Notch ligand–particle composite and use therefor

Abstract

The problem addressed by the present invention is to provide: a Notch ligand–particle composite that can be cultured in a condition maintaining undifferentiation, while also activating immune cells, and that does not need to be removed after use; an exhaustion preventer for immune cells, an activator for immune cells, and an improver for gene transfer efficiency to immune cells including the Notch ligand–particle composite; and a manufacturing method for immune cells using the Notch ligand–particle composite. The present invention provides a Notch ligand–particle composite including (a) a Notch ligand and (b) particles of a particle size of 0.1 μm or greater, and less than 10 μm, which are biodegradable, wherein the Notch ligand is conjugated to the particles.

Description

Notch ligand-particle complex and its applications

[0001] The present invention relates to a Notch ligand-particle complex in which a Notch ligand is conjugated to a particle. The present invention further relates to an agent for preventing immune cell exhaustion, an agent for activating immune cells, and an agent for improving the efficiency of gene transfer to immune cells, which include the above Notch ligand-particle complex. The present invention further relates to a method for producing immune cells using the above Notch ligand-particle complex.

[0002] Conventional immune cells, as well as processed immune cells used in adoptive immunotherapy, are activated and proliferate upon antigen stimulation. However, when mass-producing immune cells, it is known that repeated antigen stimulation induces cell differentiation and cell exhaustion, leading to a decline in quality and performance. Furthermore, in recent years, novel adoptive T-cell therapies using gene transfer technology have been shown to exhibit dramatic therapeutic effects. Specifically, in adoptive T-cell therapy, a T cell receptor (TCR) gene or a chimeric antigen receptor (CAR) gene, artificially designed to recognize tumor-associated antigens (TAAs) or therapeutic target molecules, is introduced into the patient's T cells, and the T cells, which have been cultured further through TCR stimulation, are returned to the patient (Non-Patent Literature 1). In adoptive T cell therapy, it is known that cell populations containing a higher proportion of more undifferentiated stem cell memory T cells (Tscm cells) result in better therapeutic efficacy after administration and longer-lasting effects (Non-Patent Literature 2). However, the introduction of TCR and CAR genes is known to induce cell differentiation and cell exhaustion, similar to antigen stimulation of T cells (Tonic signals), and there have been problems with the quality and performance of cells degrading during manufacturing (Non-Patent Literature 3).

[0003] Non-patent document 4 shows that signaling by Notch ligand expressed on cells or plate-coated can maintain an undifferentiated T cell population (Naive T, Tscm cells) even in T cells or CAR-T cells that have received excessive antigen stimulation. Furthermore, non-patent document 4 shows that Notch signaling enhances mitochondrial oxidative phosphorylation, a metabolic pathway characteristic of undifferentiated Tscm cells. However, non-patent document 4 indicates that Notch ligand cannot exert its effects unless immobilized on a scaffold, making it impossible to apply Notch ligand as a solubilizing additive to the 3D agitated culture system, a mass production system for CAR-T cells. Known scaffolds for Notch ligand include coating mouse cells or plastic plates with Notch ligand, as shown in non-patent document 4 and patent document 1, or coating polystyrene beads with Notch ligand, as shown in patent document 2. However, when these scaffolds are actually applied clinically, their safety cannot be guaranteed for cells, and they must be completely removed. In particular, plastic particles such as polystyrene accumulate in the body without being broken down and are harmful to the human body, so they must be completely removed.

[0004] Fesnak, A. D. , June, C. H. , and Levine, B. L. (2016). Engineered T cells: the promise and challenges of cancer immunotherapy. Nat Rev Cancer 16, 566-581. Fraietta et al. , (2018). Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nature Medicine volume 24, pages 563-571. Chen et al. , (2023). Tuning charge density of chimeric antigen receptor optimizes tonic signaling and CAR-T cell fitness. Cell Research volume 33, pages341-354Cancer Research, 2021. , 10.1158 / 2767-9764. CRC-21-0034. Rejuvenating Effector / Exhausted CAR T Cells to Stem Cell Memory-Like CAR T Cells By Resting Them in the Presence of CXCL12 and the NOTCH Ligand

[0005] International Publication No. WO2021 / 085398, Japanese Patent Publication No. 2024-016217

[0006] Autologous therapy using immune cells such as CAR-T cells requires the proliferation of a sufficient amount of immune cells for administration to the patient. Furthermore, especially when manufacturing stock cells for allogeneic therapy, a large quantity of immune cells must be produced at once. A challenge in this manufacturing process is that during the culture stage, the cells undergo differentiation from an undifferentiated state, leading to cellular exhaustion and a decline in cell performance and quality.

[0007] The present invention aims to solve the problem of providing a Notch ligand-particle complex that can be cultured while activating immune cells and maintaining their undifferentiated state, and that does not need to be removed after use. Furthermore, the present invention aims to solve the problem of providing an agent that prevents immune cell exhaustion, an agent that activates immune cells, and an agent that improves the efficiency of gene transfer to immune cells, all of which contain the above Notch ligand-particle complex, as well as a method for producing immune cells using the above Notch ligand-particle complex.

[0008] As a result of diligent research to solve the above problems, the inventors of this invention have found that by using particles of a predetermined size with confirmed safety and biodegradability as a support for Notch ligand, it is possible to prevent the exhaustion of immune cells. This invention was completed based on these findings.

[0009] In other words, the present invention provides the following inventions: <1> A Notch ligand-particle complex comprising (a) a Notch ligand and (b) biodegradable particles with a particle size of 0.1 μm or more and less than 10 μm, wherein the Notch ligand is conjugated to the particles. <2> The Notch ligand-particle complex according to <1>, wherein the specific gravity of the particles is 1.05 to 1.3. <3> The Notch ligand-particle complex according to <1> or <2>, wherein the particles are composed of at least one selected from the group consisting of biodegradable polymers and lipids. <4> The Notch ligand-particle complex according to any one of <1> to <3>, wherein the particles are composed of at least one selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), polycaprolactone, proteins and peptides. <5> A Notch ligand-particle complex according to any one of <1> to <4>, wherein the Notch ligand is at least one selected from the group consisting of DLL1, DLL4, and Jagged1. <6> A Notch ligand-particle complex according to any one of <1> to <5>, wherein the Notch ligand is conjugated to the particle via protein A. <7> An immune cell exhaustion inhibitor comprising the Notch ligand-particle complex according to any one of <1> to <6>. <8> An immune cell exhaustion inhibitor according to <7>, wherein the immune cells are cells used in adoptive immunotherapy. <9> An immune cell exhaustion inhibitor according to <7> or <8>, wherein the immune cells are CAR-T cells. <10> An immune cell exhaustion inhibitor comprising a Notch ligand-support complex, which includes (a) a Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support. <11> An immune cell activator comprising a Notch ligand-support complex, which includes (a) a Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support.<12> An agent for improving the efficiency of gene transfer to immune cells, comprising a Notch ligand-support complex comprising (a) Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support. <13> The agent according to any one of <10> to <12>, wherein the immune cells are cells used for adoptive immunotherapy. <14> The agent according to any one of <10> to <13>, wherein the immune cells are CAR-T cells. <15> The agent according to any one of <10> to <14>, wherein the support is a particle. <16> An immune cell culture kit comprising a Notch ligand-particle complex according to any one of <1> to <6> and a culture medium. <17> A method for producing immune cells, comprising culturing immune cells in the presence of a Notch ligand-particle complex according to any one of <1> to <6>. <18> The method according to <17>, further comprising introducing a gene after the culture described above. <19> The method for producing immune cells according to <17> or <18>, wherein the immune cells are cells used for adoptive immunotherapy. <20> The method for producing immune cells according to any one of <17> to <19>, wherein the immune cells are CAR-T cells.

[0010] The Notch ligand-particle complex, immune cell exhaustion prevention agent, immune cell activator, and gene transfer efficiency enhancer of the present invention can prevent immune cell exhaustion. The Notch ligand-particle complex, immune cell exhaustion prevention agent, immune cell activator, and gene transfer efficiency enhancer of the present invention can promote activation while maintaining the undifferentiated state of immune cells, thereby improving gene transfer efficiency. The immune cell manufacturing method of the present invention can produce immune cells that are not exhausted. The immune cell manufacturing method of the present invention can produce activated immune cells while maintaining their undifferentiated state. Furthermore, when gene transfer is required, immune cells with improved transfer efficiency can be produced.

[0011] Figure 1 shows the results of measuring DLL1-Fc binding to PLGA beads. Figure 2 shows the results of measuring DLL1-Fc binding to PLGA beads. Figure 3 shows the results of measuring signal activation of PLGA-DLL1 beads using Notch signal reporter Jurkat cells. Figure 4 shows the results of measuring the prevention of T cell exhaustion using PLGA-DLL1 beads. Figure 5 shows the results of measuring the prevention of T cell exhaustion using PLGA-DLL1 beads. Figure 6 shows the results of measuring the prevention of T cell exhaustion using PLGA-DLL1 beads. Figure 7 shows the results of measuring the prevention of CAR-T exhaustion using PLGA-DLL1 beads. Figure 8 shows the percentage of Tscm in CAR+CD8+ cells on Day 10 after creating CAR-T cells using PLGA-DLL1 beads. Figure 9 shows the proliferation rate (left) and tumor cell killing rate (right) of CAR-T cells prepared with and without PLGA-DLL1 beads, and tumor cells after 7 days of co-culture. Figure 10 shows the expression levels of CD25 (left) and LDLR (right) after 2 days of adding various Notch ligand beads to human primary T cells. Figure 11 shows the expression levels of TCRαβ (left), CD25 (center), and LDLR (right) after 2 days of adding various Notch ligand beads to human primary T cells that were restimulated after expansion culture. Figure 12 shows the TCR gene knockout (KO) efficiency using LNPs when PLGA-DLL1 beads were added to human primary T cells after expansion culture. Figure 13 shows the CAR introduction rate (left) and the Tscm positivity rate in CAR+ / -CD8+ cells after the addition of DLL1 and DLL4 beads to human primary T cells.

[0012] Embodiments of the present invention will be described in detail below. <Notch Ligand-Particle Complex> The Notch ligand-particle complex of the present invention is a Notch ligand-particle complex comprising (a) a Notch ligand and (b) biodegradable particles with a particle size of 0.1 μm or more and less than 10 μm, wherein the Notch ligand is conjugated to the particles.

[0013] Notch signaling is a widely present signaling pathway found in organisms ranging from fruit flies to mammals. Notch signaling is known to be involved in cell proliferation and differentiation during various developmental processes. In adults, it is also known to contribute to the maintenance of tissue and organ homeostasis by regulating the proliferation and differentiation of tissue stem cells. In the Notch signaling pathway, signals are transmitted through direct interaction between Notch receptors expressed on the cell membrane and Notch ligands (such as Delta and Jagged) expressed on the membranes of adjacent cells.

[0014] Notch receptors in human cells include Notch-1, Notch-2, Notch-3, and Notch-4. Notch ligands typically have a characteristic DSL domain (D-delta, S-serate, and L-Lag2) containing 20 to 22 amino acids at the amino terminus and 3 to 8 EGF repeats on their extracellular surface.

[0015] Notch ligands are ligands that can bind to Notch receptors, which are present on the membranes of mammalian cells such as hematopoietic stem cells / primordial cells.

[0016] Notch ligands may include anti-Notch antibodies, peptides, and aptamers (e.g., DNA aptamers) that can bind to and participate in Notch signaling.

[0017] Notch ligands that promote and maintain the differentiation and proliferation of T cell lineage cells can be used. Notch ligands may be derived from humans or from other species such as rodents, dogs, cats, pigs, sheep, cattle, goats, and primates.

[0018] Specific examples of Notch ligands include the Delta family. The Delta family includes Delta-1 (Genbank accession number AF003522, Homo sapiens), Delta-3 (Genbank accession number AF084576, Rat (Rattus norvegicus)), Delta-like 1 (DLL1; Genbank accession numbers NM_005618 and NP_005609, Homo sapiens; Genbank accession numbers X80903, 148324, House mouse (M. This includes *Musculus*, Delta-like 3 (Genbank accession numbers NM_053666, N_446118, rat), Delta-4 (Genbank accession numbers AF273454, BAB18580, mouse; Genbank accession numbers AF279305, AAF81912, Homo sapiens), and Delta-like 4 (DLL4; Genbank accession numbers Q9NR61, AAF76427, AF253468, NM_019074, Homo sapiens; Genbank accession number NM019454, mouse). Notch ligands are commercially available or can be produced by recombinant DNA techniques and can be available in varying purities.

[0019] The Notch ligand may be a homolog of a known Notch ligand. A homolog refers to a gene product that exhibits sequence identity to any known Notch ligand, either by amino acid sequence identity or nucleic acid sequence identity. A Notch ligand homolog is identical to the corresponding Notch ligand at the amino acid level by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, preferably 90%, more preferably 95%, and most preferably 98-99%.

[0020] In one example, a homolog of the Notch ligand may contain a DSL domain at the N-terminus and have 3 to 8 EGF-like repeats on its extracellular surface. A suitable homolog may also bind to the Notch receptor. Binding to the Notch receptor can be determined by various methods known in the art, including in vitro binding assays.

[0021] Notch ligands include variants or cultivars of known Notch ligands. A variant means a polypeptide having a primary amino acid sequence different from the wild-type sequence due to the addition, substitution, or deletion of one or more amino acids. Preferably, the variant has at least 90% sequence identity with respect to the wild-type sequence. Preferably, the variant has 20 or fewer mutations with respect to the entire wild-type sequence. More preferably, the variant has 10 or fewer mutations with respect to the entire wild-type sequence, most preferably 5 or fewer mutations.

[0022] The Notch ligand may optionally contain at least one protein tag. The protein tag is a peptide sequence attached to the protein of interest, such as the Notch ligand. This protein tag can be directly or indirectly linked to the protein of interest. Protein tags are known in the art. Specific examples of protein tags include Fc tags, His tags, or AviTag. Fc is the Fc domain of IgG. Another example of a protein tag is a molecule that promotes oligomerization of the Notch ligand. For example, a small domain of COMP (cartilage oligomer matrix protein) can be fused with the Notch ligand to form a pentamer. Similarly, ferritin can be used to form a multimer.

[0023] The Notch ligand is preferably at least one selected from the group consisting of DLL1, DLL4, and Jagged1.

[0024] The Notch ligand-particle complex of the present invention contains biodegradable particles with a particle size of 0.1 μm or more and less than 10 μm. The particle size is preferably 0.2 μm or more and 8 μm or less, more preferably 0.5 μm or more and 7 μm or less, and even more preferably 1 μm or more and 5 μm or less. The particles may have any shape, but are not limited, such as spheres, ovals, rods, rectangles, etc. If the particle shape is not spherical, the particle size refers to the particle size when the shape is approximated to a sphere. The particle size can be measured using methods such as laser diffraction, sedimentation, elutriation, microscopy, Coulter method, dynamic light scattering, and particle image analysis. Laser diffraction and the Coulter method are generally used for measuring microparticle size, and dynamic light scattering is often used for nanoparticles.

[0025] Using particles with a particle size of less than 10 μm in the Notch ligand-particle complex of the present invention has the advantage of making the particles more easily degradable and reducing the particle retention rate in the final product. In addition, using particles with a small size of less than 10 μm has the advantage of increasing the surface area per unit mass of the particles, improving the probability of contact with cells and the contact area.

[0026] The particles may be, for example, microbeads. Microbeads refer to spherical or substantially spherical beads having a particle size of 0.1 μm or more and less than 10 μm. The specific gravity of the particles is preferably 1.0 to 1.4, and more preferably 1.05 to 1.3.

[0027] The particles are preferably composed of at least one selected from the group consisting of biodegradable polymers and lipids. More preferably, the particles are composed of at least one selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), polycaprolactone, proteins, and peptides.

[0028] In one example of the present invention, poly(lactic acid-glycolic acid) (PLGA) can be used as the biodegradable polymer. Alternatively, polylactic acid (PLA), polycaprolactone (PCL), polyglycolic acid (PGA), etc., can also be used. In another example of the present invention, proteins and peptides may be used as the biodegradable polymer. In yet another example of the present invention, agarose, dextran, alginic acid, hyaluronic acid, other polysaccharides (e.g., glycosaminoglycans, proteoglycans, chondroitin, cellulose, carboxymethylcellulose, chitin, and chitosan), biodegradable lipids, and lipid nanoparticles (LNPs) may be used as the biodegradable polymer.

[0029] The proteins and peptides are not particularly limited, but are preferably, for example, avidin, streptavidin, neutraavidin, immunoglobulin M (IgM), immunoglobulin A (IgA), gelatin, collagen, atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin, fibrin, fibroin, entactin, thrombospongin, and retronectin, and most preferably gelatin, collagen, and atelocollagen. The gelatin is preferably natural gelatin, recombinant gelatin, or chemically synthesized gelatin, and more preferably recombinant gelatin. Natural gelatin as used herein means gelatin made from naturally derived collagen.

[0030] Biodegradable polymers commonly bind various proteins, peptides or low-molecular-weight compounds via functional groups modified on their surfaces, and are used as DDS (Drug delivery system) preparations for cells. Examples of the functional groups include a carboxyl (COOH) group, an amino (NH2) group, an N-hydroxysuccinimide (NHS) group and a maleimide group. Biodegradable particles such as PLGA may encapsulate or fill a payload or the like inside. For example, immunostimulatory proteins or drugs may be encapsulated in PLGA particles. It is known that when these biodegradable particles are taken up by cells in vivo and degraded, the payload encapsulated therein is eluted and exerts a medicinal effect.

[0031] In the Notch ligand-particle complex of the present invention, the Notch ligand is conjugated to the particle. Various means for conjugating a Notch ligand to a particle are known in the art. The Notch ligand can be conjugated to the particle directly or indirectly.

[0032] In one example, the Notch ligand can be directly conjugated to the particle. In this case, after the particles are subjected to active esterification using, for example, N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) or / and N-Hydrosulfosuccinimide Sodium Salt (NHS), the product is reacted with the Notch ligand, whereby a Notch ligand-particle complex can be prepared. In another example, the Notch ligand can be conjugated to the particle via Protein A or Protein G (preferably Protein A). In still another example, the Notch ligand can be conjugated to the particle using a biotin / streptavidin system. In that case, the Notch ligand is biotinylated, and then conjugated to streptavidin-coated particles or a support (for example, streptavidin-coated microbeads).

[0033] The Notch ligand-particle complex of the present invention can be provided as an immune cell culture kit in combination with a medium. That is, according to the present invention, there is provided an immune cell culture kit comprising the Notch ligand-particle complex of the present invention and a medium.

[0034] <Agent for Preventing Exhaustion of Immune Cells, Activator for Immune Cells, and Agent for Improving Gene Transfer Efficiency into Immune Cells> In one aspect, the agent for preventing exhaustion of immune cells of the present invention comprises the aforementioned Notch ligand-particle complex of the present invention. In one aspect, the agent for preventing exhaustion of immune cells of the present invention comprises a Notch ligand-support complex, wherein the complex comprises (a) a Notch ligand and (b) a support having biodegradability, and the Notch ligand is conjugated to the support.

[0035] In one aspect, the activator for immune cells of the present invention comprises the aforementioned Notch ligand-particle complex of the present invention. In one aspect, the activator for immune cells of the present invention comprises a Notch ligand-support complex, wherein the complex comprises (a) a Notch ligand and (b) a support having biodegradability, and the Notch ligand is conjugated to the support.

[0036] In one aspect, the agent for improving gene transfer efficiency into immune cells of the present invention comprises the aforementioned Notch ligand-particle complex of the present invention. In one aspect, the agent for improving gene transfer efficiency into immune cells of the present invention comprises a Notch ligand-support complex, wherein the complex comprises (a) a Notch ligand and (b) a support having biodegradability, and the Notch ligand is conjugated to the support.

[0037] The immune cells are preferably mammalian-derived cells, and more preferably human-derived cells. The immune cells are not particularly limited, but can be selected from, for example, lymphocytes (e.g., T cells, B cells, natural killer cells (NK cells), NKT cells, iNKT cells), monocytes, macrophages, mast cells, dendritic cells, granulocytes (e.g., neutrophils, eosinophils, and basophils), hematopoietic stem / progenitor cells, primary immune cells, CD3+ cells, CD4+ cells, CD8+ T cells, regulatory T cells (Treg), B cells, NK cells, innate lymphocytes, or dendritic cells (DCs). The immune cells may preferably be selected from peripheral blood mononuclear cells (PBMCs), lymphocytes, T cells, CD4+ cells, CD8+ cells, memory T cells, naive T cells, or stem cell memory T cells. Examples of preferred immune cells include T cells, NK cells, NKT cells, B cells, macrophages, and dendritic cells. The immune cells are preferably T cells, NK cells, or NKT cells, and more preferably T cells. Primary cells may be used, or cells differentiated from stem cells (preferably pluripotent stem cells).

[0038] Naive T cells proliferate and become activated when they encounter foreign substances such as pathogens or cancer cells, but most eventually die. However, some become memory T cells in preparation for the next reinfection or recurrence. Memory T cells include several types of cells at different stages of differentiation: stem cell memory T cells (Tscm), central memory T cells (Tcm), and effector memory T cells (TEM). Among these, TEM cells are the most differentiated and have the strongest attacking power against cancer cells. Stem cell memory T cells (Tscm) are memory T cells that are similar to naive T cells, have the longest lifespan, and are thought to retain immunological memory over a long period. Tscm can produce many advanced Tcm and TEM cells. TEM cells further differentiate into effector T cells (Teff).

[0039] Furthermore, T cells differentiate based on the expression patterns of CD4 and CD8 on their cell surface. Pro-T cells (CD4) derived from hematopoietic stem cells in the bone marrow － CD8 －T cells) migrate to the thymus and then pass through pre-T cells (CD4 ＋ CD8 ＋ T cells) to become mature T cells (CD4 ＋ T cells or CD8 ＋ T cells). T cells have antigen receptors (T cell receptors) that can bind to specific antigens. When stimulated via the antigen receptor from an antigen-presenting cell (macrophage or dendritic cell) that has captured a specific antigen, naive T cells differentiate into effector T cells (Teff) via stem cell memory T cells (Tscm), central memory T cells (Tcm) and effector memory T cells (Tem). Among effector T cells, CD4 ＋ T cells act as helper T cells to activate B cells and are involved in antibody production, while CD8 ＋ T cells act as cytotoxic T cells to kill unnecessary cells, virus-infected cells, cancerous cells, and the like. As immune cells in the present invention, CD8 ＋ T cells are preferred.

[0040] As immune cells in the present invention, cells used for adoptive immunotherapy are preferred. In adoptive immunotherapy, lymphocytes are collected from the peripheral blood of a patient (e.g., a cancer patient), the cells are expanded, activated and functionally enhanced in vitro, and then the cells are administered to the patient.

[0041] There are multiple types of effector cells capable of attacking cancer cells. For example, a T cell receptor (TCR) gene or a chimeric antigen receptor (CAR) gene that is artificially designed to recognize a cancer antigen can be introduced into the patient's T cells, and cells with enhanced specificity for the cancer antigen can be administered.

[0042] An example of the immune cell used in the present invention is CAR-T cell. CAR-T cells are T cells genetically engineered to produce chimeric antigen receptors.

[0043] Preventing immune cell exhaustion means suppressing the differentiation of immune cells. If the immune cells are T cells, this means suppressing the differentiation of T cells from stem cell memory T cells (Tscm) to central memory T cells (Tcm) to effector memory T cells (Temp) to effector T cells (Teff). As described above in this specification, when naive T cells are cultured, they differentiate into stem cell memory T cells (Tscm), central memory T cells (Tcm), effector memory T cells (Temp), and effector T cells (Teff). In other words, by preventing immune cell exhaustion, the decrease in the proportion of highly undifferentiated Tscm and Tcm cells in the cultured T cell population is suppressed, meaning that the proportion of Tscm and Tcm cells is maintained at a high value, and the increase in the proportion of Temp and Teff cells is suppressed, meaning that the proportion of Temp and Teff cells is maintained at a low value.

[0044] The total proportion of Tscm and Tcm in the T cell population is preferably 63% or more, more preferably 65% ​​or more, and even more preferably 70% or more. The proportion of Temp in the T cell population is preferably 8% or less, more preferably 2% or less. The proportion of Teff in the T cell population is preferably 6% or less, more preferably 2% or less.

[0045] Immune cell activation can be assessed by evaluating the expression status of immune cell-specific markers (for example, CD25 and RDLR in the case of T cells), using increased or decreased marker expression as an indicator to evaluate immune cell activation. Immune cell activation can lead to increased cell proliferation, increased antibody or cytokine production, increased migration to inflammatory sites, improved effector function, enhanced long-term immune memory formation, or enhanced signal transduction.

[0046] Improving the efficiency of gene transfer to immune cells means being able to reliably and efficiently introduce foreign genes into immune cells in large quantities.

[0047] In one embodiment, the immune cell exhaustion prevention agent, immune cell activator, and gene transfer efficiency improvement agent of the present invention comprises (b) a biodegradable support.

[0048] Examples of supports include, but are not limited to, particles (which may be beads or microbeads), filters, fibers, screens, meshes, tubes, hollow fibers, or combinations thereof. Particles are preferred as supports. The particles may be beads or microbeads. Details of the particles are as described herein. The materials used for the supports are the same as those used for the particles described herein.

[0049] The immune cell exhaustion inhibitor, immune cell activator, and gene transfer efficiency enhancer of the present invention may be provided as a kit for culturing immune cells. That is, according to the present invention, a kit for culturing immune cells is provided, comprising the immune cell exhaustion inhibitor, immune cell activator, or gene transfer efficiency enhancer of the present invention.

[0050] In the above kit, the Notch ligand-support complex, which comprises (a) Notch ligand and (b) a biodegradable support (preferably particles), and in which the Notch ligand is conjugated to the support, may be contained in a medium such as a buffer.

[0051] The above kit may further include a culture medium for culturing immune cells in the presence of a Notch ligand-support complex. The medium may contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or an artificial serum substitute) or may not contain serum. Specific examples of culture media include PRIME-XV T cell expansion XSFM (Irvine Scientific), PRIME-XV T cell CDM (Irvine Scientific), TexMACS Medium (Miltenyi Biotec), CTS OpTMizertm T Cell Expansion SFM (Gibco), TheraPEAK T-VIVO serum-free medium (Lonza), and RPMI1640 (Gibco).

[0052] The culture medium may optionally contain one or more cytokines that determine and promote the differentiation of immune cells. The cytokines may be of human origin or originate from other species. The concentration of cytokines in the culture medium is typically about 1 to 100 ng / mL. Specific examples of cytokines include interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-2 (IL-2).

[0053] <Method for Producing Immune Cells> The method for producing immune cells of the present invention comprises culturing immune cells in the presence of the Notch ligand-particle complex of the present invention described above. Details of the immune cells are as described herein.

[0054] Immune cells can be cultured using either suspension culture or adherent cell culture. In suspension culture, cells grow freely floating in the culture medium. In adherent cell culture, cells grow as a monolayer on a substrate.

[0055] In one example, immune cells can be cultured in suspension in a bioreactor with Notch ligand-particle complexes. Bioreactors are well known in the art and include, for example, batch bioreactors, fed-batch bioreactors, or continuous bioreactors.

[0056] The density of immune cells in the culture is not particularly limited, but for example, 1 cell / mL to 10 × 10⁶ cells per mL of culture medium. ６ The cell count is per mL, and the culture medium is 1 × 10⁶ cells per mL. ２ cells / mL ~ 1 x 10 ６ Cells / mL is also acceptable.

[0057] The ratio of Notch ligand-particle complexes to immune cells is not particularly limited, but can range from 1:1 to 10,000:1, or 10:1 to 1,000:1.

[0058] One or more cytokines that promote the differentiation of immune cells may be added to the culture. The cytokines may be of human origin or of other species. The concentration of cytokines in the culture medium is typically about 1 to 100 ng / mL. Specific examples of cytokines include interleukin-7 (IL-7), interleukin-15 (IL-15), and interleukin-2 (IL-2).

[0059] The culture medium may contain serum (e.g., bovine serum, fetal bovine serum, calf serum, horse serum, human serum, or an artificial serum substitute), or it may not contain serum. Specific examples of culture media include PRIME-XV T cell expansion XSFM (Irvine Scientific), PRIME-XV T cell CDM (Irvine Scientific), TexMACS Medium (Miltenyi Biotec), CTS OpTMizertm T Cell Expansion SFM (Gibco), TheraPEAK T-VIVO serum-free medium (Lonza), and RPMI1640 (Gibco).

[0060] The culture time is not particularly limited, but generally it can be cultured for 1 to 20 days, preferably 1 to 7 days.

[0061] The method for producing immune cells of the present invention may further include introducing a gene after culturing immune cells in the presence of the Notch ligand-particle complex of the present invention. The gene introduced after culturing immune cells in the presence of the Notch ligand-particle complex of the present invention is not particularly limited, but may be, for example, a TCR gene, a CAR gene, or a gene for genome editing (e.g., Cas9 mRNA and sgRNA for the TCR gene).

[0062] <Uses of Immune Cells> Immune cells produced by the method of the present invention are useful in treating diseases such as cancer. Immune cells produced by the method of the present invention may be modified in terms of T cell receptors (TCRs) or chimeric antigen receptors (CARs) to confer specificity to tumor-associated antigens (TAAs), as described above. Cells modified in this way are particularly useful in treating diseases such as cancer.

[0063] The immune cells produced by the method of the present invention may be used as a pharmaceutical composition comprising the immune cells and a pharmaceutically acceptable diluent or carrier. Diluents and carriers known to those skilled in the art can be used.

[0064] Examples of pharmaceutical compositions include lyophilized powders, aqueous sterile injection solutions, non-aqueous sterile injection solutions, or suspensions. Pharmaceutical compositions may also contain antioxidants, buffers, and sterilizing agents. Furthermore, pharmaceutical compositions may contain water, surfactants, alcohols, polyols, glycerin, or vegetable oils.

[0065] The pharmaceutical composition may be a cryogenic storage solution. Immune cells can be cryogenically stored in a suitable culture medium, for example, a pharmaceutically acceptable medium, and, if desired, can be formulated for administration to subjects requiring it.

[0066] A pharmaceutically acceptable carrier is preferred as the carrier. Examples of preferred carriers include water, physiological saline, glycerol solution, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioresyl-phosphotidyl-ethanolamine (DOPE), lipid nanoparticles (LNPs), and liposomes.

[0067] Preferably, immune cells are present in the pharmaceutical composition in an amount effective for treatment.

[0068] Pharmaceutical compositions can be administered via routes such as intravenous, subcutaneous, intramuscular, intra-articular, intraspinal (intrathecal), intraperitoneal, intratumoral, or intraventricular.

[0069] The present invention provides a method for treating a subject having a condition requiring an increase in immune cells, further comprising the steps of (i) (a) culturing a sample containing immune cells in the presence of the Notch ligand-particle complex of the present invention, an immune cell exhaustion inhibitor of the present invention, an immune cell activator, or an agent that improves the efficiency of gene transfer to immune cells, and (b) isolating the immune cells, and (ii) administering an effective amount of immune cells to a subject in need thereof.

[0070] The effective dose refers to the amount and duration of treatment necessary to achieve the desired effect. The effective dose can be determined based on factors such as the disease state, age, sex, and weight of the individual. Treatment can include alleviation or improvement of one or more symptoms or conditions, a decrease in the severity of the disease, a stable (or non-worsening) state of the disease, prevention of disease transmission, delay or slowing of disease progression, improvement or mitigation of the disease state, a decrease in the disease recurrence rate, or remission.

[0071] The subjects are preferably mammals, and more preferably humans. Conditions requiring an increase in immune cell count include any condition in which the immune cell level is reduced compared to a healthy animal. Conditions requiring an increase in immune cell count include, for example, immunodeficiency, cancer, genetic disorders (e.g., primary immunodeficiency disease (PID)), infectious diseases, immune disorders, and autoimmune conditions.

[0072] The present invention will be specifically described by the following examples, but the present invention is not limited to the scope of these examples.

[0073] Example 1: Preparation of Notch Ligand-Bead Complex <Preparation of PLGA Beads> PLGA beads (particle size 2 μm) were purchased from Degradex by Sigma Aldrich. The required amount of PLGA beads was weighed, and the beads were activated by adding 50 mg / mL N-(3-Dimethylaminopropyl)-N'-ethylcarbonidide hydrochloride (EDC) (Sigma Ardrich) / 50 mmol / L MES buffer (ThermoFisher) and / or 50 mg / mL N-Hydroxysulfosuccinimide Sodium Salt (NHS) (Fujifilm Wako Pure Chemical Industries) and stirring for 30 minutes at room temperature. Subsequently, the beads were washed with a 50 mmol / L MES buffer, and then protein A (AnaSpec) or DLL1-Fc (R&D) (20 μg / mg or 40 μg / mg: 20 μg / mg for all samples except Figure 2) was added, and the mixture was stirred at room temperature for 2 hours. After that, the beads were washed with PBS (ThermoFisher) containing 0.05% BSA (Miltenyi) and 0.05% Tween20 (Fujifilm Wako Pure Chemical Industries), and an appropriate amount of beads were suspended in the same buffer.

[0074] <Evaluation Method> The DLL1 protein bound to the beads was mixed with Goat anti-human IgG (Fc)-HRP (Invitrogen) diluted 1:1000 with PBS in 1 μL of prepared beads and reacted at room temperature for 2 hours. Then, the beads were washed three times with 1 mL of PBS containing 0.05% Tween 20, and 50 μL of 3,3',5,5'-Tetramethyl-benzidine (TMB) ELISA Substrate Solutions (ThermoFisher) were mixed and reacted at room temperature for 10 minutes. Subsequently, 50 μL of ELISA stop solution (Invitrogen) was added to stop the reaction, and the absorbance at 450 nm was measured using a plate reader Synergy H1 (Agilent Thechologies). It was confirmed that Goat anti-human IgG (Fc)-HRP hardly bound to Protein A and mainly bound only to DLL1-Fc.

[0075] <Evaluation Criteria> The amount of DLL1 protein bound to beads was compared based on differences in absorbance values, either for beads without protein binding or under each condition.

[0076] <Results> The results are shown in Figures 1 and 2.

[0077] The amount of DLL1 protein bound was compared under the following conditions: unbound protein beads, DLL1 bound after EDC activation, Protein A + DLL1 bound after EDC activation, DLL1 bound after EDC + NHS activation, and Protein A + DLL1 bound after EDC + NHS activation. As a result, binding was confirmed in all other groups compared to unbound beads, but the amount of DLL1 protein bound was highest under the condition of Protein A + DLL1 bound after EDC + NHS activation (Figure 1).

[0078] The amount of DLL1 protein bound was compared under the following conditions: unbound protein beads, DLL1 20 μg / mg or 40 μg / mg after EDC / NHS activation, and DLL1 20 μg / mg or 40 μg / mg after Protein A binding. As a result, binding was confirmed in all other groups compared to unbound beads, but it was confirmed that the amount of DLL1 bound was greater with Protein A binding, and that the amount of DLL1 bound was higher at 40 μg / mg than at 20 μg / mg (Figure 2).

[0079] Example 2: Reporter assay using Notch signal reporter Jurkat cells To confirm whether the prepared DLL1 beads activate the Notch signal, a reporter assay was performed using Notch signal reporter Jurkat cells.

[0080] <Sample Preparation> A lentivirus (BPS Bioscience) containing the FireFly Luciferase gene, driven by a CSL response element (CBF1 / RBPJκ / Suppressor of Hairless / Lag-1) located upstream of the TATA promoter, was used to infect Jurkat cells, an acute T-cell leukemia cell line. Subsequently, Notch signal reporter Jurkat cells were established by selecting reporter gene-transformed cells by adding Puromycin (Sigma Ardrich).

[0081] <Evaluation Method> Notch signal reporter Jurkat cells 5 x 10 ５ Cells / well cells were seeded on a 96-well plate, various DLL1 beads were added, and the cells were incubated overnight at 37°C. Subsequently, the luminescence in the cells was reacted with substrates using a 1-step Luciferase assay kit (BPS Bioscience), and the luminescence values ​​were measured using a plate reader Synergy H1 (Agilent Technologies).

[0082] <Evaluation Criteria> Notch signal intensity was compared using the relative light unit (RLU).

[0083] <Results> The results are shown in Figure 3. Compared to PLGA_NC (unbound beads), PLGA_DL1_1 (1 μL) and PLGA_DL1_3 (3 μL) showed slightly higher RLU. On the other hand, PLGA_pA (Protein A)_DL1_1 (1 μL) and PLGA_pA (Protein A)_DL1_3 (3 μL) showed higher RLU. From these results, it was confirmed that binding Protein A to DLL1 protein can strongly activate the Notch signal.

[0084] Example 3: Investigation of T cell exhaustion prevention using PLGA-DLL1 beads (2 μm) <Sample preparation> Human Pan-T cells (Stemcell Technologies), which are naive T cells, were thawed, and anti-CD3 / CD28 antibody (ImmunoCult Human CD3 / CD28 T cell activator, Stemcell Technologies) was added and stimulated according to the manual, resulting in 1 × 10⁻⁶ cells. ６ On Day 0, seeds were seeded on a 24-well plate using PRIME-XV T cell expansion XSFM (Irvine Scientific) to which 5 ng / mL of IL-7 and 5 ng / mL of IL-15 (Miltenyi) had been added to achieve a cell / mL ratio. On Day 3, the ImmunoCult stimulant was removed and the seeds were seeded again, and various DLL1 beads were added to a total protein content of 0.2 ng / 200 μL / 5 x 10⁻⁶. ４ Cells were added and cultured at 37°C. Cells were subcultured (4-fold dilution) on Day 6, and culture was terminated on Day 10. For DLL1 beads, PLGA_DL1 and PLGA_pA_DL1 were prepared as described above.

[0085] <Evaluation Method> On Day 10, cells were stained with Acridine Orange / Propidine Iodide solution (Logos Biosystems), and cell number and viability were measured using a cell counter Luna Fx-7 (Logos Biosystems). In addition, cells were stained with fluorescent dye-labeled antibodies (CD4, CD8, CD45RO, CCR7) (BD Bioscience), and the phenotype was measured using a flow cytometer Attune NxT (ThermoFisher).

[0086] <Evaluation Criteria> The toxicity of PLGA beads and DL1 protein to T cells was evaluated by assessing the proliferation efficiency and viability of T cells after culture with DL1 beads. Regarding phenotype, T cells are classified into Naive and Tscm (CD45RO-CCR7+), Tcm (CD45RO+CCR7+), Temp (CD45RO+CCR7-), and Teff (CD45RO-CCR7-) in order of highest undifferentiated state based on the expression of CD45RO and CCR7. It is generally known that after stimulated culture, the proportion of Naive and Tscm fractions decreases, and differentiation and exhaustion progress. Therefore, it was determined whether the positivity rate of Tscm and Tcm was maintained by the addition of DL1 beads.

[0087] <Results> The results are shown in Figures 4 to 6. The survival rate and proliferation rate on Day 10 were similar in the group with DLL1_PLGA beads added to the group without beads, confirming that the bead material and DLL1 protein are not toxic to T cells (Figure 4). Furthermore, phenotypic analysis showed that the group with all DLL1 beads added had a higher proportion of Tscm fraction after culture compared to the group without beads, indicating that DLL1 beads suppressed T cell differentiation and exhaustion during culture (Figures 5 and 6).

[0088] Example 4: Differentiation and exhaustion inhibitory effect of DLL1 beads in CAR-T cells <Sample preparation> Human Pan-T cells (Stemcell Technologies) were thawed, and anti-CD3 / CD28 antibody (Dynabeads Human T-Activator CD3 / CD28, ThermoFisher) was added according to the manual and stimulated to 1 × 10⁻⁶ cells. ６ Cells were seeded on a 24-well plate using PRIME-XV T cell expansion XSFM (Irvine Scientific) supplemented with 5 ng / mL of IL-7 and 5 ng / mL of IL-15 (Miltenyi) to a cell / mL ratio (Day 0). On Day 1, the cells were infected with CAR lentivirus against CD19 (CD19 ScFv-CD8-4-1BB-CD3ζ, BPS Bioscience) (MOI = 5,800x g, 60 min, 33°C). On Day 3, after destimulation and washing, the cells were diluted and reseeded in a 96-well plate, and DLL1_PLGA beads were added. On Day 6, the cells were subcultured (0.5 × 10⁻⁶). ６ Cells / mL), culture was terminated on Day 10 and evaluation was performed.

[0089] <Evaluation Method> On Day 10, cells were stained with Acridine Orange / Propidium Iodide solution (Logos Biosystems), and the cell count and viability were measured using a cell counter Luna Fx-7 (Logos Biosystems). Furthermore, cells were stained with fluorescently labeled antibodies (FITC-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) DMF Fielded (Acro Bio), BV711 Mouse Anti-Human CD4, R718 Mouse Anti-Human CD8, BV480 Mouse Anti-Human CD45RO, APC Mouse Anti-Human CCR7) (BD Bioscience), and their phenotypes were measured using an Attune NxT flow cytometer (ThermoFisher).

[0090] <Evaluation Criteria> In the CAR-T manufacturing process, the effectiveness of the differentiation exhaustion prevention mechanism of DLL1 beads was evaluated by comparing the Tscm positivity rate in phenotypic analysis after culture.

[0091] <Results> The results are shown in Figure 7. Analysis of the phenotype in CAR-T after DLL1 bead addition showed that the Tscm positivity rate was higher in the PLGA_pA_DL1 addition group than in the non-addition group.

[0092] Example 5: Performance evaluation of CAR-T cells cultured using Notch ligand beads [Sample preparation] CAR-T cells were prepared for CD19 in the same manner as in Example 4. Notch ligand beads were prepared by binding DLL1-Fc, and 200 ng / well of protein per 96-well plate was added the day after CAR Lentivus infection. The CAR-T cells prepared above were co-cultured with NALM6 cells expressing CD19 in a ratio of CAR-T:NALM6 = 1:10, and after 7 days, the number of CAR-T and NALM6 cells was measured using an Attune NxT flow cytometer.

[0093] [Evaluation Method] Anti-CD19 CAR-T cells are activated and proliferate upon binding to CD19-expressing cells, while simultaneously killing the bound CD19-expressing cells. It is also known that a higher proportion of undifferentiated Tscm and Tcm cells in CAR-T cells enhances the attack activity and proliferative capacity of CAR-T cells. Therefore, CD19 CAR-T cells treated with Notch ligand (DLL1) during manufacturing were co-cultured with NALM6 cells expressing CD19, and the number of each cell type was measured using a flow cytometer to evaluate their attack and proliferative capacity.

[0094] [Results] On Day 10, when CAR-T cell production was completed, the group with DLL1 beads added had a higher proportion of undifferentiated T cells (Tscm) compared to the group without DLL1 beads (Figure 8). Furthermore, when CD19 CAR-T cells produced with or without DLL1 beads were co-cultured with CD19-expressing tumor cells (NALM6) in a 1:10 ratio, the DLL1 bead-added group showed approximately four times the proliferation of CAR-T cells, while the group without DLL1 beads showed approximately one-fold proliferation (Figure 9). In addition, NALM6 cells were 100% killed in both groups (Figure 9). From these results, it was confirmed that DLL1 bead-added CAR-T cells have a higher proportion of undifferentiated cells and exhibit superior proliferative activity upon contact with tumor cells.

[0095] Example 6: T cell activation using Notch Ligand beads [Method] <Culture of human T cells> Anti-CD3 antibody (Invitrogen) was mixed with PBS (Gibco), and added to a 96-well plate to a concentration of 0.2 μg / mL. The plate was left to stand overnight at 4°C to coat it. Human Pan-T cells (Stemcell Technologies) were thawed and 1 × 10⁻⁶ cells were added. ６ Cells were suspended in PRIME-XV T cell CDM (Irvine Scientific) to which 10 ng / mL of IL-2 (Roche), 5 ng / mL of IL-7, and 5 ng / mL of IL-15 (Miltenyi) were added to reach a cell / mL ratio, and seeded onto plates coated with anti-CD3 antibody (Day 0). In some cases, anti-CD28 antibody (Invitrogen) was added at 1 ug / mL.

[0096] <Preparation of PLGA beads> PLGA beads (particle size 0.5 μm) were purchased from Degradex by Sigma Aldrich. Notch ligand was prepared using Recombinant Human DLL1 Fc Chimera Protein, CF (R&D systems) and (DLL4 (human):Fc (human) Recombinant (Adipogen)) in the same manner as in Example 1. The prepared Notch ligand beads were added to Day 0 to a protein content of 200 ng / well.

[0097] <Evaluation of T cell activation state> Cells were harvested on Day 2, stained with BD Pharmaingen APC Mouse Anti-Human CD25 (BD) and BD OptiBuild™ BV421 Mouse Anti-Human LDLR (BD), and fluorescence values ​​were measured using an Attune CytPix (ThermoFisher) flow cytometer. The expression level of each antigen was evaluated by fluorescence intensity (Median Fluorescence Intensity, MFI).

[0098] [Results] When the expression levels (MFI) of CD25 and LDLR were measured two days after cell stimulation, CD25 and LDLR expression were elevated in the groups treated with Notch Ligan beads DLL1, DLL4, and both, compared to the untreated group, in both CD3 antibody stimulation and co-stimulation with CD3 and CD28 antibodies (Figure 10). These results demonstrate that Notch Ligan induces T cell activation.

[0099] Example 7: Human T cell activation after expansion culture using Notch Ligand beads [Method] <Preparation of Notch Ligand PLGA beads> Prepared in the same manner as in Example 6. <Culture of human T cells> Human Pan-T cells (Stemcell Technologies) were thawed, and Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) was added according to the manual, 1 × 10⁻⁶ ６ The cells were suspended in PRIME-XV T cell CDM (Irvine Scientific) to which 5 ng / mL of IL-7 and 5 ng / mL of IL-15 (Miltenyi) had been added to reach a cell / mL ratio, seeded on plates, and stored at 37°C under 5% CO2. ２Cells were cultured under specified conditions (Day 0). On Day 3, Dynabeads were removed, and the cells were cultured in the same medium at 4x dilutions every 2-3 days. On Day 10, the cells were cryopreserved in CELLBANKER 1 (Takara). After this expanded culture, the cells were thawed again, and Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) was added according to the manual, 1 x 10⁻⁶. ６ Resuspend in the same medium to a concentration of cells / mL and store at 37°C in 5% CO2. ２ Cells were cultured under specific conditions. In the Notch Ligand bead-added group, 200 ng / 100 uL of each DLL1 and DLL4 bead were added on the day of re-thawing. Cells were harvested two days after re-stimulation and analyzed.

[0100] <Evaluation of T-cell activation state> Cells were harvested on Day 2 and stained with PE Mouse Anti-Human TCRαβ (BD), BD PharmaingenAPC Mouse Anti-Human CD25 (BD), and BD OptiBuild™ BV421 Mouse Anti-Human LDLR (BD). Fluorescence values ​​were measured using an Attune CytPix (ThermoFisher) flow cytometer. The expression level of each antigen was evaluated by fluorescence intensity (Median Fluorescence Intensity, MFI).

[0101] [Results] When the expression levels (MFI) of TCRαβ, CD25, and LDLR were measured two days after T cell restimulation following expansion culture, the expression of TCRαβ, CD25, and LDLR was elevated in the groups treated with Notch Ligand beads DLL1, DLL4, and both, compared to the group without the addition of Notch Ligand beads (Figure 11). Therefore, it was shown that Notch Ligand induces T cell activation after expansion culture.

[0102] Example 8: Improvement of transfection efficiency using Notch Ligand [Method] <Preparation of Notch Ligand PLGA beads> Prepared in the same manner as in Example 6.

[0103] <Culture of Human T Cells> Thaw Human Pan-T cells (Stemcell Technologies), add Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) according to the manual, 1 x 10⁻⁶ ６ The cells were suspended in PRIME-XV T cell CDM (Irvine Scientific) to which 10 ng / mL of IL-2 (Roche), 5 ng / mL of IL-7, and 5 ng / mL of IL-15 (Miltenyi) were added to reach a cell / mL ratio, then seeded onto plates and stored at 37°C under 5% CO2. ２ Cells were cultured under specified conditions (Day 0). On Day 3, Dynabeads were removed, and the cells were cultured in the same medium at 4x dilutions every 2-3 days. On Day 10, the cells were cryopreserved in CELLBANKER 1 (Takara). After this expanded culture, the cells were thawed again, and Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) was added according to the manual, 1 x 10⁻⁶. ６ Resuspend in the same medium to a concentration of cells / mL and store at 37°C in 5% CO2. ２ Cells were cultured under specific conditions. The day after restimulation, 200 ng / 100 uL of each DLL1 and DLL4 bead were added to the Notch Ligand bead-added group. The following day, T cells were transfected with the following lipid nanoparticles (LNPs) to obtain Cas9 mRNA and sgRNA for the TCR gene in a ratio of 4:1, resulting in a total nucleic acid amount of 1 or 2 μg / mL.

[0104] As lipid nanoparticles (LNPs), TR04 or TD05 was used as the ionized lipid, and LNPs prepared in accordance with the method described in the example for preparing nucleic acid-free lipid particles (empty LNPs) in International Publication WO2025 / 143231 were used.

[0105]

[0106]

[0107] <Evaluation of TCR knockout efficiency> Cells were harvested the day after transfection, stained with PE Mouse Anti-Human TCRαβ (BD), and the TCRαβ positivity rate was measured using an Attune CytPix (ThermoFisher) flow cytometer.

[0108] [Results] Compared to the group without DLL1 beads, the group with DLL1 beads showed an increased TCR knockout efficiency (Figure 12).

[0109] Example 9: Improvement of transduction efficiency using Notch ligand [Method] <Preparation of Notch ligand PLGA beads> Prepared in the same manner as in Example 6.

[0110] <Culture of Human T Cells> Thaw Human Pan-T cells (Stemcell Technologies), add Dynabeads Human T-Activator CD3 / CD28 (ThermoFisher) according to the manual, 1 x 10⁻⁶ ６ The cells were suspended in PRIME-XV T cell CDM (Irvine Scientific) to which 10 ng / mL of IL-2 (Roche), 5 ng / mL of IL-7, and 5 ng / mL of IL-15 (Miltenyi) were added to reach a cell / mL ratio, then seeded onto plates and stored at 37°C under 5% CO2. ２ Cells were cultured under controlled conditions (Day 0). On Day 1, Notch Ligand PLGA beads were added at a rate of 200 ng / 100 uL. On Day 2, cells were infected with a lentivirus for CD19 CAR (CD19 ScFv-CD8-4-1BB-CD3ζ, BPS Bioscience) (MOI = 20, 800x g, 60 min, 33°C). On Day 3, after removing irritants and washing, the cells were reseeded in a 96-well plate after a 4-fold dilution, and the CD19 CAR positivity rate was evaluated on Day 7.

[0111] <Evaluation of CAR Positivity Rate> Cells were harvested on Day 7 and stained with FITC-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) DMF FieldCAR (Acro Bio), BV711 Mouse Anti-Human CD4, R718 Mouse Anti-Human CD8, BV480 Mouse Anti-Human CD45RO, APC Mouse Anti-Human CCR7) (BD Bioscience), and then evaluated using an Attune flow cytometer. The CD19 CAR positivity rate and the proportion of undifferentiated T cells (Tscm, CD8+CD45-CCR7+) were measured using CytPix (ThermoFisher).

[0112] [Results] Compared to the group without Notch ligand, the group with Notch ligand showed significantly higher CAR positivity rates and a higher proportion of Tscm in CAR+ and CAR- cells (Figure 13). These results demonstrate that Notch ligand improves gene transfer efficiency while maintaining the undifferentiated state of T cells.

Claims

1. A Notch ligand-particle complex comprising (a) a Notch ligand and (b) biodegradable particles with a particle size of 0.1 μm or more and less than 10 μm, wherein the Notch ligand is conjugated to the particles.

2. The Notch ligand-particle complex according to claim 1, wherein the specific gravity of the particles is 1.05 to 1.

3.

3. The Notch ligand-particle complex according to claim 1, wherein the particles are composed of at least one selected from the group consisting of biodegradable polymers and lipids.

4. The Notch ligand-particle complex according to claim 1, wherein the particles are composed of at least one selected from the group consisting of polylactic acid, polyglycolic acid, poly(lactic acid-glycolic acid), polycaprolactone, protein, and peptide.

5. The Notch ligand-particle complex according to claim 1, wherein the Notch ligand is at least one selected from the group consisting of DLL1, DLL4, and Jagged1.

6. The Notch ligand-particle complex according to claim 1, wherein the Notch ligand is conjugated to the particle via protein A.

7. An agent for preventing immune cell exhaustion, comprising a Notch ligand-particle complex according to any one of claims 1 to 6.

8. The immune cell exhaustion prevention agent according to claim 7, wherein the immune cells are cells used in adoptive immunotherapy.

9. The immune cell exhaustion prevention agent according to claim 7, wherein the immune cells are CAR-T cells.

10. An agent for preventing immune cell exhaustion, comprising a Notch ligand-support complex, which includes (a) a Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support.

11. An immune cell activator comprising a Notch ligand-support complex, which includes (a) a Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support.

12. An agent for improving the efficiency of gene transfer to immune cells, comprising a Notch ligand-support complex, which includes (a) a Notch ligand and (b) a biodegradable support, wherein the Notch ligand is conjugated to the support.

13. The agent according to any one of claims 10 to 12, wherein the immune cells are cells used for adoptive immunotherapy.

14. The agent according to any one of claims 10 to 12, wherein the immune cells are CAR-T cells.

15. The agent according to any one of claims 10 to 12, wherein the support is particles.

16. An immune cell culture kit comprising a Notch ligand-particle complex according to any one of claims 1 to 6 and a culture medium.

17. A method for producing immune cells, comprising culturing immune cells in the presence of a Notch ligand-particle complex according to any one of claims 1 to 6.

18. The method according to claim 17, further comprising introducing a gene after the culture.

19. The method for producing immune cells according to claim 17, wherein the immune cells are cells used for adoptive immunotherapy.

20. The method for producing immune cells according to claim 17, wherein the immune cells are CAR-T cells.

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