PD-l1-targeting protac peptide, protac nanoparticles comprising same, and composition for cancer prevention or treatment containing same as active component

Abstract

The present invention relates to a PD-L1-targeting PROTAC peptide, PROTAC nanoparticles comprising same, and a composition for cancer prevention or treatment containing same as an active ingredient, wherein in cancer tissues, the PD-L1 receptor is subjected to a direct lysosomal degradation pathway and a PROTAC-mediated degradation pathway, thereby not only inducing PD-L1 protein degradation but also fundamentally blocking PD-L1 protein regeneration, thus inhibiting the transmission of immunosuppressive signals in the interaction between cancer cells and T cells to activate immune responses, so that the effect of inducing the death of cancer cells can be continuously and effectively achieved.

Description

PD-L1 targeting PROTAC peptide, PROTAC nanoparticles containing the same, and a composition for preventing or treating cancer using the same as an active ingredient

[0001] The present invention relates to a PD-L1 targeting PROTAC peptide, PROTAC nanoparticles containing the same, and a composition for preventing or treating cancer using the same as an active ingredient.

[0002]

[0003] PROTACs are an acronym for Proteolysis Targeting Chimeras, a technology that induces the degradation of target proteins (POI: protein of interest). The term TPD (Targeted Protein Degradation) is sometimes used instead of PROTAC.

[0004] PROTACs have a triple complex structure in which a target protein ligand and an E3 enzyme ligand are linked by a linker, and they induce the degradation of the target protein through the ubiquitin-proteasome system.

[0005] Recently, various PROTACs, including ARV-110 and ARV-471, have been developed and are currently undergoing Phase 2 and Phase 3 clinical trials for prostate and breast cancer. However, PROTACs targeting small molecules have high cell permeability, stability, and low cost, but they have a narrow and very limited range of targeting undruggable proteins and exhibit serious side effects.

[0006] In order to solve the aforementioned problems, PROTACs were developed using peptides as targeting warheads, but there were various issues such as low absorption and bioavailability in the body due to insolubility, difficulty in controlling the concentration distributed and maintained in the body, low cellular absorption rate, and significantly reduced therapeutic efficacy in actual cancer immunotherapy due to insufficient tumor targeting, unlike in experiments.

[0007] Therefore, there is an urgent need to develop a new PROTAC that can solve the aforementioned problems to provide sufficient therapeutic efficacy to patients, effectively degrade target substances within tumor cells, and continuously delay their regeneration, thereby improving the precision and efficacy of checkpoint immunotherapy.

[0008] Patent Document 1. Republic of Korea Registered Patent Publication No. 10-1674145

[0009]

[0010] The present invention was devised in consideration of the above-mentioned problems, and the objective of the present invention is to provide a PD-L1 targeting PROTAC peptide.

[0011] Another objective of the present invention is to provide a PROTAC nanoparticle comprising the above-mentioned PROTAC peptide and a composition for preventing or treating cancer using the same as an active ingredient.

[0012]

[0013] To achieve the above objective, the present invention provides a PD-L1 targeting PROTAC peptide comprising: a) a cancer targeting peptide; b) a linker composed of two phenylalanines; and c) an E3 enzyme ligand represented by SEQ ID NO. 26.

[0014] The above cancer target peptide may be a peptide that binds to a target specifically expressed in cancer tissue.

[0015] The targets specifically expressed in the above-mentioned cancer tissue are PD-1 (Programmed Cell Death Protein 1) protein, PD-L1 (Programmed Death-Ligand 1) protein, CP2c (Cytochrome P450 2C) protein, BRD4 (Bromodomain Containing 4) protein, c-Myc (transcriptional regulator Myc-like) protein, EGFR (Epidermal Growth Factor Receptor) protein, ALK (Anaplastic Lymphoma Kinase) protein, ROS1 (ROS Proto-Oncogene 1) protein, HER2 (Human Epidermal Growth Factor Receptor 2) protein, TROP2 (Trophoblast Cell Surface Antigen 2) protein, CTLA-4 (Cytotoxic T-Lymphocyte Associated Protein 4) protein, VEGFR (Vascular Endothelial Growth Factor Receptor) protein, BRAF (B-Raf Proto-Oncogene) protein, and KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) protein, PI3K (Phosphoinositide 3-Kinase) protein, CDK4 / 6 (Cyclin Dependent Kinase 4 / 6) protein, MET (Mesenchymal Epithelial Transition Factor) protein, mTOR (Mechanistic Target of Rapamycin) protein, PARP (Poly ADP Ribose Polymerase) protein, PARP10 (Poly ADP Ribose Polymerase 10) protein, NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1) protein, CD44 protein, CD49 protein, LAG (lymphocyte activation gene-3) protein,It may be any one selected from the group consisting of IAP (inhibitor of apoptosis protein) protein, DDX5(p68) protein, and CD19 protein.

[0016] The above cancer target peptide may be represented by any one selected from SEQ ID NOs 1 to 25.

[0017] The above cancer target peptide may be represented by SEQ ID NO. 1 or 2.

[0018] The above PROTAC peptide may be represented by SEQ ID NO. 27 or 28.

[0019] To achieve the other objectives mentioned above, the present invention provides a PROTAC nanoparticle formed by self-assembly of a plurality of PROTAC peptides.

[0020] The above self-assembly may be due to π-π interactions by the linker of the PROTAC peptide.

[0021] The average diameter of the above PROTAC nanoparticles may be 100 to 300 nm.

[0022] To achieve the above-mentioned other objective, the present invention provides a composition for cancer prevention or treatment comprising the above-mentioned PROTAC nanoparticles.

[0023] The above cancers include brain tumors, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymoma, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, small intestine cancer, colorectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumors, penile cancer, prostate cancer, female genital tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, skin cancer, hematological cancer, leukemia, malignant lymphoma, multiple myeloma. It may be one or more selected from the group consisting of aplastic anemia and resistant cancer.

[0024] To achieve the above-mentioned other objective, the present invention provides a pharmaceutical composition for cancer immunotherapy comprising the PROTAC nanoparticles as an active ingredient.

[0025] To achieve the above-mentioned other objective, the present invention provides a pharmaceutical composition for a combination therapy for cancer treatment comprising the above-mentioned PROTAC nanoparticles and an anticancer agent.

[0026] The above anticancer agent may be one or more selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, 5-fluorouracil, cisplatin, paclitaxel, doxorubicin, donorubicin, vinblastine, vincristine, actinomycin D, teniposide, etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin, and mitomycin-C.

[0027]

[0028] The present invention provides a method for preparing a composition for preventing or treating cancer, comprising the step of preparing a composition comprising the PD-L1 targeting PROTAC peptide or PROTAC nanoparticles comprising the PD-L1 targeting PROTAC peptide.

[0029] The present invention provides a therapeutic use for the manufacture of a drug comprising the PD-L1 targeting PROTAC peptide or PROTAC nanoparticles comprising the PD-L1 targeting PROTAC peptide.

[0030] The present invention provides a method for preventing or treating cancer in a subject in need, comprising administering to the subject a composition comprising the PD-L1 targeting PROTAC peptide or a PROTAC nanoparticle comprising the PD-L1 targeting PROTAC peptide.

[0031]

[0032] According to the present invention, PROTAC nanoparticles formed through the self-assembly of PROTAC peptides have specific selectivity for cancer target substances in vivo, thereby ensuring stability for normal tissues.

[0033] Furthermore, the PROTAC nanoparticles according to the present invention not only simultaneously degrade target substances present on the cell membrane surface of cancer cells through two pathways, a direct lysosomal degradation pathway and a PROTAC-mediated degradation pathway, but also fundamentally block the regeneration of target substances, thereby inhibiting the transmission of immunosuppressive signals by cancer cells during interactions with T cells and activating the immune response, so as to continuously and effectively achieve the effect of inducing apoptosis of not only cancer but also resistant cancer cells.

[0034]

[0035] FIG. 1 schematically illustrates the structure of the cancer-targeting PROTAC nanoparticles of the present invention. It is a diagram illustrating the structure of a PD-L1 targeting PROTAC peptide, which is an embodiment, and PROTAC nanoparticles (PT-NPs) formed by the self-assembly of the PROTAC peptide. The PD-L1 targeting PROTAC peptide is composed of an anti-PD-L1 peptide, a link, and an E3 enzyme ligand, which has an amphiphilic tendency and can self-assemble into nanoparticles in a liquid phase.

[0036] Figure 2a illustrates a series of processes in which cancer-targeting PROTAC nanoparticles (PT-NPs) administered intravenously accumulate at a tumor site through passive and active targeting, and the figure on the right illustrates the intracellular mechanism of cancer-targeting PROTAC nanoparticles (PT-NPs) that have reached tumor cells. The cancer-targeting PROTAC nanoparticles (PT-NPs) according to the present invention operate through two distinct mechanisms within tumor cells. One mechanism involves the simultaneous presence of multiple cancer-targeting peptides (e.g., PD-L1 binding peptides) within the cancer-targeting PROTAC nanoparticles (PT-NPs), which penetrate into the cell via the endocytosis pathway to directly induce lysosomal degradation of PD-L1. The other mechanism involves the PROTAC peptides released upon the degradation of the cancer-targeting PROTAC nanoparticles within the cytoplasm of tumor cells, which continuously inhibit and reduce recyclable PD-L1 receptors, thereby preventing the regeneration of PD-L1 receptors on the surface of tumor cells.

[0037] FIGS. 3a and 3b schematically illustrate the chemical structures of PROTAC peptides and cancer-targeting PROTAC nanoparticles prepared from Examples 1, 2, 3, and 4 and Comparative Examples 1 to 6. Specifically, FIG. 3a schematically illustrates the chemical structures of Example 1, Comparative Examples 1 and 2, and Comparative Examples 3 and 4, and FIG. 3b schematically illustrates the chemical structures of Examples 3 and 4, Comparative Examples 3 and 4, and Comparative Examples 5 and 6.

[0038] FIG. 4 is a schematic diagram showing the structure of a PROTAC peptide according to the present invention (Example 1 or 3) and a PROTAC peptide in which the linker is substituted with a DD sequence (Comparative Example 7).

[0039] Figure 5a shows the results of molecular dynamics (MD) simulation for the PROTAC peptide of Example 1.

[0040] Figure 5b shows the molecular kinetics (MD) simulation results for the PROTAC peptide of Example 3.

[0041] Figure 6 is a graph showing the radius of gyration measured from molecular dynamics (MD) simulations for the protac peptide of Example 1 and the protac peptide of Comparative Example 7.

[0042] FIG. 7 shows PROTAC nanoparticles (PT-NPs, PD-L11) prepared from Example 2(a), Comparative Example 2(b), and Comparative Example 4(c). scram -NPs and E3 scram This is the result of dynamic light scattering (DLS) analysis for -NPs. The image inset in Fig. 7 is an image taken with a cryo-transmission electron microscope (CryoTEM).

[0043] FIG. 8 shows PROTAC nanoparticles (PT-NPs, EGFR-NPs, PD-L11112) prepared from Example 2, Example 4, Comparative Example 2, Comparative Example 4, and Comparative Example 6. scram -NPs, E3 scram -NPs and EGFR scram This is the result of analyzing whether there was a change in particle size after adding -NPs) to mouse serum for 48 hours. Figure 8a shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram The results are for -NPs), and FIG. 8b shows the PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6. scram -NPs and EGFR scram This is the result for -NPs).

[0044] FIG. 9 shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram This is the result of BLI analysis on the PD-L1 binding affinity of -NPs. PD-L1 peptide (sequence 1; CLQKTPKQC) and DD linker were used as controls.

[0045] FIG. 10 shows PD-L1 antibodies on CT26 cells, PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram This is the result of evaluating cell viability after treatment with -NPs. Statistical significance was analyzed using the Tukey-Kramer test. The p-values ​​for each statistical data point are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0046] FIG. 11a shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram This is a confocal microscope image of CT26 cells treated with -NPs.

[0047] Figure 11b is the result of quantitatively analyzing the concentration of PROTAC nanoparticles (PT-NPs) prepared from Example 2 in the cell membrane and cytoplasm of CT26 cells.

[0048] FIG. 12a shows the PROTAC nanoparticles (EGFR-NPs, E3) of Example 4, Comparative Example 4, and Comparative Example 6. scram -NPs and EGFR scram This is a confocal microscope image of CT26 cells treated with -NPs), EGFR mAb, and EGFR target peptide.

[0049] Figure 12b is the result of quantitatively analyzing the concentration of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 in the cell membrane and cytoplasm of CT26 cells.

[0050] Figure 13a shows the results of confocal fluorescence microscopy analysis after treating CT26 cells pre-treated with PD-L1 mAb or PD-L1 binding peptide with PROTAC nanoparticles (PT-NPs) prepared from Example 2.

[0051] Figure 13b shows the results of confocal fluorescence microscopy analysis after treating CT26 cells pretreated with EGFR mAb or EGFR binding peptide with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4.

[0052] Figure 14a shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (PT-NPs) prepared from Example 2 were successfully introduced into the lysosomes when CT26 cells were treated.

[0053] Figure 14b quantitatively shows the fluorescence intensity measured from Figure 14a.

[0054] Figure 14c is the result of analyzing the ratio of PROTAC nanoparticles (PT-NPs) prepared from Example 2 from Figure 14a to the co-location of lysosomes.

[0055] Figure 15 shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 were successfully introduced into the lysosomes when CT26 cells were treated.

[0056] Figure 16 is a graph showing the ratios analyzed according to the location where fluorescence is activated (cell membrane (membrane), inside the lysosome (Lysosome +), and outside the lysosome (Lysosome -)) in the results of Figure 15. In Figures 16a to 16c, the graph colors represent the cell membrane (membrane), inside the lysosome (Lysosome +), and outside the lysosome (Lysosome -), which are represented in Figure 16a. Figure 16a is the result for the PROTAC nanoparticles of Example 4, Figure 16b is the result for the EGFR mAb, and Figure 16c is the result for the EGFR target peptide.

[0057] FIG. 17 shows an anti-PD-L1 antibody and PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 in mGFP-tagged, PD-L1-expressing CT26 cells. scram -NPs and E3 scramThis is an image taken with a confocal fluorescence microscope after treatment with -NPs.

[0058] FIG. 18 shows anti-PD-L1 antibodies and PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 in mGFP-tagged PD-L1-expressing CT26 cells. scram -NPs and E3 scram This is the result of a quantitative analysis of relative PD-L1 levels after treatment with -NPs.

[0059] Figure 19 shows the results of analyzing PD-L1 expression levels by Western blot after treating wild CT26 cells with an anti-PD-L1 antibody and PROTAC nanoparticles (PT-NPs) prepared from Example 2.

[0060] FIG. 20 shows the PROTAC nanoparticles of Example 4, as well as the PROTAC nanoparticles of Comparative Example 4 (E3) in wild CT26 cells. scram -NPs), PROTAC nanoparticles (EGFR) of Comparative Example 6 scram Western blot (a) showing the EGFR expression level after treatment with -NPs), EGFR target peptide (sequence 2; 0.3 nM), or EGFR antibody (APC-EGFR mAbs) (0.3 nM), and a graph (b) showing the result of quantitative analysis.

[0061] Figure 20c shows the results of analyzing the EGFR expression level over time after treating cancer cells (CT26) treated with a VHL (von Hippel Lindau) inhibitor with the PROTAC nanoparticles of Example 4.

[0062] Figure 20d shows the results of analyzing EGFR expression levels after treating cancer cells (CT26) with the PROTAC nanoparticles of Example 4 at different concentrations.

[0063] Figure 20e shows the results of analyzing the expression levels of EGFR, AKT, ERK, c-Myc, cyclin D1, etc., and the phosphorylation of EGFR and AKT after treating cancer cells (CT26) with the PROTAC nanoparticles of Example 4 at different concentrations.

[0064] Figure 21 shows non-invasive near-infrared fluorescence (NIRF) images taken over time for the experimental group, comparison group 1, comparison group 2, and control group (n=3).

[0065] Figure 22 shows the results of quantitative analysis of fluorescence intensity at the tumor site in the experimental group, comparison group 1, comparison group 2, and control group.

[0066] Figure 23 shows the results of quantitative analysis of near-infrared fluorescence images and fluorescence intensity of tumor tissues recovered from the experimental group, comparison group 1, comparison group 2, and control group after 48 hours. The p-values ​​of each statistical data are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0067] Figure 24 shows fluorescence images of tumor tissues collected from the experimental group, comparison group 1, comparison group 2, and control group after 48 hours. Statistical significance was analyzed using the Tukey-Kramer method. The p-values ​​for each statistical value are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0068] Figure 25 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram These are non-invasive near-infrared fluorescence (NIRF) images taken over time for the NPs group (n=5).

[0069] Figure 26 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scramThis is the result of quantitative analysis of fluorescence intensity in the tumor site in the -NPs group.

[0070] Figure 27 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group (also called PROTAC-NPs), and E3 after 24 hours. scram -NPs group and EGFR scram This is the result of quantitatively analyzing near-infrared fluorescence images and fluorescence intensity of tumor tissue recovered from the -NPs group.

[0071] Figure 28 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 after 24 hours. scram -NPs group and EGFR scram This is a fluorescent image of tumor tissue recovered from the -NPs group.

[0072] Figure 29 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram This is a near-infrared fluorescence image of major organ tissues isolated from the -NPs group (from left: liver, lung, spleen, kidney, heart).

[0073] Figure 30 shows the results of quantitatively analyzing the fluorescence intensity in the major organ tissues of Figure 29.

[0074] Figure 31 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram This is the result of the pharmacokinetic analysis of the drug in the -NPs group.

[0075] Figure 32 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 scram This is the result of measuring the tumor size of the -NPs group over time (days 0, 2, 4, 6, 8, and 10).

[0076] Figure 33 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is the result of TUNEL analysis of tumor tissue isolated from the -NPs group.

[0077] Figure 34 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is the result of analyzing PD-L1 expression levels in tumor tissues isolated from the -NPs group using Western blot.

[0078] Figure 35 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is an image of tumor tissue isolated from the -NPs group, stained with APC-labeled PD-L1 antibody and DAPI, taken using a confocal fluorescence microscope.

[0079] Figure 36 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram Tumor cells (CD45) from tumor tissue isolated from the -NPs group - PD-L1 + This represents the relative expression level of ).

[0080] Figure 37 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram Cytotoxic T lymphocytes (CD45) from tumor tissue isolated from the -NPs group + CD3 + CD8 + ) and regulatory T lymphocytes (CD3 + CD4 + CD25 + This represents the relative expression level of ).

[0081] Figure 38 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scramThis is an image taken with a confocal fluorescence microscope of tumor tissue isolated from the -NPs group, stained with APC-labeled CD8 antibody and DAPI.

[0082] Figure 39 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is an image taken with a confocal fluorescence microscope of tumor tissue isolated from the -NPs group, stained with PE-labeled CD25 antibody and DAPI.

[0083] Figure 40 shows the results of analyzing T cell receptor (CD274) and apoptosis and survival-related gene expression (BCL2, BRCA2, CASP3, VHL, Ube3a) in tumor tissues isolated from the anti-PD-L1 antibody group and PT-NPs group after 10 days.

[0084] Figure 41 shows the results of analyzing gene expression (Cd247, Nfatc1, Cd3g, Hras, ​​Nfkbia, Cd3e) related to the cell death signaling pathway in tumor tissues isolated from the physiological saline group, the anti-PD-L1 antibody group, and the PT-NPs group after 10 days.

[0085] Figure 42 shows the results of testing lymphocytes, red blood cells, platelet count (PLT), AST, ALT, and BUN in blood samples taken from the normal group, anti-PD-L1 antibody group, and PT-NPs group after 10 days of treatment.

[0086] Figure 43 shows the results of measuring weight changes during the treatment period in the normal group, anti-PD-L1 antibody group, and PT-NPs group.

[0087] Figure 44 shows the H&E staining results of major organ tissues isolated from the normal group, anti-PD-L1 antibody group, and PT-NPs group after 10 days of treatment.

[0088] Figure 45 shows the results of analyzing survival rates during the treatment period in the normal group, anti-PD-L1 antibody group, and PT-NPs group.

[0089] Figure 46 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of measuring tumor size over time in the -NPs group.

[0090] Figure 47 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the appearance of tumor tissue isolated from the -NPs group.

[0091] Figure 48 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of Western blot analysis of the expression levels of EGFR and proteins related to its downstream pathways (phosphorylated EGFR, Akt, phosphorylated Akt, ERK, c-Myc, Cyclin D) in tumor tissues isolated from the -NPs group.

[0092] Figure 49 shows the physiological saline group, the EGFR-NPs group (also called PROTAC-NPs), and E3 scram -NPs group, EGFR scram This is the result of immunohistochemical staining for EGFR in tumor tissue isolated from the -NPs group.

[0093] Fig. 50 is a graph showing the quantitative representation of Fig. 49.

[0094] Figure 51 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of analyzing apoptosis in tumor tissue isolated from the -NPs group.

[0095] Figures 52 and 53 show the physiological saline group, EGFR-NPs group, and E3 according to the treatment period. scram -NPs group, EGFR scram This is a graph showing the weight change and survival rate of the -NPs group.

[0096] Figure 54 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of performing blood biochemical tests on the plasma of the -NPs group.

[0097] Figure 55 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram These are the results of histopathological examinations of the liver, lungs, spleen, kidneys, and heart of the -NPs group.

[0098] FIG. 56 shows PROTAC nanoparticles (EGFR-NPs, E3) prepared from SJF 1528 (black line / dot), erlonitip (purple dot), Example 4 (light green line / dot), Comparative Example 4 (red line / dot), and Comparative Example 6 (blue line / dot) in CT26 cells. scram -NPs and EGFR scram This is the result of treating with -NPs and evaluating cell viability.

[0099] Figure 57 shows the results of analyzing EGFR and p-EGFR expression levels by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 58 is a graph quantified by image J analysis from the results of Figure 57.

[0100] Figure 59 shows the results of analyzing the expression levels of AKT, pAKT, ERK, PDL1, c-Myc, and cyclin D by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 60 is a graph quantified by image J analysis from the results of Figure 59.

[0101] Figure 61 shows the results of analyzing EGFR and p-EGFR expression levels by Western blot after treating CT25 cells and NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and erlotinib, respectively, and Figure 62 is a graph quantified from the results of Figure 61 through Image J analysis (in Figure 62, 'NanoTACs' refers to the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4).

[0102] Figure 63 shows the results of analyzing HER2 expression levels by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and cetuximab, respectively, and Figure 64 is a graph quantified by image J analysis from the results of Figure 63.

[0103] Figure 65 shows the size analysis of the fluorescently labeled PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 66 shows the results of analyzing whether there was a change in particle size after adding the fluorescently labeled PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 to mouse serum for 7 days.

[0104] Figure 67 shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 were successfully introduced into the lysosomes when CT26 cells were treated.

[0105] Figure 68 is the result of analyzing the ratio of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and lysosomes to the co-locations from Figure 67.

[0106] Figure 69 shows the results of LC-MS analysis on tumor tissue excised 9 hours after administration in an animal model treated with Cy5.5-labeled EGFR-NPs.

[0107] Below, various aspects and embodiments of the present invention will be examined in more detail.

[0108]

[0109] The objects, other objects, features, and advantages of the present invention will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0110] In this specification, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should not be understood as precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0111] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0112]

[0113] The present invention will be described in detail below.

[0114]

[0115] One aspect of the present invention relates to a cancer-targeting PROTAC peptide comprising a) a cancer-targeting peptide; b) a linker composed of two phenylalanines; and c) an E3 enzyme ligand represented by SEQ ID NO. 26.

[0116] The PROTAC peptide according to the present invention is composed of three components: a) a cancer targeting peptide; b) a linker composed of two phenylalanines; and c) an E3 enzyme ligand represented by SEQ ID NO. 26.

[0117] The cancer-targeting PROTAC peptide according to the present invention forms a nanoparticle structure in a liquid phase through self-assembly of multiple PROTAC peptides. It exhibits very low toxicity in vivo and effectively removes target substances present on the surface of tumor cells through two mechanisms, thereby demonstrating excellent anticancer effects. Furthermore, it is suitable for commercialization as it is easy to mass-produce.

[0118] The above cancer target peptide is not particularly limited to peptides that bind to a target specifically expressed in cancer tissue, and specifically may include peptides having a sequence that can block, reduce, or otherwise limit the interaction or activity of a target molecule specifically expressed in cancer tissue.

[0119] In the present invention, the target specifically expressed in cancer tissue refers to a specific receptor on the surface of cancer cells, preferably PD-1 (Programmed Cell Death Protein 1) protein, PD-L1 (Programmed Death-Ligand 1) protein, CP2c (Cytochrome P450 2C) protein, BRD4 (Bromodomain Containing 4) protein, c-Myc (transcriptional regulator Myc-like) protein, EGFR (Epidermal Growth Factor Receptor) protein, ALK (Anaplastic Lymphoma Kinase) protein, ROS1 (ROS Proto-Oncogene 1) protein, HER2 (Human Epidermal Growth Factor Receptor 2) protein, TROP2 (Trophoblast Cell Surface Antigen 2) protein, CTLA-4 (Cytotoxic T-Lymphocyte Associated Protein 4) protein, VEGFR (Vascular Endothelial Growth Factor Receptor) protein, and BRAF (B-Raf Proto-Oncogene). Protein, KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) protein, PI3K (Phosphoinositide 3-Kinase) protein, CDK4 / 6 (Cyclin Dependent Kinase 4 / 6) protein, MET (Mesenchymal Epithelial Transition Factor) protein, mTOR (Mechanistic Target of Rapamycin) protein, PARP (Poly ADP Ribose Polymerase) protein, PARP10 (Poly ADP Ribose Polymerase 10) protein, NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1) protein, CD44 protein, CD49 protein,It may be any one selected from the group consisting of LAG (lymphocyte activation gene-3) protein, IAP (inhibitor of apoptosis protein) protein, DDX5 (p68) protein, and CD19 protein.

[0120] In the present invention, "PD-L1 (Programmed death-ligand 1)" is a ligand for "Programmed Death Receptor 1 (PD-1)," an immunosuppressive receptor primarily expressed on activated T and B cells, and when PD-1 binds to the ligands PD-L1 or PD-L2, antigen receptor signaling can be negatively regulated. The ligand for PD-1 may be constitutively expressed or induced in multiple cell types, including non-hematopoietic tissues and various tumor types. PD-L1 is weakly expressed on B cells, T cells, myeloid cells, and dendritic cells (DCs), but is also expressed on peripheral cells, pseudo-microvascular endothelial cells, and non-lymphatic organs such as the heart and lungs. However, it is primarily overexpressed in tumor cells by interferon gamma in the tumor microenvironment, or expressed in immune cells present within the tumor microenvironment by the immune evasion mechanisms of tumor cells. In contrast, PD-L2 is found only on macrophages and dendritic cells. The expression patterns of PD-1 ligands suggest a role for PD-1 in maintaining peripheral tolerance and may contribute to regulating autoreactive T-cell and B-cell responses in the periphery.

[0121] In the present invention, the Epidermal Growth Factor Receptor (EGFR) is an epidermal growth factor receptor and is a group of cell membrane receptors that regulate cell growth, division, and apoptosis. As a 170 kDa type 1 membrane protein, it is known to be overexpressed in various types of tumors (solid tumors such as lung cancer, head and neck tumors, colorectal cancer, pancreatic cancer, and breast cancer), which is due to the amplification and expression of the receptor. Tumor tissues with overexpressed epidermal growth factor receptors tend to be more invasive, metastasize more easily, and be more resistant to anticancer therapy, resulting in a poor prognosis. To overcome this, targeted therapies using antibodies that target the epidermal growth factor receptor are being implemented. Cancer is treated by inhibiting the signaling and growth of cancer cells through the binding of the antibody to the epidermal growth factor receptor, thereby inhibiting the binding of epidermal growth factor.

[0122] Various activating mutations exist for EGFR, which are broadly classified into susceptibility and resistance mutations. Exon 19 deletion and the L858R point mutation in Exon 21 are the most important susceptibility mutations, accounting for approximately 85–90%, while Exon 19 del mutations are known to exhibit better sensitivity to TKIs. Conversely, the T790M point mutation in Exon 20 is the most important resistance mutation, known to be found in more than 50% of patients with acquired resistance. Therefore, EGFR may include not only wild-type but also mutant types.

[0123] In the present invention, HER2 (Human epidermal growth factor receptor 2) is a human epidermal growth factor receptor, a tyrosine phosphorylated growth factor receptor with a molecular weight of 185 kDa located on the cell surface. Although there is no ligand binding site within the HER2 molecule, it is easily activated by forming dimers with other receptors such as EGFR, HER3, and HER4. Through receptor-ligand conjugation, it exerts effects such as cell proliferation, cell survival, metastasis, and angiogenesis via various cell signaling pathways. It is overexpressed in 20–30% of breast cancers and in various cancer types including gastric cancer, ovarian cancer, lung cancer, and prostate cancer. The overexpression of these receptors further enhances the functions that promote cell survival, proliferation, angiogenesis, and metastasis.

[0124] In the present invention, VEGFR (vascular endothelial growth factor receptor) is a vascular endothelial growth factor receptor that is expressed in vascular and lymphatic endothelial cells and binds to VEGF-A, VEGF-E, etc. to promote vascular proliferation and vascular endothelial cell migration.

[0125] All of the listed targets that are specifically expressed in cancer tissue are overexpressed in cancer cells and contribute to the survival and proliferation of cancer cells.

[0126] More specifically, the cancer target peptide is not particularly limited as long as it is a peptide composed of 5 to 20, 5 to 15, 5 to 14, 5 to 12, 6 to 12, 7 to 12, 7 to 11, 7 to 10, or 7 to 9 amino acid residues, and preferably, the cancer target peptide may be represented by any one selected from SEQ ID NOs 1 to 25, and most preferably, may be represented by SEQ ID NO. 1 or 2.

[0127] Ranking

[0128]

[0129] b) The linker composed of two phenylalanines above enables self-assembly of PROTAC peptides through π-π interactions between molecules of the aromatic ring. The PROTAC peptide according to the present invention forms nanoparticles through self-assembly by the linker, and the nanoparticles formed in this way have an Enhanced Permeability and Retention (EPR) effect compared to PROTACs targeting small molecules as warheads, thus exhibiting excellent targeting efficiency against tumor tissue.

[0130] In the present invention, a linker composed of two phenylalanines can be represented as Phe-Phe. If an amino acid other than the phenylalanines is used (e.g., D(Asp), nanoparticles are not formed, and even if they are formed, the structure and function may be degraded or safety may be lowered as the radius of rotation of the protein changes, and aggregation may be induced, causing toxicity within the cell. Therefore, it is preferable that the linker used in the present invention be represented as *-FF-*.

[0131] c) The E3 enzyme ligand represented by SEQ ID NO. 26 above is for the heterobifunctionality of PROTAC and forms an E3 enzyme ligand in a ternary complex. The E3 enzyme ligand induces ubiquitination and degradation by a proteasome. The E3 enzyme ligand is not particularly limited as long as it can bind to E3 without impairing the original function of E3, but preferably it may be the E3 ligand represented by SEQ ID NO. 26.

[0132] Sequence number 26

[0133] ALAPYIP; Ala-Leu-Ala-Pro-Tyr-Ile-Pro

[0134] The above PROTAC protein may be represented by SEQ ID NO. 27 or 28.

[0135]

[0136] Another aspect of the present invention relates to PROTAC nanoparticles formed by the self-assembly of a plurality of PROTAC peptides.

[0137] The PROTAC nanoparticles in the present invention are formed by fusing at least one PROTAC peptide and are manufactured as spherical nanoparticles as shown in FIG. 1. Specifically, when the PROTAC peptide is dispersed in a liquid phase, they can be formed by self-assembly through hydrophobic interactions via a linker formed of phenylalanine. The PROTAC nanoparticles may generally have a diameter in the range of about 10 to about 1000 nm, about 10 to about 800 nm, or about 50 to 500 nm, and more preferably may have a diameter of 100 to 300 nm. Because the PROTAC nanoparticles have a uniform particle size distribution and a characteristic particle size, they possess structural stability within blood vessels while being able to easily pass into cells within the tissue of the cancer site. Therefore, since PROTAC nanoparticles having a diameter within the above range have cancer-specific effects, they can be applied to all cancer tissues throughout the body regardless of the administration route or site of administration.

[0138] When the PROTAC peptide constituting the above PROTAC nanoparticle is represented by SEQ ID NO. 27 or 28, the zeta potential of the PROTAC nanoparticle is +25 to +35 mV, and since it possesses repulsive electrical forces, it does not aggregate or precipitate, thus maintaining stability in the blood and making it easier to effectively reach target tissues. In addition, since the PROTAC nanoparticle has a positive surface charge, it forms an electrostatic attraction with the cell membrane, facilitating binding and absorption into cells, and can accumulate well in cancer tissues with high vascular permeability. In particular, due to the EPR effect, it can easily leak through the incomplete interstitial spaces of cancer blood vessels and accumulate in cancer tissues, which increases the retention time in the body and thus can lead to a more superior anticancer effect.

[0139] Conventional methods have been developed to manufacture nanostructures by aggregating hydrophobic peptides through hydrophobic interactions. However, when simply applied without designing the peptide structure, the nanostructure is formed by simple aggregation, which not only leads to the problem of easily losing the nanostructure but also makes it difficult to control the peptide nanostructure, resulting in uneven particles. Furthermore, peptide-based materials possess various chemical properties, such as excellent recognition capabilities, through tertiary structures formed by conjugation; however, in the case of undesigned sequences, problems such as reduced efficacy of nanoparticles may occur due to random self-assembly.

[0140] The PROTAC nanoparticles according to the present invention self-assemble through hydrophobic interactions by a linker via the controlled structure of the PROTAC peptide to form uniform spherical nanoparticles.

[0141] The PROTAC-NPs according to the present invention form multiple bindings to target substances, such as PD-L1 receptors on the surface of tumor cells, and are actively targeted by tumor cells. That is, the PROTAC-NPs according to the present invention form multiple complexes with target substances, which are incorporated into tumor cells through an endocytosis mechanism. In other words, the PROTAC-NPs according to the present invention can effectively degrade cancer target substances simultaneously by targeting tumor cells and simultaneously performing two mechanisms: direct lysosomal degradation and PROTAC-mediated degradation (Fig. 2). Due to this unique mechanism, the PROTAC-NPs according to the present invention have the effect of degrading cancer target substances in both the membrane and cytoplasm of tumor cells by continuously inhibiting cancer target substances without regeneration.

[0142] The above-mentioned PROTAC nanoparticles induce apoptosis in cancer cells when administered therapeutically in vivo, and block the regeneration of cancer target substances to ensure a sustained cancer cell killing effect.

[0143] In addition, since the above-mentioned PROTAC nanoparticles cause complete degradation of cancer target substances, it can be confirmed that they effectively kill even cancer cells with sensitizing and resistant mutations. Specifically, acquired resistance occurs in the form of the T790M mutation located at the gatekeeper of EGFR kinase, which is caused by first-generation inhibitors. It was confirmed that the PROTAC nanoparticles according to the present invention exhibit a therapeutic effect even in cases where existing therapeutic agents fail to show efficacy due to the occurrence of the EGFR_del19_T790M or EGFR_L858R_T790M double mutation (Experimental Examples 18, 19).

[0144] Therefore, the PROTAC nanoparticles of the present invention can exhibit high inhibitory ability against wild-type or mutant cancer cells, and thus can be usefully used in the treatment of cancer.

[0145]

[0146] In addition, the present invention provides a pharmaceutical composition for cancer treatment comprising the above-mentioned PROTAC nanoparticles as an active ingredient.

[0147] In addition, the present invention provides a pharmaceutical composition for cancer immunotherapy comprising the above-mentioned PROTAC nanoparticles as an active ingredient.

[0148] In addition, the present invention provides a pharmaceutical composition for a combination therapy for cancer treatment comprising the above-mentioned PROTAC nanoparticles and an anticancer agent as active ingredients.

[0149] The above cancers include thyroid cancer, oral cancer, oropharyngeal cancer, ovarian cancer, male genital tumors, male breast cancer, brainstem tumors, cerebral lymphoma, brain tumors, pituitary meningioma, gallbladder cancer, biliary tract cancer, colorectal cancer, intracranial tumors, head and neck tumors, bladder cancer, leukemia, abdominal tumors, nasal cavity cancer, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, ependymoma, adenoma, small cell lung cancer, small intestine cancer, kidney cancer, malignant astrocytoma, benign astrocytoma, female genital tumors, female external genital cancer, female urethral cancer, penile cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, mediastinal tumors, vaginal cancer, salivary gland cancer, skin cancer, malignant lymphoma, multiple myeloma, aplastic anemia, oligodendroglioma, hypopharyngeal cancer, anal cancer, hematological cancer, laryngeal cancer. It may be one or more selected from the group consisting of thoracic tumors, thymic cancers, and resistant cancers.

[0150] The above-mentioned resistant cancer refers to cancer that has resistance, and may be cancer that has become resistant to conventional anticancer drugs due to sensitivity mutations and resistance mutations. Specifically, it may be an EGFR mutation cancer, and the above-mentioned EGFR mutation may be one or more selected from the group consisting of EGFR Del19, EGFR L858R, EGFR Del19 / T790M, EGFR L858R / T790M, EGFR L858R / T790M / C797S, EGFR Del19 / T790M / C797S, EGFR NPH, EGFR SVD, EGFR NPG, EGFR H, EGFR ASV, EGFR FQEA, and HER2 YVMA.

[0151] The above anticancer agent may be one or more selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, 5-fluorouracil, cisplatin, paclitaxel, doxorubicin, donorubicin, vinblastine, vincristine, actinomycin D, teniposide, etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin, and mitomycin-C.

[0152] The content of the PROTAC nanoparticles in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc. For example, it may be 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The above content ratio is a value based on the dry weight after removing the solvent.

[0153] The pharmaceutical composition according to the present invention may further include a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0154] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, eye drops, oxylic agents, emulsions, suspensions, ethanol tablets, troches, fragrances, limonene adzes, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms.

[0155] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0156] When formulating, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0157] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0158] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0159] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0160] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0161] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0162] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-inflammatory agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0163] The suppository according to the present invention comprises cocoa dough, lanolin, Witepsol (H12, H15, W35, S55, E75, E85), polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroccote SP. Bases such as S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0164] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0165] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0166] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0167] The pharmaceutical composition according to 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 may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0168] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intramuscular injection, intrathecal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0169] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.

[0170] In the present invention, "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0171] In the present invention, "administration" means providing a specific composition of the present invention to an individual by any appropriate method.

[0172] In the present invention, "prevention" refers to any act of suppressing or delaying the onset of a target disease, "treatment" refers to any act of improving or beneficially altering the target disease and associated metabolic abnormality symptoms through the administration of a pharmaceutical composition according to the present invention, and "improvement" refers to any act of reducing parameters related to the target disease, such as the severity of symptoms, through the administration of a composition according to the present invention.

[0173] The cancer treatment method or immunotherapy method according to the present invention may be particularly suitable for patients with increased expression of PD-L1; or patients with cancer expressed by EGFR wild type or EGFR mutation.

[0174] Therefore, the pharmaceutical composition of the present invention has very high stability even when administered in vivo without separate additives, and specifically binds to PD-L1 expressed on the surface of cancer cells to promote PD-L1 degradation and inhibit the regeneration of PD-L1, thereby causing cancer cells to die; thus, it can be usefully utilized in pharmaceutical compositions for cancer treatment.

[0175] It can be confirmed that the pharmaceutical composition of the present invention degrades EGFR protein and successfully blocks downstream signaling pathways (RAS / RAF / MEK / ERK and PI3K / AKT pathways). Therefore, the pharmaceutical composition according to the present invention can exhibit a significant effect on cancers expressed by EGFR wild-type or EGFR mutations, and thus can be usefully used in the treatment of cancers expressed by EGFR wild-type or EGFR mutations. In particular, since it has excellent ability to inhibit the proliferation of lung cancer cell lines, it can be usefully used in the treatment of lung cancer.

[0176] Furthermore, the PROTAC nanoparticles according to the present invention act as immune checkpoint inhibitors, enabling immune cells to maintain the ability to identify cancer cells for an extended period without causing side effects to any organs of the body, including the digestive, respiratory, circulatory, endocrine, and nervous systems. Since they also resolve the issue of resistance to anticancer drugs regardless of the type of cancer, they can improve therapeutic efficacy against cancer and overcome the limitations of drugs against metastatic cancer through combination administration with conventional anticancer drugs.

[0177]

[0178] The present invention is to be explained in more detail below through examples, etc.; however, the scope and content of the present invention shall not be interpreted as being narrowed or limited by the examples, etc. below. Furthermore, based on the disclosure of the present invention including the examples below, it is evident that a person skilled in the art can easily practice the present invention even without specific experimental results presented, and it is natural that such variations and modifications fall within the scope of the appended claims.

[0179]

[0180] Examples 1 and 2. Preparation of PROTAC peptides and PROTAC nanoparticles (PT-NPs)

[0181] We intended to prepare a PROTAC peptide comprising an anti-PD-L1 peptide (CLQKTPKQC, SEQ No. 1), a self-assembled peptide link (FF, Phe-Phe), and an E3 enzyme ligand (ALAPYIP, SEQ No. 26). To this end, a PROTAC peptide (SEQ No. 27) consisting of 18 amino acids with the three components combined was designed and synthesized by Peptron (Daejeon, Korea) using a solid-phase peptide synthesis method. The PROTAC peptide according to the present invention is amphiphilic. The molecular weight and purity of the PROTAC peptide were analyzed using LC / MS and HPLC. As a result, it was confirmed that the PROTAC peptide was successfully synthesized.

[0182] The above PROTAC peptide can self-assemble in an aqueous solution through physical molecular interactions (e.g., hydrophobic interactions and π-π stacking) to form nanoparticles without a carrier molecule. That is, when the above PROTAC peptide (Example 1) is mixed with physiological saline, PROTAC nanoparticles (Example 2) with an average size of 211 nm can be obtained.

[0183]

[0184] Examples 3 and 4. Preparation of PROTAC peptides and PROTAC nanoparticles (EGFR-NPs)

[0185] Protac (PD-L1-PROTAC) peptides and protac nanoparticles were prepared in the same manner as in Examples 1 and 2, except that the protac peptide represented by SEQ No. 28 was prepared using an EGFR-binding peptide (anti-PD-L1 peptide; EHGAMEI, SEQ No. 2) instead of a PD-L1-binding peptide (anti-PD-L1 peptide; CLQKTPKQC, SEQ No. 1).

[0186]

[0187] Comparative Examples 1 and 2. Preparation of PROTAC peptides and PROTAC nanoparticles having scrambled sequences

[0188] A PROTAC peptide (SEQN 30) consisting of 18 amino acids was synthesized via a solid-phase peptide synthesis method using the PD-L1 binding peptide (KCPCTKLQQ; Lys-Cys-Pro-Cys-Thr-Lys-Leu-Gln-Gln) represented by SEQN 29 instead of the PD-L1 binding peptide represented by SEQN 1, in all cases identical to Examples 1 and 2, thereby producing a PROTAC (PD-L1 scram -PROTAC) peptide and PROTAC nanoparticles (PD-L1 scram -NPs) were synthesized. The molecular weight and purity of the PROTAC peptide were analyzed using LC / MS and HPLC. As a result, it was confirmed that the PROTAC peptide was successfully synthesized.

[0189]

[0190] Comparative Examples 3 and 4. Preparation of PROTAC peptides having scrambled sequences and PROTAC nanoparticles

[0191] A PROTAC peptide having a scrambled sequence of 18 amino acids (SEQN 32) was synthesized via a solid-phase peptide synthesis method using the E3 ligase recruiting peptide (PLPAYAI; Pro-Leu-Pro-Ala-Tyr-Ala-Ile) represented by SEQN 31 instead of the E3 enzyme ligand represented by SEQN 26, in all cases identical to Examples 1 and 2, thereby producing a PROTAC (E3333) having a scrambled sequence. scram -PROTAC) peptide and PROTAC nanoparticles (E3 scram -NPs) were synthesized. The molecular weight and purity of the PROTAC peptide were analyzed using LC / MS and HPLC. As a result, it was confirmed that the PROTAC peptide was successfully synthesized.

[0192]

[0193] Comparative Examples 5 and 6. Preparation of PROTAC peptides having scrambled sequences and PROTAC nanoparticles

[0194] A PROTAC peptide (SEQN 34) consisting of 18 amino acids was synthesized via a solid-phase peptide synthesis method using the EGFR scrambled peptide (IHEEAGM) represented by SEQN 33 instead of the PD-L1 peptide represented by SEQN 1, in all cases identical to Examples 1 and 2, thereby producing a PROTAC (EGFR) having a scrambled sequence. scram -PROTAC) peptides and PROTAC nanoparticles (EGFR scram -NPs) were synthesized. The molecular weight and purity of the PROTAC peptide were analyzed using LC / MS and HPLC. As a result, it was confirmed that the PROTAC peptide was successfully synthesized.

[0195]

[0196] Comparative Example 7. Preparation of PROTAC (DD-PROTAC) peptide having an Asp-Asp(DD) linker

[0197] A PROTAC (DD-PROTAC) peptide was prepared in the same manner as in Example 1, except that a PROTAC peptide (SEQ No. 36), using a linker indicated by SEQ No. 35 instead of a Phe-Phe (FF) linker, was synthesized via a solid-phase peptide synthesis method. The molecular weight and purity of the PROTAC peptide were analyzed using LC / MS and HPLC. As a result, it was confirmed that the PROTAC peptide was successfully synthesized.

[0198]

[0199] Experimental Example 1. Analysis of Molecular Dynamics of PROTAC Nanoparticles (PT-NPs)

[0200] To examine the effect of the linker, the structure was analyzed using molecular dynamics (MD) simulations. The PROTAC peptides of Example 1, Example 3, and Comparative Example 7 were used for the analysis of the molecular dynamics simulations. Molecular dynamics simulations predict the structure of PROTAC peptides based on general models governing interatomic interactions, and each frame was analyzed using the Desmond module of Schrödinger Suites.

[0201]

[0202] Figure 5 shows the results of molecular dynamics (MD) simulations for the PROTAC peptide of Example 1 (b) and the PROTAC peptide of Example 3 (c). Figure 6 is a graph showing the radius of gyration measured from molecular dynamics (MD) simulations for the PROTAC peptide of Example 1 and the PROTAC peptide of Comparative Example 7.

[0203] Figure 4 schematically shows the structure of the PROTAC peptide of Example 1 or 3 and the PROTAC peptide of Comparative Example 7. Although it is labeled as having 12 amino acid residues, it actually consists of 18 amino acid residues. For specific chemical structures, refer to Figures 3a and 3b.

[0204] As shown in FIGS. 4 and 5, it was confirmed that the PROTAC peptides of Examples 1 and 3, having a linker composed of two phenylalanine residues, maintained a close distance between the 16 amino acid residues present within the PROTAC peptide during 20 ns of MD simulation. In contrast, the PROTAC peptide of Comparative Example 7, having a linker composed of two aspartic acid residues, was confirmed to have an increased radius of rotation of the 16 amino acid residues present within the PROTAC peptide, unlike Examples 1 and 3.

[0205] From the results described above, it can be seen that a PROTAC peptide having a linker composed of two phenylalanine residues can self-assemble into nanoparticles (NPs) through intermolecular π-π interactions.

[0206]

[0207] Experimental Example 2-1. Analysis of Physicochemical Properties of PROTAC Nanoparticles (PT-NPs)

[0208] PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 scram -NPs and E3 scram The average diameter and surface zeta potential of the PROTAC nanoparticles were measured using dynamic light scattering with a Zetasizer-Nano ZS (Malvern Instruments, Worcestershire, UK). The PROTAC nanoparticles used were formed through self-assembly in physiological saline.

[0209]

[0210] FIG. 7 shows PROTAC nanoparticles (PT-NPs, PD-L11) prepared from Example 2(a), Comparative Example 2(b), and Comparative Example 4(c). scram -NPs and E3 scramThis is the result of dynamic light scattering (DLS) analysis for -NPs. The image inset in Fig. 7 is an image taken with a cryo-transmission electron microscope (CryoTEM).

[0211] As shown in FIG. 7, PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 scram -NPs and E3 scram It was confirmed that the -NPs had average diameters of 211.8 ± 13.5, 196.0 ± 7.7, and 214.5 ± 16.6 nm, respectively.

[0212] PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 scram -NPs and E3 scram As a result of checking the zeta potential for -NPs, it was confirmed that they exhibited positive zeta potentials of 31.24 ± 2.97 mV, 22.77 ± 9.01 mV, and 33.05 ± 2.84 mV, respectively.

[0213] According to the image inset in FIG. 7, PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 scram -NPs and E3 scram -NPs) were all confirmed to have nano-sized spherical structures.

[0214]

[0215] Experimental Example 2-2. Analysis of Physicochemical Properties of PROTAC Nanoparticles (EGFR-NPs)

[0216] PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6 scram -NPs and EGFR scramThe average diameter and surface zeta potential of the PROTAC nanoparticles were measured using dynamic light scattering with a Zetasizer-Nano ZS (Malvern Instruments, Worcestershire, UK). The PROTAC nanoparticles used were formed through self-assembly in physiological saline.

[0217]

[0218] FIG. 7d shows the results of dynamic light scattering (DLS) analysis for PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and FIG. 7e shows the results for PROTAC nanoparticles (E3) prepared from Comparative Example 4. scram This is the result of dynamic light scattering (DLS) analysis for -NPs, and Fig. 7f shows the PROTAC nanoparticles (EGFR) prepared from Comparative Example 6. scram This is the result of dynamic light scattering (DLS) analysis for -NPs. The right images in Figures 7d, e, and f are images taken with a cryo-transmission electron microscope (CryoTEM).

[0219] As shown in FIGS. 7d, e, and f, PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6 scram -NPs and EGFR scram It was confirmed that the -NPs had average diameters of 144.2 ± 20.65 nm, 132.9 ± 6.585 nm, and 143.8 ± 10.83 nm, respectively.

[0220] According to the right-hand images in FIGS. 7d, e, and f, PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6 scram -NPs and EGFR scram -NPs) were all confirmed to have nano-sized spherical structures.

[0221]

[0222] Experimental Example 3. Analysis of the structural stability of PROTAC nanoparticles in serum

[0223] PROTAC nanoparticles (PT-NPs, EGFR-NPs, PD-L11112) prepared from Examples 2, 4, Comparative Examples 2, 4, and 6 formed by self-assembly in physiological saline scram -NPs, E3 scram -NPs and EGFR scram After immersing the -NPs in mouse serum, the particle size was measured using dynamic light scattering at 1, 6, 9, 24, and 48 hours.

[0224]

[0225] FIG. 8 shows PROTAC nanoparticles (PT-NPs, EGFR-NPs, PD-L11112) prepared from Example 2, Example 4, Comparative Example 2, Comparative Example 4, and Comparative Example 6. scram -NPs, E3 scram -NPs and EGFR scram This is the result of analyzing whether there was a change in particle size after adding -NPs) to mouse serum for 48 hours. Figure 8a shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram The results are for -NPs), and FIG. 8b shows the PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6. scram -NPs and EGFR scram This is the result for -NPs).

[0226] As shown in FIG. 8, PROTAC nanoparticles (PT-NPs, EGFR-NPs, PD-L11112) prepared from Example 2, Example 4, Comparative Example 2, Comparative Example 4, and Comparative Example 6 scram -NPs, E3 scram -NPs and EGFR scram-NPs exhibited excellent structural stability in blood, as no significant change in particle size was observed in mouse serum. This indicates that storage is easy, as they can maintain their initial particle size (100-220 nm) in biological fluids for more than 2 to 7 days. Furthermore, it can be seen that they can effectively target tumor tissue through passive accumulation via the EPR effect even after long-term storage.

[0227]

[0228] Experimental Example 4. Analysis of Binding Affinity of PROTAC Nanoparticles (PT-NPs)

[0229] PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 formed by self-assembly in physiological saline scram -NPs and E3 scram -NPs) were prepared, and their binding affinity with PD-L1 was analyzed through BLI analysis.

[0230] Biolayer Interferometry (BLI) analysis was performed using a BLItz system (ForteBio, CA, USA). For the BLI analysis, 50 μg / ml of recombinant mouse PD-L1 was immobilized on a Protein A biosensor (ForteBio), the biosensor was washed twice, and PROTAC nanoparticles (PT-NPs, PD-L11111112) prepared from Example 2, Comparative Example 2, and Comparative Example 4 were immobilized. scram -NPs and E3 scram -NPs) 1 mmol was reacted. As a control, the PD-L1 peptide (sequence 1; CLQKTPKQC) and the PROTAC peptide of Comparative Example 7 having a DD linker were used, and the BLI analysis was performed in the same way.

[0231]

[0232] FIG. 9 shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scramThis is the result of BLI analysis on the PD-L1 binding affinity of -NPs. As a control, PD-L1 peptide (sequence 1; CLQKTPKQC) and the PROTAC peptide of Comparative Example 7 having a DD linker were used.

[0233] As shown in Fig. 9, the PROTAC nanoparticles (PT-NPs) prepared from Example 2 had a binding affinity for the PD-L1 receptor that was significantly more than 14 times higher than that of the PD-L1 binding peptide (CLQKTPKQC), and the PROTAC nanoparticles (PT-NPs, E3) prepared from Comparative Example 4 scram It was significantly more than twice as high as -NPs.

[0234] On the other hand, PROTAC nanoparticles (PD-L1) prepared from Comparative Example 2 scram It was confirmed that -NPs) have low binding affinity for the PD-L1 receptor. This is because the PD-L1 peptide in the PROTAC nanoparticles prepared from Comparative Example 2 is a scrambled sequence that does not have binding activity with PD-L1.

[0235] PROTAC nanoparticles (PD-L1) prepared from Comparative Examples 2 and 4 through the preceding experiments scram -NPs and E3 scram It was confirmed that the physicochemical properties of the -NPs were similar to those of the PROTAC nanoparticles (PT-NPs) of Example 2, but that the binding affinity for the PD-L1 receptor was significantly reduced due to sequence and structural differences between the PD-L1 binding peptide, linker, and E3 enzyme ligand.

[0236] Specifically, when only PD-L1 binding peptides are used, it was found that the binding affinity for the PD-L1 receptor is very low. This is considered to be a limitation of a single peptide.

[0237] When a scrambled sequence is used instead of a PD-L1 binding peptide, binding characteristics to the PD-L1 receptor do not appear even if the nanoparticle structure according to the present invention is used. From this, it was confirmed that PD-L1 receptor binding activity is not formed by the peptide-linker-E3 enzyme ligand structure alone.

[0238] When a scrambled sequence is used instead of an E3 enzyme ligand, it exhibits binding characteristics to the PD-L1 receptor, but the binding affinity to the PD-L1 receptor was significantly lower than that of the PROTAC nanoparticles of Example 1. From this, it can be seen that even if a PD-L1 sequence is included, the binding activity to the PD-L1 receptor may be reduced depending on the structure of the nanoparticles.

[0239] Therefore, it can be seen that even if the PD-L1 binding peptide and the E3 enzyme ligand are sequences with no activity when combined with an FF linker, a significant effect cannot be achieved even if a PROTAC peptide of a triple structure is prepared, and if the linker is not an FF linker, even if the PD-L1 binding peptide and the E3 enzyme ligand have excellent activity, the microstructure of the nanoparticle formed therefrom changes, so no effect can be obtained. Thus, it is preferable to include all of the triple PROTAC peptide having a PD-L1 binding peptide, an FF linker, and an E3 enzyme ligand according to the present invention.

[0240]

[0241] Experimental Example 5. Analysis of anticancer activity in vitro

[0242] PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 formed by self-assembly in physiological saline scram -NPs and E3 scram -NPs) were prepared, and cytotoxicity was analyzed. PD-L1 antibodies were used as a control.

[0243] The cytotoxicity analysis of colorectal cancer cells CT26 was performed at 1 × 10⁶ per well of a 96-well cell culture plate. 6 Canine CT26 cells were seeded. The culture medium used was DMEM supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% penicillin-streptomycin. After stabilization for 24 hours in a humid environment of 5% CO2 and 95% air at 37°C, samples were added to each cell culture medium at various concentrations (0.00 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM), respectively, and then incubated in a 37°C incubator for 48 hours. After incubation was complete, culture medium containing 10% Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated for 30 minutes, after which absorbance was measured at 450 nm using a microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, CA).

[0244]

[0245] FIG. 10 shows PD-L1 antibodies on CT26 cells, PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scram This is the result of evaluating cell viability after treatment with -NPs. Statistical significance was analyzed using the Tukey-Kramer test. The p-values ​​for each statistical data point are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0246] As shown in Fig. 10, it was confirmed that the PROTAC nanoparticles prepared from Example 2, Comparative Example 2, and Comparative Example 4 did not cause significant cytotoxicity even at high concentrations. In other words, it can be seen that all nanoparticles exhibit low cytotoxicity.

[0247]

[0248] Experimental Example 6-1. Intracellular permeability of PROTAC nanoparticles (PT-NPs)

[0249] To analyze the PD-L1 binding characteristics of the PROTAC nanoparticles according to the present invention, colon cancer cells (CT26) with overexpression of the PD-L1 receptor were used.

[0250] 1 × 10⁶ CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing for 24 hours, the PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4, labeled with Cy5.5, were administered to each cell. scram -NPs and E3 scram Cells were treated with 100 μM of -NPs and post-incubated at 4 °C for 10 minutes. Cells were washed twice with PBS (to remove unbound nanoparticles) and incubated at 37 °C for various durations (0 h, 1 h, 3 h, 6 h, 9 h). Afterward, the cells were treated with 4% paraformaldehyde for 10 minutes, followed by treatment with DAPI solution for 15 minutes to stain the cell nuclei. Cell fluorescence images were analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)).

[0251] At this time, PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 labeled with Cy5.5 scram -NPs and E3 scram -NPs) were prepared using a Cy5.5-labeled cancer-targeting peptide, wherein the Cy5.5-labeled peptide was prepared to be included in 5% by weight based on the total weight of the nanoparticles.

[0252]

[0253] FIG. 11a shows PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4.scram -NPs and E3 scram Figure 11b is a confocal microscope image of CT26 cells treated with -NPs, and is the result of quantitatively analyzing the concentration of PROTAC nanoparticles (PT-NPs) prepared from Example 2 in the cell membrane and cytoplasm of CT26 cells.

[0254] As shown in FIG. 11, PROTAC nanoparticles (PT-NPs and E3) prepared from Example 2 and Comparative Example 4 scram It was confirmed that -NPs) formed a binding with the cell membrane of CT26 cells within 1 hour. On the other hand, the PROTAC nanoparticles (PD-L1) prepared from Comparative Example 2 scram No significant binding of -NPs to the CT26 cell surface was observed.

[0255] In addition, PROTAC nanoparticles (PT-NPs and E3 prepared from Example 2 and Comparative Example 4) scram It was confirmed that the -NPs rapidly move from the cell membrane into the cytoplasm. In other words, it was confirmed that the PROTAC nanoparticles according to the present invention effectively bind to the PD-L1 receptor present on the cell membrane of tumor cells and subsequently rapidly penetrate into the cytoplasm.

[0256] As shown in Fig. 11b, the PROTAC nanoparticles according to the present invention (Example 2, PT-NPs) initially bind to PD-L1 receptors on the surface of CT26 cells and remain in the cell membrane, but as time passed, 35% to 42% penetrated into the cell within 1 hour, and after 9 hours, more than 90% moved into the cytoplasm.

[0257]

[0258] Experimental Example 6-2. Intracellular permeability of PROTAC nanoparticles (EGFR-NPs)

[0259] We intended to evaluate whether the performance is maintained when the cancer target peptide in the protac peptide constituting the cancer target protac nanoparticle of the present invention is changed to another peptide (e.g., EGFR).

[0260] The fact that EGFR-NP of Example 4 was used instead of PT-NP of Example 2, and PD-L1 of Comparative Example 2 scram EGFR of Comparative Example 6 instead of -NP scram Except for the use of -NP, the experiment was conducted in the same manner as Experimental Example 6-1. Additionally, in this experiment, EGFR mAb (0.06 μM) or EGFR target peptide (sequence 2, 200 μM) was used as a control group. The PROTAC nanoparticles of Example 4, Comparative Example 4, and Comparative Example 6 used in the experiment were prepared using a Cy5.5-labeled cancer target peptide, wherein the Cy5.5-labeled peptide was prepared to be included in 5% by weight based on the total weight of the nanoparticles.

[0261]

[0262] FIG. 12a shows the PROTAC nanoparticles (EGFR-NPs, E3) of Example 4, Comparative Example 4, and Comparative Example 6. scram -NPs and EGFR scram Figure 12b is a confocal microscope image of CT26 cells treated with EGFR mAb and EGFR target peptides, and is the result of quantitatively analyzing the concentration of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 in the cell membrane and cytoplasm of CT26 cells.

[0263] As shown in FIG. 12, PROTAC nanoparticles (EGFR-NPs and E3) prepared from Example 4 and Comparative Example 4 scram It was confirmed that -NPs) formed a binding with the cell membrane of CT26 cells within 1 hour. In contrast, the PROTAC nanoparticles (EGFR) prepared from Comparative Example 6 scram No significant binding of -NPs to the CT26 cell surface was observed.

[0264] In addition, PROTAC nanoparticles (EGFR-NPs and E3) prepared from Example 4 and Comparative Example 4 scramIt was confirmed that the -NPs rapidly move from the cell membrane into the cytoplasm. In other words, it was confirmed that the PROTAC nanoparticles according to the present invention effectively bind to the cancer target protein (EGFR) present in the cell membrane of tumor cells and subsequently rapidly penetrate into the cytoplasm.

[0265] As shown in Fig. 12b, it can be confirmed that the PROTAC nanoparticles of Example 4 initially bind to cancer target proteins on the surface of CT26 cells and remain in the cell membrane, but within 1 hour, more than 50% penetrated into the cell, and after 9 hours, more than 100% moved into the cytoplasm.

[0266] In other words, it has been confirmed that the PROTAC nanoparticles of the present invention can have their sequences modified within their structure to include other peptides having cancer-targeting functions, and that in this case, there is no problem with the function and performance of the PROTAC nanoparticles. However, even if a peptide has excellent cancer-targeting functions, if any part of the structure of the PROTAC nanoparticles of the present invention is modified (Comparative Example 4: loss of function of E3 ligand), a problem arises in which the intracellular permeability decreases by 30–50%. Therefore, it has been confirmed that the PROTAC nanoparticles of the present invention can be replaced with cancer-targeting peptides as long as the existing structure is maintained, provided that the sequence has a structural cancer-targeting function. Here, the basic structure refers to the structure of a cancer-targeting PROTAC peptide composed of a, b, and c. The sequence length of the cancer-targeting peptide is not particularly limited as long as it is a peptide having the function of binding to a target specifically expressed in cancer tissue, but reference is made to the detailed description for preferred embodiments thereof.

[0267]

[0268] Experimental Example 7. Analysis of Target Substance Degradation Mechanism in Cancer Cells

[0269] To determine whether the pathway by which the PROTAC nanoparticles according to the present invention enter tumor cells is via the PD-L1 receptor or EGFR, the entry into the cell was confirmed after competitively blocking PD-L1 or EGFR on the cell membrane. Each well of newly cultured stabilized CT26 cells (see Experimental Example 6-1) was pre-treated with PD-L1 mAb (0.06 μM), EGFR mAb (0.06 μM), PD-L1 target peptide (Sequence 1, 200 μM), or EGFR target peptide (Sequence 2, 200 μM) for 1 hour at 4°C to block target substances on the cell surface. Subsequently, PROTAC nanoparticles (PT-NPs or EGFR-NPs) (100 μM) prepared from Example 2 or Example 4 were treated and post-incubated at 37°C for 9 hours. After culturing, the cells were washed with PBS, treated with 4% paraformaldehyde for 10 minutes, and then treated with DAPI solution for 15 minutes to stain the cell nuclei. Cell fluorescence images were analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)). The PROTAC nanoparticles of Examples 2 and 4 used in the experiment were prepared using a Cy5.5-labeled cancer-targeting peptide, wherein the Cy5.5-labeled peptide was prepared to be included at 5% by weight based on the total weight of the nanoparticles.

[0270]

[0271] Figure 13a shows the results of confocal fluorescence microscopy analysis after treating CT26 cells pretreated with PD-L1 mAb or PD-L1 binding peptide with PROTAC nanoparticles (PT-NPs) prepared from Example 2, and Figure 13b shows the results of confocal fluorescence microscopy analysis after treating CT26 cells pretreated with EGFR mAb or EGFR binding peptide with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4.

[0272] As shown in Fig. 13, it was confirmed that the PROTAC nanoparticles of Example 2 and the PROTAC nanoparticles of Example 4 did not form bonds with cancer cells in which the target substance was blocked.

[0273] In other words, it can be seen that the PROTAC nanoparticles of Example 2 and Example 4 are capable of accurate recognition and penetration into cancer cells through binding to cancer targets (PD-L1 receptors or EGFR). Therefore, it can be seen that the PROTAC nanoparticles according to the present invention are very stable against normal cells because they target 'cancer' tissues by accurately recognizing targets that are specifically expressed in cancer tissues (Example 2: PD-L1 receptor, Example 4: EGFR).

[0274]

[0275] Experimental Example 8. Analysis of the intracellular entry pathway of PROTAC nanoparticles

[0276] 1 × 10⁶ PD-L1 overexpressing CT26 colorectal cancer cells in a confocal microscope cell culture plate 6After dispensing the cells and stabilizing them for 24 hours, each cell was treated with Cy5.5-labeled Example 2 PROTAC nanoparticles (PT-NPs) (100 μM) and Cy5.5-labeled Example 4 PROTAC nanoparticles (EGFR-NPs) (100 μM), respectively, and post-incubated at 4 °C for 10 minutes. The cells were washed twice with PBS (to remove unbound nanoparticles) and incubated at 37 °C for varying durations (9 h), then treated with 4% paraformaldehyde for 10 minutes, followed by treatment with LysoTracker (LysoTracker DND 99, Invitrogen, USA), which reacts specifically with lysosomes, and DAPI solution for 15 minutes. Cell fluorescence images were analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)). Additionally, in this experiment, EGFR mAb (0.06 μM) or EGFR target peptide (sequence 2, 200 μM) was used as a control group.

[0277]

[0278] Figure 14a shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (PT-NPs) prepared from Example 2 were successfully introduced into lysosomes when CT26 cells were treated, and the fluorescence intensity is shown in Figure 14b. Figure 14c shows the results of analyzing the ratio of the PROTAC nanoparticles (PT-NPs) prepared from Example 2 and the lysosomes present in the co-location from Figure 14a.

[0279] In addition, Figure 15 shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 were successfully introduced into lysosomes when CT26 cells were treated. Figure 16 shows the ratios analyzed according to the location where fluorescence is activated (cell membrane, inside the lysosome (Lysosome +), outside the lysosome (Lysosome -)) in the results of Figure 15. Figure 16a shows the results for the PROTAC nanoparticles of Example 4, Figure 16b shows the results for the EGFR mAb, and Figure 16c shows the results for the EGFR target peptide.

[0280] As shown in Figure 14, it was confirmed that the PROTAC nanoparticles (PT-NPs) prepared from Example 2 bind to the cell membrane to form a PT-NPs / PD-L1 complex, and then enter the lysosome pathway during the process of penetrating the cytoplasm. Specifically, it was confirmed that the fluorescent signals of the PROTAC nanoparticles (PT-NPs) prepared from Example 2 (red) and lysosomes (green) coexist strongly, with a Pearson correlation coefficient of 0.8606. That is, half of the PROTAC nanoparticles (PT-NPs) prepared from Example 2 were found in the lysosomes, while the rest escaped from the lysosomes, were degraded into PROTAC peptides, and strongly degraded PD-L1 through the ubiquitin-proteasome system.

[0281] Meanwhile, as shown in FIGS. 15 and 16, it can be seen that the PROTAC nanoparticles of Example 4 bind to the cell membrane upon contact with cancer cells, enter the lysosome within 1 hour, and penetrate into the cytoplasm starting from 3 hours. On the other hand, it can be seen that antibodies or peptides (Fig. 16b, c) cannot induce apoptosis in cancer cells because they are removed without penetrating into the cell even if they bind to the cell membrane, or are destroyed and removed through the intracellular foreign substance degradation pathway even if they penetrate into the lysosome or cytoplasm.

[0282] Therefore, it can be seen that the PROTAC nanoparticles of the present invention interact with cancer target substances of cancer cells, are taken up into intracellular lysosomes, escape from lysosomes without being destroyed, and induce a direct apoptotic effect on cancer cells within the cytoplasm.

[0283]

[0284] Experimental Example 9. Analysis of the PD-L1 degradation mechanism of PROTAC nanoparticles (PT-NPs)

[0285] CT26 colorectal cancer cells overexpressing PD-L1 were induced to overexpress PD-L1 labeled with GFP (green fluorescence) through lentivirus transfection with mGFP-labeled PD-L1 (hereinafter referred to as mGFP-CT26) (S. Yang, MK Shim, S. Song, H. Cho, J. Choi, SI Jeon, WJ Kim, W. Um, JH Park, HY Yoon, Liposome-mediated PD-L1 multivalent binding promotes the lysosomal degradation of PD-L1 for T cell-mediated antitumor immunity, Biomaterials 290 (2022) 121841). It was confirmed that PD-L1 labeled with mGFP was overexpressed in mGFP-CT26 cells.

[0286] 1 × 10⁶ mGFP-CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing for 24 hours, each cell was treated with a PD-L1 antibody (PD-L1 mAbs) (0.3 nM) or PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4. scram -NPs and E3 scramCells were treated with 100 μM of -NPs and post-incubated at 4°C for 10 minutes. Cells were washed twice with PBS (to remove unbound nanoparticles) and cultured at 37°C for various durations (0h, 6h, 9h, 24h, 48h), then treated with 4% paraformaldehyde for 10 minutes, and analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)).

[0287]

[0288] FIG. 17 shows an anti-PD-L1 antibody and PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 in mGFP-tagged, PD-L1-expressing CT26 cells. scram -NPs and E3 scram This is an image taken with a confocal fluorescence microscope after treatment with -NPs.

[0289] FIG. 18 shows anti-PD-L1 antibodies and PROTAC nanoparticles (PT-NPs, PD-L1) prepared from Example 2, Comparative Example 2, and Comparative Example 4 in mGFP-tagged PD-L1-expressing CT26 cells. scram -NPs and E3 scram This is the result of a quantitative analysis of relative PD-L1 levels after treatment with -NPs.

[0290] As shown in FIGS. 17 and 18, PROTAC nanoparticles (PD-L1) prepared from the PD-L1 antibody, Comparative Example 2 and Comparative Example 4 scram -NPs and E3 scram It was confirmed that in CT26 cells treated with -NPs, mGFP-tagged PD-L1 (green) present on the cell surface was significantly reduced starting from 6 hours and fully recovered within 24 to 48 hours.

[0291] In other words, it can be seen that common PD-L1 target substances, after binding to the PD-L1 receptor, move to recycling vesicles rather than lysosomes, thereby being fully regenerated without being completely degraded. Due to this PD-L1 recycling mechanism, the therapeutic effect of most cancer immunotherapies has decreased rapidly over time.

[0292] On the other hand, it was confirmed that the PROTAC nanoparticles (PT-NPs) prepared from Example 2 did not regenerate PD-L1 receptors on the surface of tumor cells even at a very low concentration of 100 μM. That is, the PROTAC nanoparticles (PT-NPs) prepared from Example 2 irreversibly and completely degraded 90% of the PD-L1 receptors on tumor cells.

[0293] The PROTAC nanoparticles according to the present invention react only with tumor cells that overexpress PD-L1 receptors. In this case, the PROTAC nanoparticles according to the present invention form multi-junction bonds with PD-L1 present on the membrane surface of the tumor cells, thereby entering the cytoplasm through lysosomes, and continuously and irreversibly degrade more than 90% of PD-L1.

[0294]

[0295] Experimental Example 10-1. Analysis of PD-L1 expression in tumor cells

[0296] 1 × 10⁶ wild CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing them for 24 hours, each cell was treated with a PD-L1 antibody (APC-PD-L1 mAbs) (0.3 nM) or PROTAC nanoparticles (PT-NPs) prepared from Example 2 (100 μM) and post-incubated at 4 °C for 10 minutes. The cells were washed twice with PBS (to remove unbound nanoparticles) and cultured at 37 °C for various durations (0h, 6h, 9h, 24h, 48h), after which PD-L1 expression was analyzed by Western blot.

[0297] For Western Blot analysis, the cytoplasmic fraction was obtained from cells, and proteins were isolated by electrophoresis. The isolated proteins were migrated to nitrocellulose membranes by treatment with 20% methanol, 25 mM Tris, and 192 mM glycine buffer. After confirming the presence of migration with Ponceau solution, the nitrocellulose membranes were treated with 5% blocking solution (non-fat milk) for 30 minutes at room temperature. The nitrocellulose membranes were then incubated with various antibodies diluted 1,000-fold with the buffer for at least 4 hours. After the reaction was complete, the membranes were washed 6 times at 5-minute intervals with Tris Tween buffered saline (TTBS), followed by incubation with rabbit or mouse IgG antibodies (secondary antibodies conjugated with horse radish peroxidase) for 2 hours and 6 washes with TTBS. The presence or degree of protein expression was confirmed by washing with distilled water, reacting with an enhanced chemiluminescence solution for 1 minute, and comparing the thickness of the bands that appeared on the film after exposure.

[0298]

[0299] Figure 19 shows the results of analyzing PD-L1 expression levels by Western blot after treating wild CT26 cells with an anti-PD-L1 antibody and PROTAC nanoparticles (PT-NPs) prepared from Example 2.

[0300] As shown in Fig. 19, when the PROTAC nanoparticles (PT-NPs) prepared from Example 2 were treated, the PD-L1 expression level in wild-type CT26 cells was continuously reduced by up to 95% by 48 hours. On the other hand, in wild-type CT26 cells treated with an anti-PD-L1 antibody, PD-L1 expression decreased rapidly by up to 90% (9 hours), then 30% of PD-L1 expression was restored after 24 hours, and PD-L1 recovered to over 100% after 48 hours. Therefore, it can be seen that there is no PD-L1 inhibitory effect.

[0301] In other words, after structural blockade by anti-PD-L1 antibodies, endocytized PD-L1 receptors are actively recycled back into the cell membrane via recycling endosomes, so the PD-L1 receptors are not completely degraded and are restored to their original state after a certain period of time.

[0302]

[0303] Experimental Example 10-2. Analysis of EGFR Expression in Tumor Cells

[0304] 1 × 10⁶ wild CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing them for 24 hours, the cells were treated with PROTAC nanoparticles (EGFR-NPs) (100 μM) prepared from Example 4 and post-incubated at 4 °C for 10 minutes. The cells were washed twice with PBS (to remove unbound nanoparticles) and cultured at 37 °C for various durations (0 h, 6 h, 9 h, 24 h, 48 h), after which EGFR expression was analyzed by Western blot.

[0305] At this time, as a comparison group, the PROTAC nanoparticles of Comparative Example 4 (E3) were used instead of the PROTAC nanoparticles of Example 4. scram -NPs), PROTAC nanoparticles (EGFR) of Comparative Example 6 scram All were prepared and analyzed in the same way, except that -NPs), EGFR target peptide (sequence 2; 0.3 nM) or EGFR antibody (APC-EGFR mAbs) (0.3 nM) were used.

[0306] For Western blot analysis, the cytoplasmic fraction was obtained from cells, and proteins were isolated by electrophoresis. The isolated proteins were migrated to nitrocellulose membranes by treatment with 20% methanol, 25 mM Tris, and 192 mM glycine buffer. After confirming the presence of migration with Ponceau solution, the nitrocellulose membranes were treated with 5% blocking solution (non-fat milk) for 30 minutes at room temperature. The nitrocellulose membranes were then incubated with various antibodies diluted 1,000-fold with the buffer for at least 4 hours. After the reaction was complete, the membranes were washed 6 times at 5-minute intervals with Tris Tween buffered saline (TTBS), followed by incubation with rabbit or mouse IgG antibodies (secondary antibodies conjugated with horse radish peroxidase) for 2 hours and 6 washes with TTBS. The presence or degree of protein expression was confirmed by washing with distilled water, reacting with an enhanced chemiluminescence solution for 1 minute, and comparing the thickness of the bands that appeared on the film after exposure.

[0307]

[0308] FIG. 20 shows the PROTAC nanoparticles of Example 4, as well as the PROTAC nanoparticles of Comparative Example 4 (E3) in wild CT26 cells. scram -NPs), PROTAC nanoparticles (EGFR) of Comparative Example 6 scram Western blot (a) showing the EGFR expression level after treatment with -NPs), EGFR target peptide (sequence 2; 0.3 nM), or EGFR antibody (APC-EGFR mAbs) (0.3 nM), and a graph (b) showing the result of quantitative analysis.

[0309] As shown in Fig. 20, the PROTAC nanoparticles of Example 4 continuously reduced EGFR expression levels (up to 90%) in wild-type CT26 cells.

[0310] In contrast, the PROTAC nanoparticles (E3) of Comparative Example 4 scramWhen treated with -NPs) or antibodies, the EGFR expression level dropped to 90% and then recovered to 100% within 15 hours. In addition, when treated only with EGFR target peptides, it was confirmed that the EGFR expression level decreased to 60% within 3 hours, then gradually recovered from 3 hours onwards, eventually exceeding 100%.

[0311] Meanwhile, the PROTAC nanoparticles (EGFR) of Comparative Example 6 scram No significant change in EGFR expression levels was observed for -NPs.

[0312] Synthesizing the results of Experimental Examples 10-1 and 10-2, it can be confirmed that the PROTAC nanoparticles according to the present invention exhibit excellent anticancer effects against not only PD-L1 but also EGFR, and also have a very excellent effect in inhibiting cancer cells from recycling EGFR. In other words, it can be seen that if the basic structure (platform) of the PROTAC peptide is maintained, the PROTAC nanoparticles of the present invention can achieve anticancer effects and the effect of inhibiting the recycling of cancer target substances even if the cancer target peptide is modified. Conversely, if either the linker or the E3 enzyme ligand sequence among the PROTAC peptide structures is modified, or if a peptide without cancer target function is used, all effects such as selectivity against cancer cells, anticancer effects, inhibition of cancer target substance expression, and inhibition of cancer target substance recycling are reduced or not activated. This implies that the mutual interactions according to the combinations of a, b, and c constituting the PROTAC peptide are essential for the anticancer effect.

[0313] In anticancer immunotherapy, although various target substances expressed on the surface of cancer cells are known, peptides or antibodies that simply target these cancer target substances have limitations in cancer immunotherapy. As confirmed by this experiment, it appears that the immunotherapeutic effect is exhibited by removing the target substances present on the surface of cancer cells in the early stages, but over time, the target substances are restored through a reuse pathway via autophagy, and it was confirmed that in some cases, the defensive mechanism is actually strengthened compared to the initial state. The PROTAC nanoparticles of the present invention penetrate cancer cells more accurately and thoroughly, thereby not only inhibiting the reuse pathway of target substances but also inducing direct cancer cell death through E3 enzyme ligands, thus possessing an anticancer effect that surpasses the effect of cancer cell survival.

[0314]

[0315] Experimental Example 10-3. Analysis of Pharmacological Mechanism of PROTAC Nanoparticles-1

[0316] 1 × 10⁶ wild CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing them for 24 hours, the cells were treated with 5 μM of the VHL (von Hippel Lindau) inhibitor (VH-298, cat No. S8449, Selleckchem) and cultured for 12 hours. Then, the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 (100 μM) were added, followed by post-incubation at 4°C for 10 minutes. The cells were washed twice with PBS (to remove unbound nanoparticles) and cultured at 37°C for various durations (0h, 6h, 9h, 24h, 48h), after which EGFR expression was analyzed by Western blot. The Western blot analysis method was based on Experimental Example 10-2.

[0317]

[0318] Figure 20c shows the results of treating cancer cells (CT26) treated with a VHL (von Hippel Lindau) inhibitor with the PROTAC nanoparticles of Example 4 and analyzing the EGFR expression levels over time. According to Figure 20c, in cancer cells where the efficacy of the E3 enzyme (ligase) was inhibited by the VHL inhibitor, the anticancer effect of the PROTAC nanoparticles of Example 4 was transient, and it was confirmed that the EGFR expression levels recovered over time.

[0319] In other words, it can be seen that the PROTAC nanoparticles of the present invention reduce the expression of the protein by promoting protein degradation through E3 ubiquitin ligase via binding with EGFR.

[0320]

[0321] Experimental Example 10-4. Analysis of Pharmacological Mechanism of PROTAC Nanoparticles-2

[0322] 1 × 10⁶ wild CT26 cells on a cell culture plate for confocal microscopy 6 After dispensing the cells and stabilizing them for 24 hours, the cells were treated with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 at various concentrations (0–1800 nM) and post-incubated at 4°C for 10 minutes. The cells were washed twice with PBS (to remove unbound nanoparticles) and cultured at 37°C for various durations (24h), after which EGFR expression was analyzed by Western blot. Refer to Experimental Example 10-2 for the Western blot analysis method.

[0323]

[0324] Figure 20d shows the results of analyzing the EGFR expression level after treating cancer cells (CT26) with the PROTAC nanoparticles of Example 4 at different concentrations, and Figure 20e shows the results of analyzing the expression levels of EGFR, AKT, ERK, c-Myc, cyclin D1, etc., and the phosphorylation of EGFR and AKT after treating cancer cells (CT26) with the PROTAC nanoparticles of Example 4 at different concentrations.

[0325] According to Fig. 20d, the PROTAC nanoparticles of Example 4 were found to have a "Hook Effect" that produces a false negative result because they eliminate the presence of the triple complex at high concentrations exceeding 1000 nM.

[0326] In addition, it was confirmed that the PROTAC nanoparticles of Example 4 exhibit selective inhibitory activity against cancer cells when treated at an appropriate concentration (200 to 1000 nM, preferably 400 to 1000 nM), thereby possessing a mechanism of action that inhibits cancer cell growth. Specifically, the PROTAC nanoparticles of the present invention inhibit Akt and Akt phosphorylation related to cancer cell survival in the cytoplasm and nucleus of cancer cells, inhibit the activation of ERK, a cell growth regulator, and ultimately inhibit the expression of G1 phase progression factors of Cyclin D1 and c-Myc, thereby inhibiting the progression of the cancer cell cycle and inducing G1 cell cycle arrest.

[0327] In other words, it can be seen that the PROTAC nanoparticles of the present invention have an anticancer effect that goes beyond the effect of cancer cell survival by inhibiting the growth of cancer cells and stopping the cell cycle to induce apoptosis through E3 ubiquitin ligase by binding to target substances such as EGFR or PD-L1, as well as blocking the recycling pathway of target substances which is a protective mechanism of cancer cells.

[0328]

[0329] Experimental Example 11. Efficacy of PROTAC nanoparticles in an animal model

[0330] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6 A cancer animal model was prepared by inoculating CT26 colorectal cancer cells via subcutaneous inoculation. The colorectal tumor volume was approximately 200 ± 20 mm². 3After rearing until reaching [the target], administer 5 mg / kg of an anti-PD-L1 antibody labeled with Cy5.5 (Comparison Group 1) intravenously, or the protac nanoparticles of Comparative Example 4 labeled with Cy5.5 (E3 scram 10 mg / kg of -NPs (comparison group 2) or 10 mg / kg of Cy5.5-labeled PROTAC nanoparticles of Example 2 (PT-NPs) (experimental group) were administered intravenously. Non-invasive near-infrared fluorescence (NIRF) imaging was analyzed using an IVIS Lumina Series III system (PerkinElmer; Waltham, MA, USA) to evaluate tumor targeting efficiency over time (1 h, 3 h, 6 h, 24 h, 48 h). As a control, the same volume of physiological saline was administered intravenously.

[0331] Next, 48 hours after treatment, the experimental group, comparison group 1, comparison group 2, and control group were anesthetized and sacrificed, and major organ and tumor tissues were isolated. Ex vivo NIRF imaging of the isolated tissues was performed using an IVIS Lumina Series III system, and the fluorescence intensity of each tissue was quantified using Living Image software (PerkinElmer, Waltham, MA, USA).

[0332]

[0333] Figure 21 shows non-invasive near-infrared fluorescence (NIRF) images taken over time for the experimental group, comparison group 1, comparison group 2, and control group (n=3). Figure 22 shows the results of quantitative analysis of fluorescence intensity at the tumor site in the experimental group, comparison group 1, comparison group 2, and control group.

[0334] Figure 23 shows the results of quantitative analysis of near-infrared fluorescence images and fluorescence intensity of tumor tissues recovered from the experimental group, comparison group 1, comparison group 2, and control group after 48 hours. Figure 24 shows the fluorescence images of tumor tissues recovered from the experimental group, comparison group 1, comparison group 2, and control group after 48 hours. Statistical significance analysis was performed using the Tukey-Kramer method. The p-values ​​of each statistical data point are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001).

[0335] As shown in Figures 21 and 22, significant accumulation and persistent drug residue were confirmed in the tumor tissue (white dotted circles) of the experimental group, comparison group 1, and comparison group 2. Specifically, the PROTAC nanoparticles of Example 2 and Comparative Example 4 used in the experimental group and comparison group 2 showed significantly superior accumulation in the tumor tissue. When examining the fluorescence intensity in the tumor tissue, it was confirmed that the experimental group and comparison group 2 were 3.03-3.31 times and 3.98-4.03 times higher, respectively, than the control group.

[0336] The PROTAC nanoparticles of Example 2 and Comparative Example 4 were maintained in tumor tissue for up to 2 days, indicating that the residual effect is significantly superior compared to small molecule drugs. On the other hand, Comparative Group 1, which used the anti-PD-L1 antibody alone, was initially accumulated in large quantities in the tumor tissue, but was rapidly expelled from the tumor tissue over time.

[0337] As shown in Figures 23 and 24, when examining the ex vivo NIRF images of tumor tissues isolated from the control group, experimental group, comparison group 1, and comparison group 2, it can be seen that the PROTAC nanoparticles of Example 2 and Comparative Example 4 exhibit approximately 2.5 times higher tumor accumulation compared to the anti-PD-L1 antibody.

[0338] In particular, it can be seen that the PROTAC nanoparticles of Example 2 were evenly distributed throughout deep within the tumor tissue, whereas the PROTAC nanoparticles and anti-PD-L1 antibody of Comparative Example 4 were mainly accumulated at the edges of the tumor tissue.

[0339] It can be seen that the PROTAC nanoparticles according to the present invention have significantly high accessibility to tumor tissues present in the body through excellent tumor targeting ability based on passive and active targeting mechanisms in the body.

[0340]

[0341] Experimental Example 12. Efficacy of PROTAC nanoparticles (EGFR-NPs) in an animal model

[0342] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6 A cancer animal model was prepared as in Experimental Example 11 by inoculating CT26 colorectal cancer cells via subcutaneous inoculation. The colorectal tumor volume was approximately 200 ± 20 mm². 3 They raised it until it reached.

[0343] The above-mentioned cancer animal models were randomly assigned to a total of six groups of five animals each, and experiments were conducted as shown in Table 1. All experiments were conducted with the approval of the Animal Ethics Committee of Ewha Womans University and in compliance with its animal experiment regulations. During treatment, drug accumulation in cancer tissues was analyzed using non-invasive near-infrared fluorescence (NIRF) imaging with an IVIS Lumina Series III system (PerkinElmer; Waltham, MA, USA). After administering the respective drugs to each group and completing 24 hours of treatment, the animals were sacrificed in accordance with animal experiment regulations, and major organs and tumor tissues were isolated.

[0344] Ex vivo NIRF imaging of isolated tissues was performed using an IVIS Lumina Series III system, and the fluorescence intensity of each tissue was quantified using Living Image software (PerkinElmer, Waltham, MA, USA). In addition, for pharmacokinetic evaluation, plasma drug concentrations were analyzed by fluorescence intensity. Based on plasma concentration data, non-compartment pharmacokinetic variables, namely the area under the curve (AUC) and the time to peak plasma concentration (Tg max ), steady-state distribution volume (Vss), mean residence time (MRT), half-life (t 1 / 2 ) was calculated for a single dose (Day 1) as well as for steady state (Day 2). Blood samples were collected in a vacuum collector pre-filled with K2 EDTA.

[0345] Statistical significance analysis was performed using the Tukey-Kramer test. The p-values ​​for each statistical data point are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0346] Group Experiment Method Experimental Animals Water Physiological Saline 10 mg / kg intravenous administration of physiological saline every 2 days 5 Anti-EGFR Antibody (EGFR Ab) 10 mg / kg intravenous administration of anti-PD-L1 antibody every 2 days 5 PEPTIDE 10 mg / kg intravenous administration of EGFR peptide (Sequence 2) every 2 days 5 EGFR-NPs 10 mg / kg intravenous administration of PROTAC nanoparticles (EGFR-NPs) of Example 4 every 2 days 5 E3 scram -NPs 10 mg / kg every 2 days PROTAC nanoparticles of Comparative Example 4 (E3 scram -NPs) Intravenous administration 5EGFR scram -NPs 10 mg / kg every 2 days PROTAC nanoparticles (EGFR) of Comparative Example 6 scram -NPs) Intravenous administration 5

[0347] Figure 25 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3scram -NPs group and EGFR scram These are non-invasive near-infrared fluorescence (NIRF) images taken at different times for the -NPs group (n=5). Figure 26 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram This is the result of quantitative analysis of fluorescence intensity in the tumor site in the -NPs group.

[0348] Figure 27 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group (also called PROTAC-NPs), and E3 after 24 hours. scram -NPs group and EGFR scram This shows the results of quantitative analysis of near-infrared fluorescence images and fluorescence intensity of tumor tissues recovered from the -NPs group. Figure 28 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 after 24 hours. scram -NPs group and EGFR scram This is a fluorescent image of tumor tissue recovered from the -NPs group.

[0349] As shown in FIGS. 25 to 28, EGFR Ab group, EGFR-NPs group, E3 scram -NPs group and EGFR scram The -NPs group was confirmed to have significant accumulation and drug residue in tumor tissue.

[0350] In particular, examination of ex vivo NIRF images of tumor tissue revealed that the EGFR-NPs group was evenly distributed throughout the tumor tissue, extending even to the center, compared to other groups. The EGFR Ab group showed low accumulation in the tumor tissue and retention time, and E3 scram -NPs group and EGFR scram The -NPs group failed to penetrate into the interior of the tumor tissue and was mainly accumulated on the outermost layer.

[0351] Therefore, it can be seen that the PROTAC nanoparticles according to the present invention exhibit significantly superior penetration and accessibility, as well as excellent persistence, through excellent tumor targeting ability based on passive and active targeting mechanisms in the body, as well as selectivity for tumor tissues present in the body.

[0352]

[0353] Figure 29 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram These are near-infrared fluorescence images of major organ tissues separated from the -NPs group (from left: liver, lung, spleen, kidney, heart). Figure 30 shows the results of quantitatively analyzing the fluorescence intensity in the major organ tissues of Figure 29.

[0354] As shown in Figures 29 and 30, the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram It was confirmed that the -NPs group did not induce damage to other organs, excluding tumor tissue.

[0355]

[0356] Figure 31 shows the physiological saline group, EGFR Ab group, PEPTIDE group, EGFR-NPs group, and E3 scram -NPs group and EGFR scram The results of the pharmacokinetic analysis of the drug in the -NPs group and the calculated pharmacokinetic parameters are shown in Table 2.

[0357] Parameter 1 / 2 (h)T max (h)AUC(mg / ml)*hMRT(h)Vss(mg / ml)Peptide0.960.0165.161.382.68EGFR-NPs3.520.5284.35.0814.75

[0358] As shown in Figure 31 and Table 2, the PROTAC nanoparticles of the present invention have excellent stability in the body of animals with cancer, and it was confirmed that they are rapidly absorbed and accumulated in cancer cells in the plasma after lasting for 5 hours following a single administration.

[0359] Specifically, in the case of the peptide form, although it is immediately distributed within the body, it is metabolized or excreted and eliminated within one hour, so it is known that repeated re-administration is required due to the short duration of effect. In contrast, the PROTAC nanoparticles of the present invention are rapidly absorbed but have a long-lasting effect within the body, and it can be confirmed that the effect persists for a long time, particularly due to their extensive tissue distribution. In other words, the PROTAC nanoparticles of the present invention can achieve a long-term therapeutic effect with a single administration.

[0360]

[0361] Experimental Example 13. Analysis of Anticancer Efficacy and Safety of PT-NPs in Animal Models

[0362] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6 A cancer animal model was prepared by inoculating CT26 colorectal cancer cells via subcutaneous inoculation. The colorectal tumor volume was approximately 200 ± 20 mm². 3 They raised it until it reached.

[0363] The above-mentioned cancer animal models were randomly assigned to groups of five animals each, resulting in a total of four groups, and experiments were conducted as shown in Table 1. All experiments were conducted with the approval of the Animal Ethics Committee of Ewha Womans University and in compliance with its animal experiment regulations.

[0364] Group Experiment Method Experimental Animals Physiological Saline 10 mg / kg intravenous administration of physiological saline every 2 days 5 Anti-PD-L1 Antibody 10 mg / kg intravenous administration of anti-PD-L1 antibody every 2 days 5 PT-NPs 10 mg / kg intravenous administration of PROTAC nanoparticles (PT-NPs) of Example 2 every 2 days 5 E3 scram -NPs 10 mg / kg every 2 days PROTAC nanoparticles of Comparative Example 4 (E3 scram -NPs) Intravenous administration 5

[0365] Next, after 10 days of treatment, the normal saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 scram The NPs group was anesthetized and sacrificed, and the following experiments were performed.

[0366] 1) Tumor size measurement

[0367] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The tumor size of the -NPs group was measured over time. Tumor size was measured using a caliper, and the tumor volume was calculated using the equation a×b 2 It was calculated based on ×0.5 (where a is the largest diameter and b is the smallest diameter).

[0368] Figure 32 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 scram This is the result of measuring the tumor size of the -NPs group over time (days 0, 2, 4, 6, 8, and 10).

[0369] As shown in Fig. 32, on day 10 after treatment, the PT-NPs group (211.19 ± 102.46 mm³) was compared to the physiological saline group (1411.41 ± 182.56 mm³), the anti-PD-L1 antibody group (624.87 ± 61.37 mm³), and E3 scram Tumor growth was significantly inhibited compared to the -NPs group (783.39 ± 164.35 mm³).

[0370] 2) TUNEL Analysis

[0371] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The -NPs group was prepared, and after 10 days, the tumor tissues were isolated and excised following anesthesia and sacrifice. Apoptosis analysis (TUNEL analysis) was performed on the tumor tissue sections. Specifically, after excising the tumor tissues from each group, terminal deoxy nucleotidyl transferase-mediated dUTP Nick end labeling (TUNEL) was performed using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore) according to the manufacturer's instructions to confirm apoptosis. DAPI staining was performed by staining in a DAPI (1 g / ml) solution for 30 minutes, followed by observation under a fluorescence microscope (Zeiss, Germany) at 372 nm.

[0372]

[0373] Figure 33 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is the result of TUNEL analysis of tumor tissue isolated from the -NPs group.

[0374] As shown in Fig. 33, severe apoptosis was induced in the tumor tissue of the PT-NPs group, whereas the remaining groups (saline group, anti-PD-L1 antibody group, E3 scram No significant apoptosis (green) was observed in the -NPs group.

[0375] That is, the PROTAC nanoparticles of Example 2 according to the present invention can effectively degrade PD-L1 overexpressed on the surface of tumor cells through a unique PD-L1 degradation mechanism and prevent regeneration within the cytoplasm through PROTAC-mediated degradation, thereby inducing and maintaining significant tumor cell death.

[0376]

[0377] 3) Western blot and fluorescence microscopy analysis

[0378] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The -NPs group was prepared, and after 10 days, the tumor tissue was isolated and excised following anesthesia and sacrifice. The tumor tissue was stained with PD-L1 antibody labeled with APC and DAPI at room temperature for 30 minutes. After washing with PBS, the tissue was analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)).

[0379] Proteins were extracted from tumor tissue, loaded onto 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gels), transferred to a PVDF membrane (Bio-Rad Laboratories), blocked, and treated with a primary antibody diluted 1:1,000 at 4°C for overnight binding. After washing, the reaction was allowed to proceed at room temperature for 1.5 hours to allow the secondary antibody to bind, followed by washing and verification using ChemiDoc (Bio-Rad Laboratories). To quantify the results, all reactions were repeated three times independently. All experimental results were expressed as Mean ± Standard Error, and statistical analysis was performed using the Tukey-Kramer post-hoc test. Significance was indicated on the plot using asterisks: *p<0.05, **p<0.01, and ***p<0.001.

[0380]

[0381] Figure 34 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scramThis is the result of Western blot analysis of PD-L1 expression levels in tumor tissues isolated from the -NPs group. Figure 35 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is an image of tumor tissue isolated from the -NPs group, stained with APC-labeled PD-L1 antibody and DAPI, taken using a confocal fluorescence microscope.

[0382] As shown in Figures 34 and 35, it was confirmed that the PT-NPs group significantly inhibited PD-L1 in tumor tissue, whereas the remaining groups (saline group, anti-PD-L1 antibody group, E3 scram In the -NPs group, PD-L1 expression was relatively restored.

[0383] Specifically, the PT-NPs group demonstrated stronger efficacy in degrading PD-L1, with PD-L1 expression levels decreasing by 90% compared to the physiological saline group, and the anti-PD-L1 antibody group, E3 scram PD-L1 expression decreased fourfold compared to the -NPs group.

[0384]

[0385] 4) Analysis of immune response

[0386] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The -NPs group was prepared, treatment was terminated on day 10, and after euthanasia according to procedure, the tumor tissue was isolated. A Tumor Dissociation Kit (Miltenyi Biotechnology, Hergisch Gladbach, Germany) was used to recover single cells from the tissue. Treatment with FcBlock was performed for 10 minutes to avoid non-specific IgG binding. Cytotoxic T lymphocytes (CTLs; CD45) present in the isolated tumor tissue were analyzed. + CD3 + CD8 +) and regulatory T cells (Treg; CD3 + CD4 + CD25 + To evaluate the level, multiparameter staining was performed for 1 hour. All experimental results were expressed as Mean ± Standard Error, and multiple comparisons were analyzed using the Tukey-Kramer post-hoc test. Significance was indicated on the plot using asterisks for *p<0.05, **p<0.01, and ***p<0.001.

[0387]

[0388] Figure 36 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram Tumor cells (CD45) from tumor tissue isolated from the -NPs group - PD-L1 + This shows the relative expression levels of ), and Figure 37 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram Cytotoxic T lymphocytes (CD45) from tumor tissue isolated from the -NPs group + CD3 + CD8 + ) and regulatory T lymphocytes (CD3 + CD4 + CD25 + This represents the relative expression level of ).

[0389] As shown in Figures 36 and 37, we intended to evaluate the anti-tumor immune response. Specifically, the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 scram PD-L1-positive tumor cells, cytotoxic T lymphocytes, and regulatory T cells in tumor tissues isolated from the -NPs group were closely analyzed.

[0390] First, the PT-NPs group consists of PD-L1 positive tumor cells (CD45 - PD-L1 +The ratio of the physiological saline group, the anti-PD-L1 antibody group, and E3 scram It can be seen that compared to the NPs group, there was a significant decrease of 70.2%, 34.5%, and 29.5%, respectively.

[0391] In addition, cytotoxic T lymphocytes (CTL; ​​CD45 + CD3 + CD8 + The ratio of the PT-NPs group to the anti-PD-L1 antibody group and E3 scram It was 1.79-2.02 times and 1.51-1.58 times better than the NPs group.

[0392] Regulatory T lymphocytes (Tregs; CD3 + CD4 + CD25 + ) is the PT-NPs group, the anti-PD-L1 antibody group, and E3 scram It decreased to levels of 52.9 ± 2.5% and 65.5 ± 5.3% compared to the -NPs group.

[0393]

[0394] 5) Histological analysis

[0395] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The -NPs group was prepared, and after 10 days, the tumor tissue was isolated and excised following anesthesia and sacrifice. The tumor tissue was stained with DAPI using APC-labeled CD8 antibody or PE-labeled CD25 antibody at room temperature for 30 minutes. After washing with PBS, the tissue was analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)).

[0396] Figure 38 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scramThis is an image taken with a confocal fluorescence microscope of tumor tissue isolated from the -NPs group, stained with APC-labeled CD8 antibody and DAPI.

[0397] Figure 39 shows the physiological saline group, anti-PD-L1 antibody group, PT-NPs group, and E3 after 10 days. scram This is an image taken with a confocal fluorescence microscope of tumor tissue isolated from the -NPs group, stained with PE-labeled CD25 antibody and DAPI.

[0398] As shown in Figures 38 and 39, the PT-NPs group showed an increase in CD8-positive cells and a decrease in CD25-positive cells in histological analysis compared to all other groups.

[0399] Synthesizing the results described above, it is confirmed that the PT-NPs group exhibits a remarkably superior tumor therapeutic effect compared to other groups. This is attributed to the effective induction of PD-L1 degradation within tumor tissue, which in turn leads to the recruitment of CTLs and the inhibition of Treg responses. Furthermore, it is confirmed that the PROTAC nanoparticles (PT-NPs) of Example 2 according to the present invention possess a therapeutic pathway different from that of other groups. To confirm this therapeutic pathway, genomic analysis was performed.

[0400]

[0401] 6) Genome analysis

[0402] Physiological saline group, anti-PD-L1 antibody group, PT-NPs group, E3 scram The -NPs group was prepared, and after 10 days, the tumor tissue was isolated and excised following anesthesia and sacrifice. Gene expression analysis was performed on the tumor tissue using RNA-Seq. Based on heatmap analysis, the expression levels of T cell receptors and genes related to apoptosis and survival in the tumor tissue were compared.

[0403]

[0404] Figure 40 shows the results of analyzing T cell receptor (CD274) and apoptosis and survival-related gene expression (BCL2, BRCA2, CASP3, VHL, Ube3a) in tumor tissues isolated from the anti-PD-L1 antibody group and PT-NPs group after 10 days, and Figure 41 shows the results of analyzing gene expression related to the apoptosis signaling pathway (Cd247, Nfatc1, Cd3g, Hras, ​​Nfkbia, Cd3e) in tumor tissues isolated from the physiological saline group, anti-PD-L1 antibody group, and PT-NPs group after 10 days.

[0405] As shown in Figures 40 and 41, compared to the anti-PD-L1 antibody group, the PT-NPs group showed an increase in proteins related to VHL and E3 enzymes (VHL and Ube3a) and apoptosis (CASP3, BCL2 and BRCA2), and a decrease in proteins related to PD-L1 downregulation (CD274).

[0406] The PT-NPs group showed lower levels of Cd247, Nfatc1, Cd3g, Hras, ​​Nfkbia, and Cd3e compared to the anti-PD-L1 antibody group, while Jak1 levels increased. Through this, it was confirmed that the PROTAC nanoparticles (PT-NPs) of Example 2 according to the present invention exhibit a potent anti-tumor immune response induced by a sustained PROTAC-mediated PD-L1 degradation mechanism. This is a simple PD-L1 lysosomal degradation mechanism (anti-PD-L1 antibody and E3 scram It can be seen that there is a difference in effect and efficacy compared to -NPs.

[0407]

[0408] Experimental Example 14. Analysis of the stability of PROTAC nanoparticles (PT-NPs) in an animal model

[0409] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6A cancer animal model was prepared by inoculating CT26 colorectal cancer cells via subcutaneous inoculation. The colorectal tumor volume was approximately 200 ± 20 mm². 3 They raised it until it reached.

[0410] Five animals from the above-mentioned cancer animal models were randomly selected for each group and divided into a total of three groups to conduct experiments as shown in Table 1. All experiments were conducted with the approval of the Animal Ethics Committee of Ewha Womans University and in compliance with its animal experiment regulations. Next, 10 days after treatment, the normal group, anti-PD-L1 antibody group, and PT-NPs group were anesthetized and sacrificed, and major organ and tumor tissues were isolated. The isolated tissues were analyzed using H&E staining. For blood biochemical testing, blood was collected on the day of autopsy, left at room temperature for 30 minutes to coagulate, and serum obtained by centrifugation (3,000 rpm, 15 min) was used for testing.

[0411] Statistical significance analysis was performed using the log-rank test. The p-values ​​of each statistical data point were indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0412] Group Experiment Method Experimental Animals Saline 10 mg / kg physiological saline intravenously administered every 2 days 5 Anti-PD-L1 Antibody 10 mg / kg anti-PD-L1 antibody intravenously administered every 2 days 5 PT-NPs 10 mg / kg PROTAC nanoparticles (PT-NPs) of Example 2 intravenously administered every 2 days 5

[0413] Figure 42 shows the results of testing lymphocytes, red blood cells, platelet count (PLT), AST, ALT, and BUN in blood samples collected from the normal group, anti-PD-L1 antibody group, and PT-NPs group after 10 days of treatment. Figure 43 shows the results of measuring changes in body weight during the treatment period in the normal group, anti-PD-L1 antibody group, and PT-NPs group. Figure 44 shows the results of H&E staining on major organ tissues isolated from the normal group, anti-PD-L1 antibody group, and PT-NPs group after 10 days of treatment. Figure 45 shows the results of analyzing survival rates during the treatment period in the normal group, anti-PD-L1 antibody group, and PT-NPs group.

[0414] As shown in Figure 42, the PT-NPs group was found to have normal hematological indicators, similar to the normal group. On the other hand, the anti-PD-L1 antibody group developed cytopenia, and AST, ALT, and BUN, which are indicators related to liver and kidney function, were significantly increased. In other words, while the PT-NPs group did not experience adverse effects such as hematological toxicity or organ dysfunction due to drug administration, the anti-PD-L1 antibody group was accompanied by extensive organ dysfunction due to strong hematological toxicity, indicating that immune-related adverse effects (irAEs) were induced by the anti-PD-L1 antibody.

[0415] As shown in Figures 43 and 44, no weight loss or major organ damage was observed in the normal group, the anti-PD-L1 antibody group, or the PT-NPs group.

[0416] As shown in Figure 45, 4 out of 5 animals in the anti-PD-L1 antibody group died due to severe toxicity on the 12th day of treatment, but all animals in the PT-NPs group survived for more than 20 days, confirming the improved therapeutic efficacy of the PT-NPs group.

[0417] It was confirmed that the PROTAC nanoparticles (PT-NPs) of Example 2 according to the present invention can exhibit excellent efficacy in precise and safe cancer immunotherapy by promoting specific and sustained PD-L1 degradation through direct PD-L1 lysosomal degradation and PROTAC-mediated PD-L1 degradation.

[0418] Experimental Example 15. Analysis of the stability of PROTAC nanoparticles (EGFR-NPs) in an animal model

[0419] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6 A cancer animal model was prepared by inoculating CT26 colorectal cancer cells via subcutaneous inoculation. The colorectal tumor volume was approximately 200 ± 20 mm². 3 They raised it until it reached.

[0420] Four animals from the above-mentioned cancer animal models were randomly selected for each group and divided into a total of four groups to conduct experiments as shown in Table 5. All experiments were conducted with the approval of the Animal Ethics Committee of Ewha Womans University and in compliance with its animal experiment regulations.

[0421] Next, treatment was carried out for a total of 16 days after drug administration, and on the last day of treatment, each group was sacrificed and major organ and tumor tissues were isolated. The isolated tissues were analyzed using H&E staining. For blood biochemical testing, blood was collected on the day of autopsy, left at room temperature for 30 minutes to coagulate, and serum obtained by centrifugation (3,000 rpm, 15 min) was tested.

[0422] Statistical significance analysis was performed using the log-rank test. The p-values ​​of each statistical data point were indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0423] Group Experiment Method Experimental Animals Physiological saline 10 mg / kg intravenous administration of physiological saline every 2 days 4EGFR-NPs 10 mg / kg intravenous administration of PROTAC nanoparticles (EGFR-NPs) of Example 4 every 2 days 4E3 scram -NPs 10 mg / kg every 2 days PROTAC nanoparticles of Comparative Example 4 (E3 scram -NPs) Intravenous administration of 4EGFR scram -NPs 10 mg / kg every 2 days PROTAC nanoparticles (EGFR) of Comparative Example 6 scram -NPs) Intravenous administration 4

[0424] Figure 46 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of measuring tumor size over time in the -NPs group. Tumor size was measured using a caliper, and Figure 47 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the appearance of tumor tissue isolated from the -NPs group. The volume of the tumor is given by the equation a×b 2 It was calculated based on ×0.5 (where a is the largest diameter and b is the smallest diameter). As a result, it was confirmed that tumor growth in the EGFR-NPs group was significantly inhibited compared to other groups.

[0425] Figure 48 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of Western blot analysis of the expression levels of EGFR and proteins related to its downstream pathways (phosphorylated EGFR, Akt, phosphorylated Akt, ERK, c-Myc, Cyclin D) in tumor tissues isolated from the -NPs group.

[0426] As shown in FIG. 48, E3 scram No significant changes in protein expression were observed in the -NPs group, and EGFR scramThe -NPs group showed a decrease in EGFR-related protein expression, but expression was still maintained. On the other hand, the EGFR-NPs group showed a complete decrease in the expression levels of proteins related to EGFR and its downstream pathways (phosphorylated EGFR, Akt, phosphorylated Akt, ERK, c-Myc, Cyclin D), and it was confirmed that they completely inhibited the phosphorylation of EGFR and Akt.

[0427] In other words, it can be seen that the PROTAC nanoparticles of the present invention exhibit an excellent anticancer effect that goes beyond the survival effect of cancer cells through a complex effect of inhibiting the growth of cancer cells and stopping the cell cycle to induce apoptosis by promoting protein degradation through E3 ubiquitin ligase by binding to target substances such as EGFR or PD-L1 in actual vivo, as well as blocking the recycling pathway of target substances which is a protective mechanism of cancer cells.

[0428] Physiological saline group, EGFR-NPs group, E3 scram -NPs group, EGFR scramTumor tissues isolated from the -NPs group were subjected to immunohistochemical staining using fluorescence and DAB (3,3'-diaminobenzidine) staining methods. Recombinant Anti-EGFR antibody (Abcam, GB) was used as the primary antibody against EGFR, and all staining procedures were performed at room temperature. After freezing, the tumor tissues were sectioned to a thickness of 10 µm, air-dried, and washed three times with PBS. For fluorescence staining, the tissues were treated with a protein blocking antibody (PBA) for 10 minutes to inhibit non-specific antibody binding. Next, the primary antibody was applied for 1 hour and washed three times with PBS for 10 minutes; subsequently, the fluorescently attached secondary antibody was applied for 1 hour and washed three times with PBS for 10 minutes. Nucleus staining was performed by treating with DAPI for 30 minutes. For DAB staining, a hydrogen peroxide blocker was applied and reacted for 10 minutes, followed by washing twice with PBS. To inhibit non-specific binding of the antibody, the sample was treated with a protein blocking antibody (PBA) for 10 minutes. Next, the sample was treated with the primary antibody for 1 hour, washed three times with PBS for 10 minutes, treated with Biotynlated Goat Anti-Polyvalent, and washed again three times with PBS for 10 minutes. Next, the sample was treated with Streptavidin peroxidase and reacted for 10 minutes, followed by washing three times with PBS for 10 minutes. Then, a mixed solution of 30 μL of DAB chromogen and 1.5 mL of DAB substrate was treated for 10 minutes and washed three times with PBS for 10 minutes to undergo the color development process, after which the sample was counterstained with Mayer's hematoxylin. The results were observed using a confocal laser microscope (CLSM) and an optical microscope and are summarized in Figures 49 and 50.

[0429] Figure 49 shows the physiological saline group, the EGFR-NPs group (also called PROTAC-NPs), and E3 scram-NPs group, EGFR scram - These are the results of immunohistochemical staining for EGFR in tumor tissue isolated from the -NPs group, and Figure 50 is a graph showing the quantitative results of Figure 49.

[0430] As shown in FIGS. 49 and 50, E3 scram -NPs group, EGFR scram It was observed that EGFR expression remained significantly maintained in the -NPs group. On the other hand, it was confirmed that the EGFR-NPs group blocked 90–99% of EGFR expression involved in the growth of cancer cells, and furthermore, EGFR expression was not observed throughout the tumor tissue, especially in the center of the tumor tissue.

[0431]

[0432] Physiological saline group, EGFR-NPs group, E3 scram -NPs group, EGFR scram TUNEL staining and DAPI staining were used to observe whether tumor tissue isolated from the -NPs group underwent apoptosis. TUNEL staining was performed by referring to Experimental Example 13. Specifically, the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore) was used according to the manufacturer's instructions, and observations were made using an optical microscope after the reaction was complete. For DAPI staining, the sample was stained in a DAPI (1 g / ml) solution for 30 minutes, and then observed at 372 nm using a fluorescence microscope (Zeiss, Germany).

[0433] Figure 51 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram As a result of analyzing apoptosis in tumor tissue isolated from the -NPs group, according to EGFR scram The -NPs group confirmed that apoptosis did not occur in the cancer tissue. E3 scramIn the cancer tissue of the -NPs group, only partial cell death was observed, and complete cell death was not observed.

[0434] In contrast, the EGFR-NPs group was found to exhibit significant apoptosis throughout the cancer tissue. It can be seen that the apoptosis induced by the PROTAC nanoparticles of the present invention is cell death resulting from physiological changes within the cell, and induces apoptosis caused by DNA fragmentation within the cell nucleus. Furthermore, the PROTAC nanoparticles of the present invention inhibit cell survival by suppressing the expression of Akt, Akt phosphorylation, ERK activity, Cyclin D1, and c-Myc, which are related to cell survival and growth; thus, they play the most significant role in hindering the growth of cancer cells and inducing apoptosis.

[0435]

[0436] Figures 52 and 53 show the physiological saline group, EGFR-NPs group, and E3 according to the treatment period. scram -NPs group, EGFR scram This is a graph showing the change in body weight and survival rate of the -NPs group; according to this, the normal saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram No significant differences in body weight change and survival rate were observed in any of the -NPs groups. In other words, it can be seen that the scaffolding of the PROTAC nanoparticles of the present invention exhibits excellent stability in vivo.

[0437]

[0438] Physiological saline group, EGFR-NPs group, E3 scram -NPs group, EGFR scram To analyze the stability of the -NPs group, blood biochemical tests and major organ tissue toxicity tests were performed. Blood tests were performed only on the physiological saline group and the EGFR-NPs group, and for the samples, serum obtained by collecting blood on the day of autopsy, leaving it at room temperature for 30 minutes to coagulate, and then centrifuging (3,000 rpm, 15 min) was used.

[0439] The blood biochemical test items are total protein (T.pro), albumin (Alb), creatinine (Crea), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), total bilirubin (T.bil), glucose (Glu), and total cholesterol (T.cho).

[0440] Major organ toxicity was physiological saline group, EGFR-NPs group, E3 scram -NPs group, EGFR scram After isolating the liver, lungs, spleen, kidneys, and heart from the NPs group, they were each fixed in a 4% paraformaldehyde solution, paraffin sections were prepared, and then analyzed by histopathological examination using H&E staining.

[0441] Figure 54 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram This is the result of performing blood biochemical tests on the plasma of the -NPs group, and Fig. 55 shows the physiological saline group, EGFR-NPs group, and E3 scram -NPs group, EGFR scram These are the results of histopathological examinations of the liver, lungs, spleen, kidneys, and heart of the -NPs group.

[0442] As shown in Figures 54 and 55, no significant difference was observed in the EGFR-NPs group in blood or major organs. In other words, it can be seen that the PROTAC nanoparticles of the present invention are very stable substances that do not cause toxicity in vivo, either visually or biochemically.

[0443]

[0444] Experimental Example 16. Analysis of in vitro anticancer activity

[0445] PROTAC nanoparticles (EGFR-NPs, E3) prepared from Example 4, Comparative Example 4, and Comparative Example 6 formed by self-assembly in physiological saline scram -NPs and EGFR scram-NPs) were prepared and cytotoxicity was analyzed. At this time, EGFR PROTAC was used as a control. 　 SJF 1528, a degrading agent, and Erlotinib, which is sold under the trade name Tarcepa, an anticancer drug, were used.

[0446] The cytotoxicity analysis of colorectal cancer cells CT26 was performed at 1 × 10⁶ per well of a 96-well cell culture plate. 6 Canine CT26 cells were seeded. The culture medium used was DMEM supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% penicillin-streptomycin. After stabilization for 24 hours in a humid environment of 5% CO2 and 95% air at 37°C, samples were added to each cell culture medium at various concentrations (0.00 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM), respectively, and then incubated in a 37°C incubator for 48 hours. After incubation was complete, culture medium containing 10% Cell Counting Kit-8 (CCK-8) solution was added to each well and incubated for 30 minutes, after which absorbance was measured at 450 nm using a microplate reader (VERSAmax™, Molecular Devices Corp., Sunnyvale, CA).

[0447]

[0448] FIG. 56 shows PROTAC nanoparticles (EGFR-NPs, E3) prepared from SJF 1528, eronitip, Example 4, Comparative Example 4, and Comparative Example 6 in CT26 cells. scram -NPs and EGFR scram This is the result of treating with -NPs and evaluating cell viability.

[0449] As shown in Figure 56, SJF 1528 and erlotinib, used as controls, showed significant anticancer effects. In addition, the PROTAC nanoparticles (EGFR-NPs, E3scram-NPs) prepared from Example 4 and Comparative Example 4 were also confirmed to exhibit anticancer effects starting from a concentration of 10 μM, similar to the control group. On the other hand, the PROTAC nanoparticles (E3scram-NPs) prepared in Comparative Example 6 did not induce significant cytotoxicity even at high concentrations of 100 μM or higher.

[0450]

[0451] Experimental Example 17. Analysis of therapeutic efficacy against resistant lung cancer cells

[0452] This experiment evaluates whether the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 of the present invention have in vitro anticancer efficacy in an NCI-H1975 cell transplantation model of non-small cell lung cancer (NSCLC) that is resistant to epidermal growth factor receptor (EGFR) targeted therapy.

[0453] For the experiment, the NCI-H1975 (ATCC, CRL-5908) cell line derived from a non-small cell lung cancer (NSCLC) patient was obtained. The NCI-H1975 cell line is a non-small cell lung cancer (NSCLC) cell line possessing double mutations in the epidermal growth factor receptor (EGFR) L858R and T790M, and the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) (Tarceva 　 , Iressa 　 It has strong resistance to ). To evaluate the in vitro anticancer efficacy in the above cells, NCI-H1975 cells were cultured in RPMI 1640 medium (HyClone, Logan, UT) containing 10% FBS (fetal bovine serum).

[0454] Prepare the NCI-H1975 cell line, and place 5 × 10⁶ cells per well in a 96-well plate. 3Cells were aliquoted according to the number of cells and incubated at 37°C for 24 hours to allow attachment. Then, PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 were applied to each well at various concentrations (0–1800 nM). After drug treatment, the cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. After washing 2–3 times with PBS, the cells were lysed using lysis buffer, and 50 mg of protein was separated using SDS-PAGE. The separated protein was transferred to a PVDF membrane, incubated in blocking buffer at room temperature for 1 hour, washed with TBST, diluted with the primary antibody, and incubated with the membrane at room temperature for 1–2 hours or at 4°C for more than 12 hours, washed with TBST, diluted with the HRP-conjugated secondary antibody, and incubated with the membrane at room temperature for 1–2 hours, washed again with TBST, and confirmed using an ECL kit.

[0455]

[0456] Figure 57 shows the results of analyzing EGFR and p-EGFR expression levels by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 58 is a graph quantified by image J analysis from the results of Figure 57.

[0457] As shown in Figures 57 and 58, it was confirmed that the expression of EGFR and p-EGFR in H1975 resistant lung cancer cells was significantly inhibited by treatment with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4. Specifically, it was found that the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 successfully induce the degradation of EGFR protein in resistant cancer cells and target EGFR through the E3 ligase-mediated ubiquitin-proteasome pathway. In addition, it was found that the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 inhibit tyrosine phosphorylation of EGFR, indicating a high likelihood of inhibiting EGFR-mediated signaling pathways (PI3K / AKT, MAPK / ERK, JAK / STAT, etc.).

[0458] Therefore, it can be seen that the PROTAC nanoparticles (EGFR-NPs) according to the present invention can effectively degrade EGFR and block signal transduction in resistant cancer cells (H1975) having the L858R / T790M double mutation. That is, since the PROTAC nanoparticles (EGFR-NPs) according to the present invention have high inhibitory ability against not only wild-type cancer but also mutations, they can be usefully used in the treatment of EGFR inhibitor-resistant cancer in which EGFR mutations are expressed.

[0459]

[0460] Figure 59 shows the results of analyzing the expression levels of AKT, pAKT, ERK, PDL1, c-Myc, and cyclin D by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 60 is a graph quantified by image J analysis from the results of Figure 59.

[0461] As shown in Figures 59 and 60, it was confirmed that the expression of AKT, pAKT, ERK, PDL1, c-Myc, and cyclin D in H1975 resistant lung cancer cells was significantly inhibited by treatment with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4.

[0462] Specifically, it can be seen that the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 successfully degrade EGFR in resistant cancer cells and also successfully block its downstream signaling pathways (RAS / RAF / MEK / ERK and PI3K / AKT pathways). Furthermore, it can be seen that EGFR-NPs can inhibit not only tumor growth but also immune evasion. Moreover, since it can be seen that EGFR-NPs inhibit cancer cell growth and block the cell cycle, it can be seen that they will exhibit excellent anticancer effects in myc-related cancers as well.

[0463] It can be seen that the EGFR-NPs according to the present invention have various anticancer mechanisms, including not only EGFR degradation but also signal transduction blockade, inhibition of immune evasion, and inhibition of cell growth.

[0464]

[0465] Experimental Example 18. Comparison of therapeutic efficacy with conventional therapeutic agents in cancer cells-1

[0466] This experiment aimed to compare and evaluate the therapeutic efficacy of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 of the present invention and erlotinib, a conventional EGFR TKI (Tyrosine kinase inhibitor), in cancer cells. An equal amount of physiological saline was used as a control.

[0467] For the experiment, NCI-H1975 non-small cell lung cancer (NSCLC) cells resistant to epidermal growth factor receptor (EGFR) targeted therapies and wild-type colorectal cancer cells CT26 were prepared to evaluate their in vitro anticancer efficacy. Each cell type was cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% penicillin-streptomycin. 5 × 10⁶ cells were cultured per well in a 96-well plate. 3 Cells were aliquoted according to the number of cells and incubated at 37°C for 24 hours to induce attachment. Then, PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 or erlotinib were added to each well at a concentration of 800 nM. After drug treatment, the cells were incubated at 37°C in a 5% CO2 incubator for 72 hours. After washing 2-3 times with PBS, the cells were lysed using lysis buffer, and protein quantification was performed to isolate 50 mg using SDS-PAGE. The isolated protein was transferred to a PVDF membrane, incubated in blocking buffer at room temperature for 1 hour, washed with TBST, diluted with the primary antibody, and incubated with the membrane at room temperature for 1-2 hours or at 4°C for more than 12 hours, washed with TBST, diluted with the HRP-conjugated secondary antibody, and incubated with the membrane at room temperature for 1-2 hours, washed again with TBST, and confirmed using an ECL kit.

[0468]

[0469] Figure 61 shows the results of analyzing EGFR and p-EGFR expression levels by Western blot after treating CT25 cells and NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and erlotinib, respectively, and Figure 62 is a graph quantified from the results of Figure 61 through Image J analysis (in Figure 62, 'NanoTACs' refers to the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4).

[0470] As shown in Figures 61 and 62, erlotinib, a first-generation TKI, does not directly degrade EGFR at CT26, but inhibits EGFR ATP binding, thereby lowering the expression of pEGFR. In H1975 cells, EGFR expression was lowered by erlotinib treatment, but no significant difference in pEGFR expression was observed compared to the control group.

[0471] It was confirmed that the EGFR-NPs of Example 4 according to the present invention directly degrade EGFR, so the reduction of EGFR and pEGFR occurs simultaneously, and the reduction of EGFR and pEGFR is successfully performed even in resistant cell H1975.

[0472]

[0473] Experimental Example 19. Comparison of therapeutic efficacy with conventional therapeutic agents in cancer cells-2

[0474] This experiment aimed to compare and evaluate the therapeutic efficacy of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 of the present invention and the monoclonal antibody cetuximab in cancer cells.

[0475] For the experiment, NCI-H1975 cells with non-small cell lung cancer (NSCLC) resistant to epidermal growth factor receptor (EGFR) targeted therapies were prepared to evaluate their in vitro anticancer efficacy. Each cell was cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% penicillin-streptomycin. 5 × 10⁶ cells were cultured per well in a 96-well plate. 3 Cells were seeded in equal numbers and incubated at 37°C for 24 hours to allow attachment, after which PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 or cetuximab were treated to 800 nM in each well. After drug treatment, the cells were incubated at 37°C in a 5% CO2 incubator for various durations (0, 3, 6, 9, 24, 48 hours). After washing 2-3 times with PBS, the cells were lysed using lysis buffer, and protein was quantified to separate 50 mg using SDS-PAGE. After transferring the isolated proteins to a PVDF membrane, they were incubated in blocking buffer at room temperature for 1 hour and washed with TBST. Then, the primary antibody was diluted and incubated with the membrane at room temperature for 1-2 hours or at 4°C for more than 12 hours and washed with TBST. After that, the HRP-conjugated secondary antibody was diluted and incubated with the membrane at room temperature for 1-2 hours and washed again with TBST, and then verified with an ECL kit.

[0476]

[0477] Figure 63 shows the results of analyzing HER2 expression levels by Western blot after treating NCI-H1975 cells with PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and cetuximab, respectively, and Figure 64 is a graph quantified by image J analysis from the results of Figure 63. HER2 (Human Epidermal Growth Factor Receptor 2) is a Receptor Tyrosine Kinase (RTK) belonging to the EGFR family and plays an important role in cell growth, proliferation, and survival.

[0478] As shown in Figures 61 and 62, cetuximab failed to inhibit HER2 expression in NCI-H1975 cells; rather, HER2 expression increased over time. This is attributed to the fact that cetuximab blocks EGFR signaling but does not degrade the EGFR protein itself. In other words, cetuximab may activate a bypass pathway via HER2 due to EGFR inhibition, which may limit its effectiveness in resistant cancer cells.

[0479] On the other hand, it was confirmed that the EGFR-NPs of Example 4 according to the present invention did not increase HER2 expression. This is believed to be because the EGFR-NPs block interaction with HER2 by directly degrading and completely removing the EGFR protein, and as a result, no increase in HER2 expression occurs. Therefore, unlike existing therapeutic agents, the EGFR-NPs of Example 4 effectively suppress HER2-dependent resistance, suggesting that they can provide a more potent therapeutic effect against wild-type and mutant cancer cells.

[0480]

[0481] Experimental Example 20. Size and stability of EGFR-NPs in Example 4

[0482] PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, conjugated with a fluorescent dye (Cy5.5), were prepared. At this time, the peptide labeled with Cy5.5 was prepared to be included in an amount of 5% by weight based on the total weight of the nanoparticles.

[0483] The particle size of the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 was measured by Dynamic Light Scattering, and the change in particle size was measured daily for 7 days using Dynamic Light Scattering after immersion in mouse serum.

[0484]

[0485] Figure 65 shows the size analysis of the fluorescently labeled PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, and Figure 66 shows the results of analyzing whether there was a change in particle size after adding the fluorescently labeled PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 to mouse serum for 7 days.

[0486] As shown in Fig. 65, it was confirmed that the fluorescently labeled PROTAC nanoparticles (EGFR-NPs) of Example 4 had an average particle diameter in the range of 100 to 160 nm.

[0487] In addition, as shown in Fig. 66, no significant change in particle size was observed for the fluorescently labeled EGFR-NPs in mouse serum. This indicates that the nanoparticles are structurally very stable in vivo.

[0488]

[0489] Experimental Example 21. Analysis of the intracellular entry pathway of EGFR-NPs from Example 4

[0490] 1 × 10⁶ PD-L1 overexpressing CT26 colorectal cancer cells in a confocal microscope cell culture plate 6After dispensing the cells and stabilizing them for 24 hours, each cell was treated with 100 μM of PROTAC nanoparticles (EGFR-NPs) prepared from Example 4, bound to a fluorescent dye (FITC or Cy5.5), and post-incubated at 4 °C for 10 minutes. At this time, the peptides labeled with Cy5.5 and FITC were prepared to contain 5 wt% each based on the total weight of the nanoparticles. The cells were washed twice with PBS (to remove unbound nanoparticles) and incubated at 37 °C for various durations (0.5, 1, 3, 6, 9 h), followed by treatment with 4% paraformaldehyde for 10 minutes, and then treatment with DAPI solution for 15 minutes. Cell fluorescence images were analyzed using a confocal fluorescence microscope (Leica TCS SP8 confocal laser-scanning microscope (Leica Microsystems GmbH; Wetzlar, Germany)).

[0491]

[0492] Figure 67 shows the results of observing, using a fluorescence microscope, whether the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 were successfully introduced into lysosomes when CT26 cells were treated. Figure 68 shows the results of analyzing the ratio of the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 and the lysosomes present in the co-locations from Figure 67.

[0493] As shown in Figures 67 and 68, it was confirmed that the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 enter the lysosome pathway during the process of penetrating from the cell surface into the cytoplasm. Specifically, it was confirmed that the fluorescence signals of the PROTAC nanoparticles (EGFR-NPs) (FITC, Cy5.5) prepared from Example 4 all coexist strongly around the cell nucleus. In other words, it can be seen that the PROTAC nanoparticles (EGFR-NPs) prepared from Example 4 decompose within the lysosome, move to the cytoplasm (Cytosol), are degraded into PROTAC peptides, and operate strongly through the ubiquitin-proteasome system.

[0494]

[0495] Experimental Example 22. Verification of the intracellular targeting of EGFR-NPs of Example 4

[0496] 1 × 10 on the flank of 5-week-old BALB / c male nude mice 6 A cancer animal model was prepared as in Experimental Example 11 by inoculating CT26 cancer cells via subcutaneous inoculation. The tumor volume was approximately 200 ± 20 mm². 3 After rearing until reaching [the target], EGFR-NPs labeled with Cy5.5 (Example 4) were administered intravenously at a dose of 10 mg / kg, and tumor tissue was excised 9 hours later to evaluate tumor targeting efficiency. All experiments were conducted with the approval of the Animal Ethics Committee of Ewha Womans University and in compliance with its animal experiment regulations. During treatment, drug accumulation in the tumor tissue was analyzed using non-invasive near-infrared fluorescence (NIRF) imaging with an IVIS Lumina Series III system (PerkinElmer; Waltham, MA, USA). The isolated tumor tissue was lysed in [the target] and then analyzed using an LC-MS system mass spectrometer with a SCIEX TripleTOF 5600+ system.

[0497]

[0498] Figure 69 shows the results of LC-MS analysis on tumor tissue excised 9 hours after administration in an animal model treated with Cy5.5-labeled EGFR-NPs. According to this, both EGFR-NPs and Cy5.5-EGFR-NPs were detected within the tumor tissue. In other words, it can be confirmed that the EGFR-NPs according to the present invention possess excellent targeting ability against cancer cells, and that there is no change in the behavior of EGFR-NPs even when a fluorescent dye is bound. The fluorescent dye is suitable for tracking the behavior of EGFR-NPs.

Claims

1. a) Cancer targeting peptide; b) a linker composed of two phenylalanines; and c) an E3 enzyme ligand represented by SEQ ID NO. 26; a cancer-targeting PROTAC peptide.

2. In Paragraph 1, The above cancer-targeting PROTAC peptide is characterized by being a peptide that binds to a target specifically expressed in cancer tissue.

3. In Paragraph 2, The targets specifically expressed in the above-mentioned cancer tissue are PD-1 (Programmed Cell Death Protein 1) protein, PD-L1 (Programmed Death-Ligand 1) protein, CP2c (Cytochrome P450 2C) protein, BRD4 (Bromodomain Containing 4) protein, c-Myc (transcriptional regulator Myc-like) protein, EGFR (Epidermal Growth Factor Receptor) protein, ALK (Anaplastic Lymphoma Kinase) protein, ROS1 (ROS Proto-Oncogene 1) protein, HER2 (Human Epidermal Growth Factor Receptor 2) protein, TROP2 (Trophoblast Cell Surface Antigen 2) protein, CTLA-4 (Cytotoxic T-Lymphocyte Associated Protein 4) protein, VEGFR (Vascular Endothelial Growth Factor Receptor) protein, BRAF (B-Raf Proto-Oncogene) protein, and KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) protein, PI3K (Phosphoinositide 3-Kinase) protein, CDK4 / 6 (Cyclin Dependent Kinase 4 / 6) protein, MET (Mesenchymal Epithelial Transition Factor) protein, mTOR (Mechanistic Target of Rapamycin) protein, PARP (Poly ADP Ribose Polymerase) protein, PARP10 (Poly ADP Ribose Polymerase 10) protein, NTRK1 (Neurotrophic Receptor Tyrosine Kinase 1) protein, CD44 protein, CD49 protein, LAG (lymphocyte activation gene-3) protein,A cancer-targeting PROTAC peptide characterized by being selected from the group consisting of IAP (inhibitor of apoptosis protein) protein, DDX5(p68) protein, and CD19 protein.

4. In Paragraph 1, A cancer-targeting PROTAC peptide characterized by the above-mentioned cancer-targeting peptide being represented by any one selected from SEQ ID NOs 1 to 25.

5. In Paragraph 1, The above cancer-targeting PROTAC peptide is characterized by being represented by SEQ ID NO. 1 or 2.

6. In Paragraph 1, Cancer-targeting PROTAC peptide characterized by the above-mentioned PROTAC peptide being represented by SEQ ID NO. 27 or 28.

7. Cancer-targeting PROTAC nanoparticles formed by self-assembly of multiple PROTAC peptides according to claim 1.

8. In Paragraph 7, Cancer-targeting PROTAC nanoparticles characterized by the above self-assembly being due to π-π interactions by a linker of a PROTAC peptide.

9. In Paragraph 7, Cancer-targeting PROTAC nanoparticles characterized by having an average diameter of 100 to 300 nm.

10. A pharmaceutical composition for the prevention or treatment of cancer comprising cancer-targeting PROTAC nanoparticles according to claim 7.

11. In Paragraph 10, The above cancers include thyroid cancer, oral cancer, oropharyngeal cancer, ovarian cancer, male genital tumors, male breast cancer, brainstem tumors, cerebral lymphoma, brain tumors, pituitary meningioma, gallbladder cancer, biliary tract cancer, colorectal cancer, intracranial tumors, head and neck tumors, bladder cancer, leukemia, abdominal tumors, nasal cavity cancer, non-small cell lung cancer, nasopharyngeal cancer, esophageal cancer, breast cancer, stomach cancer, liver cancer, pancreatic cancer, ependymoma, adenoma, small cell lung cancer, small intestine cancer, kidney cancer, malignant astrocytoma, benign astrocytoma, female genital tumors, female external genital cancer, female urethral cancer, penile cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, mediastinal tumors, vaginal cancer, salivary gland cancer, skin cancer, malignant lymphoma, multiple myeloma, aplastic anemia, oligodendroglioma, hypopharyngeal cancer, anal cancer, hematological cancer, laryngeal cancer. A pharmaceutical composition for the prevention or treatment of cancer, characterized by being one or more selected from the group consisting of thoracic tumors, thymic cancers, and resistant cancers.

12. A pharmaceutical composition for cancer immunotherapy comprising cancer-targeting PROTAC nanoparticles according to claim 7 as an active ingredient.

13. A pharmaceutical composition for combination therapy for cancer treatment comprising cancer-targeted PROTAC nanoparticles and an anticancer agent according to claim 7.

14. In Paragraph 13, A pharmaceutical composition for a combination therapy for cancer treatment, characterized in that the above anticancer agent is one or more selected from the group consisting of 7-ethyl-10-hydroxycamptothecin, 5-fluorouracil, cisplatin, paclitaxel, doxorubicin, donorubicin, vinblastine, vincristine, actinomycin D, teniposide, etoposide, cyclophosphamide, epirubicin, adriamycin, daunomycin, and mitomycin-C.

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