KR20230128690A - Composition for Preventing or Treating Lung Cancer Comprising Nucleic Acid Complex - Google Patents

Abstract

The present invention relates to a nucleic acid complex and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient, and more particularly, to a bioactive nucleic acid targeting the PD-L1 gene; and a nucleic acid complex complementary to a carrier peptide nucleic acid and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient. The nucleic acid complex of the present invention, in which a bioactive nucleic acid targeting PD-L1 and a carrier peptide nucleic acid are complementaryly bound, has excellent cell penetration and intracellular activity, and can efficiently inhibit PD-L1 expression. It is useful for preventing or treating cancer, particularly lung cancer.

Description

Composition for preventing or treating lung cancer comprising a nucleic acid complex {Composition for Preventing or Treating Lung Cancer Comprising Nucleic Acid Complex}

The present invention relates to a nucleic acid complex and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient, and more particularly, to a bioactive nucleic acid targeting the PD-L1 gene; and a nucleic acid complex complementary to a carrier peptide nucleic acid and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient.

Lung cancer is a malignant tumor with a 5-year survival rate of only 8.9%. It is divided into non-small cell lung cancer and small cell lung cancer based on the size and shape of cancer cells, and 80-85% of patients are known to have non-small cell lung cancer. Lung cancer is discovered through screening because there are no early symptoms, and PD-L1 protein is an important biomarker that is tested not only for diagnosis but also for determining specific targeted therapies and immunotherapy in lung cancer patients (Beung-Chul Ahn, et al. ., European Journal of Cancer 153 (2021) 179-189).

PD-L1 (Programmed Death-Ligand 1) is a surface protein of cancer cells, and when it binds to the PD-1 receptor, a surface protein of T cells, T cell function is lost and death is induced to incapacitate immune cells. This causes immune system evasion, which prevents immune cells from attacking cancer cells, and worsens the proliferation of cancer cells into malignant tumors (Yanyan Han, et al., Am J Cancer Res 2020;10(3):727-742) . In particular, it is known that the survival rate of lung cancer patients decreases significantly with the increase in PD-L1 expression in non-small cell lung cancer patient tissues (Konrad Pawelczyk, et al., Int. J. Mol. Sci. 2019, 20(4), 824;YAJUAN QIU, et al., ONCOLOGY LETTERS 18: 161-168, 2019). Therefore, studies on immuno-anticancer agents for the PD-L1 biomarker, which helps immune cells attack cancer cells while blocking their mutual relationship by targeting the PD-1/PD-L1 pathway, are being actively conducted (Sun Min Lim, et al., Immune Netw. 2020 Feb;20(1):e10).

Among them, anti-PD-L1 antibody drugs that restore T cell functions by binding to PD-L1 and incapacitating immune evasion mechanisms have been studied a lot, and these antibody drugs have a superior treatment effect than conventional chemotherapy for lung cancer. However, due to the nature of antibody drugs, the degree of binding to the target protein cannot be controlled, which can cause autoimmune side effects, which limits the dosage. -141, 2020). In addition, there is a disadvantage in that sufficient therapeutic effect cannot be shown in all patients because of the low number of tumor-infiltrating T cells and different immune evasion mechanisms for each tumor microenvironment (Chaoyue Su, et al., Front. Oncol, 10:554313, 2020). Therefore, there is a need for a treatment that can maximize the treatment effect and overcome the side effects of lung cancer, which has frequent recurrence and metastasis and has a low cure rate.

Accordingly, the present inventors have made diligent efforts to use a nucleic acid complex having excellent cell penetrating ability to apply it to cancer, particularly lung cancer treatment. It was found that shows an excellent effect in preventing or treating lung cancer, and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be

An object of the present invention is to provide a nucleic acid complex having cell penetrating ability and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient.

In order to achieve the above object, the present invention is a bioactive nucleic acid targeting the PD-L1 gene (Bioactive Nucleic Acid); And a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) provides a complementary nucleic acid complex.

The present invention also provides a pharmaceutical composition for preventing or treating cancer containing the nucleic acid complex as an active ingredient.

The present invention also provides a method for preventing or treating cancer comprising administering the nucleic acid complex.

The present invention also provides the use of the nucleic acid complex for preventing or treating cancer.

The present invention also provides the use of the nucleic acid complex for preparing a drug for preventing or treating cancer.

The nucleic acid complex of the present invention, in which a bioactive nucleic acid targeting PD-L1 and a carrier peptide nucleic acid are complementaryly bound, has excellent cell penetration and intracellular activity, and can efficiently inhibit PD-L1 expression. It is useful for preventing or treating cancer, particularly lung cancer.

1a and 1b are graphs showing the cell growth inhibitory effect according to the treatment of four types of nucleic acid complexes in human lung cancer cell HCC827 and H460 models.
Figure 2 is a diagram showing changes in the expression level of the target gene PD-L1 according to the treatment of four nucleic acid complexes in human lung cancer cell HCC827 and H460 models.
3a and 3b are diagrams confirming the effect of inhibiting cell migration according to treatment with four types of nucleic acid complexes in human lung cancer cell HCC827 and H460 models and graphs digitizing them.
4A and 4B are graphs comparing the colony formation ability observed in human lung cancer cells HCC827 and H460 models by treatment with 4 types of nucleic acid complexes and the number of colonies counted.
5A and 5B are diagrams showing the results of confirming PD-L1 expression changes in lung cancer cells HCC827 and H460 models according to treatment with two nucleic acid complexes using flow cytometry equipment.

Unless defined otherwise, 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. In general, the nomenclature used herein is one well known and commonly used in the art.

In the present invention, a bioactive peptide nucleic acid targeting the PD-L1 gene; and carrier peptide nucleic acid (Carrier Peptide Nucleic Acid), when administering a nucleic acid complex complementarily bound thereto, it was attempted to determine whether the prevention and treatment of lung cancer is effective.

In one embodiment of the present invention, a nucleic acid complex in which a bioactive nucleic acid targeting the PD-L1 gene and a carrier peptide nucleic acid were complementarily linked was prepared based on Korean Patent Registration No. 10-1963885 of the present inventors. When adenocarcinoma and large cell carcinoma cell lines were treated with these nucleic acid complexes, the cell viability was reduced, the expression of the target gene, PD-L1, was effectively suppressed, and the migration and colony formation ability of lung cancer cells were suppressed. It was confirmed that the treatment effect was excellent.

Accordingly, in one aspect, the present invention provides a bioactive nucleic acid targeting the PD-L1 gene; and a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

In the present invention, a nucleic acid complex in which a bioactive nucleic acid targeting the PD-L1 gene and a carrier peptide are complementaryly bound may have a structure of the following structural formula (1).

structural formula (1)

[A ≡C ⁽⁺⁾ ]

In the structural formula (1),

A is a bioactive nucleic acid having a sequence capable of binding to a gene of interest,

C is a carrier peptide nucleic acid capable of binding to a bioactive nucleic acid;

‘≡’ means a complementary bond between a bioactive nucleic acid and a carrier peptide nucleic acid,

The bioactive nucleic acid represented by A has an overall negative charge or neutral charge,

C ⁽⁺⁾ means that the carrier peptide nucleic acid has an overall positive charge,

The carrier peptide nucleic acid includes one or more peptide nucleic acid monomers modified to have a positive charge throughout the carrier peptide nucleic acid.

The bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex according to the present invention may have anti-parallel binding or parallel binding. In the present invention, the complementary binding form of the nucleic acid can be separated in the presence of a target sequence of the bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).

In the present invention, "Bioactive Nucleic Acid" binds to a target gene and a nucleotide sequence containing the target gene in vitro or in vivo to determine the unique function of the gene (e.g., For example, activating or inhibiting transcript expression or protein expression), or regulating pre-mRNA splicing (eg, exon skipping), etc. , The nucleotide sequence may be characterized as a gene regulatory sequence, a gene coding sequence, or a splicing regulatory sequence. Preferably, the bioactive nucleic acid is expressed A nucleic acid having a complementary sequence capable of binding to a target gene of interest to be reduced, in particular, a complementary sequence capable of binding to the mRNA of such a target gene of interest, and suppressing the expression of the gene. It means a nucleic acid involved in regulation, and may be a nucleic acid having a sequence complementary to a target gene whose expression is to be reduced.

Therefore, the bioactive nucleic acid in the present invention is preferably an antisense peptide nucleic acid of the PD-L1 (Programmed Death-Ligand 1) gene, which is a cancer target gene, and more preferably includes the nucleotide sequence represented by SEQ ID NO: 1. It can be done, but is not limited thereto.

The bioactive nucleic acids include DNA, RNA, or modified nucleic acids such as PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid), antisense It may be selected from the group consisting of oligonucleotide, aptamer, small interfering RNA (siRNA), short hairpin RNA (shRNA), ribozyme and DNAzyme, preferably the bioactive The nucleic acid is selected from the group consisting of DNA, RNA, or modified nucleic acid PNA (peptide nucleic acid), PMO (phosphorodiamidate morpholino oligonucleotide), LNA (locked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid) it may be

In the present invention, "Carrier Peptide Nucleic Acid" refers to a nucleic acid to which a bioactive nucleic acid and some or all bases are complementaryly combined to impart functionality, and the carrier peptide nucleic acid used in the present invention is In addition to peptide nucleic acid (PNA), modified nucleic acids similar thereto may be used, and peptide nucleic acids are preferred, but are not limited thereto.

In the present invention, the carrier peptide nucleic acid preferably includes one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so that the entire carrier peptide nucleic acid is positively charged, and the gamma- or alpha-backbone modified peptide nucleic acid It is more preferable that monomers having amino acids having a positive charge are included more than monomers having amino acids having a negative charge so that the overall charge of the carrier peptide nucleic acid is positive.

In particular, in the present invention, the carrier peptide nucleic acid preferably includes a base sequence represented by any one sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 6, but is not limited thereto.

In the present invention, the “nucleic acid complex” is capable of permeating bioactive substances into the body and ultimately into cells through extracellular processing, and specifically, the ability to deliver a bioactive nucleic acid targeting the PD-L1 gene into cells. have Accordingly, the nucleic acid complex may be characterized as having cell permeability.

In the present invention, the nucleic acid complex is a bioactive nucleic acid represented by the sequence of SEQ ID NO: 1; and a carrier peptide nucleic acid represented by any one of SEQ ID NOs: 3 to 6, but is not limited thereto. More preferably, the nucleic acid complex comprises a bioactive nucleic acid represented by the sequence of SEQ ID NO: 1; and a carrier peptide nucleic acid represented by the sequence of SEQ ID NO: 3 or 4, but is not limited thereto.

In addition, the binding ability (melting temperature, Tm) of the bioactive nucleic acid targeting the PD-L1 gene and the carrier peptide nucleic acid is lower than that of the bioactive nucleic acid and the target PD-L1 gene. can be characterized.

The binding force is obtained by parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid according to the 5'-direction and the 3'-direction of each nucleic acid, so that the bioactive nucleic acid and the carrier peptide nucleic acid The binding force (Tm) of may be lower than the binding force between the bioactive nucleic acid and the target gene of the bioactive nucleic acid.

In the present invention, the bioactive nucleic acid or carrier peptide nucleic acid may be characterized by additionally binding a substance that helps endosome escape to the 5'-end or 3'-end of each nucleic acid. That is, it may be characterized in that it has a structure of the following Structural Formula (2) by further including a material that helps the endosome escape of the bioactive nucleic acid and the carrier peptide nucleic acid.

structural formula (2)

[ mA ≡ mC ⁽⁺⁾ ]

In the structural formula (2),

‘m’ means a substance that helps endosome escape of bioactive nucleic acid and carrier peptide nucleic acid.

In the present invention, "substances that help endosomes escape" can be characterized in that they help the escape of bioactive nucleic acids from endosomes by increasing the osmotic pressure inside the endosomes or by destabilizing the membranes of endosomes. there is. It means that bioactive nucleic acids move more efficiently and rapidly to the nucleus or cytoplasm to meet and act on target genes (Daniel W Pack, et al., Nat Rev Drug Discov. 2005 Jul;4(7):581-93 ).

In the present invention, the material that helps the endosome escape is a peptide, lipid nanoparticles, conjugate nanoparticles (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanomaterials (inorganic nanoparticles), cationic lipids It may be characterized in that at least one selected from the group consisting of nanomaterials (cationic lipid-based nanoparticles), cationic polymers (cationic polymers) and pH sensitive polymers (pH sensitive polymers).

In the present invention, a peptide (GLFDIIKKIAESF, SEQ ID NO: 8) may be linked to the bioactive nucleic acid as a material that helps the endosome escape through a linker, and Histidine (10) to the carrier peptide nucleic acid as a linker. It may be characterized by combining, but is not limited thereto.

In the present invention, the lipid nanoparticles may be selected from the group consisting of Lipid, phospholipids, acetyl palmitate, poloxamer 18, Tween 85, tristearin glyceride and Tween 80.

In the present invention, the polyplex nanoparticles may be poly(amidoamine) or polyethylenimine (PEI).

In the present invention, the polymer nanospheres are selected from the group consisting of polycaprolactone, poly(lactide-co-glycolide, polylactide, polyglycolide, poly(d,l-lactide), chitosan, and PLGA-polyethylene glycol. can be characterized.

In the present invention, the inorganic nanoparticles may be selected from the group consisting of Fe ₂ O ₃ , Fe ₃ O ₄ , WO3 and WO2.9.

In the present invention, the cationic lipid-based nanoparticles are 1- (aminoethyl) iminobis [N- (oleicylcysteinyl-1-amino-ethyl) propionamide], N-alkylated derivative of PTA and 3, 5- It may be characterized in that it is selected from the group consisting of didodecyloxybenzamidine.

In the present invention, the cationic polymer may be selected from the group consisting of vinylpyrrolidone-N, N-dimethylaminoethyl methacrylate acid copolymer diethyl sulphate, polyisobutylene and poly(N-vinylcarbazole).

In the present invention, the pH sensitive polymers may be selected from the group consisting of polyacids, poly(acrylic acid), poly(methacrylic acid), and hydrolyzed polyacrylamide.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid may each contain 2 to 50, preferably 2 to 30, more preferably 5 to 20 nucleic acid monomers, but are limited thereto. it is not going to be

The bioactive nucleic acid may be characterized in that it consists of natural nucleic acid bases and/or modified nucleic acid monomers.

In the present invention, when the monomer used for the bioactive nucleic acid is PNA, it is referred to as a bioactive peptide nucleic acid, and when other monomers are used, it is referred to in the same way.

In the present invention, the bioactive nucleic acid and the carrier peptide nucleic acid are phosphodiester, 2' O-methyl, 2' methoxy-ethyl, phosphor It may be characterized by further comprising at least one functional group selected from the group consisting of amidate, methylphosphonate, and phosphorothioate.

In the present invention, the carrier peptide nucleic acid may be characterized in that a part or all of the base sequence of the bioactive nucleic acid is composed of a complementary sequence. In particular, the carrier peptide nucleic acid may include one or more universal bases, and all of the carrier peptide nucleic acids may consist of universal bases.

In the present invention, each of the bioactive nucleic acid and the carrier peptide nucleic acid in the nucleic acid complex may be a complex characterized by having a positive charge (positive), negative charge (negative) or neutral charge as a whole.

In the expression of the electrical properties, the meaning of "overall" means the electrical properties of the entire bioactive nucleic acid or carrier peptide nucleic acid as a whole, not the electrical properties of individual bases, when viewed from the outside. Even if some of the monomers in the sexual nucleic acid have a positive charge, if there are more monomers with a negative charge, the bioactive nucleic acid will have a negative charge when looking at the electrical properties “as a whole”, and some bases and/or Even if the backbone has a negative charge, when a larger number of bases and/or backbones having a positive charge are present, the carrier peptide nucleic acid has a positive charge when looking at the electrical characteristics “as a whole”.

From this point of view, the nucleic acid complex of the present invention may be characterized as having a positive charge as a whole. In the nucleic acid complex, preferably, the bioactive nucleic acid has negative or neutral electrical characteristics as a whole, and the carrier peptide nucleic acid has positive electrical characteristics as a whole. It is not limited thereto.

In the present invention, the electrical properties of the bioactive nucleic acid and the carrier peptide nucleic acid can be imparted using a modified peptide nucleic acid monomer, and the modified peptide nucleic acid monomer is a carrier peptide nucleic acid having a positive charge, such as lysine (Lysine, Lys, K) , arginine (Arg, R), histidine (Histidine, His, H), diamino butyric acid (DAB), ornithine (Orn), and any one or more positive charges selected from the group consisting of amino acid analogs It is a carrier peptide nucleic acid that includes amino acids of and has a negative charge, and may be characterized in that it includes a negatively charged amino acid such as glutamic acid (Glu, E) or an amino acid analog that is negatively charged.

In the present invention, the carrier peptide nucleic acid may be characterized by including one or more gamma- or alpha-backbone modified peptide nucleic acid monomers so as to have a positive charge as a whole.

The gamma- or alpha-backbone modified peptide nucleic acid monomer has lysine (Lysine, Lys, K), arginine (Arginine, Arg, R), histidine (His, H), diamino butyric acid (Diamino butyric acid) to have an electrical positivity. acid, DAB), ornithine (Orn), and amino acids having at least one positive charge selected from the group consisting of amino acid analogs.

In the present invention, the modification of the peptide nucleic acid monomer for charge imparting may use a peptide nucleic acid monomer having a modified nucleobase in addition to the backbone modification. Preferably, an amine, triazole, or imidazole moiety may be included in the nucleobase to have an electronegative property, or a carboxylic acid may be included in the base to have an electronegative property.

In the present invention, the modified peptide nucleic acid monomer of the carrier peptide nucleic acid may further contain a negative charge in the backbone or nucleobase, but the modified peptide nucleic acid monomer contains more positively charged monomers than monomers having negative charges, so that the carrier peptide as a whole is formed. It is preferred that the charge of the nucleic acid be positive.

Preferably, the nucleic acid complex according to the present invention has a positive charge as a whole.

In the nucleic acid complex according to the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, or a fluorescent/luminescent marker is a bioactive nucleic acid And / or may be characterized in that it is bound to a carrier peptide nucleic acid, preferably the hydrophobic moiety, the hydrophilic moiety, a target antigen-specific antibody, an aptamer, and a fluorescent / luminescent marker for imaging One or more substances selected from the group consisting of and the like may be bound to the carrier peptide nucleic acid.

In the present invention, at least one material selected from the group consisting of a hydrophobic moiety, a hydrophilic moiety, a target antigen-specific antibody, an aptamer, a quencher, a fluorescent marker, and a luminescent marker, and a bioactive nucleic acid and/or The binding of the carrier peptide nucleic acid may be characterized as a simple covalent bond or a covalent bond mediated by a linker, but is not limited thereto. Preferably, substances related to cell permeation, solubility, stability, transport and imaging (eg, hydrophobic residues, etc.) bound to the nucleic acid carrier exist independently of the bioactive nucleic acid that regulates the expression of the target gene.

In the present invention, as described above, the complementary binding form of the bioactive nucleic acid and the carrier peptide nucleic acid may be largely characterized by having the form of antiparallel binding and parallel binding. . The complementary binding form has a structure that separates in the presence of a target sequence of a bioactive nucleic acid (a sequence complementary to the bioactive nucleic acid).

The antiparallel bond and parallel bond are determined according to the 5'-direction and the 3'-direction in the binding method of DNA-DNA or DNA-PNA. Antiparallel coupling is a general DNA-DNA or DNA-PNA coupling method. Taking the nucleic acid complex according to the present invention as an example, the bioactive nucleic acid is in the 5' to 3' direction and the carrier peptide nucleic acid is in the 3' to 5' direction. It means that the shape is connected to each other in the direction. Parallel binding is a form in which binding force is somewhat lower than that of anti-parallel binding, and means that both the bioactive nucleic acid and the carrier peptide nucleic acid are bonded to each other in the 5' to 3' direction or the 3' to 5' direction.

In the nucleic acid complex according to the present invention, preferably, the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is lower than the binding force of the bioactive nucleic acid and the target gene, particularly the mRNA of the target gene. . The binding force is determined by melting temperature, melting temperature or Tm.

As an example of a specific method for making the binding strength (melting temperature, Tm) of the bioactive nucleic acid and the carrier peptide nucleic acid lower than the binding strength of the bioactive nucleic acid and the bioactive nucleic acid to the target gene, in particular, the mRNA of the target gene, the above Parallel binding or partial specific binding between the bioactive nucleic acid and the carrier peptide nucleic acid may be characterized, but is not limited thereto.

As another example, the carrier peptide nucleic acid has at least one peptide nucleic acid base selected from the group consisting of a linker, a universal base, and a peptide nucleic acid base having a base that is not complementary to a corresponding base of a bioactive nucleic acid. It can be, but is not limited thereto.

In the present invention, the universal base binds to natural bases such as adenine, guanine, cytosine, thymine, and uracil without selectivity, and is complementary to One or more bases selected from the group consisting of inosine PNA, indole PNA, nitroindole PNA, and abasic can be used as a base having a lower binding force than the binding force, preferably. It can be characterized by using inosine PNA.

In the present invention, a combination of the binding form and electrical properties of nucleic acids for function control of the nucleic acid complex is provided, and the combination of the binding form and electrical properties of the nucleic acids controls the particle size and the time point of action, cell permeability, solubility and specificity It can be characterized as improving the degree.

In the present invention, the binding force of the bioactive nucleic acid and the carrier peptide nucleic acid is controlled, in the presence of the target gene, when the bioactive nucleic acid binds to the target sequence (when the bioactive nucleic acid is substituted with the target sequence, the target specific It is possible to adjust the timing of enemy separation and joining).

In the nucleic acid complex according to the present invention, the control of the strand displacement time point and the target specific release and bind time point of the bioactive nucleic acid into the target gene is a carrier peptide for non-specific binding of the complex It can be characterized in that it can be controlled by the presence, number, and location of non-specific bases, universal bases, and linkers in nucleic acids. It may be characterized in that control is possible by a combination of the above conditions, such as a parallel or antiparallel bond, which is a form of complementary bond of the peptide complex.

In the present invention, the particle size of the nucleic acid complex may be characterized in that it is controlled by adjusting the charge balance between the bioactive nucleic acid and the carrier peptide nucleic acid. Specifically, when the positive charge of the carrier peptide nucleic acid increases, the particle size decreases, but when the positive charge of the carrier peptide nucleic acid exceeds a certain level, the particle size increases. In addition, the particle size is determined by an appropriate charge balance with the overall carrier peptide nucleic acid according to the charge of the bioactive nucleic acid forming the complex as another important factor determining the particle size.

The positive charge of the carrier peptide nucleic acid according to the present invention is 1 to 7 (meaning that 1 to 7 monomers having a positive charge are included), preferably 2 to 5, most preferably 2 to 3 And, the charge of the bioactive nucleic acid may be characterized in that the net charge of the charge balance is 0 to 5 negative charges, preferably 0 to 3.

In the present invention, the nucleic acid complex can be prepared by hybridizing a bioactive nucleic acid and a carrier peptide nucleic acid under appropriate conditions.

"Hybridization" in the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when the complementarity between the two nucleic acid strands is perfect (perfect match) or even when some mismatch bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and may be particularly controlled by binding temperature.

The “target gene” of the present invention refers to a nucleic acid sequence (base sequence) to be activated, inhibited, or labeled, and is not different from the term “target nucleic acid”, and is used interchangeably herein.

In the present invention, when a target nucleic acid (base sequence) containing a target gene is contacted (bound) with the complex in vitro or in vivo , bioactive nucleic acid is separated from the carrier peptide nucleic acid and exhibit biological activity.

In the present invention, diseases that can be prevented or treated using the nucleic acid complex may be determined according to the target gene of the bioactive nucleic acid in the nucleic acid complex. In the present invention, the target gene of the bioactive nucleic acid may be characterized in that PD-L1.

Therefore, in another aspect, the present invention provides a bioactive nucleic acid targeting the PD-L1 gene; and a pharmaceutical composition for preventing or treating cancer containing, as an active ingredient, a nucleic acid complex to which a carrier peptide nucleic acid is complementarily bound.

In the present invention, the term "cancer" or "tumor" refers to a lump that has grown abnormally due to autonomous excessive growth of body tissue. The cancer or tumor in the present invention is applicable to any type that expresses PD-L1, but is preferably non-Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia (acute- myeloid leukemia), acute-lymphoid leukemia, multiple myeloma, head and neck cancer, lung cancer (small cell lung cancer and non-small cell lung cancer), glioblastoma, large intestine (colon and rectal cancer), pancreatic cancer, breast cancer, ovarian cancer, melanoma, prostate cancer, renal cancer, and mesothelioma, but is not limited thereto.

As used herein, the term "prevention" refers to any action that prevents the onset of a disease or delays its progression by administering a composition containing the nucleic acid complex. In addition, the term “treatment” used in the present invention refers to all activities in which symptoms of a disease are improved, symptoms are alleviated, or cured by administration of a composition containing the nucleic acid complex.

A pharmaceutical composition comprising a nucleic acid complex according to the present invention may include a pharmaceutically effective amount of the nucleic acid complex alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents. The pharmacologically effective amount in the above refers to an amount sufficient to prevent, improve, and treat symptoms of cancer.

The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives.

The term "carrier" is defined as a compound that facilitates the addition of a nucleic acid complex into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into the cells or tissues of living organisms.

The term “diluent” is defined as a compound that is diluted in water which will dissolve the compound as well as stabilize the biologically active form of the subject compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

A substance containing a nucleic acid complex in the present invention can be administered to a patient by itself or as a mixed pharmaceutical composition together with other active ingredients, such as in combination therapy, or with suitable carriers or excipients.

Compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration. The dosage form may be in the form of a powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, or sterile powder.

The composition of the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on various factors such as the route of administration, age, sex, weight and severity of the patient. can be selected appropriately.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in effective amounts to achieve their intended purpose. More specifically, a therapeutically effective amount refers to an amount of a compound effective to prolong the survival of the subject being treated or to prevent, alleviate or ameliorate symptoms of a disease. Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For administration of the nucleic acid complexes of the present invention, any nucleic acid delivery method known in the art may be used. Although not limited thereto, suitable delivery reagents include, for example, Mirus Transit TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, polycations (eg, polylysine), atelocollagen, nanoplexes, and liposomes. The use of atelocollagen as a delivery vehicle for nucleic acid molecules has been described by Minakuchi et al. Nucleic Acids Res., 32(13):e109 (2004); Hanai et al. Ann NY Acad Sci., 1082:9-17 (2006); and Kawata et al. Mol Cancer Ther., 7(9):2904-12 (2008). Exemplary interfering nucleic acid delivery systems are provided in U.S. Patent Nos. 8,283,461, 8,313,772, and 8,501,930.

In the present invention, the nucleic acid complex may be administered using a delivery system such as a liposome. The liposome can help target the complex to a specific tissue, such as lymphoid tissue, or selectively target infected cells, and can also help increase the half-life of a composition containing the complex. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In such formulations, the complex to be delivered, alone or to specific cells, in combination with a molecule that binds to a receptor prevalent in lymphocytes, such as a monoclonal antibody that binds to the CD45 antigen, or in combination with other therapeutic compositions, is a liposome. incorporated as part of Thus, liposomes filled or decorated with a given complex of the present invention to deliver the nucleic acid complex composition can be directed to the site of lymphocytes.

Liposomes for use in accordance with the present invention are generally formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols such as cholesterol. In general, lipids are selected in consideration of, for example, stability of liposomes in the blood stream, acid lability, and size of liposomes. A variety of methods can be used to prepare liposomes. See, eg, Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467, 1980), and US Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

In another aspect, the present invention provides a bioactive nucleic acid targeting the PD-L1 gene; and a method for preventing or treating cancer, comprising administering to a subject a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

In another aspect, the present invention provides a bioactive nucleic acid targeting the PD-L1 gene for preventing or treating cancer; And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementarily bound.

In another aspect, the present invention provides a bioactive nucleic acid targeting the PD-L1 gene for preparing a drug for preventing or treating cancer; And it relates to the use of a nucleic acid complex in which a carrier peptide nucleic acid is complementaryly bound.

In the present invention, “subject” means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed or treated by administering a nucleic acid complex according to the present invention, and preferably means a human.

In addition, the dose of the nucleic acid complex of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and disease severity.

Toxicity and therapeutic efficacy of compositions comprising the nucleic acid complexes described herein can be measured by, for example, the LD50 (lethal dose to 50% of the population), the ED50 (the dose that is therapeutically effective in 50% of the population), the IC50 It can be estimated by standard pharmaceutical procedures in cell culture or laboratory animals to determine (the dose that has a therapeutically inhibitory effect on 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between the LD50 and the ED50 (or IC50). Compounds exhibiting high therapeutic indices are preferred. Data obtained from these cell culture assays can be used to estimate a range of human doses. The dosage or applied amount of such compounds lies preferably within a range of circulating concentrations that include the ED50 (or IC50) with little or no toxicity.

The term "administration" of the present invention refers to the act of introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration may be administered through various oral or parenteral routes as long as it can reach the target tissue. there is.

The administration route of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or intrarectally, as desired. In addition, the composition may be administered by any device capable of transporting an active substance to a target cell.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example One: vitality peptide nucleic acids and carrier Preparation of peptide nucleic acids and complexes using them

In the present invention, adenocarcinoma (HCC827) and large cell carcinoma (H460) cell lines were used to comprehensively study the expression of PD-L1, an indicator related to immunotherapy, and to confirm the anticancer effect on non-small cell lung cancer. For this purpose, antisense peptide nucleic acid (Antisense PNA) was used as a bioactive peptide nucleic acid targeting PD-L1.

In the present invention, the bioactive peptide nucleic acid (antisense PNA) used to confirm the anticancer effect on lung cancer includes the sequence represented by SEQ ID NO: 1, and the bioactive nucleic acid (antisense PNA) used as a control is represented by SEQ ID NO: 2 contains a sequence that is The carrier peptide nucleic acid used to confirm the anticancer effect in this example includes the sequences represented by SEQ ID NOs: 3 to 6, and the carrier peptide nucleic acid used as a control group includes the sequence represented by SEQ ID NO: 7. The base sequences, monomeric modifications and structures of bioactive nucleic acids and carrier peptide nucleic acids are shown in Table 1 below. All peptide nucleic acids used in the present invention were synthesized by HPLC purification method at PANAGENE (Korea).

Sequence of bioactive nucleic acid and carrier peptide nucleic acid for verification of lung cancer anti-cancer effect division sequence number base sequence monomer transformation vitality
nucleic acid One 5'-CT ^(-) CAG ⁽⁺⁾ CCT ^(-) GAC ⁽⁺⁾ AT ^(-) GT-OK-3' -+-+- 2 5'-AC ^(-) GCG ⁽⁺⁾ TTC ^(-) GCC ⁽⁺⁾ AGC ^(-) TOK-3' -+-+- carrier
peptide
nucleic acid 3 5'-GA ⁽⁺⁾ GTCGGA ⁽⁺⁾ CTGTA ⁽⁺⁾ CA-OK-3' +++ 4 5'-KO-AC ⁽⁺⁾ ATGTC ⁽⁺⁾ AGGCTG ⁽⁺⁾ AG-3' +++ 5 5'-GT ⁽⁺⁾ AC ⁽⁺⁾ AOK-3' ++ 6 5'-KO-AC ⁽⁺⁾ AT ⁽⁺⁾ G-3' ++ 7 5'-TG ⁽⁺⁾ CGCAAG ⁽⁺⁾ CGGTCG ⁽⁺⁾ AOK-3' +++

Modification of monomers converts the backbone of peptide nucleic acid to electrically positive lysine (represented by Lysine, Lys, K, ⁽⁺⁾ ) and electronegatively to glutamic acid (Glu, E, ^(-)) to impart electrical properties. notation) was constructed to have a modified peptide backbone.

Each combination of the bioactive nucleic acid and the carrier peptide nucleic acid was hybridized in the presence of DMSO, and as a result, a complex composed of the bioactive nucleic acid and the carrier peptide nucleic acid was prepared.

Example 2: Analysis of lung cancer anti-cancer effect using nucleic acid complex

According to Example 1, the lung cancer anticancer effect was analyzed using a nucleic acid complex comprising a carrier peptide nucleic acid and a bioactive peptide nucleic acid having PD-L1 as a target gene prepared with the structure shown in Table 2 below.

Combination of Nucleic Acid Complexes for Cell Viability Analysis in Human Lung Cancer Cell Lines Combination nucleic acid complex One SEQ ID NOs 1 and 3 2 SEQ ID NOs 1 and 4 3 SEQ ID NOs 1 and 5 4 SEQ ID NOs 1 and 6 5 SEQ ID NOs 2 and 7

Example 2-1: Cell culture

Human lung cancer adenocarcinoma cell line HCC827 (CRL-2868) obtained from ATCC (American Type Culture Collection, USA) and human lung cancer large cell carcinoma cell line H460 (30177) obtained from Korea Cell Line Bank (KCLB, Korea) were mixed in RPMI culture medium ( 10% (v/v) fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin were equally added to Rosewell Park Memorial Institute Medium, Wellgene, Korea), 37°C, 5% (v/v) CO It was cultured under the condition of ₂ .

Example 2-2: MTT ( MTT assay) to analyze cell viability of lung cancer cell lines

Under the experimental conditions in Example 2-1, lung cancer cell lines HCC827 and H460 were seeded in 6x10 ³ and 4x10 ³ respectively in 96 wells, cultured for 24 hours, and then complexes containing the bioactive nucleic acid and the carrier peptide nucleic acid of Table 2 were obtained. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was prepared at a concentration of 5 mg/mL in 1X PBS and treated with 20 μL per well for 4 hours. After culturing, OD (optical density) was measured and analyzed with a spectrophotometer.

As a result, as shown in FIG. 1, in the HCC827 human lung cancer adenocarcinoma cell model, the nucleic acid complex of Combination 2 reduced cell viability by about 50% at 24 hours based on 500 nM, and it was confirmed that the reduced effect lasted up to 72 hours. (Fig. 1a). In addition, in lung cancer large cell H460 cells, the nucleic acid complex of Combination 2 reduced the cell viability by about 40% by 48 hours based on 500 nM (FIG. 1B).

Example 2-3: Western blot Analysis of gene expression through Western blot assay

The human lung cancer cell lines HCC827 and H460 were seeded in 1x10 ⁵ cells in a 6-well plate, cultured for 24 hours, and the complex containing the bioactive peptide nucleic acid and carrier peptide nucleic acid of Table 2 was conducted under the conditions of Example 2-1. After treatment with 500nM each and incubation for 24, 48, 72, 96, and 120 hours, 30 μL of RIPA buffer was added to each well to obtain protein lysate. Protein lysate was quantified using a BCA assay kit (Thermo Fisher, USA), and 30 μg of protein was separated by size through electrophoresis, and the protein was transferred to a PVDF membrane, followed by primary antibody PD-L1 (Cell signaling technology , USA) at a ratio of 1:1000 and left at 4°C for one day. It was washed with 1X TBS-T, treated with a secondary antibody, Goat Anti-Rabbit (Cell signaling Technology, USA) at a ratio of 1:2000, and left at room temperature for 1 hour. The cells were treated with Supersignal ^TM West Femto Maximum Sensitivity Substrate (Thermo Fisher, USA) and analyzed for target gene expression inhibition efficiency in lung cancer cell lines using Image600 (Amersham, Germany) equipment.

As a result, as shown in Figure 2, the expression of the PD-L1 target gene in the group treated with the nucleic acid complex of SEQ ID NOs: 1 and 4 (combination 2) in human lung cancer adenocarcinoma cells HCC827 compared to the control complex of SEQ ID NOs: 2 and 7 It was confirmed that it was most effectively inhibited by day 5 (Fig. 2a). In addition, the same effect of reducing PD-L1 expression by 48 hours was confirmed by the nucleic acid complex of combination 2 in human lung cancer large-cell H460 cells (FIG. 2B).

Example 2-4: Lung cancer cells through migration assay Mobility analyze

Human lung cancer cell lines HCC827 and H460 were seeded inside the insert using a medium without fetal bovine serum at 1x10 ⁵ in a cell culture insert (BD, USA) of a 24-well plate, and 24 wells below the insert After adding a medium containing 10% fetal bovine serum to the plate and incubating for 24 hours, the experiment was conducted under the conditions of Example 2-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid of Table 2 was added at 500 nM each. processed. After culturing the cells for 24, 48, and 72 hours, respectively, the cells were fixed with 4% PFA (paraformaldehyde) for 10 minutes, stained with 0.1% crystal violet solution for 30 minutes, and the remaining cells inside the insult were removed with a cotton swab. The staining solution was washed with water. After drying the insult membrane (membrane) for about a day, images were obtained by observing the number of migrating cells under a microscope, and the number of migrating cells was measured using the Image J program.

As a result, as shown in FIG. 3, it was confirmed that the migration efficiency of lung cancer cells in the nucleic acid complex combination of SEQ ID NOs: 1 and 4 (combination 2) was suppressed by about 50% or more compared to the control group of SEQ ID NOs: 2 and 7, and this effect It was confirmed through quantitative digitization graphs using the Image J program that continuously appeared up to 72 hours (FIG. 3a, FIG. 3b).

Example 2-5: colony Formability Analysis of proliferation inhibitory effect of lung cancer cells through colony formation assay

Human lung cancer cell lines HCC827 and H460 were seeded at 5x10 ³ and 1x10 ³ per well in a 6-well plate and cultured for 24 hours, and experiments were performed under the conditions of Example 2-1 to obtain bioactive peptide nucleic acids and carrier peptide nucleic acids in Table 2 above. complexes were treated. After culturing for 7 and 14 days after complex treatment, colony-forming ability of lung cancer cells was analyzed.

On days 7 and 14 after complex treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes, stained with 0.1% crystal violet solution for 30 minutes, and then the staining solution was washed with PBS. After drying the plate for about an hour, the morphology of the colony formed by the increase in cells was observed under a microscope to obtain an image, and the area of the colony formed was measured using Image J.

As a result, as shown in FIG. 4, it was visually observed that the number of colonies formed compared to the control group of Combination 5 in Table 2 was reduced by treatment with the combination complex (Combination 2) of SEQ ID NOs: 1 and 4 in human lung cancer cells HCC827. . On the other hand, in the human lung cancer cell line H460, the colony formation ability was not relatively high due to cell characteristics, so no significant inhibitory effect was observed by the complex treatment.

Changes due to complex treatment were analyzed through a graph in which the area of the colonies was quantified using the Image J program for the images acquired under a microscope. As a result, in HCC827 cells, combination 1 reduced the colony area by about 40% on day 7, and it was confirmed that this effect increased to about 50% by day 14. In H460 cells, no significant effect was confirmed on the 7th day, but on the 14th day, a colony area reduction effect of about 20% was observed in Combination 1 (FIGS. 4a and 4b).

Example 3: flow cell Analysis (Flow cytometry ) Analysis of PD-L1 expression in lung cancer cells

Example 3-1: Cell culture

The human lung cancer adenocarcinoma cell line HCC827 obtained from ATCC (American Type Culture Collection, USA) and the large cell cancer cell line H460 obtained from Korea Cell Line Bank (KCLB, Korea) were cultured in RPMI (Rosewell Park Memorial Institute Medium, Wellgene, Korea), respectively. 10% (v/v) fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin were added in the same manner, and cultured under conditions of 37°C and 5% (v/v) CO ₂ .

Example 3-2: By flow cytometry Target gene expression change analysis through

Human lung cancer cell lines HCC827 and H460 were seeded in a 6-well plate at 1x10 ⁵ , cultured for 24 hours, and the experiment was conducted under the conditions of Example 3-1, and the complex containing the bioactive peptide nucleic acid and the carrier peptide nucleic acid was treated at 500 nM each, and then 24 , and cultured for 72 and 120 hours. After treatment with trypsin-EDTA (Thermo Fisher, USA), the pellet obtained by spinning down with the medium was mixed with 250μL of 1X BD Fixation/Permeabilization solution buffer (BD, USA) and left at 4℃ for 20 minutes to fix cells and the permeability process was performed. After washing twice with 1X BD Perm/wash buffer (BD, USA), the primary antibody, PD-L1 (Cell Signaling Technology, USA) was treated at 1:500 and left at room temperature for 1 hour. After washing twice with 1X BD Perm/wash buffer, the secondary antibody, anti-rabbit Alexa488 (abcam, England) was treated at 1:2000 and left at room temperature for 1 hour at room temperature to block light. After washing twice with 1X BD Perm/wash buffer, the efficiency of suppressing the expression of the target gene was confirmed using a flow cytometer (FACS Canto, BD, USA). When using a flow cytometer, the FSC-A / SSC-A plot was analyzed by comparing the 488nm wavelength value detected on the histogram by gating only the live cells excluding cell debris.

Nucleic acid complex combinations used in this Example are shown in Table 3 below.

Combination of Nucleic Acid Complexes for Analysis of PD-L1 Expression in Human Lung Cancer Cell Lines Combination nucleic acid complex One SEQ ID NOs 1 and 3 2 SEQ ID NOs 1 and 4 3 SEQ ID NOs 2 and 7

As a result of the analysis, as shown in FIG. 5, in HCC827 human lung cancer adenocarcinoma cells, the average measured value of PD-L1 compared to the control group of Combination 3 was reduced by about 5 to 6 times by the nucleic acid complex of Combination 2 in Table 3 for 24 and 48 hours. This effect lasted up to 5 days, and about 10-fold inhibition of PD-L1 expression was confirmed. In addition, in H460 human lung cancer large-cell cells, it was commonly observed that the combination 2 nucleic acid complex reduced the expression of the target gene PD-L1 by about 1.5 times compared to the control group at 120 hours.

Accordingly, it was confirmed that the nucleic acid complex of combination 2 inhibited the target PD-L1 most effectively in flow cytometry analysis through two lung cancer cell lines (FIGS. 5a and 5b).

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

<110> SEASUN THERAPEUTICS <120> Composition for Preventing or Treating Lung Cancer Comprising Nucleic Acid Complex <130> P22-B008 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> DNA <213> artificial sequence <220> <223> Bioactive Nucleic Acid <400> 1 ctcagcctga catgt 15 <210> 2 <211> 15 <212> DNA <213> artificial sequence <220> <223> Bioactive Nucleic Acid <400> 2 acgcgttcgc cagct 15 <210> 3 <211> 15 <212> DNA <213> artificial sequence <220> <223> Carrier Peptide Nucleic Acid <400> 3 gagtcggact gtaca 15 <210> 4 <211> 15 <212> DNA <213> artificial sequence <220> <223> Carrier Peptide Nucleic Acid <400> 4 acatgtcagg ctgag 15 <210> 5 <211> 5 <212> DNA <213> artificial sequence <220> <223> Carrier Peptide Nucleic Acid <400> 5 gtaca 5 <210> 6 <211> 5 <212> DNA <213> artificial sequence <220> <223> Carrier Peptide Nucleic Acid <400> 6 acatg 5 <210> 7 <211> 15 <212> DNA <213> artificial sequence <220> <223> Carrier Peptide Nucleic Acid <400> 7 tgcgcaagcg gtcga 15 <210> 8 <211> 13 <212> PRT <213> artificial sequence <220> <223> peptide for facilitating endosome escape <400> 8 Gly Leu Phe Asp Ile Ile Lys Lys Ile Ala Glu Ser Phe 1 5 10

Claims (11)

Bioactive Nucleic Acid targeting the PD-L1 gene; and a carrier peptide nucleic acid (Carrier Peptide Nucleic Acid) complementary nucleic acid complex.
The nucleic acid complex according to claim 1, wherein the bioactive nucleic acid targeting the PD-L1 gene comprises the nucleotide sequence represented by SEQ ID NO: 1.
The nucleic acid complex according to claim 1, wherein the carrier peptide nucleic acid comprises a nucleotide sequence represented by any one sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 6.
The method of claim 1, wherein the bioactive nucleic acid or carrier peptide nucleic acid targeting the PD-L1 gene is obtained by additionally binding a substance that helps endosome escape to the 5'-end or 3'-end of each nucleic acid. Characterized by nucleic acid complexes.
The method of claim 4, wherein the material that helps the endosomes escape is a peptide, lipid nanoparticles, conjugate nanomaterials (polyplex nanoparticles), polymer nanospheres (polymer nanospheres), inorganic nanomaterials (organic nanoparticles, cations) A nucleic acid complex, characterized in that at least one selected from the group consisting of cationic lipid-based nanoparticles, cationic polymers and pH sensitive polymers.
The nucleic acid complex according to claim 5, wherein the peptide is GLFDIIKKIAESF (SEQ ID NO: 8) or Histidine (10).
The nucleic acid complex according to claim 1, wherein the bioactive nucleic acid targeting the PD-L1 gene has a negative or neutral charge as a whole.
The nucleic acid complex according to claim 1, wherein the carrier peptide nucleic acid has an overall positive charge.
The nucleic acid complex according to claim 1, wherein the nucleic acid complex has a positive charge as a whole.
A pharmaceutical composition for preventing or treating cancer containing the nucleic acid complex according to any one of claims 1 to 9 as an active ingredient.
The method of claim 10, wherein the cancer is non-Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, Multiple myeloma, head and neck cancer, lung cancer, glioblastoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer and mesothelioma A pharmaceutical composition for preventing or treating cancer, characterized in that selected from the group consisting of.