Anticancer adjuvant for cancer immunotherapy and composition for inhibiting cancer metastasis comprising epiregulin inhibitor

Abstract

The present invention relates to an anticancer adjuvant for cancer immunotherapy and a composition for inhibiting cancer metastasis, comprising an epiregulin (EREG) inhibitor. The objective of the present invention is to provide a composition which induces polarization changes in tumor-associated macrophages (TAMs) by targeting EREG of the TAMs, particularly increases the proportion of M1 macrophages by regulating subtypes of M2 macrophages, and thus has the effect of inhibiting cancer cell proliferation and metastasis.

Description

Anticancer adjuvant for cancer immunotherapy and composition for suppressing cancer metastasis comprising an epiregulin inhibitor

[0001] This patent application claims priority to Patent Application No. 10-2024-0039969, filed with the Korean Intellectual Property Office on March 22, 2024, and Patent Application No. 10-2024-0081965, filed with the Korean Intellectual Property Office on June 24, 2024, the disclosures of which are incorporated herein by reference.

[0002] This invention was made possible with the support of the Ministry of Health and Welfare, under grant number 1465036429 and sub-grant number HR20C0025020022. The research management organization for this project is the Korea Health Industry Development Institute, and the research project name is "Research-Oriented Hospital Promotion R&D." The project implementing organization is Samsung Medical Center, and the research project titled "Development of Immune Cell Therapy Biomarkers and Novel Targets Based on an Ultra-Precision Multi-Omics Analysis System" was conducted from January 1, 2022 to December 31, 2022.

[0003] This invention was made possible by grant number 1711188753 and sub-grant number 2020R1A2C2006408 from the Ministry of Science and ICT. The research management organization for this project is the National Research Foundation of Korea, and the research project name is "Individual Basic Research." The project implementing organization is Samsung Medical Center, and the research project titled "Development of Technology to Modulate the Immune Microenvironment through Macrophage-Mediated Immune Profiling in Colorectal Cancer Patients" was conducted from March 1, 2023 to February 29, 2024.

[0004] The present invention relates to an anticancer adjuvant for cancer immunotherapy comprising an epiregulin (EREG) inhibitor, specifically, to an anticancer adjuvant for cancer immunotherapy that induces changes in the tumor microenvironment by regulating the polarization of tumor-associated macrophages (TAMs) and a composition for inhibiting cancer metastasis.

[0005] Colorectal cancer (CRC) is a malignant tumor that arises in the colonic mucosa, and is a fatal disease with a low five-year survival rate. Despite various treatments that target cancer cells directly with anticancer drugs, it remains difficult to suppress cancer cell proliferation and metastasis in cancer patients. While immunotherapy offers the advantage of extending overall survival compared to chemotherapy, its effectiveness varies from patient to patient and can cause side effects due to immune system overactivation. Various immunotherapies are being developed to induce a transition to a state where anti-tumor cells dominate. This requires an understanding of the functions of not only the tumor cells themselves but also the cells that make up the tumor microenvironment.

[0006] Colorectal cancer (CRC) harbors a microenvironment infiltrated by various immune cells, including tumor-associated macrophages (TAMs), monocytes, natural killer cells, CD4+ T cells, and CD8+ T cells. Among these, TAMs adapt to their environment by polarizing into M1 or M2 macrophages. M1 macrophages are known to decrease the viability of colorectal cancer cells through apoptosis, whereas M2 macrophages are known to significantly increase cell viability compared to M0 macrophages.

[0007] Epiregulin (EREG) is known to be a ligand for the epidermal growth factor receptor (EGFR) and the Erb-B2 tyrosine kinase receptor 4 (ErBb4). ErBb4 exists on the cell membrane and, in addition to activating various downstream signaling pathways, is cleaved by proteases such as matrix metalloproteinase 1 (MMP1) and translocates to the nucleus, where it plays an important role in regulating gene expression. However, the precise mechanism underlying the role of ErbB4 in colon cancer has not been elucidated. Therefore, there is a growing need for therapeutic agents that target EREG in cancer-associated macrophages and induce changes in the tumor microenvironment.

[0008] As a prior art document for the present invention, there is a non-patent document, Lee et al., "The FBW7-MCL-1 axis is key in M1 and M2 macrophage-related colon cancer cell progression: validating the immunotherapeutic value of targeting PI3Kγ" Experimental & Molecular Medicine volume 52 (2020): 815-831.

[0009] The present invention aims to provide a composition that targets epiregulin (EREG) of tumor-associated macrophages (TAMs) to induce changes in macrophage polarization, and an anticancer adjuvant and metastasis-suppressing composition having effects such as inhibition of cancer cell proliferation.

[0010] The present invention has revealed that EREG is involved in regulating the polarization of cancer-associated macrophages, which are one of the important cells that constitute the tumor microenvironment, and thus has elucidated that targeting EREG of macrophages may be promising in the development of combination therapy for the treatment of colon cancer.

[0011] Hereinafter, the present invention will be described in more detail.

[0012] One aspect of the present invention relates to an anticancer adjuvant for cancer immunotherapy comprising an epiregulin (EREG) inhibitor.

[0013] In the present invention, the epiregulin (EREG) inhibitor may induce changes in the tumor microenvironment, but is not limited thereto.

[0014] In the present invention, the epiregulin (EREG) inhibitor may be a small interfering RNA (siRNA) consisting of the sequence of SEQ ID NO: 4, but is not limited thereto.

[0015] In the present invention, the epiregulin (EREG) inhibitor may induce polarization of tumor-associated macrophages (TAMs) into M1 macrophages, but is not limited thereto.

[0016] In the present invention, the epiregulin (EREG) inhibitor may suppress polarization of tumor-associated macrophages (TAMs) into M2 macrophages, but is not limited thereto.

[0017] In the present invention, the epiregulin (EREG) inhibitor may induce polarization of M2 macrophages into M1 macrophages, but is not limited thereto.

[0018] In the present invention, the anticancer adjuvant for cancer immunotherapy may additionally include one or more additives selected from the group consisting of suitable carriers, disintegrants, sweeteners, coating agents, swelling agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0019] In the present invention, the anticancer adjuvant for immunotherapy can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes.

[0020] In the present invention, the dosage of the anticancer adjuvant for immunotherapy may vary depending on the condition and weight of the subject, the type and degree of the disease, the form of the drug, and the tumor progression status of the subject, and may be appropriately selected by a person skilled in the art.

[0021] In the present invention, the subject may be a mammal, for example, a human or a mouse, but is not limited thereto.

[0022] In the present invention, the cancer may be at least one selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, gastric cancer, and squamous cell carcinoma, which are cancers with high EREG expression, but is not limited thereto.

[0023] One aspect of the present invention relates to a composition for inhibiting cancer metastasis comprising an epiregulin (EREG) inhibitor.

[0024] One aspect of the present invention relates to a method for inhibiting the expression of epiregulin (EREG) in tumor-associated macrophages (TAMs).

[0025] One aspect of the present invention relates to a method for inducing polarization of tumor-associated macrophages (TAMs) into M1 macrophages by suppressing the expression of epiregulin (EREG).

[0026] One aspect of the present invention relates to a method for inhibiting polarization of tumor-associated macrophages (TAMs) into M2 macrophages by inhibiting the expression of epiregulin (EREG).

[0027] One aspect of the present invention relates to a method for inducing polarization of M2 macrophages into M1 macrophages by suppressing the expression of epiregulin (EREG).

[0028] The term "epiregulin (EREG)" in this specification refers to a gene encoding a member of the epidermal growth factor (EGF) protein family and a protein encoded therefrom, as defined by the National Institutes of Health (NIH) of the United States, and includes all ligands of the epidermal growth factor receptor (EGFR) and Erb-B2-tyrosine-kinase receptor 4 (ErBb4) encoded by the gene, proteins involved in a wide range of biological processes such as inflammation, and proteins that can promote cancer progression in human cell tissues.

[0029] The term "tumor microenvironment" in this specification refers to a concept that includes not only cells such as immune cells and stromal cells within a tumor, but also blood vessels passing through them, extracellular matrix, and hypoxic, acidic environments.

[0030] The term "tumor microenvironmental change" as used herein refers to any action that inhibits tumor progression, improves symptoms, or otherwise beneficially alters tumor microenvironment as a result of administration of a composition. For example, this may refer to, but is not limited to, adjusting the M2 / M1 ratio in cancer-associated macrophages to less than 3.

[0031] The term "siRNA" used herein refers to a double-stranded RNA molecule that causes RNA interference (RNAi). siRNA binds to specific mRNAs with complementary nucleotide sequences, thereby interfering with their translation, thereby knocking down genes and suppressing their expression.

[0032] The term "inhibition" as used herein means slowing down or interfering with the expression of a gene or chemical reaction, etc., and may mean, for example, but is not limited to, reducing, blocking, or down-regulating the expression of epiregulin (EREG).

[0033] The term "tumor-associated macrophage (TAM)" in this specification refers to an immune cell present in large numbers in the tumor microenvironment, and includes both monocyte-derived macrophages and tissue-resident macrophages.

[0034] The term "macrophage polarization" used herein refers to the activation pattern of macrophages at a specific point in time and space. Macrophages have multiple potentials and can be activated in various ways. Macrophage polarization is not fixed, as it is plastic and integrates various factors. The induction and inhibition of macrophage polarization are regulated by the tissue microenvironment, cell survival pathways, and cytokines.

[0035] The term "M1 macrophage" in this specification generally refers to a population classified as inflammatory macrophages that are activated by IFN-γ or LPS (Lipopolysaccharide) and produce pro-inflammatory cytokines. The M1 macrophage includes the concept of immune cells that are classically activated and produce nitric oxide and reactive oxygen species.

[0036] The term "M2 macrophage" as used herein refers to a population classified as anti-inflammatory macrophages that produce polyamines or proline, which induce cell proliferation. The M2 macrophage encompasses the concept of an immune cell that is alternatively activated and has pro-regenerative properties.

[0037] The term "cancer immunotherapy" used herein refers to the treatment of cancer utilizing the body's immune system. More specifically, it refers to treatment through an immune response targeting antigens in cancer cells and the tumor microenvironment. This may include antibody therapy, immunocytotherapy, immune checkpoint blockade, bispecific T-cell-engaging antibodies (BiTE), and chimeric antigen receptor T-cell (CAR-T) therapy.

[0038] The term "anticancer adjuvant" as used herein refers to a preparation that can improve or supplement the anticancer effects of an anticancer agent. It can be used in additional cancer treatments following primary treatment to reduce the risk of cancer recurrence. Such cancer treatments may include chemotherapy, hormone therapy, targeted therapy, and immunotherapy. The anticancer adjuvant of the present invention may be administered in combination with an anticancer agent. Co-administration may include simultaneous, separate, or sequential administration with the anticancer agent.

[0039] The term "metastasis" in this specification means that cancer cells separate from the original tumor, infiltrate the circulatory system and lymphatic system, etc., and proliferate while evading the immune response.

[0040] The present invention relates to an anticancer adjuvant for cancer immunotherapy comprising an epiregulin (EREG) inhibitor, which targets epiregulin (EREG) in tumor-associated macrophages (TAMs) and induces changes in the tumor microenvironment. Inhibition of epiregulin expression can reduce cancer cell proliferation and metastasis by modulating the polarization of tumor-associated macrophages.

[0041] Figure 1 shows the results of analyzing genes that are increased in colon cancer patients compared to the normal group to select genes with high expression specifically in colon cancer-associated macrophages.

[0042] Figure 2 is a schematic diagram of an experiment using macrophages differentiated from monocytes isolated from bone marrow as a control group and cancer-associated macrophages isolated from tumors as an experimental group.

[0043] Figure 3 shows the results of confirming the expression of the genes of Figure 1 in the cells of Figure 2.

[0044] Figure 4 shows the results showing the efficacy of two siRNA sequences used in gene knockdown experiments.

[0045] Figure 5 shows the results of confirming macrophage polarization by gene knockdown.

[0046] Figure 6 shows the results of confirming whether there is a change in the proliferation of cancer-associated macrophages themselves according to EREG gene knockdown.

[0047] Figure 7 shows the results of confirming the proliferation or inhibition of M1 and M2 macrophages according to EREG gene knockdown.

[0048] Figure 8 shows the results of confirming the interaction between EREG expression in cancer-associated macrophages and cancer cells. Figure 8a shows the results of confirming cancer cell migration due to suppression of EREG expression, and Figure 8b shows the results of confirming cancer cell proliferation and metastatic potential due to suppression of EREG expression.

[0049] The present invention relates to an anticancer adjuvant for cancer immunotherapy comprising an epiregulin (EREG) inhibitor.

[0050] The present invention provides an anticancer adjuvant for cancer immunotherapy and a composition for inhibiting cancer metastasis, comprising an epiregulin (EREG) inhibitor.

[0051] Hereinafter, the present invention will be described in detail through experimental examples.

[0052] [Experimental Example]

[0053] Experimental Example 1. Analysis of Differentially Expressed Genes in Cancer-Associated Macrophages Using Genomic Data

[0054] Using genomic data obtained from colorectal cancer patient tissues, we selected 1) 149 genes showing differential expression between macrophages of the normal group and macrophages of colorectal cancer patients, and 2) 62 genes increased in SPP1 (secreted pohsphoprotein 1) macrophage A. In order to select genes with high expression specifically in colorectal cancer-associated macrophages, among the genes corresponding to the intersection of 1) and 2), genes increased in colorectal cancer patients compared to the normal group were analyzed, and the results are shown in Figure 1.

[0055] As a result of the analysis, nine genes were selected, and it was confirmed that EREG was included among them.

[0056] Experimental Example 2. Validation in the CT26 Animal Model

[0057] For experimental verification of nine genes derived through genomic data analysis, CT26 mouse tumor cells were injected into mice to grow tumors of 1000 and 2000 mm in size. 3 When this was done, only cancer-associated macrophages were isolated from the tumor using a cancer-associated macrophage isolation kit (miltenyi biotec #130-096-730).

[0058] At this time, mononuclear cells were isolated from the bone marrow, treated with M-CSF (concentration), induced to differentiate into macrophages, and used as a control. A schematic diagram of the experiment is shown in Figure 2.

[0059] The expression of nine genes from Experimental Example 1 was confirmed at the mRNA level in macrophages and cancer-associated macrophages. RNA was isolated using Trizol, isopropanol, and chloroform, and cDNA was synthesized using a cDNA synthesis kit (bioneer, k-2044-B). Real-time polymerase chain reaction was performed according to the SYBR (applied biosystem, 4367659) method, and the results are shown in Figure 3.

[0060] As a result, as the tumor size increased, the expression of the nine selected genes also increased, and in particular, the expression level of EREG was confirmed to be higher than that of other genes.

[0061] Experimental Example 3. Confirmation of macrophage polarization (M1, M2) according to gene expression.

[0062] Macrophages are polarized into M1 and M2 depending on the chemokine and cell that is stimulated, and are broadly classified into M1 and M2. M1 has a pro-inflammatory role, and M2 has an anti-inflammatory role. M1 is identified by markers such as iNOS, TNF-α, and IL-6, and M2 by markers such as Arg1, CD206, and CD163. Therefore, our research team selected 4 genes suitable as development targets among 9 genes that showed high expression in cancer-associated macrophages, and conducted a knockdown experiment to confirm whether the phenotype of cancer-associated macrophages (M2) was polarized into M1 macrophages when their expression was suppressed. The knockdown experiment was performed using the siRNA described in Table 1. One day before the knockdown, 2X10 cancer-associated macrophages were seeded in a 6-well plate. 5 were cultured. Knockdown experiments were performed using lipofectamine RNAi max (Invitrogen), and RNA and protein were obtained by harvesting after 48 hours to conduct experiments. Serum-free culture medium (opti-MEM; Gibco, 31985070) was used, and lipofectamine RNAi max and 100 nM siRNA were mixed in a 1:1 ratio and reacted at room temperature for 20 minutes. At this time, siRNA was diluted in serum-free culture medium. After 20 minutes, the opti-MEM siRNA mixture was distributed to cancer-associated macrophages cultured in 6-well plates, and after 24 hours, the culture medium was replaced with new medium and cultured for 48 hours.

[0063] Sequence number Knockdown gene siRNA base sequence (5'- 3') Sequence number 1 INHBA_1GUG CAU AUG CAU AUC UGU Sequence number 2 INHBA_2GUG AAC UGG UCU UUC UAC Sequence number 3 EREG_1CAC AAC CGU GAU CCC AUC Sequence number 4 EREG_2GUC AUG GAA AUC CCU GGA Sequence number 5 TNFAIP6_1CAC AGU GGA UCC UGC AUC Sequence number 6 TNFAIP6_2GGU CAG CGA AUU CAC CUG Sequence number 7 TREM1_1GAC AGA GCG UCC CAU CCU Sequence number 8 TREM1_2UGA CCU UCA GCG GUG UCU

[0064] Figure 4 shows the efficacy of two siRNA sequences used for gene knockdown, and it was confirmed that the siRNA of sequence number 4 had superior efficiency.

[0065] Subsequent experiments were conducted with siRNA sequence 4, which exhibited superior knockdown efficiency. Figure 5 shows the results of confirming macrophage polarization by gene knockdown. The left bar graph represents the control group, and the right bar graph represents the comparison group.

[0066] As can be seen in Figure 5, especially in the case of EREG expression inhibition, M1 macrophage markers iNOS and IL-6 increased and M2 macrophage markers CD206 and Arg1 decreased.

[0067] Experimental Example 4. Changes in EREG expression when cancer-associated macrophages are cultured in normal or cancer cell culture medium.

[0068] When cancer-associated macrophages isolated from the CT26 animal model were cultured in a general culture medium, the expression of EREG was confirmed to decrease as the subculture progressed. It was thought that the expression of EREG would be maintained due to a specific substance secreted by the signal transduction system of cancer cells, and so the experiment was designed as shown in Fig. 6.

[0069] The conditions reflecting the signaling system of cancer cells were recreated in a cancer cell culture medium, and the expression pattern of EREG was confirmed by comparative culture with a normal culture medium. The cancer cell culture medium was cultured for 48 hours in a culture medium containing 1% FBS, and only the culture medium was collected, centrifuged at 500 x g for 20 minutes, and only the supernatant was filtered through a 0.2 μm syringe filter (Millipore #R1HB95073) and used immediately or stored at -80℃.

[0070] As a result, while the expression of EREG decreased when cultured in a general medium, the expression of EREG was minimal and tended to increase when cultured in a cancer cell medium. In addition, it was confirmed that EREG, which decreased due to the general medium, increased when cultured in a cancer cell medium, and conversely, EREG, which increased when cultured in a cancer cell medium, decreased when cultured in a general medium. In other words, it can be seen that EREG of cancer cells and cancer-associated macrophages are related to each other, and that the expression of EREG is regulated by cross talk with cancer cells.

[0071] Experimental Example 5. Confirmation of the correlation between EREG expression in cancer-associated macrophages.

[0072] To confirm the interaction between EREG of tumor-associated macrophages and cancer cells, a wound healing assay was performed using co-culture using inserts (Corning #0111018). 1x10 tumor-associated macrophages were seeded in a 60-pi dish. 5 The cells were cultured for one day, knocked down the next day using siRNA (100 nM) - lipofectamine RNAi max, and cultured for 24 hours. At this time, cancer cells (CT26) were seeded at 1x10 in a 6-well plate. 5and the next day, the middle part of the well was scratched using a scratcher (SPL #201906). After 24 hours, the cancer-associated macrophages with EREG knockdown were detached with trypsin in a 60-pi dish and placed in the insert, and 50 nM siRNA was added. However, the insert was soaked in advance for 2 hours so that the bottom could be submerged. After culturing for 72 hours, the degree of wound healing of the cancer cells was confirmed and the mRNA expression of the proliferation marker ki67 and the metastasis marker vimentin was confirmed. As a result, it was confirmed that the inhibition of EREG expression in cancer-associated macrophages affected the proliferation and metastasis of cancer cells (CT26). Changes in M1 and M2 macrophage markers were confirmed in cancer-associated macrophages, and the results are shown in Figs. 6, 7, 8a, and 8b.

[0073] As can be seen in Fig. 6, there was no effect on the proliferation of cancer-associated macrophages (TAM) due to suppression of EREG expression, and as can be seen in Fig. 7, similar to the previous experimental results, M1 macrophages increased (increase in iNOS, a marker of M1 macrophages), and M2 macrophages decreased (decrease in CD206, a marker of M2 macrophages). Since the cancer-associated macrophages with EREG knockdown represent M2 macrophages, the increase in M1 macrophage markers and the decrease in M2 macrophage markers under the siEREG condition compared to the control indicate that polarization from M2 to M1 macrophages was induced.

[0074] As can be seen in Figure 8a, suppression of EREG expression in tumor-associated macrophages (TAMs) reduces cancer cell migration.

[0075] As can be seen in Figure 8b, the proliferation of cancer cells was reduced (decreased proliferation marker ki67) and the metastatic ability was also reduced (decreased metastasis marker vimentin).

[0076] The scope of protection of the present invention is not limited to the description of the embodiments explicitly described above. Furthermore, the scope of protection of the present invention cannot be limited by obvious modifications or substitutions within the technical field to which the present invention pertains.

[0077] The present invention relates to an anticancer adjuvant for cancer immunotherapy comprising an epiregulin (EREG) inhibitor, which targets epiregulin (EREG) in tumor-associated macrophages (TAMs) and induces changes in the tumor microenvironment. Inhibition of epiregulin expression can reduce cancer cell proliferation and metastasis by modulating the polarization of tumor-associated macrophages.

Claims

1. An anticancer adjuvant for cancer immunotherapy, comprising an epiregulin (EREG) inhibitor.

2. In paragraph 1, The above-mentioned epiregulin (EREG) inhibitor is an siRNA consisting of the base sequence of sequence number 4, and is an anticancer adjuvant for cancer immunotherapy.

3. In paragraph 1, The above epiregulin (EREG) inhibitor is an anticancer adjuvant for cancer immunotherapy that induces polarization of tumor-associated macrophages (TAMs) into M1 macrophages.

4. In paragraph 1, The above epiregulin (EREG) inhibitor is an anticancer adjuvant for cancer immunotherapy that suppresses the polarization of tumor-associated macrophages (TAMs) into M2 macrophages.

5. In paragraph 1, An anticancer adjuvant for cancer immunotherapy, wherein the cancer is at least one selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, gastric cancer, and squamous cell carcinoma, which are cancers with high EREG expression.

6. A composition for inhibiting cancer metastasis, comprising an epiregulin (EREG) inhibitor.

7. In paragraph 6, A composition for inhibiting cancer metastasis, wherein the above-mentioned epiregulin (EREG) inhibitor is an siRNA consisting of a base sequence of sequence number 4.

8. In paragraph 6, A composition for inhibiting cancer metastasis, wherein the above-mentioned epiregulin (EREG) inhibitor induces polarization of cancer-associated macrophages (TAMs) into M1 macrophages.

9. In paragraph 6, A composition for inhibiting cancer metastasis, wherein the above-mentioned epiregulin (EREG) inhibitor inhibits the polarization of cancer-associated macrophages (TAMs) into M2 macrophages.

10. In paragraph 6, A composition for inhibiting cancer metastasis, wherein the cancer is at least one selected from the group consisting of colorectal cancer, non-small cell lung cancer, prostate cancer, gastric cancer, and squamous cell carcinoma, which are cancers with high EREG expression.

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