het IL-15 expressed in CHO cells

Abstract

This provides IL-15 / IL-15Rα heterodimers produced in CHO cell lines. [Solution] A polypeptide complex is provided comprising human interleukin 15 (IL-15) polypeptide and human interleukin 15 receptor α (IL-15Rα) polypeptide, wherein the polypeptide complex comprises N-linked glycans including FA2G2, FA2G2S1, FA2G2S2, FA3G3S1, FA2F1G2S2, FA3G2S2, and FA3G3S3.

Description

[Technical Field]

[0001] This disclosure relates to human interleukin 15 (IL-15) having unique glycosylation properties. Polypeptides and human interleukin-15 receptor α (IL-15Rα) polypeptides The present invention relates to polypeptide complexes containing such complexes, and to methods for producing such polypeptides.

[0002] Sequence List This application has been filed electronically in ASCII format and is referenced in its entirety herein. Includes sequence listings referenced by [source]. Created on January 27, 2021. The file, named PAT058680-WO-PCT_SL.txt, has a size of 37.8 It is 33 bytes. [Background technology]

[0003] The cytokine interleukin-15 (IL-15) is produced by many cells in the body. It is a member of the alpha-4 helix bundle family of lymphokines. IL-15 modulates the activity of both the innate and adaptive immune systems, for example, in invasive diseases. Maintaining the memory T cell response to the primordium, inhibiting apoptosis, activating dendritic cells, etc. Furthermore, it is extremely important in the induction of natural killer (NK) cell proliferation and cytotoxic activity. To take on a role.

[0004] The IL-15 receptor is a set of three polypeptides shared by multiple cytokine receptors. , type-specific IL-15 receptor alpha ("IL-15Rα"), IL-2 / IL-1 5 receptor beta (or CD122) ("β"), and common gamma chain (or CD13 2) It consists of ("γ"). IL-15Rα is thought to be expressed by a wide range of cell types. However, β and γ are not necessarily expressed simultaneously. IL-15 signaling is IL -15R via heterodimer complex of α, β, and γ; heterodimer complex of β and γ via accretion, or via the subunit IL-15RX found on mast cells. It has been shown that this will occur.

[0005] IL-15 is a soluble protein, but endogenous IL-15 is soluble in serum or body fluids. It is not easily detectable. This is because it is mainly due to several types of accessory details. This is because it arises as a membrane-bound form expressed or acquired by cells. For example, IL-15 mRNA is detected in both hematopoietic and non-hematopoietic cells, but T cells produce IL-15. It does not produce. Instead, IL-15 binds to IL-15Rα and forms a cell surface complex on T cells. It forms the body. IL-15 has a "sushi" in exon 2 of the extracellular domain of the receptor. It specifically binds to IL-15Rα with high affinity via the main molecule. After complete recycling and re-transport to the cell surface, these IL-15 complexes are IL- It possesses the property of activating bystander cells that express the 15Rβγ low affinity receptor complex. This leads to the induction of IL-15-mediated signaling via the Jak / Stat pathway. IL-15 is cleaved at the cleavage site in the extracellular domain immediately distal to the transmembrane domain. The wild-type soluble form of Rα ("sIL-15Rα") has been observed. Tumor necrosis factor-A TACE / ADAM17 is a protease involved in this process. They are related.

[0006] Based on its multifaceted role in the immune system, to modulate IL-15-mediated function Various therapeutic designs have been explored. Recent reports have shown that IL-15 is sIL-1 5Rα or a sushi domain, maintains its immune-enhancing function , which suggests the foregoing. Recombinant IL-15 and IL-15 / IL-15Rα complexes have been studied in various preclinical models to varying degrees promote the expansion of memory CD8 T cells and NK cells and have been shown to enhance tumor rejection. Furthermore, in mouse models, IL- 15 or tumor targeting of constructs containing IL-15 / IL-15Rα complexes leads to the engraftment of syngeneic tumors planted immunocompetent animals or T and B cell-deficient S CID mice (which retain NK cells) all resulted in an improved anti-tumor response . The improved anti-tumor activity is attributed to the enhancement of NK and / or CD8 cytotoxic T cell expansion within the tumor the increased half-life of IL-15-containing moieties that provides the foregoing, as well as IL-1 on the surface of tumor cells 5 is considered to be dependent on trans-presentation. Therefore, it is possible to express IL-15 genetically engineered tumor cells can promote T cell and NK cell recruitment, proliferation and it has also been reported that enhancing function promotes rejection of established tumors (Zhang et al, (2009) PNAS USA. 106:7513-7518; Mung er et al, (1995) Cell Immunol. 165(2):289-2 93; Evans et al, (1997) Cell Immunol. 179(1) :66-73; Klebanoff et al, (2004) PNAS USA. 10 1(7):1969-74; Sneller et al, (2011) Blood. 1 18(26):6845-6848;Zhang et al,(2012)J.Imm unol.188(12):6156-6164). [Overview of the initiative]

[0007] The biological activity of proteins containing oligosaccharide chains, known as glycoproteins, is determined by the Not only the complete structure of the protein, but also the properties of the oligosaccharides covalently attached to the protein. The dependence is well understood. Glycosylation is related to protein solubility, protein Resistance to quaternary degradative attack and thermal inactivation, quaternary structure, activity, targeting, antigenicity, functional activity , and may have an effect on half-life. The glycosylation pattern of mammals is generally Fukud a et al. (1994), Molecular Glycobiology, IR As described in L Press, New York (as incorporated herein by reference) Sialic acid N-acetylneuraminic acid (NANA) is an N- and O-linked glycan. It is the main component. Sialic acid is important for extending the half-life of protein-based therapeutic drugs. It has been shown that desialylated or low-sialylated glycoproteins significantly reduce the half-life in plasma. It is well known that this reduces the amount. Therefore, IL has its own unique glycosylation characteristics. Producing the -15 / IL-15Rα complex is advantageous.

[0008] This specification describes IL-15 / IL-15Rα heterodiodes having unique glycosylation properties. A composition of the mer is disclosed. N-acetylneuraminic acid (NANA) is N- and O-bonded It is the main component of composite glycans. NANA is a protein glycosylation event in humans. While it is the dominant form of neuraminic acid within mammals, other mammals also have N-glycolylneura It may also contain other derivatives such as methyl acid (NGNA). NANA is N in several ways. - and can be bonded to the core structure of O-glycans. Preferably α(2,3) and α(2,6) ) can be found to be bound to the subsequent sugar, but other bonds such as α(2,8) are present. Human cells, such as human embryonic kidney (HEK) cells, primarily undergo α(2,6) linkage sialylation. While generating a large number of producing cell lines, such as Chinese hamster ovary (CHO) cells, Mammalian cell lines, such as cell lines, produce α(2,3) linkage sialylation.

[0009] In CHO cells, the α(2,6)-linked NANA extension portion is generated to the coagulan structure. The enzyme involved in this (β-galactoside α-2,6-sialyltransferase 1) It has been reported that CHO cells are inactive or not expressed (for example, Ch ung et al.(2017)Biotechnol.J.12:1600502 (Reference) However, the gene in the enzyme itself is found in C. griseus. It exists. This invention relates to the IL-15 / IL-15Rα complex produced in CHO cells, The glycosylation differs from that produced by the IL-15 / IL-15Rα complex by the ophthalmic cell line. This is based on the unexpected discovery that it has a glycation pattern. Furthermore, it is an α(2,6) linked glycosylation type. This was found in the IL-15 / IL-15Rα complex produced in CHO cells. The glycosylation pattern is typically observed in proteins expressed in human cell lines. This is unique and surprising. It offers direct benefits, such as human glycosyl This results in lower immunogenicity compared to the expected CHO pattern due to its closer resemblance to the chemical form. To add. For example, N-glycolylneuraminic acid, a non-human potentially immunogenic glycol. Glycoepitopes are IL-15 produced in CHO cells. It is essentially absent in IL-15Rα. Furthermore, glycosylation is in vivo This affects the half-life and distribution of cytokines, and consequently... This may affect the therapeutic effect of glycosylated IL-15 / IL-15Rα complexes.

[0010] On the other hand, the IL-15 / IL-15Rα complex derived from HEK cells exhibits low process robustness. Low yield, use of animal-derived raw materials in the production process, and complex analytical characteristics with limited resolution. This is due to the presence of splice variants in IL-15Rα that have recently been detected. It is not considered optimal for further expression. The IL-15Rα splice mutant exhibits toxicity. This can cause additional strains of active IL-15 being administered to the patient. This method accurately measures the amount of the IL-15Rα complex, making it difficult for it to affect the efficacy of the drug. The susceptibility to viral contamination also allows the use of HEK cells to produce recombinant biologics. This is considered a potential risk at that time. Therefore, CHO cells are safer and virus It is not susceptible to contamination and is far more Due to its high yield, it is the most efficient method for producing the IL-15 / IL-15Rα complex. It is considered appropriate.

[0011] Therefore, this disclosure relates to human interleukin 15 (IL-15) polypeptide and Polypeptides containing the interleukin-15 receptor α (IL-15Rα) polypeptide The focus is on fusion. In some embodiments, the polypeptide complex is FA2G2, FA 2G2S1, FA2G2S2, FA3G3S1, FA2F1G2S2, FA3G2S2, and includes N-linked glycans including FA3G3S3. In some embodiments, IL- 15 polypeptide has the sequence of SEQ ID NO: 1 or 5, and IL-15Rα has the sequence of SEQ ID NO: 6. It has an arrangement of 7, 10, 12, 14, or 21.

[0012] In some embodiments, the N-linked glycan is at least about 10% of FA2G2S1. Includes %, approximately 12.5%, approximately 15%, approximately 17.5%, approximately 20%, or approximately 22.5%. How many? In that embodiment, the N-linked glycan is at least about 10% of FA2G2S1, about 1 1%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 1 Includes 9%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, or approximately 25%.

[0013] In some embodiments, the N-linked glycan is at least about 10% of FA2G2S2. Includes %, approximately 20%, approximately 30%, or approximately 40%. In some embodiments, N-bonded glycerides. Likan is at least about 10%, about 12.5%, about 15%, and about 17.5% of FA2G2S2. %, approximately 20%, approximately 22.5%, approximately 25%, approximately 27.5%, approximately 30%, approximately 32.5%, approximately 3 It contains 5%, approximately 37.5%, or approximately 40%.

[0014] In some embodiments, the polypeptide complex includes an O-linked glycan. In that embodiment, at least 80%, 85%, 90%, or 95% of the glycan is core- It is an O-linked glycan.

[0015] In some embodiments, the core-1 O-linked glycan is mainly monosialylated and / It is odicialized.

[0016] In addition, this specification refers to a type of glycan having both O-linked and N-linked glycans. Interleukin-15 (IL-15) polypeptide and human interleukin-15 receptor Polypeptide complexes containing the α(IL-15Rα) polypeptide are disclosed. In this embodiment, the O-linked glycan of the polypeptide complex is at least about the glycan It has 80%, approximately 85%, approximately 90%, or approximately 95%, and has a core-1 O-linked glycan structure. It has; and the polypeptide complexes are FA2G2, FA2G2S1, FA2G2S2, FA N-bonds including 3G3S1, FA2F1G2S2, FA3G2S2, and FA3G3S3 Contains type glycans. In some embodiments, the IL-15 polypeptide is SEQ ID NO: 1 or It has sequence 5, and IL-15Rα is sequence number 6, 7, 10, 12, 14 or 21 It has an array of these.

[0017] In some embodiments, the N-linked glycan is at least about 10% of FA2G2S1. Includes %, approximately 12.5%, approximately 15%, approximately 17.5%, approximately 20%, or approximately 22.5%. How many? In that embodiment, the N-linked glycan is at least 10% to 20% of FA2G2S2 It contains 30%, or 40%. In some embodiments, it contains core-1 O-bonded glycans. It is mainly monosiallylated and / or disiallylated.

[0018] In addition, this specification refers to isolated IL-15 / IL-15 produced in non-human cells. A Rα heterodimer is disclosed, where the IL-15 / IL-15Rα heterodimer is α( 2,6) Includes O-linked sialylation. In some embodiments, non-human cells are recombinant. These are Chinese hamster ovary (CHO) cells. In some embodiments, CHO cells are used. The cells are modified to impair the function of matryptase.

[0019] In some embodiments, the isolated IL-15 / IL-15Rα heterodimer is O - Containing a conjugated glycan, where at least 90% or 95% of the glycan is core-1 It is an O-linked glycan. In some embodiments, about 15% of the O-glycan is α( It has 2,6)-linked sialylation.

[0020] In addition, this specification refers to any one of the polypeptide complexes or the isolation described herein. A pharmaceutical composition comprising either one of the IL-15 / IL-15Rα heterodimers is disclosed. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. .

[0021] In addition, this specification refers to human interleukin 15 (IL-15) polypeptide and Contains nucleic acids encoding the tointerleukin-15 receptor α (IL-15Rα) polypeptide. non-human cells, wherein IL-15 and IL-15Rα are expressed by these cells. The heterodimer forms a heterodimer, and the heterodimer includes α(2,6)-linked sialylation. Non-human cells are disclosed.

[0022] In addition, this specification provides a method for treating cancer, wherein the pharmaceutical composition described herein is used A method is disclosed that includes the step of administering it to a subject who requires it.

[0023] In addition, this specification describes how to generate cells that express IL-15 / IL-15α heterodimers. A method comprising the steps of (a) preparing non-human cells and (b) adding IL to the non-human cells. The first vector, which encodes both -15Rα and IL-15, and a portion of IL-15Rα are used. Two vectors, including a second vector that will be transfected, are transfected simultaneously. (c) The steps of culturing the cells and (b) the cells encoding IL-15 The third step is to transfect the cells with the third vector and culture the transfected cells; d) Step of isolating individual clones expressing IL-15 / IL-15α heterodimers. A method including the above is disclosed.

[0024] In some embodiments, non-human cells are recombinant Chinese hamster ovaries (CHO ) These are cells. In some embodiments, CHO cells are cells that perform functions of the matryptase gene. It is modified to cause an obstruction. In some embodiments, IL-15Rα is sequence number 12 It has the sequence of . In some embodiments, IL-15 has the sequence of sequence number 5. In some embodiments, a portion of IL-15Rα is the soluble portion of IL-15Rα. In some embodiments, the soluble portion of IL-15Rα has the sequence of sequence number 10. ru.

[0025] In addition, this specification describes a method for producing IL-15 / IL-15Rα heterodimers. (a) The cells generated above express IL-15 / IL-15Rα heterodimers and Steps to culture under conditions that enable the secretion of IL-15 / IL-15Rα heterodimers (b) The step of isolating the IL-15 / IL-15α heterodimer from the cell culture. A method including the above is disclosed.

[0026] This specification refers to human interleukin 15 (IL-15) polypeptide and human inter - A polypeptide complex containing the leukin-15 receptor α (IL-15Rα) polypeptide. It is produced by recombinant Chinese hamster ovary (CHO) cells, and also IL-1 Polypeptide complexes that do not have 5Rα chain splicing variants are further disclosed.

[0027] In some embodiments, the IL-15Rα chain splicing variants are I1 to G15 It contains 159 residues, ranging from 9. In some embodiments, CHO cells are subjected to matryptase It will be modified to disable its functionality. [Brief explanation of the drawing]

[0028] [Figure 1-1] Figure 1: Vector maps of pBW1697, pBW1703, and pBW1916 are shown. Vector pBW1697 (A) contains genes for expressing IL-15 and IL-15Rα; pBW1703 (B) contains a gene for expressing IL-15Rα; and (C) pBW1916 contains a gene for expressing IL-15. All three plasmids contain a SwaI site used for vector linearization. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 2-1]Figure 2: Northern blot analysis of IL-15 / IL-15Rα-producing cells (C009) at the start and end of the stability test. mRNA expression of IL-15 (A), IL-15 SP (B) containing the propeptide, and IL-15Rα (C) in the cells was analyzed by Northern blotting. A 0.5–10 kb RNA ladder was loaded into lane M, and control RNA from parental CHO-MaKo cells was loaded into lane P. (A) The expected 1 kb band size for IL-15 mRNA was detected. (B) The expected 1 kb band size for IL-15 SP mRNA containing the propeptide was detected. (C) The expected 1 kb band size for IL-15Rα mRNA was detected. No signal was detected in parental cells. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 3-1]Figure 3: Southern blot analysis of IL-15 / IL-15Rα-producing cells (C009) at the start and end of the stability test. Genomic DNA from these cells was digested with restriction enzyme MfeI and analyzed by Southern blot using probe So465 (A) targeting the IL-15 gene sequence, probe So466 (B) targeting the SP+ propeptide sequence of IL-15 itself, and probe So467 (C) targeting the IL-15Rα gene sequence. A DNA ladder (Molecular DNA Marker VII, Roche) was loaded into lane M. Lanes 1-3 contain MfeI-digested genomic DNA from parental CHO-MaKo cells, with or without MfeI-digested pBW1697 (lane 1), pBW1703 (lane 2), and pBW1916 (lane 3) vector DNA spiked at 5 copies / genome (lane 4). (A) The expected 1.9kb band size for the IL-15 fragment is detected from pBW1697 (lane 1), from pBW1916 (lane 3), and at the start and end of stability (lanes 5 and 6). (B) The expected 1.9kb band size for the SP+ propeptide fragment of IL-15 itself is detected from pBW1697 (lane 1), and no band is detected at the start and end of stability (lanes 5 and 6). (C) The expected 2.5kb band size for the IL-15Rα fragment (IL-15Rα FL) is detected from pBW1697 (lane 1), and a 2kb band size (IL-15Rα sol) is detected from pBW1703 (lane 2), and also at the start and end of stability (lanes 5 and 6). No signal was detected in parental cells. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Figure 4]The transgene copy numbers of IL-15 / IL-15Rα-producing cells (C009) at the start and end of the stability test are shown. The gene copy numbers ( / haploid genome) of IL-15 / itself SP (black column), IL-15Rα / UTR12SP (dark gray column), and IL-15 / UTR12SP (light gray column) were measured by qPCR in IL-15 / IL-15Rα-producing cells and CHO-MaKo parental cells. [Figure 5-1] Figure 5: Shows the distinction between α2,3- and α2,6-linked sialic acids. Panel A represents the ethyl esterification of 6'-sialyl lactose, and Panel B represents the formation of lactone in 3'-sialyl lactose. [Figure 5-2] (As stated above.) [Figure 6] This shows the O-glycan distribution of het IL-15. [Figure 7] This shows the structure of Core 1 type. [Figure 8] The structure of the two core types is shown. [Figure 9] The MS spectrum of het IL-15 is shown. [Figure 10] The N-glycan properties of het IL-15 produced in HEK and CHO are shown. [Figure 11] This shows the N-glycan properties of het IL-15 produced by HEK. [Figure 12] This shows the N-glycan properties of het IL-15 produced in CHO. [Figure 13-1] Figure 13: Nomenclature for individual components of glycans. [Figure 13-2] (As stated above.) [Figure 14] The design for dose escalation and expansion studies is shown. [Figure 15] The chromatographic characteristics of the HEK293 batch and the CHO batch are shown. [Modes for carrying out the invention]

[0029] General matters To facilitate understanding of the present invention, certain terms are defined throughout the detailed description. Unless otherwise defined, all scientific and technical terms used herein refer to the inventions to which this invention belongs. It has the same meaning as it is generally understood by those skilled in the art.

[0030] Terminology Unless otherwise specified, the following terms and phrases, when used herein, have the following meanings: It is intended to have.

[0031] As used herein, the articles "a" and "an" refer to the grammatical object of the article. It refers to one or more (for example, at least one).

[0032] The term "or" in this specification means that unless the context explicitly states otherwise, the term Used to mean "and / or," and interchangeably.

[0033] "Approximately" and "about" generally take into account the nature or precision of the measurement when referring to the quantity being measured. This refers to the acceptable degree of error. An example of the degree of error is given by the value or value Within 20 percent of the range, typically within 10%, more typically within 5%. That is the case.

[0034] The terms “disease” and “disorder” are used interchangeably to refer to pathological conditions, particularly pathological conditions. In one embodiment, the terms “disease” and “disorder” refer to IL-15 signaling. Diseases affected by and / or enhancement of immune effector responses It is used interchangeably to refer to a disease.

[0035] As used herein, the terms “to treat,” “treatment,” and “to treat” are defined as follows: Reduction or improvement of the progression, severity, and / or duration of disorders, such as proliferative disorders. , or one or more symptoms of a disorder resulting from the administration of one or more treatments (preferably one or more) This refers to the improvement of the above identifiable symptoms. In specific embodiments, the term "to treat" "Treatment" and "to treat" are at least one that is not necessarily identifiable by the patient. This refers to measurable physical parameters of proliferative disorders, such as improvement in tumor growth. Other implementations In this context, the terms “to treat,” “treatment,” and “the act of treating” are distinguishable, for example, Stabilization of symptoms leads to physical improvements, for example, stabilization of physical parameters leads to physiological improvements, This refers to inhibiting the progression of proliferative disorders in both of them. In other embodiments, the term "treatment" refers to inhibiting the progression of proliferative disorders. "To treat," "to treat," and "to treat" refer to reducing or stabilizing tumor size or the number of cancerous cells. It refers to stabilization.

[0036] The terms "therapies" and "treatments" as used herein "Therapy" is used for diseases such as cancer, infectious diseases, lymphopenia, and immunodeficiency. It can be used in the prevention, treatment, management, or improvement of symptoms associated with them. This may refer to any protocol, method, composition, formulation, and / or agent. In terms of form, the terms "therapies" and "therapy" )" is a method for treating, managing, preventing, or improving diseases or symptoms known to those skilled in the art. This refers to useful biotherapies, supportive therapies, and / or other treatments.

[0037] Receptors (e.g., wild-type IL-15Rα or IL-15 receptor βγ) and ligands (For example, wild-type IL-15) The term "specific" as used herein in relation to interactions "Target binding," "specifically recognizing," and similar terms refer to the interaction between ligand and receptor. This refers to specific binding or association with the receptor. Preferably, the ligand is more specific to the receptor than other molecules. It has high affinity. In a specific embodiment, the ligand is wild-type IL-15. In another specific embodiment, the ligand It is the wild-type IL-15 / IL-15Rα complex, and its receptor is the βγ receptor complex. In a further embodiment, the IL-15 / IL-15Rα complex is a βγ receptor complex. It binds to the body and activates IL-15 mediated signaling. Gand is, for example, an immunoassay, BIAcore®, or other known to those skilled in the art. It can be identified using technology.

[0038] The terms "immunely binding" and "specific" are used herein in relation to antibodies. "Target binding" means that it specifically binds to an antigen (e.g., an epitope or immune complex) and does not bind to another antigen. This refers to molecules that do not specifically bind to the antigen. Molecules that specifically bind to the antigen are, for example, immune molecules. By assay, BIAcore® or other assays known in the art This determines that it can bind to other antigens with lower affinity. In a specific embodiment, the antigen The binding molecule does not cross-react with other antigens.

[0039] "Combination" or "in combination with" means that the treatment or therapeutic agent is administered simultaneously and delivered at the same time. This does not mean that they must be formulated together for that purpose, but their delivery methods The law is within the scope described herein. The therapeutic agent in combination is one or more other additional therapeutic agents. It can be administered simultaneously with, before, or after, a therapy or treatment agent. Therapeutic agents or treatment protocols can be administered in any order. Generally, each The drug should be administered in the dose and / or time schedule determined for that drug. The additional therapeutic agents used in this combination are administered together in a single composition, and It is further recognized that these can be administered separately in different compositions. In general, combination The additional therapeutic agents used in this context must not exceed the levels at which they are individually used. It is expected to be used in bells. In some embodiments, it is advantageous in combination The level of use is lower than that of individual users.

[0040] The term "anti-cancer effect" is not limited to a variety of means and examples, but for example, tumor Reduction in ulcer volume, reduction in cancer cell count, reduction in metastasis, extension of life expectancy, reduction in cancer cell proliferation, cancer cells Organisms that may become apparent through reduced survival or improvement of various physiological symptoms associated with cancerous pathology. It refers to the scientific effect. "Anti-cancer effect" is initially achieved by peptides, polynucleotides, cells, and antibodies. It can also be manifested by its ability to prevent the development of cancer.

[0041] The term "antitumor effect" is not limited to a variety of means and examples, but for example, This can lead to a decrease in tumor volume, a decrease in tumor cell count, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. This refers to biological effects that are more likely to become apparent.

[0042] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells are local It can spread to other parts of the body, either directly or via the bloodstream and lymphatic system. Examples of various cancers are shown here. As described in the specification, and 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 Examples include lung cancer. The terms "tumor" and "cancer" are used interchangeably in this specification. For example, both terms encompass solid and liquid tumors, such as diffuse or circulating tumors. The terms "cancer" or "tumor" as used herein refer to pre-malignant and malignant cancers. Including cancer and tumors.

[0043] The terms "immune effector" or "effector" or "function" as used herein "or 'response'" refers to improving or promoting the immune attack of target cells, for example, immune response This refers to the function or response of effector cells. For example, immune effector function or response is, The properties of T or NK cells that promote the killing of target cells or the inhibition of their growth or proliferation. It refers to the function or response of an immune effector in the case of T cells. ru.

[0044] The term "effector function" refers to the specialization function of cells. For example, the effector function of T cells. The function may be cell-lytic activity or helper activity, for example, cytokine secretion.

[0045] The compositions and methods of the present invention have a specified arrangement, or substantially the same or similar thereto. Sequences that are identical to a given sequence by at least 85%, 90%, 95%, or more. This includes polypeptides and nucleic acids having a certain sequence. Regarding amino acid sequences, the term "actual "Qualitatively identical" means that the first and second amino acid sequences share a common structural domain and / or co To enable the use of all functional activities, the second amino acid sequence is either i) identical to the second amino acid sequence, or ii) the same as the second amino acid sequence. A sufficient or minimum number of conservative substitutions of amino acid residues to be aligned in an amino acid sequence. In this specification, it is used to refer to the first amino acid containing an amino acid residue. For example, For example, at least about 85% to 90% of the reference sequence, for instance, the sequence provided herein. 91 Identity of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% An amino acid sequence containing a common structural domain.

[0046] Regarding nucleotide sequences, the term "substantially identical" refers to the first and second nucleotides. Sequences encode polypeptides that have common functional activity, or common structural polypeptides A second nucleic acid sequence that encodes a dodomain or common functional polypeptide activity. Contains a sufficient or minimum number of nucleotides identical to the aligning nucleotides inside. Used herein to refer to a first nucleic acid sequence, for example, a reference sequence, e.g. If, then, the sequences provided herein and at least about 85%, 90%, 91%, 92%, 93% nucleotides having 94%, 95%, 96%, 97%, 98%, or 99% identity array.

[0047] The term "functional variant" refers to a variant that has a substantially identical amino acid sequence to the wild-type sequence, and It is encoded by substantially the same nucleotide sequence and has one or more of the activities of the wild-type sequence. It refers to a possible workspace.

[0048] Homology or sequence identity between sequences (these terms are used interchangeably in this specification) The calculation of ( ) is performed as follows.

[0049] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, Align columns for optimal comparison purposes (e.g., gaps for optimal alignment). It can be introduced into one or both of the first and second amino acids or nucleic acid sequences, and ratio Non-homologous sequences can be ignored for comparison purposes. In a preferred embodiment, comparison The length of the reference array to be aligned for the purpose is at least 70% of the length of the reference array, preferably At least 80%, more preferably at least 90%, 95%, and even better It is at least 100%. Then, the corresponding amino acid position or nucleotide Compare amino acid residues or nucleotides at a given position. A position in the first sequence is The second sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. If present, those molecules are identical at that position (as used herein). The "identity" of an amino acid or nucleic acid is equivalent to the "homology" of an amino acid or nucleic acid.

[0050] The identity percentage between the two sequences is introduced for optimal alignment of the two sequences. Considering the number of gaps that need to be filled and the length of each gap, their arrangement This is a function of the number of identical positions shared by a column.

[0051] The comparison of two arrays and the determination of their identity percentages are performed using mathematical algorithms. This can be achieved by using. In a preferred embodiment, the sameness between two amino acid sequences. Sex percentage in the GCG software package (available from NCBI) Needleman and Wunsch (1970) incorporated into the P program. Using the algorithm (J.Mol.Biol.48:444-453), Blossu m 62 matrix or PAM250 matrix, and gap weights 16, 14, 1 Determined using 2, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. Determine. In yet another preferred embodiment, identity between two nucleotide sequences Cent uses the GAP program in the GCG software package to NWS gapdna.CMP matrix and gap weighting 40, 50, 60, 70, or 8 Determined using 0 and length weights 1, 2, 3, 4, 5, or 6. Particularly preferred parameters The meter set (and what should be used unless otherwise specified) will incur a gap penalty of 12 Bloss, Gap extension penalty 4, and Frame shift gap penalty 5 This is the UM62 scoring matrix.

[0052] The percentage of identity between two amino acid or nucleotide sequences is determined by the ALIGN program. E. Meyers and W. Miller (included in version 2.0) Using the algorithm (1989)CABIOS, 4:11-17), PAM120 Weight residue table, gap length penalty 12 And it can be decided using a gap penalty of 4.

[0053] The nucleic acid and protein sequences described herein may be used as "query sequences" in public data Perform a search of the database, for example, to find other family members or related sequences. This can be determined. Such searches are described in Altschul, et al. (1990). NBLAST and XBLAST programs in J.Mol.Biol.215:403-10 This can be performed using RAM (version 2.0). BLAST nucleotide assay The search was conducted using the NBLAST program, with a score of 100 and a word length of 12. A nucleotide sequence homologous to the nucleic acid molecule (SEQ ID NO: 2) can be obtained using BLAST. Protein search was performed using the XBLAST program, with a score of 50 and a word length of 3. This allows us to obtain an amino acid sequence homologous to the protein molecule of the present invention. To obtain a gapped alignment, use a Gapped BLAST in Altschu l et al.,(1997)Nucleic Acids Res.25:3389 -3402 can be used as described below. BLAST and Gapped BLA When using ST programs, each program (for example, XBLAST and N) You can use the default parameters for BLAST (available from NBCI). .

[0054] The terms "low stringency, medium stringency" as used herein Under conditions of high stringency or extremely high stringency, hybrids "To hybridize" describes the conditions for hybridization and cleaning. Guidelines for carrying out dilation reactions are provided in Current Protocols i n Molecular Biology,John Wiley & Sons,N. This can be found in Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods. The law is listed in its references, and any of them may be used. The specific hybridization conditions mentioned are as follows: 1) Low stri Under the conditions of the industrial hybridization, 6 × sodium chloride / quaternary chloride at approximately 45°C. Sodium phosphate (SSC) followed by 0.2 × SSC, less than 0.1% in SDS Both consist of two washes at 50°C (the washing temperature is 5°C for low stringency conditions). 1) Can be increased up to 5°C; 2) Moderate stringency hybridise Conditions: 6×SSC followed by 0.2×SSC, 0.1% SD at approximately 45°C. 3) One or more washes at 60°C in S; 4) High stringency hybridize Conditions: 6×SSC followed by 0.2×SSC, 0.1% SD at approximately 45°C. One or more washes at 65°C in S; and preferably 4) extremely high stringer The hybridization conditions were 0.5 M sodium phosphate at 65°C, 7 % SDS followed by 0.2 × SSC, one or more times at 65°C in 1% SDS This is washing. Very high stringency conditions (4) are preferred conditions, and are not particularly specified. It should be used unless otherwise specified.

[0055] The molecules of the present invention provide additional conservation or non-essential amino acids that do not substantially affect the function. It is understood that it may have no acid substitution.

[0056] The term "amino acid" refers to both amino and acid-functional amino acids, whether natural or synthetic. It includes and is intended to encompass all molecules that may be present in polymers of wild-type amino acids. Exemplary amino acids include wild-type amino acids; their analogues, derivatives, and homologues; Amino acid analogs having riant side chains; and any of the above. Examples include isomers. The term "amino acid" as used herein refers to D- or L-amino acids. -Includes both optical isomers and peptide mimetic compounds.

[0057] "Conservative amino acid substitution" is the substitution of amino acid residues with similar side chains. These are the ones that have been replaced. Families of amino acid residues with similar side chains are used in this technique. These are defined in the field of technology. These families include basic side chains (e.g., ri (Zin, 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, isopropyl alcohol) (Isine, proline, phenylalanine, methionine, tryptophan), beta branched side chain (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Examples of amino acids include phenylalanine, tryptophan, and histidine.

[0058] The terms "polypeptide," "peptide," and "protein" (when single-chain) are: In this specification, the term is used interchangeably to refer to an amino acid polymer of any length. Remers can be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. This term may be used for modifications such as disulfide bond formation, glycosylation, and lipid addition. , acetylation, phosphorylation, or any other operation, e.g., conjugation with the labeled component It also includes amino acid polymers that have undergone treatment. Polypeptides are derived from natural sources. It can be separated and produced from eukaryotic or prokaryotic hosts by recombinant technology. It can be produced or may be a product of the synthesis procedure.

[0059] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide sequence" are all related to this topic. The terms "polynucleotide" and "deoxyribonucleonucleotide" are used interchangeably. Nuclei of any length of either a ribonucleotide or an analog thereof This refers to the polymerization form of osides. Polynucleotides can be single-stranded or double-stranded. If it is a single strand, it can be a coding strand or a non-coding (antisense) strand. Otide is a modified nucleotide, for example, methylated nucleotides and nucleotide analogs. It may contain nucleotides. The nucleotide sequence is interrupted by non-nucleotide components. i. Polynucleotides, after polymerization, are conjugated, for example, with labeled components. Further modifications are possible. Nucleic acids include recombinant polynucleotides, or genomes, cDNAs, and natural Heminucleotides that do not occur in nature, or are bound to other polynucleotides in configurations not found in nature. They may be naturally occurring or synthetically derived polynucleotides.

[0060] As used herein, the term "isolated" means its original or natural environment (e.g., wild). In the case of this type, it refers to substances extracted from the natural environment. For example, wild substances present in living animals. Although type polynucleotides or polypeptides have not been isolated, human intervention has revealed that they are naturally occurring. The same polynucleotide or polypeptide isolated from some or all of the coexisting substances The nucleotide has been isolated. Such polynucleotides can be part of a vector, and and / or such polynucleotides or polypeptides may be part of the composition. In that such vectors or compositions are not part of the environment in which they are found naturally, It is isolated.

[0061] As used herein, the term "glycan" means a compound containing at least three sugar residues, such as sugars. They can be monomers or polymers, and can be linear or branched (e.g., α1,3 arms). It is a sugar (and has α1,6 arms). "Glycan" is a natural sugar residue (for example, gluco N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannol (e.g., sucrose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified Sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6) It may contain sulfo-N-acetylglucosamine, etc. The term "glycan" refers to a sugar residue. Includes polymers and heteropolymers. The term "glycan" also refers to complex carbohydrates (e.g., glycoproteins). It includes glycan components (such as lipids, glycolipids, and proteoglycans). The term also refers to complex sugars. Free glycans, including glycans that have been cleaved from the substance or otherwise released. It includes.

[0062] As used herein, the term "glycoprotein" means one or more sugar moieties (i.e., glycans). This refers to a protein having a peptide backbone covalently bonded to a sugar portion. The sugar portion is a monosaccharide. It may be in the form of a disaccharide, oligosaccharide, and / or polysaccharide. The sugar portion is a single non-fraction of sugar residues. It may contain branched chains, or one or more branched chains. Glycoproteins are O-linked sugars. It may contain a portion and / or an N-linked sugar portion. The polysaccharide is serine or threonine (O- The OH group of a glycosylated polypeptide or asparagine (N-glycosylated polypeptide) It is bound via one of the amide groups (NH2) of the glycoprotein. The glycoprotein is opposed to the host cell. They may be homologous, or preferably heterogeneous, i.e., exogenous, to the host cell expressing them. It may also be (for example, human proteins produced by CHO cells).

[0063] The term "complex carbohydrate," as used herein, means a compound carbohydrate in which at least one sugar portion is present. It also includes all molecules covalently bonded to one other part. The term is specific Examples include N-linked glycoproteins, O-linked glycoproteins, glycolipids, and proteoglycans. This includes all biomolecules that have covalently linked sugar moieties, such as n.

[0064] As used herein, the term "glycosylation pattern" refers to a pattern present on a particular sample. It refers to a set of glycan structures present. For example, certain complex carbohydrates (e.g., glycoproteins) or A set of complex carbohydrates (e.g., a set of glycoproteins) has a glycosylation pattern. In some embodiments, the glycosylation pattern of cell surface glycans is referenced. The glycosylation pattern is used to determine, for example, the identity of the glycan, or the individual glycans or The amount (absolute or relative) of a specific type of glycan, the degree of occupancy of glycosylation sites, or It can be characterized by the combination of parameters.

[0065] Various aspects of the present invention are described in more detail below. Additional definitions are provided throughout this specification. It is expressed through the body.

[0066] Glycosylation The term "glycosylation" refers to the binding of a polysaccharide to a polypeptide. Preferably, the polysaccharide is It consists of 2 to 12 monosaccharides linked together by glycosidic bonds. Glycoproteins are, It may contain O-linked sugar moieties and / or N-linked sugar moieties. It is bound to specific glycosylation sites. The structure and number of the resulting sugar moieties may be variable. Such sugar moieties may include, for example, N-acetylglucan. Lucosamine, N-acetylgalactosamine, mannose, galactose, glucose, f Coarse, xylose, glucuronic acid, iduronic acid, and / or sialic acid may also be used.

[0067] The term "N-linked glycosylation" refers to the binding of an amino acid chain of a polysaccharide to an asparagine residue. To point.

[0068] The term "O-linked glycosylation" refers to the serine or threonine residue of the amino acid chain in the sugar portion. This refers to a connection to.

[0069] The terms "sugar properties" or "glycosylation properties," as used interchangeably in this application, refer to glycosylation. The glycanity of glycosylated polypeptides is described. These properties are preferably glycosylated. Site, or occupancy of glycosylated sites, or glycan and / or non-sugar parts of polypeptides This refers to the identity, structure, composition or quantity of a portion, or the identity and quantity of a specific glycoform. .

[0070] N-acetylneuraminic acid (NANA) is the main component of N- and O-linked glycans. NANA is the dominant neuraminic acid in protein glycosylation events in humans. While morphologically, other mammals also possess substances such as N-glycolylneuraminic acid (NGNA). Other derivatives may be included. NANA is a core of N- and O-glycans in several ways. It can be bound to the structure. Preferably, α(2,3) and α(2,6) can be bound to the subsequent sugar. It can be found, but other bonds such as α(2,8) are present. Human cells, for example, human Embryonic kidney (HEK) cells primarily produce α(2,6) linkage sialylation, while numerous Producing cell lines, such as the CHO cell line, produce α(2,3) linkage sialylation.

[0071] CHO cells generate an α(2-6)-linked NANA extension to the coagulan structure. The enzyme involved in this (β-galactoside α-2,6-sialyltransferase 1) It has been reported that CHO cells are inactive or not expressed (for example, Ch ung et al.(2017)Biotechnol.J.12:1600502 (Reference) However, the gene in the enzyme itself is found in C. griseus. It exists. This invention relates to the IL-15 / IL-15Rα complex produced in CHO cells, The glycosylation differs from that produced by the IL-15 / IL-15Rα complex by the ophthalmic cell line. This is based on the unexpected discovery that it has a glycation pattern. Furthermore, it is an α(2,6) linked glycosylation type. This was found in the IL-15 / IL-15Rα complex produced in CHO cells. The glycosylation pattern is unique. This is potentially due to the human glycosylation type. Being close can sometimes yield direct benefits compared to the expected CHO pattern.

[0072] In some embodiments, the IL-15 / IL-15Rα heterodimer of this disclosure is as follows: As shown in Table 1, it contains one or more glycan species.

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3]

[0076] [Table 4]

[0077] IL-15 The terms "IL-15" and "interleukin-15" as used herein are: This refers to wild-type IL-15 or IL-15 derivatives. It relates to proteins or polypeptides. The terms "wild-type IL-15" and "wild-type interleaving" as used herein are used in this specification. "Kin-15" refers to any mammalian interleukin-15 amino acid sequence, for example, immature Alternatively, it refers to the precursor and mature forms. Various species of wild-type mammalian interleukin-15. A non-limiting example of a GeneBank accession number for an amino acid sequence is: NP_000576 (human, immature form), CAA62616 (human, immature form), NP_001009207 (Felis catus, immature form), AA B94536 (Rattus norvegicus, immature form), A AB41697 (Rattus norvegicus, immature form), NP_032383 (House mouse (Mus musculus), immature form), AA R19080 (dog), AAB60398 (Rhesus macaque (Macaca mulatta)) ), immature form), AAI00964 (human, immature form), AAH23698 (Hatsuka Mouse (Mus musculus, immature form), and AAH18149 (human) Table 2 shows the long-chain signal peptide (underlined) provided in Sequence ID No. 1 and its maturation process. Immature / precursor forms of human IL-15, including human wild-type IL-15 (italic). No acid sequence. In some embodiments, wild-type IL-15 is immature mammalian IL-15. or a precursor form. In other embodiments, IL-15 is a mammalian form of IL-15. This is the mature form. In a specific embodiment, wild-type IL-15 is a precursor to human IL-15. This is the progenitor form. In 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. It has been refined.

[0078] The terms "wild-type IL-15" and "wild-type" as used herein with respect to nucleic acids Interleukin-15 is a mammalian interleukin-15, for example, immature or Refers to any nucleic acid sequence that codes for precursor and mature forms. Non-restrictive GeneBank accession numbers for nucleotide sequences of various species Examples include NM_000585 (human) and NM_008357 (mouse). (Mus musculus), and RNU69272 (Rattus (Norway rat) Norvegicus)) is one example. The long-chain signal provided in Sequence ID No. 2 in Table 2 is a The nucleotide sequence encoding the IL-15 peptide (underlined) and mature human wild-type IL-15 The immature / precursor form of human IL-15 containing the nucleotide sequence (italicized) is shown. The encoding nucleotide sequence. In a specific embodiment, the nucleic acid is isolated or purified nucleic acid. In some embodiments, the nucleic acid is an immature or precursor form of mammalian IL-15. It encodes the state. In other embodiments, nucleic acids encode the mature form of mammalian IL-15. In a specific embodiment, the nucleic acid encoding wild-type IL-15 is human IL- It encodes 15 precursor forms. In another embodiment, it encodes wild-type IL-15. Nucleic acids encode the mature form of human IL-15.

[0079] The term "IL-15" as used herein in relation to proteins or polypeptides. The derivatives and interleukin-15 derivatives are (a) wild-type mammal IL-15 Polypeptides and at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% % identical polypeptides; (b) encoding wild-type mammalian IL-15 polypeptide Nucleic acid sequence and at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical A polypeptide encoded by a single nucleic acid sequence; (c) wild-type mammal IL-15 For the lipeptide, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 15, 16, 17, 18, 19, 20 or more amino acid mutations (that is, (d) polypeptides containing (a, an addition, a deletion and / or substitution); (d) nucleic acids encoding The polypeptides undergo high or moderate stringency hybridization. Under certain conditions, it can hybridize to nucleic acids encoding wild-type mammalian IL-15 polypeptide. (e) under high or moderate stringency hybridization conditions at least 20 consecutive amino acids, at least 30 consecutive amino acids, at least 40 consecutive amino acids A series of amino acids, at least 50 consecutive amino acids, at least 100 consecutive amino acids, or a fragment of wild-type mammalian IL-15 polypeptide consisting of at least 150 consecutive amino acids polypeptide encoded by a nucleic acid sequence that can hybridize to the nucleic acid sequence encoding the nucleic acid sequence. ; and / or; (f) refers to a fragment of the wild-type mammalian IL-15 polypeptide. IL- 15 derivatives include the amino acid sequence of the mature form of the mammalian IL-15 polypeptide and Polypeptides containing heterogeneous signal peptide amino acid sequences are also examples. Specific embodiments In this context, IL-15 derivatives are derivatives of wild-type human IL-15 polypeptides. In this embodiment, the IL-15 derivative is an immature or It is a precursor derivative. In another embodiment, the IL-15 derivative is human IL-1 5. A derivative of the mature form of polypeptide. In another embodiment, the IL-15 derivative is For example, Zhu et al., (2009), J.Immunol.183:359 8 or IL-15N72D as described in U.S. Patent No. 8,163,879. In this embodiment, the IL-15 derivative is described in U.S. Patent No. 8,163,879. The IL-15 variant is as shown. In one embodiment, the IL-15 derivative is simply It has been separated or refined.

[0080] In a preferred embodiment, the IL-15 derivative is an aggregate known in the art. i. For example, IL-1 can be measured by ELISA, BIAcore (trademark), or co-immunoprecipitation. At least one function of the wild-type mammalian IL-15 polypeptide that binds to the 5Rα polypeptide It also retains 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In a more specific embodiment, the IL-15 derivative is used in assays well known in the art, e.g. For example, it can be measured by electrophoretic mobility shift assays, ELISA, and other immunoassays. The function of wild-type mammalian IL-15 polypeptides that induce IL-15-mediated signaling. Retain at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of In specific embodiments, the IL-15 derivative is, for example, well known in the art. Evaluated by ligand / receptor binding assays for IL-15Rα and / or IL-1 It binds to 5Rβγ. The identity percentage is determined by any method known to those skilled in the art. The decision can be made as follows.

[0081] The terms "IL-15 derivative" and "inter" as used herein in relation to nucleic acids "Leukin-15 derivatives" are (a) nucleic acid derivatives encoding the mammalian IL-15 polypeptide. The column is identical to at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the original column. (b) Nucleic acid sequence; (b) Amino acid sequence of wild-type mammal IL-15 polypeptide and at least Polypeptides that are 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical. (c) Nucleic acid sequence encoding mammalian IL-15 polypeptide In contrast, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 1 6, 17, 18, 19, 20 or more nucleic acid base mutations (i.e., additions, deletions) (d) Nucleic acid sequences containing (losses and / or substitutions); (d) high or moderate stringing Nucleic acids encoding mammalian IL-15 polypeptide under C-hybridization conditions Nucleic acid sequences that hybridize to the sequence; (e) high or moderate stringency Crop fragmentation of nucleic acid sequences encoding mammalian IL-15 polypeptides under bridging conditions. Nucleic acid sequences and / or (f) mammalian IL-15 polypeptides that hybridize to one side. This refers to a nucleic acid sequence that codes for a fragment of a nucleic acid sequence that codes for [a specific nucleic acid]. In a specific embodiment, IL-15 derivatives related to nucleic acids are nucleic acid sequences that encode human IL-15 polypeptides. It is a derivative. In another embodiment, an IL-15 derivative relating to nucleic acids is human IL-1 5. A derivative of a nucleic acid sequence that encodes an immature or precursor form of polypeptide. In terms of application, IL-15 derivatives related to nucleic acids are used in the maturation of human IL-15 polypeptides. It is a derivative of a nucleic acid sequence that codes for morphology. In another embodiment, IL- 15. Derivatives are, for example, Zhu et al., (2009; cited above), or in the United States. This is the nucleic acid sequence encoding IL-15N72D as described in Specification No. 8,163,879. In another embodiment, IL-15 derivatives relating to nucleic acids are described in U.S. 8,163,87 This is a nucleic acid sequence encoding one of the IL-15 variants described in Specification No. 9.

[0082] Examples of IL-15 derivative nucleic acid sequences include mammalian IL-15 polypeptides, such as I Examples include codon-optimized nucleic acid sequences that encode mature and immature forms of the L-15 polypeptide. In other embodiments, the IL-15 derivative nucleic acid is mammalian IL-15R Potential splicing without affecting the amino acid sequence to increase the stability of NA transcripts Eliminate parts and unstable elements (e.g., A / T or A / U rich elements). Nucleic acids encoding mammalian IL-15RNA transcripts containing mutations are examples. In one embodiment, the IL-15 derivative nucleic acid sequence is as follows: International Publication No. 2007 / 08 Examples include the codon-optimized nucleic acid sequences described in pamphlet No. 4342. In one embodiment... The IL-15 derivative nucleic acid sequence is the codon-optimized sequence of sequence number 4 in Table 2 (that The amino acid sequence encoded by such a nucleic acid sequence is provided in SEQ ID NO: 5 in Table 2.

[0083] In a preferred embodiment, the IL-15 derivative nucleic acid sequence is well known in the art. The assays used to measure this, for example, ELISA, BIAcore®, and co-immunoprecipitation. At least 7 functions of wild-type mammalian IL-15 polypeptide that binds to IL-15Rα Proteins that retain 5%, 80%, 85%, 90%, 95%, 98%, or 99% This encodes a polypeptide. In another preferred embodiment, an IL-15 derivative nucleic acid compound The column represents assays well known in the art, such as electrophoretic mobility shift assays. IL-15-mediated signaling can be induced by ELISA and other immunoassays. At least 75%, 80%, and 85% of the function of wild-type mammalian IL-15 polypeptide, Encoding a protein or polypeptide that retains 90%, 95%, 98%, or 99% In a specific embodiment, the IL-15 derivative nucleic acid sequence is, for example, in the technical field. In this context, IL-15Rα and / or are evaluated by a well-known ligand / receptor binding assay. Alternatively, it encodes a protein or polypeptide that binds to IL-15Rβγ.

[0084] 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. With respect to a protein or polypeptide as used herein, the terms "wild-type IL-15Rα" and "wild-type interleu kin-15 receptor alpha" refer to any mammalian interleukin-15 receptor alpha ("IL-15Rα") amino acid sequence, including by way of example immature or precursor and mature forms and isoforms. Non-limiting examples of GenBank accession numbers for amino acid sequences of various wild-type mammalian IL-15Rα include NP_002180 (human), ABK41438 (rhesus macaque (Macaca mulatta)), NP_ 032384 (house mouse (Mus musculus)), Q60819 (house mo use (Mus musculus)), and CAI41082 (human). The amino acid sequence of the immature form of full-length human IL-15Rα comprising the signal peptide (underlined) provided in SEQ ID NO: 6 of Table 2 and mature human wild-type IL-15Rα provided in SEQ ID NO: 6 of Table 2 and mature human wild-type IL-15Rα (italic). The amino acid sequence of the immature form of soluble human IL-15Rα comprising the signal peptide (underlined) provided in SEQ ID NO: 7 of Table 2 and mature human soluble IL-15Rα provided in SEQ ID NO: 7 of Table 2 and mature human soluble IL-15Rα (it alic). In some embodiments , wild-type IL-15Rα is an immature form of a mammalian IL-15Rα polypeptide . In other embodiments, wild-type IL-15Rα is a mammalian IL-15Rα poly peptide in mature form. In one embodiment, wild-type IL-15Rα is a mammalian n IL-15Rα polypeptide in soluble form. In other embodiments, wild-type IL -15Rα is the full-length form of a mammalian IL-15Rα polypeptide. In specific embodiments thereof, wild-type IL-15Rα is the immature form of a human IL-15Rα polypeptide . In another embodiment, wild-type IL-15Rα is a human IL-15Rα polypepti de in mature form. In one embodiment, wild-type IL-15Rα is human IL-15 Rα polypeptide in soluble form. In other embodiments, wild-type IL-15Rα is the full-length form of a human IL-15Rα polypeptide. In one embodiment, the wild-type IL -15Rα protein or polypeptide is isolated or purified.

[0085] As used herein with respect to nucleic acids, the terms "wild-type IL-15Rα" and "wild type interleukin-15 receptor alpha" refer to mammalian interleukin-15 receptor alpha, for example, any nucleic acid sequence encoding immature or precursor and mature forms . Non-limiting examples of Gen Bank accession numbers for the nucleotide sequences of wild-type mammalian IL-15Rα from various species include NM_002189 (human), E F033114 (rhesus monkey (Macaca mulatta)), and NM_008 358 (house mouse (Mus musculus)). SEQ ID NO: 8 in Table 2 A nucleotide sequence encoding an immature form of wild-type human IL -15Rα comprising the nucleotide sequence encoding the signal peptide (underlined) and the nucleotide sequence encoding mature human wild -type IL-15Rα (italic) provided in . The nucleotide sequence provided in SEQ ID NO: 9 of Table 2 the nucleotide sequence encoding the signal peptide (underlined) and the nucleotide sequence encoding mature human soluble wild-type I Soluble human IL-15 containing the nucleotide sequence (italicized) encoding L-15Rα (A nucleotide sequence encoding an immature form of the Rα protein or polypeptide). Specifically In a typical embodiment, the nucleic acid is isolated or purified nucleic acid. In some embodiments, The nucleic acid encodes an immature form of the mammalian IL-15Rα polypeptide. Other embodiments In this case, nucleic acids encode the mature form of the mammalian IL-15Rα polypeptide. In embodiments, the nucleic acid encodes the soluble form of the mammalian IL-15Rα polypeptide. In other embodiments, the nucleic acid is the full-length form of the mammalian IL-15Rα polypeptide. It encodes. In a specific embodiment, the nucleic acid is a precursor of the human IL-15 polypeptide. It encodes body morphology. In another embodiment, the nucleic acid is a component of human IL-15 polypeptide. It encodes maturity. In one embodiment, the nucleic acid is the morphological representation of human IL-15Rα polypeptide. It encodes a soluble form. In other embodiments, the nucleic acid is human IL-15Rα polypeptide. This codes the overall form of the 'do'.

[0086] The term "IL-15" as used herein in relation to proteins or polypeptides. Rα derivatives and interleukin-15 receptor alpha derivatives are (a) wild type Mammalian IL-15 polypeptide and at least 75%, 80%, 85%, 90%, 95% (b) polypeptides that are 98% or 99% identical to wild-type mammalian IL-15Rα Nucleic acid sequences encoding lipeptides and at least 75%, 80%, 85%, 90%, 95% , polypeptides encoded by nucleic acid sequences that are 98% or 99% identical; (c) wild For type 1 mammalian IL-15Rα polypeptide, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more Polypeptides containing amino acid mutations (i.e., additions, deletions, and / or substitutions) (d) Wild under high or moderate stringency hybridization conditions Nuclei that can hybridize to nucleic acid sequences encoding the mammalian IL-15Rα polypeptide Polypeptides encoded by acid sequences; (e) high or moderate stringency Under hybridization conditions, at least 20 consecutive amino acids, at least 30 Continuous amino acids, at least 40 continuous amino acids, at least 50 continuous amino acids, few At least 100 consecutive amino acids, or at least 150 consecutive amino acids, in the wild type. Nucleic acids that can hybridize to nucleic acid sequences encoding fragments of the mammalian IL-15 polypeptide. Sequence-encoded polypeptide; (f) wild-type mammal IL-15Rα polypeptide (g) fragments of (g) and / or (g) the specified IL-15Rα derivatives as described herein. As for IL-15Rα derivatives, the mature form of mammalian IL-15Rα polypeptide is ami Polypeptides containing anoacid sequences and heterologous signal peptide amino acid sequences are also examples. In a specific embodiment, the IL-15Rα derivative is a wild-type human IL-15Rα polypeptide. It is a derivative of IL-15. In another embodiment, the IL-15Rα derivative is human IL-15 It is a derivative of an immature form of polypeptide. In another embodiment, IL-15Rα derivative. The body is a derivative of the mature form of the human IL-15 polypeptide. In one embodiment, I L-15Rα derivatives are soluble forms of mammalian IL-15Rα polypeptides. In other words... Thus, in one embodiment, the IL-15Rα derivative includes mammalian IL-15R α soluble forms, which do not occur in nature. Other examples of IL-15Rα deriva tives include truncated soluble forms of human IL-15Rα described herein In a specific embodiment, the IL-15Rα derivative is purified or isolated

[0087] In a preferred embodiment, an IL-15Rα derivative has an activity well known in the art for assaying, e.g., by ELISA, BIAcore™, co-immunoprecipitation, that binds to IL -15 polypeptide retains at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15Rα polypeptide In another preferred embodiment, an IL-15Rα derivative is determined by an assay well known in the art , e.g., electrophoretic mobility shift assay, ELISA and other immunoassays retains at least 75%, 80%, 85%, 90%, 95%, 98% or 99% of the function of a wild-type mammalian IL-15Rα polypeptide that induces IL-15-mediated signaling %. In a specific embodiment, the IL-15Rα derivative binds to IL-15 as assessed by methods well known in the art, e.g., ELISA

[0088] As used herein with respect to nucleic acids, the terms "IL-15Rα derivative" and "inter leukin-15 receptor alpha derivative" refer to (a) mammalian IL-15Rα polypep tide that is at least 75%, 80%, 85%, 90%, 95%, 98% ​​​Nucleic acid sequences that are % or 99% identical; (b) wild-type mammalian IL-15Rα polypeptide The amino acid sequence and at least 75%, 80%, 85%, 90%, 95%, 98%, or 9% Nucleic acid sequences encoding polypeptides that are 9% identical; (c) Mammalian IL-15Rα polynucleotide For nucleic acid sequences encoding peptides, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid protrusions Nucleic acid sequences containing natural mutations (i.e., additions, deletions and / or substitutions); (d) high Mammalian IL-15R under moderate stringency hybridization conditions Nucleic acid sequences that hybridize to a nucleic acid sequence encoding an α polypeptide; (e) high or Mammalian IL-15Rα under moderate stringency hybridization conditions Nucleic acid sequences that hybridize to fragments of nucleic acid sequences encoding lipeptides; (f) mammals Nucleic acid sequences encoding fragments of nucleic acid sequences encoding IL-15Rα polypeptide; and / or (g) a nucleic acid sequence encoding a specified IL-15Rα derivative as described herein. In a specific embodiment, the IL-15Rα derivative relating to nucleic acids is human IL-15 It is a derivative of the nucleic acid sequence encoding the Rα polypeptide. In another embodiment, the nucleic acid is The IL-15Rα derivative in question encodes an immature form of the human IL-15Rα polypeptide. It is a derivative of the nucleic acid sequence. In another embodiment, IL-15Rα derivative of nucleic acid. The body is a derivative of the nucleic acid sequence that encodes the mature form of the human IL-15Rα polypeptide. In one embodiment, the IL-15Rα derivative relating to nucleic acids is soluble in mammals. This refers to a nucleic acid sequence encoding a derivative of the L-15Rα polypeptide. In one embodiment, IL-15Rα derivatives related to nucleic acids are soluble in mammalian IL-15Rα, which is not naturally occurring. This refers to a nucleic acid sequence that codes for sex morphology. In some embodiments, IL-15 related to nucleic acids Rα derivatives are soluble forms of human IL-15Rα, which do not exist naturally. This refers to the nucleic acid sequence encoding the derivative. In a specific embodiment, the IL-15Rα derivative The nucleic acid sequences have been isolated or purified.

[0089] Examples of IL-15Rα derivative nucleic acid sequences include wild-type IL-15Rα polypeptides. Codon-optimized nucleic acids that encode mature and immature forms of IL-15Rα polypeptide. A sequence is given. In another embodiment, the IL-15Rα derivative nucleic acid is IL- To increase the stability of 15RαRNA transcripts without affecting the amino acid sequence, Price areas and unstable elements (e.g., A / T or A / U rich elements) Nucleic acids encoding IL-15RαRNA transcripts containing mutations that eliminate the mutation are among the examples. In one embodiment, the IL-15Rα derivative nucleic acid sequence is sequence number 11 in Table 2. It is a 13-codon optimized sequence (the amino acid sequence encoded by such a nucleic acid sequence is (These are provided in sequence numbers 12 and 14 of Table 2, respectively.)

[0090] In a specific embodiment, the IL-15Rα derivative nucleic acid sequence is used in the art. Measurement by well-known assays, such as ELISA, BIAcore®, and co-immunoprecipitation. The function of the wild-type mammalian IL-15Rα polypeptide that binds to IL-15 is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% , encoding a protein or polypeptide that retains 98% or 99%. Another preferred In a more specific embodiment, the IL-15Rα derivative nucleic acid sequence is well known in the art. Assays, such as electrophoretic mobility shift assays, ELISAs, and other immunoassays. The wild-type mammalian IL-15Rα that induces IL-15-mediated signaling is measured by [method]. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the functionality. Encoding a protein or polypeptide that retains 90%, 95%, 98%, or 99% In a specific embodiment, the IL-15Rα derivative nucleic acid sequence is used in the art. The proteins that bind to IL-15 are evaluated using well-known methods, such as ELISA. Codes quality or polypeptide.

[0091] The soluble form of human IL-15Rα is described herein. Specified IL-15Rα derivatives in ammonium soluble form are also described herein. The soluble forms of IL-15Rα derivatives and human IL-15Rα as defined are, in part, Based on the identification of the proteolytic cleavage site of human IL-15Rα. The soluble forms of IL-15Rα characterized by glycosylation are further described herein. It will be published.

[0092] Proteolytic cleavage of human IL-15Rα is associated with immaturity of wild-type full-length human IL-15Rα. The amino acid sequences that provide the morphology are shown in bold and underlined residues (i.e., Gl Occurs between y170 and His171: [ka] (Sequence number 6 in Table 2)

[0093] Therefore, in one embodiment, the soluble form of human IL-15Rα (for example, human IL- (Purified soluble form of 15Rα), and the amino acid sequence of the soluble form of human IL-15Rα Human I IL-15Rα terminates at the site of proteolytic cleavage of wild-type membrane-bound human IL-15Rα. A soluble form of L-15Rα is provided herein. In particular, the soluble form of human IL-15Rα The form (for example, the purified soluble form of human IL-15Rα) is the form of human IL-15Rα The amino acid sequence in the soluble form is PQG (Sequence ID 20 in Table 2) (G is Gly170) A soluble form of human IL-15Rα ending in (ru) is provided herein. Specific implementation In this state, human IL-15Rα having the amino acid sequence shown in SEQ ID NO: 7 in Table 2 is available. A soluble form (for example, a purified soluble form of human IL-15Rα) is provided herein. In the embodiment of the part, (i) at least 75%, 80%, 85% of sequence number 7 in Table 2, (ii) The amino acid sequence PQG (Table 2) is 90%, 95%, 98%, or 99% identical; (ii) the amino acid sequence PQG (Table 2) IL-15Rα derivatives (e.g., IL-1) are polypeptides that terminate at SEQ ID NO: 20. Purification and / or soluble forms of 5Rα derivatives are provided herein. Other specific applications In the application form, human IL-15Rα having the amino acid sequence of Sequence ID No. 10 in Table 2 (for example) (For example, a purified soluble form of human IL-15Rα is provided herein.) In the embodiment of the part, at least 75%, 80%, 85%, 90% of sequence number 10 in Table 2 and 90% of the component. IL-15Rα derivatives are polypeptides that are identical by %, 95%, 98%, or 99%. For example, a purified and / or soluble form of an IL-15Rα derivative, and optionally The amino acid sequence of the soluble form of the IL-15Rα derivative is PQG (Sequence ID 20 in Table 2). Terminating IL-15Rα derivatives are provided herein.

[0094] In some embodiments, the IL-15Rα derivative of human IL-15Rα is soluble. It is a sex: (a) The last amino acid at the C-terminus of the IL-15Rα derivative is an amino acid residue (b) C-terminus of the IL-15Rα derivative The last amino acid at the end is amino acid residue PQGHSDT (Sequence ID 16 in Table 2). (c) The last amino acid at the C-terminus of the IL-15Rα derivative is the amino acid residue PQG (d) At the C-terminus of the IL-15Rα derivative The last amino acid consists of amino acid residues PQGHS (Sequence ID 18 in Table 2); or (e ) The last amino acid at the C-terminus of the IL-15Rα derivative is the amino acid residue PQGH (Table 2) An IL-15Rα derivative comprising SEQ ID NO: 19) is provided herein. One embodiment In this context, the amino acid sequences of these IL-15Rα derivatives are the same as those of Sequence ID No. 21 in Table 2. No acid sequence and 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% It is one. In some embodiments, these IL-15Rα derivatives are purified.

[0095] In another embodiment, the glycosylated form of IL-15Rα (for example, the crystalline form of IL-15Rα) (Glycosylated form) The glycosylation of IL-15Rα is a technique known to those skilled in the art. Evaluated by this method, at least 20% and at least 25% of the mass (molecular weight) of IL-15Rα , at least 30%, at least 35%, at least 40%, at least 45%, less 50% for both, or 20%-25%, 20%-30%, 25%-30%, 25%-35% 30%~35%, 30%~40%, 35%~40%, 35%~45%, 40%~50% IL-15Rα accounts for 45%-50%, 20%-40%, or 25%-50% of the total. Glycosylated forms are provided herein. The glycosylated form of IL-15Rα occupies IL The proportion of the mass (molecular weight) of -15Rα (e.g., purified IL-15Rα) is, for example, limited. Although it is not possible to perform gel electrophoresis and quantitative density measurement of gels, as well as IL-15R The glycosylated form of α (for example, the purified glycosylated form of IL-15Rα) and IL-1 With the non-glycosylated form of 5Rα (e.g., the purified non-glycosylated form of IL-15Rα) This can be determined by comparing the average mass (molecular weight). In one embodiment, IL The average mass (molecular weight) of -15Rα (e.g., purified IL-15Rα) is used as the matrix. The Voyager, equipped with the CovalX HM-1 high-mass detector that uses synapic acid, This can be determined using MALDI-TOF MS spectra on De-Pro. Glycosylated forms of IL-15Rα (e.g., purified glycosylated forms of IL-15Rα) The mass of the non-glycosylated form of IL-15Rα (for example, purified non-glycosyl IL-15Rα) The proportion of the mass accounted for by glycosylation can be determined by comparing it to the mass of the sylated form. .

[0096] In another embodiment, a glycosylated form of IL-15Rα, wherein IL-15Rα is IL- Glycosylated forms of 15Rα are provided herein. In one embodiment, the following Lycosylation site; (i) Amino acid sequence in IL-15Rα [ka] (Sequence number 22 in Table 2) O-glycosylation of threonine at position 5; (ii) IL Amino acid sequence in -15Rα [ka] (Sequence number 22 in Table 2) O-glycosylation of serine at position 7; (iii) IL- Amino acid sequence in 15Rα [ka] (Sequence ID 22 in Table 2) The amino acid pair on the serine at position 8, or in IL-15Rα column [ka] N-glycosylation of serine at position 8 of (Sequence ID 23 in Table 2); (iv) IL-15Rα The amino acid sequence inside is ITCPPPMSVEHADIWVKSYSLYSRERYICNS( N-glycosylation on Ser18 of sequence number 23 in Table 2; (v) in IL-15Rα Mino acid sequence [ka] N-glycosylation of serine at position 20 of (Sequence No. 24 in Table 2); (vi) IL- Amino acid sequence in 15Rα [ka] N-glycosylation of serine at position 23 of (Sequence ID 24 in Table 2); and / or (vii) Amino acid sequence in IL-15Rα [ka] N-glycosylation 1, 2, 3, 4 on serine at position 31 (Sequence ID 24 in Table 2) Human IL-15Rα that is glycosylated in 5, 6, 7, or all of the following ways is the real thing. Provided in the details.

[0097] In a specific embodiment, glycosylated IL-15Rα is used to metabolize wild-type human IL-15Rα. It is α. In other specific embodiments, glycosylated IL-15Rα is human IL- It is an IL-15Rα derivative of 15Rα. In some embodiments, glycosylated IL- 15Rα is wild-type soluble human IL-15Rα, for example, sequence number 7 or 10 in Table 2. Yes. In other embodiments, glycosylated IL-15Rα is a convertible form of human IL-15Rα. It is a soluble form of IL-15Rα derivative. In one embodiment, glycosylated IL -15Rα has been purified or isolated.

[0098] IL-15 / IL-15Rα complex As used herein, the terms "IL-15 / IL-15Rα complex" are used interchangeably. This refers to a complex containing IL-15 and IL-15Rα, either covalently or noncovalently. In a particular embodiment, IL-15Rα has a relatively high affinity for IL-15, for example For example, technologies known in the relevant field, such as KinExA assay, plasma surface K is measured by resonance (e.g., BIAcore® assay) and ranges from 10 to 50 pM. D It has. In another preferred embodiment, the IL-15 / IL-15Rα complex is said Well-known assays in the technical field, such as electrophoretic mobility shift assays and ELISAs. and other immunoassays can be used to measure and induce IL-15-mediated signaling. In this embodiment, the IL-15 / IL-15Rα complex has the ability to specifically bind to the βγ chain. It maintains its strength. In a specific embodiment, the IL-15 / IL-15Rα complex is used by cells. It has been isolated from.

[0099] It binds to the βγ subunit of the IL-15 receptor and transmits IL-15 signal (e.g., J A complex that induces ak / Stat signaling and enhances IL-15-mediated immune function. It is shared or non-shared with the interleukin-15 receptor alpha ("IL-15Rα"). The complex containing covalently bound IL-15 ("IL-15 / IL-15Rα complex") Provided herein, the IL-15 / IL-15Rα complex binds to the βγ receptor complex. It is possible.

[0100] The IL-15 / IL-15Rα complex is composed of wild-type IL-15 or IL-15 derivatives and It may consist of wild-type IL-15Rα or IL-15Rα derivatives. In one embodiment, In this context, the IL-15 / IL-15Rα complex is used with wild-type IL-15 or IL-15-induced The body and the above IL-15Rα are included. In a specific embodiment, IL-15 / IL- The 15Rα complex contains IL-15 or an IL-15 derivative and the amino acid sequence number 10 in Table 2. Contains wild-type IL-15Rα having an acid sequence. In another embodiment, IL-15 / I The L-15Rα complex contains wild-type IL-15 or an IL-15 derivative and the above-mentioned IL-1 Includes the glycosylated form of 5Rα.

[0101] In a specific embodiment, the IL-15 / IL-15Rα complex is wild-type IL-15 Alternatively, IL-15Rα derivatives and soluble IL-15Rα (e.g., wild-type soluble human IL-15Rα) Includes L-15Rα). In another specific embodiment, IL-15 / IL-15Rα compound The compound consists of an IL-15 derivative and an IL-15Rα derivative. In another embodiment... In this context, the IL-15 / IL-15Rα complex is composed of wild-type IL-15 and IL-15Rα It is composed of derivatives. In one embodiment, the IL-15Rα derivative is IL-15Rα This is its soluble form. The above is a specific example of the soluble form of IL-15Rα. In a typical embodiment, the soluble form of IL-15Rα is the transmembrane form of wild-type IL-15Rα. It lacks the domain, and in some cases the intracellular domain of wild-type IL-15Rα. Another implementation Morphologically, IL-15Rα derivatives have the extracellular domain of wild-type IL-15Rα or This is the fragment. In one embodiment, the IL-15Rα derivative is wild-type IL-15R It is a fragment of the extracellular domain containing the sushi domain or exon 2 of α. In terms of application, the IL-15Rα derivative has the sushi domain of wild-type IL-15Rα. or a fragment of the extracellular domain including exon 2 and a small amount encoded by exon 3 It contains at least one amino acid. In one embodiment, the IL-15Rα derivative is wild-type Fragments of the extracellular domain of IL-15Rα, including the sushi domain or exon 2. and includes the IL-15Rα hinge region or a fragment thereof. In one embodiment, IL-15 Rα contains the amino acid sequence of SEQ ID NO: 10 in Table 2.

[0102] In another embodiment, the IL-15Rα derivative cleaves wild-type IL-15Rα. This includes mutations in the extracellular domain cleavage site that inhibit cleavage by endogenous proteases. In a specific embodiment, the extracellular domain cleavage site of IL-15Rα is a heterogeneous known site. It is replaced by a cleavage site that is recognized and cleaved by a protease. A non-limiting example of a heteroprotease cleavage site is recognition by furin protease. and the cleaved Arg-XX-Arg (Sequence ID 25 in Table 2); and thrombin AB-Pro-Arg-XY (sequence shown in Table 2) is recognized and cleaved by proteases. Number 26) (A and B are hydrophobic amino acids, and X and Y are non-acidic amino acids) Examples include ) and Gly-Arg-Gly.

[0103] In another embodiment, IL-15 is, for example, International Publication No. 2007 / 084342. Brochures and International Publication No. 2010 / 020047; and US Special Specification No. 5,965,726; Specification No. 6,174,666; Specification No. 6,291, Specification No. 664; Specification Nos. 6,414,132; and Specification Nos. 6,794,498 Nucleic acid sequences optimized to enhance IL-15 expression using the method described below. It is coded by.

[0104] In one embodiment, with reference to Sequence IDs 22, 23, and 24 in Table 2, the above Glico Glycosylation occurs at 1, 2, 3, 4, 5, 6, 7, or all of the sylation sites. An IL-15 / IL-15Rα complex containing human IL-15Rα is provided herein. In a specific embodiment, glycosylated IL-15Rα is compared with wild-type human IL-15Rα. It is α. In other specific embodiments, glycosylated IL-15Rα is human IL- It is an IL-15Rα derivative of 15Rα. In some embodiments, glycosylated IL- 15Rα is wild-type soluble human IL-15Rα, for example, sequence number 7 or 10 in Table 2. Yes. In other embodiments, glycosylated IL-15Rα is a convertible form of human IL-15Rα. It is a soluble form of IL-15Rα derivative. In one embodiment, IL-15 / IL The -15Rα complex has been purified or isolated.

[0105] In addition to IL-15 and IL-15Rα, the IL-15 / IL-15Rα complex is heterogeneous It may contain molecules. In some embodiments, heterologous molecules increase protein stability. A non-limiting example of such a molecule is the in vivo reaction of IL-15 or IL-15Rα. Polyethylene glycol (PEG) and IgG immunoglobulin Fc, which increase half-life. Examples include the main or a fragment thereof, or albumin. In one embodiment, IL -15Rα is found in the Fc domain or fragments of immunoglobulins (e.g., IgG1). It is fused / integrated. In a specific embodiment, IL-15RαFc fused tan The protein contains the amino acid sequence of SEQ ID NO: 27 or 28 in Table 2. In another embodiment, The IL-15RαFc fusion protein was identified by Han et al., (2011), Cyt okine 56:804-810, U.S. Patent No. 8,507,222 or U.S. This is an IL-15Rα / Fc fusion protein as described in Japanese Patent No. 8,124,084. In those IL-15 / IL-15Rα complexes containing heterologous molecules, the heterologous molecules are I It may be conjugated to L-15 and / or IL-15Rα. In one embodiment In another embodiment, the heterologous molecule is conjugated to IL-15Rα. The heterologous molecules are conjugated to IL-15.

[0106] The components of the IL-15 / IL-15Rα complex are either non-covalent or covalently bonded. By using one of the following (for example, by combining amino acid sequences via peptide bonds) They can be directly fused and / or combined using one or more linkers. This is possible. A suitable linker for preparing the IL-15 / IL-15Rα complex is one of two or more linkers. Peptides, alkyl groups, chemically substituted alkyl groups, polymers, and other components that can be bound together with the constituent elements. or any other covalent or non-covalent chemical substances. The polymer linker is said Any polymer known in the technical field, for example, polyethylene glycol (PEG) Includes. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more It is a peptide with the amino acid length of [number]. In a specific embodiment, the linker is IL-15R It is long enough to preserve the ability of IL-15 to bind to α. In other embodiments, the linker - acts as an agonist that binds to the βγ receptor complex and mediates IL-15 signaling. It is long enough to preserve the ability of the active IL-15 / IL-15Rα complex.

[0107] In certain embodiments, the IL-15 / IL-15Rα complex is as described herein. Before use in the law (for example, before contacting cells with the IL-15 / IL-15Rα complex) Pre-coupling is performed (before administration of the IL-15 / IL-15Rα complex to the target). In this embodiment, the IL-15 / IL-15Rα complex is used in the method described herein. Do not pre-couple before use.

[0108] In a specific embodiment, the IL-15 / IL-15Rα complex is used in the art. Well-known assays, such as ELISPOT, ELISA, and cell proliferation assays For immune function in subjects not administered the IL-15 / IL-15Rα complex. The immune function in the subject was reduced to at least 99%, at least 95%, at least 90%, and less 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 less At best, it improves or induces by 10%. In a specific embodiment, immune function , cytokine release (e.g., interferon-gamma, IL-2, IL-5, IL- It is 10, IL-12, or transforming growth factor (TGF)-beta. In one embodiment, IL-15-mediated immune function is NK cell proliferation, which is, for example, NK cell markers (e.g., CD56) are assayed using flow cytometry. The number of cells expressing the drug can be detected. In another embodiment, IL-15-mediated immunity Its function is antibody production, which can be assayed, for example, by ELISA. In some embodiments, IL-15-mediated immune function is an effector function, and For example, by a cytotoxicity assay or other assays well known in the art, It can be done.

[0109] In a specific embodiment, the immune response enhanced by the IL-15 / IL-15Rα complex Examples of functions include lymphocyte proliferation / expansion (e.g., increase in lymphocyte count) and lymphocyte a These include inhibition of potosis, activation of dendritic cells (or antigen-presenting cells), and antigen presentation. In certain embodiments, immunity is enhanced by the IL-15 / IL-15Rα complex. The disease function 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) T cells), B cells (e.g., plasma cells), memory T cells, memory B cells, dendritic cells ( Immature or mature cells, antigen-presenting cells, macrophages, mast cells, natural killer T cells cells (NKT cells), tumor-resident T cells, CD122 + T cells, or natural killer cells This involves the proliferation / expansion of the number of cells (NK cells) or their activation. In one embodiment, The IL-15 / IL-15Rα complex promotes the proliferation / expansion or increase in the number of lymphocyte progenitor cells. To raise. In some embodiments, the IL-15 / IL-15Rα complex is CD4 + T Cells (e.g., Th1 and Th2 helper T cells), CD8 + T cells (for example, cell damage) Harmful T lymphocytes, alpha / beta T cells, and gamma / delta T cells), B cells (e.g.) For example, 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 + Number of T cells, or natural killer cells (NK cells) This was then used as a negative control (for example, not treated with the IL-15 / IL-15Rα complex) and Approximately 1x, 2x, compared to the number of cells that are neither cultured nor in contact with each other. Increased by 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times or more Let it.

[0110] In a specific embodiment, the IL-15 / IL-15Rα complex is used to target Staphylococcus aureus. It increases the expression of IL-2 in whole blood, which is activated by rotoxin B (SEB). Furthermore, the IL-15 / IL-15Rα complex is used in the same way as when SEB is used alone, the release of IL-2 Compared to the current state, it increases IL-2 expression by at least approximately 2, 3, 4, or 5 times.

[0111] Production of the IL-15 / IL-15Rα complex Nucleic acids encoding IL-15 and / or IL-15Rα are found in mammalian cells, bacteria, It can be inserted into nucleic acid constructs for expression in yeast and viruses. IL- 15 and IL-15Rα are derived from the same nucleic acid construct (for example, a bisistronic nucleic acid construct) (using a construct) or from different nucleic acid constructs (e.g., monocistronic nucleic acid constructs) It can be recombinantly expressed using ( ). In one embodiment, IL-15 and IL -15Rα is a single open reading frame for IL-15 and IL-15Rα. Recombinant expression can be achieved from a single nucleic acid construct containing (ORF).

[0112] The nucleic acid construct is operably linked to the coding sequence of IL-15 and / or IL-15Rα. It may contain one or more combined transcriptional regulatory elements. Transcriptional regulatory elements are typically This is 5' to the code sequence and codes IL-15 and / or IL-15Rα. It aims to transcribe nucleic acids. In some embodiments, wild-type IL-15 and / or This is a transcriptional regulatory element that is naturally found to regulate the transcription of the wild-type IL-15Rα gene. One or more of these are used to control transcription. In other embodiments, wild-type IL -15 and / or one or more heterologous transcriptional moduloions compared to the wild-type IL-15Rα gene The nodal elements are used to control the transcription. Any transcription control element known to those skilled in the art Ment can be used. Non-limiting examples of types of transcriptional regulatory elements include: Examples include constitutive promoters, tissue-specific promoters, and inducible promoters. In specific embodiments, the transcription is at least partially performed on mammals (partially in some embodiments) In this state, it is controlled by a human transcriptional regulatory element. In a specific embodiment, Transcription is controlled, at least partially, by a strong promoter, such as CMV. In other embodiments, an inductive promoter can be used.

[0113] The nucleic acid construct is operably linked to the coding sequence of IL-15 and / or IL-15Rα. It may also include one or more post-transfer regulatory elements that are combined. It is on the 5' and / or 3' side relative to the D sequence, and IL-15 and / or IL-15 This could potentially lead to post-transcriptional regulation of the translation of RNA transcripts encoding Rα.

[0114] In another embodiment, nucleic acid constructs are, for example, in International Publication No. 1994 / 12650. As described in Lett and International Publication No. 2001 / 68882, the gene The existing regulatory region is replaced by a regulatory sequence isolated from a different gene or a novel regulatory sequence. It may be a gene that targets a vector. In one embodiment, the host cell is, for example Therefore, modifying the regulatory regions of the endogenous IL-15 and / or IL-15Rα genes Genetic manipulation to increase the production of endogenous IL-15 and / or IL-15Rα It can be made.

[0115] The nucleic acid constructs selected may include various factors, for example, but are not limited to transcriptional modalities. To increase the strength of the nodal elements and to express IL-15 and / or IL-15Rα It depends on the host cell to be used. Nucleic acid constructs include plasmids, phagemids, and cosmids. This could be a viral vector, a phage, or an artificial chromosome. In one embodiment, a vector This is any preferred means (transformation, translocation, conjugation, protoplast fusion). , electroporation, calcium phosphate precipitation, direct microinjection, etc. ) Episomes can be introduced into suitable host cells and transform them. These may be non-homologous embedded vectors.

[0116] Nucleic acid constructs transiently induce IL-15 and / or IL-15Rα in host cells. This may be a plasmid or stable embedding vector for stable expression. Therefore, the vector can mediate chromosomal integration at a target site or a random chromosomal site. It can be used to express IL-15 and / or IL-15Rα. Non-limiting examples of host cell-vector systems include viruses (e.g., vaccinia virus). Mammalian cell lines infected with adenoviruses, retroviruses, lentiviruses, etc. Insect cell lines infected with viruses (e.g., baculoviruses); microorganisms, e.g., yeast Yeast containing bacteria, or bacteriophages, DNA, plasmid DNA, or This refers to bacteria transformed with cosmid DNA; and transformation using a selection marker. Examples include more stable cell lines generated. In some embodiments, nucleic acid constructs are selected Selective marker genes, for example, but not limited to, neomycin (neo), Contains hydrofolate reductase (DHFR) and hygromycin (HYG).

[0117] Nucleic acid constructs can be monocistronic or multicistronic. The rhonic nucleic acid constructs may consist of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more units, It codes for a range of 2-5, 5-10, or 10-20 genes / nucleotide sequences. For example, a bisistronic nucleic acid construct is obtained in the following order: promoter, first gene It includes the offspring (e.g., IL-15) and a second gene (e.g., IL-15Rα). It is possible. In such nucleic acid constructs, the transcription of both genes is driven by the promoter. Meanwhile, the translation of mRNA from the first gene is performed by a cap-dependent scanning machine. This is due to the introduction, and the translation of mRNA from the second gene is by a cap-independent mechanism, for example. For example, it is by IRES.

[0118] The techniques for carrying out these embodiments are routinely practiced by those skilled in the art unless otherwise specified. The techniques applied utilize molecular biology, microbiology, and conventional techniques of recombinant DNA manipulation and production. Yes, for example, Sambrook, 1989, Molecular Cloning, A Laboratory Manual,Second Edition;DNA Cl oning,Volumes I and II(Glover,Ed.1985);O Ligonucleotide Synthesis(Gait,Ed.1984);N ucleic Acid Hybridization(Hames & Higgin s,Eds.1984);Transcription and Translatio n(Hames & Higgins,Eds.1984);Animal Cell Culture(Freshney,Ed.1986);Immobilized Ce lls and Enzymes(IRL Press,1986);Perbal,A Practical Guide to Molecular Cloning(19 84);Gene Transfer Vectors for Mammalian Cells(Miller & Calos,Eds.1987,Cold Sprin g Harbor Laboratory);Methods in Enzymolo gy, Volumes 154 and 155 (Wu & Grossman, respectively) , and Wu,Eds.), (Mayer & Walker,Eds.,1987) ;Immunochemical Methods in Cell and Mole cular Biology(Academic Press,London,Scop) es, 1987), Expression of Proteins in Mamma lian Cells Using Vaccinia Viral Vectors in Current Protocols in Molecular Biolog See Volume 2 (Ausubel et al., Eds., 1991).

[0119] In a specific embodiment, a nucleic acid construct encoding IL-15 or IL-15Rα It is possible to simultaneously translocate or translocate into the same host cell or different host cells. In some cases, nucleic acid constructs containing nucleic acids encoding a selection marker gene can be placed in the same cell. It is also possible to transfer the cells into the cell and select the cells that will receive the transfer. IL-15 and IL-15R When a nucleic acid construct containing the nucleic acid encoding α is transmigrated into different cells, the different cells IL-15 and IL-15Rα expressed by were isolated, and the above IL-15 / IL- They can be brought into contact with each other under conditions suitable for forming a 15Rα complex. Any known technique, for example, transformation, transmutation, conjugation, Rotoplast fusion, electroporation, calcium phosphate precipitation, direct microinfiltration Injections, and viruses, for example, but not limited to, adenoviruses Nucleic acids are used to infect host cells with nucleic acids through infection by lentiviruses and retroviruses. It is possible to introduce or transfer traits.

[0120] For stable, long-term high-yield production of recombinant IL-15 and IL-15Rα polypeptides Cell lines can be generated. For example, cell lines can be selected on the same or different nucleic acid constructs. Transformation using nucleic acid constructs described herein that may contain selective marker genes. This is possible. Selective marker genes can be introduced into the same cell by simultaneous translocation. After introducing the vector, the cells are grown in concentrated medium for 1-2 days before being transferred to selective medium. This enables the growth and harvesting of cells that express the introduced nucleic acid well. Stable transformed cells. The resistant clones utilize well-known tissue culture techniques in the art that are appropriate for the cell type. It can be used to grow the cells. In certain embodiments, the cell line is grown in serum-free medium. It is adapted for growth. In one embodiment, the cell system is a shaker frog It is adapted to grow in serum-free medium in a scoping medium. In one embodiment, The cell system is adapted for growth in a stirred or rotating flask. In this state, the cell line is cultured in a suspension. In a particular embodiment, the cell line is connected They are not attached, or are adapted to grow as non-adherent cells. Morphologically, the cell line is adapted to grow under low-calcium conditions. In some embodiments, the cell line is cultured or grown in a low-serum medium. They are combined.

[0121] In specific embodiments, particularly preferred methods for high-yield production of recombinant polypeptides of the present invention The method is described in U.S. Patent No. 4,889,803, which involves continuously increasing levels of met Dihydrofolate reductase (DH) in DHFR-deficient CHO cells due to the use of trexate. This involves the use of FR amplification. Polypeptides obtained from such cells are then processed. It may be a lycosylated form.

[0122] In one embodiment, the cell line contains wild-type human IL-15 and wild-type soluble human IL- The gene was genetically engineered to express a stable heterodimer of 15Rα, and then purified. It can be administered to the patient. In one embodiment, IL-15 / IL-15Rα heterozygotes Merger stability is determined from cell lines that recombinantly express both IL-15 and IL-15Rα. It increases when it is produced.

[0123] In a specific embodiment, host cells recombinate IL-15 and full-length IL-15Rα. In another specific embodiment, the host cell expresses IL-15 and IL-15 The soluble form of Rα is recombinantly expressed. In another specific embodiment, the host cell is IL -15 and the membrane of IL-15Rα that is not cleaved from the cell surface and remains associated with the cell. Recombinant expression of the binding form. In some embodiments, IL-15 and / or IL- Host cells that recombinantly express 15Rα (full-length or soluble form) can express another polypeptide (e.g. For example, cytokines (or fragments thereof) are also recombinantly expressed.

[0124] In one embodiment, such host cells are IL-15R In addition to α-polypeptides, IL-15 polypeptide is recombinantly expressed. IL-15 and / Alternatively, using nucleic acids encoding IL-15Rα, a single unit of IL-15 and IL-15Rα Mammals that highly recombinantly express IL-15 and IL-15Rα for release and purification. Cells can be generated, preferably IL-15 and IL-15Rα are in a complex and They associate. In one embodiment, a large amount of IL-15 / IL-15Rα complex Control cells (e.g., IL-15, IL-15Rα, or IL-15 and IL-15) Cells that have not been genetically engineered to recombinantly express both Rα, or cells containing an empty vector. Less than the amount of IL-15 / IL-15Rα complex endogenously expressed by (gray cells). Both 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, or This refers to the amount of IL-15 / IL-15Rα complex expressed by cells that is more than 20 times greater. In some embodiments, the host cells described herein are based on techniques known to those skilled in the art (e.g., Measured by ELISA, approximately 0.1 pg to 25 pg, 0.1 pg to 20 pg, 0 .1pg~15pg, 0.1pg~10pg, 0.1pg~5pg, 0.1pg~2pg It expresses 2 pg to 10 pg, or 5 to 20 pg, of IL-15. In one embodiment, Therefore, the host cells described herein are based on techniques known in the art (e.g., ELIS A) Measured, the daily dose is approximately 0.1-0.25 pg, and the daily dose is 0.25-0. 5pg, 0.5-1pg per day, 1-2pg per day, 2-5pg per day, Alternatively, it expresses 5-10 pg of IL-15 per day. In a specific embodiment, IL -15Rα is a soluble form of IL-15Rα. In a specific embodiment, IL- 15Rα is the soluble form of IL-15Rα, which is associated with IL-15 as a stable heterodimer. It is in a state that increases yield, and the bioactive heterodimer IL-15 / soluble IL-15 To simplify the production and purification of Rα cytokines.

[0125] Recombinant IL-15 and IL-15Rα are used to develop methods for recombinant protein production and purification. They can be used to purify, and these are well known in the relevant art, for example, international See Pamphlet No. 2007 / 070488. In short, polypeptides are, It can be produced within cells, in the periplasmic space, or secreted directly into the culture medium. This can be done. Cell lysates or supernatants containing polypeptides can be used, for example, hydroxyl apatate We use lithochromatography, gel electrophoresis, dialysis, and affinity chromatography. It can be purified by this method. Other techniques for protein purification, such as ion exchange coloration, Fractionation on a surface, ethanol precipitation, reverse-phase HPLC, chromatography on silica, heparin SEPHAROSE (trademark) (gel filter material; Pharmacia Inc., Pis Chromatography, anion, or color chromatography on Cataway, New Jersey. Chromatography on thion exchange resin (e.g., polyaspartate column), chromium Matfocusing, SDS-PAGE, and ammonium sulfate precipitation are also available. .

[0126] In some embodiments, IL-15 and IL-15Rα are synthesized or different Recombinant expression was performed using cells, followed by isolation, and the IL-15 / IL-15Rα complex was then combined. The mixture is formed in vitro and then administered to the subject. In other embodiments, IL-15 and IL-15Rα was synthesized or recombinantly expressed in different cells, then isolated, and then... The IL-15 / IL-15Rα complex is administered simultaneously to the target in situ or in vivo. In other embodiments, IL-15 and IL-15Rα are synthesized and Alternatively, express them together in the same cell, and the formed IL-15 / IL-15Rα complex To isolate.

[0127] The host cells selected for nucleic acid expression depend on the intended use of the cells. Cells, for example, that endogenously express IL-15 and / or IL-15Rα, Factors such as whether or not glycosylation occurs may be considered when selecting host cells.

[0128] Nucleic acid constructs available herein can be used to express proteins encoded by these constructs. Non-limited examples of host cells include mammalian cells, bacterial cells, yeast cells, primary cells, immortalized cells, and plant cells. This includes phytic cells and insect cells. In specific embodiments, the host cell is a mammalian cell line. Examples of mammalian cell lines include, but are not limited to, COS, CHO, HeLa, and NIH3. T3, HepG2, MCF7, HEK293, HEK293T, RD, PC12, Hive Ridoma, pre-B cell, 293, 293H, K562, SkBr3, BT474, A20 4, M07Sb, TFβ1, Raji, Jurkat, MOLT-4, CTLL-2, M C-IXC, SK-N-MC, SK-N-MC, SK-N-DZ, SH-SY5Y, C1 Examples include 27, N0, and BE(2)-C cells. Other cells available as hosts for expression include Mammalian cell lines are publicly known in the relevant field and are owned by the United States Cell Culture Lineage Preservation Service ( American Type Culture Collection) (ATCC)? This includes numerous immortalized cell lines that are available.

[0129] CHO cells Chinese hamster ovary (CHO) cells are most commonly used in therapeutic applications. These cells are used for the production of lycosylated polypeptides. This brings about chemical characteristics and enables the creation of genetically stable, highly productive cell lines. Furthermore, CHO Cells, in serum-free medium, have a high cell density to generate safe and reproducible biological processes. They can be cultured at a certain temperature. This is a challenge when using CHO cells as host cells for recombinant expression. One major challenge is the expression and secretion of cells in the cell culture medium, also known as "clipping." This is the proteolytic degradation of the target polypeptide.

[0130] In some embodiments, the IL-15 / IL-15Rα complex acts as a mechanism for matryptase. CHO cell lines that have been modified to impair function, for example, the function of the matryptase gene Reducing or removing active expression and recombining the cells that are expressed and secreted in the cell culture medium for the purpose of the target recombination. By significantly reducing the proteolytic degradation ("clipping") of polypeptides, Therefore, it impairs the effect of the aforementioned matryptase within the cell. Compared to corresponding vertebrate cells in which the effect of matryptase is not impaired, Clipping of the recombinant IL-15 / IL-15Rα complex is reduced. Along with tase, it is involved in the clipping of recombinantly expressed and secreted polypeptides. The major proteases were identified that impair the effect of matryptase in vertebrate cells. Modifying the target polypeptide, which is recombinantly expressed and secreted in the cell culture medium, By reducing or eliminating clipping, the recombinant production of the target polypeptide can be significantly improved. This makes improvement possible, thereby increasing the yield of the IL-15 / IL-15Rα complex. Add.

[0131] CHO cells generate an α(2-6)-linked NANA extension to the coagulan structure. The enzyme involved in this (β-galactoside α-2,6-sialyltransferase 1) It has been reported that CHO cells are inactive or not expressed (for example, Ch (See ung et al 2017) However, the gene in the enzyme itself is C. glycerin. It is present in C. griseus. However, surprisingly, it is produced in the CHO cells of this disclosure. The generated IL-15 / IL-15Rα complex is produced by human cell lines. It was discovered that it has a different glycosylation pattern compared to the / IL-15Rα complex. Furthermore, α(2-6) linked glycans produced in CHO cells were linked to IL-15 / IL- It was observed in the 15Rα complex. This glycosylation pattern is unique. It is latent. In terms of characteristics, it is closer to the human glycosylated form, compared to the expected CHO pattern. It can yield direct benefits. Details of the CHO cell line can be found in International Publication No. 2015 / 166. This can be found in Pamphlet No. 427 (which is incorporated herein by reference).

[0132] Pharmaceutical composition A composition comprising the IL-15 / IL-15Rα complex is provided herein. Therefore, bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compounds) Pharmaceutical compositions that can be used in the preparation of products and unit dosage forms (i.e., target Examples include compositions suitable for administration to patients. A composition (e.g., a pharmaceutical composition) is... An effective amount of IL-15 / IL-15Rα complex, or IL-15 / IL-15Rα complex The combination includes a pharmaceutically acceptable carrier. In specific embodiments, the composition (e.g.) For example, a pharmaceutical composition contains an effective amount of one or more IL-15 / IL-15Rα complexes and drugs Contains a scientifically acceptable carrier. In some embodiments, the composition contains additional therapeutic agents, e.g. These include anticancer drugs, antiviral drugs, anti-inflammatory drugs, and adjuvants. Non-restrictive examples are provided below.

[0133] In specific embodiments, the term "pharmaceutically acceptable" means in animals, particularly humans. Approved for use by a federal government agency or state government, or as per the United States Pharmacopeia. Or it means that it is listed in other generally recognized pharmacopoeias. Term "carrier" " refers to the therapeutic agent and the diluent administered, the adjuvant (for example, Freund's adjuvant (complete) (whole and incomplete), or more preferably MF59C.1 adjuvant, excipients This refers to a vehicle. Such pharmaceutical carriers are sterile liquids, such as water and oil, for example. , petroleum, animal, plant or synthetic origin oils, such as peanut oil, soybean oil, mineral oil, and ginger oil. This could be sesame oil, etc. In one embodiment, when the pharmaceutical composition is administered intravenously, water is carried It is a body. Physiological saline and aqueous dextrose and glycerol solutions are used as liquid carriers. In particular, it can be used for injectable solutions. Suitable pharmaceutical excipients include starch, glycerin. Glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica Gel, sodium stearate, glycerol monostearate, talc, sodium chloride Examples include milk powder, skim milk powder, glycerol, propylene glycol, water, and ethanol. The composition may optionally contain a small amount of wetting agent or emulsifier, or a pH buffer. These compositions include liquids, suspensions, emulsions, tablets, pills, capsules, powders, and saturates. It can take the form of a release compound or similar.

[0134] A pharmaceutical composition may be used with one or more pharmaceutically acceptable carriers or excipients. It can be formulated in the form of. In specific embodiments, according to the method described herein The IL-15 / IL-15Rα complex administered to the target is administered as a pharmaceutical composition. ru.

[0135] Generally, the components of a pharmaceutical composition containing the IL-15 / IL-15Rα complex are in the unit dosage form. Supplied in, for example, a sealed container, for example, an ampoule or pouch indicating the amount of activator, in a dry state. Supplied as frozen powder or anhydrous concentrate. IL-15 / IL-15Rα complex is injected. If administration is to be performed by means of sterile pharmaceutical-grade water or saline solution (e.g., PBS), It can be dispensed using an injection bottle containing IL-15 / IL-15Rα compound. When administering the body by injection, sterile water for injection is used so that the components can be mixed before administration. Alternatively, an ampoule of physiological saline solution can be provided.

[0136] In some embodiments, the IL-15 / IL-15Rα complex is an arbitrary compound known to those skilled in the art. Administration by the following methods, for example, but not limited to, parenteral administration (e.g., subcutaneous, intravenous). It can be formulated for administration (intratumor or intramuscular). In one embodiment, The L-15 / IL-15Rα complex can be administered topically or systemically as parenterally, for example, intratumorally. It is formulated for this purpose. In a specific embodiment, the IL-15 / IL-15Rα complex is Each is formulated for subcutaneous or intravenous administration. In one embodiment, IL-15 / The IL-15Rα complex is formulated in a pharmaceutically compatible solution.

[0137] The IL-15 / IL-15Rα complex is administered by injection, for example, as a bolus injection or sequential injection. It can be formulated for parenteral administration by injection. For example, an injectable formulation can be an amplifier. It can be provided in a single dosage form along with a preservative added in a container for multiple doses or in a multi-dose container. The composition is in an oily or aqueous vehicle in the form of a suspension, liquid, or emulsion. It may take on a certain form and may contain compounding agents, such as suspending agents, stabilizers, and / or dispersants. Furthermore, the active ingredient is used in a suitable vehicle, for example, sterile pyrogen-free water before use. It may be in powder form for its composition.

[0138] Therapeutic and Dose Regimen In one embodiment, a method for improving IL-15-mediated immune function, wherein the complex IL-1 A method comprising administering the 5 / IL-15Rα complex to a subject in a prescribed dose regimen. This is provided herein. IL-15-mediated immune function enhancement is used for the prevention, treatment, and prevention of certain disorders. To prevent, treat, and / or manage such disorders, as they are beneficial to management. This method involves administering the IL-15 / IL-15Rα complex to the target in need. Methods including the following are provided herein: improving IL-15 mediated immune function. Non-limited examples of conditions for which it is beneficial include cancer, lymphopenia, immunodeficiency, infectious diseases, And wounds are among them.

[0139] In one embodiment, to prevent, treat, and / or manage a disorder in a subject In this regard, improving IL-15-mediated immune function can prevent, treat, and / or manage such disorders. A method that is beneficial to the principle and targets the IL-15 / IL-15Rα complex at the same dose. A method is provided herein that includes administering the medication over the duration of a therapeutic cycle. In terms of form, the dosage ranges from 0.1 μg / kg to 0.5 μg / kg. In terms of form, the dosage ranges from 0.25 μg / kg to 1 μg / kg. In some embodiments, the doses are in the range of 0.5 μg / kg and 2 μg / kg. In the application method, the dose is 1 μg / kg to 4 μg / kg. In another embodiment, The dosage is 2 μg / kg to 8 μg / kg. In another embodiment, the dosage is 0.1 μg / kg. g / kg, 0.25μg / kg, 0.5μg / kg, 1μg / kg, 2μg / kg, 4μ The concentrations are g / kg, 5 μg / kg, 6 μg / kg, and 8 μg / kg. In specific embodiments... The dosage is 1 μg / kg. In one embodiment, the dosage is 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10 times or more, or 1-3, 1-4, 2-4, 2-5, It is administered 2-6, 3-6, 4-6, 6-8, 5-8, or 5-10 times. In terms of form, the dosage is 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7- Over a period of 21 days or 14-21 days, the numbers are 1, 2, 3, 4, 5, 6, 7, 8, 9, and 1. 0 times or more, or 1-3, 1-4, 2-4, 2-5, 1-5, 2-6, 3- It is administered 6, 4-6, or 6-8 times. In specific embodiments, the respective doses are 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days or 14- It is administered at least 1, 2, 3, 4, 5, 6 times or more over a period of 21 days. In another specific embodiment, each dose is administered at least once, and the subject is The medication is administered once a week for a period of three weeks.

[0140] In another embodiment, to prevent, treat, and / or manage a disorder in the subject In this regard, improving IL-15-mediated immune function can help prevent, treat, and / or treat such disorders. A method that is beneficial for management, targeting the IL-15 / IL-15Rα complex in the administration regimen. In the administration cycle, at least 1, 2, 4, or 6 doses are administered before the non-administration period. Methods including the following are provided herein. In specific embodiments, IL-15 / The IL-15Rα complex is administered once a week for three weeks, with no administration during the fourth week. Repeat the administration cycle.

[0141] In an alternative embodiment, to prevent, treat, and / or manage a disorder in the subject In this regard, improving IL-15-mediated immune function can help prevent, treat, and / or treat such disorders. A method that is beneficial for management, (a) at least one initial low dose of IL-15 / IL (b) Administer to target the -15Rα complex; and (b) a continuously increasing dose of IL-1 This includes administering the 5 / IL-15Rα complex to the target for the duration of the treatment cycle. Methods are provided herein. In specific embodiments, methods for preventing and curing cancer in a subject are provided. A method of treating and / or managing (a) an initial dose of IL-15 / IL-15 (b) Administer the Rα complex for the duration of the treatment cycle; and (b) Continuous Targeting the IL-15 / IL-15Rα complex at higher doses, the duration of the treatment cycle was considered. A method is provided herein that includes administering over time. In a specific embodiment, first The dose ranges from 0.1 μg / kg to 0.5 μg / kg. In this case, the initial dose is in the range of 0.25 μg / kg and 1 μg / kg. Another implementation In this context, the initial dose is in the range of 0.5 μg / kg and 2 μg / kg. In one embodiment, the initial dose is 1 μg / kg to 4 μg / kg. In another embodiment... In another embodiment, the initial doses are 2 μg / kg and 8 μg / kg. The dose is approximately 0.25 μg / kg. In another embodiment, the initial dose is approximately 0.5 It is μg / kg. In another embodiment, the initial dose is approximately 1 μg / kg. In terms of administration methods, the initial doses are 0.1 μg / kg, 0.25 μg / kg, and 0.5 μg / kg. g, 1μg / kg, 2μg / kg, 4μg / kg, 5μg / kg, 6μg / kg, 8μg The value is / kg. In one embodiment, the initial dose is 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 It is administered 4-6, 6-8, 5-8, or 5-10 times. In some embodiments, The initial dose is for 5-7 days, 5-10 days, 7-12 days, 7-14 days, and 7-21 days. Alternatively, over a period of 14 to 21 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times ku is more than that, or 1-3, 1-4, 2-4, 2-5, 1-5, 2-6, 3-6, 4- It is administered 6 or 6-8 times. In one embodiment, each of the continuously increasing doses The dosage is 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, and 1.5 times the previous dose. 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times higher, or the previous value Rather than quantity, consider 1.2-2, 2-3, 2-4, 1-5, 2-6, 3-4, 3-6, or 4- Six times higher, or twice as high as the previous dose. In some embodiments, each sequential the dose that is increased is 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% , 105%, 110%, 115%, 120%, 125%, 130%, 135%, or 140% , 145%, 150%, 155%, 160%, 165%, 170%, 175%, or 180% , 185%, 190%, 195%, or 200% higher than the previous dose. In a specific embodiment, the each dose is administered over 5 to 7 days, 5 to 10 days, 7 to 12 days, 7 to 14 days, 7 to 21 days or 14 to 21 days, at least 1, 2, 3, 4, 5, 6 or more doses are administered. In another specific embodiment, each dose is administered at least once, and the subject receives the dose 3 times per 7-day week over a 2-week period (e.g., on Monday, Wednesday and Friday).

[0142] In one embodiment, the subject is monitored for the following adverse events, for example, grade 3 or 4 thrombo cytopenia, grade 3 or 4 granulocytopenia, grade 3 or 4 leukocytosis (white blood cell ( WBC) > 100,000 mm 3 ), grade 3 or 4 WBC, absolute lymphocyte count (A LC) and / or a decrease in absolute neutrophil count (ANC), lymphocytosis, and organ function impair ment (e.g., liver or renal dysfunction). In one embodiment , the dose is not increased if the subject experiences an adverse event, for example, grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade 3 or 4 leukocytosis (white blood cell (WBC) > 1 00,000 mm 3 ), grade 3 or 4 WBC, absolute lymphocyte count (ALC) and / or a decrease in absolute neutrophil count (ANC), lymphocytosis, and organ dysfunction (e.g.) If the patient experiences hepatic or renal failure, the dosage may remain the same, or the medication may be discontinued or reduced. This can be done. According to these embodiments, IL-15 / IL- The dose of the 15Rα complex can be reduced until adverse events decrease or disappear. They may remain the same.

[0143] In another embodiment, to prevent, treat, and / or manage a disorder in the subject In this regard, improving IL-15-mediated immune function can help prevent, treat, and / or treat such disorders. A beneficial method for management is to administer IL-15 / IL-15Rα complex at a dose of 0.25 μg / Start with the first cycle, which includes an initial dose of 4 μg / kg, and in subsequent cycles... In a dose regimen in which the dose is increased by 2 to 3 times compared to the previous dose, when administered to human subjects... A method comprising the following is provided herein: Each dose is at least 1, 2, or 4 times Alternatively, administer 6 doses and then increase the dose to the next level, with a certain dose of IL-15 / IL-1 For a certain period after administration of the 5Rα complex (for example, a certain dose of IL-15 / IL-15Rα complex) Approximately 24 to 48 hours after administration, approximately 24 to 36 hours, approximately Approximately 24 hours to 72 hours, approximately 48 hours to 72 hours, approximately 36 hours to For 48 hours, or approximately 48 to 60 hours, and another dose of IL-15 / IL-15 Free IL-15 in a sample obtained from the subject (e.g., plasma sample) before administration of the Rα complex. Monitor the concentration before increasing the dose to the next level.

[0144] In another embodiment, to prevent, treat, and / or manage a disorder in the subject In this regard, improving IL-15-mediated immune function can help prevent, treat, and / or treat such disorders. A beneficial method for management is to administer the IL-15 / IL-15Rα complex in the following continuous doses. :(i)0.25μg / kg;(ii)0.5μg / kg;(iii)1μg / kg;( iv) 2 μg / kg; (v) 4 μg / kg; and (vi) 8 μg / kg dose rates A method comprising administering to a subject in Dimen is provided herein. In one embodiment In this context, the IL-15 / IL-15Rα complex is administered in the following continuous doses: (i) 1 μg / kg; (ii) 2 μg / kg; (iii) 4 μg / kg; and (iv) 8 μg / kg The drug is administered to the target population in the dose regimen. Each dose is administered in small amounts during the administration cycle. Each dose is administered 1, 2, 4, or 6 times before increasing the dose to the next level, with a certain dose of IL For a certain period after administration of the IL-15 / IL-15Rα complex (for example, a certain dose of IL-15 / IL-15Rα) Approximately 24 to 48 hours after administration of the L-15Rα complex, approximately 24 hours to Approximately 36 hours, approximately 24 hours to approximately 72 hours, approximately 48 hours to approximately 72 hours, Approximately 36 to 48 hours, or approximately 48 to 60 hours, and another dose of IL Samples obtained from subjects (e.g., plasma samples) before administration of the -15 / IL-15Rα complex. Monitor the concentration of free IL-15 in the solution before increasing the dose to the next level.

[0145] In another embodiment, a person who prevents, treats, and / or manages cancer in a subject. The method involves administering the IL-15 / IL-15Rα complex in the following continuous doses: (i) 1 μg / kg (ii) 2 μg / kg; (iii) 4 μg / kg; and (iv) 8 μg / kg This includes administering the dose to the subject in a dose regimen, with each dose being administered in a dose cycle. In this case, administer at least 1, 2, 4, or 6 times before increasing the dose to the next level. A method for increasing is provided herein. In a specific embodiment, the method involves periodic administration The procedure includes the step of administering dimen to the IL-15 / IL-15Rα complex, This cyclical administration regimen involves (a) administering the IL-15 / IL-15Rα complex every 1 to 3 weeks. During the first period, the target skin was treated with a dose of 0.1 to 10 μg / kg every 1, 2, or 3 days. (b) Administer as shown below; (b) 1 week in which the IL-15 / IL-15Rα complex is not administered to the target. After the second period of approximately 2 months, the IL-15 / IL-15Rα complex is administered during the third period of 1 to 3 weeks. The drug is administered subcutaneously to the target patient at a dose of 0.1 to 10 μg / kg every 1, 2, or 3 days. This includes,

[0146] In certain embodiments, the subject is a human subject. In one embodiment, the therapeutic site The dosage for Kuru is 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 It is administered 6, 6-8, 5-8, or 5-10 times. In some embodiments, the dosage This is for 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14 days. ~1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more over a period of 21 days Top, or 1-3, 1-4, 1-5, 2-4, 2-5, 2-6, 1-6, 3-6, 4-6 Alternatively, it is administered 6 to 8 times. In one embodiment, each dose is given in one administration cycle. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more per round, or 1-3 , 1-4, 1-5, 2-4, 2-5, 1-6, 2-6, 1-6, 3-6, 4-6, 6-8 It is administered 5 to 8 times, or 5 to 10 times. In specific embodiments, the respective doses This is for 5-7 days, 5-10 days, 7-12 days, 7-14 days, 7-21 days, or 14 days. ~At least 1, 2, 3, 4, 5, 6 times or more over a period of 21 days, 1-3, 1-4, 1-5, 2-4, 2-5, 1-6, 2-6, 1-6, 3-6, 4-6 It is administered 6-8 times, 5-8 times, or 5-10 times.

[0147] In another specific embodiment, the subject is three times per week (for example, Monday, Wednesday) The dose is administered on Sundays and Fridays. In one embodiment, the subject experiences the following adverse events: For example, grade 3 or 4 thrombocytopenia, grade 3 or 4 granulocytopenia, grade Grade 3 or 4 leukocytosis (white blood cell count (WBC) > 100,000 mm3), Grade 3 or This is a decrease in 4 WBC, absolute lymphocyte count (ALC), and / or absolute neutrophil count (ANC). Monitor for lymphocyte vasoconstriction and organ dysfunction (e.g., liver or kidney dysfunction). It is ringed. In one embodiment, the dose is not increased, and the subject is an adverse event, for example, Grade 3 or 4 thrombocytopenia, Grade 3 or 4 granulocytopenia, Grade 3 or white blood cell blockade. Hemocytosis (white blood cell count (WBC) > 100,000 mm³), grade 3 or 4 WBC, Decreased absolute lymphocyte count (ALC) and / or absolute neutrophil count (ANC), increased lymphocyte count. Furthermore, the dosage remains the same if organ dysfunction (e.g., liver or kidney failure) is experienced. It may remain as is, and can be stopped or reduced. According to these embodiments, the target The dose of IL-15 / IL-15Rα complex administered to reduce or eliminate adverse events. It can be reduced to that extent, or it may remain the same.

[0148] 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, according to the method described herein. It is administered in doses over a period of two weeks. In specific embodiments, each dose is administered according to the method described herein. It is administered once, three times a week for two, three, or four weeks. In a specific embodiment, As described in the specification, each dose is administered once, six times a week for two, three, or four weeks. In specific embodiments, each dose is given every other day according to the method described herein. It is administered in a single dose for 2, 3, or 4 weeks. Specific embodiments are described herein. According to the method, each dose is administered once daily for 2, 3, or 4 weeks.

[0149] In one embodiment, the IL-15 / IL-15Rα complex is the method described herein. It is administered subcutaneously to the target. In some embodiments, IL-15 / IL-15Rα compound The compound is administered intravenously or intramuscularly to the subject by the method described herein. In this state, the IL-15 / IL-15Rα complex is targeted by the method described herein. It is administered intratumorally. In some embodiments, the IL-15 / IL-15Rα complex is used in this The drug is administered topically to a site in the subject (e.g., the site of infection) by the method described in the specification.

[0150] In one embodiment, a sample obtained from a subject by the method described herein is a blood sample. It is a sample. In a specific embodiment, the sample is a plasma sample. IL-15 basal plasma Bell's dosage is approximately 1 pg / ml in humans and approximately 8 pg / ml in monkeys (e.g., macaques). The concentration is 10 pg / ml, and approximately 12 pg / m³ in rodents (e.g., mice). A sample can be obtained from the subject using techniques known to those skilled in the art.

[0151] In a specific embodiment, an example of the immune function improved by the method described herein is Therefore, lymphocyte proliferation / expansion (e.g., increase in lymphocyte count), lymphocyte apoptosis These include inhibition, activation of dendritic cells (or antigen-presenting cells), and antigen presentation. In embodiments, the immune function improved 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), antigens Presenting cells, macrophages, mast cells, natural killer T cells (NKT cells), tumors Resident T cells, CD122 + Increase in the number of T cells or natural killer cells (NK cells) This is reproduction / expansion or activation thereof. In one embodiment, the method described herein is To increase the proliferation / expansion or number of lymphocyte progenitor cells. In some embodiments, this The methods described in the specification are 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 cells) (Nma / 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 Ral killer T cells (NKT cells), tumor-resident T cells, CD122 + T cells, or Naci The number of normal killer cells (NK cells) was increased by approximately 1, 2, 3, and 4 times compared to the negative control. To increase by 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, or more.

[0152] In specific embodiments, the methods described herein are well known in the art. For example, using ELISA, ELISA, and cell proliferation assays, IL Subjects who have not received the combination of -15 / IL-15Rα complex and anti-PD-1 antibody molecule. The immune function in the target group is reduced by at least 0.2 times, 0.5 times, and 0. 75x, 1x, 1.5x, 2x, 2.5x, 3x, 4x, 5x, 6x, 7x, 8x, 9x or improve or induce at least a tenfold improvement. In a specific embodiment, The methods described herein are based on assays well known in the art, such as ELISPO Using ELISA and cell proliferation assays, the IL-15 / IL-15Rα complex was analyzed. In relation to immune function in subjects who have not been administered a combination of anti-PD-1 antibody molecules Immune function in at least 99%, at least 95%, at least 90%, at 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%, and 35%, at least 30%, at least 25%, at least 20%, or at least To improve or induce by only 10%. In a specific embodiment, immune function is cytotoxic IL-emission (e.g., interferon-gamma, IL-2, IL-5, IL-10, IL-2) -12, or Transforming Growth Factor (TGF)-beta. One embodiment In this context, IL-15-mediated immune function is NK cell proliferation, which is, for example, in Fluorocarbons. Cells expressing NK cell markers (e.g., CD56) are assayed using tometry. The number can be detected. In one embodiment, IL-15-mediated immune function is CD8 + T Cell proliferation, which can be assayed, for example, by flow assay. Another implementation In this state, IL-15-mediated immune function is antibody production, which is, for example, ELISA. It can be assayed more effectively. In some embodiments, IL-15-mediated immune function It is an effector function, which is used, for example, in cytotoxicity assays or in the art. This can be assayed using other well-known assays. One or more peripheral blood lymphocyte counts One or more combinations of the above IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules The effects of the administration can be monitored / evaluated using standard techniques known to those skilled in the art. The number of peripheral blood lymphocytes in mammals can be determined, for example, by taking a peripheral blood sample from the mammal. For example, using Ficoll-Hypaque (Pharmacia) gradient centrifugation. Then, other components of peripheral blood, such as lymphocytes from plasma, are separated using trypan blue. This can be determined by counting lymphocytes. Peripheral blood T cells in mammals Cell count can be determined, for example, by gradient centrifugation using the Ficoll-Hypaque (Pharmacia) method. Using separation techniques, lymphocytes can be separated from other components of peripheral blood, such as plasma, and T cells. FITC or phycoeriform antigens, such as CD3, CD4, and CD8, are targeted to cytoplasmic antigens. T cells are labeled with an antibody conjugated to thrin, and the number of T cells is measured by FACS. This can be determined by measuring the following: Furthermore, a specific subset of T cells (for example) CD2 + CD4 + CD8 + CD4 + RO + CD8 + RO + CD4 + RA + , or CD8 + RA + ) or the effect on NK cells is a standard technique known to those skilled in the art. For example, this can be determined using FACS.

[0153] Plasma levels of IL-15 and / or PD-1 were measured using standard techniques known to those skilled in the art. It can be used for evaluation. For example, plasma can be obtained from a blood sample obtained from the subject. The levels of IL-15 and / or PD-1 in the plasma can be measured by ELISA. It is possible.

[0154] Combination therapy Other therapies that can be used in combination with IL-15 / IL-15Rα are also described in this disclosure. A method for preventing, treating, and / or managing cancer is provided. , IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule or IL-15 / IL-1 A composition containing a 5Rα complex and an anti-PD-1 antibody molecule is administered in an effective amount to a target that requires it. A method including the step of giving is provided herein. The term used herein is " "Cancer" refers to malignant growth or oncogene development, regardless of histopathological type or stage of invasion. This includes all types of tumors, metastatic tissues, or malignant transformed cells, tissues, or organs. This means that, in a specific embodiment, the IL-15 / IL-15Rα complex is the same It is administered subcutaneously in either a single repeated dose or a dose-escalation regimen. In the embodiment, the anti-PD-1 antibody molecule is administered intravenously in a homogeneous dosing regimen. It will be administered.

[0155] In specific embodiments, IL-15 / IL- Administration of a combination of the 15Rα complex and an anti-PD-1 antibody molecule may involve 1, 2, or 3 doses. It achieves the following results: (1) reduction of tumor or neoplasm growth; (2) tumor formation. (3) Reduction of primary, localized and / or metastatic cancer; (4) Eradication, removal or control of primary, localized and / or metastatic cancer; (5) Reduction of metastasis and spread; (6) Reduction of mortality; (7) Increase in survival rate; (8) Increase in survival time; 8) Increase in the number of patients in remission; (9) Decrease in hospitalization rate; (10) Decrease in length of hospital stay; and (11 ) The tumor size is only more than 10%, or only more than 8%, or only more than 6%, or more than 4%. The tumor size does not increase; preferably, the tumor size does not increase by more than 2%. Maintain.

[0156] In specific embodiments, subjects having cancer by the method described herein (partial implementation) Morphologically, the IL-15 / IL-15Rα complex and in animal models of cancer The administration of a combination of anti-PD-1 antibody molecules is performed using assays well known in the art. Measurements were taken, and subjects with cancer who were administered a negative control (in some embodiments, regarding cancer) Tumor growth is at least twice as large as tumor growth in the same animal model, preferably is at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least To inhibit or reduce by 7 times, or at least 10 times. In another embodiment, Subjects having cancer according to the method described herein (in some embodiments, the movement of cancer) Combination of IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule to a physical model The dose was measured using an assay well known in the art, with a negative control or I The L-15 / IL-15Rα complex or an anti-PD-1 antibody molecule was administered as a single agent. In subjects with cancer (in some embodiments, the same animal model for cancer) Tumor growth relative to ulcer growth is at least 25%, at least 30%, and at least 35%. , at least 40%, at least 45%, at least 50%, at least 55%, less 60%, at least 65%, at least 70%, at least 75%, at least 80% %, inhibiting at least 85%, at least 90%, or at least 95%, or Reduce.

[0157] Examples of cancerous complications include, but are not limited to, solid tumors, hematological malignancies, and soft tissue tumors. Examples include ulcers and metastatic lesions. Examples of solid tumors include malignant tumors of various organ systems. For example, sarcomas and carcinomas (e.g., adenocarcinoma and squamous cell carcinoma), such as liver, lung, and breast cancer. Auricular, lymphatic, gastrointestinal tract (e.g., colon), urogenital tract (e.g., renal cells, urothelial cells), anterior Examples include those affecting the prostate and pharynx. Adenocarcinomas are malignant tumors, such as most Examples include colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Squamous cell carcinoma can occur in, for example, the lungs, esophagus, skin, head and neck region, oral cavity, anus, and Examples include malignant tumors in the neck. In one embodiment, the cancer is melanoma, for example, advanced melanoma. It is a chromoma. Metastatic lesions of the above cancer can also be treated or It can be prevented.

[0158] The IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules disclosed herein An example of cancer in which growth can be inhibited using a combination of methods is typically immunotherapy. One example of a responsive cancer is melanoma. (For example, metastatic malignant melanoma), kidney cancer (for example, clear cell carcinoma), prostate cancer (for example, Examples include refractory prostate adenocarcinoma, breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Furthermore, refractory or recurrent malignancies can be cured using the combination therapies described herein. It can be treated.

[0159] Other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, and skin cancer. Or intraocular malignant melanoma, uterine cancer, ovarian cancer, kidney cancer, anal cancer, gastroesophageal cancer, stomach cancer, testicular cancer, uterine cancer Cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Merkel cell carcinoma Hodgkin lymphoma, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer Cancers of the parathyroid gland, adrenal gland, soft tissue sarcomas, urethral cancer, penile cancer, chronic or acute Leukemia, for example, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic 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 axis tumor, brainstem glioma, pituitary adenoma, capillary gland tumor Disarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, for example, asbestos-induced Examples include those that are induced (e.g., mesothelioma) and combinations of the aforementioned cancers.

[0160] In specific embodiments, the cancer is melanoma, kidney cancer, colon cancer, or prostate cancer. In another embodiment, the cancer is metastatic. In another embodiment, the subject undergoes an immune check. Point inhibitors (CPIs), such as anti-PD-1 / PD-L1 and anti-CTLA-4, were pioneers. It is being treated, responding, and progressing.

[0161] The combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule is one or more It is administered in combination with other therapeutic agents, such as anticancer drugs, cytokines, or antihormone agents, to treat cancer. They can be treated and / or managed. Non-exclusive examples of anticancer drugs are listed below.

[0162] In one embodiment, a defect in the target, for example, a high growth state or defect in the target (e.g.) A method for preventing, treating and / or managing cancer, wherein an anti-PD-1 antibody molecule is used. A method comprising the step of administering to an elephant is provided herein. In some embodiments, The PD-1 antibody molecule is administered in doses of approximately 200 mg to 500 mg, for example, approximately 250 mg to 450 mg. Approx. 300mg~400mg, approx. 250mg~350mg, approx. 350mg~450mg, or It is administered by injection (e.g. subcutaneously) in a dose of approximately 300 mg or approximately 400 mg (e.g., a uniform dose). It is administered intravenously. The administration schedule (e.g., a uniform dosing schedule) is, for example... The frequency may vary from once a week to once every two, three, four, five, or six weeks. In terms of administration, the anti-PD-1 antibody molecule is administered at a dose of approximately 300 mg to 400 mg once every three weeks. Alternatively, it is administered once every four weeks. In one embodiment, the anti-PD-1 antibody molecule is approximately 300 mg. It is administered once every three weeks at a dose of [amount]. In one embodiment, the anti-PD-1 antibody molecule is approximately 4 The drug is administered once every four weeks, starting at a dose of 00 mg. In one embodiment, an anti-PD-1 antibody molecule is used. It is administered once every four weeks at a dose starting from approximately 300 mg. In one embodiment, anti-PD-1 The antibody molecule is administered once every three weeks at a dose starting from approximately 400 mg.

[0163] According to the method described herein, the IL-15 / IL-15Rα complex is used in pharmaceutical compositions. It may be administered to the target. In a specific embodiment, the IL-15 / IL-15Rα complex is It is administered in combination with one or more other therapies, such as anti-PD-1 antibody molecules. Combination therapy This includes simultaneous and sequential administration of IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules. When used herein, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody The molecules are such that they occur on the same day, for example, simultaneously, or within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours. When administered to a patient at intervals of 1 hour, 6 hours, 7 hours, or 8 hours, simultaneously. It is said that the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule are If they are administered to a patient on different days, they are said to be administered consecutively, for example, I The L-15 / IL-15Rα complex and anti-PD-1 antibody molecule are administered at intervals of 1, 2, or 3 days. It may be administered. In the methods described herein, the administration of the IL-15 / IL-15Rα complex is It may precede or follow the administration of an anti-PD-1 antibody molecule. When administered simultaneously, IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule are present in the same pharmaceutical composition or in different pharmaceutical compositions. It may be present in the finished product.

[0164] The combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule is, for example, x Administered in conjunction with radiotherapy, including the use of ray, gamma ray, and other radiation sources, to destroy cancer cells. It can also be destroyed. In specific embodiments, radiation therapy involves external beam radiation or It is administered as remote therapy (radiation is directed from a remote source). In other embodiments, Radiation therapy is administered as medical therapy or close-range radiation therapy, and the radioactive source is used to target cancer cells or It is located inside the body, close to the tumor mass. IL-15 / IL-15Rα complex and anti-P The D-1 antibody molecule can also be administered in combination with chemotherapy. In one embodiment, The IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule are as described herein. It can be administered before, during, or after radiotherapy or chemotherapy. In one embodiment, a combination of an IL-15 / IL-15Rα complex and an anti-PD-1 antibody molecule. It can be administered before, during, or after surgery.

[0165] In some embodiments, the IL-15 / IL-15Rα complex and anti-PD-1 antibody are used. This combination is administered to subjects who have cancer or have been diagnosed with cancer. In other embodiments... Furthermore, the combination of the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule is associated with the development of cancer. It is administered to individuals who are prone to developing the disease or suspected of developing it.

[0166] In one embodiment, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule The combinations are: 0-6 months, 6-12 months, 1-5 years, 5-10 years, 10-15 years, 1 5-20 years old, 20-25 years old, 25-30 years old, 30-35 years old, 35-40 years old, 40-45 years old 45-50 years old, 50-55 years old, 55-60 years old, 60-65 years old, 65-70 years old, 70-7 5 years old, 75-80 years old, 80-85 years old, 85-90 years old, 90-95 years old, or 95-100 years old It is administered to the target of. In other embodiments, the IL-15 / IL-15Rα complex and The combination of anti-PD-1 antibody molecules is administered to adult humans. In one embodiment, IL- The combination of the 15 / IL-15Rα complex and the anti-PD-1 antibody molecule is used in surgery, chemotherapy, and and / or administered to subjects who are receiving, scheduled to receive, or have received radiation therapy. In some embodiments, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody are used. This combination is administered to refractory patients. In one embodiment, refractory patients are standard These are patients who are refractory to conventional anticancer therapy. In one embodiment, the cancer is not significantly eradicated, and If the symptoms are not significantly relieved, the patient with cancer is refractory to treatment. The determination of whether a patient is refractory or not is recognized in the relevant technical field under such circumstances. The term "refractory" in the sense of being used in the art for assaying the effectiveness of treatment This can be performed either in vivo or in vitro by any known method. In various embodiments, if cancerous tumors do not decrease or increase, patients with cancer It is refractory.

[0167] To treat patients exposed to specific toxins or pathogens using other methods of the present invention. Therefore, another aspect of the present invention is a method for treating an infectious disease in a subject, The combinations disclosed in the details, for example, the IL-15 / IL-15Rα complex and anti-PD-1 The combination of antibody molecules is administered to the target, and as a result, the target is treated for infectious diseases. This provides a method that includes the following.

[0168] In the treatment of infections (e.g., acute and / or chronic), IL-15 / IL-15R Administration of a combination of α-complex and anti-PD-1 antibody molecules enhances innate host immune defense against infection. In addition to stimulation, or as an alternative, it can be combined with conventional treatments. Natural host immune defenses are not limited to inflammation, fever, and antibody-mediated host defenses. , T lymphocyte-mediated host defense, for example, lymphokine secretion and cytotoxic T cells (especially During viral infection, complement-mediated lysis and opsonization (facilitation of phagocytosis), and phagocytosis The effects include: the ability of anti-PD-1 antibody molecules to reactivate dysfunctional T cells, chronic It is useful for treating infections, especially those in which cell-mediated immunity is crucial for complete recovery.

[0169] Antibody-mediated PD-1 blockade is an adjuvant for IL-15 / IL-15Rα complex administration. As a combination of the IL-15 / IL-15Rα complex and / or vaccine. It can act in a way that stimulates an immune response against pathogens, toxins, and autoantigens. Examples of pathogens for which a therapeutic approach may be particularly useful include those for which no effective vaccine currently exists. These include pathogens, or pathogens for which conventional vaccines are not completely effective. However, this does not limit the scope of treatment, but includes HIV, hepatitis (A, B, and C), and influenza. Genus (Influenza), Genus (Herpes), Genus (Giardia) a) Malaria, Leishmania, Yellow Staphylococcus aureus, Pseudomonas aeruginosa Monas Aeruginosa is one example. IL-15 / IL-15Rα complex Furthermore, PD-1 blockade stimulates the immune system, which presents modified antigens throughout the course of infection. These substances are particularly useful against established infections, such as those caused by HIV. The TOP is recognized as an exogenous substance at the time of treatment, and therefore, for example, via PD-1 This induces a robust T cell response that is not attenuated by negative signals.

[0170] Prevention and treatment of diseases, such as cancer, infectious diseases, lymphopenia, immunodeficiency and wounds. For the purpose of / or control, IL-15 / IL-15Rα complex and anti-PD-1 antibody molecule Other treatments that can be used in combination include, but are not limited to, small amounts Children, synthetic drugs, peptides (including cyclic peptides), polypeptides, proteins, nucleic acids ( For example, DNA and RNA nucleotides, as an example, but not limited to, Tycens nucleotide sequences, triple helices, RNAi, and biologically active proteins (nucleotide sequences encoding polypeptides or peptides), antibodies, synthetic or natural Examples include inorganic molecules, mimics, and synthetic or natural organic molecules. Examples of therapies, though not limited to them, include immunomodulatory agents (for example, interferan (e.g., ron), anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., vec), Lometasone, budesonide, flunisolide, fluticasone, triamcinolone, methylpre Donisolone, prednisolone, prednisone, hydrocortisone, glucocorticoids, Theroids, and nonsteroidal anti-inflammatory drugs (e.g., aspirin, ibuprofen, dipropyl alcohol). Clofenac and COX-2 inhibitors), analgesics, leukotriene antagonists (e.g.) For example, montelukast, methylxanthine, zafirlukast, and xyluten. Beta-2 agonists (e.g., albuterol, biterol, f Enoterol, isoetalie, metaproterenol, pirbuterol Roll, salbutamol, terbutalineformoterol, salmeterol, and salb Tamorterbutaline), anticholinergic agents (e.g., ipratropium bromide and obromide) Xytropium, sulfasalazine, penicillamine, dapsone, antihistamines, antihistamines Lariatic agents (e.g., hydroxychloroquine), antiviral agents (e.g., nucleoside analogs) Body (for example, zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridi) (Phoscarnet, trifluridine, and ribavirin), amantadine, rimanta (Zin, saquinavir, indinavir, ritonavir, and AZT) and antibiotics (for example) For example, dactinomycin (formerly actinomycin), bleomycin, erythromycin, Examples include penicillin, mitramycin, and anthramycin (AMC).

[0171] IL-15 function / signaling and / or / immune checkpoint modulation It is known to be useful in the prevention, management, and / or treatment of diseases affected by [the substance]. , or any treatment used or currently used for that purpose, IL-15 / To be used in combination with combination therapy of IL-15Rα complex and anti-PD-1 antibody molecules. This can be done. Diseases or disorders, such as cancer, infectious diseases, lymphopenia, immunodeficiency and wounds. Treatments used or currently used for the prevention, treatment, and / or management of wounds (e.g.) For example, regarding information on preventive or therapeutic agents, see, for instance, Gilman et al. .,Goodman and Gilman's: The Pharmacologic al Basis of Therapeutics,10th ed.,McGraw -Hill,New York,2001;The Merck Manual of Diagnosis and Therapy,Berkow,MDet al.( eds.),17th Ed.,Merck Sharp & Dohme Resea rch Laboratories, Rahway, NJ, 1999;Cecil T. extbook of Medicine,20th Ed.,Bennett and Plum (eds.), W.B. Saunders, Philadelphia, 19 96,and Physicians'Desk Reference(66th ed. See (2012).

[0172] In addition to combination therapy with IL-15 / IL-15Rα complex and anti-PD-1 antibody molecules, Non-limiting examples of one or more other treatments that can be used include immunomodulators, for example Examples include, but are not limited to, chemotherapy agents and non-chemotherapy immunomodulators. Non-specific examples of therapeutic agents include methotrexate, cyclosporine A, and leflunomide. cisplatin, ifosfamide, taxanes, for example, taxol and paclitaxol Lu, topoisomerase I inhibitors (e.g., CPT11, topotecan, 9AC, and GG) 211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5 -FU), leucovorin, vinorelbine, temodal, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, teniposide, vincristine, vinbrass Tin, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione Mitoxantrone, Mitramycin, Actinomycin D, 1-Dehydrotestosterone glucocorticoids, procaine, tetracaine, lidocaine, propranolol, Examples include puromycin homologs and cytoxanes.

[0173] biological activity In one embodiment, the IL-15 / IL-15Rα complex and / or anti-PD-1 antibody Examples of immune responses include antibody responses (humoral responses) or cellular immune responses, such as cytokine responses. It may be secretion (e.g., interferon-gamma), helper activity, or cytotoxicity. It increases the immune response. In one embodiment, the increase in the immune response is due to cytokine secretion, anti- This involves increased body production, effector function, T cell proliferation, and / or NK cell proliferation. Various assays for measuring such activity are well known in the art, For example, enzyme-linked immunosorbent assay (ELISA); for instance, Current Prot ocols in Immunology,Coligan et al.(eds.) See Section 2.1 of John Wiley and Sons, Inc., 1997. ), a "tetramer staining" assay for identifying antigen-specific T cells (Altman et al.) al., (1996), Science 274:94-96), mixed lymphocyte target Culture assays (e.g., Palladino et al., (1987), Cance (See r Res.47:5074-5079) and measure cytokine release in vitro. ELISPOT assays (e.g., Scheiben) can be used to measure bogen et al.,(1997),Int.J.Cancer 71:932- (See 936) is one example.

[0174] In some embodiments, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule The immune response induced or enhanced by the combination may be any method known in the art. Assayed using a negative control, or administered as a single agent, IL-15 / IL -15Rα complex or anti-PD-1 antibody molecules have less resistance to immune responses induced by these complexes. 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, or 12x Improved or increased. In one embodiment, the IL-15 / IL-15Rα complex The immune response induced by the combination of anti-PD-1 antibody molecules is known in the art. Assay by any method for at least the immune response induced by the negative control 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 50-100 times, at least 100-200 times, at least 200-300 times ...and it will be improved by at least 300-400 times or at least 400-500 times. In a typical embodiment, the assay used to evaluate the immune response involves antibody production, The level of itokine production or cytotoxicity is measured, and such assays are performed using the appropriate technology. It is well known in the field of technology. In some embodiments, it is used to measure the immune response. The assay used is an enzyme-linked immunosorbent assay that determines antibody or cytokine levels. ELISA), ELISPOT assay to determine cytokine release, or cellular cells Determine toxicity [ 51 This is a Cr release assay.

[0175] In a specific embodiment, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody The molecular combination is activated by Staphylococcus enterotoxin B (SEB) on whole blood. It increases IL-2 expression, for example, the IL-15 / IL-15Rα complex and anti-PD. -1 antibody molecules include IL-15 / IL-15Rα complex, anti-PD-1 antibody molecule, or iso Compared to IL-2 expression when a type control (e.g., IgG4) is used alone, Increases the expression of -2 by at least approximately 2, 3, 4, or 5 times. Additive or synergistic effect. The results showed that when the IL-15 / IL-15Rα complex was administered on the same day as the anti-PD-1 antibody molecule... The IL-15 / IL-15Rα complex was administered 72 hours after the administration of the anti-PD-1 antibody molecule. It was clearer than in the previous case.

[0176] In one embodiment, the IL-15 / IL-15Rα complex and the anti-PD-1 antibody molecule The proliferation or viability of cancer cells in contact with the combination is determined by assays well known in the art. For example, cell proliferation enhancement using CSFE, BrdU, and radioactive thymidine uptake. IL-15 / IL-15Rα was measured using a negative control or as a single agent. At least 2 double, preferably at least 2.5 times, at least 3 times, at least 4 times, at least 5 times , inhibited or reduced by at least 7 times, or at least 10 times. Or, Cell viability is determined by lactate dehydrogenase (LDH) and the stable cytoplasm released during cell lysis. By assays that measure enzymes, or during cell lysis [ 51 Measured by the emission of Cr This is possible. In another embodiment, the IL-15 / IL-15Rα complex and anti-PD are used. -1 The proliferation of cancer cells in contact with a combination of antibody molecules is a well-known process in the field of autoimmunity. i. For example, cell proliferation using CSFE, BrdU, and radioactive thymidine uptake. IL-15 / IL-15R was measured using a ssey as a negative control or single agent. At least 25% of cancer cells that came into contact with the α complex or anti-PD-1 antibody molecule were affected. 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 less At best, it is inhibited or reduced by 95%.

[0177] Cancer cell lines capable of performing such assays are well known to those skilled in the art. Necrosis, Perform apoptosis and proliferation assays on primary cells, for example, tissue explants. It's also possible.

[0178] In one embodiment, necrotic cells are colored with a dye, for example, neutral red, tripandromeda. - or ALAMAR (trademark) blue (Page et al., (1993), In (tl.J.of Oncology 3:473-476) Ability of cells to take in It is measured by this method. In such assays, cells are incubated in a dye-containing medium. The cells are then washed, and residual pigments reflecting the uptake of pigments by the cells are measured by spectrophotometric method. To measure. In another embodiment, the dye is sulfohodamine B (SRB), and the protein Its binding to the substance can be used as a measure of cytotoxicity (Skehan et al. l., (1990), J. Natl Cancer Inst. 82:1107-12) In yet another embodiment, a tetrazolium salt, for example MTT, is viable and dead. Quantitative assessment of mammalian cell survival and proliferation by detecting cells that have not been destroyed. Used in colorimetric assays (e.g., Mosmann, (1983), J. Immuno l.Methods 65:55-63).

[0179] In other embodiments, apoptotic cells adhere to and "float" the culture in the "suspension" compartment. Measurements are taken in both compartments. Both compartments are cleaned to remove the supernatant and trim the adherent cells. After psin treatment and a centrifugal washing step (10 minutes, 2000 rpm), both preparations It is recovered by combining. Tumor cell culture with sulindac and related compounds Protocols for treating substances to obtain a significant amount of apoptosis have been described in the literature. For example, Piazza et al., (1995) Cancer Research (See 55:3110-16). A feature of this method is that it collects both suspended and adherent cells. Identify the optimal treatment time and dose range for observing apoptosis, and determine the optimal cell culture conditions. This includes determining. In another embodiment, apoptosis is used to measure DNA fragmentation. It is quantified by a commercially available photometric method for quantitative in vitro determination of DNA fragmentation. The method is available. An example of such an assay is TUNEL (fragmented DNA). The ELISA-based assay detects the incorporation of labeled nucleotides within the sample. ochemica,(1999),no.2,pp.34-37(Roche Mole It is described in cular Biochemicals. This allows for morphological observation of apoptosis.

[0180] Details of one or more embodiments of this disclosure are set forth in the accompanying description above. Any method and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure. However, preferred methods and materials are described here. Other features, subjects, and advantages of this disclosure are described here. The points will become clear from the description and claims. This specification and attached claims Within the scope of [the subject], unless the context clearly indicates otherwise, the singular form refers to multiple objects. Includes. Unless otherwise defined, all scientific and technical terms used herein are defined in this disclosure. It has the same meaning as it is generally understood by those skilled in the art. All patents and publications are incorporated by reference as applicable unless otherwise indicated. The following examples are provided to better illustrate preferred embodiments of the present disclosure. These embodiments are the subject matter disclosed as defined by the attached claims. It should never be interpreted as limiting the scope.

[0181] Specific embodiments, citations, and references The present invention should not be limited in scope by the specific embodiments described herein. In addition to those described herein, various modifications of the present invention may be made as described in the detailed description above and in addition to the modifications described herein. Such modifications will be apparent to those skilled in the art from the attached drawings. Such modifications are within the scope of the attached claims. It shall fit within that.

[0182] Various references, such as patent applications, patents, and scientific publications, are cited herein. Used; the disclosures of each such reference are incorporated herein by reference as a whole. To be included.

[0183] [Table 5]

[0184] [Table 6]

[0185] [Table 7]

[0186] [Table 8]

[0187] [Table 9]

[0188] [Table 10]

[0189] [Table 11] [Examples]

[0190] Example 1: Production of hetIL-15 in CHO cell lines Using the Chinese hamster ovary (CHO) parent cell line CHO-MaKo, ("he It produced the IL-15 / IL-15Rα heterodimer (also known as tIL-15). The HO-MaKo cell line was developed using Zn finger nuclease (ZFN) technology, and CHO -Induced by targeted deletion of the matryptase gene within C8TD. Lipidases are involved in the degradation of various recombinant therapeutic proteins within CHO cells. It was discovered that CHO-C8TD is a parent cell line from WCB070625, CHO-K1PD. It was derived from a single vial. CHO-K1PD was initially obtained from ATCC. - Derived from the K1 cell line (catalog number CCL-61.3). CHO-MaKo cell line For further details, please refer to International Publication No. 2015 / 166427 (see Honmyo). It can be found in (which is referenced in the detailed text).

[0191] CHO-MaKo cells are subjected to electroporation (IL-15(interruption) Line encoding Ikin 15 and IL015Rα (interleukin 15 receptor α) Cotranslate pBW1703, which encodes the ionization vector pBW1697 and IL-15Rα. The cells were fected. After a 2-day recovery period, the transfected cell pool was treated with methotrexate (M Recombinant cells were selected after being cultured for several weeks in low-folate medium supplemented with IL- To increase the expression of 15, the harvested cell pool was subjected to electroporation. The linearized vector pBW1916, which encodes IL-15, was transfected on the 2nd. After the recovery period, the transfected cell pool was treated with methotrexate (MTX) and Puroma Culturing the cells in a low-folate medium supplemented with isin for several weeks, selecting recombinant cells, and then Single cells were sorted by FACS. The selected clones were then evaluated for bioreactor performance. mRNA size and integrity (by Northern blotting), transgene copy number (qP (CR), size and completeness of the expression cassette (by Southern blotting), and sequence validation. Further characteristics were provided regarding (NGS).

[0192] The vector maps for pBW1697, pBW1703, and pBW1916 are shown in Figure 1. Table 3 provides an overview of the final expression constructs and expression cassettes.

[0193] As shown in Table 1, two different IL-15 expression cassettes were used: pBW1697 This is a 29aa natural signal peptide (SP) that precedes the 114aa IL-15 chain. ) and the natural propeptide sequence of 19aa within an open reading frame (ORF) Include. In pBW1916, the IL-15 chain is the so-called UTR12 signal peptide ( Combine with UTR12 SP.

[0194] IL-15Rα was also expressed from two different ORFs. In pBW1697, The full-length receptor (IL-15Rα FL) is used with its natural signal peptide (its own SP) They are used together. In pBW1703, the soluble version of IL-15Rα is used in UTR12 It is activated together with SP.

[0195] [Table 12]

[0196] Example 2. Production of IL-15 / IL-15Rα heterodimer IL-15 / IL-15Rα was added to recombinant Chinese hamster ovary (CHO) cell lines. It produces more. Production is done using a standard fed-batch production process in a bioreactor. implement.

[0197] Thaw one frozen vial from the master cell bank and suspend it in expanding medium. The inoculum volume is increased by passing the culture in a shaking flask. Inoculum volume and viable cell density. When the density is sufficiently high (when the live cell density is approximately 4.8 × 10⁻⁶ 6 cells / mL; viability >90%) Transfer the inoculation material to the first seed reactor.

[0198] Transfer the inoculum from the previous step to the first seed bioreactor containing the expansion medium. Then, culture further in batch mode. When the viable cell density is sufficient (when the viable cell density is approximately 5. 4 x 10 6 Use the culture (individual cells / mL) and inoculate it into the second seed bioreactor. ru.

[0199] Cultures from the first seed bioreactor are transferred to a second seed bioreactor containing expansion medium. Transfer to an Oreactor and culture further in batch mode. When the live cell density is sufficient (live Cell density is approximately 5.6 × 10 6 Using individual cells / mL, cultures are produced in a bioreactor. It will be administered.

[0200] Operate the production bioreactor in fed-batch mode. Then, transfer to a production bioreactor containing the production medium. The supply of the two feed solutions is via This is done throughout the entire run of the Oreactor. Both feed additions are approximately 2 × 10 6 Individual details Start with a live cell density of cells / mL, approximately 12.5 × 10⁶. 6 With a live cell density of individual cells / mL, The culture temperature is changed from 36.5°C to 33.0°C. When cell viability decreases to ≤75% Collection begins either at or approximately 14 days after the start of production. Bioburden and exogenous substances Therefore, each lot of bulk collectibles will be monitored.

[0201] The purification process involves two steps that inactivate / remove viruses, namely low pH ink. This includes filtration and nanofiltration. Finally, the product is concentrated in the final buffer and diaphytes. To retrace

[0202] Example 3. Determination of O-glycan composition Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) revealed that O-g Lycan was analyzed. For this purpose, O-linked glycans were removed by reductive β-removal. By the beta-elimination method, proteins are chemically removed. The material is cleaved and derivatized by permethylation before MS detection. Based on the MS data, identification and semi-transferralization are performed. Generate quantitative results. Identify and compare the relative existence of the major glycan species within all five batches. The quantities are summarized in Table 4.

[0203] Figure 6 illustrates the distribution of various species in the form of a bar chart.

[0204] Significant differences and distributions in O-glycan mutants were observed in hetIL-15 mutants from different cell lines. Observed between batches. The HEK293 batch has two core type variants (C2G, C2S1 It contains approximately 50% of C2GS1 and C2GS2. These are exclusively from CHO-derived batches. It is detected at a trace level in the CHO batch compared to the HEK293 batch. Higher levels of core 1 monosialylation mutant (C1S1) were detected (approximately 50% each). (Approximately 15%). The overall level of sialylation in all batches is very high (>97%). Due to the observed differences, batches derived from HEK293 and CHO cells are one of them. It is considered that these are not equivalent to typical O-glycan compositions.

[0205] Figure 7 shows the basic structure of the Core 1 type O-glycan ("C1"). Other glycans Extensions with n residues (e.g., sialic acid) result in more complex structures, as listed in Table 4 (e.g., For example, C1S1 and C1S2 are obtained. The structural motif of core 1 is all analyzed. It is present in the batch. Figure 8 below shows the basic O-glycan type ("C2") of Core 2 type. The structure is shown. Further extensions with other glycan residues result in more complex structures, as listed in Table 4. (For example, C2S1, C2GS1) can be obtained. This structure is limited to HEK293 batches. It has been detected at a significant level.

[0206] [Table 13]

[0207] O-glycans were further characterized in terms of the sialic acid binding structure. The β-removal method was used to further characterize the glycans. After chemical cleavage from the protein, derivatization was carried out by ethyl esterification. MS analysis of O-linked glycans after glycation revealed information about sialic acid binding (α2,3 or α2 6) was obtained, but this was done purely on a qualitative basis.

[0208] Both HEK293 and CHO batches have two types of sialic acid bonds (α2,3 and α2). This shows α2,6). This is a protein expressed in human cells (HEK293). This is conceivable, but it is not common for proteins expressed within CHO cells. Figure 9 shows the mass spectrum of CHO clinical batch BC0001. α2,6-bound cells The relative strength of alic acid is rather low compared to mutants with a more dominant α2,3-linked sialic acid. .

[0209] The properties and content of sialic acid were evaluated by reverse-phase chromatography. Chemical cleavage and After fluorescent labeling, sialic acid was separated on a column by gradient elution and quantified by fluorescence detection.

[0210] N-acetylneuraminic acid is the major sialic acid in human glycoproteins. Glycolylneuraminic acid has been found in non-human glycoproteins and is undesirable. Therefore, a high ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid is preferred. It's nice.

[0211] The HEK293 batch shows a very high ratio. N-glycolyl neutrons in these batches The amount of laminate can be considered negligible.

[0212] The CHO batch shows a high ratio, comparable to over 200. This is less than the desirable 0.5%. This indicates the absence of N-glycolylneuraminic acid in these samples.

[0213] Example 4. Determination of N-glycan composition N-glycans are enzymatically cleaved from hetIL-15 heterodimers, and RapiFlu The product was fluorescently labeled and derivatized with a tertiary amine using the (trademark) technology. After purification, the labeled N was used. - Glycans are analyzed using HILIC (Hydrophilic Interaction) with fluorescence detection associated with MS (Mass Spectrometry). Analysis was performed using liquid chromatography.

[0214] The chromatogram in Figure 10 shows a batch derived from one HEK293 and one CHO The overlay of the originating batches is shown. Between CHO batches from two different cell lines, N- Significant differences and distributions are observed in the glycan population. The major N-glycan species differ, H While the characteristics of the EK293 batch show a heterogeneous distribution, the characteristics of the CHO batch are more uniform. ru.

[0215] Table 5 shows the identity and relative peak area of ​​the major N-glycan species in all five batches. To summarize.

[0216] The chromatogram in Figure 11 shows an overlay of three HEK293 batches. The overall sugar patterns and order of major morphologies in each batch are similar, and the observed differences are assumed to be due to... This is within the range of batch-to-batch variability. The main species is (FA2B / F) related to galactosylation. A2 / FA3 / FA4) On the other hand, high levels of sialylation were observed. Numerous diverse sialylation Low levels of lylated glycans were detected. Defucosylated and high-mannose glycans, etc. No species with potential effects on pharmacokinetics or immunogenicity were detected.

[0217] The chromatogram in Figure 12 shows an overlay of two CHO batches. The sugar patterns are very similar. Two major sialylated species (FA2G2S2 and FA2G2S 1) contributes to approximately 60% of the N-glycan population. Defucosylation and high mannose glycans No species with potential effects on pharmacokinetics or immunogenicity were detected.

[0218] [Table 14]

[0219] Example 5. IL-15 / IL-15Rα complex alone or in adults with metastatic cancer Phase I / Ib trial on combination with anti-PD-1 antibody molecule This embodiment describes a solid tumor or lymphoma, either alone or in combination with an anti-PD-1 antibody molecule. Subcutaneous (SC) recombinant heterozygous cells produced by CHO cell lines are administered to human patients. Safety and tolerability of dimeric IL-15 / soluble IL-15Rα complex (hetIL-15) This section describes the tests used to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD).

[0220] the purpose The objective of this Phase I / Ib trial is to evaluate the heterodimer IL- produced by the CHO cell line. 15 / Soluble IL-15Rα complex (hetIL-15) (“CHO hetIL-15 The objective is to determine the safety characteristics of (referred to as ") and to ensure that it is safe with anti-PD-1 antibodies. If combinations are possible, determine the appropriate dosage and schedule for further testing. The goal is to determine the pharmacokinetic properties of CHO hetIL-15 as a monotherapy agent. Furthermore, characterizing it in combination with anti-PD-1 antibodies and identifying preliminary antitumor activity. It will become.

[0221] The primary objective is to treat advanced solid tumors that have previously responded to immune checkpoint inhibitors (CPIs). In patients with ulcers and lymphomas (secondary resistance patients), the safety of CHO hetIL-15 The goal is to characterize the overall suitability and tolerability of the drug as a monotherapy and in combination with an anti-PD-1 antibody.

[0222] The secondary objective is 1) to assess the preliminary antitumor activity of CHO hetIL-15 and anti-PD-1 antibodies. Evaluate 2) CHO hetIL as a monotherapy and in combination with an anti-PD-1 antibody; and 2) evaluate CHO hetIL as a monotherapy and in combination with an anti-PD-1 antibody. The objective is to characterize the pharmacokinetics (PK) of -15 and the PK of anti-PD-1 antibodies.

[0223] Test design This is an advanced stage of solid disease that progresses after obtaining a prior response to anti-PD-1 / CPI therapy. In subjects with cytoplasmic tumors and lymphomas, subcutaneous administration of CHO hetIL-15 Phase I / Ib open-label global multicenter studies of the drug alone and in combination with anti-PD-1 antibodies. This is a trial. The prior response is defined as complete remission (CR) or partial remission (PR) as measured by radiography. In addition, if the most recent regimen included CPI, the patient had stable disease (SD) lasting for ≥6 months. This also includes the subjects to be targeted. The trial consists of two parts: dose escalation and dose expansion. The selected group will be tested during the dose-reducing phase: 1) Evaluation of CHO hetIL-15 as a monotherapy agent. (Anti-PD-1 antibody may be added in the anti-PD-1 antibody for the first disease reassessment), and 2) CHO hetIL-15 and anti-PD-1 antibody as a combination starting from C1D1 Administration of [the substance].

[0224] Patient group The trial was conducted after prior treatment with CPI (anti-PD-1 / PD-L1 and / or anti-CTLA-4). The test will be administered to male and female patients aged 18 years or older who have responded earlier and are progressing. If the most recent regimen included CPI, then CR / PR (confirmation) of initial X-ray examinations lasting ≥ 6 months. Targeted scan is not required) or SD. During dose escalation, the study is conducted on advanced solid tumors and The study will be conducted in patients with pyelodysplasia. During expansion, the trial will be conducted in patients with melanoma. It will be implemented there.

[0225] Key inclusion criteria 1. Male or female patient ≥ 18 years old 2. If there is recorded progression after standard treatment, or if the principal investigator believes it is appropriate to treat the disease. Histologically confirmed and documented advanced solid tumors in cases where no urgent standard treatment exists. and lymphoma (locally advanced and not curable by surgery or radiation therapy, (Including those with metastatic disease)

[0226] Gradual dose increase: First treated with CPI (anti-PD-1 / PD-L1 and / or anti-CTLA-4), Patients who have responded earlier and are progressing. Prior response is defined as the most recent regimen including CPI, ≥ Initial X-ray examinations over a 6-month period show CR / PR (confirmatory scan not required) or SD. .

[0227] Expansion: First treated with CPI (anti-PD-1 / PD-L1 and / or anti-CTLA-4), Patients with melanoma that have responded earlier and are progressing. The prior response is that the most recent regimen was C If PI is included, CR / PR of X-ray examinations lasting ≥6 months (confirmatory scan is not required). ) or SD.

[0228] Key Exclusion Criteria 1. Patients receiving any prior IL-15 treatment. 2. Severe hypersensitivity to any component of the test drug and / or other mAbs and / or their excipients. History of symptomatic reactions 3. Exclude patients with primary CNS tumors. Symptomatic CNS metastases or focal CNS Targeted treatment (such as radiation therapy or surgery), or increased dose of corticosteroids two weeks prior to clinical trial enrollment. The presence of CNS metastases requiring id. Patients with treated symptomatic brain metastases are neurological The patient must be stable (4 weeks after treatment and before clinical trial registration), and the administration of any investigational drug is required. For at least the previous two weeks, the dose of prednisone or its equivalent was ≤10 mg / day. 4. Systemic chronic steroid therapy (>10 mg / ) within 7 days of the first dose of the investigational drug. Prednisone or equivalent per day, or any other supplemental dose other than steroids in the event of adrenal insufficiency. Immunosuppressive therapy is available. Topical, inhaled, nasal, and ophthalmic steroids are possible. 5. Malignant diseases other than those being treated in this study. Exceptions include basal cell carcinoma of the skin or skin Squamous cell carcinoma (potentially receiving curative treatment) or in-situ cervical cancer or average life expectancy Other tumors that are not affected are mentioned.

[0229] Effectiveness evaluation Tumor evaluation is based on Seymour et al. (2017) Lanc for lymphoma. et Oncol;18:e143-e152 and Cheson et al (2014 )As described in J.Clin.Oncol.32(27):3059-67 Follow RECIST 1.1, the iRECIST for solid tumors. At screening, treatment Imaging within 28 days of the start (from day -28 to day -1 prior to day 1 of cycle 1) An assessment will be conducted. All subjects will undergo composite imaging with IV contrast of the chest, abdomen, and pelvis. Undergo a CT scan. If there is clinical evidence of a disorder in the neck, CT with IV contrast will also be performed. In patients with known CNS disease, Brain imaging via line imaging is necessary. Magnetic resonance imaging (MRI) is different from CT. Therefore, it is necessary to use it to evaluate diseased areas that have not been adequately imaged. If the patient is intolerant to contrast agents, CT may be performed without contrast. IV Contrast If a CT scan without CT is insufficient, MRI may be used to evaluate the site of the disease. i. Visible skin lesions and easily palpable tumors should be examined using a ruler or caliper during a physical examination. Measurements may be taken by examination, and color photographs may be taken. Ultrasound is used to detect the response in the diseased area. It should not be used to measure position.

[0230] Results Evaluation Pharmacokinetics were observed at baseline and treatment, with CHO hetIL-15 and anti-PD-1 inhibitors. The condition is evaluated based on the body's serum concentration. CHO hetIL-15 as a monotherapy and in combination with an anti-PD-1 antibody in a cyclical therapy. Incidence of dose-limiting toxicity (DLT) in 1 (28 days) Harmful effects include changes in laboratory parameters, vital signs, and electrocardiogram (ECG). Incidence and severity of adverse events (AEs) and serious adverse events (SAEs)

[0231] Other evaluation criteria include: 1. Changes in the number and cytotoxic activity of tumor-infiltrating CD8+ T cells and NK cells, and Evaluation of changes in the abundance of T cell clones. 2. Sequencing of tumor biopsies at baseline and during treatment. PD- Evaluation of L1 expression and CD8+ T cells. 3. Gene expression profiling in baseline and treatment-time tumor samples 4. DNA of circulating free tumor DNA from baseline and treatment-time plasma samples. Sequence determination 5. Activation / proliferation markers, checkpoints on T cell and NK cell subsets in the blood. Baseline levels and / or expression of soluble immunoassay inhibitors and soluble immunoassay factors in plasma Evaluation of their changes

[0232] Investigational drug Dosage gradual increase and dose expansion Patients are treated with CHO hetIL-15 as a monotherapy and in combination with an anti-PD-1 antibody. Treatment continues until MTD is achieved or a lower RD is established in the combination. Dose escalation is a future challenge in the study, as it controls the risk of dose-limiting toxicity (DLT). To manage this, it will be guided by BHLRM, which has been adapted to follow the EWOC principles. During the dose escalation phase, evaluate the two separate dosing groups: Group 1) CHO hetIL-15 monotherapy (Target subjects are those who have undergone re-evaluation of their first disease and are anti-PD- (1) This makes it possible to initiate antibody therapy. Group 2) CHO hetIL-15 in combination with anti-PD-1 antibody, initiated at C1D1 .

[0233] Figure 14 provides the provisional dose levels to be evaluated. During the course of the trial, additional and / or Intermittent dose levels can be added. Furthermore, alternative administration of hetIL-15 Schedule, for example, hetIL-15 once or twice a week during the first two weeks of the cycle. The administration of this drug can be evaluated. The cohort was able to follow up at any dose level below the MTD. In addition, safety, PK, and / or PD can be better understood.

[0234] The treatment period begins on day 1 of cycle 1 (C1D1). Each treatment cycle lasts 28 days. CHO hetIL-15 is administered subcutaneously once a week, with a 3-week on / 1-week off cycle. When PD-1 antibodies are administered, a fixed dose of 400 mg is given once on day 1 of each cycle. This is administered intravenously.

[0235] In this study, CHO hetIL-15 alone or anti-PD-1 inhibitors were administered to subjects enrolled in the study. The starting dose when combined with other medications is 2 μg / kg, following a 3-week on / 1-week off schedule. The treatment is administered subcutaneously (SC) once a week. The starting dose of anti-PD-1 antibody is 400 mg Q4W (intermittent administration). It will be intravascular. Alternative administration schedules may be considered, and any new schedule may be... To implement this, the protocol will need to be modified.

[0236] In preclinical models, IL-15 therapy alone induces IFN-γ and exhibits antitumor effects. However, this antitumor activity is limited due to the upregulation of PD-1 on CD8+ T cells. There are also cases where the addition of checkpoint inhibitors, such as PD-1 and CTLA-4 inhibitors, is effective. As a result, IFN-γ expression is further enhanced, and PD-1 expression on CD8+ T cells is reduced. This leads to increased survival (Yu et al (2010) Clin Cancer) Res;16(24):6019-28,Yu et al(2012)Proc Na tl Acad Sci USA;109(16):6187-92). Furthermore, preliminary clinical Floor data indicates that subjects responding to CPI have higher baseline levels of IL-15. This indicates that CD8+T is present in melanoma, NSCLC, and breast cancer. Cellular tumor infiltration correlates with higher levels of IL-15 and NK gene signatures. Therefore, CHO hetIL-15, IL-15 agonist and anti-PD-1 antibody, PD-1 inhibitor The combination with harmful agents shows a response particularly quickly to checkpoint inhibitors, followed by relapses. In the target population, synergistic effects may occur, further enhancing the antitumor response.

[0237] Additional drugs may be used in combination with CHO hetIL-15± anti-PD-1 antibody. Other efficacy may be considered in the expanded portion of the study. These additional combined regimes The efficacy and / or effects will only be considered if future protocol modifications are made to this study. That will happen.

[0238] The dose expansion portion of the study involves the MTD and / or RD being combined with the anti-PD-1 antibody. When explicitly stated in HO hetIL-15, it will begin. Primary objective of the expansion group This further enhances the safety and tolerability of CHO hetIL-15 when combined with an anti-PD-1 antibody. The objective is to evaluate in the following group. The secondary goal of the expanded group is to evaluate in patients with recurrent melanoma due to CPI. By evaluating the antitumor activity of CHO hetIL-15 in combination with an anti-PD-1 antibody, be.

[0239] Provisional dose level Tables 6 and 7 show the baseline in CHO hetIL-15 that may be evaluated during this study. The dosage and provisional dose levels are listed. The anti-PD-1 antibody dose is fixed at 400 mg Q4W. Determined. With the exception of starting dose level 1, the practical dose levels are determined based on available toxicity, pharmacokinetics, and The decision will be based on pharmacodynamic data.

[0240] [Table 15]

[0241] [Table 16]

[0242] Definition of dose-limiting toxicity The dose-limiting toxicity (DLT) is CHO hetIL-15 or CHO he as a monotherapy agent. The relationship with the tIL-15 / anti-PD-1 combination cannot be ruled out, and is primarily related to disease. Within the DLT period (28 days) if it is not related to the disease, disease progression, comorbidities, or concomitant medications. This is defined as an adverse event or abnormal clinical laboratory value that occurs. Table 8 lists the criteria for DLT. For all categories, the National Cancer Institute The General Terminology Standards for Adverse Events (NCI CTCAE) version 5.0 is used. Yes. The DLT will be considered and included in the BHLRM for the purpose of determining dose escalation.

[0243] [Table 17]

[0244] biomarkers Using biomarker analysis, CHO hetIL-15 was identified at the molecular and cellular levels. The efficacy of the drug or in combination with an anti-PD-1 antibody will be investigated, as well as the marker. This will determine how the changes relate to exposure and clinical outcomes. Furthermore, it will be effective Potential sex predictor markers, as well as resistance mechanisms to hetIL-15, will also be investigated. do.

[0245] [Table 18]

[0246] [Table 19]

[0247] [Table 20]

[0248] CHO hetIL-15 promotes NK and CD8+ T cells and enhances anti-tumor immunity. Combination therapy with CHO hetIL-15 and anti-PD-1 antibodies is expected to inhibit tumor growth. It is thought that...

[0249] hetIL-15 produced by CHO cells in Example 6 was converted by HEK293. Compared to the produced hetIL-15, the IL-15Rα chain C-terminal splicing mutant It does not have.

[0250] Size-based heterogeneity of HEK293 and CHO hetIL-15, Chromatography, SDS-PAGE (natural and reduced conditions), SEC, RP-HPLC and quality The comparison was made using quantitative analysis.

[0251] As shown in Figure 15, the chromatographic characteristics of the HEK293 batch and the CHO batch are as follows: It is different from the intended meaning. The HEK293 batch is a splicing variant that is absent in the CHO batch. The presence of this leads to a double IL-15Rα peak. The location of the splicing mutant is, It is located within the C-terminal region of the IL-15 receptor peak. HEK293 and CHO batch are Distribution of the four IL-15 peaks and the peak area ratio of IL-15Rα to IL-15. They are very similar. The peak area ratio was monitored for the stoichiometry of IL-15Rα and IL-15: 2. The ratio of 0 corresponds to a 1:1 molar ratio of the two chains.

[0252] Splicing variants of the IL-15Rα chain are shown in peptide mapping: [ka] HEK293 batch consists of 159 residues (I1-G159) (shown above) determined by [method]. It was detected only in Chi.

[0253] The examples and embodiments described herein are for illustrative purposes only, and various interpretations based thereon may be found. Such modifications or changes are highly suggestive to those skilled in the art, and the spirit and scope of this application and the attached claims It is understood that it should be included within the scope of [the specified range].

[0254] Furthermore, when the features or embodiments of the present invention are described in terms of the Markush Group, The person who made this invention also has the right to any individual member of the Markush Group or any member of the group You will understand that this will be explained from the perspective of the group.

[0255] All publications, patent applications, patents, and other references mentioned herein are, in their respective terms and conditions. To the same extent as when they are individually referenced, they as a whole (or according to the contextual indication) ) Explicitly invoked by reference. In case of any inconsistency, this Specified Ordinance, including its definitions, shall not be subject to change. It will become that.

Claims

1. Human interleukin-15 (IL-15) polypeptide and human interleukin-15 A polypeptide complex containing receptor α(IL-15Rα) polypeptide, FA2G 2, FA2G2S1, FA2G2S2, FA3G3S1, FA2F1G2S2, FA3G A polypeptide complex containing N-linked glycans including 2S2 and FA3G3S3.

2. The IL-15 polypeptide has the sequence of SEQ ID NO: 1 or 5, and IL-15Rα is The polypeptide according to claim 1, having the sequence of sequence numbers 6, 7, 10, 12, 14, or 21 Tydo complex.

3. The N-linked glycan is at least 10%, 12.5%, and 15% of FA2G2S1. The polypeptide complex according to claim 1, comprising 17.5%, 20%, or 22.5%.

4. The N-linked glycan is present in an amount of at least 10%, 20%, or 30% of FA2G2S2, The polypeptide complex according to claim 1, comprising 40%.

5. Human interleukin-15 (IL-15) polypeptide and human interleukin-15 A polypeptide complex comprising receptor α (IL-15Rα) polypeptide, wherein O-binding It contains a type glycan, and at least 80%, 85%, 90%, or 95% of the glycan is Core-1 is a polypeptide complex, which is an O-linked glycan.

6. The IL-15 polypeptide has the sequence of SEQ ID NO: 1 or 5, and IL-15Rα is The polypeptide according to claim 5, having sequence number 6, 7, 10, 12, 14, or 21 Tydo complex.

7. The core-1 O-linked glycan is monosialylated and / or diciallylated. The polypeptide complex according to claim 5.

8. Human interleukin-15 (IL-15) polypeptide and human interleukin-15 A polypeptide complex comprising receptor α (IL-15Rα) polypeptide, It contains O-linked glycans, and at least 80%, 85%, 90% of the glycans or 95% of them have a core-1 O-linked glycan structure; and FA2G2, FA2G2S1, FA2G2S2, FA3G3S1, FA2F1G2S2 , comprising N-linked glycans including FA3G2S2 and FA3G3S3, Polypeptide complex.

9. The IL-15 polypeptide has the sequence of SEQ ID NO: 1 or 5, and IL-15Rα is The polypeptide according to claim 8, having sequence number 6, 7, 10, 12, 14, or 21 Tydo complex.

10. The N-linked glycan is at least 10%, 12.5%, and 15% of FA2G2S1. The polypeptide complex according to claim 8, comprising 17.5%, 20%, or 22.5%.

11. The N-linked glycan is present in an amount of at least 10%, 20%, or 30% of FA2G2S2, The polypeptide complex according to claim 8, comprising 40%.

12. The aforementioned core-1 O-linked glycan is mainly monosiallylated and / or disiallylated. The polypeptide complex according to claim 8.

13. It was an isolated IL-15 / IL-15Rα heterodimer produced in non-human cells. Therefore, isolated IL-15 / IL-15Rα containing α(2,6)O-bonded sialylation Heterodimer.

14. The non-human cells are recombinant Chinese hamster ovary (CHO) cells, according to the claim. The isolated IL-15 / IL-15Rα heterodimer described in 13.

15. The CHO cells are modified to impair the function of matryptase, as described in claim 14. The isolated IL-15 / IL-15Rα heterodimer shown.

16. It contains O-linked glycans, and at least 80%, 85%, 90% of the glycans or The isolated IL according to claim 13 is 95% core-1 O-linked glycan. -15 / IL-15Rα heterodimer.

17. Claim 1, wherein approximately 15% of the O-glycan has α(2,6)-linked sialylation. The isolated IL-15 / IL-15Rα heterodimer described in 6.

18. The polypeptide complex or isolated IL-15 / IL-15Rα according to claim 13 A pharmaceutical composition containing a telodimer.

19. The pharmaceutical composition according to claim 18, further comprising a pharmaceutically acceptable carrier.

20. Human interleukin-15 (IL-15) polypeptide and human interleukin-15 Non-human cells containing nucleic acids encoding receptor α(IL-15Rα) polypeptide, The IL-15 and IL-15Rα expressed by the aforementioned cells form a heterodimer. Furthermore, the heterodimer contains α(2,6)-linked sialylation in non-human cells.

21. Recombinant Chinese hamster ovary (CHO) cells, as described in claim 20, non-human cell.

22. The CHO cells are modified to impair the function of matryptase, as described in claim 20. Non-human cells.

23. A method for treating cancer, wherein the pharmaceutical composition described in claim 18 is used in the treatment of cancer. A method including the step of administering to an elephant.

24. A method for producing cells expressing IL-15 / IL-15α heterodimers, (a) the step of preparing non-human cells; (b) The non-human cells are given a first vector encoding both IL-15Rα and IL-15. - and two vectors simultaneously, including a second vector that encodes a portion of IL-15Rα. The steps include transfecting and culturing the transfected cells; (c) Translate the cells from step b) with a third vector encoding IL-15. The steps include: sfecting and culturing the transfected cells; (d) Isolate individual clones expressing IL-15 / IL-15α heterodimers. Top, A method that includes this.

25. The non-human cells are recombinant Chinese hamster ovary (CHO) cells, according to the claim. Method 24.

26. The CHO cells are modified in such a way that the function of the matryptase gene is impaired, The method described in item 25.

27. The IL-15Rα has the sequence of sequence numbers 6, 12, or 14, as described in claim 24. The method.

28. The method according to claim 24, wherein IL-15 has an array of sequence number 1 or 5.

29. The portion of IL-15Rα is the soluble portion of IL-15Rα, as described in claim 24. The method.

30. The claim states that the soluble portion of IL-15Rα has the sequence of SEQ ID NOs: 7, 10, or 21. The method described in item 29.

31. A method for producing IL-15 / IL-15Rα heterodimers, (a) The cells produced according to claim 24 are IL-15 / IL-15Rα heterodiplex Cultivated under conditions that enable the expression of the mer and the secretion of the IL-15 / IL-15Rα heterodimer. Steps to nourish, (b) The IL-15 / IL-15α heterodimer is isolated from the cell culture. Top and, A method that includes this.

32. Human interleukin-15 (IL-15) polypeptide and human interleukin-15 A polypeptide complex comprising receptor α(IL-15Rα) polypeptide, wherein recombinant Produced by Chinese hamster ovary (CHO) cells, and IL-15Rα chain sprung A polypeptide complex that does not contain rising variants.

33. The CHO cells are modified to impair the function of matryptase, as described in claim 32. A polypeptide complex.

34. The aforementioned IL-15Rα chain splicing mutant has 159 residues ranging from I1 to G159. A polypeptide complex according to claim 32, including the polypeptide complex described in claim 32.

Images