Polypeptides for delivering antigens to immune cells

Abstract

The present invention relates to a polypeptide for delivering an antigen to an immune cell, and more specifically, to a novel polypeptide comprising a cell membrane-permeable peptide and a peptide that binds to a surface molecule of an immune cell, a fusion polypeptide in which an antigen is bound to the polypeptide, a nucleic acid encoding the polypeptide or the fusion polypeptide, an immune cell sensitized with the fusion polypeptide or a nucleic acid encoding the same, an immunotherapeutic agent comprising the immune cell, an anti-tumor or anti-cancer vaccine, and a composition for treating a tumor or cancer.

Description

[Technical field]

[0001] The present invention relates to a polypeptide for delivering an antigen to an immune cell, and more specifically, to a novel polypeptide comprising a cell membrane-permeable peptide and a peptide that binds to a surface molecule of an immune cell, a fusion polypeptide in which an antigen is bound to the polypeptide, a nucleic acid encoding the polypeptide or the fusion polypeptide, an immune cell sensitized with the fusion polypeptide or a nucleic acid encoding the same, an immunotherapeutic agent comprising the immune cell, an anti-tumor or anti-cancer vaccine, and a composition for treating a tumor or cancer.

[0002] [Background technology]

[0003] The signal sequence (membrane transduction sequence: MTS) or cytoplasmic penetration peptide (CPP) present in FGF (Fibroblast Growth Factor) is known to have the property of penetrating the cell membrane. The signal sequence has the property of easily penetrating the cell membrane, but has the characteristic of not entering the nucleus because it does not have an NLS (nuclear localization sequence).

[0004] MTS or CPP can be used as an antigen delivery vehicle. Peptide antigens that enter the cytoplasm can be processed through the proteasome, placed on MHC class I, and presented on the cell surface. MTS or CPP can also be used to deliver therapeutic substances for delivering proteins with specific functions that have been developed to target many signal transduction processes that regulate cell death and cell growth that occur in the cytoplasm.

[0005] In this regard, the inventors of the present application have developed a cytoplasmic transduction peptide (CTP) that has the ability to permeate cell membranes but remains in the cytoplasm. Since CTP has the novel property of remaining in the cytoplasm while exhibiting excellent cell membrane permeability, it has been confirmed that it is suitable for inducing cytotoxic T lymphocyte (CTL)-response and can be used as an effective drug delivery system (DDS) into the cytoplasm (Korean Patent Registration No. 0608558).

[0006] Meanwhile, Staphylococcus aureus is highly resistant to the natural environment and is widely distributed in nature, and is not only present in suppurative lesions of humans and animals, but also constantly present on the skin of healthy humans and animals, making it highly likely to contaminate food and the human body. The pathogenic factors produced by Staphylococcus aureus include exotoxin, enterotoxin, hemolysin toxin, leucodicin, epidermolytic toxin, as well as extracellular products such as toxic shock syndrome toxin (TSST), which induce various serious diseases such as food poisoning in humans.

[0007] Staphylococcal enterotoxin type B (hereinafter referred to as "SEB") is a toxin produced by the food poisoning bacterium Staphylococcus aureus, which is distributed worldwide and mainly causes food poisoning, induces abnormal immune responses, and causes serious immune disorders.

[0008] SEB is a protein with a size of approximately 26 to 29 kDa and a small molecular weight. However, SEB is highly soluble in water, highly resistant to protease enzymes such as pepsin, trypsin, and papain, and is a very stable toxin that can withstand extreme temperatures and pH levels.

[0009] In terms of the function of SEB, it is named a superantigen (SAg) and is an antigen that acts as a link between the T-cell receptor (TCR) present on activated CD4+ or CD8+ T cells and the major histocompatibility complex class II (MHC class II) present on antigen presenting cells (APCs). SEB binds to the MHC class II of macrophages and then binds to the Vb8 site of the T cell receptor, abnormally activating T cells and inducing abnormal immune responses by secreting large amounts of inflammatory cytokines such as IL-6, IL-1β, and MIP-1 (Macrophage Inflammatory Protein-1). When the respiratory system is exposed to SEB, few people die from SEB, but more than 80% of exposed people show clinical symptoms, and it is known that they show severe symptoms that make it impossible to live daily life for one to two weeks.

[0010] Under such technical background, the inventors of the present application conducted structural studies of cell membrane-permeable peptides and SEB, and selected peptides that bind to MHC class II. They confirmed that novel polypeptides including cell membrane-permeable peptides and peptides that bind to surface molecules of immune cells can improve the yield of antigen delivery to immune cells, thereby completing the present invention.

[0011]

[0012] Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a novel polypeptide for delivering an antigen to an immune cell.

[0014] It is an object of the present invention to provide a nucleic acid encoding said polypeptide.

[0015] An object of the present invention is to provide a fusion polypeptide in which an antigen is bound to the above polypeptide.

[0016] It is an object of the present invention to provide a nucleic acid encoding said fusion polypeptide.

[0017] It is an object of the present invention to provide an immune cell comprising said fusion polypeptide or said nucleic acid.

[0018] An object of the present invention is to provide an immunotherapeutic agent comprising the above-mentioned immune cells.

[0019] An object of the present invention is to provide an anti-tumor or anti-cancer vaccine comprising the immune cells.

[0020] An object of the present invention is to provide a composition for treating tumors or cancer, comprising the immune cells.An object of the present invention is to provide a method for treating tumors or cancer, comprising administering the immune cells to an individual.An object of the present invention is to provide a use of the immune cells in the manufacture of a composition for treating tumors or cancer. [Means for solving the problem]

[0021] To achieve the above object, the present invention provides polypeptides for delivering antigens to immune cells, including cytoplasmic transduction peptides (CTPs) and peptides that bind to surface molecules of immune cells.

[0022] The present invention provides a nucleic acid encoding the polypeptide.

[0023] The present invention provides a fusion polypeptide in which an antigen is bound to the polypeptide.

[0024] The present invention provides a nucleic acid encoding the fusion polypeptide.

[0025] The invention provides an immune cell comprising said fusion polypeptide or a nucleic acid encoding said fusion polypeptide.

[0026] The present invention provides an immunotherapeutic agent comprising the immune cells.

[0027] The present invention provides an anti-tumor or anti-cancer vaccine comprising the immune cells.

[0028] The present invention provides a composition for treating tumors or cancers comprising the immune cells.The present invention provides a method for treating tumors or cancers comprising administering the immune cells to an individual.The present invention provides a use of the immune cells for producing a composition for treating tumors or cancers.

[0029] [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows the results of comparing MoDC cell membrane permeability between CTP / CTP-SEB groups.

[0031] [Diagram 2] FIG. 1 shows the difference in MoDC permeability between CTP / CTP-SEB groups.

[0032] [Diagram 3] FIG. 1 shows a vector for the production of recombinant fusion proteins.

[0033] [Figure 4]FIG. 1 shows the results of purification using Ni-NTA resin and QC analysis in the production of recombinant fusion protein.

[0034] [Diagram 5] FIG. 1 shows the results of measuring the cell permeability of fusion proteins.

[0035] [Figure 6] FIG. 1 shows the results of direct binding affinity analysis of fusion proteins.

[0036] [Figure 7] FIG. 1 shows the results of an analysis of the amount of protein remaining in a culture medium.

[0037] [Figure 8] FIG. 1 shows the results of a comparative experiment on DC phenotype and cell membrane permeability of PTD series peptides.

[0038] [Figure 9] FIG. 1 shows the results of antigen-specific T cell proliferation analysis.

[0039] [Figure 10] FIG. 1 shows the results of confirming the antitumor effect in mice inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigen.

[0040] [Figure 11] FIG. 1 shows the results of permeability analysis according to immune cell type.

[0041]

[0042] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is one that is well known and commonly used in the art.

[0044] In one aspect, the present invention relates to polypeptides for delivering antigens to immune cells, including cytoplasmic transduction peptides (CTPs) and peptides that bind to surface molecules of immune cells.

[0045] The cell membrane permeable peptide may be one or more selected from the group consisting of peptides including a protein transport domain, for example, CTP (cytoplasmic transduction peptide), HP4, Hph-1, Mph-1, Sim-2, Tat, VP22, Antp (Antennapedia), Pep-1 (peptide), PTD-5, R9 (arginine) and 7R domain, but is not limited thereto.

[0046] Specifically, the cell membrane-permeable peptide may be a cytoplasm-retentive cell membrane-permeable peptide that has cell membrane permeability, and exhibits a cell membrane permeation phenomenon even when the treated cells are treated with a protease after a certain time has elapsed since being treated with the cell, and has the property of remaining in the cytoplasm after permeating the cell membrane. It was confirmed that when the cell membrane-permeable peptide is fused with a peptide that binds to a surface molecule of an immune cell, and inhibits endocytosis and ubiquitin at the same time, the polypeptide remains in the cell without being degraded.

[0047] The cell membrane-permeable peptide can have a length of, for example, 9 aa to 20 aa, more preferably 9 aa to 15 aa, and most preferably about 11 aa.

[0048] The cell membrane-penetrating peptide may, for example, comprise a sequence selected from the group consisting of SEQ ID NOs: 5 to 7. The inventors of the present application have confirmed that when a cell membrane-penetrating peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 5 to 7 is fused with a peptide that binds to a surface molecule of an immune cell, it exhibits superior cell membrane permeability compared to the cell membrane-penetrating peptide alone.

[0049] JPEG2024546516000002.jpg35169

[0050] The peptide that binds to the surface molecule of the immune cell may be, for example, a peptide that binds to a major histocompatibility complex (MHC) molecule. Specifically, the peptide that binds to the surface molecule of the immune cell may include a peptide that binds to major histocompatibility complex (MHC) class II expressed as a surface molecule of the immune cell.

[0051] In one embodiment, the peptide that binds to a surface molecule of an immune cell can comprise a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0052] JPEG2024546516000003.jpg43170

[0053] The antigen may be a cancer or tumor antigen for activating an immune response against a cancer or tumor. The antigen may be a cancer or tumor antigen, and may induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy. The cancer or tumor antigen may be, for example, an antigen derived from self or non-self. For example, autologous cancer or tumor antigens derived from a patient's own genes may include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1), etc., cancer specific mutant antigens may include tumor suppressor or inducer genes, such as neoantigens, mutated P53, RAS, etc., and foreign cancer or tumor antigens may include cancer or tumor-inducing viral antigens, such as CMV, EBV, HPV, etc.

[0054] hTERT, a self-cancer or tumor antigen, is an enzyme that synthesizes telomeric DNA at the end of chromosomes. Cancer cells overactivate this enzyme to avoid telomere-dependent cell death, and is known as a target antigen for various solid cancers including lung cancer, gastric cancer, and pancreatic cancer (Kim NW, et al. Science. 1994; 266: 2011-2015). WT1, a gene associated with Wilms tumor, encodes a zinc finger transcription factor and is a protein involved in cell proliferation and differentiation, apoptosis, and organ development, and is known as a target antigen for brain and spinal cancer, lung cancer, etc. (Call KM, et al., Cell. 1990. 60: 509-520; Nakahara Y, et al., Brain Tumor Pathol. 2004. 21: 113-6). NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen) and is well known to be expressed mainly in various cancer cells including germ cells, sarcoma and breast cancer (Gnjatic S, et al., Adv Cancer Res. 2006; 95: 1-30). MAGE-A3 is a protein belonging to the melanoma-associated antigen family and is known to be overexpressed in various cancer cells including lung cancer, sarcoma and melanoma, and is evaluated as a target antigen suitable for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun; 23 (6): 1387-93).

[0055] In some cases, the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, carbonic anhydrase I, X (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), epithelial cell adhesion molecule (EPCAM), elongation factor 2 Mutated (ELF2M), ephrin type-A receptor 2 (EphA2), EMR2, Fms-like tyrosine kinase 3 (FLT3), FCRL5, fibulin-1, G250, GD2, glycoprotein 36 (gp36), glycoprotein 100 (gp100), glucocorticoid-induced tumor death factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (GPR20 ), GPC3, hsp70-2, high molecular weight melanoma-associated antigen (HMWMAA), hepatitis A virus cellular receptor 1 (HAVCR1), HAGE, HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / Galectin 9, HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HUB1, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, neural cell adhesion molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, breast differentiation antigen (NY-BR-1), NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19 / LKB / STK1 1, NY-REN-21, NY-REN-26 / BCR, NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglioside (OAcGD2), OGFr, PSMA, prostatic acid phosphatase (PAP), p53, prostate-cancer tumor antigen-1 (PCTA-1), prostate stem cell antigen (PSCA), serine protease 21 (testisin or PRSS21), platelet-derived growth factor receptor beta (P DGFR-beta), PLAC1, Pannexin 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, Stage-specific Embryonic Antigen-4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, Thyroglobulin, 5T4, Tumor-associated glycoprotein 72 (TAG72), Tyrosinase, Transglutaminase 5 (TGS5), TEM1, TEM7R, Thyroid-stimulating hormone receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, Uroplakin 2 (UPK2), Vimentin, or Vascular Endothelial Growth Factor Receptor 2 (VEGFR2).

[0056] The antigen may be a pathogen (bacteria, virus, etc.), a compound, pollen, cancer cell, shrimp, etc., or a peptide or protein thereof, more preferably a cancer antigen peptide, but is not limited thereto as long as it is a substance that can cause an immune response in the body. The antigen may be preferably a protein, a recombinant protein, a glycoprotein, a gene, a peptide, a polysaccharide, a lipid polysaccharide, a polynucleotide, a cell, a cell lysate, bacteria, a virus, etc., more preferably a cancer antigen peptide. The protein may be an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, an enzyme, an enzyme fragment, a transport protein, a receptor, a receptor fragment, a biodefense inducer, a storage protein, a movement protein, an exploitative protein, a reporter protein, etc. However, the antigen may be a substance that can act as an antigen in the body and induce an immune response, but is not limited thereto as long as it is a substance that can act as an antigen in the body and induce an immune response.

[0057] The antigen according to the present invention is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-associated genes produced by gene recombination, peptides, or proteins. When the antigen is obtained by gene recombination, the nucleotide sequence encoding the antigen may be a known one, and the entire length of the known sequence may be used, or a part of the entire length may be used. The nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.

[0058] The antigen can be bound to the N-terminus or C-terminus of the polypeptide. Covalent binding can be performed by methods known in the art depending on the type of antigen, for example, by cloning a gene encoding the polypeptide and expressing it in cells.

[0059] In some cases, linkers that do not interfere with the activity of the polypeptide can be used, such as, but not limited to, N-succinimidyl iodoacetate, N-maleimidobutyryloxysuccinamide ester, 1,5-difluoro-2,4-dinitrobenzene, bisdiazobenzidine, 3,3-dithio-bis-(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidyl succinate), dicyclohexylcabodiimide, etc. On the other hand, if the antigen only exhibits activity when degraded from the polypeptide, a linker that can be cleaved in vivo can be used. For example, a linker with a carboxylate ester and / or disulfide bond can be used.

[0060] In one embodiment, the immune cells may be antigen-presenting cells (APCs) that present the antigen delivered by the polypeptide of the present invention on their surface. For example, the immune cells may be, but are not limited to, dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells.

[0061] The peptide that binds to the surface molecule of the immune cell can be fused to one end of the cell membrane-permeable peptide, for example. The peptide that binds to the surface molecule of the immune cell can be bound to the N-terminus or C-terminus of the cell membrane-permeable peptide.

[0062] In some cases, the peptide that binds to the surface molecule of the immune cell may be linked to the cell membrane-penetrating peptide via a linker. The linker may be a peptide linker and may have a length of about 5 aa to 25 aa, specifically about 5 aa to 10 aa. For example, the linker may include hydrophilic amino acids such as glycine and / or serine, but is not limited thereto.

[0063] Specifically, the linker may be, for example, a glycine linker (G, Gly)p (where p is 1 to 10) or a GS linker (G n S) m (n and m each may be 1 to 10). Specifically, the linker may include GGGGS or (GGGGS)2, or may include 5aa to 10aa glycines in (G,Gly)p, where p is 5 to 10.

[0064] In another aspect, the present invention relates to a nucleic acid encoding the above polypeptide.

[0065] The term "nucleic acid" refers to a nucleic acid that includes DNA (gDNA and cDNA) and RNA molecules, and the basic building blocks of nucleic acids, nucleotides, include not only natural nucleotides but also analogues in which the sugar or base moiety is modified. The sequences of the nucleic acids according to the present invention can be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0066] The nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or further expression. Based on this, the present invention relates in a further aspect to a vector comprising the nucleic acid.

[0067] DNA encoding the first and / or second arms of the binding protein is readily isolated or synthesized using conventional procedures to sequence the DNA (e.g., by using an oligonucleotide probe capable of specifically binding to the DNA). The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0068] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and viral vectors such as adeno-associated virus vectors, etc. In the vector, the nucleic acid encoding the polypeptide is operably linked to a promoter.

[0069] "Operably linked" refers to a functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0070] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. Furthermore, for example, when a eukaryotic cell is used as the host, promoters derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and they generally have a polyadenylation sequence as a transcription termination sequence.

[0071] In some cases, the vector may be fused to other sequences to facilitate purification of the polypeptide expressed therefrom, including, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

[0072] The vectors contain antibiotic resistance genes commonly used in the art as selection markers, such as genes for resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0073] In yet another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cells used to produce the polypeptide of the present invention may be, but are not limited to, prokaryotic, yeast or higher eukaryotic cells.

[0074] Prokaryotic host cells can be used, including, but not limited to, Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus).

[0075] Examples of animal cells may include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-4, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0076] In yet another aspect, the present invention relates to a fusion polypeptide in which an antigen is bound to a polypeptide.

[0077] The antigen may be a cancer or tumor antigen for activating an immune response against a cancer or tumor. The antigen may be a cancer or tumor antigen, and may induce cancer or tumor specific immune cells, such as cytotoxic T cells, in cell therapy. The cancer or tumor antigen may be, for example, an antigen derived from self or non-self. For example, autologous cancer or tumor antigens derived from a patient's own genes may include hTERT (GenBank: BAC11010.1), WT1 (GenBank: AAO61088.1), NY-ESO1 (GenBank: CAA05908.1), MAGE-A3 (NCBI Reference Sequence: NP_005353.1), etc., cancer specific mutant antigens may include tumor suppressor or inducer genes, such as neoantigens, mutated P53, RAS, etc., and foreign cancer or tumor antigens may include cancer or tumor-inducing viral antigens, such as CMV, EBV, HPV, etc.

[0078] hTERT, a self-cancer or tumor antigen, is an enzyme that synthesizes telomeric DNA at the end of chromosomes. Cancer cells overactivate this enzyme to avoid telomere-dependent cell death, and is known as a target antigen for various solid cancers including lung cancer, gastric cancer, and pancreatic cancer (Kim NW, et al. Science. 1994; 266: 2011-2015). WT1, a gene associated with Wilms tumor, encodes a zinc finger transcription factor and is a protein involved in cell proliferation and differentiation, apoptosis, and organ development, and is known as a target antigen for brain and spinal cancer, lung cancer, etc. (Call KM, et al., Cell. 1990. 60: 509-520; Nakahara Y, et al., Brain Tumor Pathol. 2004. 21: 113-6). NY-ESO1 is one of the proteins belonging to CTA (cancer testis antigen), and is well known to be expressed mainly in various cancer cells including germ cells, sarcoma, and breast cancer (Gnjatic S, et al., Adv Cancer Res. 2006; 95: 1-30). MAGE-A3 is a protein belonging to the melanoma-associated antigen family, and is known to be overexpressed in various cancer cells including lung cancer, sarcoma, and melanoma, and is evaluated as a target antigen suitable for cancer immunotherapy (Decoster L, et al., Ann Oncol. 2012 Jun; 23(6): 1387-93).

[0079] In some cases, the antigen is, for example, A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor beta 3 (ADRB3), alpha-folate receptor, AD034, AKT1, BCMA, beta-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, decarboxylase, or cytochrome P450. Hydroxenzyme IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin 6 (CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), epithelial cell adhesion molecule (EPC AM), elongation factor 2 mutated (ELF2M), ephrin type-A receptor 2 (EphA2), EMR2, Fms-like tyrosine kinase 3 (FLT3), FCRL5, fibulin-1, G250, GD2, glycoprotein 36 (gp36), glycoprotein 100 (gp100), glucocorticoid-induced tumor death factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (Gp100), and GPR20), GPC3, hsp70-2, high molecular weight melanoma-associated antigen (HMWMAA), hepatitis A virus cellular receptor 1 (HAVCR1), HAGE, HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / galectin 9, HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HUB1, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPPl 1, MSLN, neural cell adhesion molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, breast differentiation antigen (NY-BR-1), NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19 / LKB / STK1 1, NY-REN-21, NY-REN-26 / BCR, NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglioside (OAcGD2), OGFr, PSMA, prostatic acid phosphatase (PAP), p53, prostate-cancer tumor antigen-1 (PCTA-1), prostate stem cell antigen (PSCA), serine protease 21 (testisin or PRSS21), platelet-derived growth factor receptor beta (P DGFR-beta), PLAC1, Pannexin 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJkappa, RHAMM, Stage-specific Embryonic Antigen-4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, Thyroglobulin, 5T4, Tumor-associated glycoprotein 72 (TAG72), Tyrosinase, Transglutaminase 5 (TGS5), TEM1, TEM7R, Thyroid-stimulating hormone receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, Uroplakin 2 (UPK2), Vimentin, or Vascular Endothelial Growth Factor Receptor 2 (VEGFR2).

[0080] The antigen may be a pathogen (bacteria, virus, etc.), a compound, pollen, cancer cell, shrimp, etc., or a peptide or protein thereof, more preferably a cancer antigen peptide, but is not limited thereto as long as it is a substance that can cause an immune response in the body. The antigen may be preferably a protein, a recombinant protein, a glycoprotein, a gene, a peptide, a polysaccharide, a lipid polysaccharide, a polynucleotide, a cell, a cell lysate, bacteria, a virus, etc., more preferably a cancer antigen peptide. The protein may be an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxic protein, a hormone, a hormone analog, an enzyme, an enzyme fragment, a transport protein, a receptor, a receptor fragment, a biodefense inducer, a storage protein, a movement protein, an exploitative protein, a reporter protein, etc. However, the antigen may be a substance that can act as an antigen in the body and induce an immune response, but is not limited thereto.

[0081] The polypeptide and the antigen can be linked via a covalent or non-covalent bond to form a complex, and then the complex can be contained in immune cells to sensitize the immune cells with the antigen.

[0082] The present invention relates to a nucleic acid encoding said fusion polypeptide. The fusion polypeptide, in which an antigen is linked to said polypeptide, can be delivered via a vector.

[0083] The present invention also relates to an immune cell comprising said fusion polypeptide and said nucleic acid, which can sensitize the immune cell via an antigen of said fusion polypeptide.

[0084] The sensitization means that an antigen is delivered to an immune cell and loaded on the cell surface. The antigen may be produced in the form of a recombinant antigen with a polypeptide to sensitize the immune cell. The immune cell may be sensitized by treating the immune cell with the polypeptide and the antigen bound to the polypeptide for 24 hours or less. The sensitization time may be adjusted depending on the type of antigen and the type of immune cell.

[0085] The antigen is an antigen capable of binding to the polypeptide according to the present invention, and may include inactivated tumor cells, tumor cell-associated genes produced by gene recombination, peptides, or proteins. When the antigen is obtained by gene recombination, the nucleotide sequence encoding the antigen may be a known one, and the entire length of the known sequence may be used, or a part of the entire length may be used. The nucleotide sequence encoding the antigen may be cloned into a vector to express the desired antigen.

[0086] The antigen can be bound to the N-terminus or C-terminus of the polypeptide. Covalent binding can be performed by methods known in the art depending on the type of antigen, for example, by cloning a gene encoding the polypeptide and expressing it in cells.

[0087] In some cases, linkers that do not interfere with the activity of the polypeptide can be used, such as, but not limited to, N-succinimidyl iodoacetate, N-maleimidobutyryloxysuccinamide ester, 1,5-difluoro-2,4-dinitrobenzene, bis(azobenzidine), 3,3-dithio-bis(sulfosuccinimidyl-propionate), ethylene glycol bis(sulfosuccinimidyl succinate), dicyclohexyl carbodiimide, etc. On the other hand, if the antigen only exhibits activity when degraded from the polypeptide, a linker that can be cleaved in vivo can be used. For example, a linker with a carboxylate ester and / or disulfide bond can be used.

[0088] In one embodiment, the immune cells may be antigen-presenting cells (APCs) that present the antigen delivered by the polypeptide of the present invention on their surface. For example, the immune cells may be, but are not limited to, dendritic cells, monocytes, Wrangel Hans cells, macrophages, T cells, or B cells.

[0089] In yet another aspect, the present invention relates to an immunotherapeutic agent comprising the above-mentioned immune cells. The immunotherapeutic agent according to the present invention can increase an immune response or selectively increase a part of the immune response that is favorable for the treatment or prevention of a specific disease, infection or disorder.

[0090] Based on this, in yet another aspect, the present invention relates to an antitumor or anticancer vaccine comprising the immune cells. The vaccine according to the present invention can increase the immunogenicity of a subject, thereby preventing or suppressing tumor growth and / or metastasis in the subject.

[0091] The vaccine may include both single-administration and sequential-administration immunization methods.

[0092] In yet another aspect, the present invention relates to a composition for treating tumors or cancer.

[0093] The content of the active ingredient contained in the composition of the present invention and the method of administration can be determined by a person skilled in the art based on the symptoms of a typical patient and the severity of the disease. It may be provided in a unit-dose or multi-dose container, such as a sealed ampule or bottle.

[0094] The composition of the present invention can be administered orally or parenterally. The administration route of the composition of the present invention can be, but is not limited to, bronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intestinal, sublingual or topical. The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into an appropriate dosage form using known techniques for clinical administration.

[0095] The composition of the present invention is a pharmaceutical composition and may contain a pharma- ceutical acceptable carrier. Those commonly used in formulation include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further contain, in addition to the above-mentioned components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0096] The appropriate dosage of the pharmaceutical composition of the present invention may be variously prescribed depending on factors such as the formulation method, administration method, age, weight, sex, administration time and administration route of the patient, but since no serious toxicity (Grade 3 or higher) due to the dosage has been reported, it is determined in large part by the manufacturing method and yield. Meanwhile, the intradermal or subcutaneous dosage of the pharmaceutical composition of the present invention is preferably 0.1X10 7 ~10X10 7 It is a cell.

[0097] The pharmaceutical composition of the present invention is prepared in the form of a unit dose by formulating the composition using a pharma- ceutical acceptable excipient according to a method that can be easily carried out by a person skilled in the art to which the present invention pertains. In this case, the formulation may be in the form of a suspension in a cell freezing solution or a buffer solution, and may further include a stabilizer. The immune cells according to one embodiment of the present invention can be frozen after antigen sensitization and thawed when necessary for use.

[0098] The dosage of the active ingredient can be appropriately selected depending on many factors such as the administration route, the age, sex, and weight of the patient, and the severity of the patient, and the composition can be administered in combination with a known compound having the effect of preventing, improving, or treating tumor or cancer symptoms.

[0099] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention thereto.

[0100] EXAMPLES

[0101] Example 1. Binding strength studies using peptides

[0102] 1.1 Peptide synthesis

[0103] Five types of superantigen peptides that bind to MHC class II were selected, and the peptides were synthesized by Enizen Co., Ltd., a peptide manufacturer.

[0104] JPEG2024546516000004.jpg52128

[0105] 1.1.1 ELISA-based tests

[0106] To confirm the binding strength of the synthesized peptides, MHC class ± protein was coated on a 96-well plate, treated with 50 nM peptides, and incubated for 24 hours. After that, it was washed three times with PBS, incubated with HRP-antibody (1:500) for 2 hours, washed five times with PBS, and incubated for 10 to 30 minutes after adding TMB solution. After stopping the color development using a stopping solution (2M H2SO4), the absorbance was checked at 450 nm to select peptides with good binding strength. As a result, the peptide sequence corresponding to SEB2 showed the highest binding strength among the five synthesized peptides, followed by SEB4, SEB1, and SEB3 in that order.

[0107] 1.1.2 Differentiation of monocyte-derived dendritic cells using PBMCs

[0108] After thawing the PBMC vial, the monocytes were attached to a T150 plate for 30 minutes using 1% AB RPMI medium, after which non-adherent cells were removed and cultured for 3 days in maturation medium containing hGM-CSF 30ng / ml and hIL-4 20ng / ml.

[0109] 1.1.3 Peptide cell permeability measurement

[0110] MoDC cells were plated on a cover slip at 30% to 40% confluence and then cultured at 1x10 4The cells were stored in a 6-well plate at a cell density of 10 ...

[0111] 1.1.4 Cell permeability measurement using flow cytometry

[0112] MoDCs were obtained by isolating mononuclear cells from human peripheral blood and culturing them to obtain dendritic cells. Differentiated dendritic cells were treated with different peptide concentrations for each group and then cultured for 10 minutes, after which each group was washed with PBS. To analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, anti-mouse CD11c, and peptide-FITC were stained, and then measured using FACS CantoII. First, the phenotype of the prepared MoDCs was confirmed, and then an analysis was performed to check whether the prepared DCs functioned as DCs. It was confirmed that all DCs showed a phenotype of 90% or more. Then, to analyze the peptide permeability in DCs, a double population of CD11c+ and CD80+ was obtained, and peptide-FITC was analyzed within it. As a result of the analysis, it was confirmed that a large amount of peptide was permeated in the CTP-SEB2 peptide group compared to the existing CTP peptide group, while there was almost no permeability in the case of SEB2 peptide alone, which was the same as the microscopic results. The results are shown in Figure 2.

[0113]

[0114] Example 2. Preparation of peptide fusion protein fused with CTP

[0115] 2.1 Construction of fusion protein vector

[0116] Based on the permeability results, the SEB2 sequence peptide with the best binding strength was selected, and a request was made to create a recombinant protein vector as shown in FIG. 3 (genscript, Piscataway, NJ).

[0117] 2.2 Production of recombinant fusion proteins

[0118] The recombinant gene was cultured in E. coli and produced in IB state, then solubilized and isolated / purified using Ni-NTA resin. Specifically, the seed culture was first cultured overnight at 37℃, and then grown in LB Broth Miller medium (Novagen, Yeast extract 5g, peptone from casein 10g, sodium chloride 10g) supplemented with 50μg / ml ampicillin at 37℃. When the absorbance at OD600nm reached 0.8-1.0, IPTG was added to a final concentration of 0.5mM to induce protein expression, and the cells were shaken at 180rpm for 8 hours. Each bacterial cell that had grown to an absorbance of 1.5-2.0 at OD600nm was precipitated by centrifugation at 10,000 rpm, 4℃ for 20 minutes, and then 50 ml of disintegration buffer (300 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.5% TritonX-100) was added per 2 g of precipitated cells (1 L culture volume) and stirred until the cells were completely suspended. PMSF was added to a final concentration of 1 mM, and the cells were lysed using an ultrasonic grinder. The lysed cells were centrifuged at 12,000 rpm, 4℃ for 30 minutes to separate the suspension and total lysate, and only the suspension was collected and filtered through a cellulose filter membrane with a pore size of 0.45 μm and loaded onto a nickel affinity chromatography column that had been previously equilibrated with the equilibration buffer. The purified protein was stabilized through buffer exchange and then QC was performed. The results are shown in Figure 4.

[0119] 2.3 Measurement of cell permeability of fusion proteins

[0120] MoDC cells were plated on a cover slip at 30% to 40% confluence and then cultured at 1x10 4The cells were stored in a 6-well plate at a cell density of 10 ...

[0121] 2.4 Measurement of cell permeability of fusion proteins using Western blot

[0122] After washing with cold PBS, the cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 30 mM NaF, 10 mM Na3VO4, 0.5% NP40, and Pierce (protease inhibitor cocktail). Whole cell lysates were adjusted to the same concentration using Bradford preparation (Bio-Rad), and 40 μg to 120 μg of the lysates were subjected to SDS-PAGE on 8% to 12% acrylamide gels, which were then transferred to PVDF membranes (Milipore). For immunoblotting, the membranes were blocked with 5% non-fat dry milk prepared in TBS containing 0.5% Tween 20 (TBST) at room temperature for 1 hour. The membrane was washed four times with TBST, treated with the optimal concentration of primary antibody diluted in 4% non-fat dry milk, and incubated overnight at 4°C. It was then washed four times with TBST and incubated with HRP-conjugated secondary antibody for 45 min. Bound antibodies were detected using chemiluminescent HRP substrate (Millipore, USA) and Chemi DOC (Bio-RAD, USA).

[0123] 2.5 Direct binding affinity analysis of fusion proteins

[0124] Proteins were produced by constructing genes expressing HIS-tags using the selected peptides, and analyzed by immunoprecipitation against His to examine whether they directly interact with MHC class II.

[0125] Specifically, 4X10 7The cells were harvested and washed twice with PBS, and then suspended in NP-40 lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 10% glycerol) containing a protease inhibitor cocktail [2 mM AEBSF, 1 mM EDTA, 130 μM Bestatin, 1 μM leupeptin, 14 μM E-64, 0.3 μM Aprotinin] and a phosphatase inhibitor (Roche). The cells were then rotated for 30 minutes in a rotator in a 4°C chamber (C1, 25 rpm). The cells were centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was removed. The cells were then centrifuged again at 12,000 rpm at 4°C for 10 minutes, and only the clean supernatant was removed. After quantifying the protein, the protein used for cell lysate data was sampled separately and stored at -80°C, and the protein used for immunoprecipitation was separated into 500μg or more / 700μl-1000μl, and the remaining was filled with lysis buffer solution to a total volume of 1ml. 1μg-2μg of antibody for immunoprecipitation was added to this per 100μg-500μg of protein, and the mixture was rotated for 2 hours or more or overnight in a 4°C chamber with a rotor (C1, 10 rpm). Next, about 30μl-50μl of Protein A / G beads (Pierce) were added, and the mixture was rotated for 1-2 hours in a 4°C chamber with a rotor. The mixture was washed three times with NP-40 lysis buffer solution at 1200rpm, 3min, and 4°C. After washing, the remaining buffer solution was completely removed, and 20ul of 5X sample buffer solution was added and boiled. After cooling on ice and spinning in a centrifuge for 2 minutes, the supernatant was collected and separated by 7.5%-12% SDS-PAGE. The gel was blotted again with a PVDF membrane, and the membrane to which the proteins had been transferred was coated with a primary antibody, and then coated with a secondary antibody against IgG, and observed with an ECL detection kit.

[0126] The analysis results are shown in Figure 6. As a result, when the fusion protein was bound to Protein A / G beads and western blot was performed with the target protein, it was confirmed that the protein fused with the peptide was detected together with the target protein, but the protein without the peptide was not detected.

[0127] 2.6 Isolation of mouse dendritic cells

[0128] Dendritic cells (DCs) were cultured by differentiating bone marrow stem cells into dendritic cells. More specifically, bone marrow-derived DCs (BMDCs) were isolated from the femurs and tibias of 6- to 8-week-old female C57BL / 6 mice, and then treated with ACK lysis buffer (Lonza) to remove red blood cells. The cells were washed and cultured in cell culture medium (RPMI1640 containing 10% FBS and penicillin / streptomycin) containing 10 ng / ml mGM-CSF (Creagene Inc.). After 2 days, the supernatant of the cultured cells was removed and replaced with 2 ml of fresh culture medium containing mGM-CSF. On the 4th day of culture, 1 ml of fresh culture medium containing mGM-CSF was added. On day 6 of culture, the non-adherent cells were harvested and used as immature dendritic cells (imDCs). Mature dendritic cells (mDCs) were prepared by culturing immature dendritic cells (imDCs) in the presence of LPS (100-200 ng / ml) for 24 hours.

[0129] 2.7 Cell permeability measurement using flow cytometry

[0130] BMDCs were isolated from mouse bone marrow and cultured to obtain dendritic cells. Differentiated dendritic cells were treated with different peptide concentrations in each group and cultured for 1 hour, then each group was washed with PBS. To analyze the phenotype of dendritic cells, anti-mouse CD80, anti-mouse CD86, and anti-mouse CD11c were stained, respectively, and measured using FACS CantoII, and peptide-FITC was used to confirm peptide permeability in CD11c+.

[0131] 2.8 Analysis of remaining protein in culture medium using TCA precipitation method

[0132] A 100% TCA solution was added to the culture medium sample at 1 / 10 volume, vortexed vigorously, and then left on ice for 30 minutes, followed by centrifugation at 4°C and 14,000 rpm for 10 minutes. The supernatant was then discarded, and 500 μl of cold acetone was added to the precipitate, followed by centrifugation at 4°C and 14,000 rpm for 10 minutes to remove the TCA. The precipitate was then dried, resuspended in a buffer solution, and subjected to Western blotting. As shown in Figure 7, the amount of protein remaining on the medium was confirmed, and it was confirmed that in the case of the CTP-SEB2-OVA protein to which the peptide was bound, most of the protein penetrated into the cells, and the amount of protein present in the medium was not large, while in the case of the CTP-OVA existing sequence protein, the amount of penetration was slightly low. However, it was confirmed that each protein to which CTP was not bound was present on the medium.

[0133] 2.9 Comparative experiment of cell membrane permeability of PTD peptides

[0134] The company tried to confirm that the peptide sequence it is developing has better permeability by comparing the permeability of the peptide sequence it is developing with the permeability peptide sequence of the existing PTD series. BMDC cells were cultured in a 12-well plate at 2x10 5The cells were seeded at 1000μg / ml and then cultured at 37℃ for 1 day. The cells were treated with CTP-FITC, TAT-SEB2-FITC and CTP-SEB2-FITC peptides (Enizen Co., Ltd.) attached to the cell surface at a concentration of 1μM for 4 hours, and the degree of fluorescent labeling of the cells was examined by FACS. First, the DC phenotypes of mouse BMDCs were analyzed, and all showed a yield of 90% or more, confirming that BMDCs were successfully produced. In addition, as a result of treating the corresponding DCs with peptides, it was confirmed that the effect on permeability was greater when SEB2 was bound to the CTP sequence possessed by the company than when the SEB2 sequence was bound to the existing PTD series peptide. This was also confirmed by the results of the fusion protein (Figure 8).

[0135] 2.10 Analysis of antigen-specific T cell proliferation

[0136] The spleens of transgenic mice with TCR (OT-I) specific for OVA (ovalbumin) antigen were used to analyze T cell proliferation responses through co-culture with DCs. The MLR assay to confirm the function of the cultured cells was performed by incubating spleen cells (OT-I specific T cells: 1X10 5 The ratio of DCs treated with OVA antigen (S, stimulator) to DCs treated with OVA antigen (R:S ratio) was set at 1:10 to 1:100, and these were cultured together for 96 hours, after which the proliferation of responder cells was measured. As shown in Figure 9, the group containing DCs sensitized with peptide-fused antigen showed the highest OVA-specific T cell response, while in the case of CTP-sensitized DCs, the specific T cell response increased, but the reactivity was lower than that of the peptide-fused antigen.

[0137]

[0138] Example 3. Preventive effect of dendritic cell vaccine on melanoma in mice

[0139] To investigate the possibility of melanoma prevention by dendritic cell vaccines, mice were immunized twice with dendritic cells sensitized with CTP-fused melanoma recombinant antigens, and then induction tests were performed with cancer cell lines expressing melanoma-specific antigens to investigate the formation of solid tumors. Mouse dendritic cells were prepared by differentiating bone marrow cells from femurs and tibias into dendritic cells. Both ends of the femur and tibia were cut, bone marrow cells were extracted, and the cells were collected in a 50 ml tube. The collected bone marrow cells were suspended in 0.83% ammonium chloride solution, red blood cells were removed, and the bone marrow cells were cultured in dendritic cell production medium (RPMI-1640, 10% FBS, 10 ng / ml mouse recombinant IL-4, and 10 ng / ml mouse GM-CSF) for 2 days, non-adherent cells were removed, and only cells attached to the bottom of the container were obtained. The medium was replaced with fresh medium at 2-3 day intervals to prevent cytokine depletion. On the sixth day of culture, immature dendritic cells were harvested and treated with recombinant antigens CTP-OVA and SCTP-OVA. The immature dendritic cells were sensitized by treatment with 10 μg / ml of each antigen protein (Korean Patent Registration No. 10-0647847) for 20 hours. Then, cytokines required for maturation of dendritic cells (100 μg / ml IFN-γ and 100 μg / ml TNF-α) were added for 24 hours to induce maturation of dendritic cells. 1x10 of antigen-sensitized dendritic cells were cultured at 1x10 6 The cells were injected subcutaneously into mice to induce anti-cancer immunity. Dendritic cell immunization was performed twice at a weekly interval, and 1 week after the secondary dendritic cell inoculation, B16-OVA melanoma cells were injected at 1x10 6 Cells were injected subcutaneously into the mouse. The size of the tumor (horizontal x vertical) was measured every two days. As a result, the experimental groups inoculated with dendritic cells sensitized with CTP-OVA or SCTP-OVA antigens showed antitumor effects as shown in Figure 10.

[0140]

[0141] Example 4. Immune cell permeability analysis using PBMCs

[0142] We attempted to observe the permeability of the fusion peptide to be developed in various immune cells (monocyte, T cell, B cell, dendritic cell, NK cell, etc.) present on PBMC. To this end, frozen PBMC was thawed, treated with the existing CTP peptide and the fused CTP-SEB peptide at a concentration of 1 μM each, and analyzed by FACS. For each immune cell, the amount of permeated peptide was analyzed after confirming the representative markers of each immune cell, such as CD14 (monocyte), CD3 (T cell), CD19 (B cell), and HLA-DR (dendritic cell). As a result of examining the peptide permeability by immune cell, as shown in Figure 11, it was confirmed that the permeability of the fusion peptide was increased compared to the existing CTP peptide in monocytes, B cells, and dendritic cells, which are known to express HLA-DR. Furthermore, the permeability was also confirmed to increase in T cells. Through this, it was confirmed that the permeability of the fusion peptide was generally increased in immune cells.

[0143] [Industrial Applicability]

[0144] The polypeptide of the present invention has excellent cell membrane permeability of immune cells, allowing antigens to be delivered with excellent efficiency through direct targeting to immune cells, which may be effective in the proliferation of T cells.

[0145]

[0146] Although the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0147] [Sequence List Free Text]

[0148] Electronic file attached.

Claims

1. Polypeptides for delivering antigens to immune cells, including cytoplasmic transduction peptides (CTPs) and peptides that bind to surface molecules of immune cells.

2. The polypeptide according to claim 1 , wherein the surface molecule of the immune cell is major histocompatibility complex (MHC) class II.

3. The polypeptide according to claim 1 , wherein the immune cell is a dendritic cell, a monocyte, a Wrangel Hans cell, a macrophage, a T cell or a B cell.

4. The polypeptide of claim 1, wherein the peptide that binds to a surface molecule of an immune cell comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 4.

5. The polypeptide of claim 1, wherein the cell membrane-permeable peptide comprises a sequence selected from the group consisting of SEQ ID NOs: 5 to 7.

6. The polypeptide according to claim 1 , wherein the end of the cell membrane-penetrating peptide is fused with a peptide that binds to a surface molecule of an immune cell.

7. The polypeptide of claim 1 , wherein the antigen is a cancer or tumor antigen for activating an immune response against a cancer or tumor.

8. A nucleic acid encoding the polypeptide according to any one of claims 1 to 7.

9. A fusion polypeptide comprising the polypeptide according to any one of claims 1 to 7 bound to an antigen.

10. The fusion polypeptide of claim 9 , wherein the antigen is a cancer or tumor antigen for activating an immune response against a cancer or tumor.

11. A nucleic acid encoding the fusion polypeptide of claim 9.

12. An immune cell comprising the fusion polypeptide of claim 9.

13. The immune cell of claim 12, which is a dendritic cell, a monocyte, a Wrangel Hans cell, a macrophage, a T cell or a B cell.

14. An immunotherapeutic agent comprising the immune cells according to claim 12.

15. An anti-tumor or anti-cancer vaccine comprising the immune cell of claim 12.

16. A composition for treating tumors or cancer, comprising the immune cells described in claim 12.

17. An immune cell comprising the nucleic acid of claim 11.

18. The immune cell of claim 17, which is a dendritic cell, a monocyte, a Wrangel Hans cell, a macrophage, a T cell or a B cell.

19. An immunotherapeutic agent comprising the immune cells of claim 17.

20. 18. An anti-tumor or anti-cancer vaccine comprising the immune cell of claim 17.

21. A composition for treating tumors or cancer comprising the immune cells described in claim 17.

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