Membrane anchored il-2 and uses thereof

Abstract

The problem of the present invention is to make the IL-2 signal persist in vivo after immune cell administration. A molecule consisting of interleukin-2 (IL-2) - polyethylene glycol (PEG) - A is used. A is a moiety for anchoring to the cell membrane, which comprises a phospholipid moiety, or a group capable of forming a bond with a protein on the cell membrane. The PEG moiety preferably has a molecular weight of 3000 to 8000. Such a molecule is used for the modification of IL-2 to immune cells. Examples of immune cells are activated NK cells or NK-like cells.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell membrane-anchored interleukin-2 (IL-2) and use thereof. The present application is useful for high-functioning of immune cells, and is useful in the field of cell therapy using immune cells and the like. BACKGROUND

[0002] Interleukin-2 (IL-2) has an effect of activating and proliferating cells by transmitting a signal into the cells through binding to IL-2 receptors present on the surface of cells such as T cells, B cells, and NK cells. In addition, IL-2 is known to have an anti-tumor effect and is used as an effective ingredient of a drug for treating specific cancers. However, it is difficult to say that the stability of IL-2 in vivo is high. For example, when a genetically recombinant IL-2 preparation (generic name: tisagenlecleucel (genetically recombinant)) is accurately and constantly intravenously injected into four patients with malignant tumors (adults) at 700,000 units once for 2 hours, the concentration in the serum is highest at the end of administration, but returns to the baseline within several hours thereafter (Non-Patent Literature 1).

[0003] Non-Patent Literature 2 reports that RMA T lymphoma cells having IL-2 on the surface were produced by giving a diphtheria toxin transmembrane domain (T) as a membrane anchor without giving IL-2, thereby causing a defensive anti-tumor immune response in mice. In addition, Non-Patent Literature 3 reports a new membrane-bound protein (MBP) technology, and reports that MBP NK cells in which IL-2 is expressed on the surface of NK-92 cells not only improved proliferation under IL-2-deficient conditions, but also showed strong secretion of cytolytic granules, resulting in enhanced anti-tumor activity both in vitro and in vivo.

[0004] On the other hand, it is known that poly(ethylene glycol) conjugated phospholipid (PEG-lipid) derivatives spontaneously incorporate into lipid bilayer membranes, and for example, Non-Patent Literature 4 investigated the interaction of a product obtained by labeling PEG-lipid with fluorescein isothiocyanate with cells, and reported that FITC-PEG-lipid spontaneously settled on the cell membrane without losing cell viability. In addition, Non-Patent Literature 5 reports how the density and molecular weight of PEG molecules affect the immobilization and incorporation of physiologically active substances into cells.

[0005] Prior Art Documents

[0006] Patent Literature

[0007] Patent Literature 1: Japanese Patent Laid-Open No. 2018-193303 (Japanese Patent No. 6647240)

[0008] Non-Patent Literature

[0009] Non-patent literature 1: Imunace Note 35 Annex, revised in January 2024 (4th edition), https: / / www.info.pmda.go.jp / go / pack / 6399411D1022_1_21 / 6399411D1022_1_21?view=body&lang=ja

[0010] Non-patent literature 2: J Immunother. 2003 Jan-Feb; 26(1): 63-71.

[0011] Non-patent literature 3: Theranostics 2023; 13(5): 1506-1519.

[0012] Non-patent literature 4: Colloids Surf B Biointerfaces. 2015 Nov 1; 135: 765-773.

[0013] Non-patent literature 5: Acta Biomater. 2016 Jan; 30: 135-143. SUMMARY

[0014] Problems to be solved by the Invention

[0015] The present inventors have studied high activity NK-like cells (patent literature 1) and found that, regarding the cells, when the IL-2 concentration in the system decreases to a certain level or less, the aggregation and infiltration ability to solid tumors decreases. Therefore, if the IL-2 signal can be sustained in the body after the administration of high activity NK-like cells, a significant improvement in the remission rate of solid tumors can be expected.

[0016] In addition, high activity NK-like cells are produced using IL-2 and functionally maintained by IL-2 in experiments, and further, since the expression amount of IL-2 receptor alpha is low, internalization is slow, but these are common characteristics present in NK cells after activation culture. Therefore, it is considered that by anchoring IL-2 on the cell membrane of immune cells after activation culture, the cells can autonomously receive the IL-2 signal.

[0017] Means for solving the problems

[0018] The present inventors have attempted to use a phospholipid such as DSPE as a site for insertion into a cell membrane, and to use PEG to link the site for insertion into a cell membrane with IL-2, as a method for anchoring IL-2 to the membrane of an immune cell. Furthermore, by using such a molecule having IL-2 linked thereto, it is possible to anchor IL-2 to various immune cells, and to design the molecule so as to be of a size suitable for the anchored IL-2 to bind to an IL-2 receptor on the cell membrane, thereby completing the present invention. In addition, it was confirmed that using a group capable of forming a bond with a protein on a cell membrane as a method for anchoring IL-2 to the membrane of an immune cell is also effective, thereby completing the present invention.

[0019] [1] A molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-A,

[0020] A is a moiety for anchoring to a cell membrane, which comprises a group capable of forming a bond with a protein on a cell membrane, or a phospholipid moiety.

[0021] [2] The molecule according to [1], wherein the PEG moiety has a molecular weight of 3000 to 8000.

[0022] [3] The molecule according to [1], wherein A is a moiety comprising a group capable of forming a bond with a protein on a cell membrane, and comprises a maleimide reactive group.

[0023] [4] The molecule according to [1] or [2], wherein A is a phospholipid moiety, and the phospholipid moiety is a group derived from a glycerophospholipid.

[0024] [5] The molecule according to any one of [1] to [3], wherein A is a phospholipid moiety, and the phospholipid moiety is a group derived from 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0025] [6] Use of a molecule according to any one of [1] to [5] for the modification of immune cells by IL-2.

[0026] [7] The use according to [6], wherein the immune cells are activated NK cells or NK-like cells.

[0027] [8] An immune cell modified by a molecule according to any one of [1] to [5].

[0028] [9] An activated NK cell or NK-like cell modified by a molecule according to any one of [1] to [5].

[0029]

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

[0030]

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

[0031] The present application provides each of the following.

[0032] [1] A molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)ylated phospholipid.

[0033] [2] The molecule according to [1], wherein the phospholipid moiety is a group derived from a glycerophospholipid.

[0034] [3] The molecule according to [1] or [2], wherein the PEG moiety has a molecular weight of 4500 to 5500.

[0035] [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).

[0036] [5] Use of a molecule, which is a molecule according to any one of [1] to [4], for modification of an immune cell by IL-2.

[0037] [6] The use according to [5], wherein the immune cell is an activated NK cell or NK-like cell.

[0038] [7] An immune cell modified by a molecule according to any one of [1] to [4].

[0039] [8] An activated NK cell or NK-like cell modified by a molecule according to any one of [1] to [4].

[0040] [9] A pharmaceutical composition comprising the cell according to [7] or [8].

[0041] Effects of the Invention

[0042] By the molecule provided by the present application, IL-2 can be anchored to the surface of an immune cell, whereby the immune cell can autonomously receive an IL-2 signal.

[0043] The cell provided by the present application can cause IL-2 signal to persist in vivo after administration, whereby it is expected to exert higher antitumor activity. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is a study of IL-2 anchoring conditions for cell modification.

[0045] Figure 2This study enhanced the invasive ability of IL-2-anchored NK-like cells against solid tumors. A comparison was made between the results of adding IL-2 to the culture medium and those of inserting IL-2 into the cell membrane of NK-like cells for anchoring.

[0046] Figure 3 This refers to the enhanced killing activity of IL-2-anchored NK-like cells against solid tumors. Target cells: NCI-H1975 (3000 cells / sphere), effector cells: revived GAIA-102, PKH26, Sphere×5 vs NK 6×10 4 Each cell.

[0047] Figure 4 This involves determining the number of IL-2-anchored modified molecules.

[0048] Figure 5 This study aimed to enhance the efficiency of host immune cell aggregation in an in vivo solid tumor model by anchoring IL-2. The study included: a group receiving IL-2-anchored modified cells (modified at 100 nM or 10 nM); a group receiving unmodified cells; a group receiving both unmodified cells and IL-2 (10,000 U / cell); and a control group (no treatment).

[0049] Figure 6 This study aimed to enhance the therapeutic efficacy of IL-2 anchoring in an in vivo solid tumor model. The study included: a group receiving IL-2-anchored modification (modified at 100 nM); a group receiving no modification; a group receiving both no modification and IL-2 (10000 U / animal); and a control group (no treatment). n=4 / group, **P<0.01 (vs. control), *P<0.05 (vs. control).

[0050] Figure 7 It is IL-2 anchoring structure 1 (DSPE type) vs structure 2 (maleimide type).

[0051] Figure 8 GAIA-102 was revived and IL-2 anchored overnight at 37°C. The number of IL-2 molecules was determined using anti-human IL-2. The molecular weight of the DSPE type was 3.4kJ, and the molecular weight of the maleimide type was 5kJ.

[0052] Figure 9 Extraction by structure Figure 8 The right-hand xCelligence graph shows that the DSPE type has a molecular weight of 3.4kJ, while the maleimide type has a molecular weight of 5kJ. Detailed Implementation

[0053] In this specification, unless otherwise stated, the descriptions of certain implementation schemes, schemes and embodiments using the same terminology also apply to other implementation schemes, schemes and embodiments.

[0054] [(IL-2)-PEG-A]

[0055] The present embodiment relates to a molecule consisting of interleukin-2 (IL-2)-polyethylene glycol (PEG)-A. A is a moiety for anchoring to a cell membrane, which comprises a phospholipid moiety, or a group capable of forming a bond with a protein on a cell membrane.

[0056] A is a moiety for anchoring to a cell membrane. In one aspect, A comprises a phospholipid moiety derived from a phospholipid.

[0057] (phospholipid moiety)

[0058] The phospholipid moiety is not particularly limited as long as it is incorporated into the cell membrane of an immune cell. The phospholipid moiety can be a glycerophospholipid having glycerol as a backbone, or a sphingomyelin having sphingosine as a backbone. More specifically, the phospholipid moiety can be phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or sphingomyelin.

[0059] In one aspect, the phospholipid moiety is a glycerophospholipid, more specifically, a phosphatidic acid having a fatty acid ester-bonded at the C1 and C2 positions of glycerol, and a phosphoric acid ester-bonded at the C3 position. The groups ester-bonded at the C1 and C2 positions are each independently an acyl group having 10 to 20 carbons, preferably an acyl group having 14 to 18 carbons, which can be the same or different.

[0060] In one aspect, the phospholipid moiety is a group derived from a phospholipid selected from the group consisting of:

[0061] 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium (DOPG), dipalmitoyl phosphatidylglycerol (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-undecanoyl-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-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), palmitoyloleoyl phosphatidylcholine; 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, 1,1 -dibehenoyl-sn-glycero-3-phosphoethanolamine, palmitoyloleoyl phosphatidyl ethanolamine (POPE), distearoyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphatidyl ethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearophosphoethanolamine (SOPE).

[0062] (group capable of forming a bond with a protein on a cell membrane)

[0063] In one aspect, the moiety for anchoring to a cell membrane, i.e., A, comprises a group capable of forming a bond with a protein on a cell membrane.

[0064] The group capable of forming a bond with a protein on a cell membrane is not particularly limited, as long as it is capable of reacting with any of the immune cell membranes.

[0065] In one aspect, the group capable of forming a bond with a protein on the cell membrane can form a bond by reacting with a group of a membrane protein.

[0066] A primary amino group (-NH2) is present at the N-terminus of each protein or in the side chain of a lysine residue. The primary amine is present on the surface of the protein structure due to the positive charge and can be bound without denaturing the protein structure.

[0067] A carboxyl group (-COOH) is present at the C-terminus of a protein or in the side chain of an aspartic acid or glutamic acid. Like the primary amine, the carboxyl group is present on the surface of the protein structure.

[0068] A sulfhydryl group (-SH) is present in the side chain of a cysteine. Normally, cysteines form disulfide bonds inside the protein. Therefore, in order to use in the reaction with the group contained in A in the present aspect, the disulfide bond is reduced to a sulfhydryl group.

[0069] An aldehyde group (-CHO) can be prepared from a ketone or aldehyde group in a glycoprotein by post-translational modification (glycosylation) oxidation of a polysaccharide using sodium periodate.

[0070] In one aspect, the group capable of forming a bond with a protein on the cell membrane is selected from the following chemical groups: carbodiimide (e.g., EDC), NHS ester, imine ester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, haloacetic acid (bromoacetic acid or iodoacetic acid), pyridyl disulfide, thio-sulfone, vinyl sulfone, hydrazide, alkoxy amine, diazirine, aryl azide, isocyanate.

[0071] In one particularly preferred aspect, the group capable of forming a bond with a protein on the cell membrane is a maleimide. The maleimide is capable of reacting with an SH group of a protein on the cell membrane.

[0072] The protein on the NK cell membrane is not particularly limited as long as it is capable of reacting with the specified group. As the protein on the NK cell membrane, the following can be exemplified: NKp46, NKp30, NKG2D, IL-15R, IL-2R, KIR3DS1, NKG2C, NKp80, NKp65, NKp44, LALRA1, LILRA2, DNAM-1, and 2B4.

[0073] (PEGylated moiety)

[0074] The size (length) of the PEG moiety of the IL-2-PEGylated phospholipid of the present embodiment is not particularly limited, provided that the bound IL-2 can bind to the IL-2 receptor on the cell membrane. The molecular weight of the PEG moiety is, for example, 550 or more, preferably 700 or more, more preferably 1000 or more, further preferably 1500 or more, and can be 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more. In addition, the molecular weight of the PEG moiety is, for example, 10000 or less, preferably 9500 or less, more preferably 9000 or less, further preferably 8500 or less, and can be 8000 or less, 7500 or less, 7000 or less, 6500 or less, 6000 or less, 5500 or less. In one of the preferred embodiments, the molecular weight of the PEG moiety of the IL-2-PEGylated phospholipid is 4000 to 6000, more preferably 4500 to 5500, and further preferably 3000 to 4000, for example, 3400. In addition, the molecular weight of the PEG is usually expressed as an average value.

[0075] In the IL-2-PEGylated phospholipid of the present embodiment, the PEG moiety and the phospholipid moiety can be bound via a linker moiety L1.

[0076] L1 is, for example, -O-, -C(=O)-, -C(=O)-O-, -O-C(=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 -O-C(=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 carbons, and is preferably an aliphatic group having 1 to 3 carbons.

[0077] In one of the preferred embodiments, the phospholipid moiety is a phosphatidylethanolamine, L1 is -C(=O)-, and the PEGylated phospholipid moiety can be represented as follows.

[0078] [Chemical Formula 1]

[0079]

[0080] In the formula, each of the R1CO group and the R2CO group is independently an acyl group having a carbon number of 14 to 18, and the R1CO group and the R2CO group can be the same or different.

[0081] In one of the preferred aspects, the PEGylated phospholipid moiety is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol).

[0082] In the present embodiment, by using PEG instead of an amino acid to link the phospholipid moiety and the IL-2 moiety, the length of the linking moiety can be adjusted, and antigenicity can be maintained low. In addition, it can be said that by using PEG, the flexibility of the linking moiety can be maintained high, and the length can be adjusted according to the characteristics of the cell to be modified.

[0083] (IL-2 moiety)

[0084] In the present embodiment, IL-2 is bound to the end of the PEGylated phospholipid. IL-2 can be bound to the end of the PEGylated phospholipid, and the source thereof or the like is not limited as long as the target function can be exerted. In one aspect, the IL-2 is a mammalian wild-type IL-2, or a variant thereof, preferably the IL-2 is a human IL-2, or a variant thereof.

[0085] The variant of IL-2 can be a fragment, an analog, and the like. The fragment refers to a polypeptide that includes only a part of the full-length sequence of IL-2. The analog refers to a polypeptide that includes a substitution, an insertion, or a deletion of one or more amino acids in the full-length sequence of IL-2. The derivative includes any modified IL-2 polypeptide, a fragment thereof, and an analog thereof, which is modified by, for example, glycosylation, phosphorylation, fusion with another polypeptide or molecule, polymerization, and the like, or which is added by chemical or enzymatic modification, for the improvement of the properties of IL-2 (e.g., stability, specificity, and the like).

[0086] IL-2 can be produced by a recombinant technique. The host organism for expression of the gene encoding IL-2 can be a prokaryote (bacteria such as Escherichia coli), or a eukaryote (e.g., yeast, fungi, plant, or mammalian cells). The method for producing IL-2 by culturing such cells is well known to those skilled in the art. IL-2 can 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.

[0087] IL-2 is commercially available for pharmaceutical use, and is approved for use in human patients. The forms of the commercially available IL-2 drugs include, for example, the following:

[0088] • Teceleukin (Genetical Recombination), Aldesleukin (USAN / INN), Interleukin-2.

[0089] • Aldesleukin, Interleukin-2, Aldesleukin (USAN / INN), Interleukin-2.

[0090] In one of the preferred aspects, the IL-2 is Teceleukin, or Aldesleukin. Teceleukin is a 134-amino acid polypeptide produced by recombinant DNA technology in E. coli using IL-2 mRNA derived from human spleen lymphocytes as starting material. Aldesleukin is a nonglycosylated variant of mature human IL-2, which contains two amino acid modifications (deletion of the first amino acid (alanine) and substitution of cysteine at position 125 with serine) compared to mature human IL-2.

[0091] The IL-2 used in one aspect has a specific activity of 1.2 to 24 million international units (MIU) per 1 mg of protein, preferably 8 to 18 MIU per 1 mg of protein.

[0092] In the (IL-2)-PEGylated phospholipid of the present embodiment, the PEG moiety can be bound to the IL-2 via a linker moiety L2.

[0093] L2is, for example, -O-, -C(=O)-, -C(=O)-O-, -O-C(=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 -O-C(=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 carbons, preferably an aliphatic group having 1 to 3 carbons.

[0094] (Preparation method)

[0095] The interleukin-2 (IL-2)-polyethylene glycol (PEG)-A of the present embodiment can be prepared using various reactions that are already known.

[0096] For example, it can be easily prepared by reacting a moiety for anchoring to the cell membrane - PEG-NHS in which one end of PEG is esterified with NHS, with IL-2. Maleimide-PEG-NHS is commercially available in PEG moieties of various molecular weights.

[0097] In addition, for example, it can be easily prepared by reacting a PEGylated phospholipid in which one end of PEG is esterified with NHS, with IL-2.

[0098] NHS ester is a reactive group formed by carbodiimide activation of a carboxyl molecule. NHS ester reacts with the primary amine of IL-2 under physiological conditions to weakly basic conditions (pH 7.2-9) to form a stable amide bond. By this reaction, N-hydroxysuccinimide (NHS) is released.

[0099] [Chemical Formula 2]

[0100]

[0101] The conditions for reacting a moiety for anchoring to the cell membrane - PEG-NHS, represented by maleimide-PEG-NHS, with IL-2 can be appropriately carried out by those skilled in the art.

[0102] More specifically, the reaction of a PEGylated phospholipid in which one end of PEG is esterified with NHS, with IL-2 can be obtained by adding 1-20 equivalents of a PEGylated phospholipid esterified with NHS to IL-2 dissolved in an appropriate solvent, such as DMSO, and allowing it to react.

[0103] As a PEGylated phospholipid in which one end of PEG is esterified with NHS, commercially available products can be used. For example, the following are commercially available: DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-PEG20k-NHS, DSPE-PEG10k-NHS, DSPE-PEG5k-NHS, DSPE-PEG3k-NHS, DSPE-PEG2k-NHS, DSPE-PEG1k-NHS, DSPE-PEG-NHS, MW 5000 (Nanocs Inc.); DSPE-PEG-NHS, MW 600, DSPE-PEG-NHS, MW 1000, DSPE-PEG-NHS, MW 2000, DSPE-PEG-NHS, MW 5000 (BroadPharm); DSPE-PEG(2000) Carboxy NHS (Avanti Polar Lipids); DSPE-PEG-NHS (MW 3400) (MedChemExpress); DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine)-PEG5k-NHS, DPPE-PEG3k-NHS.

[0104] Methods for preparing PEGylated lipids are well known to those skilled in the art. Those skilled in the art can refer to, for example, Japanese Patent Publication No. 2007-8933, International Publication WO2005 / 026372 (Japanese Patent Publication No. 2007-505954, Japanese Patent No. 4842821), and the like.

[0105] 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 the present embodiment.

[0106] ((Function of (IL-2)-PEG-A)

[0107] The (IL-2)-PEG-A of the present embodiment can be used to anchor IL-2 to an immune cell.

[0108] For the group of one aspect that forms a bond with the (IL-2)-PEG-cell membrane protein, the group can react with a prescribed group of the protein on the membrane of the immune cell, and anchor IL-2 to the membrane of the cell.

[0109] For the phospholipid of the (IL-2)-PEGylated phospholipid of another aspect, the phospholipid portion can be incorporated into the immune cell, and anchor IL-2 to the membrane of the cell.

[0110] IL-2 acts through a tetrameric receptor signaling complex comprising a (IL-2Rα), β (IL-2Rβ), common γ chain (γc) receptor (Science. 2005 Nov 18; 310(5751): 1159-63. doi: 10.1126 / science.1117893.). In order to be able to form such a complex on the membrane of a cell, the length of the PEGylated portion can be set to be, for example, 4 nm or more, preferably 6 nm or more, and more preferably 8 nm or more.

[0111] IL-2 anchored to the membrane of a cell can bind to the IL-2 receptor on the same membrane as long as the PEG portion is an appropriate length, and thus the cell can autonomously receive the IL-2 signal, and can receive it continuously.

[0112] In consideration of the binding to the IL-2 receptor, it is considered that the PEGylated portion of the (IL-2)-PEGylated phospholipid of the present embodiment is preferably about 8 nm in length.

[0113] [Modified immune cells]

[0114] The present embodiment relates to an immune cell having IL-2 anchored to the membrane of the cell by (IL-2)-PEG-A. The immune cell having IL-2 anchored is sometimes referred to as a modified immune cell.

[0115] The immune cells express the IL-2 receptor on the cell membrane, and as long as the IL-2 signal can be received, there is no particular limitation. One of the immune cells to which the present embodiment can be favorably applied is a cell administered for immunotherapy. Preferred is a cell administered to a human for immunotherapy and subjected to an activation operation in vitro. The activation operation is preferably those performed using IL-2. The activation operation is typically performed by culturing the cell using a culture medium containing IL-2.

[0116] Such immune cells include NK cells, highly active NK-like cells (cells of Patent Literature 1), cells described in Japanese Patent Application Publication No. 2019-170176 (Japanese Patent No. 6543375) that exhibit extremely high cytotoxic activity against solid cancer forming a tumor mass, NK-92 cells, T cells, effector T cells, CAR-T cells. In addition, in the present specification, the case where the present application and the present embodiment are applied to highly active NK-like cells is sometimes exemplified for explanation, but the person skilled in the art can also appropriately understand the case where the present application is applied to cells other than these based on the explanation.

[0117] (NK cells, highly active NK-like cells, etc.)

[0118] In one aspect, the immune cells are NK cells, highly active NK-like cells (cells of Patent Literature 1), cells described in Japanese Patent Application Publication No. 2019-170176 (Japanese Patent No. 6543375) that exhibit extremely high cytotoxic activity against solid cancer forming a tumor mass, NK-92 cells. The highly active NK-like cells include GAIA-102 (see Example of the present specification). These cells are sometimes collectively referred to as NK cell species.

[0119] In general, NK cells are large granular lymphocytes that do not express T cell receptors (TCRs), CD3 as a pan-T cell marker, and B cell receptors as membrane immunoglobulins, and in humans, are usually CD16-positive and CD56-positive. As to whether or not they are NK cells, the person skilled in the art can easily judge based on the expression pattern of cell surface markers and the like. NK cells have cytotoxic activity, and the presence or absence or degree of the cytotoxic activity can be determined by various methods known per se. The NK cells can include primary NK cells, cultured NK cells, activated NK cells.

[0120] NK-92 cells are IL-2-dependent NK cell lines obtained from peripheral blood mononuclear cells of non-Hodgkin lymphoma patients (Non Patent Literature 3).

[0121] The raw material of the NK cell group is not particularly limited, and can be peripheral blood, umbilical cord blood, bone marrow and / or lymph node, blood collected by apheresis (apheresis blood). In addition, the raw material can be one prepared from any cell selected from the group consisting of hematopoietic stem cells derived from any stem cell selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent (iPS) stem cells, hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem cells derived from peripheral blood, hematopoietic stem cells derived from bone marrow blood, umbilical cord blood mononuclear cells, peripheral blood mononuclear cells (PBMC). The donor of the raw material is sometimes the patient himself / herself who receives immunotherapy using high-activity NK cells and the like, a close relative of the patient, or a healthy person who has no blood relationship with the patient. The donor can be plural.

[0122] In one of the preferred aspects, the immune cell is any one of the following [1], [2], [3], and [4].

[0123] [1] NK-like cell having the following (1) and (2) characteristics:

[0124] (1) CD16 positive, CD56 highly expressed, and CD57 negative.

[0125] (2) NKG2C positive, NKG2A negative to low expression, and CD94 positive.

[0126] The NK-like cell of [1] can be CD16 highly expressed. In addition, the NK-like cell of [1] can be a high-activity NK-like cell further having the following characteristics, regardless of whether it is CD16 highly expressed or not.

[0127] (3) Cytotoxic activity of 50% or more when co-cultured with NK cells as effector cells (E) and K562 cells as target cells (T) at a mixing ratio (E:T) of 1:1.

[0128] The high-activity NK-like cell of [1] can also be as follows:

[0129] From peripheral blood mononuclear cells derived from a healthy person, CD3 positive cells are removed using CD3 magnetic beads (e.g., CliniMACS CD3, Miltenyi Biotec, Catalog No. 130-017-601, or an equivalent product thereof), an LD column (e.g., Miltenyi Biotec, Catalog No. 130-042-901, or an equivalent product thereof), and a separation buffer (e.g., PBS containing 0.5% human AB serum (inactivated), 2 mM EDTA), and a cell population is obtained, which is cultured in an appropriate medium (e.g., COSMEDIUM 008 added with 5% human AB serum (inactivated)) for 14 days, and NK cells having the following (1) and (3) characteristics are obtained:

[0130] (1) is CD16 positive, CD56 highly expressed, and CD57 negative.

[0131] (3) has a cytotoxic activity of 50% or more when co-cultured as effector cells (E) with K562 cells as target cells (T) at a mixed ratio (E:T) of 1:1.

[0132] Details of the characteristics of the highly active NK cells of [1], or a more specific method of production, can be found in Patent Document 1.

[0133] [2] the following cell:

[0134] is CCR5 positive, CCR6 positive, and CXCR3 positive, and CD3 negative.

[0135] The cell of [2] can further be CD11c highly expressed.

[0136] The cell of [2] can also be as follows:

[0137] is CCR5 positive, CCR6 positive, CXCR3 positive, integrin alpha 1 positive, integrin alpha 3 positive, and integrin beta 3 negative, and CD3 negative. Or the following cell: is CCR5 positive, CCR6 positive, CXCR3 positive, CD11a highly expressed, and CD11c highly expressed, and CD3 negative, the high expression being judged by comparison with the expression in a population of NK cells obtained from peripheral blood without substantial culture.

[0138] According to the research by the present inventors, the cell of [2] shows extremely high cytotoxic activity against solid cancer that forms a tumor mass. Details of the characteristics of the cell of [2], or a more specific method of production, can be found in Japanese Patent Publication No. 2019-170176 (Japanese Patent No. 6543375).

[0139] [3] a highly active NK cell that can be obtained by the following method:

[0140] To mononuclear cells obtained from fresh peripheral blood, or from frozen apheresis blood, CD3 magnetic beads (e.g., CliniMACS CD3, Miltenyi Biotec, 130-017-601 (5 μL per 1 x 10 7 cells) are added, and when frozen apheresis blood is used, CD34 magnetic beads (e.g., CliniMACS CD34, Miltenyi Biotec, 130-017-501 (5 μL per 1 x 10 7cells) and suspended, after incubation at 4°C for 15 minutes, separation buffer (e.g., PBS containing 0.5% human AB serum (resultant of inactivation treatment at 56°C for 30 minutes), 2 mM EDTA) was added to suspend well, and centrifugation was performed. The supernatant was removed, and the cells were suspended in 0.5 mL of separation buffer at a cell number of 1 x 10 8 cells) and suspended, after pre-addition of 2 mL of separation buffer, the cell suspension was added to the LD column, and the eluate from the LD column was recovered. Further, 1 mL of separation buffer was added to the LD column, and the eluate was recovered. The recovered liquid was subjected to centrifugal separation, and after removal of the supernatant, the cells were suspended in a suitable medium (e.g., KBM501 medium containing either 5% human AB serum (resultant of inactivation treatment at 56°C for 30 minutes) or 2 U / mL of heparin sodium added to 5% UltraGRO (AventaCell, HPCPLCRL10)) at a cell number of 5 x 10 5 cells) and suspended, after pre-addition of 2 mL of separation buffer, the cell suspension was added to the LD column, and the eluate from the LD column was recovered. Further, 1 mL of separation buffer was added to the LD column, and the eluate was recovered. The recovered liquid was subjected to centrifugal separation, and after removal of the supernatant, the cells were suspended in a suitable medium (e.g., KBM501 medium containing either 5% human AB serum (resultant of inactivation treatment at 56°C for 30 minutes) or 2 U / mL of heparin sodium added to 5% UltraGRO (AventaCell, HPCPLCRL10)) at a cell number of 5 x 10 6 cells) and suspended, after pre-addition of 2 mL of separation buffer, the cell suspension was added to the LD column, and the eluate from the LD column was recovered. Further, 1 mL of separation buffer was added to the LD column, and the eluate was recovered. The recovered liquid was subjected to centrifugal separation, and after removal of the supernatant, the cells were suspended in a suitable medium (e.g., KBM501 medium containing either 5% human AB serum (resultant of inactivation treatment at 56°C for 30 minutes) or 2 U / mL of heparin sodium added to 5% UltraGRO (AventaCell, HPCPLCRL10)) at a cell number of 5 x 10

[0141] The specific preparation method of the high-activity NK cells of [3] can be found in the Examples of the present specification.

[0142] [4] In obtaining the cells of [1] to [3], the cells obtained by culturing with the addition of any one selected from the group consisting of IL-12, IL-15, and IL-18 at a concentration that can achieve the object of the present application, simultaneously with or instead of IL-2. The specific preparation method of such cells can be found in Non-Patent Literature 2 (Leong JW et al. Biol Blood Marrow Transplant 20 (2014) 463-473).

[0143] For the present application, when the activity or cytotoxic activity is mentioned with respect to NK cells, unless otherwise specified, it refers to the ability of the subject cells (effector cells, E) to lyse target cells (T). The cytotoxic activity can be expressed as the percentage (%) of target cells killed by the effector cells, and is calculated by the following formula.

[0144] (cell death when co-cultured with effector cells - natural cell death (negative control)) / (maximum cell death (positive control) - natural cell death (negative control)) x 100

[0145] In measuring cytotoxic activity, the mixing ratio of effector cells to target cells (E:T), and the time for which effector cells and target cells are co-cultured can be appropriately set depending on the kind of cells used or the strength of activity, in general, according to the degree of cytotoxic activity of effector cells. When NK cells are used as effector cells, the target cells are sometimes K562 cells, acute myeloid leukemia cells, chronic myeloid leukemia cells, but are not limited thereto. Effector cells and target cells, living cells and dead cells can be distinguished and quantified by reagents such as antibodies labeled with radioactive substances, fluorescent pigments, and the like. Cytotoxic activity when NK cells are used as effector cells can be measured, for example, under the following conditions, i.e., K562 cells are used as target cells, the E:T is set to 1:0.05 to 10, preferably 1:0.1 to 5, and the incubation time is set to 0.5 to 18 hours, preferably 1 to 12 hours.

[0146] For the present application, when referring to a high NK cell-like activity, unless otherwise specified, it means that the cytotoxic activity is 50% or more when K562 cells are used as target cells, mixed at an E:T 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.

[0147] (Effector T cells, CAR-T cells)

[0148] In one aspect, the immune cells are effector T cells. Effector T cells are T cells that enhance the attack on cancer cells, and in general, are T cells taken out of a patient to the outside of the body, and introduced into a gene for attacking cancer cells, and returned to the patient again. CAR-T cells are a type of effector T cells, and as a gene for distinguishing tumor antigens, a chimeric antigen receptor (CAR) gene can be used. IL-2 is known to function as a T cell growth factor indispensable for the proliferation and survival of T cells, and the production of effector T cells. In addition, it has been reported that CAR-T cells that react with tumor-specific antigens and can secrete a certain level of IL-2 can infiltrate into solid cancers to suppress tumor growth (SCIENCE. 16 Dec 2022. Vol 378, Issue 6625. DOI:10.1126 / science.aba16).

[0149] (Anchoring, modification)

[0150] In the modified immune cells of the present embodiment, the phospholipid portion of the above (IL-2)-PEGylated phospholipid is incorporated into the membrane of the immune cells, anchoring IL-2 to the membrane of the cells. The anchoring can be easily performed. For example, it can be achieved by incubation at a prescribed temperature and for a prescribed time after mixing the immune cells with the (IL-2)-PEGylated phospholipid. The temperature is not particularly limited, and is, for example, 15 to 42°C, preferably 20 to 40°C, more preferably 22 to 38°C, and can also be room temperature (about 25°C). The time is not particularly limited, and is, for example, 1 minute to several hours, preferably 2 to 60 minutes, more preferably 3 to 30 minutes, and further preferably 5 to 20 minutes.

[0151] The number of IL-2 anchored to the immune cells can be appropriately set by adjusting the cell density and the concentration of the (IL-2)-PEGylated phospholipid at the time of culture.

[0152] For the cell density of the immune cells at the time of culture, it can be set to, for example, 1 x 10 5 or more, preferably 2 x 10 5 or more, more preferably 3 x 10 5 or more, and further preferably 5 x 10 5 or more, under any other condition. It can be set to, for example, 1 x 10 8 or less, preferably 5 x 10 7 or less, more preferably 2 x 10 7 or less, and further preferably 1 x 10 7 or less, under any other condition.

[0153] For the concentration of the (IL-2)-PEGylated phospholipid at the time of culture, it can be set to, 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, 1000 nM or more, 1500 nM or more, 1800 nM or more, under any other condition. It can be set to, for example, 10000 nM or less, 9000 nM or less, 8000 nM or less, 7000 nM or less, 6000 nM or less, 5000 nM or less, 4000 nM or less, 3000 nM or less, 2000 nM or less, under any other condition.

[0154] In the modified immune cells of the present embodiment, the number of IL-2 molecules anchored to the cell surface of one immune cell is, in one aspect, one or more, for example, two or more, four or more, eight or more, ten or more, 20 or more, 30 or more, 50 or more, 100 or more. According to the research by the present inventors, effectiveness was confirmed for the case where the number of IL-2 molecules anchored to one immune cell was eight. One of the preferable aspects is an immune cell having 50 to 3000 IL-2 molecules anchored to the cell surface per cell. In addition, the quantification of the number of IL-2 molecules anchored per cell can be appropriately performed by those skilled in the art, and can be performed by referring to the method shown in the examples of the present specification.

[0155] According to the research by the present inventors, when IL-2 is anchored at 3000 per cell, if all of the IL-2 is eluted, it is calculated that 200 IU equivalent of IL-2 is eluted per 2 x 10 8

[0156] If necessary, the modified immune cells can be adjusted by a step of removing (IL-2)-PEGylated phospholipids that are not anchored to the immune cells.

[0157] The modified immune cells of the present embodiment can autonomously and continuously receive IL-2 signals. It is difficult to say that the stability of the previous IL-2 in vivo is high. For example, when a recombinant IL-2 preparation (generic name: tisagenlecleucel (recombinant)) is administered to four patients with malignant tumors (adults) at 700,000 units once by intravenous injection at a constant rate for exactly 2 hours, the concentration in the serum is highest at the end of administration, and then returns to the baseline within several hours (non-patent literature 1). According to the present embodiment, such a disadvantage can be overcome.

[0158] One of the particularly preferable aspects is a high-activity NK-like cell to which IL-2 is anchored (patent literature 1). With respect to the high-activity NK-like cell (patent literature 1), when the IL-2 concentration in the system decreases to a certain level or less, the ability to aggregate and infiltrate into a solid tumor decreases, but by anchoring IL-2 to the cell membrane, the high-activity NK-like cell can continuously receive IL-2 signals, and thus a significant improvement in the remission rate for a solid tumor can be expected.

[0159] [Pharmaceutical composition]

[0160] The present embodiment relates to a pharmaceutical composition. The pharmaceutical composition of the present embodiment includes the above-described modified immune cell group and a pharmaceutically acceptable additive. The pharmaceutical composition of the present embodiment including the immune cells can be a product for regenerative medicine or the like.

[0161] With respect to the pharmaceutically acceptable additive, for example, isotonic agents, pH adjusting agents, buffers, stabilizers, cryoprotective agents, antibiotics, and the like can be exemplified. Specifically, water, ethanol, sodium chloride, glucose, albumin, and the like can be exemplified.​

[0162] The pharmaceutical composition of the present embodiment can contain an antibody. The antibody is preferably one that can be used as an antibody drug. In addition, one that can induce Antibody-Dependent-Cellular-Cytotoxicity (ADCC) is preferred. The antibody used in the pharmaceutical composition of the present invention can be any one of a murine antibody, a chimeric antibody, a humanized antibody, and a human antibody, and is preferably a humanized antibody or a human antibody. In addition, the antibody used can be one that has been modified or one that has been produced by the Potelligent technology (removal of fucose from the IgG Fc region). The antibody-containing scheme is not limited to the case where the pharmaceutical composition contains NK cells, and is also one of the preferred schemes in the case where it contains effector T cells, CAR-T cells.

[0163] As specific examples of antibodies used as antibody drugs and which can be used in the pharmaceutical compositions of the present embodiment, the following can be cited: ibritumomab tiuxetan, iodine 131, catumaxomab, blinatumomab, muromonab-CD3, abciximab, rituximab, basiliximab, infliximab, cetuximab, brentuximab, siltuximab, dinutuximab, obiltoxaximab, daclizumab, palivizumab, trastuzumab, gemtuzumab, alemtuzumab, omalizumab, efalizumab, bevacizumab, natalizumab, tocilizumab, ranibizumab, eculizumab, certolizumab pegol, adalimumab, ustekinumab, ipilimumab, ofatumumab, pertuzumab, denosumab, tositumomab, panitumumab, ocrelizumab, secukinumab, sarilumab, clivatuzumab, farletuzumab, elotuzumab, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatumomab mafenatox, anatummogamulizumab, pertuzumab, trastuzumab, obinutuzumab, vedolizumab, pembrolizumab, idarucizumab, mepolizumab, elotuzumab, daratumumab, ixekizumab, reslizumab, adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab, ipilimumab, belimumab, raxibacumab, ramucirumab, nivolumab, secukinumab, evolocumab, alirocumab, and necitumumab.

[0164] The antibody used in the pharmaceutical composition of the present embodiment is preferably one having high affinity to CD16. In addition, in a protocol including an NK cell group (NK cells, NK-like cells, etc.) as immune cells, it is preferable that at least a part of the antibody binds to the NK cell group. According to the research by the present inventors, when mogamulizumab prepared using the Potelligent technology is used as the antibody, in a system in which the antibody is present in co-culture of highly active NK-like cells and target cells, and in a system in which highly active NK-like cells and the antibody are mixed in advance, and the NK-like cells are washed after co-culture with target cells (to remove the antibody that does not bind to the cells) (Patent Literature 1), higher cytotoxic activity is observed in the latter. On the other hand, when dinutuximab is used under the same conditions, the effect of mixing highly active NK-like cells and the antibody in advance is not observed. This suggests that, as long as the antibody is one having high affinity to CD16, the antibody binds to the NK cell group by being mixed with the NK cell group in advance, and thereby higher ADCC activity can be exerted. In addition, depending on the research conditions, it is considered that even dinutuximab can have the same effect.

[0165] The pharmaceutical composition of the present embodiment is sometimes prepared at the time of use. For example, the pharmaceutical composition of the present embodiment can be prepared by thawing frozen high-activity NK-like cells just before administration to a subject, and providing a molecule for anchoring IL-2-PEG-DSPE or the like IL-2 to the modified immune cells.

[0166] The pharmaceutical composition of the present embodiment is sometimes of a different HLA genotype from the NK cells prepared by the preparation method of the present embodiment, and is administered to a patient.

[0167] The pharmaceutical composition of the present embodiment is typically in a form in which the immune cells are suspended in a solution. The solution for suspending the immune cells is usually, for example, a cryoprotective solution containing DMSO, physiological saline, phosphate-buffered physiological saline (PBS), a culture medium, serum, or the like. The solution sometimes contains a pharmaceutically acceptable carrier as a drug product and a quasi-drug.

[0168] The pharmaceutical composition of the present embodiment is suitable for use in the treatment of cancer, or infection. In addition, the pharmaceutical composition of the present embodiment can be used in the treatment of various diseases to which immune cells are sensitive. In the present invention, when treatment is mentioned with respect to a disease or state, it includes: reduction in risk of onset, delay in onset, prevention, treatment, stop of progression, delay. Treatment includes curative treatment (treatment to remove the cause) and symptomatic treatment (treatment to improve symptoms).

[0169] The disease includes, for example, oral cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, liver cancer, large intestinal cancer, kidney cancer, bladder cancer, leukemia, or infection caused by a virus, bacteria, or the like, but is not limited thereto. The cell therapy of the present embodiment is sometimes performed alone, or in combination with surgical treatment, chemical treatment, radiation treatment, an antibody drug, or the like. In the cell therapy using the pharmaceutical composition of the present embodiment, the NK cells are sometimes administered to, for example, a vein, an artery, subcutaneously, intraperitoneally, or the like.

[0170] The production of the pharmaceutical composition of the present embodiment is preferably performed under conditions complying with the rules for the production and quality management of drug products and quasi-drug products (good manufacturing practice (GMP)) and the standards for the production and quality management of products such as regenerative medicine (Good Gene, Cellular, and Tissue-based Products Manufacturing Practice (GCTP)).

[0171] In the present embodiment, the collection of umbilical cord blood and peripheral blood whole blood, the preparation of autologous serum, the preparation of mononuclear cells from whole blood, the measurement of cell number before and after culturing the mononuclear cells, the measurement of the constituent 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 expansion fold, and the statistical analysis regarding the measurement error or significance can be performed using any method known to those skilled in the art.

[0172] Hereinafter, the present application will be further specifically described using examples, but the examples do not limit the technical scope of the present application. The technical scope of the present application is only limited by the recitations of the claims.

[0173] Example

[0174] [Methods common to the examples]

[0175] Throughout all of the examples, the following methods were used, unless otherwise specified.

[0176] A). Culture method of highly active NK-like cells GAIA-102

[0177] Using frozen apheresis blood (Cellero) as a raw material, after recovery, washing and concentration were performed using a Lovo cell processing system (FRESENIUS KABI) to obtain PBMCs.

[0178] Using CliniMACS Prodigy (registered trademark) (Miltenyi Biotec), CD3-positive cells and CD34-positive cells were removed from the obtained PBMCs, and the cells were cultured in KBM501 medium ※1 Elution. The number of cells in the eluate was counted, and the total number of cells was calculated. Culture was performed using a 640 cm2adherent culture bag (product name GICB640NA, Fukoku Co., Ltd.) or a 75 cm2flask, and was prepared in KBM501 medium at 5 x 10 5 Elution. The number of cells in the eluate was counted, and the total number of cells was calculated. Culture was performed using a 640 cm2adherent culture bag (product name GICB640NA, Fukoku Co., Ltd.) or a 75 cm2flask, and was prepared in KBM501 medium at 5 x 10

[0179] ※1: KBM501 (Kohjin Bio) to which 5% UltraGRO (AventaCell, HPCPLCRL10) and 2 U / mL heparin sodium (Nipro) were added

[0180] B). Recovery method of highly active NK-like cells GAIA-102

[0181] The culture solution was recovered on day 14 of the culture, and then 1 mM EDTA was added to the culture bag to detach the adherent cells. After centrifugation of the whole cell recovery solution including the detached cells, washing with KBM501 medium and resuspension were performed.

[0182] C). Freezing method of highly active NK-like cells GAIA-102

[0183] The number of viable cells of GAIA-102 obtained by the order described in the highly active NK-like cell culture method and the recovery method was counted, 2 x 10 8 cells were suspended in 5 mL of HSC-BANKER (ZENOAQ, CB071) and frozen at -80°C. The recovery was performed according to the method described in the examples of WO2023 / 038037 and PCT / JP2024 / 009978, and diluted 41 times with PlasmaLyte-A.

[0184] D). Viability determination method using 7-AAD staining

[0185] GAIA-102 after recovery under each condition was prepared at 1 x 10 5 cells / well in a 96-well plate (IWAKI, 4870-800SP), and centrifuged. After removing the supernatant, 7-AAD solution (Beckman Coulter, A07704) diluted with PBS (Nacalai Tesque, 14249-95) was added and suspended, and incubated at room temperature for 20 minutes. The stained cells were determined using a flow cytometer (BD LSR Fortessa, BD Biosciences), and analyzed using FlowJo software, and the viability was calculated from the 7-AAD positive rate.

[0186] E). Determination method of killing activity against tumor cells

[0187] In order to determine the cytotoxic activity, a group in which the recovered GAIA-102 was reacted with K562 cells, a group of only K562 cells as a negative control, and a group in which K562 cells were treated with 10% formalin as a positive control were prepared.

[0188] <GAIA-102>

[0189] For GAIA-102 resuscitated under each condition, IL-2 anchor-modified, and diluted, the necessary amount was taken based on the number of viable cells at the time of freezing, and a concentration of 1 x 10 6 cells / mL was prepared with 10% FBS / RPMI1640.

[0190] K562

[0191] K562 cells were suspended in serum-free RPMI1640 medium, stained using a PKH26 red fluorescent cell linker kit (Sigma-Aldrich), and then prepared at a concentration of 2 x 10 6 cells / mL with 10% FBS / RPMI1640.

[0192] GAIA-102 and K562 cells were added to a 96-well plate (IWAKI, 4870-800SP) at a cell ratio of 1:1, 2:1, and 8:1 and mixed so as to react at 37°C under 5% CO2 for 2 hours. After the reaction, centrifugal separation was performed (500 x g, 5 minutes), and after removing the supernatant, a 7-AAD solution diluted with PBS was added to suspend, and incubation was performed at room temperature for 20 minutes. Flow cytometry was used for measurement, and the results were analyzed using FlowJo software to calculate the cytotoxic activity rate (lysis rate) ※2 .

[0193] ※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

[0194] F) Method for calculating correction value using statistical analysis of killing activity rate

[0195] Based on the cytotoxic activity rate calculated when the ET ratio was 4 points (including 0) or more, the calculated values for ET ratios of 1 and 2 were calculated by nonlinear regression analysis using JMP (registered trademark) Pro statistical analysis software.

[0196] G) Measurement of the number of IL-2 anchor molecules per cell using PE magnetic beads

[0197] Using a BD Quantibrite™ PE phycoerythrin fluorescent quantification kit (Cat. no. 340495, BD, a PE molecule number counting kit using PE-labeled magnetic beads of a known molecule number), based on the standard curve thereof, the number of molecules of IL-2 on the cell surface was calculated based on the fluorescence intensity of the cells after staining with a PE-labeled anti-human IL-2 antibody (clone MQ1-17H12, Cat No. 500307, Biolegend).

[0198] [Example 1: Study of IL-2 anchoring on cell modification conditions]

[0199] The following reagents were used.

[0200] • IL-2 (recombinant human IL-2, Peprotech, AF-200-02)

[0201] • NHS-PEG 5K - DSPE (NANOCS, PG2-DSNS-5k)

[0202] IL-2 anchoring was prepared in the following order.

[0203] That is, 1-20 equivalents of NHS-PEG-DSPE (10 mg / mL-DMSO) were added to IL-2 (2 mg / mL-DMSO), and PBS was added to a DMSO concentration of 50%, and the resultant was allowed to react at room temperature overnight. A part thereof was preserved as a crude sample, and another part was added with PBS to a final volume of 1 mL, and buffer exchange was performed using a PD Miditrap G-25, and was preserved refrigerated (4°C) or frozen (-80°C) as a purified sample.

[0204] [Table 1]

[0205]

[0206] IL-2 anchoring was added to GAIA-102 (1 x 10 6 cells / mL) so as to become a final concentration of 10 nM, 100 nM, 1000 nM, respectively, and was incubated at room temperature for 10 minutes. Thereafter, staining was performed with a PE-labeled anti-human IL-2 antibody, and quantification of the number of IL-2 anchoring per cell was performed using a PE phycoerythrin fluorescence quantification kit.

[0207] The results are shown in Figure 1 . IL-2 anchoring was on the cells in a manner dependent on the amount of IL-2 anchoring added. When IL-2 was anchored at 3000 / cell, if all was eluted, it was calculated that 200 IU equivalent of IL-2 / 2 x 10 8 cells.

[0208] [Example 2: Improvement of solid tumor infiltration ability of IL-2 anchoring NK-like cells]

[0209] IL-2 anchoring was prepared in the following order.

[0210] [Table 2]

[0211]

[0212] The cryopreserved high activity NK-like cells were recovered, stained with CytoTell™ UltraGreen, and added to spheroids prepared with HsOs-1 cells. In detail, HsOs-1 cells were detached with trypsin EDTA, prepared in 100 μL so as to be 3 x 10 3 cells, inoculated to an EZ-Bind Shut II (registered trademark) microplate (IWAKI, Cat. 4870-800LP), and incubated at 37°C for 48 to 72 hours to form spheroids. The experiment used a 384-well plate, 1 spheroid per well, and 2.5 x 10 4 cells per well. For the group in which IL-2 was anchored to the high activity NK-like cells, IL-2 was anchored in the same manner as in Example 1, added to the high activity NK-like cells (1 x 10 6 cells / mL) so as to be a final concentration of 1000 nM, and anchored, and for the group in which IL-2 was added to the culture medium, set to Imunace 100 units / mL. After incubation at 37°C for 48 hours, observation was performed with a fluorescence microscope. The cryopreservation and recovery were performed according to the methods described in Japanese Patent Publication No. 2018-050502 (Japanese Patent No. 6989907), Japanese Patent Publication No. 2021-136883.

[0213] The results are shown in Figure 2 By using the membrane-insertion type IL-2 in the cryopreserved high activity NK-like cells, the infiltration of the NK-like cells into the solid tumor was more observed.

[0214] [Example 3: Enhancement of solid tumor killing activity of IL-2-anchored NK-like cells]

[0215] The cryopreserved high activity NK-like cells were recovered, stained with PKH26, and added to spheroids prepared with NCI-H1975 cells (3000 cells / spheroid). The experiment used a 96-well plate, 5 spheroids per well, and 6.0 x 10 4 cells per well. After incubation at 37°C for 22 hours, observation was performed with a fluorescence microscope. In addition, after the end of the incubation, all the cells in the well were recovered, and the number of viable cells negative for PKH26 (Zombee staining negative) was measured using a flow cytometer, whereby the killing activity was measured. The control was set to NK-like cells to which IL-2 anchoring was not performed (IL-2 was also not added to the reaction system).

[0216] The results are shown in Figure 3By using the membrane-inserting type IL-2 in the cryopreserved high-activity NK-like cells, infiltration of the NK-like cells into the solid tumor was more observed and cytotoxic activity was higher even for the spheroids prepared with NCI-H1975 cells.

[0217] [Example 4: Measurement of the number of IL-2 anchor-modified molecules]

[0218] The high-activity NK-like cells modified with the IL-2 anchor were stained with a PE-labeled anti-human IL-2 antibody (MQ1-17H12, Cat no. 500306), and the number of modifications per cell was measured using a PE phycoerythrin fluorescence quantification kit. The IL-2 anchor was modified at 0.54 μM by reacting with 1 x 10 6 cells / mL of the high-activity NK-like cells at room temperature for 10 minutes.

[0219] The results are shown in Figure 4 The average number of 1748.4 (= 2350.8 - 602.4) was present on the cell surface. When the IL-2 1748 / cell was completely eluted, it was calculated as 100 IU equivalent of IL-2 / 2 x 10 8 cells.

[0220] [Example 5: Enhancement of host immune cell aggregation efficiency by IL-2 anchor in a solid tumor model in vivo]

[0221] CT26-GFP cells were transplanted (intraperitoneally) into Balb / c mice at 1 x 10 6 cells / each, and from 3 days later to 5 days later, administration of GAIA-102 (in the present application, the group to which the IL-2 anchor-modified was administered intraperitoneally (modified at 100 nM or 10 nM), the group to which the unmodified was administered, and the group to which the unmodified and IL-2 (10000 U / each) were administered intraperitoneally at the same time) was performed. The number of cells to be administered with GAIA-102 was set to 1 x 10 7 cells / each for each administration in all groups. The IL-2 anchor-modified GAIA-102 was confirmed to have been modified with the IL-2 anchor by flow cytometry using an anti-IL-2 antibody.

[0222] The mesentery was photographed with a fluorescence microscope on day 7 after transplantation of the CT26-GFP cells, whereby the nodules formed by the CT26-GFP cells were confirmed as GFP-positive regions. In addition, the nodules were collected, chopped, and digested with Liberase, whereby a single cell suspension was obtained, staining with anti-mouse CD4, anti-mouse CD8, anti-mouse NKp46, and anti-human CD56 was performed, and analysis was performed by flow cytometry. The CT26-GFP cells were detected as GFP-positive cells, and the ratio of the number of each antigen-positive cells to the number of GFP-positive cells was calculated.

[0223] Results are shown in Figure 5 GAIA-102 administration experiment to CT26 cell-inoculated mouse peritoneal cavity model. In the group in which IL-2 was administered simultaneously or in which IL-2-anchored GAIA-102 was administered, a decrease in the amount of nodules on the peritoneum and an increase in the amount of immune cell infiltration in the mouse were confirmed. The left of the figure shows images of the tumor nodules in the peritoneum after administration of GAIA-102. Differences in the green-colored tumor region were observed depending on the presence or absence of IL-2 anchoring modification. In particular, when administration of GAIA-102 modified with 100 nM of IL-2 anchor was performed, a case in which almost no tumor remained was confirmed (column for day 7). In addition, it was also confirmed that IL-2 anchor was modified in a concentration-dependent manner in the administered GAIA-102 (columns for days 3 to 5, in which administration was performed three times for each group).

[0224] [Example 6: Improvement of therapeutic effect of IL-2 anchor in solid tumor model in vivo]

[0225] CT26-GFP cells were transplanted (into the peritoneum) into Balb / c mice at 1 x 10 6 cells / mouse, and from 3 days after to 7 days after the transplantation, administration of GAIA-102 (in the present application, administration of a group modified with IL-2 anchor (modified with 100 nM), administration of a group not modified, and administration of a group in which both the unmodified and IL-2 (10000 U / mouse) were administered) was performed. A non-treatment group was set as a comparative control. The number of cells administered with GAIA-102 was set to 1 x 10 7 cells / mouse for each administration in all groups.

[0226] IL-2 anchor-modified GAIA-102 was confirmed to have been modified with IL-2 anchor by flow cytometry using an anti-IL-2 antibody.

[0227] Results are shown in Figure 6 GAIA-102 administration experiment to CT26 cell-inoculated peritoneal cavity model. Five administrations were performed from 3 days after inoculation of CT26 cells. Although the treatment period was extremely short, a decrease in mortality was confirmed in the IL-2 anchor GAIA-102 administration group.

[0228] In Examples 7 and 8 below, unless otherwise specified, the same reagents and apparatus as in Examples 1 to 6 were used.

[0229] [Example 7: Comparison between DSPE type and maleimide type]

[0230] The frozen high-activity NK-like cells were recovered, and 0-10,000 nM of each of DSPE-PEG3.4K-FITC and maleimide-PEG5K-FITC was added thereto, and incubated at room temperature for 10 minutes. Thereafter, the cells were washed and centrifuged with a medium, suspended in HSC-BANKER, and stored at -80°C for 3 days. After the recovery of the cells, the FITC was detected by a flow cytometer.

[0231] [Chemical 3]

[0232]

[0233] [Chemical 4]

[0234]

[0235] As a result, the FITC was detected before and after the freezing regardless of the structure, and it was confirmed that the intensity changed in a reaction concentration-dependent manner. Figure 7 In addition, the dead cell rate was measured using 7-AAD after the recovery, and as a result, no clear cytotoxicity was confirmed in any condition. Figure 7

[0236] [Example 8: Study on IL-2 anchoring conditions for NK cell modification]

[0237] The IL-2 anchor was prepared in the following order.

[0238] • 5:1 molar ratio of DSPE-PEG3.4K-NHS and maleimide-PEG5K-NHS, and recombinant human IL-2 were mixed, respectively

[0239] • DMSO was adjusted to a final concentration of 50% in the reaction solution

[0240] • The mixture was left to stand at room temperature for 1 hour

[0241] The IL-2 anchor (DSPE-PEG3.4K-IL2 and maleimide-PEG5K-IL2) obtained above was added to the recovered GAIA-102 at a final concentration of 100 nM, 1,000 nM, and 10,000 nM, respectively, and incubated at room temperature for 10 minutes. Thereafter, 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 quantification kit. In addition, the killing activity against H1975 cell spheres was measured using xCelligence (real-time cell analyzer, Agilent Technologies, Inc.).

[0242] As a result, it was confirmed that both the DSPE type and the maleimide type had IL-2 modification ability for GAIA-102 and maintained the killing activity​Figure 8 、 Figure 9 )。

Claims

1. A molecule comprising interleukin-2 (IL-2)-polyethylene glycol (PEG)-A, A is the portion used to anchor to the cell membrane, which contains groups or phospholipid portions that can form bonds with proteins on the cell membrane.

2. The molecule according to claim 1, wherein the molecular weight of the PEG portion is 3000 to 8000.

3. The molecule according to claim 1, wherein A is a portion containing a group capable of forming a bond with proteins on the cell membrane, and contains a maleimide reactive group.

4. The molecule according to claim 1, wherein A is a phospholipid moiety, and the phospholipid moiety is a group derived from glycerophospholipids.

5. The molecule according to claim 1, wherein A is a phospholipid moiety, and the phospholipid moiety is a group derived from 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE).

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

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

8. An immune cell having been molecularly modified according to any one of claims 1 to 5.

9. An activated NK cell or NK-like cell, having undergone molecular modification according to any one of claims 1 to 5.

10. A pharmaceutical composition comprising the cells according to claim 8.

11. A pharmaceutical composition comprising the cells according to claim 9.

Images