Pharmaceutical composition for preventing or treating cancer, containing monocells or macrophages having inhibited expression or activity of CD244

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition containing, as an active ingredient, monocytes or macrophages in which the expression or activity of CD244 is inhibited. It has been determined that CD244 inhibits the differentiation of monocytes into macrophages, and the monocytes or macrophages in which the expression or activity of CD244 is inhibited increase phagocytosis and antigen presentation to inhibit tumor growth and induce antigen-specific activation of T cells, and thus the present invention can be usefully used as a composition for preventing or treating cancer.

Description

Technical Field

[0001] The present disclosure relates to a pharmaceutical composition for preventing or treating cancer, comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient. Background Art

[0002] Immune exhaustion is caused by an excessive immune response or an immunosuppressive environment, and it is known that anti-tumor immune responses and reduced expression of inflammatory cytokines can lead to the formation of an immunosuppressive tumor microenvironment. Immune exhaustion occurs mainly through molecular mechanisms of immune checkpoint receptors PD-1, CTLA-4, LAG-3, and TIM-3 expressed by T cells. Recently, antibody therapeutics that inhibit PD-1, PD-L1, and CTLA-4 signaling have been developed for anticancer therapy. However, due to a variety of factors (such as the immunosuppressive tumor microenvironment of solid tumors, tumor cell polymorphism, and lack of tumor-infiltrating immune cells), these therapeutics are only effective in a small number of patients who meet limited genotypes and tumor microenvironments.

[0003] CD244 (SLAMF4, 2B4) is an immune cell receptor that belongs to the signaling lymphocyte activation molecule family (SLAMF). CD244 is a unique receptor that is able to perform two functions, such as activation and inhibition of immune responses, depending on which signaling molecule it binds on NK cells and CD8 T cells. Recently conducted studies have shown that in the tumor microenvironment, CD244 inhibits NK cells and CD8 T cells to maintain their exhausted state. The intracellular domain of CD244 consists of four immunoreceptor tyrosine-based switch motifs (ITSMs), which are able to bind to signaling molecules such as SAP, EAT-2, SHP1, SHP2, and SHIP-1. CD244 is known to transmit immune activation signals when bound to SAP and transmit immune inhibition signals when bound to SH2 phosphatases. The signals transmitted by CD244 depend on the relative expression levels of the signaling molecules and their competitive binding.

[0004] Monocytes and macrophages are important cells that combine innate and adaptive immune responses, sense the surrounding environment, and determine innate and adaptive immune responses. Many recent studies have focused on monocytes and macrophages as important cells in the formation of "cold tumors," which refer to tumors that have little response to immunotherapy. In a study of immune cells in patients with advanced liver cancer, it was found that a large number of monocytes and macrophages differentiated from monocytes infiltrated the tissues around the tumor, which was associated with a decrease in the number of NK cells and immune-active cytokines such as TNF-a and IFN-g in the tumor. In addition, the same study showed that when CD48, which is highly expressed in tumor-infiltrating monocytes, binds to CD244 in NK cells, NK cells become severely exhausted and cannot function properly. Although CD244 is expressed not only in CD8 T cells and NK cells, but also in a variety of myeloid immune cells such as monocytes and macrophages, its role in these cells is still unclear.

[0005] Therefore, in contrast to previous studies, the present inventors attempted to find out what function CD244 expressed in monocyte-macrophages exerts through interaction with CD48 expressed by peripheral immune cells. Summary of the invention Technical goals

[0006] An object of the present disclosure is to provide a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient. Technical Solution

[0007] In order to achieve the above objectives, the present disclosure provides a pharmaceutical composition for preventing or treating cancer, comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

[0008] Furthermore, the present disclosure provides a co-administered pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising monocytes or macrophages having suppressed CD244 expression or activity and an anticancer drug as active ingredients.

[0009] Furthermore, the present disclosure provides a composition for enhancing the efficacy of an anticancer drug, the composition comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

[0010] Furthermore, the present disclosure provides a method for preventing or treating cancer, comprising administering monocytes or macrophages having suppressed CD244 expression or activity to a subject, including a human, alone or in combination with an anti-cancer drug.

[0011] Furthermore, the present disclosure provides a composition for inducing differentiation of monocytes into macrophages, the composition comprising a CD244 inhibitor as an active ingredient.

[0012] Furthermore, the present disclosure provides a method for screening an inducing agent for differentiation of monocytes into macrophages, the method comprising treating cells with a candidate substance to analyze the suppression of the expression or activity of CD244. Beneficial Effects

[0013] According to the present disclosure, it was found that CD244 suppresses the differentiation of monocytes into macrophages, and monocytes and macrophages with suppressed CD244 expression or activity induce antigen-specific activation of T cells by increasing phagocytosis and antigen presentation, thereby inhibiting tumor growth, and thus the present disclosure can be used as a composition for preventing or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the results of analyzing changes in CD244 expression in monocytes after tumor induction and the anticancer effect using a monocyte-specific CD244-deficient mouse model.

[0015] FIG. 2 shows the results of analyzing intratumoral T cell activity and tumor antigen specificity between Flox and cKO mice.

[0016] FIG. 3 shows the results of analyzing whether there are changes in the intratumoral monocyte-macrophage ratio between Flox and cKO mice and monocyte-macrophage differentiation under CD244 deficiency.

[0017] FIG. 4 shows the results of analyzing whether CD244 affects functions such as M1 macrophage polarization, antigen presentation, and phagocytosis using a CD244-deficient mouse model.

[0018] FIG. 5 shows the results of analyzing whether the expression of CD244 in monocytes is induced by ER stress through single-cell transcriptome analysis data and cell experiments.

[0019] FIG. 6 shows the results of analyzing whether CD244 regulates monocyte-macrophage differentiation through autophagy.

[0020] FIG. 7 shows the results of analyzing whether the efficacy of immune checkpoint inhibitors is increased when CD244 is depleted in a monocyte-macrophage-specific manner or CD244-deficient macrophages are administered.

[0021] FIG. 8 shows the results of analyzing whether there are differences in the functions and survival rates of monocyte-macrophages and T cells between patients with high and low CD244 expression levels in melanoma patients through single-cell transcriptome analysis data.

[0022] FIG. 9 shows the results of analyzing whether CD244 regulates monocyte-macrophages in patients with colorectal cancer, non-small cell lung cancer, and glioblastoma as in melanoma by single-cell transcriptome analysis data. DETAILED DESCRIPTION

[0023] The present disclosure will be described in more detail below.

[0025] The present disclosure provides a pharmaceutical composition for preventing or treating cancer, comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

[0026] The pharmaceutical composition can suppress cancer growth by increasing phagocytosis or antigen presentation.

[0027] In addition, the pharmaceutical composition can induce antigen-specific activation of T cells, thereby increasing the expression of IFN-g.

[0028] The monocytes may contain one or more markers selected from the group consisting of CD11b+, CD14+, CD16-, Ly6G-, and Ly6Chi, but are not limited thereto.

[0029] The macrophages may contain one or more markers selected from the group consisting of CD11b+, CD14+, CD16+, CD68+, Ly6G-, Ly6Clo and F4 / 80+, but are not limited thereto.

[0030] The cancer may be one or more selected from the group consisting of, but not limited to, gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal pelvis cancer, blood cancer, brain cancer, central nervous system (CNS) tumors, spinal cord tumors, brain stem gliomas, and pituitary adenomas.

[0031] According to a method that can be easily performed by those skilled in the art to which the present disclosure pertains, the pharmaceutical composition of the present disclosure can be prepared in the form of a unit dose by formulating with a pharmaceutically acceptable carrier, or by packaging into a multi-volume container.

[0032] Pharmaceutically acceptable carriers are usually used in the preparation, including but not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate / ester, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present disclosure may also include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives.

[0033] In the present disclosure, the content of the additives included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the content range used in conventional preparations.

[0034] The pharmaceutical composition can be formulated into one or more forms of external skin agents selected from the group consisting of, but not limited to: injectable preparations (e.g., aqueous solutions, suspensions and emulsions), pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastas and cataplasmas.

[0035] The pharmaceutical composition of the present disclosure may include additional pharmaceutically acceptable carriers and diluents for preparation. Pharmaceutically acceptable carriers and diluents include, but are not limited to, excipients (e.g., starch, sugar, and mannitol), fillers, and extenders (e.g., calcium phosphate); cellulose derivatives (e.g., carboxymethyl cellulose and hydroxypropyl cellulose), adhesives (e.g., gelatin, alginate / ester, and polyvinyl pyrrolidone), lubricants (e.g., talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol), disintegrants (e.g., polyvidone and cross-linked polyvidone), and surfactants (e.g., polysorbate, cetyl alcohol, and glycerol). Pharmaceutically acceptable carriers and diluents may be biologically and physiologically friendly to a subject. Examples of diluents may include, but are not limited to, saline, water-soluble buffers, solvents, and / or dispersion media.

[0036] The pharmaceutical composition of the present disclosure can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) according to the desired method. When administered orally, it can be formulated into tablets, troches, lozenges, water-soluble suspensions, oil-based suspensions, powder preparations, granules, emulsions, hard capsules, soft capsules, syrups, and elixirs. When administered parenterally, it can be formulated into injection solutions, suppositories, powders for respiratory inhalation, aerosols for spraying, ointments, powders for coating, oils, and creams.

[0037] The dosage of the pharmaceutical composition of the present disclosure may vary according to the patient's condition, body weight, age, sex, health status, dietary structure specificity, nature of the preparation, severity of the disease, administration time of the composition, administration mode, duration or interval of administration, excretion rate and drug form, and may be appropriately selected by those skilled in the art. For example, the dosage may be in the range of about 0.1 mg / kg to 10,000 mg / kg, but is not limited thereto, and may be administered once to several times a day in divided doses.

[0038] The pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or locally) according to the desired method. The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present disclosure can vary according to the preparation method, administration mode, administration duration and administration route of the pharmaceutical composition, and those skilled in the art can easily determine and prescribe the effective dose for the desired treatment. The administration of the pharmaceutical composition of the present disclosure can be performed once a day or several times in divided doses.

[0040] In addition, the present disclosure also provides a co-administered pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising monocytes or macrophages having suppressed CD244 expression or activity and an anticancer drug as active ingredients.

[0041] The anticancer drug may be one or more anticancer drugs selected from the group consisting of cytotoxic anticancer drugs, targeted anticancer drugs and immunotherapy drugs, but is not limited thereto.

[0042] The cytotoxic anticancer drug may be one or more selected from the group consisting of, but not limited to, doxorubicin, cyclophosphamide, temozolomide, irinotecan, cisplatin, vinblastine, vincristine, actinomycin-D, 5-fluorouracil, docetaxel, cabazitaxel, paclitaxel and pembrolizumab.

[0043] The targeted anticancer drug may be one or more selected from the group consisting of, but not limited to: trametinib, vemurafenib, alpelisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, dabrafenib, trastuzumab, cetuximab, bevacizumab, panitumumab, ipilimumab, pertuzumab, tofacitinib, imatinib, bortezomib, ofatumumab and alemtuzumab.

[0044] The immunotherapy drug may be one or more selected from the group consisting of, but not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIGIT, anti-TIM-3, anti-GITR, CAR-T and NK cells.

[0046] Furthermore, the present disclosure provides a composition for enhancing the efficacy of an anticancer drug, the composition comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

[0048] Furthermore, the present disclosure provides a method for preventing or treating cancer, comprising administering to a subject (including a human) monocytes or macrophages having suppressed CD244 expression or activity, alone or in combination with an anti-cancer drug.

[0050] Furthermore, the present disclosure provides a composition for inducing differentiation of monocytes into macrophages, the composition comprising a CD244 inhibitor or an inhibitor of CD48 which is a ligand of CD244 as an active ingredient.

[0051] The CD244 inhibitor or CD48 inhibitor can be selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural substances that specifically bind to CD244 or CD48 proteins, or from the group consisting of small interfering RNA (siRNA), antisense nucleotides and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the CD244 or CD48 gene.

[0052] The CD244 inhibitor can be, but is not limited to, a human anti-CD244 monoclonal antibody [ThermoFisherScientific: #16-2449-81, BioLegend: #329502, R&D SYSTEMS: MAB10393-SP, BioVision: A1073-100, BD Biosciences: #550814] or a mouse anti-CD244 monoclonal antibody [ThermoFisherScientific: #14-2441-82, BioLegend: #133501, BD Biosciences: #557451].

[0053] The CD48 inhibitor can be, but is not limited to, a human anti-CD48 monoclonal antibody [ThermoFisher Scientific: #16-0489-81, BioLegend: #336702, R&D SYSTEMS: MAB36441-SP, BD Biosciences: #555758] or a mouse anti-CD48 monoclonal antibody [BioXcell: #BE0147, BioLegend: #103453, BD Biosciences: #553682].

[0055] Furthermore, the present disclosure provides a method for screening an inducing agent for differentiation of monocytes into macrophages, the method comprising treating cells with a candidate substance to analyze the suppression of the expression or activity of CD244 or CD48 as its ligand. Embodiments of the present invention

[0056] The present disclosure will be described in more detail below by examples to help understand the present disclosure. However, the following examples are only intended to illustrate the present invention, and the scope of the present disclosure is not limited to the following examples. The embodiments of the present disclosure are provided to more completely explain the present disclosure to those of ordinary skill in the art.

[0058] [Experimental Example 1] Preparation of animal model

[0059] All experiments using animals were approved by the Korea University Institutional Animal Care and Use Committee (approval number: KUIACUC-2019-0101) and followed the guidelines and regulations of the Korea University Institutional Animal Care and Use Committee.

[0060] Wild-type (WT) C57BL / 6 mice were used as a control group for CD244-deficient mice and were purchased from Orient Bio (Orient Bio. Inc., Seongnam, South Korea). - / - , and the mice were C57BL / 6CD244 - / - , CD244-deficient in all cells. C57BL / 6LysM + / + -CD244 fl / fl Flox mice were used as a control group, which had the same CD244 expression as WT mice and were custom-made by Cyagen Biosciences. cre / + -CD244 fl / fl and monocyte-macrophage-specific CD244-deficient mice, CD244 fl / fl Mice and Lyz2 purchased from Jackson Laboratorycre + (B6.129P2-Lyz2tm1(cre)Ifo / J) mice were crossed to generate CD244 fl / fl and CD244 fl / fl -Lyz2 cre+ / - Mice, and littermate control mice were used in CD244 conditional KO mouse experiments. Female mice aged 5 to 10 weeks were used in the experiments, and all mice were bred and maintained in a specific pathogen-free facility at Korea University.

[0061] B16F10 melanoma cell line was purchased from ATCC. For subcutaneous injection, 1 × 10 6 B16F10 cells were injected into the flank of mice, and tumor formation with a diameter of 1 cm was observed within 1 to 3 weeks after injection. For the co-administration experiment of bone marrow-derived macrophages (BMDM) and anti-PD-L1 antibody, bone marrow cells from WT and CD244 KO mice were cultured in RPMI medium containing 50 ng / mL recombinant M-CSF (Peprotech) for 6 days. 1×10 B16F10 cells were injected intraperitoneally into WTC57BL / 6 mice 7 and 10 days after B16F10 tumor induction, respectively. 6 WT and CD244 KO BMDM with 200 μg of anti-PD-L1 (BioXcell: #BE0101) and isotype rat IgG2ak antibodies (BioXcell).

[0063] [Experimental Example 2] Cell preparation

[0064] After obtaining single cell suspensions from spleen and bone marrow, red blood cells were removed using ammonium chloride lysis buffer. From single cell suspensions of tumor tissues, immune cells were isolated and used in experiments using a mouse tumor dissociation kit and percoll density gradient separation according to the manufacturer's recommendations (Miltenyi Biotec and GE Healthcare).

[0066] [Experimental Example 3] Isolation of CD11b+ cells and monocytes

[0067] For the isolation of CD11b+ cells, tumor single cell suspensions were labeled with biotin-anti-CD11b antibody (Biolegend) and streptavidin-magnetic beads (Miltenyi biotec) and isolated using MACS LS columns (Miltenyi Biotec). Similarly, for the isolation of monocytes, a monocyte isolation kit (Miltenyi Biotec) was used in bone marrow cells.

[0069] [Experimental Example 4] Differentiation of BMDM and tumor-invading monocytes

[0070] CD11b+ cells isolated from isolated monocytes, total bone marrow cells or tumor lysates were cultured by treatment with 50 ng / mL recombinant M-CSF (Peprotech) in RPMI (Wellgene) supplemented with 10% FBS, 1% penicillin / streptomycin, 1% non-essential amino acids (NEAA), 10 mM HEPES and 20 μM 2-mercaptoethanol. In some experiments, B16F10 tumor explant supernatant was used at 20% v / v.

[0072] [Experimental Example 5] Flow cytometry

[0073] Generally speaking, up to 1×10 6 Each cell was incubated at room temperature for 5 minutes with Fc blocking antibody (anti-CD16 / CD32 antibody, BioXcell: #BE0307), surface protein staining was performed in a dark room at 4°C for 30 minutes, and each centrifugation of washing was performed at 1700 rpm for 5 minutes. For intracellular IFN-γ staining, CD45+ or CD8+ cells isolated from tumors or TDLN were reactivated by co-culturing with B16F10 melanoma cells irradiated with γ at 10gy and Brefeldin A for 16 hours. Cells were then subjected to surface staining, punched using BD fixation / permeabilization kit as recommended by the manufacturer, and then intracellular staining was performed with PE-anti-IFN-g antibody (BD Biosciences). Data collected using FACS Canto II flow cytometry (BD Biosciences) were analyzed by FlowJo software (Tristar).

[0075] [Experimental Example 6] Quantitative PCR

[0076] Total RNA from tissues and cells was extracted using TRIzol reagent (Invitrogen). TM cDNA was synthesized using a cDNA synthesis kit (Enzynomics). Real-time PCR was performed using SYBR green (Bio-rad) on a StepOne Plus TM (Applid Biosystems) instrument. Gene expression was normalized based on the expression level of GAPDH mRNA, and relative expression levels were calculated using the ΔΔCT method.

[0078] [Experimental Example 7] ELISPOT

[0079] On the third day of differentiation, 5×10 4 Lymph node cells were co-cultured with BMDM and OVA peptide (SIINFEKL; 1 μg / mL) at different ratios. The cells were inoculated in the Mouse IFN-g ELISPOT PLUS Kit (ALP) (MABTECH), and then IFN-g spots were detected by ELISPOT reading system (Autoimmun DiagnostikaGmbH) after 48 hours.

[0081] [Experimental Example 8] Statistical Analysis

[0082] All data are presented as mean ± standard error (SEM). Statistical significance was determined by unpaired two-tailed t-test and two-way ANOVA. Significance was defined as *p<0.05, **p<0.01, and ***p<0.001. All analyses were performed using Prism 7.0 software (GraphPad Software, Inc).

[0084] [Experimental Example 9] Antigen presentation assay

[0085] Two days after BMDM differentiation, 1 mg / mL whole OVA protein was added. MHC I complex and MHC II bound to OVA peptide were labeled with anti-H-2kb-SIINFEKL antibody (Biolegend: #141604) and anti-MHC II antibody (Biolegend: #107622), and then the expression level was measured by flow cytometry.

[0087] [Experimental Example 10] Phagocytosis assay

[0088] Two days after BMDM differentiation, gamma-irradiated B16F10 cells stained with 5 μM CFSE were co-cultured for 24 hours. Using flow cytometry, CFSE fluorescence was detected in CD45+, CD11b+, Ly6Chi and F4 / 80low monocytes and CD45+, CD11b+, Ly6Clow and F4 / 80high macrophages through the 488 / 519 channel.

[0090] [Experimental Example 11] Fluorescence microscopy

[0091] BMDM cells cultured in confocal dishes were fixed, cell membranes were perforated with 4.2% paraformaldehyde solution, and stained with anti-LC3B antibody (abcam: #ab51520) (1:200) and Hoechst 33342 (Invitrogen: #H3570) (1000:1). After incubation for 2 hours, the cells were washed, stained with Alexa555-rabbit IgG secondary antibody (Invitrogen: #A27039), and incubated for 1 hour at room temperature. All images were recorded with a LSM700 confocal laser scanning microscope (Carl Zeiss) equipped with a 63X oil immersion lens.

[0093] [Experimental Example 12] Analysis of single-cell RNA sequencing data

[0094] Data from scRNA-seq studies were analyzed for responsiveness to immune checkpoint blockade therapy and changes in immune cells. 5,928 cells from the pre-immune checkpoint blockade cohort were clustered using the Seurat v4 R package. Afterwards, patients were divided into high and low CD244 expressers based on their mean expression levels of CD244 in their monocyte-macrophages. Only genes with relatively high mean expression and large differences in expression between cells were selected for downstream clustering analysis. Dimensionality reduction was performed using principal component analysis (PCA), and the number of principal components was calculated using the built-in JackStraw function. Two-dimensional data visualization was performed using T-distributed stochastic neighbor embedding (t-SNE) and unified manifold approximation and projection (UMAP). Differentially expressed genes were identified using the FindMarkers function provided in the Seurat package. From the results of the Seurat package, genes with a P value of less than 0.05 were considered differentially expressed genes. For each population of differentially expressed genes and gene expression levels, signaling pathways and gene signatures were analyzed using IPA software (QIAGEN Inc.) and the fgsea R package.

[0096] [Example 1] Analysis of the anticancer effect of CD244 inhibition in a mouse melanoma model

[0097] To confirm whether CD244 expressed in monocytes has an impact on tumor progression, we determined whether CD244 expression in monocytes in tumors is increased. 6The tumor model was established by subcutaneously injecting mouse allogeneic melanoma cells B16F10 into the flank of C57BL / 6 mice, and 14 days later, the CD244 expression levels of intratumoral immune cells were compared with those of intradermal immune cells of normal C57BL / 6 mice. As a result, natural killer cells (hereinafter referred to as NK cells) and dendritic cells (hereinafter referred to as DCs) showed high CD244 expression regardless of the presence of tumors, while monocytes rarely showed CD244 expression in normal skin tissues, and CD244 expression increased significantly after tumor induction ( Figure 1a -A).

[0098] In addition, to determine whether the elevated expression of CD244 in monocytes after tumor induction affects tumor growth, 1 × 10 6 B16F10 was injected subcutaneously into C57BL / 6 or C57BL / 6CD244 - / - The tumor model was established by excising the flank of mice and observing tumor growth for 14 days. The tumor size was calculated using a vernier caliper as the width × length × height / 2 of the tumor tissue. - / - Tumor growth was significantly inhibited in mice ( Figure 1a -B).

[0099] Furthermore, since CD244 is expressed in a variety of immune cells other than monocytes, in order to determine the more specific role of CD244 expressed in monocytes, monocyte-macrophage-specific CD244-deficient mice were constructed by crossing LysM-cre mice (in which Cre recombinase and monocyte-macrophage-specific LysM gene are expressed together) with CD244 flox mice (in which a gene sequence flox sequence that can be cleaved by Cre recombinase is attached before and after the CD244 gene) Figure 1b -C). In the control group CD244 fl / fl (hereinafter referred to as Flox) and experimental group LysM cre -CD244 fl / fl (hereinafter referred to as cKO) Figure 1a -B induced melanoma in the same way. The results showed that tumor growth was significantly inhibited in the experimental group of monocyte-macrophage-specific CD244-deficient mice ( Figure 1b -D).

[0101] [Example 2] Analysis of changes in T cell activity in CD244-deficient monocytes / macrophages

[0102] To confirm how CD244 expressed in monocyte-macrophages suppresses anti-tumor immune responses, the activity of T cells, which is the most representative anti-tumor immune response, was determined. Figure 1b -D Melanoma was induced in Flox and cKO mice in the same manner, and tumors were extracted and analyzed by flow cytometry 14 days later. As a result, there was no difference in the ratio of CD8 and CD4 T cells relative to NK cells, but it was found that CD8 and CD4 T cells expressed more IFN-g in cKO mice compared with the control group (Flox), and IFN-g plays a key role in anti-tumor immune response ( Figure 2a In addition, it was found that the expression of the protein granzyme b, which directly kills tumor cells, was significantly increased in the CD8 T cells of cKO mice ( Figure 2b -C). By staining with H-2Db gp100 tetramer, it was determined that this difference was due to an increase in antigen-specific CD8 cells in cKO mice, which express T cell receptors specific for the melanoma antigen gp100 ( Figure 2b -D). In addition, it was found that the expression of memory T cell marker CD44 was higher in CD8 T cells of cKO mice ( Figure 2b -E).

[0104] [Example 3] Analysis of monocyte / macrophage distribution and differentiation changes in CD244-deficient mice

[0105] After extracting tumors from the control group Flox mice and the experimental group cKO mice, the distribution of tumor-infiltrating immune cells was determined by flow cytometry. As a result, there was no difference in the proportion of CD11b+ cells (which are total myeloid immune cells), neutrophils, and DC cells, but a significant decrease in monocytes and a significant increase in macrophages were observed in cKO mice ( Figure 3a -A).

[0106] To determine whether CD244 regulates the differentiation of monocytes into macrophages, BMDM experiments were performed in which C57BL / 6 or C57BL / 6CD244 - / - The bone marrow cells of mice were induced to differentiate. After 3 days, the proportion and number of macrophages were measured. CD244 - / - The number of macrophages in BMDM increased significantly compared with the control group (WT) ( Figure 3a -B). In addition, given that the physiological characteristics of monocytes in the bone marrow are significantly different from those in the tumor, it was found that after the monocytes in the tumor were separated, when they were treated with M-CSF, the proportion and number of macrophages also increased significantly ( Figure 3b ).

[0107] Furthermore, to directly investigate whether CD244 regulates monocyte-macrophage differentiation even in the tumor microenvironment, we isolated 144-positive C57BL / 6CD45.1WT and 144-positive C57BL / 6CD45.2CD244WT cells from the tumor microenvironment. - / - Monocytes were isolated from the bone marrow of mice, stained with Celltrace Far Red, mixed at a 1:1 ratio, and injected directly into tumors of C57BL / 6CD45.2 mice ( Figure 3c -D). 24 hours later, the CD45.1 WT and CD45.2CD244- / - cells in the tumor were examined by Cell trace far red staining, and a large number of CD244 - / - Monocytes differentiate into macrophages ( Figure 3c -E).

[0108] To determine whether CD244 inhibits monocyte-macrophage differentiation by interacting with its ligand CD48 and signaling, bone marrow-derived macrophages were treated with anti-CD48 antibody (Biolegend: #103453) that blocks CD244 ligand CD48 in a differentiation assay. - / - There was no significant change in WT mice, but a significant increase in WT mice ( Figure 3c -F).

[0110] [Example 4] Analysis of the mechanism by which CD244-deficient monocytes / macrophages suppress adaptive immune responsiveness

[0111] To determine how CD244 inhibits the adaptive immune responsiveness of monocytes / macrophages, tumors from the control group Flox mice and the experimental group cKO mice were extracted, and then the markers of M1 and M2 macrophages were analyzed by flow cytometry and real-time PCR. The results showed that the expression of M1 macrophage markers (CD80, IL-6, NOS2, IFNB1) was increased in the experimental group (cKO) compared with the control group (Flox). Figure 4a -A and Figure 4a -B), while M2 macrophage markers (CD206, ARG1, TGFB1) showed no significant changes in expression ( Figure 4a -C and Figure 4a -D).

[0112] In addition, to determine whether antigen presentation, a key function of M1 macrophages, is regulated by CD244, BMDM differentiated for 2 days were treated with ovalbumin (hereinafter referred to as OVA) protein and cultured for 24 hours to determine the extent of antigen presentation. As a result, the expression of MHCI-ova complex was significantly increased in CD244-deficient macrophages ( Figure 4a -E), and the number of macrophages expressing MHCI-ova complexes was significantly increased in BMDMs of CD244-deficient mice compared with WT mice, accompanied by an increase in the number of macrophages in CD244-deficient mice ( Figure 4a In addition, when examining whether CD244 regulates the expression of co-stimulatory molecules necessary for antigen presentation, it was determined that CD80 expression was high in CD244-deficient macrophages ( Figure 4b To confirm whether the increase in antigen-specific CD8 T cells in tumors of cKO mice was due to the inhibition of antigen presentation by macrophages by CD244, OT-1 T cells that recognized OVA were co-cultured with bone marrow-derived macrophages differentiated in vitro, and when OVA was added, it was found that T cell-specific IFN-g secretion was increased when co-cultured with CD244-deficient macrophages ( Figure 4b -H).

[0113] Next, to determine the extent of phagocytosis (an important mechanism by which macrophages participate in antitumor immune responses and must precede antigen presentation), 2-day-differentiated BMDMs were irradiated with 10 gy of gamma rays and co-cultured with CFSE-fluorescently labeled B16F10 for 24 hours. Subsequently, CFSE fluorescence expressed in BMDMs was measured by flow cytometry, and the results showed that phagocytosis was significantly increased in both monocytes and macrophages in CD244-deficient cells compared with the control group (WT). Figure 4b -I and Figure 4b -J).

[0115] [Example 5] Analysis of the reasons for the increase in CD244 expression in monocytes after tumor induction

[0116] exist Figure 1a -A, in order to determine the reasons for the increased expression of CD244 in monocytes after tumor induction and the molecular mechanism by which CD244 inhibits monocyte-macrophage differentiation and function, the single-cell transcriptome analysis data of mouse tumor models in the public database (GSE121861) were analyzed. Seven clusters of CD45 (PTPRC) + immune cells were distinguished using representative marker expression patterns, and the expression levels of known markers were identified to determine the subclusters of four monocytic lineage cells, such as monocytes (LY6C2, CCR2 and IL1B), immature monocytes (S100A9 and OLR1), macrophages (ADGRE1 and CSF1R), M1 (H2-Aa, H2Ab1, CX3CR1, CD86 and APOE) and M2 (ARG1, TREM 2 and SPP1) macrophages ( Figure 5a -A and Figure 5a-B). Pathway enrichment analysis revealed that immature monocytes expressed genes related to immune response, differentiation, and protein folding at a lower rate than normal monocytes ( Figure 5a -C). As a result of determining CD244 expression in four monocyte lineage cell subsets, it was confirmed that CD244 expression in immature monocytes was significantly higher than that in normal monocytes and M1 / M2 macrophages ( Figure 5a -D and Figure 5a -E). In addition, the ER stress pathway was found to be upregulated in immature monocytes with high expression of CD244 compared to immature monocytes with low CD244 expression (Figure 5f). Assuming that the ER stress pathway, one of the main mechanisms for the development of immunosuppressive immature monocytes, also regulates CD244 expression in immature monocytes, when BMDMs were treated with the ER stress inducer Thapsigargin, CD244 expression in monocytes increased, and differentiation into macrophages decreased. On the other hand, when treated with the ER stress inhibitor tauroursodeoxycholic acid, CD244 expression in monocytes was found to be reduced, while differentiation into macrophages increased (Figure 5f). Figure 5b -G and Figure 5b -H).

[0118] [Example 6] Analysis of the molecular mechanism of CD244 regulation

[0119] To confirm how CD244 inhibits monocyte-macrophage differentiation, antigen presentation and phagocytosis, the expression of CD244 signaling molecules was determined in the single-cell transcriptome analysis data of Example 5. The results showed that compared with T cells and NK cells, the expression of SAP, EAT-2, ERT and SHP-2 in monocyte-macrophages was low, while the expression of Beclin-1 was high ( Figure 6a -A). In addition, the incidence of autophagy in WT and CD244-deficient BMDMs was examined. First, to determine the lipidation of LC3, a process required for the initiation of autophagy, WT and CD244 KO BMDMs were stained with anti-LC3B antibody (abcam: #ab51520) and then observed by fluorescence microscopy and flow cytometry. The results showed that LC3 lipidation was significantly increased in CD244 KO BMDMs ( Figure 6a -B and Figure 6a -C). The results of quantifying the amount of autophagosomes actually formed by LC3B by cyto-ID staining showed that it was also significantly increased in CD244 KO BMDM ( Figure 6a -D).

[0120] Next, to confirm whether the degree of differentiation of BMDMs changes when autophagy is controlled, they were treated with autophagy-regulating drugs. When treated with chloroquine (which interferes with the fusion of autophagosomes and lysosomes, thereby inhibiting the final stage of autophagy), the number of macrophages in both WT and CD244 KO BMDMs was significantly reduced ( Figure 6b -E and Figure 6b Furthermore, even when treated with Ly294002 (an inhibitor of vps-34, another molecule that binds to CD244), the number of macrophages was found to be reduced, and there was no significant difference between WT and CD244 - / - The difference in LC3B expression between BMDMs disappeared ( Figure 6b -G and Figure 6b -H). The above results confirmed that CD244 inhibits the differentiation of monocytes into macrophages by inhibiting autophagy.

[0122] [Example 7] Analysis of the anti-cancer effect of co-administration of CD244-deficient monocytes / macrophages and anti-PD-L1 antibodies

[0123] Based on the above results, it was confirmed that CD244 suppresses anti-tumor immune responses by accumulating monocytes in the tumor microenvironment and subsequently reducing differentiation, antigen presentation, and phagocytosis to macrophages, so the possibility of applying CD244-deficient macrophages as a therapeutic method to immunologically convert "cold" tumors into "hot" tumors was experimented. First, B16F10 melanoma was induced in monocyte lineage-specific CD244-deficient mice (cKO) and control group Flox mice, and then treated with anti-PD-L1 antibodies. As a result, tumors in Flox mice did not show significant changes in anti-PD-L1 antibody treatment. However, compared with isotype antibody treatment, anti-PD-L1 antibody treatment further reduced tumor size in cKO mice ( Figure 7a -A). When measuring the proportion of T cells within the tumor, it was found that the proportion of CD8 T cells increased significantly only when cKO mice were treated with anti-PD-L1 antibodies ( Figure 7a -B). In addition, as a result of analyzing the memory phenotype of T cells in TDLN of cKO mice treated with anti-PD-L1 antibodies, the populations of central memory (CD62L+, CD44+) CD8 T cells and effector memory (CD62L-, CD44+) CD4 T cells were significantly increased compared to when Flox mice were treated with anti-PD-L1 antibodies ( Figure 7b ). In addition, only in the tumors of cKO mice injected with anti-PD-L1 antibodies, the proportion of T cells expressing PD-1 but not T cell immunoreceptor with Ig and ITIM domains (TIGIT) increased significantly, while the proportion of PD-1 / TIGIT double-positive CD8 T cells showed no significant change ( Figure 7c In general, since PD-1 is known to be an immune checkpoint receptor expressed in all antigen-specific activated T cells, and TIGIT is expressed only in severely exhausted T cells, the above results suggest that treatment of cKO mice with anti-PD-L1 antibodies increased the activity of CD8 T cells in tumors but did not increase immune exhaustion.

[0124] To investigate the potential of CD244-deficient macrophages as anti-tumor immune cell therapeutics, WT mice bearing B16F10 melanoma were co-injected with anti-PD-L1 antibody and CD244 - / - BMDM cells. When isotype antibodies and CD244 - / - When BMDM were treated together, tumor growth was slightly inhibited, but CD244 - / - Co-administration of BMDM and anti-PD-L1 antibody significantly reduced the growth of B16F10 tumors, and the same results were observed in another cold tumor model, Lewis lung cancer (LL2). Figure 7c -E). The above results show that CD244-deficient macrophages have enhanced T cell activity and memory phenotype, increased intratumoral CD8 T cells, and enhanced responsiveness to immune checkpoint inhibitors in tumor models including melanoma.

[0126] [Example 8] Analysis of the anticancer effect of CD244-deficient monocytes / macrophages on melanoma patients

[0127] To confirm the clinical significance of CD244 as an immune checkpoint receptor for monocytes / macrophages in the tumor microenvironment, a single-cell RNA sequencing dataset of CD45+ immune cells isolated from human melanoma tissue from a public database (SCP398) was analyzed. Of the 16,291 cells reported in previous studies, 1,391 cells classified as monocytes and macrophages were used. To identify monocytes / macrophages that did not express CD244 among these cells, they were further divided into seven subgroups using the shared nearest neighbor clustering method in Seurat ( Figure 8a -A). They were then divided into three groups according to CD244 expression: 1) C0-1, with a relatively high proportion of CD244-expressing cells; 2) C2-3, with a relatively low proportion of CD244-expressing cells; and 3) C4-6, with no CD244 expression ( Figure 8a -B). Among these groups, C0-1, which has a large number of CD244+ and CD244- monocytes / macrophages, was used for a fair comparison between the two cell types. A total of 511 genes and 221 genes were found to be up-regulated and down-regulated in CD244+ and CD244- monocytes / macrophages, respectively ( Figure 8a -C). 221 genes expressed primarily in CD244- monocytes / macrophages were highly related to phagosomes, antigen processing and presentation, and autophagy ( Figure 8a -D), which is consistent with the findings in the mouse model. Next, we tried to confirm whether the expression of CD244 in monocytes / macrophages has an impact on patient survival. To this end, we obtained a bulk RNA-seq dataset generated from tumor tissues from 470 melanoma patients (103 primary tumors and 367 metastatic tumors) from The Cancer Genome Atlas database (TCGA-SKCM, Figure 8b Then, in the bulk RNA-seq dataset, to estimate the proportions of the four cell types identified in the previous scRNA-seq analysis, the genes (DEGs) specifically upregulated in the four cell types were identified and deconvolution analysis was performed on the TCGA dataset by CIBERSORTx ( Figure 8b -F). By comparing the survival rates of patients with and without monocytes / macrophages without CD244 expression, it was found that the overall survival rate of patients with monocytes / macrophages without CD244 expression was significantly higher than that of patients without monocytes / macrophages without CD244 expression in both the primary cancer cohort and the metastatic cancer cohort ( Figure 8b -G).

[0128] Next, we aimed to determine whether monocytes / macrophages with low CD244 expression could enhance the antigen-specific activity of T cells even in humans as they did in the mouse model. In the previous scRNA-seq dataset, individual patients were divided into CD244 high and CD244 low groups according to the average expression level of CD244 in monocytes / macrophages, and CD8 T cells ( Figure 8c -H). Thus, 207 genes that were mainly upregulated in CD8 T cells in the CD244 low group were obtained, showing that these genes were mainly related to cytokine signaling, TCR signaling, and the adaptive immune system ( Figure 8c -I).

[0130] [Example 9] Analysis of the Effect of CD244 on Monocyte-Macrophage Function in Different Cancers

[0131] Finally, we examined whether the results based on CD244 expression in monocytes determined from the melanoma model and transcriptome profiling data could be extended to other tumor models. To this end, we analyzed monocytes with colorectal cancer (SCP1162) ( Figure 9a )、Non-small cell lung cancer (GSE127465)( Figure 9b ) and glioblastoma (GSE131928) ( Fig.9c As a result of pathway enrichment analysis using DEGs of monocytes / macrophages that do not express CD244, it was found that CD244 regulates the innate immune system, phagocytosis, antigen presentation, and autophagy in monocyte-macrophages in cancers other than melanoma.

[0133] Although the specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that this specific description is only a preferred exemplary embodiment, and the scope of the present disclosure is not limited thereto. In other words, the essential scope of the present disclosure is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition inhibits cancer growth by increasing phagocytosis or antigen presentation.

3. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition increases the expression of IFN-g by inducing antigen-specific activation of T cells.

4. The pharmaceutical composition according to claim 1, wherein The monocytes contain one or more markers selected from the group consisting of CD11b+, CD14+, CD16-, Ly6G- and Ly6Chi.

5. The pharmaceutical composition according to claim 1, wherein The macrophages contain one or more markers selected from the group consisting of CD11b+, CD14+, CD16+, CD68+, Ly6G-, Ly6Clo and F4 / 80+.

6. The pharmaceutical composition according to claim 1, wherein The cancer is selected from one or more of the group consisting of: gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, head and neck cancer, melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal pelvis cancer, blood cancer, brain cancer, central nervous system (CNS) tumors, spinal cord tumors, brain stem gliomas and pituitary adenomas.

7. A co-administered pharmaceutical composition for preventing or treating cancer, comprising the following as active ingredients: monocytes or macrophages having suppressed CD244 expression or activity; and an anticancer drug.

8. The pharmaceutical composition according to claim 7, wherein The anticancer drug is one or more anticancer drugs selected from the group consisting of cytotoxic anticancer drugs, targeted anticancer drugs and immunotherapy drugs.

9. The pharmaceutical composition according to claim 8, wherein The cytotoxic anticancer drug is selected from one or more of the group consisting of doxorubicin, cyclophosphamide, temozolomide, irinotecan, cisplatin, vinblastine, vincristine, actinomycin-D, 5-fluorouracil, docetaxel, cabazitaxel, paclitaxel and pembrolizumab.

10. The pharmaceutical composition according to claim 8, wherein The targeted anticancer drug is selected from one or more of the group consisting of trametinib, vemurafenib, apellisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, dabrafenib, trastuzumab, cetuximab, bevacizumab, panitumumab, ipilimumab, pertuzumab, tofacitinib, imatinib, bortezomib, ofatumumab and alemtuzumab.

11. The pharmaceutical composition according to claim 8, wherein The immunotherapy drug is selected from one or more of the following groups: anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIGIT, anti-TIM-3, anti-GITR, CAR-T and NK cells.

12. A composition for enhancing the efficacy of an anticancer drug, the composition comprising monocytes or macrophages having suppressed CD244 expression or activity as an active ingredient.

13. A method for preventing or treating cancer, the method comprising administering monocytes or macrophages having suppressed CD244 expression or activity to a subject, including a human, alone or in combination with an anticancer drug.

14. A composition for inducing differentiation of monocytes into macrophages, the composition comprising a CD244 inhibitor or an inhibitor of CD48 which is a ligand of CD244 as an active ingredient.

15. The composition for inducing monocytes to differentiate into macrophages according to claim 14, wherein: The CD244 inhibitor or CD48 inhibitor is selected from the group consisting of compounds, peptides, peptidomimetics, aptamers, antibodies and natural substances that specifically bind to CD244 or CD48 proteins, or is selected from the group consisting of small interfering RNA (siRNA), antisense nucleotides and short hairpin RNA (shRNA) that complementarily bind to the mRNA of the CD244 or CD48 gene.

16. A method for screening an inducing agent for differentiation of monocytes into macrophages, the method comprising treating cells with a candidate substance to analyze the suppression of the expression or activity of CD244 or CD48 as its ligand.

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