wherein the stabilizer is present at a concentration of approximately 260 mM.

4. The composition according to any one of claims 1 to 3, wherein the concentration of the IL-15 / IL-15Rα complex is about 0.1 mg / mL to about 50 mg / mL.

5. A composition according to any one of claims 1 to 4, comprising approximately 0.1 mg / mL to approximately 10 mg / mL of IL-15 / IL-15Rα complex, approximately 0.2% to approximately 0.3% (w / v) of poloxamer 188, approximately 220 mM to approximately 260 mM of sucrose, and approximately 10 mM to approximately 30 mM of a sodium acetate buffer.

6. The composition according to any one of claims 1 to 5, comprising approximately 1 mg / mL of IL-15 / IL-15Rα complex, approximately 0.2% (w / v) of poloxamer 188, approximately 260 mM of sucrose, and approximately 20 mM of a sodium acetate buffer.

7. The composition according to any one of claims 1 to 5, comprising approximately 1 mg / mL of IL-15 / IL-15Rα complex, approximately 0.2% (w / v) of poloxamer 188, approximately 260 mM of sucrose, and approximately 20 mM of a sodium acetate buffer, wherein the composition has a pH of approximately 5.

0.

8. The liquid composition is a. After storage at 2-8°C for 24 weeks, the total amount of aggregates is approximately 0.1% to approximately 0.25%, b. After storage at 25°C for 12 weeks, the total amount of aggregates is approximately 0.15% to approximately 0.35%, c. After storage at 40°C for 6 weeks, the total amount of aggregates is approximately 0.3% to approximately 0.6%, d. After storage at 2-8°C for 24 weeks, the total amount of fragments is approximately 0.25% to approximately 0.75%. e. After 12 weeks of storage at 25°C, the total amount of fragments is approximately 1.5% to approximately 2.0%. f. After storage at 40°C for 6 weeks, the total amount of fragments is approximately 2.5% to 3.5%. g. After 24 weeks of storage at 2-8°C, the total amount of basic variants is approximately 42.5% to approximately 45%. h. After 24 weeks of storage at 2-8°C, the total amount of acidic variants is approximately 55% to 57.5%. i. After 12 weeks of storage at 25°C, the total amount of basic variants is approximately 38% to 42%, j. After 12 weeks of storage at 25°C, the acidic variant is approximately 55% to 60%. k. After storage at 2-8°C for 24 weeks, IL-15Rα was found to be approximately 65% ​​to 67% according to CE-SDS.

1. After storage at 2-8°C for 24 weeks, IL-15 levels were approximately 17% to 19% according to CE-SDS. m. After storage at 2-8°C for 24 weeks, IL-15 HMW was found to be approximately 7% to 8% according to CE-SDS. n. After storage at 2-8°C for 24 weeks, CE-SDS indicates that the amount of aglycosylated IL-15 is approximately 5% to 6%. o. After storage at 2-8°C for 24 weeks, RP-HPLC determined that the total amount of impurities was approximately 2% to 4%. p. After storage at 25°C for 12 weeks, RP-HPLC determined that the total amount of impurities was approximately 3% to 5%. q. After storage at 40°C for 6 weeks, is the total amount of impurities less than 5% by RP-HPLC? r. After storage at 2-8°C for 24 weeks, PAMAS tests showed that there were virtually no SVPs >2 μm. s. After storage at 2-8°C for 24 weeks, PAMAS tests showed that there were virtually no SVPs >10 μm. t. After storage at 2-8°C for 24 weeks, the turbidity should be less than 1.0 NTU. u. After being subjected to five freeze / thaw cycles or overnight shaking, the total amount of aggregates assessed by SEC is less than 0.25%, or v. After being subjected to five freeze / thaw cycles or overnight shaking, the total amount of fragments evaluated by SEC is less than 0.35%. A composition according to any one of claims 1 to 7, which maintains the following:

9. The composition according to any one of claims 1 to 8, wherein the IL-15 / IL-15Rα complex comprises an IL-15 polypeptide containing the amino acid sequence of SEQ ID NO: 2, an IL-15Rα polypeptide containing the amino acid sequence of SEQ ID NO: 5, or an IL-15 polypeptide containing the amino acid sequence of SEQ ID NO: 2 and an IL-15Rα polypeptide containing the amino acid sequence of SEQ ID NO:

5.

10. The composition according to any one of claims 1 to 9, wherein the buffering agent has a concentration of about 15 mM to about 30 mM.

11. The composition according to any one of claims 1 to 10, wherein the buffering agent has a concentration of about 20 mM.

12. The composition according to any one of claims 1 to 11, wherein the concentration of the IL-15 / IL-15Rα complex is about 0.1 mg / mL to about 10 mg / mL.

13. The composition according to any one of claims 1 to 12, wherein the composition has a pH of about 4.7 to about 5.

0.

14. The composition according to any one of claims 1 to 13, wherein the composition has a pH of about 4.

7.

15. A composition according to any one of claims 1 to 14 for use in the treatment of a disorder, wherein the disorder is selected from the group consisting of cancer, lymphopenia, immunodeficiency, infectious disease, and wound.

16. Use of the composition according to any one of claims 1 to 14 in the manufacture of a pharmaceutical product in the treatment of a disorder, wherein the disorder is selected from the group consisting of cancer, lymphopenia, immunodeficiency, infectious disease, and wound.

17. The aforementioned cancers include melanoma, kidney cancer, colorectal cancer, prostate cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, kidney cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, for example, acute myeloid leukemia, chronic bone cancer The composition according to claim 15, selected from the group consisting of myelin leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in infants, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, kidney or ureter cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, vertebral axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancer, and combinations thereof; and / or the cancer is metastatic.

18. The cancer is melanoma, kidney cancer, colorectal cancer, prostate cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, kidney cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Merkel cell carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, for example, acute myeloid leukemia, chronic The use according to claim 16, selected from the group consisting of myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in infants, lymphocytic lymphoma, bladder cancer, multiple myeloma, myelodysplastic syndrome, kidney or ureter cancer, renal pelvis carcinoma, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, vertebral axial tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environment-induced cancer, and combinations thereof; and / or the cancer is metastatic.

19. A vial, syringe, or autoinjector comprising the pharmaceutical composition according to any one of claims 1 to 14, wherein the syringe is optionally included in the autoinjector.

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