ADP-ribose-binding peptide having anticancer activity, and application of the same

Abstract

To provide an ADP (adenosine diphosphate)-ribose-binding peptide having anticancer activity.SOLUTION: Provided are an ADP-ribose-binding peptide of a specific amino acid sequence and a denatured object thereof, and a pharmaceutical composition for preventing or treating cancer and a pharmaceutical composition for assisting anti-cancer activity which contain them as an active ingredient. The ADP-ribose-binding peptide breaks equilibrium of a cell to kill a cancer cell by accumulating ADP-ribose in a cancer cell, and has excellent anti-cancer effect that does not express toxicity in a normal cell, and in the case where the peptide is administered in combination with administration of other anticancer agent or anti-cancer radiotherapy, it enhances responsiveness to the anticancer agent and radiotherapy, and has extremely excellent effect as an anti-cancer adjuvant.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an ADP (adenosine diphosphate)-ribose binding peptide having anti-cancer activity, and specifically, to an ADP-ribose binding peptide having a specific amino acid sequence and its variants, and to a pharmaceutical composition for preventing or treating cancer and a pharmaceutical composition for adjuvant anti-cancer treatment, which contain the same as an active ingredient.

Background Art

[0002] PARylation (poly ADP-ribosylation) is one of the post-translation modification processes, and refers to the process in which an ADP-ribose polymer (poly(adenosine diphosphate-ribose)) is covalently attached to a protein by a PAR polymerase. Through PARylation, a high-molecular-weight ADP-ribose chain is generated, and it can induce a unique intracellular biochemical action that does not show a form such as ubiquitination or SUMOylation, rather than a small molecular dimension modification such as acetylation or methylation. The balance of PARylation plays an important part in DNA damage repair, transcriptional regulation, chromatin structure alteration, redox homeostasis, diverse intracellular signal transduction, non-membrane structure formation, host-pathogen interaction, and RNA metabolism regulation (Juan et al., Cancers, 2020, 12(3):739).

[0003] PARylation is involved in the development of systemic diseases including cancer, viral infection, and neurodegeneration. In particular, since PARylation is derived from the activation of PARP-1, the anti-cancer efficacy of targeting ovarian cancer, prostate cancer, breast cancer, and other cancers by a method of suppressing the activity of PARP-1 is already well known (J Mateo et al., Ann Oncol., 2019, 30(9):1437).

[0004] Information on the biochemical mechanisms associated with PARylation-mediated cell death has only recently become known, and activation of PARylation can induce cell death through typically three major pathways. (Rebecca Gupte et al., Genes Dev. 2017, 31(2):101).

[0005] NAD + Depletion can damage cell metabolism that occurs in a cellular energy crisis (ATP depletion), particularly the oxidative phosphorylation process. PARylation activation induced by extensive DNA damage consumes NAD + and it has been proposed that the subsequent effects of this action can kill cells.

[0006] In addition, a pathway can be activated in which PAR polymers released from the nucleus after DNA repair are bundled onto the mitochondrial membrane in the cytoplasm to release cell death-inducing factors and mobilize cell death-inducing factors inside the nucleus. When the cell death factors move to the nucleus, they mediate large-scale DNA fragmentation and induce cell death. Energy depletion and cell death-inducing factors by PARylation are known to be related to PAR signaling that controls protein kinase-phosphatase pathways such as the PI3K-Akt pathway or the MAP kinase pathway, and such PAR-dependent cell death processes contribute significantly to the complexity of the death mechanism.

[0007] And although the exact biochemical mechanism has not been clarified yet, it has been reported that PARP-1 itself can be degraded through autoPARylation of PARP-1 to induce cell death. However, cancer can cleverly avoid this death effect by activating various proteolytic enzymes to degrade excess PAR polymers involved in various biochemical actions necessary for cancer survival.

[0008] Therefore, contrary to attempts for PARylation inhibition such as existing PARP-1 inhibitors, activating PARylation or inhibiting the degradation of PAR polymers can also be a strategy for discovering effective anti-cancer therapies. In particular, PARylation activation or inhibition of PAR polymer degradation can be seen as a very promising target in cancer cell-specific regions that undergo metabolic processes in a manner different from normal cells. Summary of the Invention Problems to be Solved by the Invention

[0009] As a result of intensive efforts to develop a novel anti-cancer agent, the inventors of the present invention have found that a peptide having the novel amino acid sequence of the present invention can ultimately prevent the utilization of ADP-ribose or PAR polymers from the action of various intracellular degrading enzymes and signal transduction proteins through binding to ADP-ribose, overactivate PARylation, and disrupt the degradation process of PAR polymerase. As a result of this disruption, it has been confirmed that cancer cell death can be induced, and an epoch-making anti-cancer efficacy can be achieved, thus completing the present invention. Means for Solving the Problems

[0010] One object of the present invention is to provide an ADP (adenosine diphosphate)-ribose binding peptide having any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14.

[0011] Another object of the present invention is to provide a polynucleotide encoding the peptide.

[0012] Another object of the present invention is to provide a vector and a transformant containing the polynucleotide.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, a pharmaceutical composition for adjuvant cancer treatment that enhances the reactivity to a second anticancer agent, and a pharmaceutical composition for adjuvant cancer treatment that enhances the reactivity to radiotherapy, which contain the peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide the use of the peptide or a pharmaceutically acceptable salt thereof for preventing or treating cancer, and a method for preventing or treating cancer, which includes the step of administering to an individual in need of the peptide or a pharmaceutically acceptable salt thereof.

Advantages of the Invention

[0015] The ADP-ribose binding peptide of the present invention has an excellent anticancer effect of disrupting the cell balance to cause cancer cell death by accumulating ADP-ribose in cancer cells and showing no toxicity to normal cells. When it is co-administered during the administration of other anticancer agents or radiotherapy, it enhances the reactivity to the anticancer agent and radiotherapy, and also has an excellent effect as an adjuvant anticancer agent.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0017] Specifically explaining this, it is as follows. On the one hand, each of the explanations and embodiments disclosed in the present invention can also be applied to each of the other explanations and embodiments. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, it is not considered that the scope of the present invention is limited by the specific descriptions described later.

[0018] One aspect of the present invention for achieving the above object is an ADP (adenosine diphosphate)-ribose binding peptide having any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 14.

[0019] In the present invention, the term "ADP-ribose" is used as a concept including both isolated ADP-ribose itself and ADP-ribose polymer (poly ADP-ribose), and "ADP-ribose binding peptide" means all peptides having an activity of binding to ADP-ribose or ADP-ribose polymer and suppressing degradation. In the present invention, the peptide not only is formed simply by peptide bonds between amino acids constituting it, but also includes all forms of peptide analogs, derivatives, etc. in a partially modified form in order to improve characteristics such as stability and efficacy from the perspective of protein pharmaceuticals.

[0020] The inventors of the present invention have noted that, in a completely different aspect from the many reported anti-cancer effects in terms of the suppression of PARylation (poly ADP-ribosylation) by conventional PARP-1 inhibitors, an anti-cancer effect can be expected through the activation of PARylation or the suppression of PAR polymer degradation. In particular, since cancer cells continue to divide at a faster rate and have active metabolic activities compared to normal cells, it was expected that by hyperactivating PARylation or suppressing the degradation of PAR polymers, cancer cells could be specifically killed without any special effect on normal cells that do not hyperactivate PARylation.

[0021] Any ADP (adenosine diphosphate)-ribose binding peptide having any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 14 (Table 1) of the present invention is derived from the WWE domain. The WWE domain is a globular domain conserved in a number of proteins such as deltex, Trip12, and poly-ADP-ribose polymerase homologs, and is named after the name of the most conserved residue within the domain (L. Aravind, TRENDS in Biochemical Sciences, 2001, 26(5):273). It is known that an ADP-ribose binding motif exists within the domain in some proteins having the WWE domain. The WWE domain of intracellular enzymes has been reported to mainly bind to ADP-ribose and induce degradation. That is, the binding of a specific enzyme to PAR or ADPR through the WWE domain is considered to be of great purpose for cancer cell survival (PNAS August 23, 2011 108(34)14103-14108).

[0022] However, when a peptide produced by separating a part of the WWE domain as in the present invention is treated with cells, it has not been reported at all that, conversely, ADP-ribose degradation is suppressed, intracellular ADP-ribose accumulates, and as a result, it has an excellent anti-cancer effect.

[0023] In a specific embodiment of the present invention, fragments were synthesized from WWE domains present in various types of proteins, and their anti-cancer activities were irradiated. As a result, when the ADP-ribose binding peptides (SEQ ID NOs: 1 to 14) of Examples 1 to 14 were treated with various types of cancer cells, it was confirmed that the level of ADP-ribose in the cancer cells increased significantly (Figs. 1 to 4). Furthermore, through in vitro experiments, it was confirmed that the treatment with the peptide disrupted the intracellular ADP-ribose balance, suppressed the growth of cancer cells, and almost killed them (Figs. 5 to 11). When the ADP-ribose binding peptides of Examples 1 to 14 were administered subcutaneously or orally from a tumor xenograft animal model, it was confirmed that the growth of in vivo tumor tissues decreased rapidly (Figs. 19 and 20).

[0024] In another specific embodiment of the present invention, typically, the peptide of SEQ ID NO: 7 was treated with normal cells to evaluate the cytotoxicity against normal cells. As a result, it was confirmed that no toxicity was exhibited against normal cells (Figs. 21 and 22).

[0025] Therefore, the ADP-ribose binding peptides of SEQ ID NOs: 1 to 14 of the present invention have a very excellent anti-cancer effect regardless of the type of cancer and do not exhibit any cytotoxicity against normal cells, so they can be usefully used as a composition for cancer prevention or treatment.

[0026] The ADP-ribose binding peptide is not only a peptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 14, but also any sequence in which one or more amino acids are added, substituted, or deleted from these sequences, as long as it falls within the equivalent range, all belong to the scope of the present invention.

[0027] For example, a peptide having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with any one of the amino acid sequences of SEQ ID NOs: 1 to 14 of the present invention, and corresponding efficacy to the peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 14, that is, a peptide showing ADP-ribose binding activity and anti-cancer activity, even if it has an amino acid sequence in which a partial sequence is added, substituted or deleted from the amino acid sequences of SEQ ID NOs: 1 to 14, it is obvious that it is included within the scope of the present invention.

[0028] Also, if it has an activity corresponding to the peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 14, meaningless sequence addition before and after the amino acid sequence, naturally occurring mutations, or its potential silent mutations may also be included within the scope of the present invention.

[0029] In the present invention, the term "homology" means the degree of identity with a given amino acid sequence or nucleotide sequence and can be expressed as a percentage. In this specification, its homologous sequence having the same or similar activity as the given amino acid sequence or nucleotide sequence is expressed as "% homology". For example, it can be confirmed by using standard software for calculating parameters such as score, identity, and similarity, specifically BLAST 2.0, or by comparing sequences through Southern hybridization experiments under defined stringent conditions. The appropriate hybridization conditions defined are within the scope of the relevant art and can be determined by methods well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0030] In addition, the peptide of the present invention can include modifications such as chemical derivatization with one or more amino acids constituting the peptide in order to additionally improve desired properties. Despite such modifications, as long as it has anti-cancer activity equivalent to that of the peptide of the present invention, it is obvious to those skilled in the art that all fall within the scope of the present invention. The said derivatization can include, but is not limited to, acetylation, hydroxylation, methylation, amidation, pegylation, and the addition of carbohydrate or lipid components, cofactors, etc.

[0031] As a specific embodiment of the present invention, the peptide can be used in a form fused with a cell-penetrating peptide in order to enhance cell permeability. That is, the ADP-ribose binding peptide can further include a cell-penetrating peptide at the N-terminus, C-terminus, or both termini. At this time, a linker can be further included between the ADP-ribose binding peptide and the cell-penetrating peptide, which can be appropriately carried out by those skilled in the art.

[0032] In the present invention, the term "cell-penetrating peptide" refers to a peptide having the property of promoting the intracellular uptake / absorption of various substances such as nanoparticles, compounds, DNA, proteins, etc. Specifically, the cell-penetrating peptide may be, but is not limited to, a peptide derived from TAT, maurocalcine, penetratin, Poly-arginine, Antennapedia, Transportan, VP22, Hph-1, Poly-arginine, R11 (R9), Pep-1, HP4, LAH4, Vetofusing-1, a signal sequence-based peptide, or an amphipathic peptide. As long as it can promote the intracellular movement of the ADP-ribose binding peptide of the present invention, it can be appropriately selected by those skilled in the art.

[0033] In a specific embodiment of the present invention, TAT, a cell-penetrating peptide, was fused to the N-terminus of the ADP-ribose binding peptides of SEQ ID NOs: 1 to 14 of the present invention, and its anti-cancer activity was evaluated (SEQ ID NOs: 15 to 24, Table 2). As a result, it was confirmed that it has anti-cancer activity that is even more excellent than when the ADP-ribose binding peptide alone was used (Figs. 5 to 12, Figs. 17 and 18). Therefore, not only the ADP-ribose binding peptides of SEQ ID NOs: 1 to 14 of the present invention, but also the form in which a cell-penetrating protein is fused to the peptide can be very usefully used as a composition for preventing or treating cancer.

[0034] Furthermore, those skilled in the art can appropriately modify and use the ADP-ribose binding peptide of the present invention in order to apply it depending on the type of cell-penetrating peptide used. That is, the ADP-ribose binding peptide of the present invention is not limited to only the amino acid sequence presented in the present invention even when it is used by fusing it to a cell-penetrating peptide, and within a range obvious to those skilled in the art, that is, within an equivalent range, the amino acid sequence can be added / substituted / removed in a form appropriate for the application of the cell-penetrating peptide and used.

[0035] In addition, the ADP-ribose binding peptide of the present invention can be used together with reagents known in the art that can transfer proteins to cells or enhance the transfer efficiency in order to enhance cell permeability. The reagents are, for example, Chariot TM (Active motif, Cat.30025), Xflect TM (Takara, Cat.631324), Pierce TM (ThermoFisher Scientific, Cat.89850), ProteoJuice TM (Merck, Cat.71281), PULSin TMIt includes not only commercially available reagents such as (Poylplus transfection), but also other non-commercial reagents, and is not particularly limited to the above examples as long as the ADP-ribose binding peptide of the present invention can be transferred to cells.

[0036] Another aspect of the present invention is a polynucleotide encoding the ADP-ribose binding peptide.

[0037] Another aspect of the present invention is a vector containing the polynucleotide.

[0038] Another aspect of the present invention is a transformant containing the polynucleotide.

[0039] The ADP-ribose binding peptide is as described above.

[0040] The polynucleotide may have a nucleotide sequence encoding the ADP-ribose binding peptide of the present invention, or a nucleotide sequence having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology thereto, and as long as the polypeptide translated therefrom exhibits the corresponding efficacy of the ADP-ribose binding peptide of the present invention, addition of a nonsense sequence to the 5'- and / or 3'-ends of the nucleotide sequence, or deletion, modification, or substitution of a partial sequence may all be included within the scope of the present invention. The polynucleotide can be used in the form of an expression cassette operably linked to a known promoter sequence, or a vector containing the polynucleotide, and the production of the polynucleotide, expression cassette, or vector can be appropriately carried out through methods known to those skilled in the art. The types of promoters and vectors are not particularly limited and can be appropriately selected by those skilled in the art according to the purpose. Further, the polynucleotide, expression cassette, or vector can be transformed into a host cell to produce and use a transformant, and at this time, the transformation method can also be used without limitation by methods known to those skilled in the art. The transformant is an object for expressing the ADP-ribose binding peptide of the present invention, and may be a microorganism, plant, or animal, and may be one excluding humans, but is not limited thereto.

[0041] Those skilled in the art can produce and use a polynucleotide encoding the same, a vector containing the same, or a transformant containing the vector for applying / producing the ADP-ribose binding peptide for various purposes. For example, the polynucleotide or the vector can be directly used for therapeutic applications against cancer, and the peptide can be produced or used for therapeutic purposes by using a transformant expressing the ADP-ribose binding peptide including the same, but is not limited thereto.

[0042] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, comprising the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0043] Another aspect of the present invention is a method for preventing or treating cancer, comprising the use of the peptide or a pharmaceutically acceptable salt thereof for preventing or treating cancer, and the step of administering to an individual in need of the peptide or a pharmaceutically acceptable salt thereof.

[0044] As described above, the ADP-ribose binding peptide of the present invention has excellent effects on cancer prevention and / or treatment.

[0045] In the present invention, the term "cancer" refers to a disease related to the regulation of cell death, which occurs when the normal cell death balance is disrupted and cells proliferate excessively. In the present invention, the cancer includes all malignant tumors and benign tumors. For example, it may be brain cancer, head and neck cancer, lung cancer, breast cancer, thymoma, esophageal cancer, colorectal cancer, liver cancer, gastric cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma or skin cancer, but the types of cancer in the present invention are not limited by the above examples. The pharmaceutical composition for preventing or treating cancer of the present invention has a therapeutic effect on all cancers in which cell death can occur due to the accumulation of intracellular ADP-ribose.

[0046] As an example, the cancer may be a solid cancer such as brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, gastric cancer or ovarian cancer, but is not limited thereto.

[0047] In the present invention, the term "treatment" refers to intervening to alter the natural processes of an individual or cell having a disease, which can be performed during or to prevent the progression of a pathological condition. The intended therapeutic effects include preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the disease progression rate, alleviating or temporarily relieving the disease state, and improving the convalescence or prognosis. In particular, in the present invention, it includes all acts of improving the course of cancer by administering a composition containing an ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient. Also, the term "prevention" refers to all acts of suppressing or delaying the onset of cancer by administering the composition.

[0048] The weight percentage of the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof contained in the pharmaceutical composition is not particularly limited thereto, but may be contained in an amount of 0.0001 to 90% by weight, specifically 0.001 to 50% by weight, more specifically 0.01 to 20% by weight based on the total weight of the final composition.

[0049] The pharmaceutical composition can be further manufactured by including suitable carriers, excipients or diluents commonly used in the manufacture of drugs. Specifically, the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral patches, etc., external preparations, external patches, suppositories and sterile injection solutions by ordinary methods.

[0050] When the pharmaceutical composition is used for oral administration, it can be manufactured as a sustained-release preparation through appropriate encapsulation, enteric coating, blending of polymers, etc.

[0051] In one embodiment, the sustained-release preparation can be manufactured as a long acting preparation.

[0052] In one embodiment, in the long-acting preparation, the polymer and the lipid can be mixed at an appropriate ratio.

[0053] The pharmaceutical composition of the present invention can be administered to an individual who has developed cancer or is at risk of developing cancer. In the present invention, the term "individual" means all animals including humans.

[0054] The pharmaceutical composition of the present invention can be administered to a subject individual in a pharmaceutically effective amount. In the present invention, the term "administer" means introducing the pharmaceutical composition of the present invention to a subject individual by an appropriate method, and the administration route can be administered through various oral or parenteral routes as long as the target tissue can be reached. Examples of administration routes include oral, intramuscular, intravenous, arterial, subcutaneous, intraperitoneal, pulmonary, and nasal, and for example, it may be administered subcutaneously or orally, but is not limited thereto.

[0055] In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to prevent and / or treat cancer at a reasonable benefit / risk ratio applicable to medical use. Appropriate dosages and dosing frequencies can be selected by methods known in the art, and the amount and dosing frequency of the pharmaceutical composition of the present invention actually administered can be appropriately determined by various factors such as the type of symptoms to be treated, the administration route, gender, health status, diet, age, weight of the individual, and the severity of the disease.

[0056] In the present invention, a pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry. For example, there are salts of inorganic ions such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc., and salts of inorganic acids such as hydrochloric acid, phosphoric acid, and sulfuric acid. In addition, there are salts of organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, acetylsalicylic acid, etc., and amino acid salts such as lysine, arginine, guanidine, etc. Also, there are salts of organic ions such as tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, benzyltrimethylammonium, benzetonium, etc., which can be used in pharmaceutical reactions, purification, and separation processes. However, the types of salts meant in the present invention are not limited by these listed salts.

[0057] Since the ADP - ribose binding peptide of the present invention has excellent anti - cancer activity, it can be produced not only in the form of a pharmaceutical composition but also in the form of a functional food composition.

[0058] When the composition of the present invention is produced in the form of a food composition, the food composition can contain additional components that are commonly used in foods and can improve odor, taste, visual appearance, etc. For example, food additives can be added. The additives are selected according to the type of food and used in appropriate amounts.

[0059] The food composition can be produced as a health - functional food. Here, a health - functional food is the same term as a food for special health use (FoSHU), and it means a food with high medical and therapeutic effects that is processed so that its biological regulatory function can be efficiently shown in addition to nutrient supply. The health - functional food can be produced in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain useful effects for the improvement of cancer.

[0060] Another aspect of the present invention is a pharmaceutical composition for enhancing the reactivity to a second anti-cancer agent, which contains the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0061] Another aspect of the present invention is a pharmaceutical composition for preventing or treating cancer, which contains (i) the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof, and (ii) a second anti-cancer agent as active ingredients.

[0062] The ADP-ribose binding peptide and a pharmaceutically acceptable salt thereof are as described above.

[0063] In addition to having an anti-cancer effect alone, the ADP-ribose binding peptide of the present invention also has a very excellent effect as a pharmaceutical composition for anti-cancer adjuvant for the purpose of enhancing the reactivity to a second anti-cancer agent. Therefore, together with the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof, a second anti-cancer agent can be used as an active ingredient to form a pharmaceutical composition for preventing or treating cancer.

[0064] In the present invention, the term "second anti-cancer agent" refers to any drug having anti-cancer activity excluding the ADP-ribose binding peptide of the present invention. In the present invention, the scope of the second anti-cancer agent is not particularly limited, and those skilled in the art can appropriately select and use an appropriate type according to the type of cancer and the degree of progression for the purpose of complete cure, regulation, symptom relief, etc. of cancer. The second anti-cancer agent may be, for example, a cytotoxic anti-cancer agent, a targeted anti-cancer agent, an immune anti-cancer agent or a metabolic anti-cancer agent, but is not limited thereto.

[0065] In the present invention, the cytotoxic anti-cancer agent is a drug that attacks cancer cells that divide indiscriminately at a faster rate than normal cells and exhibits an anti-cancer effect, and its meaning is the same as that commonly used in the technical field to which the present invention belongs. The cytotoxic anti-cancer agent includes alkylating agents, antimetabolites, and natural anti-cancer agents.

[0066] The alkylating agents include, but are not limited to, nitrogen mustards (e.g., cyclophosphamide, chloromethine, uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, etc.), alkyl sulfonates (e.g., busulfan, procarbazine, etc.), nitrosoureas (e.g., carmustine, lomustine, streptozocin, etc.), platinum-based alkylating agents (e.g., cisplatin, carboplatin, dicycloplatin, eptaplatin, lobaplatin, miloplatin, nedaplatin, oxaliplatin, picoplatin, satraplatin, triplatin tetranitrate, etc.). Alkylating agents can induce the destruction of cancer cells by binding to DNA in cancer cells and damaging the DNA structure.

[0067] The antimetabolites include, but are not limited to, pyrimidine derivatives (e.g., 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine, etc.), folate derivatives (e.g., methotrexate, pemetrexed, etc.), purine derivatives (e.g., mercaptopurine, etc.). Antimetabolites can induce cancer cell death by suppressing the metabolism necessary for DNA replication and cell survival.

[0068] The natural product anticancer agents include, but are not limited to, topoisomerase inhibitors (e.g., camptothecin, epipodophyllotoxin, taxane series drugs (docetaxel, paclitaxel)), antibiotics (e.g., dactinomycin, doxorubicin, daunorubicin, mitomycin, bleomycin, idarubicin, mitoxantrone HCl, etc.).

[0069] In the present invention, the target anti-cancer agent is an anti-cancer agent that induces the death of cancer cells by suppressing a target protein (receptor or enzyme) involved in cancer growth, and its meaning is the same as that commonly used in the technical field to which the present invention pertains. The cytotoxic anti-cancer agent includes a target protein (such as tyrosine kinase) inhibitory low molecular compound and a monoclonal antibody.

[0070] As an example, the target anti-cancer agent may be a receptor tyrosine kinase inhibitor that targets one or more targets selected from the group consisting of VEGF-A and EGFR.

[0071] In one embodiment, the target anti-cancer agent that can be co-administered with the ADP-ribose binding peptide is a VEGF-A inhibitor. In the present invention, examples of VEGF-A inhibitors include monoclonal antibodies such as bevacizumab, ranibizumab, aflibercept, ramucirumab, and low molecular compounds such as sunitinib, pazopanib, sorafenib, axitinib, but are not limited thereto.

[0072] In one embodiment, the target anti-cancer agent that can be co-administered with the ADP-ribose binding peptide is an EGFR inhibitor. In the present invention, examples of EGFR inhibitors include low molecular compounds such as osimertinib, gefitinib, erlotinib, afatinib, brigatinib, icotinib, vandetanib, and monoclonal antibodies such as cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab, but are not limited thereto.

[0073] In addition, the target anti-cancer agent of the present invention also includes HER2 target anti-cancer agents such as lapatinib, neratinib, and afatinib; Bcr-Abl target anti-cancer agents such as imatinib, dasatinib, and nilotinib; Src target anti-cancer agents such as bosutinib; JAK target anti-cancer agents such as lestaurtinib, ruxolitinib, and pacritinib; MAP2 Kinase target anti-cancer agents such as cobimethinib, selumetinib, trametinib, and binimetinib; MEL4-ALK target anti-cancer agents such as ceritibin and crizotinib, etc., and there is no particular limitation on the type thereof.

[0074] Further, the second anti-cancer agent of the present invention may be a combination of one or more cytotoxic anti-cancer agents and / or target anti-cancer agents, and these may be administered simultaneously or at different times.

[0075] In the present invention, an immune anti-cancer agent refers to a drug that activates the human immune system to fight cancer cells. In the present invention, the immune anti-cancer agent includes immune checkpoint inhibitors, immune cell therapy agents, anti-cancer vaccines, and antibody-drug conjugates, and appropriate types can be selected for the complete cure, regulation, and symptom relief of cancer according to the type and progression degree of cancer.

[0076] In one embodiment, the immune anti-cancer agent may be an immune checkpoint inhibitor, and may be one or more selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, a CD28 antibody, a KIR antibody, a TCR antibody, a LAG-3 antibody, a TIM-3 antibody, a TIGIT antibody, an A2aR antibody, an ICOS antibody, an OX40 antibody, a 4-1BB antibody, and a GITR antibody. For example, the immune checkpoint inhibitor may be a PD-1 antibody such as nivolumab, pembrolizumab, cemiplimab, pidilizumab, toripalimab; a PD-L1 antibody such as atezolizumab, avelumab, duralumab; a CTLA-4 antibody such as ipilimumab, tremelimumab, but is not limited thereto.

[0077] In one embodiment, the immune anti-cancer agent may be an immune cell therapy agent, and may be a CAR-T therapy agent such as tisagenlecleucel, axicabtagene ciloleucel, or a CAR-NK therapy agent, but is not limited thereto.

[0078] In the present invention, a metabolic anti-cancer agent refers to a drug that participates in various metabolic actions related to the growth and survival of cancer cells, such as supplying nutrients to cancer cells, and kills cancer cells by participating in such actions. The metabolic anti-cancer agent may be, for example, IM-156, 3-bromopyruvic acid (3BP), NYH817100, WZB117, GNE-140, AZ93, AZD3965, CPI-613, MKT-077, CB-839, CB-1158, CPI-444, TVB-2640, NDI-010976, TCD-717, ADI-PEG20, Epacadostat, Indoximod, PX478, CPI-0610, RTA402, APO866, GMX1778, AG-221 or AG-120, etc., but is not limited thereto.

[0079] Since the ADP-ribose binding peptide of the present invention has the effect of disrupting the balance of ADP-ribose in cancer cells and enhancing the reactivity to the second anti-cancer agent, it can be used as an anti-cancer adjuvant, and is not particularly limited by the type of the second anti-cancer agent used or the type of cancer. As an example, the cancer may be a solid cancer such as brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, stomach cancer or ovarian cancer, but is not limited thereto.

[0080] The ADP-ribose binding peptide of the present invention can be administered in combination in a form that exists independently of the second anti-cancer agent, and depending on the purpose, it can be administered in a state where a physical / chemical bond is formed with the second anti-cancer agent by any known method. For example, the ADP-ribose binding peptide can be used in a state directly bound to the second anti-cancer agent, or can be used in a state linked to the second anti-cancer agent through a known linker. As long as the ADP-ribose binding peptide of the present invention acts together with the second anti-cancer agent to exhibit a synergistic anti-cancer effect, the application method thereof is not particularly limited.

[0081] In a specific embodiment of the present invention, when the ADP-ribose binding peptide of the present invention is treated with low concentrations of bevacizumab, osimertinib, gemcitabine, and docetaxel in different cancer types, it was confirmed that the reactivity to the anticancer agent was enhanced in all cancer cells (FIGS. 13 and 14). Therefore, the ADP-ribose binding peptide of the present invention can enhance the reaction of the second anticancer agent and can be very usefully used as an anticancer adjuvant. Since it has excellent anticancer activity even when the second anticancer agent is treated at a low concentration, it is possible to minimize the side effects that may be caused by the second anticancer agent.

[0082] Another aspect of the present invention is an anticancer adjuvant pharmaceutical composition for enhancing reactivity to radiation anticancer therapy, which contains the ADP-ribose binding peptide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0083] The ADP-ribose binding peptide and its pharmaceutically acceptable salt are as described above.

[0084] In the present invention, the term "radiation anticancer therapy" refers to a therapeutic act of irradiating cancer cells or tumor tissues with radiation for the purpose of killing cancer cells. Generally, it is a standard treatment method for controlling tumors that are inoperable or surgically inaccessible, or tumor metastases, based on the principle that the radiation transmitted to the target site causes the death of reproductive cells. In the present invention, the radiation anticancer therapy may be, but is not limited to, ionizing radiation therapy, electromagnetic radiation, brachytherapy, or external beam radiation therapy.

[0085] The composition containing the ADP-ribose binding peptide according to the present invention or a pharmaceutically acceptable salt thereof exhibits a synergistic anti-cancer effect when used in combination with radiotherapy, and thus can be usefully utilized as an anti-cancer adjuvant for radiotherapy or a radiosensitizer for improving radiation sensitivity, and is not particularly limited to the type of cancer applicable. As an example, the cancer may be a solid cancer such as brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, stomach cancer, and ovarian cancer, but is not limited thereto.

[0086] In one embodiment, the solid cancer may be resistant to radiotherapy. Since the anti-cancer effect of radiotherapy is shown by the formation of DNA breaks, the resistance to radiotherapy is generally determined by the mechanism capable of repairing DNA damage induced by radiotherapy. As a method of synergizing the anti-cancer efficacy of radiotherapy, there have been many attempts to suppress the DNA repair mechanism, and when the repair of the broken DNA strand is blocked, the sensitivity to radiotherapy can be increased. Two main forms of DNA damage are SSB (Single Strand Breaks) and DSB (Double Strand Breaks). Therefore, it can be explained in the scope of two repair pathways targeting SSB and DSB. BER (Base Excision Repair) is one of the various pathways involved in the repair of selected types of DNA SSB.

[0087] PARP1 plays an important role in the BER of DNA SSB through a process known as ADP-ribosylation. In the nucleus, PARP1 senses the damage of SSB DNA and recruits DNA repair complexes to the SSB site through ADP-ribosylation for repair. The over-accumulation of poly-ADP-ribose synthesized through ADP-ribosylation by PARP-1 activity ultimately leads to cell death. Therefore, cancer cells activate the degradation of poly-ADP-ribose through proteasomes such as PARG and ARH3 to prevent this phenomenon and activate survival signaling activities. The inventors focused on the fact that the accumulation of ADP-ribose and ADP-ribose polymers in cancer cells can act as mediators that can disrupt such biochemical survival mechanisms of cancer cells and can be utilized as important anti-cancer adjuvants to overcome radiation therapy resistance.

[0088] In a specific embodiment of the present invention, when the ADP-ribose binding peptide of the present invention was co-treated with low-dose radiation in different cancer types, it was confirmed that the responsiveness to radiation therapy was enhanced in all cancer types (FIGS. 15 and 16). Therefore, the ADP-ribose binding peptide of the present invention can enhance the response to radiation therapy, can be very usefully used for anti-cancer adjuvant applications, and has excellent anti-cancer activity even when irradiated with a resistant dose of radiation, so it can minimize the side effects that can be caused by radiation.

[0089] Embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. Also, the embodiments of the present invention are provided to more fully explain the present invention to those with average knowledge in the relevant technical field. Furthermore, throughout the specification, stating that a certain component "includes" means that, unless otherwise specifically stated to the contrary, it does not exclude other components and can further include other components.

[0090] [Embodiments for Carrying Out the Invention] Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are merely for exemplifying the present invention, and the scope of the present invention is not limited by these examples.

[0091] Example 1 - 14. Production of ADP (adenosine diphosphate)-ribose binding peptides The ADP-ribose binding peptides of Examples 1 to 14 were synthesized from WWE domains present in various types of proteins and used in experiments. Specific information on the peptides used is shown in Table 1 below.

[0092] [Table 1]

[0093] In addition, a cell-penetrating peptide was bound to the N-terminus of the peptides of Examples 1 to 14 to produce the peptides of Examples 15 to 28. The peptide sequences of Examples 15 to 28 are shown in Table 2.

[0094] [Table 2]

[0095] Experimental Example 1: Confirmation of changes in ADP (adenosine diphosphate)-ribose levels in cancer cells The peptides of Examples 1 to 28 of the present invention were designed to be able to suppress the enzymatic action of poly ADP-ribose degradation. In Experimental Example 1, through cell experiments, it was confirmed whether the degradation of poly ADP-ribose was suppressed by each peptide and accumulated in cancer cells.

[0096] 1-1. Changes in ADP-ribose levels in U-87MG cells 5×10 5 cells of U-87MG were cultured at 37 °C in 5% CO 2The example peptides were divided into a group not treated with the peptides and groups treated with the peptides of Example 1 (SEQ ID NO: 1) or Example 8 (SEQ ID NO: 8) at 0.2, 1, 2, 4, 8, 16, and 32 μM, and cultured in EMEM (Eagle's Minimum Essential Medium) medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin for 24 hours under the conditions. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0097] The upper left of Figure 1 and the lower right of Figure 2 show the fold change in the increased amount of ADP-ribose in the control group compared to the treatment with the peptides of Example 1 or 8 in U-87MG cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner for both of the two treated peptides compared to the non-treated group ( ** p < 0.001).

[0098] 1-2. Changes in ADP-ribose levels in H1975 cells 5×10 5 individual H1975 cells were divided into a group not treated with the example peptides and a group treated with the peptide of Example 2 (SEQ ID NO: 2) at 0.2, 1, 2, 4, 8, 16, and 32 μM, and cultured in RPMI-1640 medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin for 24 hours at 37°C and 5% CO 2 conditions. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0099] The upper right of Figure 1 and the upper left of Figure 3 show the fold change in the increased amount of ADP-ribose in the control group compared to the treatment with the peptides of Example 2 or Example 9 in H1975 cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner for the treated peptide compared to the non-treated group (**p < 0.001).

[0100] 1-3. Changes in ADP-ribose levels in Aspc-1 cells 5×10 5 Aspc-1 cells were divided into a group not treated with the example peptide and groups treated with 0.2, 1, 2, 4, 8, 16, and 32 μM of the peptide of Example 3 (SEQ ID NO: 3) in RPMI-1640 medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin under conditions of 37 °C and 5% CO 2 and cultured for 24 hours. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0101] The lower left of FIG. 1 and the upper right of FIG. 3 show the fold change in the increase in ADP-ribose in the Aspc-1 cells by the peptide treatment of Example 3 or Example 10 compared to the control group, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner with the peptide concentration compared to the non-treated group ( ** p < 0.001).

[0102] 1-4. Changes in ADP-ribose levels in Hep G2 cells 5×10 5 Hep G2 cells were divided into a group not treated with the example peptide and groups treated with 0.2, 1, 2, 4, 8, 16, and 32 μM of the peptide of Example 4 (SEQ ID NO: 4) in EMEM medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin under conditions of 37 °C and 5% CO 2 and cultured for 24 hours. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0103] The lower right of FIG. 1 and the lower left of FIG. 3 show the fold change in the increase in ADP-ribose compared to the control group by peptide treatment in Example 4 or Example 11 in Hep G2 cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner with the peptide being treated compared to the untreated group ( ** p < 0.001).

[0104] 1-5. Changes in ADP-ribose levels in MDA-MB-231 cells 5×10 5 MDA-MB-231 cells were divided into a group untreated with the peptide of the example and groups treated with the peptides of Example 5 (SEQ ID NO: 5) at 0.2, 1, 2, 4, 8, 16, 32 μM, and cultured for 24 hours in Leibovitz’s L-15 medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37 °C and 5% CO 2 conditions. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0105] The upper left of FIG. 2 and the lower right of FIG. 3 show the fold change in the increase in ADP-ribose compared to the control group by peptide treatment in Example 5 or Example 12 in MDA-MB-231 cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner with the peptide being treated compared to the untreated group ( ** p < 0.001).

[0106] 1-6. Changes in ADP-ribose levels in HCT116 cells 5×10 5 HCT116 cells were cultured at 37 °C and 5% CO 2The group not treated with the example peptide and the groups treated with the peptides of Example 6 (SEQ ID NO: 6) at 0.2, 1, 2, 4, 8, 16, and 32 μM were divided and cultured in McCoy's 5A medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin under the conditions for 24 hours. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0107] The upper right of Figure 2 and the left side of Figure 4 show the fold change in the increased amount of ADP-ribose in the control group compared to the peptide treatment of Example 6 or Example 13 in HCT116 cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner with the peptide concentration compared to the untreated group ( ** p < 0.001).

[0108] 1-7. Changes in ADP-ribose levels in Caki-1 cells 5 × 10 5 individual Caki-1 cells were divided into a group not treated with the example peptide and groups treated with the peptides of Example 7 (SEQ ID NO: 7) at 0.2, 1, 2, 4, 8, 16, and 32 μM and cultured in McCoy's 5A medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin at 37°C and 5% CO 2 conditions for 24 hours. The medium of the cultured cells was removed and treated with RIPA buffer and 1% SDS. The prepared supernatant was utilized for ELISA analysis to detect and analyze ADP-ribose.

[0109] The lower left of Figure 2 and the right side of Figure 4 show the fold change in the increased amount of ADP-ribose in the control group compared to the peptide treatment of Example 7 or Example 14 in Caki-1 cells, indicating that the amount of ADP-ribose increased significantly in a concentration-dependent manner with the peptide concentration compared to the untreated group ( ** p < 0.001).

[0110] Experimental Example 2: Changes in Cancer Cell Viability by Peptide Treatment of Examples Cells maintain biochemical homeostasis and balance the production and decomposition of ADP-ribose. Since cancer cells continuously divide and grow rapidly, if such a balance is disrupted, they can be significantly affected in terms of survival compared to normal cells.

[0111] According to the results of Experimental Example 1, when the example peptides of the present invention are treated on cancer cells, the amount of intracellular ADP-ribose increases significantly, and thus the homeostasis balance of cancer cells may be disrupted through this. Therefore, in Experimental Example 2, the viability of cancer cells by peptide treatment of Examples 1 to 28 was measured.

[0112] 2-1. Changes in viability of U-87MG cells treated with ADP-ribose binding peptide 3×10 3 U-87MG cells were cultured at 37 °C and 5% CO 2 for 24 hours under the conditions, and then either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM, respectively. Then, after further culturing at 37 °C and 5% CO 2 for 24 hours under the conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the viability of the cells.

[0113] The upper left of FIG. 5 and the upper right of FIG. 8 are representative microscopic photographs of the groups treated with the peptides of Examples 1, 15, 8, and 22 on U-87MG cells and the untreated group. Commonly, in the untreated group of peptides, a form in which U-87MG cells are growing rapidly can be observed, while in the group treated with the example peptides of the present invention, the growth of U-87MG cells is suppressed and a form in which they are dead can be observed. In particular, it was confirmed that cancer cells were completely killed in the groups treated with the peptides of Examples 15 and 22 to which cell-penetrating peptide (CPP) was attached.

[0114] 2-2. Changes in viability of H1975 cells treated with ADP-ribose binding peptide 3×10 cells of H1975 were seeded in a 96-well plate and cultured at 37 °C and 5% CO 3 for 24 hours. After that, the cells were either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM. Then, the cells were cultured at 37 °C and 5% CO 2 for an additional 24 hours. After that, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability. 2 The upper right of FIG. 5 and the upper left of FIG. 9 are representative microscopic photographs of the groups treated with the peptides of Examples 2, 16, 9, and 23 and the untreated group in H1975 cells. Commonly, in the untreated group, a morphology in which H1975 cells grow rapidly can be observed, while in the group treated with the example peptides of the present invention, a morphology in which the growth of H1975 cells is suppressed and the cells are dead can be observed. In particular, it was confirmed that the cancer cells were completely killed in the groups treated with the peptides of Examples 16 and 23 to which CPP was attached.

[0115] The upper right of FIG. 5 and the upper left of FIG. 9 are representative microscopic photographs of the groups treated with the peptides of Examples 2, 16, 9, and 23 and the untreated group in H1975 cells. Commonly, in the untreated group, a morphology in which H1975 cells grow rapidly can be observed, while in the group treated with the example peptides of the present invention, a morphology in which the growth of H1975 cells is suppressed and the cells are dead can be observed. In particular, it was confirmed that the cancer cells were completely killed in the groups treated with the peptides of Examples 16 and 23 to which CPP was attached.

[0116] 2-3. Changes in viability of Aspc-1 cells treated with ADP-ribose binding peptide 3×10 cells of Aspc-1 were seeded in a 96-well plate and cultured at 37 °C and 5% CO 3 for 24 hours. After that, the cells were either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM. Then, the cells were cultured at 37 °C and 5% CO 2 for an additional 24 hours. After that, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability. 2 for an additional 24 hours. After that, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability.

[0117] The upper left of FIG. 6 and the upper right of FIG. 9 are representative micrographs of the groups treated with the peptides of Examples 3, 17, 10, and 24 and the untreated group in Aspc-1 cells. Commonly, in the untreated group, a form in which Aspc-1 cells are growing rapidly can be observed, while in the group treated with the example peptides of the present invention, a form in which the growth of Aspc-1 cells is suppressed and they are dying can be observed. In particular, it was confirmed that cancer cells were completely killed in the groups treated with the peptides of Examples 17 and 24 to which the cell-penetrating peptide CPP was attached.

[0118] 2-4. Changes in viability of Hep G2 cells treated with ADP-ribose binding peptide 3×10 3 Hep G2 cells were cultured in a 96-well plate at 37 °C and 5% CO 2 for 24 hours under conditions, and then either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM, respectively. Then, after further culturing at 37 °C and 5% CO 2 for 24 hours under conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0119] The upper right of FIG. 6 and the upper left of FIG. 10 are representative micrographs of the groups treated with the peptides of Examples 4, 18, 11, and 25 and the untreated group in Hep G2 cells. Commonly, in the untreated group, a form in which Hep G2 cells are growing rapidly can be observed, while in the group treated with the example peptides of the present invention, a form in which the growth of Hep G2 cells is suppressed and they are dying can be observed. In particular, it was confirmed that cancer cells were completely killed in the groups treated with the peptides of Examples 18 and 25 to which the cell-penetrating peptide CPP was attached.

[0120] 2-5. Changes in viability of MDA-MB-231 cells treated with ADP-ribose binding peptide 3×10 in a 96-well plate 3 MDA-MB-231 cells were cultured at 37 °C and 5% CO 2 for 24 hours under the conditions, and then either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM. Subsequently, after culturing for an additional 24 hours at 37 °C and 5% CO 2 conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0121] The upper left of FIG. 7 and the upper right of FIG. 10 are representative micrographs of the groups treated with the peptides of Examples 5, 19, 12, and 26 and the untreated group in MDA-MB-231 cells. Commonly, in the group without peptide treatment, it is possible to observe the morphology in which MDA-MB-231 cells are growing rapidly, while in the group treated with the example peptides of the present invention, it is possible to observe the morphology in which the growth of MDA-MB-231 cells is suppressed and the cells are dying. In particular, it was confirmed that the cancer cells were completely killed in the groups treated with the peptides of Examples 19 and 26 to which CPP was attached.

[0122] 2-6. Changes in viability of HCT116 cells treated with ADP-ribose binding peptide 3×10 in a 96-well plate 3 HCT116 cells were cultured at 37 °C and 5% CO 2 for 24 hours under the conditions, and then either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM. Subsequently, after culturing for an additional 24 hours at 37 °C and 5% CO 2 conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0123] The upper right of Figure 7 and the upper left of Figure 11 are representative microscopic photographs of the groups treated with the peptides of Examples 6, 20, 13, and 27 and the untreated group in HCT116 cells. Commonly, in the untreated group, a morphology in which HCT116 cells are growing rapidly can be observed, while in the group treated with the example peptides of the present invention, a morphology in which the growth of HCT116 cells is suppressed and the cells are dying can be observed. In particular, it was confirmed that cancer cells were completely killed in the groups treated with the peptides of Examples 20 and 27 to which the cell-permeable peptide CPP was attached.

[0124] 2-7. Changes in viability of Caki-1 cells treated with ADP-ribose binding peptide 3×10 3 Caki-1 cells were cultured in a 96-well plate at 37 °C and 5% CO 2 for 24 hours under conditions, and then either left untreated or treated with the peptides of Examples 1 to 28 at a concentration of 16 μM, respectively. Then, after further culturing at 37 °C and 5% CO 2 for 24 hours under conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0125] The upper left of Figure 8 and the upper right of Figure 11 are representative microscopic photographs of the groups treated with the peptides of Examples 7, 21, 14, and 28 and the untreated group in Caki-1 cells. Commonly, in the untreated group, a morphology in which Caki-1 cells are growing rapidly can be observed, while in the group treated with the example peptides of the present invention, a morphology in which the growth of Caki-1 cells is suppressed and the cells are dying can be observed. In particular, it was confirmed that cancer cells were completely killed in the groups treated with the peptides of Examples 21 and 28 to which CPP was attached.

[0126] 2-8. Changes in viability of SNU-1 cells and OVCAR-3 cells treated with ADP-ribose binding peptide 3×10 3Individual SNU-1 cells or OVCAR-3 cells were cultured at 37°C and 5% CO 2 After culturing for 24 hours under the conditions, either no treatment was performed, or each of the peptides of Examples 15 to 28 at a concentration of 16 μM was treated. Then, after further culturing for 96 hours at 37°C and 5% CO2, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0127] As a result, it was confirmed that the groups treated with the peptides of Examples 15 to 28 almost killed cancer cells in both SNU-1 cells and OVCAR-3 cells (Figure 12).

[0128] Experimental Example 3: Change in cancer cell viability by combined treatment of the peptide of the example and a low concentration of anticancer agent Since the anticancer effect represented by the decrease in cancer cell survival by existing anticancer agents can be synergistically enhanced by the signal of ADP-ribose, it was expected that when an existing anticancer agent was treated to induce the accumulation of ADP-ribose, even if the existing anticancer agent was treated at a concentration below the required concentration in cancer cells, a synergistic anticancer effect would be shown. As confirmed in Experimental Example 1 above, since the peptide of the example of the present invention significantly increases the amount of ADP-ribose in cancer cells, in Experimental Example 3, when the peptide of the example and a low concentration of anticancer agent were treated in combination, it was attempted to confirm whether a synergistic anticancer effect was shown.

[0129] 3-1. Changes in viability of U-87MG cells treated with the combined treatment of the example peptide and low-concentration bevacizumab 3×10 3 Individual U-87MG cells were cultured at 37°C and 5% CO 2 After culturing for 24 hours under the conditions, they were treated with 18 mM bevacizumab alone or in combination with the peptides of Examples 1 to 14 at concentrations of 0.2, 1, or 8 μM. Then, at 37°C and 5% CO 2After further culturing for 24 hours under the conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0130] As a result, when U-87MG cells were treated with bevacizumab alone, about 49% of all cancer cells survived. However, in all cases of combined treatment with the example peptides, it was confirmed that the viability of cancer cells was significantly reduced even with a low concentration of bevacizumab ( * p < 0.05, ** p < 0.001, *** p < 0.001; the left side of Figure 13).

[0131] 3-2. Changes in viability of H1975 cells treated with the combined treatment of the example peptide and low-concentration osimertinib 3×10 3 individual H1975 cells were cultured at 37 °C and 5% CO 2 for 24 hours under the conditions, and then treated with 1 nM osimertinib alone or in combination with the peptides of Examples 1 to 14 at concentrations of 0.2, 1, or 8 μM. Then, after further culturing for 24 hours at 37 °C and 5% CO 2 under the conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0132] As a result, when H1975 cells were treated with 1 nM osimertinib alone, about 49% of all cancer cells survived. However, in all cases of combined treatment with the example peptides, it was confirmed that the viability of cancer cells was significantly reduced even with a low concentration of osimertinib ( * p < 0.05, ** p < 0.001, *** p < 0.001; the right side of Figure 13).

[0133] 3-3. Changes in viability of Aspc-1 cells treated with the combined treatment of the example peptide and low-concentration gemcitabine 3×10 cells were seeded in a 96-well plate and cultured at 37 °C in 5% CO 3 for 24 hours. After that, the cells were treated with 1 μM gemcitabine alone or in combination with the peptides of Examples 15 to 28 at concentrations of 0.2, 1 or 8 μM. Then, the cells were cultured at 37 °C in 5% CO 2 for an additional 24 hours. After that, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability. 2

[0134] As a result, when aspc-1 cells were treated with gemcitabine alone, about 50% of all cancer cells survived. However, in all cases of treatment in combination with the example peptides, it was confirmed that the viability of cancer cells was significantly reduced even with low concentrations of gemcitabine ( * p < 0.05, ** p < 0.001, *** p < 0.001; left side of Figure 14).

[0135] 3-4. Changes in viability of MDA-MB-231 cells treated with the combined treatment of the example peptide and low-concentration docetaxel 3×10 cells were seeded in a 96-well plate and cultured at 37 °C in 5% CO 3 for 24 hours. After that, the cells were treated with 50 μM docetaxel alone or in combination with the peptides of Examples 15 to 28 at concentrations of 0.2, 1 or 8 μM. Then, the cells were cultured at 37 °C in 5% CO 2 for an additional 24 hours. After that, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and allowed to react for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and the absorbance was measured to confirm the cell viability. 2

[0136] ​​As a result, when MDA-MB-231 cells were treated with docetaxel alone, approximately 52% of all cancer cells survived. However, in all cases of combined treatment with the example peptides, it was confirmed that even at a low concentration of docetaxel, the survival rate of cancer cells was significantly reduced ( * p < 0.05, ** p < 0.001, *** p < 0.001; on the right side of Figure 14).

[0137] Experimental Example 4: Change in cancer cell survival rate by combined treatment of the example peptide and radiation Radiation therapy can be expected to have an anti-cancer effect through a killing effect due to damage to genetic material. However, cancer cells continuously repair DNA strands through a process known as ADP-ribosylation in order to overcome this. However, it was expected that if it was assisted to continuously accumulate ADP-ribose, which only temporarily increases due to the repair action by radiation irradiation, there would be a synergistic anti-cancer effect even when irradiated with a resistant dose of radiation. As confirmed in Experimental Example 1 above, since the example peptide of the present invention significantly increases the amount of ADP-ribose in cancer cells, in Experimental Example 4, an attempt was made to confirm whether a synergistic anti-cancer effect is shown when the example peptide is combined with a resistant dose of radiation irradiation.

[0138] 4-1. Changes in viability of H1975 cells treated with the combined treatment of the example peptide and resistant-dose radiation 3 × 10 3 H1975 cells were cultured in a 96-well plate at 37°C and 5% CO 2 for 24 hours, and then treated with radiation alone at a dose of 2 Gy or in combination with peptides of SEQ ID NOs: 1 to 14 at 1.6 or 3.2 μM. Then, after further culturing at 37°C and 5% CO 2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the survival rate of the cells.

[0139] As a result, when H1975 cells were treated with a radiation dose of 2 Gy alone, about 76% of the cancer cells showed resistance to survive compared to the untreated group. However, in all cases of combined treatment with the example peptides, it was confirmed that the survival rate of cancer cells decreased significantly even with radiation irradiation at the resistant dose ( ** p < 0.001, *** p < 0.001; the left side of Figure 15).

[0140] 4-2. Changes in viability of Aspc-1 cells treated with the combined treatment of the example peptide and resistant-dose radiation 3 × 10 3 Aspc-1 cells were cultured in a 96-well plate at 37 °C and 5% CO 2 for 24 hours. After that, they were treated with radiation alone at a dose of 2 Gy or in combination with peptides of SEQ ID NOs: 1 to 14 at 1.6 or 3.2 μM. Then, after further culturing at 37 °C and 5% CO 2 for 24 hours, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell survival rate.

[0141] As a result, when Aspc-1 cells were treated with a radiation dose of 2 Gy alone, about 86% of the cancer cells showed resistance to survive compared to the untreated group. However, in all cases of combined treatment with the example peptides, it was confirmed that the survival rate of cancer cells decreased significantly even with radiation irradiation at the resistant dose ( ** p < 0.001, *** p < 0.001; the right side of Figure 15).

[0142] 4-3. Changes in viability of MDA-MB-231 cells treated with the combined treatment of the example peptide and resistant-dose radiation 3 × 10 3 MDA-MB-231 cells were cultured in a 96-well plate at 37 °C and 5% CO 2 for 24 hours. After that, they were treated with radiation alone at a dose of 2 Gy or in combination with peptides of SEQ ID NOs: 15 to 28 at 1.6 or 3.2 μM. Then, after further culturing at 37 °C and 5% CO 2After further culturing for 24 hours under the conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0143] As a result, when MDA-MB-231 cells were treated with 2 Gy of radiation alone, about 86% of the cancer cells showed resistance to survive compared to the untreated group. However, in all cases of combined treatment with the example peptide, it was confirmed that the viability of cancer cells decreased significantly even with radiation at the resistant dose ( ** p < 0.001, *** p < 0.001; on the left side of Figure 16).

[0144] 4-4. Changes in survival rate by combined treatment of the example peptide and a tolerable dose of radiation in Caki-1 cells 3×10 3 Caki-1 cells were cultured in a 96-well plate at 37 °C and 5% CO 2 for 24 hours under the conditions, and then treated with 2 Gy of radiation alone or in combination with 1.6 or 3.2 μM of the peptides of SEQ ID NOs: 15-28. Then, after further culturing at 37 °C and 5% CO 2 for 24 hours under the conditions, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to each well and reacted for 1 hour. After removing the reacted reagent, 200 μl of dimethyl sulfoxide was added to each well, and then the absorbance was measured to confirm the cell viability.

[0145] As a result, when Caki-1 cells were treated with 2 Gy of radiation alone, about 96% of the cancer cells showed resistance to survive compared to the untreated group. However, in all cases of combined treatment with the example peptide, it was confirmed that the viability of cancer cells decreased significantly even with radiation at the resistant dose ( ** p < 0.001, *** p < 0.001; on the right side of Figure 16).

[0146] Experimental Example 5: Comparison and Verification of Anti-Cancer Efficacy and Effects According to the Administration Route of the Example Peptide Using an Animal Model 5-1. Changes in tumor volume from an animal model injected with the example peptide via the subcutaneous route Aspc-1 cells (1×10 7 ) were inoculated into the back of the flanks of 5-week-old balb / c nude mice, and classified into 15 groups: an untreated control group (control) and groups injected subcutaneously with the peptides of Examples 15 to 28, respectively. When the tumor volume grew to approximately 150 mm 3 , each example peptide was subcutaneously injected at a dose of 20 mg / kg three times a week. The tumor size was measured with a digital caliper, and the change results of the tumor volume were compared by group.

[0147] As a result, the final volume of the control group tumor after the final dosing grew significantly to approximately 3136 mm 3 , but it was confirmed that tumor growth was significantly suppressed in all groups injected subcutaneously with the peptides of Examples 15 to 28 ( ** p<0.001; Figure 17).

[0148] Figure 18 shows representative tumor photographs obtained after autopsy in all groups. When compared with the control group, it was observed that tumor tissue growth was significantly suppressed in all groups injected subcutaneously with the example peptides of the present invention.

[0149] 5-2. Changes in tumor volume from an animal model injected with the example peptide via the oral administration route Aspc-1 cells (1×10 7 ) were inoculated into the back of the flanks of 5-week-old balb / c nude mice, and classified into 15 groups: an untreated control group (Control) and groups orally administered with the peptides of Examples 1 to 14, respectively. When the tumor volume grew to approximately 150 mm 3 , each example peptide was orally administered at a dose of 20 mg / kg five times a week. The tumor size was measured with a digital caliper, and the change results of the tumor volume were compared by group.

[0150] As a result, the final volume of the control group tumor after the final dosing was approximately 3517 mm 3It grew significantly, but it was confirmed that tumor growth was significantly suppressed in all groups orally administered with the peptides of Examples 1 to 14 ( ** p < 0.001; Figure 19).

[0151] Figure 20 shows representative tumor photographs obtained after autopsy in all groups, and it was observed that tumor tissue growth was significantly suppressed in all groups orally administered with the example peptides of the present invention when compared with the control group.

[0152] Experimental Example 6: Confirmation of cytotoxicity of the example peptides in normal cells Next, it was additionally confirmed whether the peptides of the present invention exhibit cytotoxicity even in normal cells. For the test, CCD-18Co, which is a human colon fibroblast, and HDPC, which is a human dermal papilla cell, were used as normal cells, and the peptide of SEQ ID NO: 7 was typically used as the example peptide of the present invention.

[0153] First, 5 × 10 3 individual normal cells of each type were inoculated into a 96-well plate and then cultured in DMEM medium at 37°C under 5% CO 2 conditions for 24 hours. Each well was divided into a group not treated with the peptide (untreated) and a group treated with the peptide of SEQ ID NO: 7 (25, 50, and 100 μM, respectively), and the experiment was conducted.

[0154] As a result of the experiment, no significant difference was found between the untreated control group and all groups treated with the peptide (24 hours, 48 hours, or 72 hours after treatment) (Figures 21 and 22). Therefore, it was confirmed that the peptides of the examples of the present invention have a very excellent effect as an anticancer use because they exhibit strong anticancer activity in cancer cells while showing no cytotoxicity in normal cells.

[0155] From the above description, those skilled in the art to which the present invention pertains should be able to understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. In this regard, it must be understood that the above-described embodiments are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the claims described below rather than the above detailed description, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims

1. An ADP (adenosine diphosphate)-ribose binding peptide having any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

14.

2. The ADP-ribose binding peptide of claim 1, further comprising a cell-penetrating peptide at the N-terminus, C-terminus or both termini.

3. The ADP-ribose binding peptide according to claim 2, wherein the cell-penetrating peptide is at least one selected from the group consisting of TAT, maurocalcine, penetratin, poly-arginine-derived peptide, antennapedia, transportan, VP22, Hph-1, poly-arginine, R11 (R9), Pep-1, HP4, LAH4, Vetofusing-1, signal sequence-based peptides, and amphipathic peptides.

4. The ADP-ribose binding peptide according to claim 2, wherein the peptide has any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 15 to 24.

5. A polynucleotide encoding the ADP-ribose binding peptide according to any one of claims 1 to 4.

6. A vector comprising the polynucleotide of claim 5.

7. A transformant comprising the polynucleotide of claim 5.

8. A pharmaceutical composition for preventing or treating cancer, comprising the ADP-ribose binding peptide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 4 as an active ingredient.

9. The pharmaceutical composition for preventing or treating cancer according to claim 8, wherein the cancer is one or more solid cancers selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, gastric cancer and ovarian cancer.

10. The pharmaceutical composition for preventing or treating cancer according to claim 8 , wherein the composition is administered subcutaneously or orally.

11. 5. An anti-cancer adjuvant pharmaceutical composition for enhancing reactivity to a second anti-cancer drug, comprising the ADP-ribose binding peptide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4 as an active ingredient.

12. The anti-cancer adjuvant pharmaceutical composition according to claim 11, wherein the anti-cancer drug is a cytotoxic anti-cancer drug, a targeted anti-cancer drug, or a combination thereof.

13. The anti-cancer adjuvant pharmaceutical composition according to claim 11, wherein the cancer targeted by the second anti-cancer drug is one or more solid cancers selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, kidney cancer, gastric cancer and ovarian cancer.

14. (i) an ADP-ribose binding peptide according to any one of claims 1 to 4 or a pharma- ceutically acceptable salt thereof; and (ii) A pharmaceutical composition for preventing or treating cancer, comprising a second anticancer agent as an active ingredient.

15. 5. An adjuvant anti-cancer pharmaceutical composition for enhancing responsiveness to anti-cancer radiation therapy, comprising the ADP-ribose binding peptide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 4 as an active ingredient.

16. The anti-cancer adjuvant pharmaceutical composition according to claim 15, wherein the cancer targeted by the radiation anti-cancer therapy is one or more solid cancers selected from the group consisting of brain cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, colon cancer, kidney cancer, gastric cancer and ovarian cancer.

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