Antisense oligonucleotide capable of suppressing or inhibiting expression of PD-1 protein or CTLA-4 protein

Abstract

The present invention provides a means or the like capable of temporarily inhibiting expression of PD-1 or CTLA-4 protein. The present invention relates to an antisense oligonucleotide or the like capable of hybridizing with a nucleic acid sequence of an mRNA precursor and / or mature mRNA of Programmed Cell Death 1 (PD-1) protein or Cytotoxic T-lymphocyte Antigen-4 (CTLA-4) protein and suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein.

Description

Antisense oligonucleotides that can suppress or inhibit the expression of PD-1 protein or CTLA-4 protein.

[0001] The present invention relates to an antisense oligonucleotide, etc., that can suppress or inhibit the expression of PD-1 protein or CTLA-4 protein.

[0002] The programmed cell death 1 (PD-1) protein is a type I transmembrane protein that is preferentially expressed in immune cells such as T, B, and NK cells. The ligand, programmed cell death 1 ligand 1 (PD-L1), is known as a member of the B7 family of co-stimulatory / co-inhibitory molecules for antigen presentation, which is expressed by a wide range of cell types including cancer cells. As a result of the interaction between the receptor PD-1 and PD-L1, it strongly interferes with T cell receptor (TCR) signaling through an intracellular molecular mechanism. The protein structure of PD-1 consists of an extracellular immunoglobulin-like binding domain, a transmembrane region, and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Interference with PD-1 signaling by immune checkpoint inhibitors enhances T cell function by enhancing signaling from the TCR signalosome. PD-1 plays a particularly important role in suppressing T cell responses. After activation of immune cells, the expression of PD-1 is induced on the surface of T cells. In the framework of antigen-specific T cell responses, various activating factors are phosphorylated by binding to the T cell receptor. However, as a result of the interaction between PD-1 and PD-L1, this phosphorylation is canceled, the secretion of cytokines decreases, and cell division is suppressed, resulting in a decrease in the expression of lymphocyte growth factors. This mechanism is thought to potentially lead to a decrease in treatment efficiency due to lymphocyte cell exhaustion. In recent years, T cell therapy has proven to be a promising treatment option for patients with various diseases, especially in the form of chimeric antigen receptor transgenic T cells (CAR-T) for the treatment of cancer patients (Non-Patent Documents 1 and 2). However, there is a possibility that cancer cells cannot be normally eliminated because the activation of T cells is not sufficiently efficient or the persistence in the patient's body decreases. As an alternative method, permanent removal of PDCD1 (for example, via CRISPR / CAS) is considered. However, permanent knockout by genome editing of PDCD1 in therapeutic T cells bears a high risk such as the development of cell tumors associated with genome invasion. Therefore, methods to avoid such risks are being studied and demanded.

[0003] Furthermore, CTLA-4 (Cytotoxic T-lymphocyte Antigen-4) protein, like PD-1 protein, is known to be one of the immune checkpoint molecules that play an important role in the immune system. It is mainly involved in regulating T cell activity and has an inhibitory function in controlling the immune response. Therefore, the same challenges and risks as those described above for PD-1 may arise. (Prior Art References)

[0004] Lei, W. , Zhao, A. , Liu, H. , Yang, C. , Wei, C. , Guo, S. , Chen, Z. , Guo, Q. , Li, L. , Zhao, M. , Wu, G. , Ouyang, G. , Liu, M. , Zhang, J. , Gao, J. , & Qian, W. (2024). Safety and feasibility of anti-CD19 CAR T cells expressing inducible IL-7 and CCL19 in patients with collapsed or refractory large B-cell lymphoma. Cell discovery, 10(1), 5. https: / / doi. org / 10.1038 / s41421-023-00625-0Wang, Z. , Li, N. , Feng, K. , Chen, M. , Zhang, Y. , Liu, Y. , Yang, Q. , Nie, J. , Tang, N. , Zhang, X. , Cheng, C. , Shen, L. , He, J. , Ye, X. , Cao,W. , Wang, H. , & Han, W. (2021). Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors. Cellular & molecular immunology, 18(9), 2188-2198. https: / / doi. org / 10.1038 / s41423-021-00749-x

[0005] Under these circumstances, there was a need for the development of a means to temporarily inhibit the expression of PD-1 protein and other proteins in order to avoid the aforementioned risks.

[0006] The present invention has been made in consideration of the above circumstances and provides the following antisense oligonucleotides, pharmaceutical compositions, immune cells, and methods for treating or preventing malignant or benign tumors and / or infectious diseases and / or kidney diseases.

[0007] (1) An antisense oligonucleotide that can hybridize with the nucleic acid sequence of the mRNA precursor and / or mature mRNA of the PD-1 (Programmed Cell Death 1) protein and suppress or inhibit the expression of the PD-1 protein. (2) The antisense oligonucleotide according to (1) above, which can inhibit the expression of the PD-1 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. (3) The antisense oligonucleotide according to (1) above, wherein the mRNA precursor of the PD-1 protein consists of a nucleic acid sequence comprising the base sequence of Sequence ID No. 17, and the antisense oligonucleotide can hybridize with the base sequences from the 141st to the 2131st, the 4929th to the 6078th, or a portion of the 6092nd to the 6584th of the nucleic acid sequence.

[0008] (4) The antisense oligonucleotide according to (1) above, which can induce exon 2 skipping in the mRNA precursor of the PD-1 protein. (5) An antisense oligonucleotide that can hybridize with the nucleic acid sequence of the mRNA precursor and / or mature mRNA of the CTLA-4 (Cytotoxic T-lymphocyte Antigen-4) protein and suppress or inhibit the expression of the CTLA-4 protein. (6) The antisense oligonucleotide according to (5) above, which can inhibit the expression of the CTLA-4 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

[0009] (7) The antisense oligonucleotide according to (1) or (5) above, wherein the antisense oligonucleotide consists of 10 to 25 nucleotides. (8) The antisense oligonucleotide according to (1) or (5) above, wherein the antisense oligonucleotide includes a modified nucleotide. (9) The antisense oligonucleotide according to (8) above, wherein the modified nucleotide is an LNA (Locked Nucleic Acid) modified nucleotide, a cET (Constrained Ethyl) modified nucleotide, an ENA modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-O-methyl modified nucleotide, or a combination thereof.

[0010] (10) The antisense oligonucleotide according to (8) above, wherein the modified nucleotide is included in the 5' and / or 3' end of the antisense oligonucleotide. (11) The antisense oligonucleotide according to (1) above, comprising any of the base sequences of SEQ ID NOs: 1 to 13 and 21, or a base sequence having at least 80%, 90%, 95%, 98%, or 99% identity with said base sequence. Furthermore, the following are examples of guide RNAs (gRNAs) that are preferred to be used when screening the antisense oligonucleotide described in (1) above (specifically, the antisense oligonucleotide of Sequence ID No. 1). (12) The antisense oligonucleotide according to (5) above, comprising a base sequence of any of sequence numbers 14 to 16, or a base sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with said base sequence.

[0011] (13) The antisense oligonucleotide according to (2) or (6) above, wherein the immune cell is at least one selected from the group consisting of peripheral blood T cells, malignant ascites T cells, inflammatory ascites T cells, malignant pleural effusion T cells, inflammatory pleural effusion T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, and B cells. (14) A pharmaceutical composition comprising the antisense oligonucleotide according to (1) above, and a pharmaceutically acceptable carrier, excipient and / or diluent. (15) A pharmaceutical composition comprising the antisense oligonucleotide according to (5) above, and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0012] (16) The pharmaceutical composition according to (14) or (15) above, for use in suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein expressed on the surface of immune cells. (17) The pharmaceutical composition according to (14) or (15) above, for use in immunotherapy. (18) A pharmaceutical composition comprising immune cells into which the antisense oligonucleotide described in (1) above has been introduced.

[0013] (19) A pharmaceutical composition comprising immune cells into which the antisense oligonucleotide described in (5) above has been introduced. (20) Immune cells into which the antisense oligonucleotide described in (1) above has been introduced. (21) Immune cells into which the antisense oligonucleotide described in (5) above has been introduced.

[0014] (22) A pharmaceutical composition according to any one of the above paragraphs (14), (15), (18), and (19) for use in the treatment or prevention of malignant or benign tumors, and / or infectious diseases, and / or kidney diseases, or an immune cell according to the above paragraph (20) or (21). (23) The tumor is a group consisting of solid tumors, congenital hematological malignancies, leukemia, tumor metastases, hemangiomas, tumor metastases, hemangiomas, acoustic neuromas, neurofibromas, trachoma, pyogenic granuloma, psoriasis, astrocytoma, blastoma, Ewing's tumor, craniopharyngioma, ependymoma, medulloblastoma, glioma, hemangioblastoma, Hodgkin lymphoma, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, pinealoma, retinoblastoma, sarcoma, seminoma, The pharmaceutical composition or immune cells described in (22) above, wherein the infection is at least one selected from the group consisting of Wilms' tumor, bile duct cancer, bladder cancer, brain tumor, breast cancer, head cancer, liver cancer, lung cancer, medullary carcinoma, cervical cancer, non-small cell bronchial / lung cancer, ovarian cancer, pancreatic cancer, papillary carcinoma, papillary adenocarcinoma, prostate cancer, small intestine cancer, prostate cancer, stomach cancer, rectal cancer, renal cell carcinoma, skin cancer, small cell lung cancer, squamous cell carcinoma, sebaceous carcinoma, testicular cancer, and uterine cancer; the infection is at least one selected from the group consisting of hepatitis A infection, cytomegalovirus infection, Epstein-Barr virus infection, and adenovirus infection; and the kidney disease is at least one selected from the group consisting of kidney disease involving glomerular dysfunction and age-related nephropathy. Specifically, kidney diseases involving the glomerular damage mentioned above include glomerulonephritis and nephrotic syndrome. Examples of glomerulonephritis include IgA nephropathy (IgAN), acute glomerulonephritis (acute nephritis), rapidly progressive glomerulonephritis (RPGN), chronic glomerulonephritis, and membranoproliferative glomerulonephritis (MPGN). Examples of nephrotic syndrome include focal segmental glomerulosclerosis (FSGS) and other focal segmental glomerulosclerosis, minimal change nephrotic syndrome (MCD), and membranous nephropathy (MN). Furthermore, age-related nephropathy refers to a condition in which the structure and function of the kidneys change with age, leading to a decline in kidney function. More specifically, it refers to a condition in which the glomerular filtration rate (GFR) of the kidneys decreases as the glomeruli, which are the functional units of the kidneys, become harder (hyalinized) with age.(24) A method for treating or preventing malignant or benign tumors and / or infectious diseases and / or kidney diseases, comprising administering a pharmaceutical composition according to any one of paragraphs (14), (15), (18), and (19) above, or immune cells according to paragraph (20) or (21) above, to a subject suffering from or likely to suffer from malignant or benign tumors and / or infectious diseases and / or kidney diseases.

[0015] The present invention provides antisense oligonucleotides capable of suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein, pharmaceutical compositions containing the antisense oligonucleotide, immune cells into which the antisense oligonucleotide has been introduced, pharmaceutical compositions containing the immune cells, and methods for treating or preventing malignant or benign tumors and / or infectious diseases and / or kidney diseases. The treatment or prevention methods also include methods for treating or preventing pathological conditions caused by the tumors, such as ascites and pleural effusion (malignant ascites, inflammatory ascites, malignant pleural effusion, and inflammatory pleural effusion).

[0016] This figure shows an overview of immune cell (T cell) therapy. This is a schematic diagram showing normal splicing from PD-1 pre-mRNA to mRNA. This is a schematic diagram showing artificial splicing from PD-1 pre-mRNA using an antisense oligonucleotide (ASO) that suppresses the expression of the extracellular receptor. This is a schematic diagram showing the structural prediction of the PD-1 pre-mRNA Exon 2 region, which was referenced when designing the antisense oligonucleotide (ASO) (CLV-301 (SEQ ID NO: 1) and CLV-301-2 (SEQ ID NO: 2)) according to the present invention as a nucleic acid drug. This figure shows an example of an antisense oligonucleotide (ASO) according to the present invention as a nucleic acid drug that suppresses the expression of PD-1 extracellular receptor expression or a nucleic acid drug that suppresses the expression of CTLA-4 extracellular receptor. The first figure shows the results of measuring the number of cells expressing the PD-1 protein receptor domain in a total of 25 nM human lymphocyte cell lines cultured for 5 to 21 days after introducing either CLV-301 (SEQ ID NO: 1), CLV-301-2 (SEQ ID NO: 2), or control ASO (antisense oligonucleotide) via electroporation. The second figure shows the results of measuring the number of cells expressing the PD-1 protein receptor domain in a total of 25 nM human lymphocyte cell lines cultured for 5 to 21 days after introducing either CLV-302 (SEQ ID NO: 14) or control ASO (antisense oligonucleotide) via electroporation. The third figure shows the results of measuring the number of cells expressing the CTLA-4 protein receptor domain in a total of 25 nM human lymphocyte cell lines cultured for 5 to 21 days after introducing either CLV-302 (SEQ ID NO: 14), or control ASO (antisense oligonucleotide) via electroporation. The fourth figure shows the results of measuring the number of cells expressing the CTLA-4 protein receptor domain in a total of 25 nM human lymphocyte cell lines cultured for 5 to 21 days after immunostaining the extracellular receptor region of the CTLA-4 protein. This figure shows the results of ELISA analysis performed on the supernatant of the culture medium after centrifugation of human lymphocyte cell lines cultured for 5 days after introducing either CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) at a concentration of 25 nM each by electroporation, using the Luminex® Multiplex Immunoassays kit.This figure shows the results of ELISA analysis performed on the supernatant of the culture medium after centrifugation of human lymphocyte cell lines cultured for 5 days after introducing 25 nM each of CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) by electroporation. This figure shows the overview of local immunotherapy for malignant ascites. This figure shows the results of co-culturing lymphocytes from malignant ascites patients introduced with CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) with cancer cells (PDC) cultured and grown from the same ascites fluid. This figure shows the results of evaluating the cytotoxicity of CLV-301 (SEQ ID NO: 1) introduced lymphocytes in co-culturing with cancer cells. This figure shows the results of evaluating the cytotoxicity of CLV-301 (SEQ ID NO: 1) introduced lymphocytes in co-culturing with cancer cells. The first figure shows the results of measuring fluorescence by adding doxycycline (Dox) and alamarBlue to culture medium in which mouse-derived podocytes expressing normal PD-1 (WT) or exon-skipping PD-1 (Mut) under Tet-ON (tetracycline-responsive regulatory system (Tet-ON system)) were cultured. The second figure shows the results of measuring by flow cytometry using CytoFLEX after adding anti-mouse PD-1-PE to mouse-derived podocytes into which eight types of ASOs (ASO19 to ASO26) targeting PD-1 pre-mRNA were introduced.

[0017] The present invention will now be described in detail. The scope of the present invention is not limited to this description, and modifications can be made as appropriate, insofar as they do not impair the spirit of the invention, in addition to the examples given below. This specification encompasses the entirety of Japanese Patent Application No. 2024-176800 (filed October 8, 2024), which forms the basis of this application's priority claim. Furthermore, all publications cited herein, such as prior art documents, and published gazettes, patent gazettes, and other patent documents, are incorporated herein by reference.

[0018] 1. Outline of the Invention The antisense oligonucleotide according to the present invention can inhibit only the expression of extracellular factors of PD-1 without controlling lymphocyte function and while maintaining cell proliferation ability, by extracting PD-1 splice elements in vitro.

[0019] The antisense oligonucleotide according to the present invention is, for example, an antisense oligonucleotide comprising 10 to 25 nucleotides, wherein at least one of the nucleotides may be modified. The antisense oligonucleotide hybridizes with the nucleic acid sequence of PD-1 or CTLA-4 (the nucleic acid sequence of the mRNA precursor and / or mature mRNA) and inhibits intracellular expression of PD-1 or CTLA-4 by at least 60% compared to untreated cells. The present invention further provides a pharmaceutical composition comprising such an antisense oligonucleotide, which can be used, for example, in methods for preventing or treating malignant tumors, malignant ascites, malignant pleural effusion, and benign tumors, as well as infections and / or kidney diseases. The antisense oligonucleotide and pharmaceutical composition according to the present invention can also be used to reduce or inhibit the expression of extracellular receptors such as PD-1 protein and CTLA-4 protein in immune cells isolated for cell therapy.

[0020] 2. Antisense oligonucleotides, pharmaceutical compositions, etc. One embodiment of the antisense oligonucleotide according to the present invention is an antisense oligonucleotide that hybridizes with the nucleic acid sequence of the mRNA precursor (pre-mRNA; e.g., SEQ ID NO: 17) and / or mature mRNA (e.g., SEQ ID NO: 18) of the PD-1 (Programmed Cell Death 1) protein, and can suppress or inhibit the expression of the PD-1 protein (protein expression as an extracellular receptor for cells such as immune cells).

[0021] Preferably, the antisense oligonucleotide can hybridize with the 141st to 2131st base sequence, the 4929th to 6078th base sequence, or a portion of the 6092nd to 6584th base sequence of the nucleic acid sequence consisting of the base sequence of Sequence ID No. 17, when the mRNA precursor of the PD-1 protein consists of the nucleic acid sequence consisting of the base sequence of Sequence ID No. 17.

[0022] Furthermore, it is preferable that the antisense oligonucleotide is capable of inducing exon 2 skipping (exclusion of exon 2) in the mRNA precursor of the PD-1 protein.

[0023] The antisense oligonucleotide is preferably capable of inhibiting the expression of PD-1 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. Here, examples of immune cells include at least one selected from the group consisting of peripheral blood T cells, malignant ascites T cells, inflammatory ascites T cells, malignant pleural effusion T cells, inflammatory pleural effusion T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, and B cells.

[0024] Another embodiment of the antisense oligonucleotide of the present invention is an antisense oligonucleotide that hybridizes with the nucleic acid sequence of the mRNA precursor and / or mature mRNA of the CTLA-4 (Cytotoxic T-lymphocyte Antigen-4) protein and can suppress or inhibit the expression of the CTLA-4 protein (protein expression as an extracellular receptor for cells such as immune cells).

[0025] The antisense oligonucleotide is preferably capable of inhibiting the expression of CTLA-4 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. Here, examples of immune cells include at least one selected from the group consisting of peripheral blood T cells, malignant ascites T cells, inflammatory ascites T cells, malignant pleural effusion T cells, inflammatory pleural effusion T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, and B cells.

[0026] The antisense oligonucleotide of the present invention preferably consists of 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 19 nucleotides, or 15 to 18 nucleotides, and more specifically, it is preferable that it consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.

[0027] The antisense oligonucleotide of the present invention may contain modified nucleotides. Preferred modified nucleotides include, for example, LNA (Locked Nucleic Acid) modified nucleotides (e.g., 2',4'-LNA), cET (Contraindicated Ethyl) modified nucleotides, ENA modified nucleotides, 2'-fluoro modified nucleotides, or 2'-O-methyl modified nucleotides, or combinations thereof (such as bridge nucleotides). The modified nucleotides may be of the same type or different types. The modified nucleotides may, for example, be included in the 5' and / or 3' ends of the antisense oligonucleotide of the present invention.

[0028] The protein expression inhibitory activity of the antisense oligonucleotides of the present invention is, more specifically, capable of inhibiting (knockdown) the expression of human or cynomolgus monkey PD-1 or CTLA-4 protein (extracellular receptor protein) by at least 25% to 99%, 30% to 95%, 35% to 90%, 40% to 85%, 45% to 80%, 50% to 75%, 55% to 70%, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to untreated cells. Here, the protein expression inhibitory activity can be evaluated by the activity level of PD-1 or CTLA-4 protein, and this activity level can be measured by any commonly known standard method, such as immunohistochemical analysis (including FACS analysis), cytokine quantitative analysis, Western blot analysis, or quantitative real-time PCR analysis.

[0029] The antisense oligonucleotides of the present invention are immunosuppressive reversal oligonucleotides that inhibit and reverse immunosuppression in cells, tissues, organs, or subjects, respectively. The antisense oligonucleotides of the present invention can inhibit the expression of PD-1 or CTLA-4 proteins at concentrations of 0.1, 1, 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1, 10 or 100 μM. The antisense oligonucleotides of the present invention can be used at concentrations of, for example, 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6, or 10 μM. The antisense oligonucleotides of the present invention may be in any of the following forms: antisense oligonucleotide DNA, siRNA, sdRNA, and aptamers.

[0030] As antisense oligonucleotides that can suppress or inhibit the expression of the aforementioned PD-1 protein, for example, those consisting of the following nucleic acid sequences (the base sequences of SEQ ID NOs: 1 to 13 and 21) are preferred. Also preferred are base sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following base sequences. Any base sequence within this range of identity may have the same suppressive or inhibitory activity on PD-1 protein expression as the following nucleic acid sequences.

[0031]

[0032] Some or all of the bases in the above nucleic acid sequence may be modified nucleotides as described above, for example, 2'-O-methyl-modified nucleotides are preferred.

[0033] Furthermore, as antisense oligonucleotides that can suppress or inhibit the expression of the aforementioned CTLA-4 protein, for example, those consisting of the following nucleic acid sequences (nucleotide sequences of SEQ ID NOs. 14 to 16) are preferred. Also preferred are those consisting of nucleotide sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the following nucleotide sequences. Any nucleotide sequence within this range of identity may have the same suppressive or inhibitory activity against CTLA-4 protein expression as the following nucleic acid sequences.

[0034]

[0035] Some or all of the bases in the above nucleic acid sequence may be modified nucleotides as described above, for example, 2'-O-methyl-modified nucleotides are preferred.

[0036] In the present invention, there is also provided a pharmaceutical composition comprising the antisense oligonucleotide of the present invention described above, and a pharmaceutically acceptable carrier, excipient and / or diluent. The pharmaceutical composition of the present invention is preferably for use in suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein expressed on the surface of immune cells, or for use in immunocyte therapy (for example, ex vivo step).

[0037] In the present invention, there may also be provided immune cells (transduced immune cells) into which the antisense oligonucleotide of the present invention described above has been introduced, and a pharmaceutical composition containing the immune cells. The pharmaceutical composition of the present invention and the immune cells (transduced immune cells) of the present invention described above are preferably for use in the treatment or prevention of malignant or benign tumors, and / or infectious diseases, and / or renal diseases.

[0038] In the present invention, there may also be provided a method for treating or preventing malignant or benign tumors, and / or infectious diseases, and / or renal diseases, which includes administering the pharmaceutical composition of the present invention and the immune cells (transduced immune cells) of the present invention described above to a subject suffering from or likely to suffer from malignant or benign tumors, and / or infectious diseases, and / or renal diseases. In addition, the above treatment or prevention also includes the treatment or prevention of pathological conditions caused by the tumor, such as ascites and pleural effusion (for example, malignant ascites, inflammatory ascites, malignant pleural effusion, and inflammatory pleural effusion).

[0039] Hereinafter, the pharmaceutical composition, antisense oligonucleotide, etc. of the present invention will be illustrated and described in more detail.

[0040] Embodiments of the pharmaceutical composition of the present invention (and consequently the antisense oligonucleotide of the present invention; the same applies hereinafter) may further include chemotherapy, radiotherapy, another disease-specific activator such as another oligonucleotide of the present invention, or hybridization with PD-1 pre-mRNA or a different target, an antibody (PD-1 antibody or CTLA-4 antibody), a HERA fusion protein, a ligand trap, a fab fragment, a nanobody, BiTe, a small molecule or a combination thereof. The radiotherapy is preferably further combined with chemotherapy (e.g., platinum, gemcitabine).

[0041] The pharmaceutical compositions of the present invention are administered topically (e.g., orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, percutaneously, and / or rectally) or systematically. The pharmaceutical compositions of the present invention are used in methods for the prevention and / or treatment of solid tumors or hematological malignancies. Examples of cancers that can be prevented and / or treated include breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, laryngeal cancer, gallbladder, pancreas, testes, rectum, parathyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney, basal cell carcinoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, retinal cell sarcoma, liposarcoma, myeloma, giant cell tumor, small cell lung tumor, small cell tumor, pancreatic islet cell tumor, primary brain tumor, meningioma, acute and Examples include diffuse lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, pilosarcoma, adenoma, hyperplasia, myelin carcinoma, enteric ganglia, Wilm's tumor, ovarian tumor, leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant cancer, focal skin lesions, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, multicellular tumor, adenocarcinoma, astrocytoma, glioblastoma, leukemia, epidermal carcinoma, cancer with malignant ascites, and cancer with malignant pleural effusion.

[0042] The pharmaceutical composition of the present invention is also used in a method for preventing and / or treating infectious diseases. Infectious diseases include, for example, hepatitis B infection, hepatitis A infection, cytomegalovirus infection, Epstein-Barr (EB) virus infection, adenovirus infection, or a combination thereof. The pharmaceutical composition of the present invention is also used in a method for preventing and / or treating kidney diseases. Kidney diseases include, for example, kidney diseases involving glomerular disorders and various kidney diseases such as senile nephropathy. Specifically, kidney diseases involving glomerular disorders include disease groups such as glomerulonephritis and nephrotic syndrome. Examples of glomerulonephritis include IgA nephropathy (IgAN), acute glomerulonephritis (acute nephritis), rapidly progressive glomerulonephritis (RPGN), chronic glomerulonephritis, membranoproliferative glomerulonephritis (MPGN), etc. Examples of nephrotic syndrome include focal segmental glomerulosclerosis (FSGS) and other focal glomerulosclerosis, minimal change nephrotic syndrome (MCD), membranous nephropathy (MN), etc. In addition, senile nephropathy refers to a pathological condition (state) in which the structure and function of the kidney change with aging and kidney function declines. Specifically, with aging, sclerosis (hyalinization) of glomeruli, which are the functional units of the kidney, progresses, resulting in a pathological condition (state) in which the blood filtration ability of the kidney (glomerular filtration rate: GFR) decreases. All of the above diseases listed as uses of the pharmaceutical composition of the present invention can be caused by or affected by, for example, an imbalance of PD-1 or CTLA-4.

[0043] For example, two or more antisense oligonucleotides of the present invention are administered together at the same time, for example, in a pharmaceutical composition, separately, or at staggered intervals. In other embodiments, one or more antisense oligonucleotides of the present invention are administered together with another oligonucleotide (i.e., not part of the present invention), an antibody, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, BiTe, a small molecule, and / or a chemotherapeutic agent, for example, in a pharmaceutical composition, separately, or at staggered intervals. In some embodiments of these combinations, the antisense oligonucleotides of the present invention inhibit the expression, function, and / or activity of immunosuppressive factors and other oligonucleotides (i.e., not part of the present invention), antibodies, HERA fusion proteins, ligand traps, Fab fragments, nanobody, BiTe, and / or small molecules (antagonists), or inhibit the same and / or other immunosuppressive factors and / or immunostimulators (agonists). Immunosuppressive factors are selected from the group consisting of, for example, IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, 2B4, CD304, PQR-prot, PERK, FOXP3, GMCSF, INFg, TNFa, TGFb, IL-1, IL-2, IL-6, IL-10, IL-12, IL-17, IL-9, STAT3, IL-6 receptor, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, Chop, Xbp1, and combinations thereof. Immunostimulators encode proteins that affect the expansion and / or survival of immune cells, selected from groups such as 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and combinations thereof, or from groups such as BID, BIM, BAD, NOXA, PUMA, BAX, BAX, BAK, BOK, BCL-rambo, BCL-Xs, Hrk, Blk, BMf, p53 and combinations thereof.

[0044] Immunosuppressive factors are factors whose expression, function, and / or activity increase in, for example, cells, tissues, organs, or subjects. Immunostimulating factors are factors whose levels increase or decrease in, depending on the cells, tissues, organs, or subjects and their individual conditions.

[0045] Antisense oligonucleotides or antibodies combined with the pharmaceutical composition of the present invention include, for example, anti-PD-1 antibodies (e.g., semiprimab, CT-011, nivolumab, pembrolizumab), anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab), CTLA-4 antibodies (e.g., ipilimumab), or bispecific antibodies. Small molecules combined with antisense oligonucleotides or the pharmaceutical composition of the present invention include, for example, epacadostat, vemurafenib, or tyrosine kinase inhibitors.

[0046] The antisense oligonucleotides or pharmaceutical compositions of the present invention are intended for use in humans and other animals. Examples of non-human animals include mammals such as monkeys, horses, cattle, pigs, lambs, cats, dogs, guinea pigs, and hamsters. Furthermore, the antisense oligonucleotides of the present invention are used in cell therapies such as T-cell therapy. Antisense oligonucleotides are significantly more advantageous than, for example, antibodies, siRNA, sdRNA, and Antisense GapmeRs, respectively. Antisense oligonucleotides are administered in vivo and excivo without delivery systems such as delivery agents or electroporation. They do not promote RNA degradation by RNAseH, etc., and therefore do not adversely affect cell viability, which can lead to negative side effects in cell therapy.

[0047] The present invention further relates to a method for reducing the expression, function, and / or activity of the PD-1 extracellular receptor in isolated cells, such as immune cells, in preparation for cell therapy. This method comprises the step of incubating isolated cells, such as immune cells, containing PD-1 pre-mRNA with an antisense oligonucleotide, without the use of transfection means. The antisense oligonucleotide is administered to the isolated cells, such as immune cells, at least once during the period from day 0 to day 21. The antisense oligonucleotide hybridizes with the PD-1 pre-mRNA, reducing the expression, function, and / or activity of the PD-1 extracellular receptor from day 0 to up to 8 weeks of incubation with the antisense oligonucleotide. Since the administration of the antisense oligonucleotide does not permanently block the expression, function, and / or activity of PD-1, side effects based on permanent blockage of RNA expression, function, and / or activity are avoided. Furthermore, the administration of antisense oligonucleotides without transfection significantly reduces stress on cells, meaning that side effects caused by other transfection means are mitigated or avoided.

[0048] Isolated cells include, for example, immune cells, stem cells, pluripotent stem cells such as induced pluripotent stem cells, embryonic stem cells, skin stem cells, umbilical cord blood stem cells, mesenchymal stem cells, neural stem cells, or combinations thereof. Immune cells include, for example, T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, B cells, and combinations thereof. T cells are genetically modified to express antigen-specific receptors, such as chimeric antigen receptors and T cell receptors. These cells can exert antitumor function by recognizing antigens on the surface of tumor cells via antigen-specific receptors, leading to T cell activation. Activated T cells release tumor cytotoxic cytokines.

[0049] PD-1 RNA is mRNA, pre-mRNA, lncRNA, and / or miRNA. Oligonucleotides hybridize with specific sequences of PD-1 RNA, thereby regulating the expression, modifying the function, and / or reducing the activity of a portion of the PD-1 (RNA or protein) domain consisting of or constituting this sequence. Cells used in methods to reduce PD-1 RNA extracellular receptor expression are isolated from human or non-human animals. Non-human animals include mammals such as monkeys, horses, cattle, pigs, lambs, cats, dogs, guinea pigs, and hamsters.

[0050] Isolated cells are optionally genetically modified by gene transfer techniques including 1) chemical (biochemical) transfection, 2) physical transfection, and 3) virus-mediated transduction. Chemical (biochemical) methods include, for example, calcium phosphate transfection, DEAE dextran transfection, or lipofection. Physical methods include, for example, electroporation, nuclear ofection, microinjection, particle bombardment transfection, or sonication transfection. Virus-mediated transduction uses, for example, adenoviruses for short-term infection with high levels of transient expression, herpesviruses for long-term expression, or retroviruses or lentiviruses for stable integration of DNA into the host cell genogenome. After genetic modification, cells are cultured and grown. Genetic modification can be permanent or transient.

[0051] The isolated cells are incubated with the antisense oligonucleotide of the present invention, for example, before or after genetic recombination and / or before or after the culture and proliferation of genetically modified cells. Optionally, the isolated cells are purified by one or more washing steps before or after culture with the antisense oligonucleotide. The present invention optionally includes a cell purification and concentration step, in which the isolated cells are concentrated before and / or after culture with the antisense oligonucleotide via any concentration method. The antisense oligonucleotide is administered, for example, to the purified and concentrated cells after the concentration step.

[0052] Furthermore, the isolated cells are cryopreserved after any purification or concentration step, or a combination thereof, before or after incubation with the introduction of antisense nucleotides. Isolation according to the present invention means obtaining cells from a source, such as immune cells from blood, stem cells from bone marrow, or umbilical cord blood, and / or obtaining a subpopulation of cells from a previously isolated cell or cell population. The method for reducing PD-1 RNA expression optionally includes an activation step, and the isolated cells are activated via any activation method of the technique, for example, by stimulating the cells on the surface of T cells with monoclonal antibodies specific to CD3 and CD23 before and / or after incubation with antisense oligonucleotides of the present invention. The antisense oligonucleotides are then administered to the isolated cells again, for example, after the activation step.

[0053] Methods for reducing PD-1 RNA expression optionally include an extended step, and any proliferation method is used for isolated cells, for example, by adding basic fibroblast growth factor (FGF2) to mesenchymal stem cells before and / or after incubation with oligonucleotides, or by adding interleukin-2 (IL-2) and / or interleukin-15 (IL-15) to NK cells before and / or after incubation with oligonucleotides.

[0054] For isolated cells, PD-1 antisense oligonucleotides can be administered once, or in any combination of dosing patterns during the period or on days 2, 3, 4, 5, 6, 7, 8, 9, and 10. PD-1 antisense oligonucleotides are administered daily, every 5 days, or every 2 days between day 0 and day 9. PD-1 antisense oligonucleotides are added to isolated cells at concentrations between 0.1 nmol and 1000 μmol, 0.5 nmol and 900 μmol, 1 nmol and 800 μmol, 50 nmol and 700 μmol, 100 nmol and 600 μmol, 200 nmol and 500 μmol, 300 nmol and 400 μmol, 500 nmol and 300 μmol, 600 nmol and 200 μmol, 700 nmol and 100 μmol, or 800 nmol and 50 μmol.

[0055] PD-1 antisense oligonucleotides control the splicing of target RNA for at least 10 weeks, at least 8 weeks, at least 6 weeks, at least 4 weeks, and at least 2 weeks from day 0 after introduction. Isolated cells are introduced with different antisense oligonucleotides according to the present invention or 2, 3, 4, 5, 6, 7, 8, 9, or 10, in combination with other oligonucleotides that hybridize with the same (PD-1) or different target RNA. Oligonucleotides that hybridize with different target RNAs are administered to isolated cells at the same time in the same period, at the same time in different periods, at different time in the same period, or at different time in different periods.

[0056] The different target RNAs are target RNAs other than PD-1 pre-mRNA, and in parallel with the antisense oligonucleotide of the present invention, they hybridize to multiple target RNAs, and the different oligonucleotides directly and / or indirectly affect the target factor, reducing the expression of the different target RNAs.

[0057] The present invention is further directed toward isolated cells obtained by a method of reducing PD-1 RNA expression. The isolated cells are used, for example, in methods of preventing and / or treating diseases. The cells are isolated, for example, from a patient suffering from a disease or a healthy subject, and the isolated cells are incubated ex vivo with an antisense oligonucleotide or a pharmaceutical composition of the present invention that hybridizes with PD-1 RNA according to the method of the present invention. After incubation of the isolated cells with the antisense oligonucleotide, the isolated cells are reintroduced into the patient from whom they were isolated. Alternatively, cells isolated from a healthy subject and incubated ex vivo with the antisense oligonucleotide of the present invention that hybridizes with PD-1 RNA according to a method of reducing PD-1 RNA expression are introduced into a patient suffering from a disease based on PD-1 imbalance. Thus, the present invention includes allogeneic cell therapy. Antisense oligonucleotide-treated immune cells are reintroduced or introduced into the patient, for example, intravenously, intraperitoneally, intramuscularly and / or subcutaneously.

[0058] Cells, such as immune cells, used in methods for preventing and / or treating disease consist of cells isolated from a patient, a healthy subject, or a combination thereof, and incubated ex vivo with an antisense oligonucleotide that hybridizes with PD-1 target RNA according to the present invention. In methods for reducing PD-1 RNA expression, either an antisense oligonucleotide or / or a pharmaceutical composition containing such an antisense oligonucleotide is used.

[0059] [Examples] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these.

[0060] [Example 1] Referring to the schematic diagram in Figure 4A, which shows the structural prediction of the PD-1 pre-mRNA Exon 2 region, an antisense oligonucleotide (ASO) as a nucleic acid drug to suppress PD-1 extracellular receptor expression was prepared as follows (see Figure 4B). In addition to these, we also created the following ASOs as nucleic acid drugs that suppress PD-1 extracellular receptor expression.

[0061] Furthermore, antisense oligonucleotides (ASOs) used as nucleic acid drugs to suppress CTLA-4 extracellular receptor expression were also prepared as follows (see Figure 4B). In addition to this, we also created the following ASOs as nucleic acid drugs that suppress CTLA-4 extracellular receptor expression.

[0062] [Example 2] Human lymphocyte cell lines were introduced with 25 nM each of CLV-301 (SEQ ID NO: 1), CLV-301-2 (SEQ ID NO: 2), or a control ASO (antisense oligonucleotide) by electroporation and cultured for 5 to 21 days. The extracellular receptor region of the PD-1 protein was immunostained, and then FACS analysis was performed to measure the number of cells expressing the PD-1 protein receptor domain in the total number of cells. As shown in Figure 5A, a decrease in the number of cells expressing the PD-1 protein receptor domain was observed in cell lines introduced with either CLV-301 or CLV-301-2 ASO (CLV-301: 43.1% → 17%, CLV-301-2: 43.1% → 17.9%).

[0063] Similarly, human lymphocyte cell lines were introduced with 25 nM of CLV-302 (SEQ ID NO: 14) or control ASO (antisense oligonucleotide) by electroporation and cultured for 5 to 21 days. The extracellular receptor region of the CTLA-4 protein was immunostained, and then FACS analysis was performed to measure the number of cells expressing the CTLA-4 protein receptor domain in the total number of cells. As shown in Figure 5B, a decrease in the number of cells expressing the CTLA-4 protein receptor domain was observed in the cell lines introduced with CLV-302 ASO (CLV-302: 43.1% → 38.3%). No decrease in the number of cells expressing the PD-1 protein receptor domain was observed.

[0064] [Example 3] Human lymphocyte cell lines were introduced with 25 nM of CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) by electroporation and cultured for 5 days. ELISA analysis was performed on the supernatant of the culture medium after centrifugation using the Luminex® Multiplex Immunoassays kit. The kit used was Invitrogen (Procartaplex human th1 / th2 cytokine panel 11 plex from Invitrogen, cat # epx110-10810-901), and the experiment was carried out according to the manufacturer's instructions. The experiment was performed in 3 replicates. The results are shown in Figures 6A and 6B.

[0065] [Example 4] Local immunotherapy was performed for malignant ascites (see Figure 7A). Specifically, ex vivo cell therapy was performed using highly tumor-specific antitumor lymphocytes, in which activation of lymphocytes was induced by introducing immune checkpoint-targeted nucleic acid drugs, and these lymphocytes were used for treatment, according to the following procedure.

[0066] 1) Mix lymphocytes and cancer cells from patients with malignant ascites for 5 days, and increase the size of the flask as the cell count increases. 2) 10 suspended cells in the mixed culture medium ６ For each individual cell, CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) is introduced by electroporation, and the cells are subsequently cultured. 3) The proliferation activity and antitumor activity of lymphocytes from cancerous ascites patients into which CLV-301 (SEQ ID NO: 1) has been introduced are evaluated. In this example, it was found that the proliferation activity and antitumor activity were maintained for at least one month.

[0067] Lymphocytes from cancerous ascites patients to which CLV-301 (SEQ ID NO: 1) or control ASO (antisense oligonucleotide) had been introduced were co-cultured with cancer cells (PDCs) cultured and grown from the same ascites fluid. As a result, although the cancerous ascites cells were derived from colorectal cancer patients who had shown resistance to immune checkpoint inhibitors, the CLV-301-introduced lymphocytes exhibited a remarkable antitumor effect (see Figure 7B).

[0068] Next, the cytotoxicity of CLV-301 (SEQ ID NO: 1)-introduced lymphocytes in co-culture with cancer cells was evaluated. On day 5 after co-culture, the number of adherent cancer cells per unit area was measured (6 tests), and no adherent cancer cells were observed in CLV-301 (SEQ ID NO: 1)-introduced lymphocytes (see Figures 7C and 7D).

[0069] [Example 5] Mouse-derived podocytes were used. Culture was performed by adding 10 FBS, 1% penicillin / streptomycin, and IFN-γ to RPMI-1640 and culturing at 33°C with 5% CO2. ２ The cells were maintained under the following conditions. Mouse-derived podocytes expressing normal PD-1 (WT) or exon-skipping PD-1 (Mut) under Tet-ON (tetracycline-responsive regulatory system (Tet-ON system)) control were established. Doxycycline (Dox) (0, 0.1, 1, 5 μg / mL) was added to the culture medium, and alamarBlue was added on day 5 to measure fluorescence (Ex / Em = 560 / 590). The fluorescence value of the cell-free medium alone was subtracted as the background value to obtain each measured value. The results of Test 1 and Test 2, which were performed independently, are shown in Figure 8. In WT, the normalized alamarBlue value decreased in a Dox concentration-dependent manner in both Test 1 and Test 2. In Mut, the degree of decrease at the same concentration was smaller than in WT. It was confirmed that induction of normal PD-1 can reduce podocyte metabolic activity, while exon-skipping PD-1 can mitigate this reduction.

[0070] Next, eight types of ASOs (ASO19 to ASO26, shown below), targeting PD-1 pre-mRNA, were introduced into mouse-derived podocytes by electroporation. Using NEPA21 (NEPAGENE), 1 × 10⁻⁶ samples were taken. ６ Cells were suspended in 100 μL of Opti-MEM, 1 μg of ASO was added, and electroporation was performed. After introduction, the cells were cultured, and measurements were taken by flow cytometry on day 10.

[0071] The sequences of ASO19 to ASO26 used are as follows:

[0072] 5 μL / 10 of anti-mouse PD-1-PE (BioLegend, Cat. 114117) ６ Cells were added, the reaction was carried out at 4°C for 30 minutes, washed with PBS + 2% FBS, and measured using CytoFLEX. As a result of ASO introduction, a decrease in the PE positive rate was observed when, for example, ASO25 or ASO26 was introduced (see Figure 9). This demonstrated that ASO can reduce the amount of the extracellular domain of PD-1. From the results of this example, it is suggested that the ASO according to the present invention is useful for the treatment or prevention of various kidney diseases, such as various kidney diseases involving glomerular damage and age-related nephropathy.

[0073] Sequence IDs 1-16 and 19-21: Synthetic constructs

[0074] The present invention provides an antisense oligonucleotide capable of suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein, a pharmaceutical composition containing the antisense oligonucleotide, immune cells into which the antisense oligonucleotide has been introduced, a pharmaceutical composition containing the immune cells, and a method for treating or preventing malignant or benign tumors and / or infectious diseases and / or kidney diseases.

Claims

1. An antisense oligonucleotide that can hybridize with the nucleic acid sequence of the mRNA precursor and / or mature mRNA of the PD-1 (Programmed Cell Death 1) protein and suppress or inhibit the expression of the PD-1 protein.

2. The antisense oligonucleotide according to claim 1, which can inhibit the expression of PD-1 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

3. The antisense oligonucleotide according to claim 1, wherein the mRNA precursor of the PD-1 protein consists of a nucleic acid sequence comprising the base sequence of Sequence ID No. 17, and the antisense oligonucleotide can hybridize with the base sequences from the 141st to the 2131st, the base sequences from the 4929th to the 6078th, or a portion of the base sequences from the 6092nd to the 6584th of the nucleic acid sequence.

4. The antisense oligonucleotide according to claim 1, which can induce exon 2 skipping in the mRNA precursor of the PD-1 protein.

5. Antisense oligonucleotides that hybridize with the nucleic acid sequence of the mRNA precursor and / or mature mRNA of the CTLA-4 (Cytotoxic T-lymphocyte Antigen-4) protein and can suppress or inhibit the expression of the CTLA-4 protein.

6. The antisense oligonucleotide according to claim 5, which can inhibit the expression of CTLA-4 protein expressed on the surface of immune cells by at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%.

7. The antisense oligonucleotide according to claim 1 or 5, wherein the antisense oligonucleotide consists of 10 to 25 nucleotides.

8. The antisense oligonucleotide according to claim 1 or 5, wherein the antisense oligonucleotide comprises a modified nucleotide.

9. The antisense oligonucleotide according to claim 8, wherein the modified nucleotide is an LNA (Locked Nucleic Acid) modified nucleotide, a cET (Constrained Ethyl) modified nucleotide, an ENA modified nucleotide, a 2'-fluoro modified nucleotide, or a 2'-O-methyl modified nucleotide, or a combination thereof.

10. The antisense oligonucleotide according to claim 8, wherein the modified nucleotide is contained in the 5' and / or 3' end of the antisense oligonucleotide.

11. The antisense oligonucleotide according to claim 1, comprising any of the base sequences of Sequence IDs 1 to 13 and 21, or a base sequence having at least 80%, 90%, 95%, 98%, or 99% identity with said base sequence.

12. The antisense oligonucleotide according to claim 5, comprising a base sequence of any of sequence numbers 14 to 16, or a base sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with said base sequence.

13. The antisense oligonucleotide according to claim 2 or 6, wherein the immune cell is at least one selected from the group consisting of peripheral blood T cells, malignant ascites T cells, inflammatory ascites T cells, malignant pleural effusion T cells, inflammatory pleural effusion T cells, dendritic cells, natural killer (NK) cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, and B cells.

14. A pharmaceutical composition comprising the antisense oligonucleotide described in claim 1, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

15. A pharmaceutical composition comprising the antisense oligonucleotide described in claim 5, and a pharmaceutically acceptable carrier, excipient, and / or diluent.

16. The pharmaceutical composition according to claim 14 or 15, for use in suppressing or inhibiting the expression of PD-1 protein or CTLA-4 protein expressed on the surface of immune cells.

17. The pharmaceutical composition according to claim 14 or 15, for use in immunotherapy.

18. A pharmaceutical composition comprising immune cells into which the antisense oligonucleotide described in claim 1 has been introduced.

19. A pharmaceutical composition comprising immune cells into which the antisense oligonucleotide described in claim 5 has been introduced.

20. Immune cells into which the antisense oligonucleotide described in claim 1 has been introduced.

21. Immune cells into which the antisense oligonucleotide described in claim 5 has been introduced.

22. A pharmaceutical composition according to any one of claims 14, 15, 18, and 19, or an immune cell according to claim 20 or 21, for use in the treatment or prevention of malignant or benign tumors, and / or infectious diseases, and / or kidney diseases. twenty three. The tumor is at least one of the following: a group consisting of solid tumors, congenital hematological malignancies, leukemia, tumor metastases, and hemangiomas; a group consisting of tumor metastases, hemangiomas, acoustic neuromas, neurofibromas, trachoma, pyogenic granulomas, psoriasis, astrocytomas, blastomas, Ewing's tumors, craniopharyngiomas, ependymomas, medulloblastomas, gliomas, hemangioblastomas, Hodgkin lymphomas, mesotheliomas, neuroblastomas, non-Hodgkin lymphomas, pineal gland tumors, retinoblastomas, sarcomas, seminomas, Wilms' tumors, bile duct cancers, bladder cancers, brain tumors, breast cancers, head cancers, liver cancers, lung cancers, medullary carcinomas, cervical cancers, non-small cell bronchial / lung cancers, ovarian cancers, pancreatic cancers, papillary carcinomas, papillary adenocarcinomas, prostate cancers, small intestine cancers, prostate cancers, gastric cancers, rectal cancers, renal cell carcinomas, skin cancers, small cell lung cancers, squamous cell carcinomas, sebaceous gland cancers, testicular cancers, and uterine cancers. The pharmaceutical composition or immune cells according to claim 22, wherein the infectious disease is at least one selected from the group consisting of hepatitis A infection, cytomegalovirus infection, Epstein-Barr virus infection, and adenovirus infection, and the kidney disease is at least one selected from the group consisting of kidney disease involving glomerular damage and age-related nephropathy.

24. A method for treating or preventing malignant or benign tumors and / or infectious diseases and / or kidney diseases, comprising administering a pharmaceutical composition according to any one of claims 14, 15, 18, and 19, or immune cells according to claim 20 or 21, to a subject suffering from or likely to suffer from malignant or benign tumors and / or infectious diseases and / or kidney diseases.

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