Membrane-anchored il-2 and use thereof

Abstract

The present invention addresses the problem of sustaining IL-2 signaling in the body after the administration of immune cells. In the present invention, a molecule comprising Interleukin-2 (IL-2)-polyethylene glycol (PEG)-A is used. The A is a moiety for anchoring to a cell membrane, and contains a group that can form a bond with a phospholipid moiety or a protein on the cell membrane. The PEG moiety preferably has a molecular weight of 3,000-8,000. Such a molecule is used for the modification of immune cells with IL-2. Examples of the immune cells are activated NK cells and NK-like cells.

Description

Membrane-anchored IL-2 and its uses

[0001] The present invention relates to a cell membrane-anchored interleukin-2 (IL-2) and its use. The present invention is useful for enhancing the function of immune cells and in fields such as cell therapy using immune cells.

[0002] Interleukin-2 (IL-2) binds to IL-2 receptors present on the surface of cells such as T cells, B cells, and NK cells, transmitting signals into the cells, activating and promoting their proliferation. IL-2 is also known to have antitumor effects and is used as an active ingredient in pharmaceuticals for the treatment of certain cancers. However, IL-2 is not very stable in vivo. For example, when a recombinant IL-2 preparation (generic name: teceleukin (recombinant)) was administered intravenously at a constant rate of 700,000 units over a precise two-hour period to four adult patients with malignant tumors, serum concentrations peaked at the end of administration but returned to baseline within several hours (Non-Patent Document 1).

[0003] Non-Patent Document 2 reports that instead of administering IL-2, RMA T lymphoma cells bearing IL-2 on their surface were prepared using the diphtheria toxin transmembrane domain (T) as a membrane anchor, which induced a protective antitumor immune response in mice. Non-Patent Document 3 reports on a new membrane-bound protein (MBP) technology, reporting that MBP NK cells, in which IL-2 was expressed on the surface of NK-92 cells, not only showed improved proliferation under IL-2-deficient conditions but also exhibited strong secretion of cytolytic granules, leading to enhanced antitumor activity both in vitro and in vivo.

[0004] On the other hand, poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives are known to spontaneously incorporate into lipid bilayers. For example, Non-Patent Document 4 investigated the interaction between fluorescein isothiocyanate-labeled PEG-lipids and cells and reported that FITC-PEG-lipids spontaneously anchored to the cell membrane without losing cell viability. Non-Patent Document 5 also reported how the density and molecular weight of PEG molecules affect the immobilization and cellular uptake of bioactive substances.

[0005] JP 2018-193303 A (Patent No. 6647240)

[0006] Immunace Injection 35 package insert, revised January 2024 (4th edition), https: / / www.info.pmda.go.jp / go / pack / 6399411D1022_1_21 / 6399411D1022_1_21?view=body&lang=jaJ Immunother. 2003 Jan-Feb;26(1):63-71. Theranostics 2023; 13(5):1506-1519. Colloids Surf B Biointerfaces. 2015 Nov 1;135:765-773. Acta Biomater. 2016 Jan;30:135-143.

[0007] The present inventors have studied highly active NK-like cells (Patent Document 1) and found that the accumulation and infiltration ability of these cells into solid tumors decreases when the IL-2 concentration in the system falls below a certain level. Therefore, if IL-2 signaling can be sustained in the body after administration of highly active NK-like cells, a significant improvement in the response rate to solid tumors can be expected.

[0008] Furthermore, highly activated NK-like cells are generated using IL-2, and their function is maintained experimentally by IL-2. Furthermore, their low expression of IL-2 receptor α results in slow internalization, which are characteristics commonly shared by activated NK cells. Therefore, we hypothesized that anchoring IL-2 on the cell membrane of activated immune cells would enable the cells to autonomously receive IL-2 signals.

[0009] The present inventors attempted to anchor IL-2 on the cell membrane of immune cells by using a phospholipid such as DSPE as a site for insertion into the cell membrane and linking IL-2 to the site for insertion into the cell membrane using PEG. The present inventors then completed the present invention by using such an IL-2-linked molecule to anchor IL-2 on various immune cells and designing the molecule to a size suitable for the anchored IL-2 to bind to the IL-2 receptor on the cell membrane. Furthermore, the present inventors confirmed that using a group capable of forming a bond with a protein on the cell membrane is also effective as a method for anchoring IL-2 on the cell membrane of immune cells, thereby completing the present invention.

[0010] [1] A molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-A, where A is a moiety for anchoring to a cell membrane and is a group capable of forming a bond with a protein on the cell membrane or a moiety containing a phospholipid moiety. [2] The molecule according to 1, wherein the PEG moiety has a molecular weight of 3,000 to 8,000. [3] The molecule according to 1, wherein A is a moiety containing a group capable of forming a bond with a protein on the cell membrane and contains a maleimide reactive group. [4] The molecule according to 1 or 2, wherein A is a phospholipid moiety, which is a group derived from a glycerophospholipid. [5] The molecule according to any one of 1 to 3, wherein A is a phospholipid moiety, which is a group derived from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). [6] Use of the molecule according to any one of 1 to 5 for modulating IL-2 delivery to immune cells. [7] The use according to 6, wherein the immune cells are activated NK cells or NK-like cells. [8] Immune cells modified with the molecule according to any one of 1 to 5. [9] Activated NK cells or NK-like cells modified with the molecule according to any one of 1 to 5.

[10] A pharmaceutical composition comprising the cells according to 8.

[11] A pharmaceutical composition comprising the cells according to 9.

[0011] The present invention provides the following: [1] A molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-modified phospholipid. [2] The molecule according to [1], wherein the phospholipid moiety is a group derived from a glycerophospholipid. [3] The molecule according to [1] or [2], wherein the PEG moiety has a molecular weight of 4500 to 5500. [4] The molecule according to any one of [1] to [3], wherein the phospholipid moiety is a group derived from 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). [5] Use of the molecule according to any one of [1] to [4] for the delivery of IL-2 to immune cells. [6] The use according to [5], wherein the immune cells are activated NK cells or NK-like cells. [7] Immune cells modified with the molecule according to any one of [1] to [4]. [8] Activated NK cells or NK-like cells modified with the molecule according to any one of [1] to [4]. [9] A pharmaceutical composition comprising the cells according to [7] or [8].

[0012] The molecules provided by the present invention enable IL-2 to be anchored on the surface of immune cells, thereby enabling the immune cells to autonomously receive IL-2 signals.

[0013] The cells provided by the present invention are expected to be able to sustain IL-2 signaling in the body after administration in therapy, thereby exerting high anti-tumor activity.

[0014] Examination of cell modification conditions using IL-2 anchors. IL-2 anchors improve the infiltration ability of NK-like cells into solid tumors. Addition of IL-2 to the culture medium was compared with insertion of IL-2 anchors into the cell membrane of NK-like cells. IL-2 anchors improve the cytotoxic activity of NK-like cells against solid tumors. Target: NCI-H1975 (3,000 cells / sphere), Effector: Thawed GAIA-102, PKH26, Sphere x 5 vs. NK 6 x 10 4Measurement of the number of modified IL-2 anchor molecules. IL-2 anchors improve the efficiency of host immune cell accumulation in an in vivo solid tumor model. Groups were set up to administer IL-2 anchor-modified (100nM or 10nM), unmodified, unmodified and IL-2 (10,000U / head) simultaneously, and a control group was untreated. IL-2 anchors improve treatment outcomes in an in vivo solid tumor model. Groups were set up to administer IL-2 anchor-modified (100nM), unmodified, unmodified and IL-2 (10,000U / head) simultaneously, and a control group was untreated. n=4, each, ** P<0.01 (vs. Control), * P<0.05 (vs. Control) IL-2 anchor Structure 1 (DSPE type) vs. Structure 2 (Maleimide type). GAIA-102 was thawed and IL-2 anchoring was performed overnight at 37°C. The number of IL-2 molecules was measured using anti-human IL-2. The molecular weight of the DSPE type is 3.4kJ, and that of the maleimide type is 5kJ. The xCelligence graph on the right of Figure 8 has been extracted and displayed by structure. The molecular weight of the DSPE type is 3.4kJ, and that of the maleimide type is 5kJ.

[0015] In this specification, descriptions of certain embodiments, aspects, and examples also apply to other embodiments, aspects, and examples where the same terminology is used, unless otherwise stated.

[0016] [(IL-2)-PEG-A] This embodiment relates to a molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-A, where A is a moiety for anchoring to a cell membrane, and is a phospholipid moiety or a moiety containing a group capable of forming a bond with a protein on the cell membrane.

[0017] A is a moiety for anchoring to a cell membrane. In one embodiment, A consists of a phospholipid moiety derived from a phospholipid.

[0018] (Phospholipid portion) The phospholipid portion is not particularly limited as long as it is incorporated into the cell membrane of immune cells. The phospholipid portion may be a glycerophospholipid having a glycerin backbone or a sphingosine phospholipid having a sphingosine backbone. More specifically, the phospholipid portion may be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or sphingomyelin.

[0019] In one embodiment, the phospholipid portion is a glycerophospholipid, more specifically, a phosphatidic acid in which a fatty acid is ester-linked to the C1 and C2 positions of glycerin and a phosphoric acid is ester-linked to the C3 position. The groups ester-linked to the C1 and C2 positions are each independently an acyl group having 10 to 20 carbon atoms, preferably an acyl group having 14 to 18 carbon atoms, and may be the same or different.

[0020] In one embodiment, the phospholipid moiety is a group derived from a phospholipid selected from: 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium (DOPG), dipalmitoylphosphatidylglycerol (DPPG); 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), palmitoyloleoylphosphatidylcholine; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16).0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE).

[0021] Groups Capable of Forming Bonds with Proteins on a Cell Membrane In one aspect, the moiety for anchoring to a cell membrane, A, comprises a group capable of forming a bond with a protein on a cell membrane.

[0022] The group capable of forming a bond with a protein on a cell membrane is not particularly limited as long as it can react with any of the cell membranes of immune cells.

[0023] In one embodiment, the group capable of forming a bond with a protein on a cell membrane can form a bond by reacting with the following groups on the membrane protein: Primary amine groups (-NH2) are present at the N-terminus of each protein or in the side chains of lysine groups. Primary amines are often present on the surface of the protein structure due to their positive charge, allowing for binding without denaturing the protein structure. Carboxyl groups (-COOH) are present at the C-terminus of proteins or in the side chains of aspartic acid and glutamic acid. Like primary amines, carbonyls are often present on the surface of the protein structure. Sulfhydryl groups (-SH) are present in the side chains of cysteine. Cysteine ​​generally forms disulfide bonds within proteins. Therefore, to be used in the reaction with the group included in A in this embodiment, the disulfide bond must be reduced to form a sulfhydryl substrate. Carbonyl groups (-CHO) can be generated in glycoproteins from ketone or aldehyde groups by oxidation of polysaccharide post-translational modifications (glycosylation) with sodium metaperiodate.

[0024] In one embodiment, the group capable of forming a bond with a protein on a cell membrane is selected from the following chemical groups: carbodiimide (e.g., EDC), NHS ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, maleimide, haloacetic acid (bromoacetic acid or iodoacetic acid), pyridyl disulfide, thiosulfone, vinyl sulfone, hydrazide, alkoxyamine, diazirine, aryl azide, isocyanate.

[0025] In one particularly preferred embodiment, the group capable of forming a bond with a protein on a cell membrane is maleimide, which is capable of reacting with an SH group of a protein on a cell membrane.

[0026] The protein on the cell membrane of NK cells is not particularly limited as long as it can react with a predetermined group, and examples of proteins on the cell membrane of NK cells include the following: NKp46, NKp30, NKG2D, IL-15R, IL-2R, KIR3DS1, NKG2C, NKp80, NKp65, NKp44, LALRA1, LILRA2, DNAM-1, and 2B4.

[0027] (PEGylated Moiety) The size (length) of the PEGylated moiety of the IL-2-PEGylated phospholipid of this embodiment is not particularly limited, as long as the bound IL-2 can bind to the IL-2 receptor on the cell membrane. The molecular weight of the PEG moiety may be, for example, 550 or more, preferably 700 or more, more preferably 1,000 or more, even more preferably 1,500 or more, 2,000 or more, 2,500 or more, 3,000 or more, 3,500 or more, 4,000 or more, or 4,500 or more. The molecular weight of the PEG moiety may be, for example, 10,000 or less, preferably 9,500 or less, more preferably 9,000 or less, even more preferably 8,500 or less, 8,000 or less, 7,500 or less, 7,000 or less, 6,500 or less, 6,000 or less, or 5,500 or less. In a preferred embodiment, the PEG moiety of the IL-2-PEGylated phospholipid has a molecular weight of 4,000 to 6,000, more preferably 4,500 to 5,500, and even more preferably 3,000 to 4,000, for example, 3,400. The molecular weight of PEG is usually expressed as an average value.

[0028] In the IL-2-PEGylated phospholipid of this embodiment, the PEG moiety and the phospholipid moiety may be linked via a linker moiety L1.

[0029] L1 is, for example, -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, -S(=O)2-, -R 3 -O-, -R 3 -C(=O)-, -R 3 -C(=O)-O-, -R 3 -OC(=O)-, -R 3 -C(=O)-NH-, -R 3 -NH-C(=O)-, -R 3 -S-, -R 3 -S(=O)- or -R 3 -S(=O)2-. R 3 is an aliphatic group having 1 to 6 carbon atoms, preferably an aliphatic group having 1 to 3 carbon atoms.

[0030] In one preferred embodiment, the phospholipid moiety is phosphatidylethanolamine, L1 is -C(=O)-, and the PEGylated phospholipid moiety can be represented as follows:

[0031]

[0032] In the formula, R 1 CO group and R 2 Each CO group is independently an acyl group having 14 to 18 carbon atoms, and R 1 CO group and R 2 The CO groups may be the same or different.

[0033] In one preferred embodiment, the PEGylated phospholipid moiety is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol).

[0034] In this embodiment, PEG is used instead of amino acids to link the phospholipid moiety to the IL-2 moiety, allowing for adjustment of the length of the linking moiety while maintaining low antigenicity. Furthermore, the use of PEG allows for high flexibility of the linking moiety, making it possible to adjust the length depending on the characteristics of the modified cells.

[0035] (IL-2 Moiety) In this embodiment, IL-2 is bound to the terminus of the PEGylated phospholipid. The origin of IL-2 is not limited as long as it can be bound to the terminus of the PEGylated phospholipid and can exert its intended function. In one aspect, IL-2 is mammalian wild-type IL-2 or a variant thereof, and preferably, IL-2 is human IL-2 or a variant thereof.

[0036] IL-2 variants may be fragments, analogs, and derivatives thereof. A fragment refers to a polypeptide consisting of only a portion of the full-length IL-2 sequence. An analog refers to a polypeptide consisting of a sequence having one or more amino acid substitutions, insertions, or deletions in the full-length IL-2 sequence. Derivatives include any modified IL-2 polypeptide, fragments, and analogs thereof, which have been modified by chemical or enzymatic modification or addition, such as glycosylation, phosphorylation, fusion to another polypeptide or molecule, or polymerization, to improve the properties of IL-2 (e.g., stability, specificity, etc.).

[0037] IL-2 may be produced by recombinant technology. The host organism used to express the gene encoding IL-2 may be a prokaryote (a bacterium such as E. coli) or a eukaryote (e.g., yeast, fungus, plant, or mammalian cell). Methods for producing IL-2 by such cell culture are well known to those skilled in the art. IL-2 may also be produced by chemical peptide synthesis. For example, IL-2 can be produced by parallel synthesis of shorter peptides. The total synthesis of IL-2 is exemplified, for example, in Asahina et al., Angewandte Chemie International Edition, 2015, Vol. 54, Issue 28, 8226-8230.

[0038] IL-2 is commercially available for pharmaceutical use and approved for use in human patients. Commercially available pharmaceutical forms of IL-2 include, for example: Teceleukin (Genetical Recombination) Aldesleukin (USAN / INN), Interleukin-2

[0039] In one preferred embodiment, the IL-2 is teceleukin or aldesleukin. Teceleukin is a polypeptide consisting of 134 amino acids produced in Escherichia coli by genetic recombination technology using IL-2 mRNA obtained from lymphocytes derived from human spleen as the starting material. Aldesleukin is a non-glycosylated variant of mature human IL-2 that contains two amino acid alterations compared to mature human IL-2: deletion of the first amino acid (alanine) and substitution of cysteine ​​at position 125 with serine.

[0040] The IL-2 used in one embodiment has a specific activity of 1.2 to 24 million international units (MIU) / mg of protein, preferably 8 to 18 MIU / mg of protein.

[0041] In the (IL-2)-PEGylated phospholipid of this embodiment, the PEG moiety and IL-2 may be linked via a linker moiety L2.

[0042] L 2 For example, -O-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -C(=O)-NH-, -NH-C(=O)-, -S-, -S(=O)-, -S(=O)2-, -R 3 -O-, -R 3 -C(=O)-, -R 3 -C(=O)-O-, -R 3 -OC(=O)-, -R 3 -C(=O)-NH-, -R 3 -NH-C(=O)-, -R 3 -S-, -R 3 -S(=O)- or -R 3 -S(=O)2-. R 3 is an aliphatic group having 1 to 6 carbon atoms, preferably an aliphatic group having 1 to 3 carbon atoms.

[0043] (Production Method) Interleukin-2 (IL-2)-polyethylene glycol (PEG)-A of this embodiment can be produced by applying various existing reactions. For example, it can be easily produced by reacting IL-2 with a moiety, PEG-NHS, in which one end of PEG is NHS-esterified, for anchoring to a cell membrane. Maleimide-PEG-NHS, in which the PEG moiety has various molecular weights, is commercially available. Alternatively, it can be easily produced by reacting IL-2 with a PEGylated phospholipid in which the PEG end is NHS-esterified.

[0044] NHS esters are reactive groups formed by carbodiimide activation of carboxyl molecules. NHS esters react with the primary amines of IL-2 under physiological to slightly alkaline conditions (pH 7.2-9) to form stable amide bonds. This reaction liberates N-hydroxysuccinimide (NHS).

[0045]

[0046] Those skilled in the art can appropriately determine the conditions for the reaction of IL-2 with a moiety-PEG-NHS, such as maleimide-PEG-NHS, for anchoring to a cell membrane. More specifically, the reaction of IL-2 with a PEGylated phospholipid in which the PEG terminus is NHS-esterified can be achieved by adding 1 to 20 equivalents of the NHS-esterified PEGylated phospholipid to IL-2 dissolved in an appropriate solvent, such as DMSO, and allowing the reaction to proceed.

[0047] Commercially available PEGylated phospholipids with NHS esterification at the PEG end are available. For example, DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine)-PEG 20k -NHS, DSPE-PEG 10k -NHS, DSPE-PEG 5k -NHS, DSPE-PEG 3k -NHS, DSPE-PEG 2k -NHS, DSPE-PEG 1k-NHS, DSPE-PEG-NHS, MW 5000 (Nanocs Inc.); DSPE-PEG-NHS, MW 600, DSPE-PEG-NHS, MW 1,000, DSPE-PEG-NHS, MW 2,000, DSPE-PEG-NHS, MW 5,000 (BroadPharm); DSPE-PEG(2000) Carboxy NHS (Avanti Polar Lipids); DSPE-PEG-NHS (MW 3400) (MedChemExpress); DPPE (1,2-Dipalmitoryl-sn-Glycero-3-Phosphoethanolamine) -PEG 5k -NHS, DPPE-PEG 3k -NHS is available commercially.

[0048] Methods for producing PEGylated lipids are well known to those skilled in the art, and those skilled in the art can refer to, for example, Japanese Patent Laid-Open No. 2007-8933, International Publication WO2005 / 026372 (JP Patent Publication No. 2007-505954, Japanese Patent No. 4842821), etc.

[0049] In addition, PEG having a carboxylic acid and an amino group at both ends is commercially available and can be used to obtain the (IL-2)-PEGylated phospholipid of this embodiment.

[0050] (Function of (IL-2)-PEG-A) The (IL-2)-PEG-A of this embodiment can be used to anchor IL-2 to immune cells.

[0051] In one embodiment, the (IL-2)-PEG-group capable of forming a bond with a protein on a cell membrane reacts with a predetermined group on a protein on the membrane of an immune cell, thereby anchoring IL-2 to the cell membrane. In another embodiment, the phospholipid of the (IL-2)-PEGylated phospholipid is taken up by an immune cell, allowing the phospholipid portion to anchor IL-2 on the cell membrane.

[0052] IL-2 acts through a quaternary receptor signaling complex that includes the α (IL-2Rα), β (IL-2Rβ), and common γ chain (gc) receptors (Science. 2005 Nov 18;310(5751):1159-63. doi: 10.1126 / science.1117893.) To enable the formation of such a complex on the cell membrane, the length of the PEGylated moiety can be, for example, 4 nm or longer, preferably 6 nm or longer, and more preferably 8 nm or longer.

[0053] IL-2 anchored on the cell membrane can bind to the IL-2 receptor on the membrane of the same cell if the PEG moiety is of appropriate length, allowing the cell to receive the IL-2 signal autonomously and continuously.

[0054] Considering the binding to the IL-2 receptor, it is thought that the PEGylated portion of the (IL-2)-PEGylated phospholipid of this embodiment preferably has a length of around 8 nm.

[0055] [Modified Immune Cells] This embodiment relates to immune cells having IL-2 anchored on the cell membrane by (IL-2)-PEG-A. Immune cells having anchored IL-2 may be referred to as modified immune cells.

[0056] The immune cells are not particularly limited as long as they express IL-2 receptors on their cell membranes and can receive IL-2 signals. One type of immune cell to which this embodiment is preferably applied is a cell administered for immunotherapy. Preferably, the cell is administered to a human for immunotherapy and has undergone an ex vivo activation procedure. The activation procedure is preferably performed using IL-2. The activation procedure is typically performed by incubating the cells in a medium containing IL-2.

[0057] Such immune cells include NK cells, highly active NK-like cells (cells of Patent Document 1), cells that exhibit extremely high cytotoxic activity against solid cancers that form tumor masses, as described in JP 2019-170176 A (Patent No. 6543375), NK-92 cells, T cells, effector T cells, and CAR-T cells. Note that, although the present specification may explain the present invention and this embodiment by using highly active NK-like cells as an example, those skilled in the art will be able to understand the application of the present invention to other cells based on this explanation.

[0058] (NK cells, highly active NK-like cells, etc.) In one embodiment, the immune cells are NK cells, highly active NK-like cells (cells described in Patent Document 1), or NK-92 cells, which exhibit extremely high cytotoxic activity against solid cancers that form tumor masses, as described in JP 2019-170176 A (Patent No. 6543375). Highly active NK-like cells include GAIA-102 (see the Examples section of the present specification). These cells are sometimes collectively referred to as NK cells.

[0059] Generally, NK cells are large granular lymphocytes that do not express the T cell receptor (TCR), the universal T cell marker CD3, or the membrane immunoglobulin B cell receptor, and in humans, they are usually CD16-positive and CD56-positive. Those skilled in the art can easily determine whether a cell is an NK cell based on the expression pattern of cell surface markers, etc. NK cells have cytotoxic activity, and the presence or absence and level of this cytotoxic activity can be measured by various known methods. NK cells may include primary NK cells, cultured NK cells, and activated NK cells.

[0060] NK-92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a patient with non-Hodgkin's lymphoma (Non-Patent Document 3).

[0061] The source of NK cells is not particularly limited and may be peripheral blood, umbilical cord blood, bone marrow and / or lymph nodes, or blood collected by apheresis (apheresis blood). The source may also be prepared from at least one type of cell selected from the group consisting of hematopoietic stem cells derived from any stem cell selected from the group consisting of embryonic stem cells, somatic stem cells, and induced pluripotent stem (iPS) cells, hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem cells derived from peripheral blood, hematopoietic stem cells derived from bone marrow, umbilical cord blood mononuclear cells, and peripheral blood mononuclear cells (PBMCs). The donor of the source may be the patient receiving immunotherapy with highly active NK cells, a close relative of the patient, or a healthy individual unrelated to the patient. Multiple donors may be used.

[0062] In one preferred embodiment, the immune cells are any of the following [1], [2], [3], and [4].

[0063] [1] NK-like cells have the following characteristics: (1) They are CD16 positive, have high CD56 expression, and are CD57 negative. (2) They are NKG2C positive, have low or negative NKG2A expression, and are CD94 positive.

[0064] The NK-like cells of [1] may be highly CD16 expressing cells. Furthermore, the NK-like cells of [1] may be highly active NK-like cells that further have the following characteristics, regardless of whether they are highly CD16 expressing cells: (3) When the NK cells are used as effector cells (E) and K562 cells as target cells (T) and co-cultured at a mixing ratio (E:T) of 1:1, the cytotoxic activity is 50% or more.

[0065] The highly active NK-like cells of [1] can also be described as follows: NK cells obtained by removing CD3-positive cells from peripheral blood mononuclear cells derived from healthy individuals using CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, catalog number 130-017-601, or an equivalent), an LD column (e.g., Miltenyi Biotec, catalog number 130-042-901, or an equivalent), and a separation buffer (e.g., PBS containing 0.5% human AB type serum (heat-inactivated) and 2 mM EDTA), and culturing the resulting cell population in an appropriate medium (e.g., Cosmedium 008 supplemented with 5% human AB type serum (heat-inactivated)) for 14 days, and which have the following characteristics (1) and (3): (1) CD16-positive, highly CD56-expressing, and CD57-negative. (3) When the NK cells are used as effector cells (E) and K562 cells as target cells (T) and co-cultured at a mixing ratio (E:T) of 1:1, the cytotoxic activity is 50% or more.

[0066] For details of the characteristics of the highly active NK cells [1] and a more specific method for producing them, refer to Patent Document 1.

[0067] [2] The following cells: CCR5-positive, CCR6-positive, and CXCR3-positive cells, and CD3-negative cells.

[0068] The cells of [2] may further highly express CD11c.

[0069] The cells of [2] can also be described as follows: CCR5-positive, CCR6-positive, CXCR3-positive, integrin α1-positive, integrin α3-positive, integrin β3-negative, and CD3-negative cells, or CCR5-positive, CCR6-positive, CXCR3-positive, highly expressing CD11a and CD11c, and CD3-negative cells, the high expression levels being determined by comparison with the expression levels in a population of NK cells obtained from peripheral blood that have not been substantially cultured.

[0070] According to the inventors' investigations, the cells of [2] exhibit extremely high cytotoxic activity against solid cancers that have formed tumor masses. For details of the characteristics of the cells of [2] and more specific manufacturing methods, please refer to JP 2019-170176 A (Patent No. 6543375).

[0071] [3] Highly active NK cells can be obtained by the following method: Mononuclear cells obtained from fresh peripheral blood or frozen apheresis blood are mixed with CD3 beads (e.g., CliniMACS CD3, Miltenyi Biotec, 130-017-601 (1x10 7 If frozen apheresis blood was used, additional CD34 beads (e.g., CliniMACS CD34, Miltenyi Biotec, 130-017-501 (1 x 10 7 Add 2.5 μL of PBS containing 0.5% human AB type serum (heat-inactivated at 56°C for 30 minutes) and 2 mM EDTA to the cells, suspend the cells, and then centrifuge. Remove the supernatant and load the cells onto an LD column (e.g., Miltenyi Biotec, 130-042-901) at a maximum of 1 x 10 per column. 8 The cells were suspended in 0.5 mL of separation buffer to a cell count of up to 10 cells. 2 mL of separation buffer was added beforehand, and the cell suspension was then added to the LD column, and the eluate was collected. 1 mL of separation buffer was then added to the LD column, and the eluate was collected. The collected liquid was centrifuged, and after removing the supernatant, 5 x 10 cells were collected. 5 cells / mL, 1x10 when using frozen apheresis blood 6 The cells are suspended in an appropriate medium (e.g., KBM501 medium containing either 5% human AB type serum (heat-inactivated at 56°C for 30 minutes) or 5% UltraGRO (AventaCell, HPCPLCRL10) supplemented with 2 U / mL heparin sodium) to a concentration of 100 cells / mL, and cultured for up to 14 days, with appropriate medium changes.

[0072] For specific methods for producing the highly active NK cells of [3], please refer to the Examples section of this specification.

[0073] [4] Cells obtained by culturing the cells of [1] to [3], adding any one selected from the group consisting of IL-12, IL-15, and IL-18 at a concentration sufficient to achieve the object of the present invention, either simultaneously with or instead of IL-2. Non-Patent Document 2 (Leong JW et al. Biol Blood Marrow Transplant 20 (2014) 463-473) is a specific example of a method for producing such cells.

[0074] In the present invention, when referring to NK cells, activity or cytotoxic activity refers to the ability of target cells (effector cells, E) to lyse target cells (T), unless otherwise specified. Cytotoxic activity can be expressed as the percentage (%) of target cells killed by effector cells, and is calculated using the following formula:

[0075] (Cell death when co-cultured with effector cells - spontaneous cell death (negative control)) / (maximum cell death (positive control) - spontaneous cell death (negative control)) x 100

[0076] When measuring cytotoxic activity, the mixing ratio of effector cells to target cells (E:T) and the duration of co-culture of effector and target cells can generally be adjusted depending on the type of cells used and the strength of their activity, as well as the degree of cytotoxic activity of the effector cells. When NK cells are used as effector cells, the target cells may be, but are not limited to, K562 cells, acute myeloid leukemia cells, or chronic myeloid leukemia cells. Effector and target cells, and live and dead cells, can be distinguished and quantified using reagents such as antibodies labeled with radioactive substances or fluorescent dyes. When NK cells are used as effector cells, cytotoxic activity can be measured, for example, using K562 cells as target cells at an E:T ratio of 1:0.05-10, preferably 1:0.1-5, with an incubation time of 0.5-18 hours, preferably 1-12 hours.

[0077] In the present invention, unless otherwise specified, the term "high activity of NK cells" refers to a cytotoxic activity of 50% or more when the target cells are K562 cells, mixed at an E:T ratio of 2:1, and co-cultured for 1 to 3 hours, more specifically 2 hours. The activity is preferably 60% or more, and more preferably 70% or more.

[0078] (Effector T Cells, CAR-T Cells) In one embodiment, the immune cells are effector T cells. Effector T cells are T cells with enhanced ability to attack cancer cells. They are typically derived from a patient's own T cells, explanted, transfected with genes that attack cancer cells, and then reintroduced into the patient. CAR-T cells are a type of effector T cell, and utilize a chimeric antigen receptor (CAR) gene to recognize cancer antigens. IL-2 is known to act as a T cell growth factor essential for the proliferation and survival of T cells and the generation of effector T cells. It has also been reported that CAR-T cells, which can secrete a certain level of IL-2 in response to cancer-specific antigens, can infiltrate solid tumors and suppress cancer growth (SCIENCE. 16 Dec 2022. Vol. 378, Issue 6625. DOI: 10.1126 / science.aba16).

[0079] (Anchoring, Modification) In the modified immune cells of this embodiment, the phospholipid moiety of the (IL-2)-PEGylated phospholipid described above is incorporated into the cell membrane of the immune cells, and IL-2 is anchored on the cell membrane. Anchoring can be easily achieved. For example, it can be achieved by mixing the immune cells with the (IL-2)-PEGylated phospholipid and then incubating them at a predetermined temperature for a predetermined time. The temperature is not particularly limited, but is, for example, 15 to 42°C, preferably 20 to 40°C, more preferably 22 to 38°C, or may be room temperature (around 25°C). The time is not particularly limited, but is, for example, 1 minute to several hours, preferably 2 to 60 minutes, more preferably 3 to 30 minutes, and even more preferably 5 to 20 minutes.

[0080] The number of IL-2 molecules anchored to immune cells can be adjusted appropriately by adjusting the cell density and the concentration of (IL-2)-PEGylated phospholipid during incubation.

[0081] The density of immune cells during incubation should be, for example, 1 x 10, regardless of other conditions. 5 cells / ml or more, preferably 2 x 10 5 cells / ml or more, more preferably 3x10 5 cells / ml or more, more preferably 5x10 5 cells / ml or more. Regardless of other conditions, for example, 1x10 8 cells / ml or less, preferably 5x10 7 cells / ml or less, more preferably 2x10 7 cells / ml or less, more preferably 1x10 7 cells / ml or less.

[0082] The concentration of (IL-2)-PEGylated phospholipid during incubation can be, for example, 1 nM or more, 10 nM or more, 50 nM or more, 100 nM or more, 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, 600 nM or more, 700 nM or more, 800 nM or more, 900 nM or more, 1,000 nM or more, 1,500 nM or more, or 1,800 nM or more, regardless of other conditions. It can also be, for example, 10,000 nM or less, 9,000 nM or less, 8,000 nM or less, 7,000 nM or less, 6,000 nM or less, 5,000 nM or less, 4,000 nM or less, 3,000 nM or less, or 2,000 nM or less, regardless of other conditions.

[0083] In the modified immune cells of this embodiment, the number of IL-2 molecules anchored on the cell surface of a single immune cell is, in one aspect, 1 or more, for example, 2 or more, 4 or more, 8 or more, 10 or more, 20 or more, 30 or more, 50 or more, or 100 or more. Studies by the present inventors have confirmed efficacy when the number of IL-2 molecules anchored on a single immune cell is 8. One preferred aspect is an immune cell in which 50 to 3,000 IL-2 molecules are anchored on the cell surface per cell. The number of IL-2 anchors per cell can be quantified by those skilled in the art, as appropriate, and can be determined by reference to the method described in the Examples herein.

[0084] According to the study by the present inventors, when IL-2 is anchored at 3,000 / cell, elution of all IL-2 results in 200 IU of IL-2 / 2x10 8 It is calculated as cells.

[0085] If necessary, the modified immune cells may be prepared through a step of removing (IL-2)-PEGylated phospholipids that have not been anchored to the immune cells.

[0086] The modified immune cells of this embodiment can autonomously and continuously receive IL-2 signals. Conventional IL-2 is not very stable in vivo. For example, when a recombinant IL-2 preparation (generic name: teceleukin (recombinant)) was administered intravenously at a constant rate of 700,000 units over a period of exactly two hours to four adult patients with malignant tumors, the serum concentration reached its highest point at the end of administration but returned to baseline within several hours (Non-Patent Document 1). This embodiment overcomes these drawbacks.

[0087] One particularly preferred embodiment is highly active NK-like cells anchored with IL-2 (Patent Document 1). The ability of highly active NK-like cells (Patent Document 1) to accumulate and infiltrate into solid tumors decreases when the IL-2 concentration in the system falls below a certain level. However, by anchoring IL-2 on the cell membrane, the highly active NK-like cells can sustain the reception of IL-2 signals, which is expected to significantly improve the response rate to solid tumors.

[0088] [Pharmaceutical Composition] This embodiment relates to a pharmaceutical composition. The pharmaceutical composition of this embodiment contains the above-described population of modified immune cells and a pharmaceutically acceptable additive. The pharmaceutical composition of this embodiment containing immune cells may be a regenerative medicine product.

[0089] Examples of pharmaceutically acceptable additives include isotonicity agents, pH adjusters, buffers, stabilizers, cryoprotectants, antibiotics, etc. Specific examples include water, ethanol, sodium chloride, glucose, albumin, etc.

[0090] The pharmaceutical composition of this embodiment may contain an antibody. The antibody is preferably one that can be used as an antibody pharmaceutical. Furthermore, it is preferably one that can induce antibody-dependent cellular cytotoxicity (ADCC). The antibody used in the pharmaceutical composition of the present invention may be any of a mouse antibody, a chimeric antibody, a humanized antibody, and a human antibody, but is preferably a humanized antibody or a human antibody. The antibody used may also be modified, or may be prepared by Potelligent technology (removal of fucose from the Fc region of IgG). The embodiment containing an antibody is not limited to cases where the pharmaceutical composition contains NK cells, but is also one of the preferred embodiments when the pharmaceutical composition contains effector T cells or CAR-T cells.

[0091] Specific examples of antibodies that are used as antibody pharmaceuticals and can be used in the pharmaceutical composition of this embodiment include ibritumomabtiuxetan, iodine131, catumaxomab, blinatumomab, muromonab-CD3, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, siltuximab, dinutuximab, obiltoxaximab, daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, and eculizumab. b, certolizumab pegol, mogamulizumab, pertuzumab, trastuzumab, obinutuzumab, vedolizumab, pembrolizumab, idarucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, pa Mention may be made of nitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.

[0092] The antibody used in the pharmaceutical composition of this embodiment preferably has high affinity for CD16. Furthermore, in embodiments in which immune cells include NK cells (NK cells, NK-like cells, etc.), it is preferable that at least a portion of the antibody binds to NK cells. According to studies by the present inventors, when mogamulizumab, produced using Potelligent® technology, was used as the antibody, a system in which the antibody was present during co-culture of highly active NK-like cells and target cells was compared with a system in which highly active NK-like cells and the antibody were pre-mixed and the NK-like cells were washed (to remove unbound antibody) before co-culture with target cells, resulting in higher cytotoxic activity in the latter system (Patent Document 1). On the other hand, when dinutuximab was used under the same conditions, no effect was observed from pre-mixing the highly active NK-like cells with the antibody. This suggests that an antibody with high affinity for CD16 can exert high ADCC activity by pre-mixing with NK cells, allowing the antibody to bind to the NK cells. It is possible that similar effects may be obtained with dinutuximab if the conditions are considered.

[0093] The pharmaceutical composition of this embodiment may be prepared immediately before use. For example, the pharmaceutical composition of this embodiment may be prepared by thawing frozen highly active NK-like cells immediately before or several hours before administration to a subject, and providing a molecule for anchoring IL-2, such as IL-2-PEG-DSPE, to prepare modified immune cells.

[0094] The pharmaceutical composition of this embodiment may be administered to a patient having an HLA genotype different from that of the NK cells prepared by the preparation method of this embodiment.

[0095] The pharmaceutical composition of this embodiment is typically in the form of immune cells suspended in a solution. Typical solutions for suspending immune cells include, for example, cryoprotectants containing DMSO, saline, phosphate-buffered saline (PBS), culture media, serum, etc. The solution may contain a pharmaceutically acceptable carrier for use as a drug or quasi-drug.

[0096] The pharmaceutical composition of this embodiment is suitable for use in treating cancer or infectious diseases. The pharmaceutical composition of this embodiment can also be used to treat various diseases sensitive to immune cells. In the context of the present invention, the term "treatment" for a disease or condition includes reducing the risk of onset, delaying onset, prevention, treatment, and halting or delaying progression. Treatment includes radical treatment (treatment that removes the cause) and symptomatic treatment (treatment that improves symptoms).

[0097] Diseases include, but are not limited to, oral cancer, gallbladder cancer, bile duct cancer, lung cancer, liver cancer, colon cancer, kidney cancer, bladder cancer, leukemia, and infectious diseases caused by viruses, bacteria, etc. The cell therapy of this embodiment may be performed alone or in combination with surgical therapy, chemotherapy, radiation therapy, antibody drugs, etc. In cell therapy using the pharmaceutical composition of this embodiment, NK cells may be administered, for example, intravenously, arterially, subcutaneously, intraperitoneally, etc.

[0098] The pharmaceutical composition of this embodiment is preferably produced under conditions that comply with the regulations for manufacturing control and quality control of drugs and quasi-drugs (good manufacturing practice, GMP) and the standards for manufacturing control and quality control of regenerative medicine products (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice, GCTP).

[0099] In this embodiment, the collection of whole blood from umbilical cord blood and peripheral blood, the preparation of autologous serum, the preparation of mononuclear cells from the whole blood, the measurement of the cell count before and after culturing the mononuclear cells, the measurement of the composition ratios of NK cells, T cells, hematopoietic progenitor cells, and other cell types in the mononuclear cells before and after culturing, the calculation of the NK cell amplification fold, and the statistical analysis of the measurement error and significance may be performed using any method known to those skilled in the art.

[0100] The present invention will be described in more detail below using examples, but the examples do not limit the technical scope of the present invention, which is limited only by the claims.

[0101] [Methods common to all Examples] The following methods were used throughout the Examples section unless otherwise specified. A) Method for culturing highly active NK-like cells GAIA-102 Frozen apheresis blood (Cellero) was used as the raw material. After thawing, the blood was washed and concentrated using the Lovo Cell Processing System (FRESENIUS KABI) to obtain PBMCs.

[0102] CD3-positive cells and CD34-positive cells were removed from the obtained PBMCs using CliniMACS Prodigy (registered trademark) (Miltenyi Biotec), and the cells were cultured in KBM501 medium. ※1 The number of cells in the eluate was counted and the total number of cells was calculated. 2 (Product name GICB640NA, Fukoku Co., Ltd.) or 75cm 2 Use flasks, 5x10 5 The cells were cultured in KBM501 medium at a final concentration of 1 μg / mL and incubated in a CO2 incubator (37°C, 5% CO2). Additional KBM501 medium was added on days 7-9 of culture, and the cells were incubated until days 14-24. Urelumab (CreativeBioLab) and IL-21 (PeproTech) were added during culture. Urelumab and IL-21 were added at final concentrations of 1 μg / mL and 1 ng / mL, respectively.

[0103] *1: KBM 501 (Kohjin Bio) supplemented with 5% UltraGRO (AventaCell, HPCPLCRL10) and 2U / mL heparin sodium (Nipro)

[0104] B). Method for recovering highly active NK-like cells, GAIA-102. The culture medium was recovered on the 14th day of culture, and 1 mM EDTA was added to the culture bag to detach the adhered cells. All recovered cell medium, including the detached cells, was centrifuged, washed, and resuspended in KBM501 medium.

[0105] C) Method for freezing highly active NK-like cells GAIA-102 The number of viable GAIA-102 cells obtained by the procedure described in the highly active NK-like cell culture and collection method was counted and frozen at 2 x 10 8The cells were suspended in 5 mL of HSC-BANKER (ZENOAQ, CB071) and frozen at −80° C. Thawing was performed by 41-fold dilution with PlasmaLyte-A according to the methods described in the Examples of WO2023 / 038037 and PCT / JP2024 / 009978.

[0106] D) Viability measurement by 7-AAD staining GAIA-102 cells thawed under each condition were placed in a 96-well plate (IWAKI, 4870-800SP) at 1x10 5 After the supernatant was removed, 7-AAD solution (Beckman Coulter, A07704) diluted with PBS (Nacalai Tesque, 14249-95) was added and suspended, followed by incubation at room temperature for 20 minutes. The stained cells were measured using a flow cytometer (BD LSRFortessa, BD Biosciences) and analyzed using FlowJo software. Viability was calculated from the 7-AAD positivity rate.

[0107] E). Method for measuring cytotoxic activity against tumor cells To measure cytotoxic activity, we prepared a group in which thawed GAIA-102 was reacted with K562 cells, a group containing only K562 cells as a negative control, and a group in which K562 cells were treated with 10% formalin as a positive control.

[0108] GAIA-102: Thawed, IL-2 anchored, and diluted GAIA-102 under each condition was aliquoted based on the number of viable cells at the time of freezing, and then diluted to 1x10 in 10% FBS / RPMI 1640. 6 The cells were prepared at a concentration of 1000 cells / ml.

[0109] K562 cells were suspended in serum-free RPMI 1640 medium, stained with PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich), and then cultured at 2 x 10 cells in 10% FBS / RPMI 1640. 6 The concentration was adjusted to cells / mL.

[0110] GAIA-102 and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) at cell ratios of 1:1, 2:1, or 8:1, mixed, and incubated at 37°C for 2 hours in 5% CO2. After incubation, the plate was centrifuged (500 x g, 5 minutes), the supernatant was removed, and 7-AAD solution diluted with PBS was added and suspended. The mixture was then incubated at room temperature for 20 minutes. Measurements were performed using a flow cytometer and analyzed using FlowJo software to determine the cytotoxicity rate (% Lysis). ※2 was calculated.

[0111] *2: Cytotoxic activity rate = (K562 cell death rate - negative control cell death rate) / (positive control cell death rate - negative control cell death rate) x 100 F). Method for calculating corrected values ​​by statistical analysis of cytotoxic activity rate ET ratios of 1 and 2 were calculated by nonlinear regression analysis using JMP (registered trademark) Pro statistical analysis software from cytotoxic activity rates calculated at ET ratios of 4 or more points (including 0).

[0112] G) Measurement of the number of IL-2 anchor molecules per cell using PE-beads (BD Quantibrite) TM The number of IL-2 molecules on the cell surface was calculated from the fluorescence intensity of cells stained with a PE-labeled anti-human IL-2 antibody (clone MQ1-17H12, Cat. No. 500307, ​​Biolegend) using a PE Phycoerythrin Fluorescence Quantitation Kit (Cat. No. 340495, BD, a PE molecule counting kit using PE-labeled beads with a known number of molecules) based on the calibration curve.

[0113] Example 1: Study of cell modification conditions using IL-2 anchor The following were used: IL-2 (recombinant human IL-2, Peprotech, AF-200-02) NHS-PEG 5K -DSPE (NANOCS, PG2-DSNS-5k)

[0114] The IL-2 anchor was prepared as follows: IL-2 (2 mg / mL in DMSO) was mixed with 1-20 equivalents of NHS-PEG-DSPE (10 mg / mL in DMSO), and PBS was added to a DMSO concentration of 50%. The mixture was incubated overnight at room temperature. A portion of this mixture was kept as a crude sample, and another portion was added with PBS to a total volume of 1 mL, subjected to buffer exchange using a PD Miditrap G-25, and stored refrigerated (4°C) or frozen (-80°C) as a purified sample.

[0115]

[0116] GAIA-102 (1x10 6 IL-2 anchors were added to the cells (200 cells / ml) at final concentrations of 10 nM, 100 nM, and 1000 nM, respectively, and incubated at room temperature for 10 minutes. Afterwards, the cells were stained with a PE-labeled anti-human IL-2 antibody, and the number of IL-2 anchors per cell was quantified using a PE Phycoerythrin Fluorescence Quantitation Kit.

[0117] The results are shown in Figure 1. IL-2 was anchored onto the cells in a manner dependent on the amount of IL-2 anchor added. When IL-2 was anchored at 3,000 cells / cell, eluting all of it resulted in 200 IU of IL-2 / 2 x 10 8 It is calculated as cells.

[0118] Example 2: IL-2 anchor enhances NK-like cell infiltration into solid tumors

[0119] The IL-2 anchor was prepared according to the following procedure.

[0120]

[0121] Thaw cryopreserved highly active NK-like cells and incubate them in CytoTell TM The cells were stained with UltraGreen and added to the spheroids prepared with HsOs-1 cells. Specifically, HsOs-1 cells were detached with trypsin-EDTA and diluted to 3 × 10 cells per 100 μL in RPMI (10% FBS). 3The cells were prepared and seeded on EZ-Bind Shut II® microplates (IWAKI, Cat. 4870-800LP) and cultured at 37°C for 48 to 72 hours to form spheroids. The experiment was performed using a 384-well plate, with 2.5 x 10 cells per spheroid per well. 4 For the group in which IL-2 was anchored to the highly active NK-like cells, the highly active NK-like cells (1 x 10 6 IL-2 anchor was added to a final concentration of 1000 nM to the medium (cells / ml), and for the group in which IL-2 was added to the medium, Immunase was added at 100 units / ml. Incubation was carried out at 37 ° C, and observation was carried out under a fluorescent microscope after 48 hours. Cryopreservation and thawing were carried out in accordance with the methods described in JP 2018-050502 A (Patent No. 6989907) and JP 2021-136883 A.

[0122] The results are shown in Figure 2. The use of membrane-integrated IL-2 with cryopreserved highly active NK-like cells resulted in increased infiltration of NK-like cells into solid tumors.

[0123] Example 3: IL-2 anchor enhances NK-like cell activity in cytotoxicity to solid tumors. Cryopreserved highly active NK-like cells were thawed, stained with PKH26, and added to spheroids (3,000 cells / sphere) prepared from NCI-H1975 cells. The experiment was performed using a 96-well plate, with 6.0 x 10 cells per spheroid (5 cells / well). 4 The cells were incubated at 37°C and observed under a fluorescent microscope after 22 hours. After incubation, all cells in the wells were collected and the number of viable PKH26-negative cells (Zombie staining negative) was counted using a flow cytometer to measure cytotoxicity. NK-like cells without IL-2 anchoring were used as a control (no IL-2 was added to the reaction system).

[0124] The results are shown in Figure 3. By using membrane-inserted IL-2 with cryopreserved highly active NK-like cells, we observed greater infiltration of NK-like cells into solid tumors and higher cytotoxic activity, even against spheroids made with NCI-H1975 cells.

[0125] Example 4: Measurement of the number of IL-2 anchor-modified molecules Highly active NK-like cells were modified with IL-2 anchor, stained with PE-labeled anti-human IL-2 antibody (MQ1-17H12, Cat. No. 500306), and the number of modified molecules per cell was measured using a PE Phycoerythrin Fluorescence Quantitation Kit. The IL-2 anchor was 1 x 10 6 The highly active NK-like cells (cells / ml) were reacted with 0.54 μM of the antibody at room temperature for 10 minutes to perform modification.

[0126] The results are shown in Figure 4. An average of 1748.4 (=2350.8-602.4) IL-2 particles were present on the cell surface. When all of the IL-2 particles were eluted, 1748 IL-2 particles / cell was equivalent to 100 IU of IL-2 / 2 x 10 8 It is calculated as cells.

[0127] Example 5: Improvement of host immune cell accumulation efficiency in an in vivo solid tumor model by IL-2 anchor. CT26-GFP cells were cultured in Balb / c mice at 1 x 10 6 The mice were transplanted (intraperitoneally) at a rate of 1 x 10 cells / mouse, and 3 to 5 days later, GAIA-102 was administered (intraperitoneally to one group modified with the IL-2 anchor of the present invention (modified at 100 nM or 10 nM), to another group administered unmodified GAIA-102, or to another group administered unmodified GAIA-102 and IL-2 (10,000 U / mouse) simultaneously). The number of GAIA-102 cells administered per administration was 1 x 10 cells in all groups. 7 The IL-2 anchor-modified GAIA-102 was confirmed to be modified with an IL-2 anchor by flow cytometry using an anti-IL-2 antibody.

[0128] Seven days after CT26-GFP cell transplantation, the mesentery was photographed under a fluorescent microscope to confirm the formation of CT26-GFP cell nodules as GFP-positive areas. The nodules were then harvested, minced, and digested with Liberase to obtain single-cell suspensions. The suspensions were then stained with anti-mouse CD4, anti-mouse CD8, anti-mouse NKp46, and anti-human CD56 and analyzed by flow cytometry. CT26-GFP cells were detected as GFP-positive cells, and the ratio of the number of GFP-positive cells to the number of antigen-positive cells was calculated.

[0129] The results are shown in Figure 5. GAIA-102 administration experiment in a mouse peritoneal dissemination model using CT26 cells. Groups administered with IL-2 or IL-2-anchored GAIA-102 showed a decrease in the number of peritoneal nodules and an increase in the amount of immune cell infiltration in mice. Images of intraperitoneal tumor nodules after GAIA-102 administration are shown on the left. Differences in tumor area, shown in green, can be seen between the presence and absence of IL-2 anchor modification. In particular, administration of GAIA-102 modified with 100 nM IL-2 anchors revealed almost no residual tumor (day 7 column). Furthermore, we confirmed that the IL-2 anchor was modified on the administered GAIA-102 in a concentration-dependent manner (three administrations per group, days 3-5 columns).

[0130] Example 6: IL-2 anchor improves therapeutic outcomes in an in vivo solid tumor model. 1x10 CT26-GFP cells were cultured in Balb / c mice. 6 The mice were transplanted (intraperitoneally) at a rate of 1 x 10 cells / head, and 3 to 7 days later, GAIA-102 was administered (a group receiving GAIA-102 modified with the present IL-2 anchor (modified at 100 nM), a group receiving unmodified GAIA-102, and a group receiving unmodified GAIA-102 and IL-2 (10,000 U / head) simultaneously). An untreated group was used as a control. The number of GAIA-102 cells administered per administration was 1 x 10 in all groups. 7 The number was set at 1 / head.

[0131] The IL-2 anchor-modified GAIA-102 was confirmed to be modified with an IL-2 anchor by flow cytometry using an anti-IL-2 antibody.

[0132] The results are shown in Figure 6. GAIA-102 was administered to a CT26 cell peritoneal dissemination model. Five doses were administered, beginning three days after CT26 cell inoculation. Despite the extremely short treatment period, a reduction in mortality was confirmed in the group administered the IL-2 anchor GAIA-102.

[0133] In the following Examples 7 and 8, the same reagents and equipment as in Examples 1 to 6 were used unless otherwise specified.

[0134] Example 7: Comparison of DSPE type and Maleimide type. Cryopreserved highly active NK-like cells were thawed and then incubated with DSPE-PEG 3.4K -FITC, Maleimide-PEG 5K FITC was added at concentrations ranging from 0 to 10,000 nM and incubated at room temperature for 10 minutes. Afterwards, the cells were washed with culture medium, centrifuged, suspended in HSC-BANKER, and frozen at -80°C for 3 days. After thawing, FITC was detected using a flow cytometer.

[0135]

[0136]

[0137] As a result, FITC was detected in both structures before and after freezing, and it was confirmed that the intensity changed depending on the reaction concentration (Fig. 7). In addition, the cell death rate was also measured by 7-AAD staining after thawing, and no clear cytotoxicity was observed under either condition (Fig. 7).

[0138] [Example 8: Examination of conditions for modifying NK cells with IL-2 anchor] The IL-2 anchor was prepared according to the following procedure: DSPE-PEG 3.4K -NHS and Maleimide-PEG 5K-NHS and recombinant human IL-2 were mixed at a molar ratio of 5:1. DMSO was added to the reaction mixture at a final concentration of 50%. The mixture was left at room temperature for 1 hour.

[0139] The thawed GAIA-102 was treated with the IL-2 anchor (DSPE-PEG 3.4K -IL2, and Maleimide-PEG 5K IL-2) was added to final concentrations of 100 nM, 1,000 nM, and 10,000 nM, respectively, and incubated at room temperature for 10 minutes. Afterwards, the cells were stained with a PE-labeled anti-human IL-2 antibody, and the number of IL-2 anchors per cell was quantified using a PE Phycoerythrin Fluorescence Quantitation Kit. Cytotoxicity against H1975 cell spheres was also measured using xCelligence (Agilent Technologies, Inc., a real-time cell analyzer).

[0140] As a result, it was confirmed that both the DSPE type and the maleimide type had the ability to modify IL-2 on GAIA-102 and maintain its cytotoxic activity (Figures 8 and 9).

Claims

1. A molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-A, where A is a moiety for anchoring to a cell membrane, and is a group capable of forming a bond with a protein on the cell membrane, or a moiety containing a phospholipid moiety.

2. The molecule of claim 1, wherein the PEG moiety has a molecular weight of 3,000 to 8,000.

3. The molecule of claim 1, wherein A is a moiety containing a group capable of forming a bond with a protein on a cell membrane, and includes a maleimide reactive group.

4. The molecule of claim 1, wherein A is a phospholipid moiety, the phospholipid moiety being a group derived from a glycerophospholipid.

5. The molecule of claim 1, wherein A is a phospholipid moiety, and the phospholipid moiety is a group derived from 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

6. Use of a molecule according to any one of claims 1 to 5 for the modulation of IL-2 to immune cells.

7. The use according to claim 6, wherein the immune cells are activated NK cells or NK-like cells.

8. An immune cell modified with a molecule according to any one of claims 1 to 5.

9. Activated NK cells or NK-like cells modified with a molecule according to any one of claims 1 to 5.

10. A pharmaceutical composition comprising the cells of claim 8.

11. A pharmaceutical composition comprising the cells of claim 9.

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