KR20230167741A - Pharmaceutical composition for preventing or treating cancer comprising monocytes or macrophages that inhibit CD244 expression or activity as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising monocytes or macrophages inhibiting CD244 expression or activity as an active ingredient, wherein CD244 inhibits differentiation from monocytes to macrophages and inhibits CD244 expression or activity. By confirming that the monocytes and macrophages inhibit tumor growth by increasing phagocytosis and antigen presentation and induce antigen-specific activation of T cells, it can be usefully used as a composition for preventing or treating cancer.

Description

Pharmaceutical composition for preventing or treating cancer comprising monocytes or macrophages that inhibit CD244 expression or activity as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing monocytes or macrophages suppressed in CD244 expression or activity as an active ingredient.

Immune exhaustion is caused by an excessive immune response or an immunosuppressive surrounding environment, and is known to reduce the expression of anti-tumor immune response and inflammatory cytokines, forming an immunosuppressive tumor microenvironment. Immune exhaustion occurs mainly through a molecular mechanism caused by immune checkpoint receptors PD-1, CTLA-4, LAG-3, and TIM-3 expressed on T cells. Recently, antibody treatments that inhibit PD-1, PD-L1, and CTLA-4 signaling have been developed and are being used in anticancer treatment. However, these treatments are only effective in a small number of patients who meet the limited genotype and tumor microenvironment due to various factors such as the tumor microenvironment of solid tumors, polymorphism of tumor cells, and lack of tumor infiltrating immune cells.

CD244 (SLAMF4, 2B4) is an immune cell receptor belonging to the signaling lymphocyte activation molecule family (SLAMF). CD244 is a unique receptor that can perform two functions, activating and suppressing immune responses, depending on which signaling molecule binds to NK cells and CD8 T cells. Recent studies have revealed that CD244 suppresses NK cells and CD8 T cells in the tumor microenvironment to maintain their exhausted state. The intracellular domain of CD244 consists of four immunoreceptor tyrosine-based switch motifs (ITSM) and can bind to signaling molecules such as SAP, EAT-2, SHP1, SHP2, and SHIP-1. CD244 is known to transmit an immune activation signal when it binds to SAP and an immunosuppression signal when it binds to SH2 phosphatase enzymes. The relative expression level and competitive binding of signaling molecules play an important role in determining what signal CD244 transmits.

Monocytes and macrophages are important cells that connect the innate and adaptive immune responses, sense the surrounding environment, and determine the innate and adaptive immune responses. Many recent studies have focused on monocytes and macrophages as important cells forming “cold tumors,” which refer to tumors that do not respond to immunotherapy. In a study on the immune cells of patients with advanced liver cancer, the tissue surrounding the tumor was heavily infiltrated with monocytes and macrophages differentiated from monocytes, which resulted in a decrease in the number of NK cells inside the tumor and a decrease in immune-activating cytokines such as TNF-a and IFN-g. It was discovered that it was related to the phenomenon. Additionally, the same study showed that when CD48, which is expressed at high levels on tumor-infiltrating monocytes, binds to CD244 on NK cells, the NK cells are severely exhausted and cannot perform their function. CD244 is expressed not only in CD8 T cells and NK cells, but also in various myeloid immune cells such as monocytes and macrophages, but the role it plays in these cells has not been revealed.

Therefore, contrary to previous studies, the present inventor sought to find out what function CD244 expressed in monocyte-macrophages performs through its interaction with CD48 expressed in surrounding blood cells.

US published patent US 2019-0054090 (published on February 21, 2019)

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing monocytes or macrophages with suppressed CD244 activity as an active ingredient.

To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer containing monocytes or macrophages in which CD244 expression or activity is suppressed as an active ingredient.

In addition, the present invention relates to monocytes or macrophages that suppress CD244 expression or activity; and a combination administration pharmaceutical composition for preventing or treating cancer, comprising an anticancer agent as an active ingredient.

In addition, the present invention provides a composition for enhancing the efficacy of an anticancer agent comprising monocytes or macrophages in which CD244 expression or activity is suppressed as an active ingredient.

Additionally, the present invention provides a method for preventing or treating cancer, which includes administering monocytes or macrophages in which CD244 expression or activity is suppressed to subjects, including humans, alone or in combination with an anticancer agent.

Additionally, the present invention provides a composition for inducing differentiation from monocytes to macrophages containing a CD244 inhibitor as an active ingredient.

In addition, the present invention provides a screening method for an agent that induces differentiation from monocytes to macrophages, comprising the step of treating cells with a candidate substance and analyzing CD244 expression or inhibition of activity.

According to the present invention, CD244 suppresses the differentiation of monocytes into macrophages, monocytes and macrophages that suppress CD244 expression or activity increase phagocytosis and antigen presentation to suppress tumor growth, and antigen specificity of T cells. By confirming that it induces activation, it can be usefully used as a composition for preventing or treating cancer.

Figure 1 shows the changes in CD244 expression on monocytes during tumor induction and the results of analyzing the anticancer effect using a monocyte-specific CD244-deficient mouse model.
Figure 2 shows the results of analyzing the activity and tumor antigen specificity of intratumoral T cells between Flox and cKO mice.
Figure 3 shows the results of analyzing the ratio of monocyte-macrophages in tumors between Flox and cKO mice and whether monocyte-macrophage differentiation changes in CD244 deficiency.
Figure 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.
Figure 5 shows the results of analyzing whether the expression of CD244 is induced by ER stress in monocytes through single cell transcriptome analysis data and cell experiments.
Figure 6 shows the results of analyzing whether CD244 regulates monocyte-macrophage differentiation through autophagy.
Figure 7 shows the results of analyzing whether the efficacy of immune checkpoint inhibitors increases when CD244 is specifically removed from monocyte-macrophages or when CD244-deficient macrophages are administered.
Figure 8 shows the results of analyzing whether there are differences in the function and survival rate of monocyte-macrophages and T cells between the patient group with high and low CD244 expression in melanoma patients through single cell transcriptome analysis data.
Figure 9 shows the results of analyzing whether CD244 regulates monocyte-macrophages in patients with colorectal cancer, non-small cell lung cancer, and glioblastoma through single-cell transcriptome analysis data, as in melanoma.

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating cancer containing monocytes or macrophages whose expression or activity is suppressed as an active ingredient.

The pharmaceutical composition can inhibit cancer growth by increasing phagocytosis or antigen presentation.

Additionally, the pharmaceutical composition can induce antigen-specific activation of T cells and increase the expression of IFN-g.

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

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

The above cancers include stomach 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, colon cancer, small intestine cancer, rectal cancer, anal cancer, and fallopian tube carcinoma. , endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or It may be one or more selected from the group consisting of acute leukemia, lymphocytic lymphoma, renal cancer, ureteral cancer, renal pelvic cancer, hematological cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma. , but is not limited to this.

The pharmaceutical composition of the present invention is prepared in unit dose form or in a multi-dose container by formulating it using a pharmaceutically acceptable carrier according to a method that can be easily performed by a person skilled in the art. It can be manufactured by internalizing it.

The pharmaceutically acceptable carriers are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Includes, but is not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

In the present invention, the content of additives included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

The pharmaceutical compositions include injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, paste agents, and cataplasmase agents. It may be formulated in the form of one or more external skin preparations selected from the group consisting of, but is not limited to this.

The pharmaceutical composition of the present invention may additionally contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginate, polyvinyl pyrrolidone. It includes, but is not limited to, binders such as talc, calcium stearate, lubricants such as hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, and surfactants such as polysorbate, cetyl alcohol, glycerol, etc. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically friendly to the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method. For oral administration, it can be formulated as tablets, troches, lozenges, aqueous suspensions, oily suspensions, powders, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, etc. In the case of parenteral administration, it can be formulated as an injection, suppository, powder for respiratory inhalation, aerosol for spray, ointment, powder for application, oil, cream, etc.

The dosage of the pharmaceutical composition of the present invention is determined by the patient's condition, weight, age, gender, health, dietary constitution specificity, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and The range may vary depending on the drug form and can be appropriately selected by a person skilled in the art. For example, it may range from about 0.1 to 10,000 mg/kg, but is not limited and may be administered once to several times a day.

The pharmaceutical composition may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally, or topically applied) depending on the desired method. The pharmaceutically effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time, administration route, etc. of the pharmaceutical composition, and those skilled in the art will know that it is effective for the desired treatment. Dosage can be easily determined and prescribed. The pharmaceutical composition of the present invention may be administered once a day, or may be administered in several divided doses.

In addition, the present invention relates to monocytes or macrophages that suppress CD244 expression or activity; and a combination administration pharmaceutical composition for preventing or treating cancer, comprising an anticancer agent as an active ingredient.

The anticancer agent may be one or more anticancer agents selected from the group consisting of cytotoxic anticancer agents, targeted anticancer agents, and immune anticancer agents, but is not limited thereto.

The cytotoxic anticancer drugs include doxorubicin, cyclophosphamide, temozolomide, irinotecan, cisplatin, vinblastine, vincristine, and actino. May be one or more selected from the group consisting of actinomycin-D, 5-fluouracil, docetaxel, cabazitaxel, paclitaxel, and pembrolizumab. However, it is not limited to this.

The targeted anticancer drugs include Trametinib, vemurafenib, alpelisib, dactolisib, Gefitinib, Erlotinib, Lapatinib, Sunitinib, Sorafenib, Crizotinib, Dabrafenib, Trastuzumab, Cetuximab, Bevacizumab, Panitumumab (Panitumumab), Ipilimumab, Pertuzumab, Tofacitinib, Imatinib, Bortezomib, Ofatumumab and Alemtuzumab. It may be one or more selected from the group, but is not limited thereto.

The immunotherapy agent consists of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIGIT, anti-TIM-3, anti-GITR, CAR-T and NK cell. It may be one or more selected from the group, but is not limited thereto.

In addition, the present invention provides a composition for enhancing the efficacy of an anticancer agent comprising monocytes or macrophages suppressing CD244 expression or activity as an active ingredient.

Additionally, the present invention provides a method for preventing or treating cancer, which includes administering monocytes or macrophages in which CD244 expression or activity is suppressed to individuals, including humans, alone or in combination with an anticancer agent.

In addition, the present invention provides a composition for inducing differentiation from monocytes to macrophages, comprising a CD244 inhibitor or a CD48 inhibitor, a ligand of CD244, as an active ingredient.

The CD244 inhibitor or CD48 inhibitor is selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the CD244 or CD48 protein, or bind complementary to the mRNA of the CD244 or CD48 gene. It may be selected from the group consisting of small interfering RNA (siRNA), antisense nucleotides, and short hairpin RNA (shRNA).

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

The CD48 inhibitor is 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. It may be [BioXcell: #BE0147, BioLegend: #103453, BD Biosciences: #553682], but is not limited thereto.

In addition, the present invention provides a screening method for an agent that induces differentiation from monocytes to macrophages, comprising the step of treating cells with a candidate material to analyze the inhibition of expression or activity of CD244 or its ligand, CD48. .

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[Experimental Example 1] Animal model production

All experiments using animals were approved by the Korea University Animal Experiment Ethics Committee (approval number: KUIACUC-2019-0101) and followed the guidelines and regulations of the Korea University Animal Experiment Ethics Committee.

Wild-type (WT) C57BL/6 mice were used as controls for CD244-deficient mice and were purchased from Orient Bio (Orient Bio. Inc., Seongnam, Korea). KO is a CD244 ^-/- mouse on a C57BL/6 background, which is a C57BL/6 CD244 ^-/- mouse, which is a mouse lacking CD244 in all cells. Flox is a C57BL/6 LysM ^+/+ -CD244 ^fl/fl mouse and was used as a control with the same CD244 expression as the WT mouse, and was custom-made by Cyagen Biosciences. cKO is LysM ^cre/+ -CD244 ^fl/fl , a monocyte-macrophage-specific CD244-deficient mouse, and CD244 ^{fl /fl} mice are Lyz2 ^{cre +} (B6.129P2-Lyz2tm1(cre)Ifo/J) purchased from Jackson Laboratory. CD244 ^{fl /fl} and CD244 ^{fl /fl} -Lyz2 ^{cre +/-} mice were generated by mating with mice, and littermate control mice were used in CD244 conditional KO mouse experiments. Female mice aged 5 to 10 weeks were used in the experiment, and all mice were bred and maintained in a specific pathogen free facility at Korea University.

B16F10 melanoma cell line was purchased from ATCC. For subcutaneous injection, ¹ For the combined administration experiment with bone marrow-derived macrophage (BMDM) and anti-PD-L1 antibody, bone marrow cells from WT and CD244 KO mice were cultured in RPMI medium containing 50ng/ml recombinant M-CSF (Peprotech) for 6 days. . Seven and ten days after B16F10 tumor induction, WT C57BL/6 mice were intraperitoneally injected with 1 × ¹⁰⁶ WT and CD244 KO BMDM and 200 μg of anti-PD-L1 (BioXcell: #BE0101) and Isotype Rat IgG2ak antibodies (BioXcell), respectively. Injected.

[Experimental Example 2] Cell preparation

After obtaining a single cell suspension from the spleen and bone marrow, red blood cells were removed using ammonium chloride lysis buffer. Immune cells were isolated from a single cell suspension of tumor tissue using the Mouse Tumor Dissociation Kit and percoll density gradient separation according to the manufacturer's recommendations (Miltenyi Biotec and GE Healthcare) and used in the experiment.

[Experimental Example 3] Separation of CD11b+ cells and monocytes

To isolate CD11b+ cells, tumor single cell suspension was labeled with biotin-anti-CD11b antibody (Biolegend) and streptavidin-magnetic beads (Miltenyi biotec) and separated using MACS LS column (Miltenyi Biotec). Similarly, to isolate monocytes, a monocyte isolation kit (Miltenyi Biotec) was used from bone marrow cells.

[Experimental Example 4] Differentiation of BMDM and tumor infiltrating monocytes

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

[Experimental Example 5] Flow cytometry

Typically, up to ¹ Staining was performed, and all centrifugation for washing was performed at 1700 rpm for 5 minutes. For intracellular IFN-gamma staining, isolated CD45+ or CD8+ cells from the tumor or TDLN were reactivated by co-culturing with B16F10 melanoma cells gamma-irradiated with 10gy and brefeldin-A for 16 hours. Afterwards, cells were surface stained, perforated using a BD fixation/permeabilization kit according to the manufacturer's recommendations, and intracellularly stained with PE-anti-IFN-g antibody (BD Biosciences). Data collected using a FACS Canto II flow cytometer (BD Biosciences) were analyzed with FlowJo software (Tristar).

[Experimental Example 6] Quantitative PCR

Total RNA from tissues and cells was extracted with TRIzol reagent (Invitrogen). cDNA was synthesized using aTOPscript™ cDNA synthesis kit (Enzynomics). Real-time PCR was performed using SYBR green (Bio-rad) on a StepOne Plus TM (ApplidBiosystems) instrument. Gene expression was normalized based on the GAPDH mRNA expression level, and the relative expression level was calculated according to the ΔΔCT method.

[Experimental Example 7] ELISPOT

5 × ¹⁰⁴ lymph node cells obtained from OT-1 transgenic mice were cocultured with BMDM on day 3 of differentiation and OVA peptide (SIINFEKL; 1 μg/ml) at various ratios. Cells were plated on mouse IFN-g ELISPOT PLUS kit (ALP) (MABTECH), and 48 hours later, IFN-g spots were detected using the ELISPOT reader system (Autoimmun Diagnostika GmbH).

[Experimental Example 8] Statistical analyzes

All data were expressed as mean ± standard error of the mean (S.E.M). Statistical significance was determined by unpaired two-tailed t-test, two-way analysis of variance. Significance was defined as *p<0.05, **p<0.01, ***p<0.001. All analyzes were performed using Prism 7.0 software (GraphPadSoftware, Inc).

[Experimental Example 9] Antigen presentation assay

After 2 days of BMDM differentiation, 1 mg/ml whole-OVA protein was added. The MHCI complex and MHCII bound to the OVA peptide were labeled with anti-H-2kb-SIINFEKL antibody (Biolegend: #141604) and anti-MHCII antibody (Biolegend: #107622), and the expression level was measured using flow cytometry.

[Experimental Example 10] Phagocytosis assay

After 2 days of BMDM differentiation, they were co-cultured with gamma-irradiated B16F10 cells stained with 5 μM CFSE for 24 hours. CFSE fluorescence was detected through the 488/519 channel in CD45+, CD11b+, Ly6Chi, and F4/80low monocytes and CD45+, CD11b+, Ly6Clow, and F4/80high macrophages through flow cytometry.

[Experimental Example 11] Fluorescence microscopy

BMDM cells cultured in a confocal dish were fixed, the cell membrane perforated with 4.2% paraformaldehyde solution, and then incubated with anti-LC3B antibody (abcam: #ab51520) (1:200) and Hoechst 33342 (Invitrogen: #H3570) (1000: It was stained with 1). After 2 hours of incubation, 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 an LSM700 confocal laser scanning microscope (Carl Zeiss) equipped with a 63X oil immersion lens.

[Experimental Example 12] Single-cell RNA sequence data analysis

Data from a scRNA seq study on responsiveness to immune checkpoint blockade treatment and changes in immune cells were analyzed. Clustering of 5,928 cells was performed in the cohort before immune checkpoint blockade administration using the Seurat v4 R package. Afterwards, patients were classified into CD244 high and low expressors according to the average expression level of CD244 on their monocyte-macrophages. Only genes with relatively high average expression and high expression differences between cells were selected and used for downstream clustering analysis. Principal component analysis (PCA) was used for dimensionality reduction, and the number of important principal components was calculated using the built-in JackStraw function. T-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP) were used for two-dimensional data visualization. The FindMarkers function built into the Seurat package was used to identify differentially expressed genes. From the results of the Seurat package, genes with a P value of less than 0.05 were considered differentially expressed genes. Signal transduction pathway and gene signature analysis were performed on differentially expressed genes and gene expression rank of each population using IPA software (QIAGEN Inc.) and fgsea R package.

[Example 1] Analysis of anticancer effect through CD244 inhibition targeting mouse melanoma model

To determine whether CD244 expressed on monocytes affects tumor progression, we confirmed whether CD244 expression was elevated on monocytes within the tumor. A tumor model was constructed by subcutaneously injecting ¹ Compared to my immune cells. As a result, natural killer cells (hereinafter referred to as NK cells) and dendritic cells (hereinafter referred to as DC) have high CD244 expression regardless of the presence or absence of tumor, while monocytes have almost no CD244 expression in normal skin tissue. It was confirmed that CD244 expression was significantly increased after tumor induction (Figure 1a-A).

In addition, to determine whether the increase in CD244 expression on monocytes after tumor induction affects tumor growth, 1 × 10 ⁶ B16F10 was subcutaneously injected into the flanks of C57BL/6 or C57BL/6 CD244 ^−/− mice to model the tumor. After construction, tumor growth was observed for 14 days. The size of the tumor was calculated as the width × length × height of the tumor tissue / 2 using a vernier caliper. As a result, it was confirmed that tumor growth was significantly suppressed in CD244 ^-/- mice (Figure 1A-B).

In addition, since CD244 is expressed on various immune cells in addition to monocytes, in order to confirm the more specific effect of CD244 expressed on monocytes, the LysM gene, which is expressed specifically on monocytes and macrophages, and the LysM-gene, which is co-expressed with Cre recombinase, Monocyte-macrophage-specific CD244-deficient mice were created by crossing cre mice with CD244 flox mice with flox sequences, a gene sequence that can be cut by Cre recombinase, attached to the front and rear of the CD244 gene (Figure 1B-C). Melanoma was induced in the control group, CD244 ^{fl /fl} (hereinafter referred to as Flox) and the experimental group, LysM ^cre -CD244 ^{fl /fl} (hereinafter referred to as cKO), in the same manner as in Figure 1A-B. As a result, it was confirmed that tumor growth was significantly suppressed in the experimental group, monocyte-macrophage specific CD244 deficient mice (Figure 1b-D).

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

To determine how CD244 expressed in monocyte-macrophages suppresses the anti-tumor immune response, the activity of T cells, the most representative anti-tumor immune response, was confirmed. Melanoma was induced in Flox and cKO mice in the same manner as Figure 1B-D, and 14 days later, tumors were extracted and analyzed through flow cytometry. As a result, there was no difference in the ratio of CD8 and CD4 T cells and NK cells, but CD8 and CD4 T cells in cKO mice expressed more IFN-g, which plays a central role in anti-tumor immune response, compared to the control group (Flox). It was confirmed that (Figure 2a). In addition, it was confirmed that the expression of granzyme-b, a protein that directly kills tumor cells, was significantly elevated in CD8 T cells of cKO mice (Figure 2b-C). It was confirmed through staining using H-2Db gp100 tetramer that this difference was due to an increase in antigen-specific CD8 cells expressing T cell receptors specific for gp100, a melanoma antigen, in cKO mice (Figure 2B-D). In addition, the expression of CD44, a marker of memory T cells, was confirmed to be higher in CD8 T cells of cKO mice (Figure 2B-E).

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

Tumors from Flox mice, the control group, and cKO mice, the experimental group, were extracted and the distribution of tumor-infiltrating immune cells was confirmed through flow cytometry. As a result, there was no difference in the ratio of total myeloid immune cells, CD11b+ cells, neutrophils, and DC cells, but it was confirmed that monocytes were significantly reduced and macrophages were significantly increased in cKO mice (Figure 3A-A ).

To determine whether CD244 regulates the differentiation process of monocytes into macrophages, a BMDM experiment was conducted in which bone marrow cells of C57BL/6 or C57BL/6 CD244 ^-/- mice were treated with M-CSF to differentiate. As a result of measuring the ratio and number of macrophages after 3 days, the number of macrophages in CD244 ^-/- BMDM was significantly increased compared to the control group (WT) (Figure 3A-B). In addition, considering that the physiological characteristics of monocytes in the bone marrow and monocytes in the tumor are significantly different, it was confirmed that the ratio and number of macrophages also significantly increased when the monocytes in the tumor were isolated and treated with M-CSF (Figure 3b).

In addition, to directly confirm whether CD244 regulates monocyte-macrophage differentiation in the tumor microenvironment, monocytes were isolated from the bone marrow of C57BL/6 CD45.1 WT and C57BL/6 CD45.2 CD244 ^-/- mice, and then Cell After staining with trace far red dye, the mixture was mixed at a 1:1 ratio and injected directly into the tumor of C57BL/6 CD45.2 mice (Figure 3c-D). After 24 hours, CD45.1 WT and CD45.2 CD244 ^-/- cells stained with Cell trace far red were confirmed within the tumor, and it was confirmed that CD244 ^-/- monocytes were significantly differentiated into macrophages (Figure 3c- E).

To determine whether CD244 inhibits monocyte-macrophage differentiation through interaction with its ligand, CD48, and downstream signaling, a bone marrow-derived macrophage differentiation experiment was performed using an anti-CD48 antibody (Biolegend: #103453) was processed. As a result, it was confirmed that the number of macrophages did not change significantly in CD244 ^-/-, but significantly increased in WT mice (Figure 3C-F).

[Example 4] Analysis of the mechanism of inhibition of adaptive immune response activity of CD244-deficient monocytes/macrophages

To determine how CD244 inhibits the adaptive immune response activity of monocyte-macrophages, tumors from Flox mice as a control group and cKO mice as an experimental group were extracted, and then flow cytometry and real-time PCR were used to determine markers of M1 and M2 macrophages. was analyzed. As a result, the expression of M1 macrophage markers (CD80, IL-6, NOS2, and IFNB1) increased in the experimental group (cKO) compared to the control group (Flox) (Figures 4A-A and 4A-B), while the expression of M2 macrophage markers (Figures 4A-B) CD206, ARG1, TGFB1) did not show significant expression changes (Figures 4A-C and 4A-D).

In addition, to determine whether antigen presentation, a core 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 degree of antigen presentation. was confirmed. As a result, the expression of the MHCI-ova complex was significantly increased in CD244-deficient macrophages (Figure 4A-E), and this, coupled with the increase in the number of macrophages in CD244-deficient mice, resulted in a significant increase in the expression of the MHCI-ova complex. The number of macrophages was significantly increased in BMDM of CD244-deficient mice compared to WT mice (Figure 4A-F). In addition, when it was confirmed that CD244 regulates the expression of costimulatory molecules essential for antigen presentation, it was confirmed that CD80 expression was high in CD244-deficient macrophages (Fig. 4B-G). To confirm whether the increase in antigen-specific CD8 T cells within the tumor of cKO mice was due to CD244 suppressing antigen presentation by macrophages, in vitro differentiated bone marrow-derived macrophages and OT-1 T cells that recognize ova were used. When co-cultured and ova peptide was added, it was confirmed that T cell-specific IFN-g secretion increased when co-cultured with CD244-deficient macrophages (Figures 4B-H).

Next, to confirm the degree of phagocytosis, which is an important mechanism in which macrophages participate in the anti-tumor immune response and must precede antigen presentation, BMDM differentiated for 2 days and 10gy were irradiated with gamma rays and then fluorescently labeled with CFSE. B16F10 was co-cultured for 24 hours. Afterwards, CFSE fluorescence expressed in BMDM was measured using flow cytometry, and it was confirmed that the phagocytosis of both CD244-deficient monocytes and macrophages was significantly increased compared to the control group (WT) (Figures 4b-I and 4b-J).

[Example 5] Analysis of the cause of increased CD244 expression in monocytes during tumor induction

In order to determine the cause of the increase in CD244 expression on monocytes during tumor induction in Figure 1A-A and the molecular mechanism by which CD244 suppresses monocyte-macrophage differentiation and function, single cell transcriptome analysis of a mouse tumor model in a public database was performed. Data (GSE121861) was analyzed. Representative marker expression patterns distinguish seven CD45 (PTPRC)+ immune cell clusters, among which monocytes (LY6C2, CCR2, and IL1B), immature monocytes (S100A9 and OLR1), macrophages (ADGRE1 and CSF1R), and M1 (H2). By checking the expression levels of known markers such as -Aa, H2Ab1, CX3CR1, CD86, and APOE) and M2 (ARG1, TREM 2, and SPP1) macrophages, we identified four subclusters of monocyte lineage cells (Figures 5A-A and Figure 5A-B). Pathway enrichment analysis showed that immature monocytes expressed genes related to immune response, differentiation, and protein folding at lower levels than normal monocytes (Figure 5A-C). As a result of confirming CD244 expression in the four subgroups of monocyte lineage cells, it was confirmed that CD244 expression was significantly elevated in immature monocytes compared to normal monocytes and M1/M2 macrophages (Figures 5A-D and 5A-E). In addition, compared to immature monocytes expressing low CD244, the ER stress pathway was confirmed to be upregulated in immature monocytes expressing high CD244 (Figure 5f). The ER stress pathway is one of the major mechanisms in the development of immunosuppressive immature monocytes. It is assumed that it also regulates CD244 expression in immature monocytes. When BMDMs were treated with Thapsigargin, an ER stress inducer, CD244 expression increased in monocytes. and differentiation into macrophages decreased. On the other hand, when treated with Tauroursodeoxycholic acid, an ER stress inhibitor, it was confirmed that CD244 expression in monocytes decreased and differentiation into macrophages increased (Figures 5B-G and 5B-H) ).

[Example 6] Analysis of molecular mechanism regulated by CD244

In order to determine how CD244 inhibits monocyte-macrophage differentiation, antigen presentation, and phagocytosis, the expression of signaling molecules of CD244 was confirmed in the single cell transcriptome analysis data of Example 5. As a result, it was confirmed that the expression of SAP, EAT-2, ERT, and SHP-2 was low and the expression of Beclin-1 was high in monocyte macrophages compared to T cells and NK cells (Figure 6a-A). Additionally, the degree of autophagy occurred in WT and CD244-deficient BMDMs. First, to confirm lipidation of LC3, which is a necessary process for the initiation of autophagy, WT and CD244 KO BMDM were stained with anti-LC3B antibody (abcam: #ab51520) and observed through fluorescence microscopy and flow cytometry. As a result, CD244 It was confirmed that LC3 lipidation was significantly increased in KO BMDM (Figures 6A-B and 6A-C). The amount of autophagosomes actually formed by LC3B was quantified through cyto-ID staining, and it was confirmed that it was significantly increased in CD244 KO BMDM (Figures 6A-D).

Next, to confirm that the degree of differentiation of BMDM changes when autophagy is controlled, the cells were treated with an autophagy-regulating drug. When treated with chloroquine, which inhibits the final step of autophagy by interfering with the fusion of autophagosomes and lysosomes, the number of macrophages was significantly reduced in both WT and CD244 KO BMDMs (Figures 6B-E and 6B-F). . In addition, when treated with Ly294002, an inhibitor of vps-34, another molecule that binds to CD244, the number of macrophages decreased, and the difference in LC3B expression between WT and CD244 ^-/- BMDMs disappeared (Figures 6B-G and Figure 6b-H). From the above results, it was confirmed that CD244 inhibits the differentiation of monocytes into macrophages by inhibiting autophagy.

[Example 7] Analysis of anticancer effect through combined administration of CD244-deficient monocytes/macrophages and anti-PD-L1 antibody

Through the above results, it was confirmed that CD244 suppresses the anti-tumor immune response by accumulating monocytes in the tumor microenvironment and reducing subsequent differentiation into macrophages, antigen presentation, and phagocytosis. Accordingly, CD244-deficient macrophages were identified as immunological agents. Experiments were conducted on the possibility of using the treatment as a treatment method to convert “cold” tumors into “hot” tumors. First, B16F10 melanoma was induced in monocyte lineage-specific CD244-deficient mice (cKO) and control Flox mice, and then treated with anti-PD-L1 antibody. As a result, the tumors of Flox mice showed no significant changes upon anti-PD-L1 antibody treatment. However, anti-PD-L1 antibody treatment further reduced tumor size in cKO mice compared to isotype antibody treatment (Figure 7A-A). When measuring the proportion of T cells inside the tumor, it was confirmed that the proportion of CD8 T cells increased significantly only when cKO mice were treated with anti-PD-L1 antibody (Figures 7A-B). In addition, as a result of analyzing the memory phenotype of T cells in the TDLN of cKO mice treated with anti-PD-L1 antibody, the central memory (CD62L+, CD44+) CD8 T cells and effector cells were compared to when Flox mice were treated with anti-PD-L1 antibody. The memory (CD62L-, CD44+) CD4 T cell population significantly increased (Figure 7b). In addition, the proportion of T cells that express PD-1 and do not express TIGIT (T cell immunoreceptor with Ig and ITIM domains) was significantly increased only in tumors of cKO mice injected with anti-PD-L1 antibody, whereas PD-1/ The proportion of TIGIT double-positive C D8 T cells did not show significant change (Figure 7c-D). In general, PD-1 is an immune checkpoint receptor expressed on all antigen-specific activated T cells, whereas TIGIT is known to be expressed only on severely exhausted T cells. Therefore, based on the above results, anti-PD-L1 antibodies were administered to cKO mice. When treated with , it was confirmed that the activity of CD8 T cells in the tumor increased, but the phenomenon of immune exhaustion did not increase.

To investigate the potential of CD244-deficient macrophages as an anti-tumor immune cell therapy, anti-PD-L1 antibody and CD244 ^-/- BMDM cells were simultaneously injected into WT mice with B16F10 melanoma. When isotype antibody and CD244 ^-/- BMDM were treated together, tumor growth was slightly inhibited, but co-administration of CD244 ^-/- BMDM and anti-PD-L1 antibody significantly reduced the growth of B16F10 tumors and LL2, another cold tumor model. The same result was confirmed in (Lewis lung carcinoma) (Figure 7c-E). The above results confirmed that CD244-deficient macrophages increase T cell activity and memory phenotype, increase intratumoral CD8 T cells, and improve responsiveness to immune checkpoint inhibitors in tumor models including melanoma. did.

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

To determine the clinical significance of CD244 as an immune checkpoint receptor for monocytes/macrophages in the tumor microenvironment, we analyzed a single cell RNA sequencing data set (SCP398) of CD45+ immune cells isolated from human melanoma tissues in a public database. Among the 16,291 cells reported in previous studies, 1,391 cells classified as monocytes and macrophages were used. To identify monocytes/macrophages that do not express CD244 in these cells, they were further classified into seven subgroups using the shared nearest neighbor clustering method in seurat (Figure 8A-A). Next, according to CD244 expression, 1) C0-1 with a relatively high proportion of cells expressing CD244, 2) C2-3 with a relatively low proportion of cells expressing CD244, and 3) C4-6 with no CD244 expression. were classified into three groups (Figure 8a-B). Among these groups, C0-1 with significant numbers of CD244+ and CD244- monocytes/macrophages was used to ensure a fair comparison between the two cell types. A total of 511 genes and 221 genes were confirmed to be up- and down-regulated in CD244+ and CD244- monocytes/macrophages, respectively (Figures 8A-C). The 221 genes mainly expressed in CD244 monocytes/macrophages were mainly related to phagosome, antigen processing and presentation, and autophagy (Figure 8A-D), which was consistent with the findings in the mouse model. Next, we sought to determine whether the expression level of CD244 on monocytes/macrophages affected the patient's survival rate. For this purpose, a bulk RNA-seq dataset generated from tumor tissues of 470 melanoma patients (103 primary tumors, 367 metastatic tumors) was obtained from The Cancer Genome Atlas database (TCGA-SKCM, Figure 8B-E). We then identified specifically upregulated genes (DEGs) in the four cell types to estimate the proportion of the four cell types identified in the preceding scRNA-seq analysis in the bulk RNA-seq dataset and TCGA via CIBERSORTx. The dataset was subjected to deconvolution analysis (Figure 8B-F). We then compared the survival rates of patients with and without non-CD244-expressing monocytes/macrophages, showing that in both primary and metastatic cancer cohorts, patients with non-CD244-expressing monocytes/macrophages had better overall survival than those without. It was confirmed that this was significantly high (Figure 8b-G).

Next, we sought to determine whether monocytes/macrophages with low CD244 expression could improve the antigen-specific activity of T cells in humans, similar to the mouse model. In the previous scRNA-seq data set, individual patients were classified into CD244 high and CD244 low groups according to the average CD244 expression level on monocytes/macrophages, and CD8 T between CD244 high and low groups regardless of CD244 expression on T cells. Cells were compared (Figure 8C-H). Through this, 207 genes that were mainly upregulated in CD8 T cells of the CD244 low group were obtained, and it was confirmed that these genes were mainly associated with cytokine signaling, TCR signaling, and the adaptive immune system (Figure 8c-I).

[Example 9] Analysis of the effect of CD244 on the function of monocyte-macrophages in other carcinomas

Lastly, we confirmed whether the results on CD244 expressed in monocyte-lineage cells confirmed through the melanoma model and transcriptome analysis data could be extended to other tumor models. For this purpose, single-cell transcriptome analysis datasets from patients with colorectal cancer (SCP1162) (Figure 9a), non-small cell lung cancer (GSE127465) (Figure 9b), and glioblastoma (GSE131928) (Figure 9c) were analyzed. Pathway enrichment analysis was performed using DEGs from monocytes/macrophages that do not express CD244, and it was confirmed that CD244 regulates the innate immune system, phagocytosis, antigen presentation, and autophagy in monocyte-macrophages even in carcinomas other than melanoma. .

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (16)

A pharmaceutical composition for preventing or treating cancer, comprising monocytes or macrophages suppressing CD244 expression or activity as an active ingredient. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits cancer growth by increasing phagocytosis or antigen presentation. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition induces antigen-specific activation of T cells and increases the expression of IFN-g. The pharmaceutical composition according to claim 1, wherein the monocytes include one or more markers selected from the group consisting of CD11b+, CD14+, CD16-, Ly6G-, and Ly6Chi. The pharmaceutical composition according to claim 1, wherein the macrophages include one or more markers selected from the group consisting of CD11b+, CD14+, CD16+, CD68+, Ly6G-, Ly6Clo, and F4/80+. The method of claim 1, wherein the cancer is stomach 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, colon cancer, small intestine cancer, rectal cancer, and anus. Peripheral cancer, fallopian tube carcinoma, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, prostate cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, In the group consisting of penile cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney cancer, ureteral cancer, renal pelvic cancer, hematological cancer, brain cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma. A pharmaceutical composition characterized by one or more selected substances. Monocytes or macrophages with suppressed CD244 expression or activity; and a pharmaceutical composition for combined administration for the prevention or treatment of cancer, comprising an anticancer agent as an active ingredient. The pharmaceutical composition according to claim 7, wherein the anticancer agent is one or more anticancer agents selected from the group consisting of cytotoxic anticancer agents, targeted anticancer agents, and immunological anticancer agents. The method of claim 8, wherein the cytotoxic anticancer agent is doxorubicin, cyclophosphamide, temozolomide, irinotecan, cisplatin, vinblastine, vincristine ( A group consisting of vincristine, actinomycin-D, 5-fluouracil, docetaxel, cabazitaxel, paclitaxel, and pembrolizumab A pharmaceutical composition characterized in that it is one or more selected from . The method of claim 8, wherein the targeted anticancer agent is Trametinib, vemurafenib, alpelisib, dactolisib, Gefitinib, Erlotinib, and Lapatinib. (Lapatinib), Sunitinib, Sorafenib, Crizotinib, Dabrafenib, Trastuzumab, Cetuximab, Bevacizumab ), Panitumumab, Ipilimumab, Pertuzumab, Tofacitinib, Imatinib, Bortezomib, Ofatumumab and Alemtuzumab A pharmaceutical composition comprising at least one selected from the group consisting of (Alemtuzumab). The method of claim 8, wherein the immunotherapy agent is anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIGIT, anti-TIM-3, anti-GITR, CAR-T And a pharmaceutical composition characterized in that it is one or more selected from the group consisting of NK cells. A composition for enhancing the efficacy of an anticancer drug comprising monocytes or macrophages suppressing CD244 expression or activity as an active ingredient. A method for preventing or treating cancer, comprising administering monocytes or macrophages whose expression or activity of CD244 is suppressed to individuals, including humans, alone or in combination with an anticancer agent. A composition for inducing differentiation from monocytes to macrophages, comprising a CD244 inhibitor or a CD48 inhibitor, a CD244 ligand, as an active ingredient. The method of claim 14, wherein the CD244 inhibitor or CD48 inhibitor is selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies and natural products that specifically bind to CD244 or CD48 protein, or mRNA of the CD244 or CD48 gene. In monocytes, characterized in that they are selected from the group consisting of small interfering RNA (siRNA), antisense nucleotides, and short hairpin RNA (shRNA) that bind complementary to macrophages. A composition for inducing differentiation. A screening method for an agent that induces differentiation from monocytes to macrophages, comprising the step of treating cells with a candidate material and analyzing the inhibition of expression or activity of CD244 or its ligand, CD48.