Modified T cells for adoptive immunotherapy

Abstract

The present invention relates to a population of T cells having reduced expression of SIGLEC15, wherein the T cells are derived from a sentinel lymph node in a subject with cancer. The present invention also relates to methods for obtaining such T cells and their use in therapy, and to pharmaceutical compositions comprising such T cells.

Description

Technical Field

[0001] The present invention relates to the field of therapeutic treatment. In particular, the present invention relates to cancer treatment based on the administration of autologous cells to patients who need it, cells useful in such treatment, and methods for preparing such cells.

Background Art

[0002] Adoptive immunotherapy using autologous lymphocytes isolated from sentinel lymph nodes in the tumor inflow region and amplified in vitro is known in the art (1). Also, adoptive immunotherapy including the collection and amplification of autologous tumor-reactive lymphocytes and reinfusion into patients has been explored in melanoma (2).

[0003] WO2018 / 234516 teaches a method for amplifying anti-tumor T cells together with phagocytosable particles having one or more tumor neoantigen constructs firmly associated therewith.

[0004] Adoptive T cell transfer therapy, in which autologous or allogeneic T cells are injected into a patient having cancer, has recently been shown to be quite promising (3).

[0005] [[ID=二十四]]Siglec is a vertebrate cell surface receptor that recognizes sialic acid-added glycans. SIGLEC15 is a type I transmembrane protein consisting of (i) two immunoglobulin (Ig)-like domains, (ii) a transmembrane domain containing a lysine residue, and (iii) a short cytoplasmic tail. SIGLEC15 is expressed on macrophages and / or dendritic cells in human pancreas and lymph nodes (4). SIGLEC15 messenger RNA expression is minimal in most normal human tissues and various immune cell subsets, but can be found in macrophages and most abundantly in M2 macrophages (4). SIGLEC15 can not only regulate osteoclast differentiation but also suppress T cell responses (5).

[0006] US2019 / 202912 suggests the use of antibodies that bind to SIGLEC15 in cancer treatment. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Surprisingly, the inventors have found that downregulation of SIGLEC15 in T cells derived from sentinel lymph nodes can mitigate the immune compromise caused by cancer-derived immunosuppressive factors, and thus reduce the tumor's ability to invade and metastasize. [Means for solving the problem]

[0008] Therefore, in a first aspect, the present invention is a method for obtaining a population of T cells in which SIGLEC15 expression is reduced, S1. A step of identifying the lymph nodes into which lymphatic fluid from cancerous tumors flows in the subject; S2. The process of extracting cells from the identified lymph nodes; S3. A step to reduce the expression of SIGLEC15 in extracted cells; S4. A step of amplifying extracted cells in which SIGLEC15 expression is reduced under conditions that support T cell amplification. Regarding methods including

[0009] In a second aspect, the present invention relates to a method for obtaining a population of T cells in which SIGLEC15 expression is reduced, S1'. A step of providing T cell-containing lymph node tissue removed from a subject, wherein the lymph node tissue is obtained from a lymph node identified in the subject as a lymph node into which lymphatic fluid from a cancerous tumor flows; S2. The process of extracting cells from the identified lymph nodes; S3. A step to reduce the expression of SIGLEC15 in extracted cells; S4. A step of amplifying extracted cells in which SIGLEC15 expression is reduced under conditions that support T cell amplification. Regarding methods including

[0010] In some embodiments, SIGLEC15 expression is reduced by downregulation of the expression of the gene encoding SIGLEC15, mediated by siRNA transfection, shRNA transfection, or CRISPR-mediated gene editing.

[0011] In some embodiments, SIGLEC15 expression is reduced by downregulation of the expression of the gene encoding SIGLEC15 by siRNA transfection using an siRNA molecule having the nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0012] In some embodiments, conditions supporting T cell amplification include maintaining cells in the presence of interleukin-2.

[0013] In a further embodiment, the present invention relates to a population of T cells in which SIGLEC15 expression is reduced, which can be obtained by the method described in the present invention.

[0014] In a further embodiment, the present invention relates to a population of T cells described in the present invention for use in pharmaceuticals.

[0015] In a further embodiment, the present invention relates to a population of T cells described in the present invention for use in a method for treating cancer.

[0016] In some embodiments, cancer is a solid tumor.

[0017] In some embodiments, cancer is a cancerous tumor of a target originating from T cells, or a metastasis thereof.

[0018] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, melanoma, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC).

[0019] In a further aspect, the present invention is a method for treating a cancerous tumor in a subject, comprising: S1. identifying a lymph node into which lymph fluid from the cancerous tumor in the subject flows; S2. extracting cells from the identified lymph node; S3. reducing the expression of SIGLEC15 in the extracted cells; S4. amplifying the extracted cells in which the expression of SIGLEC15 is reduced under conditions that support T cell amplification; S5. administering the amplified T cells in which the expression of SIGLEC15 is reduced. The present invention relates to a method comprising the above steps.

[0020] In some embodiments, the expression of SIGLEC15 is reduced by down-regulation of the gene encoding SIGLEC15 by siRNA transfection, shRNA transfection, or CRISPR-mediated gene editing.

[0021] In some embodiments, the expression of SIGLEC15 is reduced by down-regulation of the gene encoding SIGLEC15 by siRNA transfection using an siRNA molecule having the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0022] In some embodiments, the cancer is a solid tumor.

[0023] In some embodiments, the cancer is a cancerous tumor of the subject from which the T cells are derived, or a metastasis thereof.

[0024] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, malignant melanoma, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC).

[0025] In a further embodiment, the present invention relates to the use of a population of T cells according to the present invention in the preparation of a pharmaceutical composition for use in a treatment method according to the present invention.

[0026] In a further embodiment, the present invention relates to a pharmaceutical composition comprising a population of T cells described in the present invention, and optionally pharmaceutically acceptable excipients and / or carriers. [Brief explanation of the drawing]

[0027] [Figure 1] This figure shows a flowchart illustrating a method for obtaining a population of T cells as described in the present invention. [Figure 2] This figure shows a flowchart illustrating the treatment method described in the present invention. [Figure 3A] This figure shows the relative expression of SIGLEC15 in sentinel lymph nodes and non-sentinel lymph nodes, respectively. [Figure 3B] This figure shows the relative expression of SIGLEC15 in sentinel lymph nodes before and after siRNA knockdown. [Figure 4A] This figure shows SIGLEC15 protein expression in various cell types. It compares sentinel node (SN) and non-sentinel node (NSN) expression across cell types. [Figure 4B] This figure shows the protein expression of SIGLEC15 in various cell types. It provides a comparison of sentinel node (SN) expression across different cell types. [Figure 5] This figure shows the release of functional cytokines from T cells after SIGLEC15 knockdown. [Modes for carrying out the invention]

[0028] Lymph nodes that receive influx from the primary tumor are crucial for initiating an effective anti-tumor T-cell immune response. However, cancer-derived immunosuppressive factors impair sentinel lymph node (SN) immunity, allowing tumors to invade and metastasize.

[0029] The inventors have studied the various mechanisms underlying this immune evasion in order to devise therapeutic interventions that can halt tumor spread in the early clinical stages. Accordingly, this invention utilizes an understanding of the microenvironmental regulation of transcriptional levels in the lymph nodes of cancer patients, such as those with colorectal cancer.

[0030] Several cytokines restrict tumor cell proliferation either through direct antiproliferative or pro-apoptotic activity, or indirectly by stimulating the cytotoxic activity of immune cells against tumor cells. Interleukin-2 (IL-2)2 is considered a key cytokine in promoting the amplification of natural killer (NK) cells and T lymphocytes. NK cells and T cells are the primary lymphocyte subsets responsible for tumor death. Tumor necrosis factor-alpha (TNF-α) is primarily considered a mediator of the anti-tumor immune response. Interferon-gamma (IFN-γ) is a multifaceted molecule with associated antiproliferative, pro-apoptotic, and antitumor mechanisms. IFN-γ release is, for example, an important potency indicator in the quality assessment of tisagenlecleucel, a CAR-T cell product (6).

[0031] T cells isolated from tumor-inflow lymph nodes and treated to reduce SIGLEC15 expression have been found to exhibit improved antitumor efficacy, as assessed by the effector cytokine release profiles described above. As disclosed in the Experiments section below, T cells with reduced SIGLEC15 expression show increased release of cytokines IL-2, TNF-α, and IFN-γ.

[0032] The present invention relates, in part, to the use of autologous T cells treated to reduce SIGLEC15 expression in cancer treatment, and to methods of corresponding treatment. The present invention also relates to methods for preparing and obtaining such T cells, T cells obtained or obtainable by such methods, and pharmaceutical compositions comprising such T cells.

[0033] Therefore, in a first aspect, the present invention is a method for obtaining a population of T cells in which SIGLEC15 expression is reduced, S1. A step of identifying the lymph nodes into which lymphatic fluid from cancerous tumors flows in the subject; S2. The process of extracting cells from the identified lymph nodes; S3. A step to reduce the expression of SIGLEC15 in extracted cells; S4. A step of amplifying extracted cells in which SIGLEC15 expression is reduced under conditions that support T cell amplification. Regarding methods including

[0034] In a further embodiment, the present invention relates to a method for obtaining a population of T cells in which SIGLEC15 expression is reduced, which does not involve any surgical procedure performed on the body of a human or animal. S1'. A step of providing T cell-containing lymph node tissue removed from a subject, wherein the lymph node tissue is obtained from a lymph node identified in the subject as a lymph node into which lymphatic fluid from a cancerous tumor flows; S2. The process of extracting cells from the identified lymph nodes; S3. A step to reduce the expression of SIGLEC15 in extracted cells; S4. A step of amplifying extracted cells in which SIGLEC15 expression is reduced under conditions that support T cell amplification. This includes methods.

[0035] The above method is schematically shown in Figure 1.

[0036] Identification of lymph nodes as those into which lymphatic fluid from a cancerous tumor flows can be carried out as is well known in the art, for example, as described by Dahl and his collaborators (7). Briefly, a detectable and physiologically acceptable dye is injected into or around the tumor. The lymphatic fluid carries the dye from the injection site to the aspiration lymph nodes, so that the aspiration lymph nodes are stained with the dye and identified as aspiration lymph nodes. Exemplary dyes include patent blue, Evans blue, and Alexa Fluor® 488 dye.

[0037] Cell extraction from lymph nodes identified as inflow lymph nodes can be performed by various methods. The entire lymph node may be removed from the patient by excision. Alternatively, it may be possible to obtain only a single piece of lymph node tissue, for example, through biopsy. The extracted cells from the lymph node tissue may be made into a single-cell suspension, as is well known in the art (8).

[0038] Subsequently, SIGLEC15 expression is reduced in the extracted cells.

[0039] In some embodiments, SIGLEC15 expression is reduced by downregulation of the expression of the gene encoding SIGLEC15 via siRNA transfection. Briefly, siRNA transfection can be performed on single lymph node cells. 1 μmol / L of siRNA may be transfected for 72 hours in a 96-well tissue culture plate containing Accell siRNA delivery medium (GE Dharmacon) according to the manufacturer's instructions. Typically, RT-PCR is used to evaluate SIGLEC15 mRNA expression in the siRNA-transfected and untransfected groups. Other protocols are well known in the art (9) and will not be described in detail here. Furthermore, protocols and reagents for downregulating the expression of specific genes with siRNA are commercially available from several companies, including ThermoFisher Scientific, Sigma-Aldrich, Qiagen, and GE Dharmacon.

[0040] In some embodiments, SIGLEC15 expression is reduced by downregulation of the expression of the gene encoding SIGLEC15 by short hairpin RNA (shRNA) transfection. Such protocols are well known in the art (10) and will not be described in detail here. Briefly, a pGPU6 vector containing SIGLEC15 shRNA may be transfected into lymph node single cells obtained as described above, using a transfection kit, for example, a kit from Shanghai GenePharma Company (Shanghai, China), according to the manufacturer's instructions for use. After 48 hours of incubation, knockdown of SIGLEC15 expression is confirmed by RT-PCR. Other protocols and reagents for downregulating the expression of specific genes with shRNA are also commercially available from several companies, including ThermoFisher Scientific and Sigma-Aldrich, without prejudice.

[0041] In some embodiments, SIGLEC15 expression is reduced by downregulation of the expression of the gene encoding SIGLEC15 via CRISPR-mediated gene editing. The CRISPR / Cas9 vector is constructed to target selected specific sites and regions within the SIGLEC15 gene. sgRNA is designed using CRISPRdirect (http: / / crispr.dbcls.jp / ). sgRNA oligomers are synthesized and cloned into the pU6gRNACas9EGFP vector. Lymph node single cells are initially seeded in 6-well plates and then transfected using lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's instructions. After incubating the cells for a further 48 hours, knockout of SIGLEC15 expression was confirmed by RT-PCR and flow cytometry.

[0042] After reducing SIGLEC15 expression, the extracted cells are cultured ex vivo under conditions that support T cell amplification. Such protocols and reagents are publicly known and, in particular, available under the trade names Gibco® CTS® OpTmizer®, CTS AIM V medium and CTS Immune Cell SR (ThermoFisher Scientific), and as disclosed in Technical Bulletin 27143 from Stem Cell Technologies Inc. (Cambridge, MA, USA). Protocols for T cell amplification are also disclosed in WO2018234516 and Chinese Patent Application No. 108220234.

[0043] Generally, a suspension of single cells with reduced SIGLEC15 expression is resuspended in serum-free cell culture medium in the presence of interleukin-2 and / or other cytokines, and then transferred to a growth vessel such as a flask or plate. The cells are then amplified at 37°C in a 5% CO2-rich atmosphere and re-stimulated with tumor antigens along with antigen-presenting cells during cell culture.

[0044] The following protocol (8) is provided as one possible protocol: A single cell suspension obtained from SLN is placed in X-VIVO® 15 serum-free cell culture medium (LONZA) containing 4 × 10 cells. 6 Resuspend the cells at a density of cells / ml in the presence of 1000 IU / ml recombinant human interleukin-2 (Shuanglu, China). Seed these cells in a flask or plate and maintain at 37°C in a humidified atmosphere containing 5% CO2. Add autologous tumor lysate to the initial culture at a dilution of (1) 1 / 100 (v / v) as previously described. Restimulation is performed to induce highly tumor-specific SLN-T cells by adding autologous tumor lysate together with irradiated autologous PBMCs during SLN-T cell culture. One week before transfusion, remove 5 ml of culture medium for bacterial and fungal contamination testing using BACTEC 9120 (Becton-Dickinson) and measure endotoxin levels based on the Limulus reaction. On the day of transfusion, repeat these assays to detect any bacterial, fungal, or endotoxin contamination. Analyze the lymphocyte subset of SLN-T cells. Furthermore, analyze 1 × 10⁶ cells. 6 The samples were used for flow cytometry analysis of the tumor surface marker epithelial cell adhesion molecule (EpCAM) to rule out the presence of tumor cells.

[0045] In a further embodiment, the present invention relates to a population of T cells in which SIGLEC15 expression is reduced, which can be obtained or obtained by the method described in the above embodiment.

[0046] In a further embodiment, the present invention relates to a population of T cells having reduced expression of SIGLEC15, which can be obtained or obtained by the method of the present invention for pharmaceutical use.

[0047] In one embodiment of this aspect, the present invention relates to a population of T cells having reduced expression of SIGLEC15, which can be obtained or obtained by the method described in the above embodiment for use in the treatment of cancer, for example, the treatment of solid tumors.

[0048] In one embodiment, the present invention relates to the use of autologous T cells in a method for treating cancerous tumors. In this embodiment, a population of T cells obtained or obtainable by the method described above can be used in the treatment of cancerous tumors located in close proximity to lymph nodes identified as tumor inflow lymph nodes according to the method described above in a subject from which tumor inflow lymph nodes were obtained.

[0049] Cancerous tumors can be any type of solid tumor. The solid tumors described in this invention are abnormal masses of tissue that occur in organs. Solid tumors typically do not contain cysts or fluid areas. Solid tumors can be malignant. Different types of solid tumors are named based on the type of cells that form them. Types of solid tumors include sarcomas, carcinomas, and lymphomas. This invention provides compositions comprising T cells obtained or obtainable by the method of this invention.

[0050] Examples of solid tumors include adrenal cancer, anal cancer, anaplastic large cell lymphoma, angioimmunoblastic T-cell lymphoma, B-cell lymphoma, cholangiocarcinoma, bladder cancer, brain / CNS tumors, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, Ewing's tumor, eye cancer, gallbladder cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, hepatosplenic T-cell lymphoma, Hodgkin lymphoma, intravascular large B-cell lymphoma, renal cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer (non-small cell and small cell), pulmonary carcinoid, lymphomatous granulomatosis, and malignant cancer. Examples include uterine mesothelioma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, nodular marginal zone B-cell lymphoma, non-Hodgkin lymphoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, primary humoral lymphoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (basal cells and squamous cells, melanoma and Merkel cells), small intestine cancer, gastric cancer, testicular cancer, thymic cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor. The T cells of the present invention are particularly effective in the treatment of solid tumors. Therefore, subjects treated by the therapeutic method of the present invention may have solid tumors. The T cells of the present invention are particularly effective in the treatment of solid cancers selected from the group consisting of anal cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, liver cancer, lung cancer (non-small cell and small cell), pulmonary carcinoid, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, gastric cancer, testicular cancer, uterine sarcoma, vaginal cancer, and vulvar cancer, and more particularly in the treatment of breast cancer, colon cancer, liver cancer, lung cancer (non-small cell and small cell), pulmonary carcinoid, pancreatic cancer, prostate cancer, ovarian cancer, and bladder cancer.

[0051] In one embodiment, the cancer is selected from the group consisting of colorectal cancer, malignant melanoma, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC).

[0052] In one embodiment, the present invention relates to a method for treating cancerous tumors in a subject, S1. A step of identifying the lymph nodes into which lymphatic fluid from a cancerous tumor flows in the subject; S2. The process of extracting cells from the identified lymph nodes; S3. A step to reduce the expression of SIGLEC15 in extracted cells; S4. A step of amplifying extracted cells in which SIGLEC15 expression is reduced under conditions that support T cell amplification; S5. A step of administering amplified T cells in which SIGLEC15 expression is reduced. Regarding methods including

[0053] Process S1 can be replaced with process S1' as described above.

[0054] The treatment method described in the present invention is schematically shown in Figure 2.

[0055] Processes S1, S2, S3, and S4 may be carried out as described above.

[0056] The amplified T cells may be administered by intravenous administration as is known in the art (11). The administration may also be intra-arterial, intrathecal, or intraperitoneal.

[0057] The following protocol (8) is provided as one useful protocol for T cell administration. The final SLN-T cells are collected, washed twice in saline, and transferred to a sterile plastic bag containing 200 ml of saline and 1% human serum albumin (CSL Behring GmbH, Germany). The cells are transfused intravenously at 60-minute intervals according to hospital transfusion guidelines. After cell transfusion, transfusion-related toxicity is assessed using the Common Terminology Criteria for Adverse Events (CTCAE) 3.0 criteria.

[0058] Furthermore, the present invention relates to the use of a population of T cells obtained or obtainable by the method described above in the manufacture of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a pharmaceutical composition for use in the treatment of cancer, for example, in the treatment of solid tumors.

[0059] The present invention also relates to a pharmaceutical composition comprising a population of T cells obtained or obtainable according to the method described above. In one embodiment, the pharmaceutical composition is a pharmaceutical composition for use in the pharmaceutical described above, for example, in the treatment of cancer, for example, in the treatment of solid tumors.

[0060] The pharmaceutical composition may contain pharmaceutically acceptable excipients that are common in the art. The pharmaceutical composition is preferably formulated as a liquid suitable for injection, for example, intravenous, intra-arterial, intrathecal, or intraperitoneal administration.

[0061] A “pharmaceutical excipient” or “pharmaceutically acceptable excipient” is a carrier, usually a liquid, in which an active therapeutic agent is formulated. In one embodiment of the present invention, the active therapeutic agent is a population of T cells obtained or obtainable by the method of the present invention. Excipients generally do not provide any pharmacological activity to the formulation, but they may provide chemical and / or biological stability. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition, edited by Grennaro, A., 1995, incorporated by reference.

[0062] Where used herein, “pharmaceutically acceptable carrier” or “excipient” includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration. pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions for the immediate preparation of sterile injection solutions or dispersions. The use of such media and agents for pharmaceutically effective substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is intended. Furthermore, auxiliary active compounds may be incorporated into the composition.

[0063] In carrying out the present invention, a person skilled in the art may use methods and protocols known in the art, which non-exclusively include the methods and protocols disclosed in the patent documents and scientific papers referenced herein and the references in such documents, all of which are incorporated herein by reference. [Examples]

[0064] experiment Gene expression analysis Sentinel and non-sentinel lymph nodes were identified as previously described (7). Briefly, a lymphotropic dye (patent blue, Sigma-Aldrich) was injected subserosaally around the primary tumor. Lymph nodes in the tumor inflow area, i.e., sentinel nodes, were stained blue. Lymph nodes that were not stained were considered to be non-sentinel nodes.

[0065] Two lymph nodes, one non-metastatic sentinel lymph node (SN) and one non-sentinel lymph node (NSN), were obtained from each of 23 patients diagnosed with colorectal cancer and used to obtain gene expression profiles using high-throughput RNA sequencing and bioinformatics analysis.

[0066] Gene expression data was obtained by sequencing lymph node tissue using Ilumina's RNA-SEQ technology.

[0067] A total of 16 genes, including 9 upregulated and 7 downregulated genes, were differentially expressed in sentinel lymph nodes compared to non-sentinel lymph nodes. IL1RL1, STON2, TPSAB1, GATA2, DNAJB4, NR2F1, and DIPK2A were found to be downregulated, while PLA2G2D, ZBED6CL, SIGLEC15, KCNC3, ATP2A1, MMP2-AS1, FBXO41, DSC2, and TFEC were found to be upregulated.

[0068] Confirmation by RT-PCR Total RNA was extracted from SN and NSN pairs from 8 patients randomly selected from over 23 patients, using TRIzol reagent according to the manufacturer's instructions. cDNA synthesis was performed using the PrimeScript® RT reagent kit and gDNA Eraser (Takara, Japan). Primers for SIGLEC15 and the endogenous reference gene β-actin were designed using Primer5 software. RT-qPCR was performed using TB Green® Premix Ex Taq® II (Takara, Japan) according to the manufacturer's instructions. After PCR, melting curve analysis was performed to confirm primer specificity, and the relative level of SIGLEC15 was calculated using the 2-ΔΔCt method (12). The relative expression of SIGLEC15 in sentinel lymph nodes before and after siRNA knockdown was analyzed in three sentinel lymph nodes. The results are shown in Tables 1 and 2, and Figure 3.

[0069] [Table 1]

[0070] [Table 2]

[0071] RNAscope in situ hybridization To further explore the expression pattern of SIGLEC15 in lymph nodes, we investigated the expression of SIGLEC15 and CD163 in SN and NSN pairs from four patients using RNAscope in situ hybridization technology (13), which can provide information about the spatial expression of RNA in tissue cells. The results are disclosed in Table 3, showing that mRNA expression was higher in sentinel lymph nodes (p=0.042, Student's t-test).

[0072] [Table 3]

[0073] Based on the double staining results for SIGLEC15 and CD163 in sentinel and non-sentinel lymph node slices, we can further confirm that SIGLEC15 is highly expressed in SNs compared to NSNs. In addition, SIGLEC15 was expressed not only in M2 macrophages but also in other cell types.

[0074] These results further confirm that SIGLEC15 is highly expressed in SNs compared to NSNs, and that SIGLEC15 is expressed not only in M2 macrophages but also in other cells.

[0075] Flow cytometry analysis We detected SIGLEC15 protein expression on the surface of various cell subsets using flow cytometry.

[0076] Single-cell suspensions from SN or NSN and tumor tissue were obtained immediately postoperatively by applying gentle pressure using a loose-fit glass homogenizer.

[0077] For SIGLEC15 analysis of cells from SN or NSN cells, fluorescently labeled monoclonal antibodies (mAbs) against CD45, CD86, CD11c, CD163, CD11b, CD15, CD14, CD3, CD4, CD8, CD16, CD56, CD19, and SIGLEC15 (Biolegend) were used. Cells were incubated in the presence of mAbs at room temperature (18-25°C) for 20 minutes, protected from light, according to the manufacturer's recommendations. After incubation, the cell suspension was washed with phosphate-buffered saline (PBS), and the cell pellet was resuspended in 0.5 ml of PBS for analysis. Samples were further analyzed using a Navios flow cytometer (Beckman Coulter). At least 50,000 events in total were collected and analyzed using Flowjo software (Flowjo LLC).

[0078] SIGLEC15 was expressed at relatively high levels in almost all subgroups of sentinel lymph nodes (Figure 4A). In sentinel lymph nodes, SIGLEC15 was expressed at relatively high levels in M2 macrophages (Figure 4B), and the same trend was observed in non-sentinel lymph nodes (data not shown).

[0079] SIGLEC15 Functional Analysis To evaluate the role of SIGLEC15 in sentinel lymph nodes, we knocked down SIGLEC15 expression in sentinel lymph nodes via siRNA technology. Briefly, transfection of single lymph node cells with siRNA was performed according to the manufacturer's instructions for use (GE Dharmacon).

[0080] To confirm the knockout effect, we synthesized a total of three siRNA sequences. The siRNAs were designed according to the RNA sequence of the target gene (SIGLEC15) and according to siRNA design principles using siRNA design software (Invivogen, https: / / www.invivogen.com / sirnawizard / guidelines.php).

[0081] The following siRNA sequence was designed (including a complementary strand and a 3'-TT overhang to prevent degradation by 3'-exonuclease).

[0082] [Table 4]

[0083] 1 μmol / L siRNA was transfected into 96-well tissue culture plates containing Accell siRNA delivery medium for 72 hours. SIGLEC15 mRNA expression was evaluated in the siRNA-transfected and untransfected groups using RT-PCR.

[0084] Flow cytometry was used to detect the mean fluorescence intensity (MFI) of SIGLEC15 protein on the surface of all living cells in lymph nodes to evaluate the effect of siRNA interference on reducing SIGLEC15 protein expression. The results are shown in Table 5.

[0085] [Table 5]

[0086] Flow cytometry data showed that antitumor functional cytokines released by T cells, such as IL-2, TNFα, and IFNγ, were upregulated in cells from sentinel lymph nodes after SIGLEC15 knockdown (Table 6 and Figure 5).

[0087] [Table 6]

[0088] Following SIGLEC15 blockade, functional T cell cytokines increase, which is recognized as an improvement in the antitumor effect of T cells.

[0089] IL2 was primarily secreted by Th1 T cells. IL2 is a marker for activated Th cells, cytotoxic T cells, and NK cells, and it is also an essential element for the proliferation of activated T cells.

[0090] TNFα is primarily produced by activated T lymphocytes and natural killer (NK) cells. It induces hemorrhagic necrosis in certain sets of tumor types and is used in the local treatment of locally advanced soft tissue sarcomas and metastatic melanomas. TNFα also triggers an inflammatory response, which induces more immune cells to kill tumor cells.

[0091] IFNγ plays a crucial role in activating cellular immunity and subsequently stimulating anti-tumor immune responses. It acts as a cytotoxic cytokine in conjunction with granzyme B and perforin to initiate apoptosis in tumor cells, but also enables the synthesis of immune checkpoint inhibitory molecules and indoleamine-2,3-dioxygenase (IDO), thus stimulating other immunosuppressive mechanisms.

[0092] As a result, the cytokine release profile indicates the antitumor effect of amplified T cells with reduced SIGLEC15 expression.

[0093] References 1. Pilot Study of Sentinel-Node-Based Adoptive Immunotherapy in Advanced Colorectal Cancer. Karlsson, M., et al. 2010, Ann Surg Oncol, Vol. 17, pp. 1747-1757. 2. Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients. Dudley, M.E., et al.,. 4, 2003, Journal of Immunotherapy, Vol. 26, pp. 332-342. 3. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Waldman, A.D., et al. 2020, Nature Reviews Immunology, Vol. 20, pp. 651-668. 4. SIGLEC15: an immune system Siglec conserved throughout vertebrate evolution. Angata, T., et al. 8, 2007, Glycobiology, Vol. 17, pp. 838-846. 5. SIGLEC15 as an immune-suppressor and potential target for normalization cancer immunotherapy. Wang, J. et al. 4, 2019, Nature Medicine, Vol. 25, pp. 656-666. 6. Optimizing CAR-T Cell Manufacturing Process during Pivotal Clinical Trials. Tyagarajan, S., et al. March 2020, Molecular Therapy: Methods & Clinical Development, Vol. 16, pp. 136-144. 7. Identification of sentinel nodes in patients with colon cancer. Dahl, K., et al. 4, May 2005, Eur J Surg Oncol, Vol. 31, pp. 381-385. 8. Phase I / II study of adjuvant immunotherapy with sentinel lymph node T lymphocytes in patients with colorectal cancer. Zhen, Y-H, et al. s.l. : Springer, 20 May 2015, Cancer Immunol Immunother. 9. RNA Interference to Knock Down Gene Expression. Han, H. 2018, Methods Mol Biol, Vol. 1706, pp. 293-302. 10. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment. Moore, C.B., et al. 2010, Methods Mol Biol. , Vol. 629, pp. 141-158. 11. Tumor-infiltrating lymphocytes for the treatment of metastatic cancer. Foppen, M.H.G. et al.,. 2015, M O L E C U L A R ON C O L O G Y, Vol. 9, pp. 1918-1935. 12. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCT Method. Livak, K.J., et al. 4, December 2001, Methods, Vol. 25, pp. 402-408. 13. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, Paraffin-embedded tissues. Wang, F., et al. 1, 2012, The Journal of Molecular Diagnostics, Vol. 14, pp. 22-29.

Claims

1. A method for obtaining a population of T cells in which SIGLEC15 expression is reduced, S1. A step of providing T cell-containing lymph node tissue that has been previously removed from a subject, wherein the lymph node tissue is obtained from a lymph node that has been identified in the subject as a lymph node into which lymphatic fluid from a cancerous tumor flows; S2. The step of extracting cells from the provided lymph nodes; S3. A step of reducing the expression of SIGLEC15 in the extracted cells; S4. A step of amplifying the extracted cells in which the expression of SIGLEC15 is reduced under conditions that support T cell amplification. A method including, A method wherein the expression of SIGLEC15 is reduced compared to the expression of SIGLEC15 in the extracted cells before downregulation, by downregulation of the expression of the gene encoding SIGLEC15 by siRNA transfection, shRNA transfection, or CRISPR-mediated gene editing.

2. The method according to claim 1, wherein the expression of SIGLEC15 is reduced compared to the expression of SIGLEC15 in the extracted cells before downregulation by downregulation of the expression of the gene encoding SIGLEC15 by siRNA transfection using a siRNA molecule having the nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

3. The method according to claim 1 or 2, wherein the conditions supporting T cell amplification include maintaining the cells in the presence of interleukin-2.

4. A population of T cells having reduced expression of SIGLEC15, obtained by the method according to any one of claims 1 to 3, for use in pharmaceuticals.

5. A population of T cells having reduced expression of SIGLEC15, obtained by the method according to any one of claims 1 to 3, for use in a method for treating cancer.

6. The population of T cells for use according to claim 5, wherein the cancer is a solid tumor.

7. The population of T cells for use according to claim 5 or 6, wherein the cancer is the target cancerous tumor or its metastasis from which the T cells originate.

8. A population of T cells for use according to any one of claims 5 to 7, wherein the cancer is selected from the group consisting of colorectal cancer, malignant melanoma, cervical cancer, head and neck squamous cell carcinoma (HNSCC), and non-small cell lung cancer (NSCLC).

9. A pharmaceutical composition comprising a population of T cells having reduced expression of SIGLEC15, obtained by the method according to any one of claims 1 to 3, and optionally a pharmaceutically acceptable excipient and / or carrier.

10. A population of T cells in which SIGLEC15 expression is reduced, administered to subjects for the treatment of cancerous tumors, The aforementioned population of T cells, S1. A step of identifying the lymph nodes into which lymphatic fluid from the cancerous tumor flows in the subject; S2. The step of extracting cells from the identified lymph nodes; S3. A step of reducing the expression of SIGLEC15 in the extracted cells; S4. A step of amplifying the extracted cells in which the expression of SIGLEC15 is reduced under conditions that support T cell amplification, Obtained by a method including, A group of T cells, A population of T cells in which the expression of SIGLEC15 is reduced compared to the expression of SIGLEC15 in the extracted cells before downregulation, by downregulation of the expression of the gene encoding SIGLEC15 by siRNA transfection, shRNA transfection, or CRISPR-mediated gene editing.

11. The population of T cells according to claim 10, wherein the expression of SIGLEC15 is reduced compared to the expression of SIGLEC15 in the extracted cells before downregulation by downregulation of the expression of the gene encoding SIGLEC15 by siRNA transfection using a siRNA molecule having the nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

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