CELLS INCUBATED WITH CD1d LIGAND PRIOR TO CD1d INTRODUCTION

Abstract

The present disclosure provides a composition having NKT cell activation ability that contains cells incubated with a CD1d ligand prior to CD1d introduction. The present disclosure also provides a composition comprising cells, wherein the cells are human-derived cells and express a complex of CD1d and CD1d ligand on the cell surface and in the cell, respectively, and the amount of the complex in the cells is significantly greater than the amount of the complex on the cell surface. The present disclosure further provides a composition capable of further expressing an antigen and further inducing antigen-specific immunity.

Description

Cells incubated with CD1d ligand before CD1d transfection

[0001] The present disclosure provides cells incubated with a CD1d ligand prior to CD1d introduction, and methods for producing such cells.

[0002] Natural killer T (NKT) cells are immune cells that have the characteristics of T cells and natural killer (NK) cells, and are activated by CD1d ligands, which consist of glycolipids loaded onto CD1d, a major histocompatibility complex (MHC)-like molecule on antigen-presenting cells (APCs). Activated NKT cells are known to directly enhance cytotoxicity via perforin / granzyme B and the like, as well as induce their own differentiation and proliferation through the production of cytokines such as interferon gamma (IFN-γ) and interleukin-4 (IL-4), thereby activating T cells, NK cells, and B cells.

[0003] NKT cells activated by dendritic cells (DCs) pulsed with α-GalCer, a CD1d ligand, exhibit cytotoxicity against various cancer cells. It has also been reported that α-GalCer-pulsed DCs induce more IFN-γ-producing NKT cells than DCs administered with free α-GalCer, demonstrating a strong antitumor effect (Non-Patent Documents 1 and 2), and clinical trials targeting lung cancer patients have also been conducted (Non-Patent Documents 3 and 4).

[0004] Fujii et al. exogenously expressed CD1d and a cancer antigen in human-derived cells, and then pulsed the cells with α-GalCer to produce aAVC (Patent Documents 1 to 5). The aAVC activated NKT cells via the CD1d / α-GalCer complex, and the activated NKT cells produced cytokines such as IFN-γ, activating NK cells and other cells. In mouse models, administration of aAVC has demonstrated an NK cell-dependent antitumor effect (Patent Documents 1 to 4, Non-Patent Documents 5 and 6). Furthermore, aAVC administered to mice was rapidly killed by activated NKT cells in vivo, and the resulting cell fragments were taken up by dendritic cells. Dendritic cells that had taken up the aAVC fragments presented the cancer antigen incorporated into MHC on their cell surface, inducing cancer antigen-specific T cells. In mouse models, administration of aAVC also demonstrated an antitumor effect via the induction of cancer antigen-specific T cells (Patent Documents 1 to 3, Non-Patent Documents 5 and 6). Thus, it has been shown that aAVC can potently induce two immune mechanisms: activation of innate immunity by NK cell activation via NKT cell activation, and induction of adaptive immunity by induction of antigen-specific T cells.

[0005] International Publication No. 2007 / 097370 International Publication No. 2010 / 061930 International Publication No. 2013 / 018778 International Publication No. 2021 / 112056 International Publication No. 2021 / 112055

[0006] The Journal of Immunology, 1999; 163 (5): 2387-2391 Nature Immunology, 2002; 3 (9): 867-874 The Journal of Immunology, 2009;182(4):2492-2501Clinical Cancer Research, 2005;11(5):1910-1917Cancer Research, 2013;73(1):62-73Cancer Research, 2016;76(13):3756-3766

[0007] Conventionally, in the production of aAVC, cells expressing CD1d were incubated in the presence of a CD1d ligand to obtain cells presenting CD1d bound to the CD1d ligand. Such cells were beneficial for activating NKT / NKT cells. This was because it was believed that CD1d ligands could not bind to cells without CD1d on the cell surface. The present inventors found that cells capable of activating NKT cells could be effectively obtained by incubating cells in the presence of a CD1d ligand before introducing or inducing the expression of CD1d. It was a surprising result that this method could confer NKT cell activation ability even to cells that were originally CD1d-negative.

[0008] According to the present disclosure, for example, the following inventions are provided: (1) A composition comprising cells, wherein the cells are derived from a human and express a complex of CD1d and a CD1d ligand on the cell surface and intracellularly, respectively, and the amount of the complex intracellularly is significantly greater than the amount of the complex on the cell surface. (2) The composition according to (1) above, wherein the cells are further incubated (post-incubated) in the presence of a CD1d ligand after introduction or induction of expression of CD1d. (3) The composition according to (1) or (2) above, wherein the cells have a higher NKT cell activation ability compared to control cells, and the control cells are cells that were not pre-incubated in the presence of a CD1d ligand before introduction or induction of expression of CD1d, but were introduced or induction of expression of CD1d and then post-incubated in the presence of a CD1d ligand, wherein the conditions for incubation with the CD1d ligand are the same for the cells in the composition and the control cells, except for whether the incubation with the CD1d ligand is pre-incubation or post-incubation. (4) The composition according to any one of (1) to (3) above, wherein the cells are CD1d-negative before introduction of CD1d or induction of expression. (5) The composition according to any one of (1) to (4) above, wherein the cells express an antigenic peptide. (6) A method for producing a composition containing cells capable of activating NKT cells, comprising: incubating human-derived cells in the presence of a CD1d ligand (pre-incubation); introducing CD1d into the cells or inducing CD1d expression; and incubating the obtained cells in the presence or absence of a CD1d ligand (post-incubation), thereby obtaining cells that express CD1d protein and have NKT cell activity. (7) The method according to (6) above, wherein the post-incubation is performed in the absence of a CD1d ligand. (8) The method according to (6) above, wherein the post-incubation is performed in the presence of a CD1d ligand. (9) The method according to any one of (6) to (8) above, wherein the cells are CD1d-negative before introduction of CD1d or induction of expression.

[0009] (21) Any of the above-mentioned inventions, wherein the introduction or induction of expression of CD1d comprises introduction of a nucleic acid encoding CD1d into cells. (22) Any of the above-mentioned inventions, wherein the introduction or induction of expression of CD1d comprises introduction of an mRNA encoding CD1d into cells. (23) The invention according to (21) above, wherein the introduction of a nucleic acid encoding CD1d into cells is performed by electroporation. (24) The invention according to (22) above, wherein the introduction of an mRNA encoding CD1d into cells is performed by electroporation. (25) The invention according to (23) above, wherein the introduction of a nucleic acid encoding CD1d into cells is performed under serum-free conditions. (26) The invention according to (24) above, wherein the introduction of an mRNA encoding CD1d into cells is performed under serum-free conditions.

[0010] (31) Any of the above methods, further comprising introducing an antigen peptide into the cell or inducing expression of an antigen peptide. (32) Any of the above methods, not comprising introducing an antigen peptide into the cell or inducing expression of an antigen peptide.

[0011] (41) The cell according to any one of the above, which has the ability to activate NKT cells. (42) The cell according to any one of the above, which expresses an antigen peptide and has the ability to activate NKT cells and induce specific immunity against the antigen peptide.

[0012] (51) A composition comprising cells, wherein the cells express at least an extracellular complex of CD1d and a CD1d ligand, and the expression of the complex is at or above the detection limit of flow cytometry {e.g., the composition may be the composition (1) above}. That is, the expression level of the complex is greater than or equal to the value measured using an isotype antibody. For example, in Figure 7B (left), 4 hours after EP, the MFI of the surface is 71.1, and the MFI of the intracellular is 323 or greater. Also, for example, in Figure 7B (right), 24 hours after EP, the MFI of the surface is 101, and the MFI of the intracellular is 516 or greater. (52) The composition according to (51) above, wherein the cells are derived from a human. (53) The composition according to (51) above, wherein the cells further comprise an antigen. (54) The composition according to (52) above, wherein the cells further comprise an antigen. (55) The composition according to (53) above, wherein the antigen is an antigenic peptide. (56) The composition according to (54) above, wherein the antigen is an antigenic peptide. (57) The composition according to any one of (51) to (56) above, for use in activating NKT cells. (58) The composition according to any one of (53) to (56) above, for use in inducing antigen-specific acquired immunity.

[0013] The figure shows the effect of medium composition at the time of nucleic acid transfection on protein expression. The figure shows CD1d expression in the cells used (HEK293sf cells). This figure shows a schematic diagram of an experiment in which cells were incubated in the presence or absence of a CD1d ligand (α-GalCer in this case) with or without electroporation (EP), and then recovered and co-cultured with NKT cells to measure the activation of NKT cells and the amount of interferon-γ (IFN-γ) produced by the NKT cells. The figure shows the results of the experiment in Figure 3A. This figure shows a schematic diagram of an experiment in which cells were pre-incubated in the presence of a CD1d ligand (α-GalCer in this case) before EP, subjected to EP, and then post-incubated in the absence of a CD1d ligand (α-GalCer in this case) for 6 hours, recovered, and co-cultured with NKT cells to measure the activation of NKT cells and the amount of interferon-γ (IFN-γ) produced by the NKT cells. The figure shows the results of the experiment in Figure 4A. This is a schematic diagram of an experiment in which cells are pre-incubated in the presence of a CD1d ligand (here, α-GalCer) before EP, then post-incubated for 6 hours in the presence of a CD1d ligand (here, α-GalCer), recovered, and co-cultured with NKT cells to measure the activation of NKT cells and the amount of interferon-γ (IFN-γ) produced by the NKT cells. The results of the experiment in Figure 5A are shown. The conditions and presence / absence of pre-incubation before EP, the presence / absence of EP, and the post-incubation conditions for each experimental condition are shown. The expression levels of human CD1d (hCD1d) on the cell surface and the expression levels of intracellular antigen proteins (here, OVA) for each experimental condition in Figure 6A are shown. An outline of the experimental conditions is also shown. In condition 1, the CD1d-encoding gene (EP) was not introduced, but pretreatment (48 hours) and posttreatment (4 hours or 24 hours) with a CD1d ligand were performed; in condition 2, the gene was introduced, followed by pretreatment and posttreatment with a CD1d ligand; and in condition 3, the gene was introduced, followed by posttreatment with a CD1d ligand. Net mean fluorescence intensity (netMFI) was calculated and displayed relative to the expression level (same below). NetMFI was normalized by subtracting the netMFI of the isotype control antibody (same below).Figure 7A shows the cell surface expression (Surface) and intracellular expression (Intracellular) of the CD1d / CD1d ligand complex under conditions 1 to 3 shown in Figure 7A. In this case, mouse CD1d (mCD1d) was used instead of human CD1d (hCD1d). Figure 7A shows the cell surface expression and intracellular expression of mCD1d under conditions 1 to 3 shown in Figure 7A. An outline of the experimental conditions is shown. In condition 1, transfection of a gene encoding CD1d (EP) was followed by post-treatment with a CD1d ligand (6 hours). In condition 2, post-treatment was performed without transfection. In condition 3, transfection was performed but without post-treatment. In condition 4, neither gene transfection nor post-treatment was performed. Figure 8A shows the cell surface expression and intracellular expression of the CD1d / CD1d ligand complex under conditions 1 to 4 shown in Figure 8A. An outline of the experimental conditions is shown. Under condition 1, cells were transfected with a gene encoding CD1d (EP), followed by pretreatment (48 hours) and posttreatment (6 hours) with a CD1d ligand. Under condition 2, cells were transfected with a CD1d ligand and then pretreated with a CD1d ligand (48 hours). Under condition 3, cells were transfected with a CD1d ligand and then pretreated with a CD1d ligand (6 hours). Under condition 4, cells were transfected with a CD1d ligand and then pretreated with a CD1d ligand (2 hours). Figure 9A shows the cell surface and intracellular expression of the CD1d / CD1d ligand complex under conditions 1 to 4. Figure 9A shows the cell surface and intracellular expression of CD1d under conditions 1 to 4. An overview of the experimental conditions is shown below. Under condition 1, cells were pretreated with a CD1d ligand (48 hours), transfected with a gene encoding CD1d (EP), and then cultured in the absence of CD1d ligand for 16 hours (condition 1), 24 hours (condition 2), 48 hours (condition 3), 72 hours (condition 4), and 96 hours (condition 5). 10A shows the cell surface expression and intracellular expression of CD1d / CD1d ligand complex under conditions 1 to 5. FIG. 10B shows the cell surface expression and intracellular expression of CD1d under conditions 1 to 5. FIG.

[0014] As used herein, "mammal" includes, for example, non-human primates such as chimpanzees, gorillas, orangutans, monkeys, marmosets, and bonobos; non-human mammals (e.g., Carnivora, Artiodactyla, Perissodactyla, and Rodents) such as pigs, rats, mice, cows, sheep, goats, horses, cats, and dogs.

[0015] As used herein, "artificial adjuvant vector cells" (aAVCs) refer to cells that express CD1d and an antigenic peptide, with CD1d forming a complex with a CD1d ligand. aAVCs can activate natural killer T cells (NKT cells) and are taken up by dendritic cells (DCs), which are antigen-presenting cells. Activated NKT cells mature dendritic cells that have taken up antigen, and the mature dendritic cells migrate to lymphoid tissues to induce antigen-specific T cells (e.g., CD4+ T cells, CD8+ T cells, etc.). aAVCs drive or trigger this series of processes (NKT cell activation, phagocytosis by dendritic cells, maturation of dendritic cells after phagocytosis by activated NKT cells, and migration of dendritic cells to lymphoid tissues). Although aAVCs express antigenic peptides, they are beneficial because they have the ability to activate NKT cells even in the absence of antigenic peptides. aAVC expresses antigenic peptides and thus induces specific immunity (acquired immunity) against the antigenic peptides. In this way, aAVC can stimulate both innate and antigen-specific adaptive immunity, thereby promoting the immune elimination of cancer cells and pathogens.

[0016] As used herein, "antigen" refers to a substance having antigenicity. An antigen is usually a substance against which specific immunity should be induced. An antigen may preferably be an antigen accessible to the immune system from outside a cell. An antigen may be exogenous or endogenous. Exogenous antigens include, but are not limited to, pathogens (e.g., viruses, bacteria, fungi, parasites, etc.). An antigen may be, for example, an endogenous factor that is selectively present in a specific tissue or specific cell that is the target of specific immune attack, or an endogenous factor that is selectively expressed in a pathological state. An endogenous factor includes, but is not limited to, a cancer antigen. A cancer antigen is an antigen that is specifically expressed in cancer and preferably does not show substantial expression in normal tissues or normal cells.

[0017] As used herein, an "antigenic peptide" refers to an antigenic protein or a fragment thereof. Antigens can be, for example, nucleic acids, lipids, glycolipids, sugar chains, peptides, etc. Antigenic peptides can be, for example, proteins selectively expressed in cancer cells (e.g., cancer antigens), or antigens selectively expressed in pathogen-infected cells (e.g., proteins constituting pathogens, proteins selectively expressed in target cells, or fragments thereof). Antigens (e.g., antigenic peptides) are present intracellularly or on cell membranes. This is because they are taken up by dendritic cells and presented as antigens. When presented as antigens, they are presented as short protein fragments, so antigenic peptides may be proteins or protein fragments as long as they can be presented as antigens by dendritic cells. Antigenic peptides can be, for example, membrane proteins or portions of their extracellular domains.

[0018] As used herein, the term "starting cells" refers to cells prior to the treatment of the present invention, which comprises at least preincubating the starting cells in the presence of a CD1d ligand and introducing a nucleic acid encoding CD1d into the cells obtained above.

[0019] As used herein, "preincubation" refers to incubating (culturing) cells under conditions suitable for maintaining the cells before introducing or inducing expression of CD1d into the cells. Incubation can be performed, for example, in the presence or preferably in the absence of serum (preferably under xeno-free conditions). As used herein, "xeno-free" refers to not containing components of a species different from the cells.

[0020] As used herein, "post-incubation" refers to incubating (culturing) cells under conditions suitable for maintaining the cells after introducing or inducing expression of CD1d into the cells. Incubation can be performed, for example, in the presence or preferably in the absence of serum (preferably under xeno-free conditions).

[0021] As used herein, "CD1d" may refer to naturally occurring CD1d and its functional variants. CD1d may be CD1d endogenously expressed in the human-derived cells used, or CD1d exogenously expressed in the human-derived cells used. In one embodiment, "expressed" means expressed somewhere on the cell, and in one embodiment, means expressed on the cell surface. In one embodiment, aAVC expresses exogenous CD1d. In one embodiment, CD1d used in the present invention is CD1d derived from a mammal (e.g., human, monkey, mouse, rat, dog, chimpanzee, etc.). In one embodiment, CD1d used in the present invention is human CD1d. As used herein, "CD1d negative" means not expressing CD1d. CD1d negativity can be determined by flow cytometry, taking into account the background signal.

[0022] In one embodiment, human CD1d is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, human CD1d is a protein consisting of an amino acid sequence obtained by deleting, substituting, inserting, and / or adding one or several, in some embodiments, 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 amino acids in the amino acid sequence shown in SEQ ID NO: 1, and having the function of CD1d. In one embodiment, human CD1d is a protein consisting of an amino acid sequence that has at least 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 1, and having the function of CD1d.

[0023] The function of CD1d includes the ability to bind to CD1d ligands (for example, α-galactosylceramide (α-GalCer), α-C-galactosylceramide (α-C-GalCer), 7DW8-5, and isoglobotrihexosylceramide (iGb3)). The binding ability of CD1d to CD1d ligands can be easily evaluated by those skilled in the art using known methods. Furthermore, the function of CD1d can also be evaluated using as an index the ability to activate human NKT cells by aAVC. This ability to activate human NKT cells can be evaluated by the method described in Patent Document 1 or Example 4 of the present application. α-GalCer is a compound having CAS RN: 158021-47-7, molecular formula: C ５０ H ９９ NO ９ and molecular weight: 858.34, and is one of the CD1d ligands. α-GalCer may be synthesized according to techniques known in the art, or commercially available α-GalCer (e.g., α-galactosylceramide (Funakoshi, Cat. KRN7000)) may be used. Cells may be loaded with α-GalCer by culturing CD1d-expressing cells in a medium containing α-GalCer.

[0024] As used herein, the terms "exogenous" and "exogenous" are used interchangeably to refer to the artificial introduction of a gene or nucleic acid into a target cell by genetic engineering, gene transfer, or other manipulation, as well as the gene or nucleic acid artificially introduced into a target cell and the protein expressed therefrom. An exogenous gene may be operably linked to a promoter sequence that drives expression of the gene.

[0025] As used herein, "endogenous" or "intrinsic" means something that is naturally present in a cell.

[0026] As used herein, the term "derived" refers to the animal species from which the cells are obtained. For example, human-derived cells refer to cells obtained from a human or a cell line obtained by subculturing the cells, e.g., human cells. In one embodiment, the human-derived cells used in the present invention are immortalized cells or cell lines derived from human tissue. Immortalized cells and cell lines can be prepared by methods known to those skilled in the art.

[0027] "Identity" as used herein refers to the Identity value obtained using EMBOSS Needle (Nucleic Acids Res.; 2015; 43: W580-W584) with default parameters. The parameters are as follows: Gap Open Penalty = 10, Gap Extend Penalty = 0.5, Matrix = EBLOSUM62, End Gap Penalty = false.

[0028] <Cells of the present disclosure and methods for producing said cells> The cells of the present disclosure are mammalian cells (preferably human cells). The cells of the present disclosure have at least the ability to activate NKT cells. Cells that present CD1d complexed with a CD1d ligand have the ability to activate NKT cells. Therefore, the cells of the present disclosure have CD1d complexed with a CD1d ligand. The cells of the present disclosure can induce antigen-specific acquired immunity by expressing an antigen. The present disclosure also provides cells (aAVC) that further express such an antigen.

[0029] Conventionally, CD1d cells complexed with a CD1d ligand have been prepared by contacting cells with a CD1d ligand after CD1d introduction or expression induction (or by incubating (postincubation) in the presence of a CD1d ligand). This was because it was thought that contacting cells with a CD1d ligand before CD1d expression on the cells was meaningless unless CD1d, which receives the ligand, was expressed. However, as shown in the Examples below, even if CD1d-negative cells were preincubated in the presence of a CD1d ligand and, after CD1d introduction or expression induction, were not postincubated in the presence of a CD1d ligand, they still had the ability to activate NKT cells. Furthermore, a longer preincubation time resulted in a superior ability to activate NKT cells. This is thought to be the result of CD1d-negative cells taking up the CD1d ligand as a result of the long preincubation. Furthermore, it has been shown that preincubation in the presence of a CD1d ligand can enhance NKT cell activation more than postincubation. Therefore, it is suggested that when cells express CD1d de novo (newly) in a state where CD1d ligand has been incorporated, CD1d can efficiently form a complex with the CD1d ligand. CD1d does not activate NKT cells without a ligand, and CD1d ligands do not activate NKT cells without CD1d. This supports the idea that even CD1d-negative cells can incorporate CD1d ligands into cells by contacting them with a CD1d ligand, and when CD1d is expressed on the cell surface, CD1d can efficiently form a complex with the CD1d ligand, thereby promoting NKT cell activation. WO2021 / 112056A reveals that the amount of CD1d ligand bound to CD1d in aAVC, rather than the number of cells, is associated with high immune induction ability, and it is believed that efficient binding of CD1d ligands to CD1d-expressing cells is important for achieving high immune induction ability.

[0030] The present disclosure may include incubating starting cells in the presence of a CD1d ligand and then expressing CD1d on the cell surface. The starting cells may be CD1d-positive or CD1d-negative, but the presence of CD1d presented de novo on the cell surface after incubation in the presence of a CD1d ligand is considered important. Those skilled in the art can utilize various methods to present CD1d de novo on the cell surface at that timing, such as introducing CD1d into cells, introducing a nucleic acid (e.g., DNA or RNA) encoding CD1d into cells, or inducing expression of a nucleic acid (e.g., DNA) encoding CD1d that has already been introduced into cells and is operably linked to an inducible promoter by providing an inducer.

[0031] Thus, the present invention is based on the fact that by preincubating cells in the presence of a CD1d ligand before introduction or induction of CD1d expression, and allowing the cells to incorporate the CD1d ligand, efficient binding of the CD1d ligand to CD1d can be achieved after introduction or induction of CD1d expression. Therefore, as long as cells are preincubated in the presence of a CD1d ligand before introduction or induction of CD1d expression, postincubation in the presence of a CD1d ligand after introduction or induction of CD1d expression is not necessary, and preferably, postincubation in the presence of a CD1d ligand after introduction or induction of CD1d expression can be performed. By preincubating in the presence of a CD1d ligand and then postincubating in the presence of a CD1d ligand, the ability of cells to activate NKT cells can be improved. Furthermore, it is believed that it is important for CD1d to be presented on the cell membrane surface after preincubation. As long as CD1d expression is induced after preincubation, the timing of introduction or induction of CD1d expression is not important, and as long as CD1d is generated de novo in the cells, introduction of CD1d may not be necessary.

[0032] Preincubation of cells in the presence of a CD1d ligand is thought to induce uptake of the CD1d ligand by the cells, as evidenced by the fact that even when the cells were washed after preincubation and all subsequent steps were carried out in the absence of a CD1d ligand, the cells retained the ability to activate NKT cells.

[0033] Previous methods of culturing cells in the presence of a CD1d ligand only after gene transfer have failed to produce cells expressing the complex at levels detectable by flow cytometry. In contrast, according to the present disclosure, the resulting cells express a complex of CD1d and a CD1d ligand at least extracellularly, and the expression of the complex is above the detection limit of flow cytometry. According to the present disclosure, the resulting cells further express a complex of CD1d and a CD1d ligand intracellularly, and the expression of the complex is above the detection limit of flow cytometry. In one aspect of the present disclosure, a composition is provided that exhibits significantly increased expression of the complex by flow cytometry compared to cells obtained by culturing cells in the presence of a CD1d ligand only after gene transfer for 24 hours (provided that all conditions except the timing of CD1d ligand treatment are the same). CD1d is introduced by introducing a nucleic acid encoding CD1d into the cells. The CD1d ligand can be α-GalCer (e.g., 500 ng / mL).

[0034] According to the present disclosure, cells and compositions comprising the cells are provided. The cells are preferably cells of the same species as the animal species to which the cells are to be administered. When administered to humans, the cells are preferably derived from humans. The cells take up a CD1d ligand and then present CD1d on the cell surface. It is believed that CD1d forms a complex with the CD1d ligand before being presented on the cell surface. The cells obtained in this manner present both CD1d complexed with the CD1d ligand and free CD1d on the cell surface, and it is believed that incubation with additional CD1d ligand can cause the free CD1d to form a complex with the CD1d ligand.

[0035] In a preferred embodiment, uptake of a CD1d ligand into cells can be achieved, for example, by incubating cells in the presence of a CD1d ligand. There are also various other methods for uptake of a CD1d ligand into cells.

[0036] In a preferred embodiment, the incubation can be carried out in the presence of serum or under serum-free conditions.

[0037] In a preferred embodiment, the cells are incubated (preincubated) in the presence of a CD1d ligand before introduction or induction of CD1d expression, and then present CD1d on the cell surface by introduction or induction of CD1d expression. In a preferred embodiment, the cells may have a stronger NKT cell activation ability than conventional cells, i.e., control cells that are postincubated in the presence of a CD1d ligand after introduction or induction of CD1d expression. All conditions except the timing of incubation are the same for the two types of cells compared. Furthermore, extending the preincubation time can improve NKT cell activation. This is believed to be because extending the preincubation time increases the amount of CD1d ligand taken up by the cells. Therefore, in a preferred embodiment, preincubation can be performed for a time sufficient for cellular uptake of the CD1d ligand (e.g., 12 to 80 hours, 24 to 72 hours, or 36 to 60 hours). CD1d introduction can be by introduction of a CD1d protein or a nucleic acid encoding CD1d. In a preferred embodiment, the nucleic acid is mRNA. The mRNA has a structure suitable for translation of the CD1d protein (e.g., a cap structure, a change of uracil to pseudouridine, polyA, etc.). Induction of CD1d expression can be achieved by expression of CD1d from an inducible promoter. The CD1d protein preferably has a signal sequence for cell membrane localization, which enables efficient expression on the cell membrane.

[0038] In one aspect of the present disclosure, the cells contain CD1d / CD1d ligand complexes on the cell surface and intracellularly. In a preferred aspect, the amount of intracellular CD1d / CD1d ligand complexes is significantly greater than the amount of cell-surface CD1d / CD1d ligand complexes, and preferably, the ratio (amount of intracellular complexes / amount of cell-surface complexes) can be 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more. Alternatively, for example, the ratio can be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, or 4 or less. The ratio can be, for example, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5. Such cells are preferably obtainable in the context of preparing cells by introduction of mRNA encoding CD1d.

[0039] In one aspect of the present disclosure, the cells contain intracellular CD1d / CD1d ligand complexes 48 hours, 72 hours, or preferably 96 hours after the initiation of culture in the absence of CD1d ligand following gene transfection (preferably mRNA transfection) and medium replacement. The intracellular CD1d / CD1d ligand complexes may comprise an effective amount of the complex. In one aspect, the amount of intracellular CD1d / CD1d ligand complexes is significantly greater than the amount of CD1d / CD1d ligand complexes on the cell surface, and may be preferably 5 to 100 times, 10 to 80 times, 15 to 60 times, or 20 to 50 times the amount of CD1d / CD1d ligand complexes on the cell surface. The gene transfection can be performed in a serum-free medium or a chemically defined medium, as described above. The replacement medium can also be a serum-free medium or a chemically defined medium. The cells can be obtained, for example, by culturing cells in the presence of a CD1d ligand before gene transfection. The culture time of the cells in the presence of a CD1d ligand before gene transfer can be, for example, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, 48 hours or more, 72 hours or more, or 96 hours or more. The culture time of the cells in the presence of a CD1d ligand before gene transfer can be, for example, 120 hours or less, 96 hours or less, 84 hours or less, 72 hours or less, 60 hours or less, or 48 hours or less. The culture time of the cells in the presence of a CD1d ligand before gene transfer can be, for example, 24 to 120 hours, 36 to 72 hours, or 36 to 60 hours. The upper and lower limits of this time can be as described above. The cells contain CD1d / CD1d ligand complexes intracellularly even after administration, which can contribute to prolonged NKT cell activation.

[0040] Intracellular CD1d / CD1d ligand complexes can be detected by flow cytometry using BD's Cytofix / Cytoperm Fixation / Permeabilization Kit (#554714) and an anti-CD1d / α-GalCer complex antibody. Preferably, the anti-CD1d / α-GalCer complex antibody has affinity for the CD1d and α-GalCer complex but has substantially no affinity for CD1d or α-GalCer. Examples of such antibodies include anti-CD1d / α-GalCer complex antibody (clone L363, #12-2019-82; e-Bioscience).

[0041] An inducible promoter is a promoter that inducibly promotes expression and is capable of inducing expression of a polynucleotide operably linked to the promoter in the presence of an inducer that drives the promoter (also referred to herein as an "inducer of an inducible promoter" or simply as an "inducer"). A nucleic acid encoding CD1d operably linked to an inducible promoter may be introduced into cells either before or after preincubation, but de novo expression of CD1d on the cell surface can be induced by driving expression with an inducer after preincubation.

[0042] Examples of inducible promoters include thermo-inducible promoters (e.g., heat shock promoters) and drug-inducible promoters. In one embodiment, the inducible promoter is a drug-inducible promoter. As used herein, "drug-inducible promoter" refers to a promoter in which the expression of a polynucleotide operably linked to the promoter is regulated by a drug as an inducer. Examples of drug-inducible promoters include a cumate operator sequence, a lambda operator sequence (e.g., 12×λOp), and an inducible promoter of a tetracycline gene expression induction system (hereinafter referred to as a "tetracycline-inducible promoter"). The cumate operator sequence is inactive in the presence of the CymR repressor, but in the presence of the inducer Cumate, it dissociates from the CymR repressor and induces the expression of a polynucleotide operably linked to the promoter. The λ operator sequence induces expression of a polynucleotide operably linked to the promoter in the presence of an inducer (e.g., coumermycin) that dimerizes an activator (λRep-GyrB-AD) that has transcriptional activation ability through dimerization. The tetracycline-inducible promoter induces expression of a polynucleotide operably linked to the promoter in the presence of inducers such as tetracycline or a derivative thereof (e.g., doxycycline) and a reverse tetracycline-controlled transactivator (rtTA) (e.g., Tet-On 3G). An example of a tetracycline-inducible promoter is the TRE3G promoter. In one embodiment, the drug-inducible promoter is a tetracycline-inducible promoter, and in another embodiment, the tetracycline-inducible promoter is the TRE3G promoter.

[0043] Constitutive promoters include, but are not limited to, promoters derived from viruses such as CMV (cytomegalovirus), RSV (respiratory syncytial virus), and SV40 (simian virus 40), actin promoters, and EF (elongation factor) 1α promoters. A constitutive promoter is a promoter that always drives transcription from a nucleic acid to which it is operably linked. A nucleic acid encoding CD1d operably linked to a constitutive promoter is preferably introduced into cells after preincubation. Of course, the timing of introduction is not necessarily limited to after preincubation, as long as it is possible to induce de novo expression of CD1d on the surface of cells into which a CD1d ligand has been incorporated.

[0044] When the nucleic acid is DNA, the DNA is usually operably linked to a promoter. The promoter may be an inducible promoter or a constitutive promoter. When it is desired to induce expression induction at a desired timing, an inducible promoter may be used as the promoter, and an inducer may be allowed to act at the desired timing to drive the inducible promoter to induce expression.

[0045] According to the present disclosure, introduction or induction of expression of CD1d into cells (particularly introduction of a nucleic acid (preferably mRNA) encoding CD1d and / or an antigen or antigenic peptide) is preferably carried out under conditions of a serum concentration of 5% or less, or under serum-free conditions. Nucleic acid introduction can be carried out by conventional methods, but in one embodiment, it can be carried out by transfection, and in one preferred embodiment, it can be carried out by electroporation. In the production of cells capable of activating NKT cells, cells have traditionally been incubated with α-GalCer in the presence of serum, and nucleic acid introduction into cells has also been carried out in the presence of serum. Therefore, according to the present disclosure, there is provided a method for producing cells capable of activating NKT cells, comprising introducing a nucleic acid encoding CD1d and / or a nucleic acid encoding an antigen or antigenic peptide into cells under serum-free conditions, and incubating the cells in the presence of a CD1d ligand, thereby obtaining cells that present CD1d complexed with a CD1d ligand. In one embodiment, all steps from obtaining cells capable of activating NKT cells from starting cells to obtaining aAVC are carried out under serum-free conditions. The serum-free conditions may preferably be xeno-free conditions.

[0046] The aAVC must express an antigen. If the cells already express the antigen, it is not necessary to introduce a nucleic acid encoding the antigen peptide again, but the production process may include introducing a nucleic acid encoding the antigen peptide again. For example, if the starting cells are cancer cells or pathogen-infected cells, the cells already express the antigen, so there is no need to additionally express the antigen again.

[0047] In one embodiment, the aAVC used in the present invention expresses a cancer antigen. The cancer antigen used in the present invention may be any protein that is expressed in cancer cells, such as Wilmus Tumor 1 (WT-1), Human Carbohydrate Antigen 125 (CA-125), Carcinoembryonic Antigen (CEA), Human Telomerase Reverse Transcriptase (hTERT), Mucin-1 (Muc-1), Mucin-2 (Muc-2), Cancer / Testis antigen 1B (CTAG1B / NY-ESO-1), Prostatic Acid Phosphatase (PAP), and Prostate Specific Antigen (PSA). Examples of cancer antigens used in the present invention include PSA, prostate specific membrane antigen (PSMA), survivin b, mutant ras, and mutant p53. In one embodiment, the cancer antigen used in the present invention includes Wilmus tumor 1 (WT-1). In one embodiment, WT-1 is human WT-1. In one embodiment, the aAVC expresses one type of cancer antigen. In one embodiment, the aAVC expresses multiple types of cancer antigens. The cancer antigen may be a naturally occurring cancer antigen or a modified form thereof, as long as the aAVC expressing it exhibits an anti-tumor effect through immune action against the cancer antigen. The cancer antigen may be an antigen endogenously expressed in the human-derived cells used, or an antigen exogenously expressed in the human-derived cells used. In one embodiment, the aAVC expresses an exogenous cancer antigen. In one embodiment, the aAVC expresses multiple exogenous cancer antigens.

[0048] In the method of the present disclosure, cell culture or incubation is carried out under conditions suitable for cell maintenance. The conditions suitable for cell maintenance may be conditions suitable for cell maintenance, culture, or proliferation. Cell culture for cell maintenance or proliferation is carried out by a known method. Examples of basal media include MEM medium (Science; 1952; 122: 501), DMEM medium (Virology; 1959; 8: 396-397), RPMI1640 medium (J. Am. Med. Assoc.; 1967; 199: 519-524), 199 medium (Proc. Soc. Exp. Biol. Med.; 1950; 73: 1-8), FreeStyle ＴＭ 293 Expression Medium (Thermo Fisher Scientific, Cat. 12338022), CD 293 Medium (Thermo Fisher Scientific, Cat. 12338022) Scientific, Cat. 11913019), Expi293 ＴＭ Expression Medium (Thermo Fisher Scientific, Cat. A1435101), Eagle's Medium, Eagle's Minimum Essential Medium, and modified media thereof can be used. The culture medium can contain, for example, serum (e.g., fetal bovine serum), serum replacement (e.g., KnockOut Serum Replacement: KSR), fatty acids or lipids, amino acids, vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, antibiotics, etc. In one embodiment, the medium used for culturing is a serum-free medium or, preferably, a chemically defined medium. The medium has a composition suitable for culturing or incubating cells.

[0049] Culture conditions (e.g., culture time, temperature, pH of the medium, CO ２ The culture conditions (e.g., concentration) can be appropriately selected by those skilled in the art. The pH of the medium is preferably about 6 to 8, and the culture temperature is not particularly limited, but is, for example, about 30 to 40°C, preferably about 37°C. ２The concentration is about 1 to 10%, preferably about 5%. The culture time is not particularly limited, but is about 15 to 336 hours. Aeration and stirring can be performed as necessary.

[0050] Pharmaceutical Compositions of the Present Disclosure The present disclosure provides a composition comprising the cells of the present disclosure. In one embodiment, the pharmaceutical composition is for use in treating cancer. In one embodiment, the CD1d ligand is α-GalCer. The pharmaceutical composition can be prepared by a commonly used method using excipients commonly used in the art, i.e., pharmaceutical excipients, pharmaceutical carriers, etc. When formulating a pharmaceutical composition, excipients, carriers, additives, etc. appropriate for the dosage form can be used within a pharmaceutically acceptable range. Examples of dosage forms of these pharmaceutical compositions include parenteral preparations such as injections and infusions. In one embodiment, the pharmaceutical composition of the present disclosure may comprise a frozen product of the cells of the present disclosure. In one embodiment, the pharmaceutical composition of the present disclosure may comprise a frozen product of the cells of the present disclosure and a cryoprotectant. In one embodiment, the pharmaceutical composition of the present disclosure may comprise a suspension of the cells of the present disclosure. In one embodiment, the pharmaceutical composition of the present disclosure may comprise a suspension of the cells of the present disclosure and a cryoprotectant. The present invention also provides use of the cells of the present disclosure for the manufacture of a pharmaceutical composition for use in treating cancer, wherein the cells express CD1d and an antigen (e.g., an antigenic peptide) and are loaded with a CD1d ligand on their surface. In one embodiment, the CD1d ligand is α-GalCer. The present invention also provides use of the cells of the present disclosure for the manufacture of a pharmaceutical composition for use in treating cancer, wherein the cells express CD1d and an antigen (e.g., an antigenic peptide) and are loaded with a CD1d ligand on their surface. In one embodiment, the CD1d ligand is α-GalCer.

[0051] The present disclosure also provides a method for treating cancer, comprising administering the cells of the present disclosure to a subject. In this method, the cells of the present disclosure express CD1d and an antigen (e.g., an antigenic peptide) or a fragment thereof, and carry a CD1d ligand on their surface. In one embodiment, the CD1d ligand is α-GalCer. As used herein, the term "subject" refers to a mammal (e.g., a human, a monkey, a mouse, a rat, a dog, a chimpanzee, etc.), and in one embodiment, the subject is a human. As used herein, the term "treatment" includes therapeutic treatment and prophylactic treatment. When the cells of the present disclosure are administered to a subject, they can be administered to the subject in the form of a pharmaceutical composition containing the cells of the present disclosure and a pharmaceutically acceptable excipient. The dosage and frequency of administration of the cells of the present disclosure to a subject can be adjusted appropriately depending on the type, location, and severity of the cancer, as well as the age, weight, and condition of the subject to be treated. The dosage of the cells of the present disclosure to a subject can be, for example, 1 x 10 3 cells / kg~1×10 9 The cells of the present disclosure can be administered to a subject at a dose of 1000 cells / kg. The cells can be administered to a subject, for example, by intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, or intraarterial injection or infusion. The treatment method of the present invention can be used in combination with other cancer treatment methods. Examples of other cancer treatment methods include surgery, radiation therapy, hematopoietic stem cell transplantation, or treatment with other anticancer agents.

[0052] Cancers that can be treated in the present disclosure include, but are not limited to, blood cancers such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, multiple myeloma, and T-cell lymphoma, myelodysplastic syndrome, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, non-small cell lung cancer, small cell lung cancer, mesothelioma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, head and neck cancer, uterine cancer, and uterine Examples of cancers that can be treated include solid cancers such as cervical cancer, liver cancer, gallbladder cancer, bile duct cancer, kidney cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, small intestine cancer, stomach cancer, esophageal cancer, testicular cancer, ovarian cancer, and brain tumors, as well as cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue, as well as sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma. In one embodiment, the cancer that is the target of treatment according to the present invention is NY-ESO-1-positive cancer.

[0053] The pharmaceutical compositions of the present disclosure can be used in combination with other pharmaceutical compositions for treatment, for example, other pharmaceutical compositions. For example, to treat cancer, the pharmaceutical compositions of the present disclosure can be used in combination with other pharmaceutical compositions for use in treating cancer. Such other pharmaceutical compositions include anticancer agents, immune checkpoint inhibitors, and cancer immunotherapeutic agents (e.g., T cells, chimeric antigen receptor-expressing immune cells (T cells, NK cells, etc.)). In one embodiment, the immune checkpoint inhibitor administered to the subject is a PD-1-based immune checkpoint inhibitor or a CTLA-4-based immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor administered to the subject is a PD-1-based immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor administered to the subject is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody. In one embodiment, the immune checkpoint inhibitor administered to the subject is nivolumab, pemprolizumab, avelumab, atezolizumab, or durvalumab. In one embodiment, the immune checkpoint inhibitor administered to the subject is nivolumab or pemprolizumab. In one embodiment, the immune checkpoint inhibitor used in combination with the aAVC-NY-ESO-1 cells of the present invention is pemprolizumab. In yet another embodiment, the immune checkpoint inhibitor that can be used in combination with the aAVC-NY-ESO-1 cells of the present invention is a CTLA-4-based immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor administered to the subject is an anti-CTLA-4 antibody, an anti-CD80 antibody, or an anti-CD86 antibody. In one embodiment, the immune checkpoint inhibitor administered to the subject is ipilimumab or tremelimumab. In one embodiment, the immune checkpoint inhibitor administered to the subject is nivolumab or pemprolizumab. In one embodiment, the immune checkpoint inhibitor administered to the subject is ipilimumab. The immune checkpoint inhibitor may be administered simultaneously, sequentially, consecutively, or overlappingly with the pharmaceutical composition of the present disclosure.

[0054] Example 1: Introduction of antigen into mammalian cells In this example, a peptide was used as the antigen, and the effects of the conditions for introducing nucleic acid encoding the antigen peptide into cells on the expression level of the antigen peptide were examined. Ovalbumin (OVA), a model antigen molecule, was used as the antigen peptide. In this example, nucleic acid encoding OVA (10 μg; OVA mRNA (OZ Biosciences)) was transfected into HEK293sf cells (5 × 10 6 OVA was transfected into 1000 cells (cells) by electroporation (500V, 30ms). After 6 hours, cells were harvested, and cell lysates were prepared and the amount of OVA protein was measured. Four different media were used for transfection: 1) OPTI-MEM phenol red-free; 2) OTPI-MEM phenol red +; 3) AIM-V (serum-free medium); and 4) R10 (RMPI containing 10% fetal calf serum (FCS)). AIM-V is a mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 medium. AIM-V contains purified human albumin, human transferrin, human insulin, cholesterol, L-glutamine, and an antibiotic (gentamicin sulfate).

[0055] The results are shown in Figure 1. As shown in Figure 1, antigen protein expression by cells was good in the absence of serum (i.e., conditions 1) to 3) but was weak in the presence of FCS. Previously, CD1d ligands (especially α-galactosylceramide (α-GalCer)) were loaded into cells in FCS-containing R10 medium, and although this was effective, the transfection conditions and the timing and conditions for loading the CD1d ligands need to be reconsidered. Furthermore, the presence or absence of phenol red did not have any particular effect. Based on these experimental results, we decided to use condition 1) from now on.

[0056] The expression of CD1d on the surface and intracellularly of these cells was measured using an anti-human CD1d antibody (clone 51.1 #350308; Biolegend) and flow cytometry (LSRFortessa X-20, BD) by standard methods. The results are shown in Figure 2. In Figure 2, "Iso" indicates the negative control using an isotype antibody, and the expression level is indicated by the gray peak. The expression level of CD1d is indicated by the red peak. As shown in Figure 2, no CD1d expression was observed on the cell surface or intracellularly in these cells. This result is consistent with previous results, particularly in 293 cells, where CD1d expression is not observed.

[0057] Example 2: NKT cell activation Under condition 1), cells were transfected with genes and loaded with α-GalCer. HEK293sf cells were used, and mRNA encoding human CD1d (5 μg) and mRNA encoding OVA (10 μg) were transfected into HEK293sf cells (5 × 10 6 The cells were transfected by electroporation (500 V, 30 ms). After that, α-GalCer was added as a CD1d ligand to the medium (AIM-V, the same applies below) at a final concentration of 500 ng / mL, and the cells were cultured for 6 hours. The cells were harvested and cultured at 1 × 10 4 Each cell was seeded in a 96-well plate and the human NKT cell line (1 × 10 5 The cells were then added to the culture medium and co-cultured for 24 hours. The supernatant was collected and the IFN-γ concentration was measured by ELISA. The experimental scheme was as shown in Figure 3A.

[0058] The results are shown in Figure 4. As shown in Figure 3B, strong IFN-γ production was observed only in the experiment with both electroporation and α-GalCer loading. This indicates that loading a ligand onto CD1d is essential for NKT cell activation (IFN-γ production).

[0059] Example 3: Effect of pre-incubation with CD1d ligand CD1d-negative HEK293sf cells were pre-incubated in the presence of a CD1d ligand (500 ng / mL α-GalCer) 48 hours, 6 hours, or 2 hours before electroporation. Then, in the absence of a CD1d ligand, mRNA (5 μg) encoding human CD1d and mRNA (10 μg) encoding OVA were transfected into HEK293sf cells (5 × 10 6 The cells were transfected by electroporation (500V, 30ms). After culturing for 6 hours, 1 × 10 4 Each cell was seeded in a 96-well plate and the human NKT cell line (1 × 10 5 The cells were then added to the culture medium and co-cultured for 24 hours. The supernatant was collected and the IFN-γ concentration was measured by ELISA. The experimental scheme is shown in Figure 4A.

[0060] The results are shown in Figure 4B. As shown in Figure 4B, the amount of IFN-γ produced by NKT cells increased in a manner dependent on the pre-incubation time of CD1d-negative HEK293sf cells in the presence of a CD1d ligand. In other words, pre-incubation of CD1d-negative HEK293sf cells in the presence of a CD1d ligand promoted NKT cell activation. It is clear from Figure 3B (EP(-) and Gal(+)) that incubation with CD1d does not promote NKT cell activation in cells that do not express CD1d. Therefore, it was unexpected that pre-incubation of negative HEK293sf cells in the presence of a CD1d ligand affected NKT cell activation.

[0061] Furthermore, it has been suggested that pre-incubation of CD1d-negative HEK293sf cells in the presence of CD1d ligand for 48 hours results in higher NKT cell activation potential with aAVC than culture in the presence of CD1d ligand after CD1d gene transfer (see Figures 3B and 4B).

[0062] Example 4: Effect of pre-culture and post-transfection culture in the presence of CD1d ligand In Figure 5A, HEK293sf cells were pre-cultured in the presence of CD1d ligand (500 ng / mL), followed by electroporation (EP) of mRNA (5 μg) encoding human CD1d and mRNA (10 μg) encoding OVA, followed by further culture of HEK293sf cells in the presence of CD1d ligand (500 ng / mL), and then co-culture with NKT cells after seeding.

[0063] The results are shown in Figure 5B. As shown in Figure 5B, pre-incubation of HEK293sf cells in the presence of CD1d ligand (500 ng / mL) and further incubation of HEK293sf cells in the presence of CD1d ligand (500 ng / mL) after EP significantly activated NKT cells, even compared to Figures 3B and 4B. Therefore, pre-incubation of HEK293sf cells in the presence of CD1d ligand (500 ng / mL) and further incubation of HEK293sf cells in the presence of CD1d ligand (500 ng / mL) after EP is important for the production of aAVCs.

[0064] Example 5: Gene Expression Under each of the conditions (up to co-culture with NKT cells) in Examples 2 to 4 above, hCD1d protein expression was measured by flow cytometry, and OVA protein expression was measured by ELISA, and the results were compared (see Figure 6A). As shown in Figure 6B, no significant differences in hCD1d and OVA expression were observed under any of the conditions. This suggests that the activation of NKT cells in Examples 3 to 4 is not due to differences in the amount of hCD1d expression, but rather to the amount of CD1d ligand binding to hCD1d (in other words, the amount of hCD1d complexed with the CD1d ligand).

[0065] Example 6: Detection of CD1d / CD1d ligand complex The effect of pretreatment of non-CD1d-expressing cells with a CD1d ligand on the amount of CD1d / CD1d ligand complex was examined.

[0066] Specifically, as shown in Figure 7A, we investigated the effects of pretreatment with CD1d ligand (Gal) and electroporation (EP) on the amount of CD1d / CD1d ligand complexes. In Figure 7A, solid arrows indicate treatment in the presence of CD1d ligand, while dotted arrows indicate treatment in the absence of CD1d ligand. Pretreatment of CD1d-nonexpressing cells with CD1d ligand was performed by pre-incubating CD1d-negative HEK293sf cells in the presence of CD1d ligand (500 ng / mL α-GalCer) for 48, 6, or 2 hours before electroporation. Electroporation (EP) was performed by injecting human CD1d-encoding mRNA (5 μg) and OVA-encoding mRNA (10 μg) into HEK293sf cells (5 × 10 6 The cells were cultured for 4 or 24 hours and then harvested.

[0067] The expression levels of CD1d / CD1d ligand complexes on the cell surface and intracellularly were confirmed by flow cytometry using an anti-mCD1d / αGalCer complex antibody (clone L363, #12-2019-82; e-Bioscience). Intracellular complex staining was performed using BD's Cytofix / Cytoperm Fixation / Permeabilization Kit (#554714). Specifically, cells were punctured with Fixation / Permeabilization solution, and anti-mCD1d / GalCer complex Ab or anti-mouse CD1d Ab was added in the presence of Perm / Wash buffer. After incubation on ice, the cells were washed and the amount of antibody binding was measured. CD1d expression was confirmed by flow cytometry using an anti-mCD1d antibody (anti-mouse CD1d (clone 1B1 #553846 BD)).

[0068] The results are shown in Figures 7B and 7C. As shown in Figure 7C, cell surface expression of CD1d was confirmed under conditions 2 and 3. In contrast, as shown in Figure 7B, pretreatment with CD1d ligand significantly increased the CD1d / CD1d ligand complex on the cell surface (see condition 2 in Figure 7B). It is noteworthy that this increase in complex was detected by flow cytometry as a significant difference from measurements using an isotype antibody. The increase in cell surface complex expression after 24 hours, when compared with 4 hours after treatment, is thought to be due to the formation of a complex between the CD1d ligand accumulated in cells by CD1d ligand pretreatment and the CD1d molecules generated in cells after electroporation. This complex was expressed on the cell surface, but was recycled without being immediately degraded, resulting in accumulation on the cell surface. In contrast, when CD1d was expressed but no CD1d ligand treatment was performed, as in Figure 7B, condition 3, no CD1d / CD1d ligand complex was detected either intracellularly or on the cell surface (see Figure 7B, condition 3, and Figures 8A-8C; conditions 2-4 in Figure 8 are negative controls). Furthermore, under condition 1, in which no gene transfer by electroporation was performed, neither CD1d nor CD1d complex expression was observed on the cell surface (see Figures 7B and 7C, condition 1). When CD1d ligand was added after CD1d mRNA electroporation, as in condition 1 in Figures 8A-C, both CD1d and CD1d complex expression were observed. However, under conditions 2, 3, and 4 in Figure 8, in which either or both were absent, as in Figure 7, conditions 1 and 3, complex expression was below the detection limit. Thus, specificity was confirmed for the detection of complex formation by the antibody. Condition 1 in Figure 8A is a condition in which CD1d ligand was pulsed only after EP. Compared to Figure 9B, the amount of CD1d / CD1d ligand complex in the cells is significantly different. This suggests that the amount of the above complex not only extracellularly but also intracellularly may contribute to NKT cell activation. A long-term (e.g., 48-hour) pulse with a CD1d ligand before gene transfer is considered effective, especially when the culture period after gene transfer is short. Figure 7B suggests that the increase in the amount of intracellular complex is dependent on the expression level of the CD1d molecule.As a general trend, extending the time of pulsing with CD1d ligand before gene transfection appears to be effective (see Figure 4B). However, Figure 9B suggests that extending the time of pulsing with CD1d ligand before gene transfection is particularly effective when the culture period after gene transfection is short. Furthermore, Figure 8B shows that when CD1d ligand pulsing was performed only after gene transfection, the amount of CD1d / CD1d ligand complexes on the cell surface was equal to or greater than the amount of CD1d / CD1d ligand complexes within the cells. However, pulsing with CD1d ligand for a sufficient period before gene transfection significantly increased the amount of CD1d / CD1d ligand complexes within the cells relative to the amount of CD1d / CD1d ligand complexes on the cell surface (see Figures 7B, 9B, and 10B). Thus, pulsing with CD1d ligand before gene transfection clearly increases the amount of CD1d ligand complexes within the cells. Cells (e.g., aAVC) containing a greater amount of intracellular complexes than the amount on the cell surface have not been known to date. This suggests that it may take time for CD1d ligand to penetrate into the cells.

[0069] The effect of pretreatment time on the amount of CD1d / CD1d ligand complexes on the cell surface was further evaluated using the same conditions as above. As shown in Figure 9A, cells were pretreated with CD1d ligand for 2, 6, or 48 hours before electroporation (EP). After EP, cells were cultured for an additional 6 hours in the presence or absence of CD1d ligand, and then harvested. The conditions for pretreatment, EP, and post-EP culture were the same as above.

[0070] As shown in Figures 9B and 9C, the same amount of CD1d expression was observed under conditions 1 to 4 (see Figure 9C). In contrast, the amount of CD1d / CD1d ligand complex on the cell surface increased with increasing pretreatment time (see Figure 9B, conditions 2 to 4). Further incubation in the presence of CD1d ligand after EP further increased the amount of complex (see Figure 9B, conditions 1 and 2). Thus, pretreatment of cells with CD1d ligand is thought to improve the efficiency of ligand loading onto newly expressed CD1d, thereby increasing the amount of complex on the cell surface. Furthermore, as shown in Figure 9B, the amount of intracellular complex expression also increased depending on the pretreatment time with CD1d ligand. This indicates that the amount of intracellular CD1d ligand increases both on the cell surface and within the cells by expressing CD1d in cells that have taken up CD1d ligand. Considering the significant effect of pretreatment on the increase in complex expression, it is believed that CD1d ligand is internalized within cells, where CD1d and CD1d ligand can form a complex within the cells, and that this intracellular complex contributes significantly to the immunostimulatory activity of aAVC. The intracellular CD1d / CD1d ligand complex is known to recycle to the cell surface, and the cytoplasm may function as a reservoir for the complex. In particular, the long-term presence of the complex within the cells may be associated with its prolonged presentation on the cell surface. Furthermore, the intracellular CD1d / CD1d ligand complex itself is thought to activate NKT cells. Therefore, the increase in intracellular CD1d / CD1d ligand complex and its long-term maintenance are suggested to favorably contribute to NKT cell activation. Although specific NKT cell responses could also be induced under conditions 3 and 4 in Figure 9A (see Figure 4B), flow cytometry failed to detect clear complex formation signals, likely due to the trace amounts of complex formation (NKT cells were activated, and complex formation was considered sufficient for activation). Therefore, the fact that the complex is below the detection limit in flow cytometry does not mean that a complex is not formed, nor does it mean that an antigen-specific NKT cell response cannot be induced.

[0071] Furthermore, after 48 hours of pretreatment and electroporation, cells were cultured in the absence of CD1d ligand for 16, 24, 48, 72, or 96 hours and then harvested (see Figure 10A). Pretreatment, electroporation, and subsequent culture were performed under the same conditions as above.

[0072] The results are shown in Figures 10B and 10C. As shown in Figure 10B, CD1d / CD1d ligand complexes were highest on the cell surface and intracellularly when the incubation time after EP was 16 to 48 hours, and tended to decrease when the incubation time after EP exceeded 72 hours. As shown in Figure 10C, mCD1d expression was confirmed on the cell surface and intracellularly under all conditions 1 to 5. However, mCD1d expression on the cell surface tended to decrease when the incubation time after EP exceeded 72 hours, and this decrease was thought to be related to complex expression. After gene transfer of a target antigen mRNA of interest, e.g., a cancer antigen mRNA, the expression level of antigen protein from mRNA is often highest between 6 and 16 hours. When prioritizing target antigen protein expression, the time available for pulsing with CD1d ligand after EP is limited to a maximum of 16 hours. However, in this case, when the ligand was pulsed only after EP, complex expression 16 hours after gene transfer was limited. In contrast, according to the present disclosure, pulsing cells with a CD1d ligand prior to EP allows the cells to incorporate the CD1d ligand in advance, significantly increasing the amount of complex on the cell surface and intracellularly after 16 hours of culture after EP. Furthermore, according to the present results, the amount of intracellular complex, which is thought to contribute to NKT cell activation, was maintained even after 96 hours (see Figure 10B). This is the first time that intracellular complexes have been observed 96 hours after mRNA transfection in cells obtained with a CD1d pulse prior to transfection; previously, intracellular complexes were below the detection limit 96 hours after mRNA transfection. The present cells obtained with a CD1d pulse prior to transfection may continue to express the complex intracellularly for a long period of time even after administration, suggesting further utility as aAVC.

[0073] These results demonstrate that incubation of cells with a CD1d ligand before and / or after CD1d gene transfer improves the NKT activation ability of aAVC.

Claims

1. A composition comprising cells, wherein the cells are derived from a human and express a complex of CD1d and a CD1d ligand on the cell surface and intracellularly, respectively, and the amount of the complex intracellularly is significantly greater than the amount of the complex on the cell surface.

2. A composition comprising cells, wherein the cells are derived from a human and have been pre-incubated in the presence of a CD1d ligand before introduction or induction of expression of CD1d, and then CD1d is introduced or induction of expression is carried out, resulting in the expression of CD1d protein.

3. The composition according to claim 2, wherein after introduction or induction of expression of CD1d, the cells are further incubated (post-incubated) in the presence of a CD1d ligand.

4. A composition according to claim 2 or 3, wherein the cells have a higher NKT cell activation ability compared to control cells, and the control cells are cells that have not been pre-incubated in the presence of a CD1d ligand before introduction of or induction of expression of CD1d, but have been introduced with or induced to express CD1d, and then the cells are post-incubated in the presence of a CD1d ligand, and the conditions other than whether the incubation with the CD1d ligand is pre-incubation or post-incubation are the same for the cells in the composition and the control cells.

5. The composition according to any one of claims 2 to 4, wherein the cells are CD1d-negative before introduction or induction of expression of CD1d.

6. The composition of any one of claims 1 to 5, wherein the cells express an antigen.

7. A method for producing a composition containing cells capable of activating NKT cells, comprising: incubating (pre-incubating) human-derived cells in the presence of a CD1d ligand; introducing CD1d into the cells or inducing CD1d expression; and incubating (post-incubating) the resulting cells in the presence or absence of a CD1d ligand, thereby obtaining cells that express CD1d protein and have the ability to activate NKT cells.

8. The method of claim 7, wherein the post-incubation is carried out in the absence of a CD1d ligand.

9. The method of claim 7, wherein the post-incubation is carried out in the presence of a CD1d ligand.

10. The method according to any one of claims 7 to 9, wherein the cells are CD1d-negative before introduction or induction of expression of CD1d.

11. The method of any one of claims 7 to 10, wherein the cells contain an antigen.

12. The method of claim 11, further comprising expressing an antigen in said cells.

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