Asymmetric siRNA that inhibits PD-1 expression

Abstract

The present invention relates to an asymmetric siRNA that inhibits the expression of PD-1, namely Programmed Cell Death protein 1, and its use, and more particularly to an asymmetric siRNA comprising an antisense strand containing a sequence complementary to the mRNA encoding PD-1 and a sense strand that forms a complementary bond with the antisense strand, a pharmaceutical composition for preventing or treating cancer comprising the asymmetric siRNA, an immune cell in which the expression of PD-1 is inhibited by treatment with the siRNA, and an immunocytotherapy agent for treating cancer comprising the immune cell.

Description

Technical Field

[0001] The present invention relates to an asymmetric siRNA that inhibits the expression of PD-1, that is, Programmed Cell Death protein 1 (PD-1), and uses thereof, and more particularly to an asymmetric siRNA including an antisense strand containing a sequence complementary to the mRNA encoding PD-1 and a sense strand that forms a complementary bond with the antisense strand, a pharmaceutical composition for preventing or treating cancer including the asymmetric siRNA, an immune cell in which the expression of PD-1 is inhibited by treatment with the siRNA, and an immune cell therapeutic agent for treating cancer including the immune cell. This patent application claims the priority of Korean Patent Application No. 10-2019-0058805, filed with the Korean Intellectual Property Office on May 20, 2019, the disclosure of which is incorporated herein by reference. Background Art

[0002] PD-1, that is, Programmed Cell Death protein 1 (PD-1) is one of the receptors expressed on the cell surface and has the effect of inhibiting the activity of immune cells. When T lymphocytes, which play a major role in the adaptive immune response, are activated, the expression of PD-1 is induced. The interaction with PD-L1, that is, Programmed Death Ligand 1 and / or PD-L2, that is, Programmed Death Ligand 2, which are its ligands, results in the inhibition of the signal transduction pathway initiated at the T cell receptor (TCR), which receives a stimulation signal from an antigen. This is accomplished by PD-1 using SHP-2, which is a phosphatase, to inactivate Zap70, which is an effector molecule of the TCR.

[0003] This PD-1 / PD-L interaction inhibits the occurrence of autoimmune reactions in a normal state, and in a disease state such as a viral infection, it plays a role in preventing unintentional damage to cells in normal tissues due to overactivation of immune cells. Therefore, PD-1 / PD-L plays a role in regulating immune checkpoints.

[0004] PD-1 is usually expressed on most immune cells, such as activated T cells, B cells, and macrophages, etc., while PD-L1 / PD-L2 is expressed on the surface of antigen-presenting cells or stromal cells.

[0005] In the case of cancer cells, this immunosuppressive mechanism is exploited to evade immune system surveillance, creating a favorable environment for cancer cell growth and infiltration. A representative method is the overexpression of PD-L1 in cancer cells and the utilization of the PD-1 / PD-L1 interaction to render T cells anergic. Anergic T cells no longer proliferate, no longer secrete cytokines, and do not mediate cytotoxic responses. By providing this PD-L1, cancer cells evade the immune action of immune cells that play an important role in the immune system and grow and invade tissues, thereby leading to the ineffectiveness of inhibitory cell therapy.

[0006] For example, in an in vivo mouse model of a study, it was confirmed that tumor cells expressing PD-L1 were less sensitive to the cytolytic response induced by cytotoxic T cells and had increased tumorigenicity and invasiveness (Iwai, Yoshiko, et al. “Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade.” Proceedings of the National Academy of Sciences 99.19 (2002): 12293-12297). Additionally, in some cases, it was confirmed that the activity of cytotoxic T cells could be significantly reduced solely by PD-L1 expression (Juneja, Vikram R., et al. “PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity.” Journal of Experimental Medicine (2017): jem-20160801).

[0007] Studies have been conducted to inhibit the PD-1 / PD-L interaction in these cancer cells, and the results have been reported. Cancer is treated by antibodies that bind complementarily to PD-1 or PD-L in a way that competitively or non-competitively inhibits the PD-1 / PD-L1 interaction (Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, et al. (April 2015). “Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer”. Science. 348(6230): 124-128).

[0008] However, as side effects of these PD-1 / PD-L1 inhibitory antibody therapies, dermatologic toxicities, digestive system diseases, endocrine toxicities, hepatic toxicities, pneumonia, neurologic syndromes, ocular toxicity, renal toxicity, pancreatic toxicities, etc. have been reported (J. Naidoo, et al. “Toxicities of the Anti-PD-1 and Anti-PD-L1 Immune Checkpoint Antibodies” Annals of Oncology, mdv383. doi: 10.1093 / annonc / mdv383).

[0009] On the other hand, treating diseases using the RNA interference phenomenon can treat diseases more safely because small interfering RNA (siRNA) that targets mRNA and regulates gene expression at the translational level is used. Small interfering RNA (siRNA) consists of a sense strand with the same sequence as the target mRNA and an antisense strand with its complementary sequence. Conventional siRNA has a short duplex of 19 - 21 bp and has 2 nucleotides protruding from the 3' of both strands. siRNA enters the cell, attaches to the target mRNA, degrades the target mRNA, and inhibits the expression of the target gene. Since all mRNAs can be targeted by changing the sequence of the oligonucleotide, the expression of proteins with complex structures can also be inhibited, so diseases that are currently difficult to treat, such as cancer, viral infections, and genetic diseases, can be treated. However, siRNA introduced into the cell may cause side effects, such as inducing an immune response and inhibiting non-target genes. Among them, the delivery system for introducing siRNA into the cell is the biggest problem in developing therapeutic agents using siRNA.

[0010] siRNA is negatively charged due to its phosphate backbone. Since it has a repulsive force against the negatively charged cell membrane, a delivery system is needed to introduce siRNA into the cell. As an example of a delivery system, a method of introducing siRNA into the cell by offsetting the negative charge by wrapping siRNA with a positively charged liposome or polymer is widely used. However, the positively charged carrier may cause various side effects, such as unwanted toxicity by attaching to the negatively charged cell membrane, or forming unwanted complexes by interacting with various types of proteins in the cell, etc. In addition, since siRNA is rapidly degraded by nucleases in the blood, the amount of siRNA reaching the target cell may not be sufficient to significantly reduce the expression of the target gene. Therefore, a method that can safely and effectively deliver siRNA to the target cell is needed.

[0011] For this reason, the present inventors have developed a technology related to asymmetric siRNA structures (asymmetric shorter duplex siRNA, asiRNA) (WO 2009 / 078685). AsiRNA is an asymmetric RNAi-inducing structure with a shorter double helix length compared to the 19+2 structure. This is a technology that overcomes problems such as off-target effects of siRNA, saturation of the RNAi mechanism, and Toll-like receptor 3 (TLR3) immune responses, and it can be a powerful platform for developing new RNAi drugs. Additionally, due to the addition of chemical and structural modifications, it can be self-delivered into cells without a separate delivery system, which overcomes the problems of existing siRNA delivery systems and thus enables the development of effective next-generation therapies.

[0012] Therefore, the present inventors predicted that by suppressing the expression of PD-1 through RNA interference, the immunosuppressive mechanism caused by overexpression of PD-L1 in cancer cells could be disrupted, and thus they endeavored to invent a nucleic acid molecule that has intracellular permeability to be effectively delivered into cells by targeting PD-1 and has the ability to induce RNA interference. As a result, asymmetric shorter duplex small interfering RNA (asiRNA) was developed, which induces RNA interference to inhibit the expression of target genes while reducing off-target effects. Furthermore, through various chemical modifications of the asymmetric double-stranded nucleic acid molecule, it was confirmed to have excellent target gene inhibition efficiency in vitro without a carrier, thus completing the present invention.

[0013] The above information provided in this background art is only for understanding the background of the present invention and may not include information on the prior art known to those of ordinary skill in the art to which the present invention pertains. Summary of the Invention

[0014] Technical Problem

[0015] An object of the present invention is to provide an asymmetric siRNA (asymmetric shorter duplex siRNA, asiRNA) that specifically inhibits the expression of PD-1, i.e., Programmed cell Death protein 1.

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the above asymmetric siRNA.

[0017] Another object of the present invention is to provide an immune cell that inhibits PD-1 expression using the asymmetric siRNA.

[0018] Another object of the present invention is also to provide an immune cell therapeutic agent for treating cancer, which comprises the above-mentioned immune cells.

[0019] Technical Solution

[0020] To achieve the above object, the present invention provides an asymmetric siRNA, which is characterized by comprising: an antisense strand containing a sequence complementary to the mRNA encoding PD-1, i.e., Programmed Cell Death protein 1; and a sense strand forming a complementary bond with the antisense strand, wherein the 5'-end of the antisense strand and the 3'-end of the sense strand form a blunt end.

[0021] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned asymmetric siRNA.

[0022] The present invention also provides an immune cell that inhibits PD-1 expression using the asymmetric siRNA.

[0023] The present invention also provides an immune cell therapeutic agent for treating cancer, which comprises the above-mentioned immune cells.

[0024] Beneficial Effects

[0025] According to the present invention, an asymmetric siRNA capable of causing an RNA interference effect by targeting the mRNA of PD-1, which is closely related to the immunosuppression caused by the interaction of PD-1 / PD-L and the resulting growth and metastasis of cancer by encoding immune cells, is screened out. The siRNA is introduced into cells without the help of a carrier and is made resistant to nucleases by chemical modification, thereby eliminating the cytotoxicity caused by the carrier, and can be effectively used as a prophylactic or therapeutic agent for cancer by more effectively inhibiting gene expression in vivo. Description of the Drawings

[0026] Figure 1 It is a schematic structural diagram of an asymmetric siRNA targeting PD-1, which is composed of a sense strand (16mer) and an antisense strand (21mer).

[0027] Figure 2Histogram of PD-1 mRNA levels (n = 3) obtained by transfecting asymmetric siRNA at a concentration of 10 nM, showing the expression levels (%) based on untreated samples (control group: NT) using tubulin as a housekeeping gene. siOCT4 was used as a negative control.

[0028] Figure 3 Histogram of PD-1 mRNA levels (n = 3) obtained by transfecting asymmetric siRNA at a concentration of 1 nM, showing the expression levels (%) based on untreated samples (control group: NT) using tubulin as a housekeeping gene. siOCT4 was used as a negative control.

[0029] Figure 4 Histogram of PD-1 protein levels (n = 4) obtained by enzyme-linked immunosorbent assay (ELISA) after transfection of three asymmetric siRNAs (asiPD-1-93, asiPD-1-95, asiPD-1-89) with good PD-1 expression inhibitory effects.

[0030] Figure 5 Photograph of the Western blot result of PD-1 protein levels (n = 3), showing the PD-1 expression inhibitory efficacy against 12 cp-asiRNAs specified in Table 3.

[0031] Figure 6 Photograph of the Western blot result of PD-1 protein levels (n = 3), showing the PD-1 expression inhibitory efficacy against 11 cp-asiRNAs specified in Table 4.

[0032] Figure 7 Photograph of the Western blot result of PD-1 protein levels (n = 3), showing the duration test results (from 2 days to 5 days) of two types (cp-asiPD-1-22, cp-asiPD-1-23) that showed excellent efficiency among the 11 cp-asiRNAs specified in Table 4.

[0033] Figure 8 Histogram showing the luciferase luminescence measurement values (n = 3) expressed as relative values (average relative light unit) after treating with cp-asiRNA at concentrations of 0.5 μM, 1 μM, and 3 μM for 4 days (96 hours), showing Figure 7 the increased T cell activity confirmed by the PD-1 expression inhibition of cp-asiPD-1-22 and cp-asiPD-1-23.

[0034] Figure 9 Histogram showing the luciferase luminescence measurement values (n = 3) expressed as relative values (average relative light unit) after treatment with a concentration of 1 μM for 3 days under the same conditions as Figure 8 that shown, demonstrating increased T cell activity due to PD-1 expression inhibition by cp-asiPD-1-22 and asiPD-1-23.

[0035] Figure 10 Based on the expression level (%) of PD-1 mRNA in untreated samples (control group: NT), showing the PD-1 expression inhibition efficacy against 8 asymmetric siRNAs specified in Table 5.

[0036] Figure 11 Photograph of the Western blot results of the PD-1 protein level, showing the PD-1 expression inhibition efficacy of 6 types (8-039, 8-046, 8-048, 8-053, 8-068, and 8-070) that showed excellent efficiency among the 8 asymmetric siRNAs specified in Table 5.

[0037] Figure 12 Based on the expression level (%) of PD-1 mRNA in untreated samples (control group: NT), showing the PD-1 expression inhibition efficacy against 12 cp-asiRNAs specified in Table 6.

[0038] Figure 13 Based on the expression level (%) of PD-1 mRNA in untreated samples (control group: NT), showing the PD-1 expression inhibition efficacy of 4 types (cp-8-068-3, 8-068-13, 8-039-8, and 8-039-13) that showed excellent efficiency among the 12 cp-asiRNAs specified in Table 6.

[0039] Figure 14 Photograph of the Western blot results of the PD-1 protein level, showing the PD-1 expression inhibition efficacy of 4 types (8-068-3, 8-068-13, 8-039-8, and 8-039-13) that showed excellent efficiency among the 12 cp-asiRNAs specified in Table 6.

[0040] Figure 15 Based on the expression level (%) of PD-1 mRNA in untreated samples (control group: NT), showing the PD-1 expression inhibition efficacy in T cells against 4 cp-asiRNAs (8-068-3, 8-068-13, 8-039-8, and 8-039-13).

[0041] Figure 16 Histogram showing luciferase luminescence measurements relative to untreated samples (control: NT) after treatment with cp-asiRNA at 1 μM and 2 μM concentrations, showing Figure 15 the increased T cell activity confirmed in [reference] through the PD-1 expression inhibition by cp-asiRNAs (8-068-3, 8-068-13, 8-039-8, and 8-039-13). Detailed implementation

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is the same as that known and commonly used in the art.

[0043] The definitions of key terms used in the detailed description of the present invention and the like are as follows.

[0044] "RNA interference (RNAi)" refers to a mechanism that induces the degradation of target gene mRNA by introducing double-stranded RNA (dsRNA) composed of a strand having a sequence homologous to the mRNA of the target gene and a strand having a sequence complementary thereto into cells, etc., thereby inhibiting the expression of the target gene.

[0045] "siRNA (small interfering RNA)" refers to short double-stranded RNA (dsRNA) that mediates sequence-specific and effective gene expression inhibition (gene silencing).

[0046] "Antisense strand" refers to a polynucleotide that is substantially or 100% complementary to a target nucleic acid of interest, and can be complementary to messenger RNA (mRNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences, either in whole or in part.

[0047] "Sense strand", having the same nucleic acid sequence as the target nucleic acid, refers to a polynucleotide that is identical, either in whole or in part, to messenger RNA (mRNA), non-mRNA RNA sequences (e.g., microRNA, piwiRNA, tRNA, rRNA, and hnRNA), or coding or non-coding DNA sequences.

[0048] "Gene" should be understood in the broadest sense and can encode a structural protein or a regulatory protein. At this time, regulatory proteins include proteins involved in transcription factors, heat shock proteins, or DNA / RNA replication, transcription, and / or translation.

[0049] "PD-1 (PD-1)" is the abbreviation of Programmed Cell Death protein 1, which belongs to the type I transmembrane proteins of the CD28 family. The human PD-1 gene is located on chromosome 2q37.35 and encodes a transmembrane protein of approximately 55 kD. PD-1 is widely expressed on the surfaces of activated T cells, B cells, monocytes, and dendritic cells. The structure of PD-1 has 30% homology with cytotoxic T lymphocyte-associated protein 4 (CTLA-4). There are two tyrosine residues in the intracellular region, which are respectively involved in constructing an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N-terminus and an immunoreceptor tyrosin-based switch motif (ITSM) at the C-terminus. The extracellular region consists of an IgV-like domain, including multiple glycosylation sites and being highly glycosylated. This domain can bind to ligands, thereby exerting the function of inhibiting T cell activation. PD-L1 is overexpressed in most cancer tissues, including non-small cell lung cancer (NSCLC), melanoma, breast cancer, glioma, lymphoma, leukemia, and various urinary tract cancers, gastrointestinal cancers, reproductive system tumors, etc. High expression of PD-L1 can regulate the expression of cell cycle checkpoint proteins and cell proliferation-related proteins by inhibiting the RAS and PI3K / AKT signaling pathways, ultimately resulting in the inhibition of T cell proliferation. It has been found in in vitro experiments and mouse models that the activation of the PD-1 / PD-L1 signaling pathway can induce apoptosis of specific cytotoxic lymphocytes (CTL), reduce the sensitivity of the cytotoxic killing effect of CTL, and promote immune escape of tumor cells.

[0050] The present invention mainly has two objectives. One is to design asymmetric siRNAs targeting the mRNA encoding PD-1 and screen out the most effective asymmetric siRNAs for inhibiting PD-1 expression. The other is to deliver siRNAs into cells without a specific vector and introduce chemical modifications to increase resistance to nucleases. To cross the cell membrane composed of phospholipids, it should be very small or hydrophobic. However, due to the negatively charged phosphate backbone, siRNAs are difficult to penetrate the cell membrane. In addition, it is necessary to increase their resistance to nucleases to extend their lifespan in serum and ensure that the amount reaching the target is sufficient to induce effective RNAi. Therefore, by introducing chemical modifications, the delivery problem of siRNAs is overcome.

[0051] In one embodiment of the present invention, an asiRNA targeting PD-1 mRNA was designed, and the asiRNA with the best knockdown efficiency was identified and screened. In addition, through chemical modification, without the aid of a vector, the asiRNA was modified to have cell-penetrating ability and resistance to nucleases, thereby screening out cp-asiRNA that can effectively inhibit PD-1 expression. The following four modifications were introduced into the selected siRNA to endow the asiRNA with cell-penetrating ability and resistance to nucleases. First, cholesterol was added to the 3'-end of the sense strand to enable the siRNA to penetrate the cell membrane. Second, by replacing the phosphate backbone near the 5'-end or 3'-end of the sense strand and the antisense strand with phosphorothioate, the siRNA was made resistant to exonucleases, could be absorbed by cells, and the bioavailability of the siRNA in vivo was increased. Third, by modifying the 2'-OH group of the sugar with 2'-O-methyl, resistance to nucleases was conferred, the immunogenicity of the siRNA was reduced, and the off-target effect was decreased. Fourth, by fluorinating the 2' of the sugar, stability was provided for the double strand duplex, stability in serum was increased, and silencing could be effectively carried out in vitro and in vivo. By performing the above modifications on the siRNA, the siRNA can have cell-penetrating ability, stay in serum for a longer time, and a sufficient number of siRNAs are delivered to target cells, thereby more effectively inhibiting the gene.

[0052] Therefore, according to one aspect of the present invention, there is provided an asymmetric siRNA, characterized by comprising: an antisense strand containing a sequence complementary to the mRNA encoding PD-1, namely Programmed Cell Death protein 1 (PD-1); and a sense strand forming a complementary bond with the antisense strand, wherein the 5'-end of the antisense strand and the 3'-end of the sense strand form a blunt end.

[0053] The siRNA in the present invention is a concept that includes all substances having the general RNAi (RNA interference) effect. Therefore, it is not necessarily limited to synthetic siRNA, and can also be applied to siRNA or shRNA expressed using an expression vector or the like in cells. RNAi is a method of intracellular gene regulation first discovered by the Fire research group in Caenorhabditis elegans in 1998. Its mechanism of action is known to induce degradation of the target gene by complementary binding of the antisense strand in the double-stranded RNA introduced into the cell to the mRNA of the target gene. Among them, synthetic RNA interference (siRNA) is one of the methods for "in vitro" inhibition of gene expression. 19-21bp siRNA can theoretically selectively inhibit almost all genes, and it is a technology that can be developed for the treatment of various gene-related diseases such as cancer, rare diseases, fibrosis, and viral infections. In the mid-2000s, the first attempt was made to use siRNA for in vivo treatment in mammals, and since then, many attempts at applied research have been made. In particular, in 2018, as the first siRNA-based RNAi therapeutic agent for hereditary transthyretin-mediated amyloid polyneuropathy (hATTR), Alnylam Pharmaceuticals' Onpattro was approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA), and before that, it was designated as an innovative therapeutic agent or rare drug. In addition, many global pharmaceutical companies are investing more than billions of dollars to develop RNAi therapy projects, including siRNA for various refractory diseases. However, existing RNA therapeutic agents still have obstacles to overcome: 1) lack of an effective delivery system; 2) off-target effects; 3) induction of immune responses; and 4) saturation of the intracellular RNAi mechanism. Although siRNA is an effective method for directly regulating target gene expression, due to the above problems, it is difficult to develop therapeutic agents using such siRNA.

[0054] To solve these problems, the present invention provides an asymmetric siRNA, which includes an antisense strand and a sense strand complementary to the antisense strand. The siRNA according to the present invention does not cause problems such as off-target effects and saturation of the RNAi mechanism, so that high delivery efficiency can be stably maintained, and the expression of the PD-1 target gene can be effectively inhibited to the required level.

[0055] In the siRNA of the present invention, the length of the sense strand can be 15 nt to 17 nt, and the length of the antisense strand can be more than 16 nt. However, it is not limited thereto. The length of the antisense strand can be 16 nt to 31 nt, and the preferred length can be 19 nt to 25 nt. More preferably, the length of the sense strand can be 16 nt, and the length of the antisense strand complementary thereto can be 19 nt, 21 nt or 25 nt, but it is not limited thereto. The 3'-end of the sense strand and the 5'-end of the antisense strand form a blunt end. The 3'-end of the antisense strand can include, for example, an overhang of 1 nt to 16 nt.

[0056] In one embodiment of the present invention, 36 asiRNAs were designed to inhibit PD-1 expression, and inhibition at the mRNA level and protein level was confirmed in HeLa-PD-1 cells expressing PD-1.

[0057] According to the present invention, it is characterized in that the sense strand can be selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 77, 79, 81, 83, 85, 87, 89 and 91. Preferably, it has been confirmed that siRNAs including a sense strand selected from SEQ ID NO: 43, 47, 49, 77 and 89 and an antisense strand complementary to the sense strand have the best effect of inhibiting PD-1 expression.

[0058] According to the present invention, it is characterized in that the antisense strand can be selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 78, 80, 82, 84, 86, 88, 90 and 92. Preferably, it is characterized in that the antisense strand can be selected from SEQ ID NO: 44, 48, 50, 78 and 80.

[0059] More preferably, the sense strand of the siRNA according to the present invention is SEQ ID NO: 43, 47, 49, 77 or 89, and the antisense strand is SEQ ID NO: 44, 48, 50, 78 or 90.

[0060] In the present invention, the sense strand or antisense strand of the siRNA can include one or more chemical modifications.

[0061] Due to its phosphate backbone structure, general siRNA has a high negative charge and high molecular weight, so it cannot pass through the cell membrane, and it is rapidly degraded and removed in the blood, making it difficult to deliver a sufficient amount of RNAi induction to the actual target site. Currently, for in vitro delivery, many efficient delivery methods using cationic lipids and cationic polymers have been developed. However, for in vivo delivery, it is difficult to achieve the same high level of siRNA as in vitro delivery, and the siRNA delivery efficiency is reduced due to interactions with various proteins present in the living body.

[0062] Accordingly, the present inventors chemically modified the asymmetric siRNA structure and developed an asiRNA structure (cp-asiRNA) with self-delivery ability, which can effectively perform intracellular delivery without a separate carrier.

[0063] In the present invention, the chemical modification in the sense strand or antisense strand may include one or more of the following: the -OH group at the 2'-carbon position of the sugar structure in the nucleotide is substituted by -CH3 (methyl), -OCH3 (methoxy), -NH2, -F (fluorine), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl; the oxygen in the sugar structure of the nucleotide is substituted by sulfur; the phosphoester bond of the nucleotide is modified by phosphorothioate, phosphorodithioate, boranophosphate or methyl phosphonate; the nucleotide is substituted by peptidenucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA); and the binding of a phosphate group, a lipophilic compound or a cell-penetrating peptide.

[0064] In the present invention, the lipophilic compound may be selected from cholesterol, tocopherol, docosahexaenoic acid (DHA), palmitic acid and long-chain fatty acids having more than 10 carbon atoms. Preferably, it may be cholesterol, but is not limited thereto.

[0065] Specifically, it may include one or more modifications selected from the following: the -OH group at the 2'-carbon position of the sugar structure in at least one or more nucleotides of the sense strand or the antisense strand is replaced by -OCH3 (methoxy) or -F (fluoro); at least one or more nucleotide bonds of the sense strand or the antisense strand are modified with phosphorothioate bonds; a lipophilic compound is bound to the 3'-end of the sense strand; and a phosphate group is bound to the 5'-end of the antisense strand.

[0066] The sense strand of the siRNA according to the present invention is any one of (a) to (c) selected from the following table, and the antisense strand is any one of (d) to (o) selected from the following table.

[0067]

[0068] In the above table, * represents a phosphorothioated bond, m represents 2'-O-methyl, / chol / represents cholesterol. Specifically, "*" represents a form in which the existing phosphodiester bond is replaced by a phosphorothioate bond, and "m" represents a form in which the existing 2'-OH is replaced by 2'-O-methyl. In addition, "2'-F-" means, for example, for 2'-FG, a form in which the 2'-OH of the existing G (guanine) is replaced by fluorine, and "Chol" represents a form in which cholesterol is added to the 3'-end. One to three phosphate groups may be further bound to the 5'-end of the antisense strand.

[0069] Preferably, the sense strand may be (b) in the above table, and the antisense strand may be (i) or (m) in the above table.

[0070] More specifically, the sense strand may be any one of (a) to (c) selected from the following table, and the antisense strand may be any one of (d) to (h) selected from the following table.

[0071]

[0072] In the above table, * represents a phosphorothioated bond, m represents 2'-O-methyl, 2'-F- is 2'-fluoro, / chol / represents cholesterol, and 5'P represents a 5'-phosphate group.

[0073] Most preferably, the sense strand may be (c) in the above table, and the antisense strand may be (g) or (h) in the above table.

[0074] Preferably, the sense strand can be any one of (A) to (D) selected from the following table, and the antisense strand can be any one of (E) to (H) selected from the following table. More preferably, the siRNA can be sense strand (A) and antisense strand (E); sense strand (B) and antisense strand (F); sense strand (C) and antisense strand (G); or sense strand (D) and antisense strand (H).

[0075]

[0076] In the above table, * can represent a phosphorothioated bond, m can represent 2'-methyl, 2'-F- can represent 2'-F (fluorine), / chol / can represent cholesterol, and 5'P can represent a 5'-phosphate group.

[0077] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the siRNA.

[0078] In the present invention, the cancer can include but is not limited to multiple myeloma, non-epithelial tumors, non-small cell lung cancer, melanoma, breast cancer, glioma, lymphoma, leukemia, urinary tract cancer, digestive tract cancer, reproductive system tumors, refractory classical Hodgkin lymphoma, prostate cancer, metastatic melanoma, renal cell carcinoma, Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial cell carcinoma, refractory B-cell precursor acute lymphoblastic leukemia, and diffuse large B-cell lymphoma. As a composition for preventing or treating cancer known to express PD-1 ligands represented by PD-L1, it can be used without limitation.

[0079] In addition to the active ingredient siRNA, the pharmaceutical composition can be prepared by including one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier should be compatible with the active ingredient of the present invention and can be used by mixing with one or more of the components such as saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and other conventional additives can be added as needed, such as antioxidants, buffers, bacteriostatic agents, etc. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to form injectable dosage forms, such as aqueous solutions, suspensions, emulsions, etc. In particular, it is preferably provided in a lyophilized form. The preparation of the lyophilized dosage form can adopt methods well known in the art to which the present invention pertains, and a lyophilization stabilizer can be added.

[0080] Those skilled in the art can determine the method of administering the pharmaceutical composition according to the symptoms of the patient and the severity of the disease. Additionally, it can be formulated into various forms, such as powders, tablets, capsules, liquids, injections, ointments, syrups, etc., and can be provided using unit-dose or multi-dose containers, such as sealed ampoules and bottles, etc.

[0081] The pharmaceutical composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited thereto, but can be, for example, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration. The dose of the composition according to the present invention varies according to the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or the severity of the disease, and can be easily determined by those of ordinary skill in the art. Additionally, for clinical administration, the composition of the present invention can be formulated into a suitable dosage form using known techniques.

[0082] Another aspect of the present invention relates to a method for preventing or treating cancer, which comprises administering the siRNA to an individual. The configuration included in the improvement or treatment method according to the present invention is the same as the configuration included in the above-mentioned present invention, and thus the above description equally applies to the improvement or treatment method.

[0083] Another aspect of the present invention relates to immune cells with inhibited PD-1 expression obtained by treating immune cells with the siRNA.

[0084] As used herein, the term "immune cell" refers to a cell involved in the immune response that expresses PD-1. Preferably, it can be an immune cell for tumor immunotherapy, but is not limited thereto.

[0085] In the present invention, the immune cells may be selected from natural killer cells (NK cells), chimeric antigen receptor-modified NK cells (CAR-NK cells), Universal-NK cells, cytotoxic T lymphocytes (CTL), tumor infiltrating lymphocytes (TIL), peripheral blood mononuclear cells (PBMC), T cell receptor-modified T cells (TCR-T), and chimeric antigen receptor-modified T cells (CAR-T). Preferably, it may be human CD8+ T cells, but is not limited thereto.

[0086] As used herein, the term "chimeric antigen receptor-modified T cell (CAR-T cell)" or "chimeric antigen receptor-modified NK cell (CAR-NK cell)" refers to a T cell or NK cell expressing a "chimeric antigen receptor".

[0087] As used herein, the term "chimeric antigen receptor (CAR)" is a recombinant polypeptide construct that provides a target cell and intracellular signal generation to cells in immune effector cells. The chimeric antigen receptor is a molecule that binds to the receptor-activating intracellular domain of a T cell or NK cell to produce a chimeric protein that exhibits antibody-based specificity and specific anti-tumor cell immune activity against a target antigen (e.g., a tumor antigen).

[0088] As used herein, the term "chimeric" means composed of some different proteins or DNAs from different sources. The chimeric antigen receptor at least includes an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain.

[0089] This immune cell-based tumor immunotherapy has been successfully applied clinically and shown unprecedented clinical efficacy. Despite the significant effects of immune cell-based tumor immunotherapy, various difficulties have been presented during the actual tumor treatment process. One of the most important reasons is the PD-1 / PD-L1 inhibitory immune checkpoint, which inhibits the immune activity of immune cells by binding to inhibitory signal transduction, thus greatly reducing the effect of the immune cell-based tumor immunotherapy.

[0090] Therefore, the present inventors developed immune cells with inhibited PD-1 expression obtained by treating with the siRNA, and prevented the effect of the immune cell-based tumor immunotherapy from being reduced by the inhibitory immune checkpoint of the PD-ligand expressed by cancer cells.

[0091] In one embodiment of the present invention, when effector T cells expressing PD-1 are treated with cp-asiRNA, it can be confirmed that excellent activity recovery effects are exhibited even in an environment where PD-L1-expressing cells are present. Accordingly, even for any of the immune cells, such as NK cells, chimeric antigen receptor-modified natural killer cells, chimeric antigen receptor-modified T cells (CAR-T), and T cell receptor-modified T cells (TCR-T), etc., the recovery of immune cell activity can be expected by inhibiting PD-1 expression using siRNA.

[0092] On the other hand, the present invention relates to an immunocyte therapeutic agent for cancer comprising the immune cells with inhibited PD-1 expression.

[0093] As used herein, the term "immunocyte therapeutic agent" refers to a drug for treating diseases by activating the in vivo immune response using immune cells such as dendritic cells, natural killer cells, and T cells. The immunocyte therapeutic agents under development mainly target cancer treatment as an indication, which activate the immune function by directly administering immune cells to patients to obtain therapeutic effects, and have a treatment mechanism and efficacy different from the existing surgical therapies, anticancer agents, and radiotherapy for cancer treatment, thus occupying a major position in biopharmaceuticals.

[0094] According to the characteristics of the immune cells used and the genes introduced into the cells during the preparation process, immune cell therapeutics can include, but are not limited to, dendritic immunomodulatory cell therapeutics, natural killer cells (NK cells), chimeric antigen receptor-modified NK cells, universal natural killer cells, lymphokine-activated killer (LAK) cells, tumor-infiltrating T lymphocytes (TIL), T cell receptor-modified T cells (TCR-T), and chimeric antigen receptor-modified T cells (CAR-T).

[0095] In the present invention, the cancers can include, but are not limited to, breast cancer, multiple myeloma, non-epithelial tumors, non-small cell lung cancer (NSCLC), melanoma, glioma, lymphoma, leukemia, urinary tract cancer, digestive tract cancer, reproductive system tumors, refractory classic Hodgkin's lymphoma, prostate cancer, metastatic melanoma, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, urothelial cell carcinoma, refractory B cell precursor acute lymphoblastic leukemia, and diffuse large B cell lymphoma. As immune cell therapeutics for cancers known to express PD-1 ligands represented by PD-L1, they can be used without limitation.

[0096] The present invention will be described in more detail below by way of examples. It is obvious to those of ordinary skill in the art that these examples are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited by these examples.

[0097] Example 1: Screening of 36 RNAi-Inducing Double-Stranded Nucleic Acid Molecules Targeting PD-1

[0098] 1.1. Confirmation of PD-1 Expression in HeLa-PD-1 Cell Line

[0099] Before designing asiRNA and verifying its inhibitory effect on PD-1 mRNA expression, the expression of PD-1 in the HeLa-PD-1 cell line was confirmed using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The characteristics of HeLa-PD-1 can be that after transfecting the plasmid encoding PD-1 mRNA into HeLa (ATCC), a cell line screened using the selection marker hygromycin B (Gold Biotechnology Inc) is used to stably overexpress PD-1. This overcomes the limitation that transcription is induced only by stimulation rather than by a protein that always expresses PD-1, and screening can be carried out smoothly.

[0100] 1.2. Screening of Targeted PD-1 Gene and Design and Preparation of 36 asiRNAs

[0101] To obtain a double-stranded nucleic acid molecule capable of inducible and highly efficient RNAi interference targeting PD-1, asiRNA was designed after selecting the target sequence for the PD-1 gene. Compared with well-known siRNA, the asiRNA structure has different secondary structures, GC content (%), 5' end stability differences, etc. Therefore, it may be somewhat difficult to design optimized asiRNA when using a conventional siRNA design program to design the base sequence of asiRNA. Therefore, the design of the candidate asiRNA of the present invention was carried out as follows.

[0102] PD-1 gene information (mRNA accession number: NM_005018.2) was obtained by retrieving through the NCBI database. Considering animal experiments, first, a base sequence showing 100% homology was selected based on the base sequence having a common target with mice, and then in the antisense strand except for the seed region (2-8 base sequence based on the 5'), a base sequence allowing mismatch was additionally selected to design a total of 36 asiRNAs, which were then synthesized at a scale of 10 nmole by OliX Pharmaceuticals, Inc. (South Korea). The base sequences of the designed 36 asiRNAs are shown in Table 1. The asiRNAs were diluted to an appropriate concentration in 5x siRNA duplex (Bioneer), annealed through a process of 5 minutes at 95 °C and incubation at 37 °C for 1 hour, and after 12% polyacrylamide gel electrophoresis (PAGE), QC was carried out using a ChemiDoc ultraviolet transilluminator (Biorad). The base sequences of the screened 36 asiRNAs are shown in Table 1 below.

[0103]

Table 1

[0104]

[0105]

[0106] 1.3. Confirmation of Inhibitory Effect of 36 asiRNAs on PD-1 mRNA Expression

[0107] To confirm the gene silencing efficiency at the mRNA level, after transfecting the selected 36 asiRNAs into the HeLa-PD-1 cell line at concentrations of 10 nM and 1 nM, qRT-PCR was performed to measure the expression level of PD-1 mRNA.

[0108] Specifically, HeLa-PD-1 cells were cultured in Dulbecco's modified Eagle's Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.2 g / L hygromycin B. HeLa-PD-1 cells cultured 24 hours before transfection were seeded into 12-well plates. Then, according to the protocol provided by the manufacturer, 36 asiRNAs (Table 1) at 1 nM or 10 nM were transfected using RNAimax (Invitrogen). After 24 hours, total RNA was extracted using Tri-RNA reagent (FAVORGEN), and then 500 ng of the RNA was used for cDNA synthesis. According to the manufacturer's protocol, a high-capacity cDNA reverse transcription kit (Applied Biosystems) was used to synthesize cDNA. Then, qRT-PCR was performed on the synthesized cDNA using a StepOne real-time PCR system (Applied Biosystems). Using the primers in Table 2 and SYBR Green PCR master mix (Applied Biosystems), the PD-1 expression levels of asiRNA-transfected samples relative to untreated samples (NT) were converted to percentages ( Figure 2 (10 nM), Figure 3 (1 nM)). Tubulin was used as a housekeeping gene (Table 2).

[0109]

Table 2

[0110]

[0111] The experiment was independently performed 3 times, and the gene silencing efficiencies of asiRNAs for the 36 sequences targeting PD-1 mRNA are shown as Figure 2 (10 nM) and Figure 3 (1 nM). The values represent the mean ± standard deviation of independent experiments. The results of screening a total of 36 base sequences are shown as Figure 2As shown, 25 types were confirmed to inhibit the expression of PD-1 mRNA by more than 50% at a concentration of 10 nM. When the concentration was reduced to 1 nM, as Figure 3 shown, 14 out of a total of 36 base sequences were confirmed to inhibit the expression of PD-1 mRNA with an efficiency of 50% or higher. In particular, based on the IC50 measurement results for asiRNAs with a gene inhibition efficiency of 80% or higher, it was confirmed that asiPD-1-93 and asiPD-1-95 had the lowest IC50. Among them, asiPD-1-89 was also selected as a candidate group for chemical modification and self-delivery experiments.

[0112] 1.4. Confirmation of Protein Expression Inhibitory Effect by Quantifying PD-1 Protein Levels of 3 asiRNAs

[0113] Based on the screening results of 36 asiRNAs, two of the most effective types (asiPD-1-93, asiPD-1-95) were selected from the aiRNAs with a gene inhibition efficiency of 80% or higher. Additionally, asiPD-1-89 was selected. After transfection at a concentration of 1 nM, the PD-1 protein level was confirmed using a Human Programmed Death 1 (PD-1) Enzyme-Linked Immunosorbent Assay Kit (Mybiosoure).

[0114] Specifically, HeLa-PD-1 cells were cultured in Dulbecco's modified Eagle's Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.2 g / L hygromycin B in a 100 mm cell culture dish. The HeLa-PD-1 cells cultured 24 hours before transfection were seeded into a 12-well plate. Then, according to the protocol provided by the manufacturer, 1 nM of 3 asiRNAs (asiPD-1-89, asiPD-1-93, asiPD-1-95) were transfected using RNAimax (Invitrogen). The medium was changed to a new medium 24 hours after transfection and passaged again using a 12-well plate 48 hours later. After 96 hours, harvesting was performed using 5X RIPA (self-made) buffer to prepare cell lysates. After quantification by BCA assay (Pierce), the PD-1 protein level was confirmed using a Human Programmed Death 1 (PD-1) Enzyme-Linked Immunosorbent Assay Kit (Mybiosoure) according to the manufacturer's protocol.

[0115] The experiment was independently conducted four times, and the PD-1 protein levels of the 3 selected candidate groups (% of the control group) were as Figure 4As shown, compared with the control group (NT), asiPD-1-93 and asiPD-1-95 with the highest gene silencing efficiency showed PD-1 protein levels below 20%, and asiPD-1-89 also showed a PD-1 protein level of approximately 40%, thus confirming that each candidate group had a high PD-1 expression inhibition efficiency.

[0116] Example 2: Screening of RNAi-induced asymmetric double-stranded nucleic acid molecules (cp-asiRNAs) with cell-penetrating ability targeting PD-1

[0117] 2.1. Design and Preparation of 12 cp-asiRNAs with Cell-Penetrating Ability

[0118] For three candidate asiRNAs targeting PD-1 (asiPD-1-89, asiPD-1-93, and asiPD-1-95), chemical modifications that promote cell penetration and contribute to the stability of the RNA structure were selected and applied in two steps. In the first chemical modification, cholesterol was conjugated to the nucleotide present at the 3'-end of the 16-nt sense strand, and the three phosphate backbones present in the 5'-direction were replaced with phosphorothioate, and the hydroxyl group at the 2'-position of the ribose of 8 nucleotides out of the 16 nucleotides of the sense strand was replaced with -O-methyl. Additionally, the length of the antisense strand was 19 nt or 25 nt, the hydroxyl group at the 2'-position of the ribose of the nucleotides from 2 nt to 11 nt was replaced with -O-methyl, and the phosphate backbones of the 4 nucleotides present in the 3'-end direction were replaced with phosphorothioate, thereby designing 12 cp-asiRNAs. The base sequences of the 12 cp-asiRNAs designed in this way are shown in Table 3. cp-asiPD-1-1 to cp-asiPD-1-5 were designed based on asiPD-1-95 in Table 1, cp-asiPD-1-6 to cp-asiPD-1-10 were designed based on asiPD-1-93 in Table 1, and cp-asiPD-1-11 to cp-asiPD-1-12 were designed based on asiPD-1-89 in Table 1. The 12 cp-asiRNAs designed in this way were diluted to an appropriate concentration in OptiMEM (Gipco), annealed through a process of 5 minutes at 95 °C and 1 hour of incubation at 37 °C, and after performing 12% polyacrylamide gel electrophoresis (PAGE), QC was performed using a ChemiDoc ultraviolet transilluminator (Biorad).

[0119]

Table 3

[0120]

[0121] 2.2. Toxicity Verification of 12 cp-asiRNAs and Verification of Cell Permeability and PD-1 Expression Inhibitory Efficacy at Protein Level Figure 5

[0122] To confirm the gene silencing efficiency at the protein level, after treating with cp-asiRNA at a concentration of 1 μM for 24 hours, the toxicity caused by cp-asiRNA was confirmed, and the expression level of PD-1 protein was measured 96 hours after the treatment.

[0123] Specifically, first, HeLa-PD-1 cells were cultured in Dulbecco's modified Eagle's Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.2 g / L hygromycin B (Gold Biotechnology Inc) in a 100 mm petri dish. The HeLa-PD-1 cells cultured 24 hours before the treatment were seeded into a 12-well plate. After seeding, 12 cp-asiRNAs were treated at a concentration of 1 μM in the absence of a separate vector.

[0124] To confirm the toxicity, the toxicity caused by cp-asiRNA was confirmed 24 hours after the treatment. After 24 hours, the medium was replaced with a new medium, and MTT (Sigma-Aldrich) dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS (Gibco)) was added to a final concentration of 5 mg / ml. After culturing at 37 °C for 2 hours, all the solutions were removed, DMSO (Sigma-Aldrich) was added, and then after culturing at 37 °C for 10 minutes again, the absorbance at 570 nm was measured. The results confirmed that the prepared cp-asiRNA did not induce cytotoxicity.

[0125] To measure the PD-1 protein expression, the cells were harvested 96 hours after the treatment to obtain cell lysates. After quantification using the Thermo Scientific Pierece BCA Protein Assay Kit (Thermo Scientific), the PD-1 protein level was confirmed by Western blotting. The anti-PD1 antibody (Abcam) was used as the primary antibody, and goat anti-rabbit IgG H&L (HRP) (Abcam) was used as the secondary antibody.

[0126] The protein level was independently examined 3 times. Figure 5 For its representative results. The experimental results are as 3.1. Design and Preparation of 11 cp-asiRNAs with Further Chemical Modification of cp-asiPD-1-6 to cp-asiPD-1-10As shown, gene expression inhibitory effects at the protein level were confirmed in 7 out of 12 cp-asiRNAs. Different from asiRNAs, for chemically modified cp-asiRNAs, it was confirmed that excellent effects could be shown even without a vector and only with treatment.

[0127] Example 3: Screening of RNAi-induced asymmetric double-stranded nucleic acid molecules (cp-asiRNAs) with cell-penetrating ability targeting PD-1

[0128] Figure 5 3.2. Toxicity Verification of Additional 11 Prepared cp-asiRNAs and Verification of Cell Permeability and

[0129] For PD-1 Expression Inhibitory Efficacy at Protein Level Among the 7 types with the lowest PD-1 protein expression levels among the 12 cp-asiRNAs with confirmed effects, chemical modifications were further introduced into cp-asiPD-1-6 to cp-asiPD-1-10, where cp-asiPD-1-6 to cp-asiPD-1-10 were further chemically modified on asiPD-1-93. At this time, the further modifications included: binding a phosphate to the nucleotide at the 5'-end of the antisense strand; or in both strands, substituting the hydroxyl group at the 2'-position of 10 to 12 nucleotides of ribose with an -O-fluorine group or with an -O-methyl group (methoxy). A total of 11 cp-asiRNAs were designed and prepared, and their base sequences are shown in Table 4.

[0130]

Table 4

[0131]

[0132] Figure 6 Figure 6

[0133] In the same manner as in Example 2.2, toxicity verification and verification of cell permeability and PD-1 expression inhibitory efficacy at the protein level were carried out for the additionally prepared 11 cp-asiRNAs. The experiments were independently conducted 3 times. Figure 6 These are the representative results. As Figure 7 shown, from Figure 7 it can be seen that cp-asiPD-1-22 and cp-asiPD-1-23 have the most excellent gene expression inhibitory efficiency at the protein level.

[0134] Example 4: Duration test for two cp-asiRNAs (cp-asiPD-1-22, cp-asiPD-1-23)

[0135] As shown in Example 3, in order to confirm the increased ability of the maintenance effect caused by chemical modification, duration tests were performed on cp-asiPD-1-22 and cp-asiPD-1-23, which showed the most excellent PD-1 expression inhibitory effect. asiPD-1-93 was used as a control, and cp-asiPIN1 was used as a negative control, which is a cp-asiRNA targeting a gene other than PD-1.

[0136] Specifically, HeLa-PD-1 cells were cultured in Dulbecco's modified Eagle's Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 0.2 g / L hygromycin B. HeLa-PD-1 cells cultured 24 hours before transfection were seeded into a 12-well plate. 24 hours after seeding, asiRNA (asiPD-1-93) was transfected using RNAimax (Invitrogen) according to the protocol provided by the manufacturer, and cp-asiPD-1-22 and cp-asiPD-1-23 were treated without a separate vector. Thereafter, in the same manner as in Example 2.2, the PD-1 protein level was confirmed by Western blotting ( Figure 7 ). The experiment was independently performed 3 times, Figure 7 and a representative photograph thereof is shown.

[0137] As 5.1. Functional Assay of T Cell Activation Ability of Two cp-asiRNAs (cp-asiPD-1-22, cp-asiPD-1-23) (96 hours) shown, it has been confirmed that the gene expression inhibitory effect of No. 22, which has the most excellent efficiency, began to be confirmed at the protein level between the 2nd and 3rd days after treatment and was maintained until the 4th day, and then the expression of the target gene PD-1 began to recover. Therefore, it was confirmed that the effect maintenance time of cp-asiPD-1 No. 22 was longer than that of asiPD-1 and cp-asiPD-1 No. 23.

[0138] Example 5: Functional assay of the T cell activation ability of two cp-asiRNAs (cp-asiPD-1-22, cp-asiPD-1-23)

[0139] It was confirmed that the use of Figure 8The gene expression inhibition efficiency of two cp-asiRNAs (cp-asiPD-1-22, cp-asiPD-1-23) Whether the gene expression inhibition causes an increase in T cell activity. Two cp-asiRNAs (cp-asiPD-1-22, cp-asiPD-1-23) and asiPD-1-93 were used. To confirm the effect, the currently commercially available PD-1 blocking antibody Nivolumab was also used. cp-asiPIN1 was used as a negative control, which is a cp-asiRNA targeting a gene other than PD-1. The following principle was adopted in this experiment: When T cells recognize an antigen bound to an MHC molecule on the surface of an antigen-presenting cell through the TCR, one of the results is the promotion of NFAT expression. When PD-1 is expressed, it interferes with the signal transduction system stimulated by the TCR, resulting in the inhibition of NFAT expression. Therefore, when NFAT is expressed, effector cells (T cells) genetically engineered to express luciferase when NFAT is expressed were used, and a PD-1 / PD-L1 blocking bioassay (Promega) was used, which can quantitatively confirm the effect of PD-1 activity inhibition or expression reduction.

[0140] Figure 8 5.2. Functional Assay of T Cell Activation Ability of Two cp-aiRNAs (cp-asiPD-1-22, cp-asiPD-1-23) (72 hours)

[0141] Specifically, effector cells expressing PD-1 were treated with asiPD-1-93 at concentrations of 3 μM, 1 μM, and 0.5 μM in the same manner as in Example 1.3, and the same concentrations of cp-asiPD-1-22 and cp-asiPD-1-23 were also treated in the same manner as in Example 2.2. After 96 hours of treatment, they were co-cultured with PD-L1 cells for 6 hours, and then luminescence was measured. The luminescence measurement value of each sample compared to the untreated sample (NT) was expressed as a relative value (average relative light unit, RLU). These measurements were performed using a PD-1 / PD-L blocking bioassay (Promega) and the protocol recommended by the manufacturer. Nivolumab used as a positive control was used at a concentration of 1 μg / mL. The experiment was independently performed 3 times, Figure 7 showing the mean ± standard deviation values of each experiment.

[0142] As Figure 8 shown, in terms of restoring T cell activity, cp-asiPD-1-22 demonstrated an effect similar to or better than that of the currently commercially available PD-1 antibody Nivolumab.

[0143] Figure 9 Figure 9

[0144] As in Example 4 ( 6.1. Design and Preparation of 8 asiRNAs) As confirmed, the gene expression efficiency of cp-asiPD-1-22 persisted from 3 days (72 hours) to 4 days (96 hours) at the protein level, as shown in Example 5.1( 6.2. Confirmation of Inhibitory Effect on PD-1 mRNA Expression ) As confirmed, cp-asiPD-1-22 had the best T cell activation ability, with the most significant increase in T cell activity at a concentration of 1 μM. Thus, the functional assay of the same method as in Example 5.1 was independently repeated 3 times under the condition of 72 hours. Figure 10 It is a graph showing the luminescence measurement values of each sample relative to the untreated sample (NT) of the experiment, expressed as relative values (average relative light unit, RLU).

[0145] As 6.3. Confirmation of Inhibitory Effect on PD-1 Protein Expression shown, the effect of cp-asiPD-1 No. 22 was about 2 times higher than the effect of Nivolumab in restoring T cell activity, and the statistical significance of this difference can be confirmed by the p-value of the t-test.

[0146] Example 6: Screening of 8 RNAi-induced double-stranded nucleic acid molecules targeting PD-1

[0147] Figure 11

[0148] In this example, RNAi-induced double-stranded nucleic acid molecules targeting PD-1 were designed and prepared in the same method as in Example 1.2. The nucleic acid molecules consisted of a 16-nt sense strand and a 19-nt antisense strand. The 8 asiRNA base sequences and target regions screened are shown in Table 5 below.

[0149] [Table 5]

[0150]

[0151] 7.1. Design and Preparation of 12 cp-asiRNAs with Further Chemical Modification of 8-039 and 8-068

[0152] In this example, to confirm the gene inhibition efficiency at the mRNA level, after transfecting the HeLa-PD-1 cell line with 8 asiRNAs at a concentration of 1 nM using the same method as in Example 1.3, qRT-PCR was performed to measure the expression level of PD-1 mRNA. Specifically, 24 hours after inoculating HeLa-PD-1 cells into a 12-well plate, according to the protocol provided by the manufacturer, asiRNA was transfected at a concentration of 1 nM using RNAimax (Thermo). 24 hours after transfection, RNA was extracted using Tri-RNA reagent (FAVORGEN), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Thereafter, the PD-1 expression inhibitory efficacy of asiRNA was evaluated at the mRNA level by qRT-PCR.

[0153] As a result, as 7.2. Confirmation of Inhibitory Effect on PD-1 mRNA Expression confirmed, all RNAi-induced double-stranded nucleic acid molecules reduced the expression of PD-1 mRNA.

[0154] Figure 12

[0155] In this example, after transfecting asiRNAs (8-039, 8-046, 8-048, 8-053, 8-068, and 8-070) showing a gene silencing efficiency of more than about 70% in the results of Example 6.2 into the HeLa-PD-1 cell line, for the cells obtained 48 hours later, the PD-1 expression inhibitory efficacy of the asiRNAs was evaluated at the protein level by the same method as in Example 1.4.

[0156] As a result, as Figure 13 confirmed, all RNAi-induced double-stranded nucleic acid molecules reduced the expression of PD-1 protein.

[0157] Example 7: Screening of RNAi-induced asymmetric double-stranded nucleic acid molecules (cp-asiRNAs) with cell-penetrating ability targeting PD-1

[0158] 7.3. Confirmation of Inhibitory Effect on PD-1 mRNA Protein Expression

[0159] For 8-068 and 8-039, which showed excellent PD-1 expression inhibitory efficacy among the 6 asiRNAs whose effects were confirmed in Example 8, chemical modifications were introduced by the same method as in Example 3.1. A total of 12 cp-asiRNAs were further designed and manufactured, and their base sequences are shown in Table 6.

[0160]

Table 6

[0161]

[0162]

[0163] Figure 14

[0164] In this example, to confirm the gene silencing efficiency at the mRNA level, after treating the HeLa-PD-1 cell line with 12 cp-asiRNAs at a concentration of 1 μM, qRT-PCR was performed to measure the expression level of PD-1 mRNA. Specifically, 24 hours after inoculating HeLa-PD-1 cells in a 12-well plate, the cp-asiRNAs were treated without a separate vector. After 24 hours of treatment, RNA was extracted using Tri-RNA reagent (FAVORGEN), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Thereafter, the PD-1 expression inhibitory efficacy of cp-asiRNAs was evaluated at the mRNA level by qRT-PCR. In addition, four of the 12 cp-asiRNAs, namely 8-068-3, 8-068-13, 8-039-8, and 8-039-13, were selected. After treating the HeLa-PD-1 cell line with a concentration of 1 μM or 2 μM, the PD-1 expression inhibitory efficacy was evaluated again at the mRNA level.

[0165] As a result, as Figure 15 confirmed, among the RNAi-inducing double-stranded nucleic acid molecules with cell-penetrating ability, 8-068-2, 8-068-3, 8-068-5, 8-068-7, 8-068-8, 8-068-13, 8-068-15, 8-039-3, and 8-039-13 reduced the expression of PD-1 mRNA. In addition, as Figure 16 shown, the expression inhibitory efficacy of 8-068-3, 8-068-13, 8-039-3, and 8-039-13 among them was significant.

[0166] ​

[0167] In this example, after treating the HeLa-PD-1 cell line with the 4 cp-asiRNAs showing excellent gene silencing efficiency in the results of Example 7.1, for the cells obtained after culturing for 24 hours therefrom, the PD-1 expression inhibitory efficacy of cp-asiRNAs was evaluated at the protein level by the same method as in Example 1.4.

[0168] As a result, as ​ confirmed, all RNAi-inducing double-stranded nucleic acid molecules with cell-penetrating ability (8-068-3, 8-068-13, 8-039-8, and 8-039-13) reduced the expression of PD-1 protein.

[0169] Example 8: Functional assay for the T cell activation ability of cp-asiRNA

[0170] In this example, when treating T cells with 8-068-3, 8-068-13, 8-039-8, and 8-039-13, which showed excellent PD-1 expression inhibitory efficacy among the cp-asiRNAs whose effects were confirmed in Example 7, it was confirmed whether the inhibition of PD-1 gene expression caused an increase in T cell activity. Specifically, cryopreserved human T cells (purchased from Stem Cell Technologies) were allowed to recover for 18 to 20 hours. Then, the recovered human T cells were treated with CD3 / Cd28 magnetic beads (Dynabeads) and 200 IU IL-2 in CTS OpTmizer (Thermo) containing 2% human serum to activate the T cells for 72 hours. After treating 20,000 activated T cells with cp-asiRNA in a 12-well plate for 72 hours, RNA was extracted using Tri-RNA reagent (FAVORGEN), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Thereafter, the PD-1 expression inhibitory efficacy of the treated T cells was evaluated at the mRNA level by qRT-PCR.

[0171] Then, effector cells (T cells) genetically engineered to express luciferase were used, and a Promega bioluminescence assay kit (product number: J4011) was used, which could quantitatively confirm the effect of PD-1 activity inhibition or expression reduction.

[0172] As a result, as ​ confirmed, all RNAi-inducing double-stranded nucleic acid molecules with cell-penetrating ability (8-068-3, 8-068-13, 8-039-8, and 8-039-13) reduced the expression of PD-1 mRNA in T cells. Additionally, as ​ confirmed, the expression inhibitory efficacy of the group treated with the RNAi-inducing double-stranded nucleic acid molecule with cell-penetrating ability was approximately 3 times superior compared to the recovery of T cell activity in the untreated group (NT).

[0173] The above has described in detail specific parts of the present invention. For those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents. Sequence Listing (SEQUENCE LISTING) <110> OriGene Technologies Inc. <120> Asymmetric siRNA for Inhibiting PD-1 Expression <130> 2120823KR08 <150> KR 2019-0058805 <151> 2019-05-20 <160> 92 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 1 cuguggggcc aucucc 16 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 2 ggagauggcc ccacagaggu a 21 <210> 3 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 3 ucuguggggc caucuc 16 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 4 gagauggccc cacagaggua g 21 <210> 5 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 ccuucaccug cagcuu 16 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 6 aagcugcagg ugaagguggc g 21 <210> 7 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 7 accuucaccu gcagcu 16 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 8 agcugcaggu gaagguggcg u 21 <210> 9 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 9 caccuucacc ugcagc 16 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 10 gcugcaggug aagguggcgu u 21 <210> 11 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 11 cucugugggg ccaucu 16 <210> 12 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 12 agauggcccc acagagguag g 21 <210> 13 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 13 ccucuguggg gccauc 16 <210> 14 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 14 gauggcccca cagagguagg u 21 <210> 15 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 15 accucugugg ggccau 16 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 16 auggccccac agagguaggu g 21 <210> 17 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 17 ccaccuucac cugcag 16 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 18 cugcagguga agguggcguu g 21 <210> 19 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 19 gccccagcaa ccagac 16 <210> 20 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 20 gucugguugc uggggcucau g 21 <210> 21 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 21 agccccagca accaga 16 <210> 22 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 22 ucugguugcu ggggcucaug c 21 <210> 23 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 23 ggcaccuacc ucugug 16 <210> 24 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 24 cacagaggua ggugccgcug u 21 <210> 25 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 25 cggcaccuac cucugu 16 <210> 26 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 26 acagagguag gugccgcugu c 21 <210> 27 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 27 gcggcaccua ccucug 16 <210> 28 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 28 cagagguagg ugccgcuguc a 21 <210> 29 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 29 agcggcaccu accucu 16 <210> 30 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 30 agagguaggu gccgcuguca u 21 <210> 31 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 31 cgugacuucc acauga 16 <210> 32 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 32 ucauguggaa gucacgcccg u 21 <210> 33 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 33 gggcgugacu uccaca 16 <210> 34 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 34 uguggaaguc acgcccguug g 21 <210> 35 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 35 acuaugggga gcugga 16 <210> 36 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 36 uccagcuccc cauaguccac a 21 <210> 37 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 37 gacacugcuc uuggcc 16 <210> 38 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 38 ggccaagagc aguguccauc c 21 <210> 39 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 39 gccaccuuca ccugca 16 <210> 40 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 40 ugcaggugaa gguggcguug u 21 <210> 41 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 41 cgccaccuuc accugc 16 <210> 42 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 42 gcaggugaag guggcguugu c 21 <210> 43 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 43 caugagcccc agcaac 16 <210> 44 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 44 guugcugggg cucaugcggu a 21 <210> 45 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 45 gcaugagccc cagcaa 16 <210> 46 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 46 uugcuggggc ucaugcggua c 21 <210> 47 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 47 agggugacag agagaa 16 <210> 48 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 48 uucucucugu cacccugagc u 21 <210> 49 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 49 cagggugaca gagaga 16 <210> 50 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 50 ucucucuguc acccugagcu c 21 <210> 51 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 51 ucagggugac agagag 16 <210> 52 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 52 cucucuguca cccugagcuc u 21 <210> 53 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 53 gacuaugggg agcugg 16 <210> 54 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 54 ccagcucccc auaguccaca g 21 <210> 55 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 55 aguggcgaga gaagac 16 <210> 56 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 56 gucuucucuc gccacuggaa a 21 <210> 57 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 57 caguggcgag agaaga 16 <210> 58 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 58 ucuucucucg ccacuggaaa u 21 <210> 59 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 59 ccaguggcga gagaag 16 <210> 60 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 60 cuucucucgc cacuggaaau c 21 <210> 61 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 61 uccaguggcg agagaa 16 <210> 62 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 62 uucucucgcc acuggaaauc c 21 <210> 63 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 63 ucugcagacc cuccac 16 <210> 64 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 64 guggaggguc ugcagaacac u 21 <210> 65 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 65 gcctggctcc tattgtccct c 21 <210> 66 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 66 caggugaagg uggcguuguc c 21 <210> 67 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 67 aacgccaccu ucaccu 16 <210> 68 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 68 aggugaaggu ggcguugucc c 21 <210> 69 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 69 uucugcagac ccucca 16 <210> 70 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 70 uggagggucu gcagaacacu g 21 <210> 71 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 71 ggacuauggg gagcug 16 <210> 72 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 72 cagcucccca uaguccacag a 21 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Foward Primer (Housekeeping gene Tublin) <400> 73 gaccaagcgt accatccagt 20 <210> 74 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (Housekeeping gene Tublin) <400> 74 acgtttggca tacatcagg 19 <210> 75 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Foward Primer (PD-1 mRNA qPCR primer) <400> 75 ccctggtggt tggtgtcgt 19 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer (PD-1 mRNA qPCR primer) <400> 76 gcctggctcc tattgtccct c 21 <210> 77 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 77 cuaaacuggu accgca 16 <210> 78 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 78 ugcgguacca guuuagcac 19 <210> 79 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 79 ggagagcuuc gugcua 16 <210> 80 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 80 uagcacgaag cucuccgau 19 <210> 81 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 81 cggagagcuu cgugcu 16 <210> 82 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 82 agcacgaagc ucuccgaug 19 <210> 83 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 83 ucugggcggu gcuaca 16 <210> 84 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 84 uguagcaccg cccagacga 19 <210> 85 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 85 cgcagaucaa agagag 16 <210> 86 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 86 cucucuuuga ucugcgccu 19 <210> 87 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 87 gcagaucaaa gagagc 16 <210> 88 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 88 gcucucuuug aucugcgcc 19 <210> 89 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 89 ggaguaugcc accauu 16 <210> 90 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 90 aaugguggca uacuccguc 19 <210> 91 <211> 16 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 91 ccauugucuu uccuag 16 <210> 92 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 92 cuaggaaaga caauggugg 19

Claims

1. A siRNA, characterized in that, Comprising: An antisense strand containing a sequence complementary to the mRNA encoding PD-1, i.e., programmed cell death protein 1; and a sense strand forming complementary bonds with the antisense strand, with the 5'-end of the antisense strand and the 3'-end of the sense strand forming blunt ends. The sense strand consists of the sequence of SEQ ID NO: 77, and the antisense strand consists of the sequence of SEQ ID NO:

78.

2. The siRNA according to claim 1, wherein The sense strand or the antisense strand includes one or more chemical modifications selected from the following (a) to (e): (a) The -OH group at the 2'-carbon position of the sugar structure in the nucleotide is substituted with -CH3, -OCH3, -NH2, -F (fluorine), -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl; (b) The oxygen in the sugar structure of the nucleotide is substituted with sulfur; (c) The phosphate backbone of the nucleotide is modified with phosphorothioate, boranophosphate or methylphosphonate; (d) The nucleotide is substituted with peptide nucleic acid PNA, locked nucleic acid LNA or unlocked nucleic acid UNA; and (e) The binding of a phosphate group, a lipophilic compound or a cell-penetrating peptide.

3. The siRNA according to claim 2, wherein The lipophilic compound is selected from cholesterol, tocopherol, docosahexaenoic acid DHA, palmitic acid and long-chain fatty acids with 10 or more carbon atoms.

4. The siRNA according to claim 2, wherein Including one or more chemical modifications selected from the following (a) to (d): (a) A modification in which the -OH group at the 2'-carbon position of the sugar structure in at least one or more nucleotides of the sense strand or the antisense strand is substituted with -OCH3 or -F; (b) A modification in which at least one or more nucleotide bonds of the sense strand or the antisense strand are modified with phosphorothioate bonds; (c) Binding cholesterol to the 3'-end of the sense strand; and (d) Binding a phosphate group to the 5'-end of the antisense strand.

5. The siRNA according to claim 2, wherein The sense strand and the antisense strand of the siRNA are selected from the following table, and the combinations of the sense strand and the antisense strand are selected from the following combinations: sense strand (a) and antisense strand (d); sense strand (b) and antisense strand (d); sense strand (c) and antisense strand (d); sense strand (c) and antisense strand (e); and sense strand (c) and antisense strand (f):

6. A pharmaceutical composition for preventing or treating cancer, which comprises the siRNA according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, wherein The cancer is selected from multiple myeloma, non-epithelial tumors, non-small cell lung cancer NSCLC, melanoma, breast cancer, glioma, lymphoma, leukemia, urinary tract cancer, digestive tract cancer, reproductive system tumors, renal cell carcinoma and head and neck squamous cell carcinoma.

8. The pharmaceutical composition according to claim 7, characterized in that, The melanoma is metastatic melanoma; the lymphoma is selected from Hodgkin lymphoma and diffuse large B-cell lymphoma; the leukemia is refractory B-cell precursor acute lymphoblastic leukemia; the urinary tract cancer is urothelial cell carcinoma; the reproductive system tumor is prostate cancer.

9. The pharmaceutical composition according to claim 8, wherein The Hodgkin lymphoma is refractory classical Hodgkin lymphoma.

10. An immune cell with inhibited expression of PD-1, characterized in that, It is an immune cell obtained by treating immune cells with the siRNA according to any one of claims 1 to 5.

11. The immune cell with inhibited expression of PD-1 according to claim 10, wherein The immune cells are selected from natural killer cells, namely NK cells, tumor-infiltrating T lymphocytes TIL, peripheral blood mononuclear cells PBMC, T cell receptor-modified T cells TCR-T, and chimeric antigen receptor-modified T cells CAR-T.

12. The immune cell with inhibited PD-1 expression according to claim 11, characterized in that, The natural killer cells are CAR-NK cells, namely chimeric antigen receptor-modified natural killer cells or universal natural killer cells.

13. An immune cell therapeutic agent for treating cancer, which comprises the immune cells with inhibited expression of PD-1 as claimed in claim 10.

14. Use of the siRNA as claimed in any one of claims 1 to 5 in the manufacture of a composition for preventing or treating cancer, wherein the cancer is selected from multiple myeloma, non-epithelial tumors, non-small cell lung cancer NSCLC, melanoma, breast cancer, glioma, lymphoma, leukemia, urinary tract cancer, digestive tract cancer, reproductive system tumors, renal cell carcinoma, and head and neck squamous cell carcinoma.

15. The application according to claim 14, wherein The melanoma is metastatic melanoma; the lymphoma is selected from Hodgkin lymphoma and diffuse large B-cell lymphoma; the leukemia is refractory B-cell precursor acute lymphoblastic leukemia; the urinary tract cancer is urothelial cell carcinoma; the reproductive system tumor is prostate cancer.

16. The application according to claim 15, characterized in that, The Hodgkin lymphoma is refractory classical Hodgkin lymphoma.

Images