Modified mitochondria comprising prodrug converting enzyme and use thereof

Abstract

The present invention relates to modified mitochondria comprising an antibody binding to a cancer cell-specific protein and cytosine deaminase, in the mitochondrial outer membrane It was confirmed that, when a pancreatic cancer cell line was co-treated with the mitochondria according to the present invention and 5-FC, cytotoxicity induced by 5-FC treatment was significantly increased. The effect was confirmed to result from the conversion of 5-FC to 5-FU by cytosine deaminase expressed on the mitochondrial outer membrane, inducing apoptosis of pancreatic cancer cells. Therefore, it is considered that the modified mitochondria of the present invention can be effectively used in anticancer treatment using 5-FC as an anticancer agent.

Description

Modified mitochondria containing prodrug converting enzymes and uses thereof

[0001] The present invention provides a modified mitochondrion in which a prodrug converting enzyme exists in the outer membrane of the mitochondria and a pharmaceutical composition for preventing and treating cancer comprising the same as an active ingredient.

[0002] Prodrug converting enzymes (PCEs) are enzymes that convert harmless or mildly toxic prodrugs into highly cytotoxic drugs. Therefore, when administered, prodrugs can induce cell death around the enzyme. One such prodrug converting enzyme, cytosine deaminase (CD), is known to convert 5-fluorocytosine (5-FC), a harmless prodrug for mammalian cells, into 5-fluorouracil (5-FU), a highly cytotoxic drug.

[0003] 5-FU is known to induce apoptosis by inhibiting protein synthesis and DNA synthesis. Within cells, 5-FU is converted to 5-FUTP by intracellular enzymes, which inhibits protein synthesis by inhibiting mRNA biosynthesis within the cell. 5-FU is also known to be converted to 5-fluoro-dUTP (5-FdUTP) by intracellular enzymes, which inhibits DNA replication by inducing dTTP depletion by inhibiting the activity of thymidylate synthase.

[0004] The most common method of using cytosine deaminase as an anticancer agent is gene therapy using viral genes. However, the use of viruses as delivery vehicles has safety issues, so its use is very limited. Another method is to express the suicide gene cytosine deaminase in mesenchymal stem cells that have a homing effect on cancer cells and then inject the mesenchymal stem cells. During this process, 5-FC is converted to 5-FU within the mesenchymal stem cells and is secreted outside the cells, resulting in a bystander effect that kills adjacent cancer cells (Korean Patent Publication No. 10-2007-0036289).

[0005] However, the homing effect of mesenchymal stem cells on cancer cells is very weak, and there is still a drawback that it is difficult to overcome the problem of cytotoxicity.

[0006] The present invention aims to provide a substance capable of treating cancer by binding an antibody and a prodrug converting enzyme that bind to a cancer cell-specific protein on the outer membrane of mitochondria, thereby transporting the prodrug converting enzyme to the periphery of cancer cells in a cancer cell-specific manner, and activating an anticancer drug in a cancer cell-specific manner by administering the prodrug, a composition for treating cancer using the substance, and a treatment method.

[0007] To solve the above problem, one aspect of the present invention provides a fusion protein comprising a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0008] Another aspect of the present invention provides a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, and a transformed cell into which the vector has been introduced.

[0009] Another aspect of the present invention provides modified mitochondria in which a prodrug converting enzyme or a fragment thereof is present in the mitochondrial outer membrane.

[0010] Another aspect of the present invention provides a modified mitochondrion, wherein a prodrug converting enzyme or a fragment thereof, and an antibody or a fragment thereof that specifically binds to a tumor-associated antigen are present in the mitochondrial outer membrane.

[0011] Another aspect of the present invention provides a pharmaceutical composition for preventing and treating cancer comprising the modified mitochondria as an active ingredient.

[0012] Another aspect of the present invention provides the use of the modified mitochondria for preparing a pharmaceutical composition for treating cancer.

[0013] Another aspect of the present invention provides a method for treating cancer comprising administering the modified mitochondria to a subject.

[0014] Another aspect of the present invention provides a kit for treating cancer comprising the modified mitochondria and a prodrug.

[0015] When modified mitochondria expressing cytosine deaminase, a prodrug converting enzyme, and an antibody that binds to a cancer cell-specific protein according to the present invention were treated with 5-FC to pancreatic cancer cells, they exhibited higher cytotoxicity compared to the unmodified mitochondrial treatment group. Based on the above results, modified mitochondria containing the prodrug converting enzyme of the present invention may be useful in anticancer treatment using 5-FC as an anticancer agent.

[0016] Figure 1 is a schematic diagram of an expression vector (pCMV-yCD-myc-TOM7) for expressing a yeast-derived cytosine deaminase fragment in a fused form to mitochondria.

[0017] Figure 2 is a diagram showing the results of Western blotting to confirm the yeast-derived cytosine deaminase fragment expressed in human fetal kidney cells (HEK293) transformed with pCMV-yCD-myc-TOM7. Lane 1 is HEK293 cells, Lane 2 is empty vector-transformed cells (HEK293-EV), and Lane 3 is vector-transformed cells loaded with a gene encoding yeast-derived cytosine deaminase (HEK293-CD).

[0018] Figure 3 is a graph showing the absorbance at each concentration as a standard curve by measuring the absorbance of 5-FC (5-fluorucytosine) and 5-FU (5-Fluoruacil) at 314 nm.

[0019] Figure 4 is a graph showing the results of measuring the enzyme activity of cytosine deaminase expressed in HEK293-CD cells to convert 5-FC into 5-FU.

[0020] Figure 5 is a drawing showing the results of observing changes in cell morphology using an optical microscope after treating HEK293 cells and HEK293-CD cells transfected with a vector loaded with a gene encoding cytosine deaminase with 5-FC, a precursor of the anticancer drug 5-FU.

[0021] Figure 6 is a graph showing the results of measuring cytotoxicity after treating HEK293 cells and HEK293-CD cells transfected with a vector loaded with a gene encoding cytosine deaminase with 5-FC, a precursor of the anticancer drug 5-FU.

[0022] Figure 7 is a drawing showing the results of confirming the expression of yCD-myc-TOM7 by Western blot to select a HEK293-CD cell line (Stable cell line) that stably expresses yeast-derived cytosine deaminase (yCD-myc-TOM7).

[0023] Figure 8 is a drawing showing the results of Western blot confirmation that yeast-derived cytosine deaminase (yCD-myc-TOM7) expressed in the HEK293-CD cell line exists in the same location as intracellular mitochondria.

[0024] Figure 9 is a drawing showing the results of confirming the intracellular location of yeast-derived cytosine deaminase (yCD-myc-TOM7) expressed in the HEK293-CD cell line using a confocal microscope.

[0025] Figure 10 shows the expression of modified mitochondria (MT) conjugated with MT or yeast-derived cytosine deaminase in human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2 and MIA Paca-2). yCD-myc-TOM7 ) and then treated with 5-FC to observe morphological changes and cell death of cancer cells using an optical microscope. In this case, MT is a mitochondrion isolated from HEK293 cells to which yeast-derived cytosine deaminase is not bound.

[0026] Figure 11 shows the expression of MT or MT in human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2). yCD-myc-TOM7 This is a graph showing the results of measuring cytotoxicity by injecting 5-FC at different concentrations.

[0027] Figure 12 shows the expression of MT or MT in human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2 and MIA Paca-2). yCD-myc-TOM7 This is a graph showing the results of measuring cytotoxicity by treating with 5-FC at different concentrations after injection.

[0028] Figure 13 shows the expression of MT or MT in human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2). yCD-myc-TOM7 This is a diagram showing the results of Western blot analysis of cell death caused by 5-FC treatment after injection.

[0029] Hereinafter, the present invention will be described in detail.

[0030] fusion protein

[0031] One aspect of the present invention provides a fusion protein comprising a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0032] The term "prodrug" as used herein refers to a drug that has no or weak pharmacological effect on its own, but is converted into an active metabolite with pharmacological effect through enzymatic or chemical changes when absorbed into the body.

[0033] As used herein, the term "prodrug-conversion enzyme" refers to an enzyme that has the activity of converting a non-toxic prodrug into a toxic drug. Such prodrug-conversion enzymes are commonly used in enzyme / prodrug therapy.

[0034] In the present invention, the prodrug conversion enzyme may be any one selected from the group consisting of cytosine deaminase (CD), carboxyl esterase (CE), simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine-nucleoside-phosphorylase (PNP), and horseradish peroxidase (HRP), but is not limited thereto as long as it is an enzyme having an activity of converting a prodrug into a toxic drug.

[0035] In the present invention, the prodrug conversion enzyme includes a native form, a mutant form, and fragments thereof. The native form generally refers to a polypeptide comprising the amino acid sequence of each native prodrug conversion enzyme, and the amino acid sequence of the native prodrug conversion enzyme generally refers to the amino acid sequence found in each naturally occurring prodrug conversion enzyme. In addition, as long as it has the same activity as the native prodrug conversion enzyme or the gene encoding the enzyme on the chromosome is the same, the protein may be composed of a sequence in which one or more amino acids of the protein are added, deleted, or substituted.

[0036] Specifically, the prodrug conversion enzyme of the present invention may have a sequence identity of at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95% when compared with the amino acid sequence and sequence identity of each natural enzyme.

[0037] As used herein, the term "prodrug converting enzyme fragment" refers to a fragment of each enzyme that has enzymatic activity for converting a prodrug into a toxic drug, in a form in which the N-terminus, C-terminus, or portions of the N-terminus and C-terminus are deleted (truncated). Here, the activity of the prodrug converting enzyme can be measured using a method known to those skilled in the art. In this case, the prodrug converting enzyme may include a variant.

[0038] The term "variant" as used herein refers to a form in which a portion of the above-described prodrug converting enzyme or a fragment thereof has been substituted, and has the function of activating a prodrug into a toxic anticancer agent. In other words, a prodrug converting enzyme variant may have a sequence different from the native prodrug converting enzyme, and refers to an enzyme that has the function of activating a prodrug. Here, the activity of the prodrug-activating enzyme can be measured using methods known to those skilled in the art.

[0039] In one specific embodiment of the present invention, the prodrug conversion enzyme may be cytosine deaminase. Cytosine deaminase (CD) is an enzyme that catalyzes a reaction that generates uracil by hydrolyzing an amino group of cytosine as a substrate. Cytosine deaminase generates 5-fluorouracil (5-FU) through a deamination reaction using 5-fluorocytosine (5-FC), a non-toxic prodrug, as a reactant, and the generated 5-FU exhibits specific toxicity to rapidly dividing cancer cells by inhibiting DNA synthesis. The cytosine deaminase of the present invention may be derived from yeast (SEQ ID NO: 1) or bacteria (SEQ ID NO: 2). Preferably, it may be a yeast-derived cytosine deaminase.

[0040] Information on the above cytosine deaminase can be obtained from known databases such as GenBank of the National Institutes of Health of the United States, and may have an amino acid sequence of SEQ ID NO: 1 as a specific example, but is not limited thereto. In addition, the cytosine deaminase of the present invention may include or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 1. In one specific example, the cytosine deaminase may have the amino acid sequence of SEQ ID NO: 2, and may comprise or consist of an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or 100% identity with the amino acid sequence of SEQ ID NO: 2. In addition, the cytosine deaminase of the present invention includes variants and fragments thereof. Herein, the variants and fragments are the same as described above.

[0041] As used herein, the term "membrane protein" refers to a protein that is part of or interacts with a biological membrane. As used herein, membrane proteins may include both integral membrane proteins that are part of or permanently attached to a membrane, and extrinsic membrane proteins that are temporarily attached to a phospholipid bilayer or other integral membrane proteins.

[0042] As used herein, the term "mitochondrial outer membrane protein" refers to a protein capable of being located in the outer membrane of mitochondria. A "mitochondrial outer membrane targeting protein" is a protein comprising the N-terminus or C-terminus of a protein present in the outer membrane of mitochondria, and may have an amino acid sequence that allows it to be specifically located in the outer membrane of mitochondria. In this case, the mitochondrial outer membrane targeting protein allows the fusion protein disclosed in the present invention to be attached to the outer membrane of mitochondria. In addition, the mitochondrial outer membrane targeting protein prevents the fusion protein disclosed in the present invention from entering the interior of mitochondria.

[0043] The above mitochondrial outer membrane targeting protein may be selected from proteins present in mitochondria within eukaryotic cells. For example, it may be selected from proteins present in the mitochondrial outer membrane within yeast, animal cells, or human cells.

[0044] A specific example of the above mitochondrial outer membrane targeting protein may be a protein comprising an N-terminus or C-terminus of any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[0045] In particular, when the mitochondrial outer membrane targeting protein is derived from any one selected from the group consisting of TOM20, TOM70, and OM45, it may include an N-terminal region of TOM20, TOM70, or OM45. A specific example of the mitochondrial outer membrane targeting protein may be yeast-derived TOM70 represented by SEQ ID NO: 8, or human-derived TOM70 represented by SEQ ID NO: 9. Another specific example may be yeast-derived TOM20 represented by SEQ ID NO: 10, or human-derived TOM20 represented by SEQ ID NO: 11. Another specific example may be yeast-derived OM45 represented by SEQ ID NO: 12.

[0046] In addition, when the mitochondrial outer membrane targeting protein is derived from any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, it may include any one C-terminal region selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. A specific example of the mitochondrial outer membrane targeting protein may be yeast-derived TOM5 represented by SEQ ID NO: 13, or human-derived TOM5 represented by SEQ ID NO: 14. Another specific example may be yeast-derived TOM7 represented by SEQ ID NO: 15, or human-derived TOM7 represented by SEQ ID NO: 16. Another specific example may be yeast-derived TOM22 represented by SEQ ID NO: 17, or human-derived TOM22 represented by SEQ ID NO: 18. Another specific example may be yeast-derived Fis1 represented by SEQ ID NO: 19, or human-derived Fis1 represented by SEQ ID NO: 20. Another specific example may be human-derived Bcl-2 alpha represented by SEQ ID NO: 21. Another specific example may be yeast-derived VAMP1 represented by SEQ ID NO: 22, or human-derived VAMP1 represented by SEQ ID NO: 23.

[0047] The prodrug converting enzyme or a fragment thereof may be bound to the outer membrane of a mitochondrion by a mitochondrial outer membrane targeting protein. The mitochondrial outer membrane targeting protein and the prodrug converting enzyme may be linked from the N-terminus to the C-terminus.

[0048] These fusion proteins may be combined in the following order:

[0049] <Structural formula 1>

[0050] N-terminal-mitochondrial outer membrane targeting protein-prodrug convertase or fragment thereof-C-terminal.

[0051] or

[0052] <Structural formula 2>

[0053] N-terminal-prodrug converting enzyme or fragment thereof-mitochondrial outer membrane targeting protein-C terminus.

[0054] In the above structural formula 1, the mitochondrial outer membrane targeting protein may be an N-terminal sequence of a protein selected from the group consisting of TOM20, TOM70, and OM45.

[0055] In the above structural formula 2, the mitochondrial outer membrane targeting protein may be a C-terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAMP1B. In this case, the prodrug converting enzyme or a fragment thereof is the same as described above.

[0056] In addition, a linker may be further included between the prodrug converting enzyme or a fragment thereof and the mitochondrial outer membrane targeting protein. The linker may be composed of 1 to 150 amino acids, 10 to 100 amino acids, or 20 to 50 amino acids, but is not limited thereto. The linker may be appropriately selected from 20 amino acids, and preferably may be composed of glycine and / or serine. A specific example of the linker may be 5 to 50 amino acids composed of glycine and serine. A specific example of the linker is (G4S)n (SEQ ID NO: 24), wherein n is an integer from 1 to 10, and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0057] These fusion proteins may be combined in the following order:

[0058] <Structural formula 3>

[0059] N-terminal-mitochondrial outer membrane targeting protein-linker-prodrug convertase or fragment thereof-C-terminal.

[0060] or

[0061] <Structural formula 4>

[0062] N-terminal-prodrug convertase or fragment thereof-linker-mitochondrial outer membrane targeting protein-C terminus.

[0063] In the above structural formula 3, the mitochondrial outer membrane targeting protein may be an N-terminal sequence of a protein selected from the group consisting of TOM20, TOM70, and OM45.

[0064] In the above structural formula 4, the mitochondrial outer membrane targeting protein may be a C-terminal sequence of a protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAMP1B.

[0065] Another aspect of the present invention provides a polynucleotide encoding a fusion protein comprising a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein. The prodrug converting enzyme or a fragment thereof, the mitochondrial outer membrane targeting protein, and the fusion protein are the same as described above.

[0066] The polynucleotide may be modified by substitution, deletion, insertion, or a combination thereof of one or more bases, as long as they encode the same polypeptide. When producing a polynucleotide sequence by chemical synthesis, synthetic methods widely known in the art can be used, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), and examples thereof include triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on solid supports.

[0067] In one specific example, the polynucleotide may comprise a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identity to a base sequence of a polynucleotide encoding a fusion protein comprising a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0068] Another aspect of the present invention provides a vector loaded with a polynucleotide encoding the fusion protein. The fusion protein is the same as described above.

[0069] As used herein, the term "vector" refers to a vector capable of being introduced into a host cell and recombined and integrated into the host cell genome. Alternatively, the vector is understood to be a nucleic acid vehicle comprising a nucleotide sequence capable of autonomously replicating as an episome. The vector includes linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, mini-chromosomes, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0070] Specifically, the vector may be a plasmid DNA, a phage DNA, etc., and may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.), etc. Since the above vector exhibits different protein expression levels and expression patterns depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0071] Additionally, the plasmid may contain a selection marker, such as an antibiotic resistance gene, and the host cell harboring the plasmid may be cultured under selective conditions.

[0072] As used herein, the terms "gene expression" or "expression" of a protein of interest are understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or a variant thereof. The expression vector may include a human cytomegalovirus (CMV) promoter to promote continuous transcription of the gene of interest in mammalian cells and a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of post-transcriptional RNA.

[0073] Another aspect of the present invention provides a transformed cell into which a vector carrying a polynucleotide encoding the fusion protein is introduced.

[0074] As used herein, the term "transformed cell" refers to prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. Such transformed cells can be produced by introducing the vector into a host cell and transforming it. Furthermore, the polynucleotide contained in the vector can be expressed to produce the fusion protein of the present invention.

[0075] The above transformation can be performed by various methods. As long as the fusion protein of the present invention can be produced, it is not particularly limited thereto. Specifically, the transformation method may include a CaCl2 precipitation method, a Hanahan method that increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, a calcium phosphate precipitation method, a protoplast fusion method, a stirring method using silicon carbide fibers, an Agrobacterium-mediated transformation method, a transformation method using PEG, a dextran sulfate, lipofectamine, and a drying / inhibition-mediated transformation method. In addition, a target product can be delivered into a cell using a virus particle by means of infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc.

[0076] In addition, the host cell used for producing the transformed cell is not particularly limited as long as it can produce the fusion protein of the present invention. Specifically, the host cell may include, but is not limited to, prokaryotic cells, eukaryotic cells, mammals, plants, insects, fungi, or cells of cellular origin. An example of the prokaryotic cell may be Escherichia coli. In addition, an example of the eukaryotic cell may be yeast. In addition, the mammalian cell may be CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid, NSO cells, HT-1080 cells, PERC.6 cells, HEK 293 cells, or HEK293T cells, but is not limited thereto, and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.

[0077] As described above, to optimize the therapeutic properties of the fusion protein or for other purposes, the glycosylation-related genes of the host cell can be manipulated using methods known to those skilled in the art to adjust the sugar chain pattern (e.g., sialic acid, fucosylation, glycosylation) of the fusion protein.

[0078] modified mitochondria

[0079] Another aspect of the present invention provides modified mitochondria in which a prodrug converting enzyme or a fragment thereof is present in the mitochondrial outer membrane.

[0080] As used herein, the term "mitochondria" refers to eukaryotic cell organelles involved in the synthesis and regulation of adenosine triphosphate (ATP), the energy source within cells, and are involved in various metabolic pathways in the body, such as cell signaling, cell differentiation, apoptosis, as well as the control of the cell cycle and cell growth.

[0081] Therefore, it has been reported that mitochondrial dysfunction or dysfunction due to genetic, environmental, or unknown causes is related to the development of various diseases such as mitochondrial-related genetic diseases, inflammatory diseases such as rheumatoid arthritis, ischemic diseases, infectious diseases, heart diseases, muscle diseases, degenerative diseases such as Parkinson's disease and Alzheimer's disease, and the development and metastasis of various cancers.

[0082] The mitochondria may be obtained from a mammal or a human. Specifically, the mitochondria may be isolated from cells or tissues, and may be isolated from blood cells or platelets. In addition, the mitochondria may be normal mitochondria obtained from cells with normal mitochondrial biological activity. In this case, the mitochondria may be separated and used after concentrating and disrupting tissues or cells, or may be disrupted and isolated from tissue or cell samples that have been frozen and then thawed. In addition, the mitochondria may be isolated by culturing cells and tissues in vitro, or from samples that have been frozen and then thawed. For example, the mitochondria may be obtained from somatic cells, germ cells, or stem cells.

[0083] Specifically, the somatic cells may be muscle cells, hepatocytes, nerve cells, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells.

[0084] In addition, the stem cells are undifferentiated cells capable of differentiating into various types of tissue cells, and may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells, but are not limited thereto. In this case, the mesenchymal stem cells may be obtained from any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, synovial fluid, testis, amniotic membrane, and placenta.

[0085] In addition, the mitochondria may be obtained from an autologous, allogenic, or xenogenic source. Specifically, autologous mitochondria refer to mitochondria obtained from tissues or cells of the same individual. In addition, allogeneic mitochondria refer to mitochondria obtained from an individual that belongs to the same species as the individual but has a different genotype for an allele. In addition, xenogenic mitochondria refer to mitochondria obtained from an individual that belongs to a different species from the individual. In addition, xenogenic mitochondria refer to mitochondria obtained from an individual that belongs to a different species from the individual.

[0086] Additionally, the mitochondria may be mitochondria isolated from cells. Additionally, the mitochondria may be intact and have mitochondrial activity.

[0087] Meanwhile, when isolating the mitochondria from a specific cell, the mitochondria can be isolated using various known methods, including modified methods such as using a specific buffer solution or utilizing a potential difference and a magnetic field.

[0088] The above mitochondrial separation can be obtained by disrupting and centrifuging tissues or cells in order to maintain mitochondrial activity. In one specific example, the method may include the steps of culturing cells, centrifuging a composition containing the cells a first time to produce a pellet, resuspending the pellet in a buffer solution and homogenizing it, centrifuging the homogenized solution a second time to produce a supernatant, and centrifuging the supernatant a third time to purify mitochondria. At this time, it is preferable to adjust the time for performing the second centrifugation to be shorter than the times for performing the first and third centrifugations in order to maintain cell activity, and the speed can be increased from the first to the third centrifugation.

[0089] Specifically, the first to third centrifugations may be performed at a temperature of about 0°C to about 10°C, preferably about 3°C ​​to about 5°C. In addition, the time for performing the centrifugation may be performed for about 1 minute to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations and the content of the sample.

[0090] In addition, the first centrifugation may be performed at a speed of about 100xg to about 1,000xg, about 200xg to about 700xg, or about 300xg to about 450xg. In addition, the second centrifugation may be performed at a speed of about 1xg to about 2,000xg, about 25xg to about 1,800xg, or about 500xg to about 1,600xg. In addition, the third centrifugation may be performed at a speed of about 100xg to about 20,000xg, about 500xg to about 18,000xg, or about 800xg to about 15,000xg.

[0091] Additionally, pharmaceutically acceptable sugars may be used to stabilize the obtained mitochondria. Specifically, sugars such as sucrose, mannitol, and trehalose may be used, but are not limited thereto. Furthermore, pharmaceutically acceptable pH buffers such as Tris, HEPES, and phosphate may be used, but are not limited thereto.

[0092] Meanwhile, additives such as chelators and antioxidants can be used to remove damage caused by ion outflow after obtaining the mitochondria and to suppress oxidative stress. These additives can include, without limitation, reagents widely known in the art. Examples thereof include, but are not limited to, EDTA, EGTA, Citrate, Glycine, Taurine, ATP, etc.

[0093] Additionally, the above-described mitochondrial isolation can be obtained by thawing, disrupting, and centrifuging frozen cells or tissues to maintain mitochondrial activity. The above-described method for obtaining mitochondria can be performed by the steps of freezing cells or tissues, thawing the cells or tissues, and disrupting the thawed cells and tissues.

[0094] As used herein, the term "modified mitochondrion" refers to a mitochondrion in which a foreign protein is bound to the mitochondrial outer membrane. Specifically, the foreign protein may be a recombinant fusion protein comprising a mitochondrial outer membrane targeting protein and / or a target protein. Here, the mitochondrial outer membrane targeting protein is the same as described above.

[0095] At this time, the target protein may be any one selected from the group consisting of a prodrug conversion enzyme that exhibits activity inside and outside of a cell and a protein capable of binding to a receptor or ligand present in the cell membrane. Here, the fusion protein comprising the prodrug conversion enzyme and the mitochondrial outer membrane targeting protein is as described above.

[0096] In the present invention, the target protein may be a protein capable of binding to a receptor or ligand present in the cell membrane. In this case, the protein capable of binding to a receptor or ligand present in the cell membrane may be an antibody or fragment thereof that specifically binds to a tumor-associated antigen.

[0097] The above tumor-related antigen may be any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine related protein 1 (tyrp1), tyrosine related protein 1 (tyrp2), Brachyury, Mesothelin, Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, Nectin-3 and a combination thereof, but if it is a protein specifically present on the tumor cell surface, It is not limited to this.

[0098] As used herein, the term "antibody" refers to an immunoglobulin molecule that immunologically reacts with a specific antigen, and is a protein molecule that specifically recognizes the antigen. In this case, an antibody fragment refers to a fragment that has the same antigenic determining region (CDR) as the antibody and has antigen-binding activity. Specifically, it may be Fab, scFv, F(ab')2, or a combination thereof.

[0099] When the target protein is an antibody or fragment thereof that binds to a tumor-associated antigen, the target protein may be bound to a mitochondrial outer membrane targeting protein comprising a C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, and may be bound in the following order:

[0100] <Structural formula 5>

[0101] Mitochondrial outer membrane targeting protein-C terminus comprising a C-terminal region of any one of the group consisting of N-terminal-targeting proteins-TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

[0102] At this time, the target protein may be an antibody or a fragment thereof that binds to a tumor-associated antigen.

[0103] Additionally, the foreign protein may further comprise a linker between the target protein and the C-terminal region of any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In this case, the linker is the same as described above and may be linked in the following order:

[0104] <Structural formula 6>

[0105] A mitochondrial outer membrane targeting protein-C terminus comprising a C terminus region selected from the group consisting of N terminus-targeting protein-linker-TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

[0106] At this time, the target protein may be an antibody or a fragment thereof that binds to a tumor-associated antigen.

[0107] Modified mitochondria with these target proteins can be easily introduced into specific targets, allowing efficient entry of mitochondria into specific cells.

[0108] Accordingly, another aspect of the present invention provides a modified mitochondrion comprising a prodrug converting enzyme or a fragment thereof, and an antibody or a fragment thereof that specifically binds to a tumor-associated antigen present in the mitochondrial outer membrane. The prodrug converting enzyme, the tumor-associated antigen, the antibody or a fragment thereof, and the modified mitochondrion are the same as those described above.

[0109] Pharmaceutical composition comprising modified mitochondria as an active ingredient

[0110] Another aspect of the present invention provides a pharmaceutical composition for the prevention and treatment of cancer, comprising the modified mitochondria as an active ingredient. The pharmaceutical composition may be used for the prevention or treatment of cancer.

[0111] The term "cancer" used in this specification is classified as a disease in which normal tissue cells proliferate without limit for some reason and continue to develop rapidly regardless of the living phenomenon of the body or the condition of the surrounding tissues, and the cancer in the present invention may be any one cancer selected from the group consisting of various cancers of the human body, such as breast cancer, lung cancer, pancreatic cancer, glioma, stomach cancer, liver cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma, but is not limited to the above types.

[0112] For the pharmaceutical composition, mitochondria may be included in a concentration of, but is not limited to, about 0.1 μg / mL to about 500 μg / mL, about 0.2 μg / mL to about 450 μg / mL, or about 0.5 μg / mL to about 400 μg / mL. By including mitochondria in the above range, it is easy to adjust the mitochondrial dosage upon administration, and the degree of improvement in the patient's symptoms may be improved. At this time, the dosage of mitochondria can be quantified by quantifying the membrane protein of the isolated mitochondria. Specifically, the isolated mitochondria can be quantified through the Bradford Protein Assay. Specifically, the isolated mitochondria can be quantified using the Bradford protein assay (Paper written by James D. McCully (J Vis Exp. 2014;(91): 51682.).

[0113] In addition, for the pharmaceutical composition, the prodrug conversion enzyme that binds to mitochondria may be included in a concentration of, but is not limited to, 0.1 μg / mL to 500 μg / mL, 0.2 μg / mL to 450 μg / mL, or 0.5 μg / mL to 400 μg / mL. By including the prodrug conversion enzyme in the above range, it is easy to adjust the dosage of the prodrug conversion enzyme upon administration, and the degree of improvement in the patient's symptoms may be improved. The prodrug conversion enzyme is the same as described above.

[0114] In addition, for the pharmaceutical composition, an antibody or fragment thereof that binds to a tumor-associated antigen that enables the transfer of mitochondria to specific cells may be included at a concentration of, but is not limited to, about 0.1 μg / mL to about 500 μg / mL, about 0.2 μg / mL to about 450 μg / mL, or about 0.5 μg / mL to about 400 μg / mL. By including the antibody or fragment thereof in the above range, it is easy to adjust the dosage of the tumor-associated antigen-binding antibody upon administration, and the degree of improvement in the patient's symptoms may be improved.

[0115] For the pharmaceutical composition, when the modified mitochondria include both a prodrug conversion enzyme as a foreign protein and an antibody or fragment thereof that binds to a tumor-associated antigen, the prodrug conversion enzyme and the antibody may be present in the outer membrane of the mitochondria at an appropriate ratio. For example, the ratio of the prodrug conversion enzyme and the antibody or fragment thereof based on the weight ratio may be about 1:10 to about 10:1, about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1, about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, about 1:2 to about 2:1, or about 1:1. Preferably, it may be about 1:5. In this case, the prodrug conversion enzyme may be a prodrug conversion enzyme or a fragment thereof.

[0116] As used herein, the term "prevention" refers to any action that inhibits or delays the development of cancer through the administration of the pharmaceutical composition. Furthermore, "treatment" refers to any action that improves or beneficially alters cancer symptoms through the administration of the pharmaceutical composition.

[0117] As used herein, the term "efficacy" may be determined by one or more parameters, such as survival or disease-free survival over a period of time, such as one year, five years, or ten years. In addition, the parameters may include suppression of the size of at least one tumor in the subject.

[0118] The preferred dosage of the pharmaceutical composition above varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical composition for preventing or treating cancer of the present invention, the active ingredient may be included in any amount (effective amount) depending on the intended use, formulation, and mixing purpose, as long as it can exhibit anticancer activity. Here, "effective amount" refers to the amount of the active ingredient capable of inducing an anticancer effect. This effective amount can be experimentally determined within the ordinary capabilities of those skilled in the art.

[0119] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any non-toxic substance suitable for delivery to a patient. Examples of such carriers include distilled water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0120] Specifically, the pharmaceutical composition may be prepared as a parenteral formulation according to the route of administration by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the tolerable level of the intended subject.

[0121] When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated into the form of an injection, a transdermal administration agent, or a nasal inhaler using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or an isotonic solution such as 5% dextrose.

[0122] The pharmaceutical composition of the present invention may be an injectable formulation. Therefore, to ensure product stability during distribution of the injectable formulation, the pharmaceutical composition of the present invention can be manufactured into a physically and chemically very stable injectable formulation by adjusting the pH using a buffer solution, such as an acid solution or phosphate solution suitable for injectable formulations.

[0123] Specifically, the pharmaceutical composition of the present invention may include water for injection.

[0124] The above-mentioned water for injection is distilled water made for dissolving solid injections or diluting water-soluble injections, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactate-Ringer's solution, or a phosphate buffer solution or sodium dihydrogen phosphate-citrate buffer solution having a pH range of about 3.5 to 7.5.

[0125] The pharmaceutical composition of the present invention may further comprise a stabilizer or a solubilizer. For example, the stabilizer may be pyrosulfite or ethylene diaminetetraacetic acid, and the solubilizer may be hydrochloric acid, acetic acid, potassium phosphate hydroxide, potassium bicarbonate, potassium carbonate, or Tris. In one specific example, the pharmaceutical composition may comprise a mixed preservative solution such as a trehalose-tris-glycine (TTG) solution, which may be commonly used in pharmaceutically acceptable drug preparations.

[0126] Specifically, the pharmaceutical composition of the present invention may include an injectable liquid composition in addition to modified mitochondria. In this case, the modified mitochondria are the same as those described above. By including the injectable liquid composition, the pharmaceutical composition of the present invention is a composition for the prevention or treatment of diseases related to mitochondrial function, and can suppress thrombosis that may be caused by mitochondrial aggregation, platelet reduction and aggregation, etc. when administering mitochondria via injection, and can maintain and / or enhance mitochondrial stability and also stably maintain mitochondrial activity.

[0127] Here, the liquid composition may include glycine, sugar, and a buffer.

[0128] The glycine may be present in the injectable liquid composition at a concentration of about 15 mM or more, but is not limited thereto, and specifically, may be present at a concentration of about 15 mM to about 150 mM, about 17 mM to about 130 mM, about 20 mM to about 120 mM, about 22 mM to about 110 mM, or about 25 mM to about 100 mM. In addition, the glycine may be used together with an amino acid selected from the group consisting of, but is not limited to, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0129] In addition, the sugar included in the liquid composition for injection may be at least one selected from the group consisting of, but not limited to, sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin. In particular, the sugar may be trehalose, mannitol, or sucrose. Preferably, the sugar may be trehalose.

[0130] The buffer included in the above injectable liquid composition may be selected from the group consisting of, but not limited to, a tris buffer, a HEPES (hydroxyethyl piperazine ethane sulfonic acid) buffer, a MOPS (3-(N-morpholino)propane sulfonic acid) buffer, and an acetate or phosphate-containing buffer. Preferably, the buffer may be an injectable tris buffer.

[0131] At this time, the pH of the buffer is not limited thereto, but may be in the range of about 7.0 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5.

[0132] Additionally, the buffer may be present in the injectable liquid composition at a concentration of, but not limited to, about 5 mM to about 50 mM, about 8 mM to about 40 mM, about 10 mM to about 35 mM, about 13 mM to about 30 mM, or about 15 mM to about 25 mM.

[0133] The above-described liquid composition for injection may have an osmolarity in the range of about 200 to about 400 mOsm, about 230 to about 380 mOsm, about 250 to about 350 mOsm, about 260 to about 320 mOsm, about 270 to about 330 mOsm, or about 280 to about 300 mOsm. In this case, the osmolarity in the above range facilitates long-term storage at a temperature of 2°C to 8°C or higher, while making the composition suitable for parenteral administration, for example, intravascular, intramuscular, or subcutaneous injection, without causing side effects to a subject.

[0134] The term "osmolarity" as used herein refers to the moles of solute contributing to the osmotic pressure of a solution per kilogram of solvent, and osmolarity is determined by measuring the freezing point depression of a sample using an osmometer.

[0135] Additionally, the above injectable liquid composition may further comprise a chelating agent.

[0136] The chelating agent may be, but is not limited to, one or more selected from the group consisting of injectable grades of EGTA (ethylene glycol tetraacetic acid), EDTA (ethylene diamine tetraacetic acid), and BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). The chelating agent may eliminate damage caused by ion outflow after obtaining mitochondria included in the injectable liquid composition.

[0137] In addition, the pharmaceutical composition may include, as additives, antioxidants, ATP, magnesium, etc., which are effective in maintaining the function and activity of mitochondria.

[0138] The pharmaceutical composition of the present invention may be stored in a container selected from the group consisting of a vial, a cartridge, a syringe, and an autoinjector. Furthermore, the container containing the pharmaceutical composition may be stored at room temperature, a refrigerated temperature of 2°C to 8°C, or a temperature of 25°C to 40°C until administered to a subject in need of treatment.

[0139] The above object may be a mammal such as, but not limited to, a human, a dog, a cow, a horse, a pig, a sheep, a goat, a cat, a mouse, a rabbit, a rat, and preferably a human.

[0140] Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "therapeutically effective amount" or "pharmaceutically effective amount" refers to an amount of a compound or composition that is effective in preventing or treating a target disease, and is sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects. The level of the effective amount may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the excretion rate, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. In one specific embodiment, the therapeutically effective amount refers to an amount of a drug that is effective in treating cancer.

[0141] The term "administration" as used herein means introducing a given substance into an individual by an appropriate method, and the route of administration of the composition may be through any common route as long as it can reach the target tissue. Examples of such routes include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, and rectal administration.

[0142] The preferred dosage of the pharmaceutical composition of the present invention is about 0.01 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 4 mg / kg, or about 0.25 mg / kg to about 2.5 mg / kg of mitochondria per dose, based on the body weight of the subject to be administered, but is not limited thereto. That is, in terms of cell activity, it is most preferable that the modified mitochondria containing the anticancer agent are administered in the above range of amounts based on the body weight of the subject in which cancer tissue exists. In addition, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, and preferably 5 times. At this time, the administration interval can be 1 to 7 days or 2 to 5 days, and preferably 3 days. Such dosage should not be construed as limiting the scope of the present invention in any aspect.

[0143] At this time, the pharmaceutical composition may be administered parenterally using an 18G to 32G needle in a volume of, but not limited to, about 5 mL or less, about 3 mL or less, or about 2 mL or less.

[0144]

[0145] The term "subject" above refers to a subject to which the composition of the present invention can be applied (prescribed), and may be a subject suffering from cancer. In addition, the subject may be a mammal such as a rat, mouse, or livestock, including a human, but may preferably be a human. In addition to the modified mitochondria, the composition of the present invention may further include any compound or natural extract that has already been verified for safety and is known to have anticancer activity, in order to increase or enhance anticancer activity. In this case, the pharmaceutical composition and the compound or natural extract having anticancer activity may be administered simultaneously or sequentially.

[0146] Uses of modified mitochondria

[0147] Another aspect of the present invention provides the use of the modified mitochondria for preparing a pharmaceutical composition for treating cancer.

[0148] Here, the cancer, treatment, and modified mitochondria are identical to those described above. The modified mitochondria can be used as a means for delivering a foreign protein. Specifically, the modified mitochondria can be used as a means for delivering a prodrug converting enzyme or a fragment thereof capable of functioning intracellularly as a target protein into the cell. Furthermore, by incorporating an antibody or a fragment thereof that binds to the modified mitochondrial tumor-associated antigen into the mitochondrial outer membrane, tumor cells can be effectively targeted, allowing the target protein, the prodrug converting enzyme, to be introduced into the cell.

[0149] Therefore, the modified mitochondria can be used as an effective protein delivery system. Here, the prodrug converting enzyme or fragment thereof is the same as described above.

[0150] Cancer treatment using modified mitochondria

[0151] Another aspect of the present invention provides a method for treating cancer comprising administering the modified mitochondria to a subject.

[0152] At this time, the modified mitochondria can be administered in combination with an anticancer prodrug, and the anticancer prodrug can be any one selected from the group consisting of 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazaphosphorine, 6-methylpurine deoxyriboside, and indole-3-acetic acid (IAA). However, any compound that is converted from a non-toxic compound to a toxic anticancer agent by the prodrug conversion enzyme can be included.

[0153] Additionally, when administering modified mitochondria containing one or more prodrug conversion enzymes or fragments thereof, one or more of the prodrugs may be combined and co-administered, depending on the combined prodrug conversion enzymes. In this case, the modified mitochondria may contain individual prodrug conversion enzymes, or may be mitochondria modified to contain one or more prodrug conversion enzymes.

[0154] The modified mitochondria, cancer and treatment are the same as described above.

[0155] The subject may be a subject suffering from cancer. In addition, the subject may be a mammal, preferably a human.

[0156] Cancer treatment kit containing modified mitochondria

[0157] Another aspect of the present invention provides a kit for preventing and treating cancer, comprising the modified mitochondria and a prodrug. The modified mitochondria, prodrug, cancer, and prevention and treatment are as described above.

[0158] In addition, when the kit includes modified mitochondria comprising one or more prodrug conversion enzymes or fragments thereof, the kit may include one or more combinations of the prodrugs depending on the combined prodrug conversion enzymes or fragments thereof. In this case, the modified mitochondria may be those comprising each prodrug conversion enzyme or fragment thereof, or may be mitochondria comprising one or more prodrug conversion enzymes or fragments thereof.

[0159] Method for producing modified mitochondria

[0160] Another aspect of the present invention provides a method for producing modified mitochondria in which a prodrug converting enzyme or a fragment thereof is expressed on the outer membrane of a mitochondrion, comprising the steps of: expressing a prodrug converting enzyme or a fragment thereof on the outer membrane of a mitochondrion within a cell; and isolating the mitochondria. Here, the prodrug converting enzyme and the modified mitochondria are the same as those described above.

[0161] In addition, another aspect of the present invention provides a method for producing modified mitochondria in which a prodrug conversion enzyme or a fragment thereof and an antibody or a fragment thereof that specifically binds to a tumor-associated antigen are expressed on the outer membrane of mitochondria, comprising the steps of: expressing a prodrug conversion enzyme or a fragment thereof, and an antibody or a fragment thereof that specifically binds to a tumor-associated antigen on the outer membrane of mitochondria within a cell; and isolating the mitochondria. Here, the prodrug conversion enzyme, the tumor-associated antigen, the antibody, and the modified mitochondria are the same as those described above.

[0162] At this time, the prodrug conversion enzyme or fragment thereof and the antibody or fragment thereof that specifically binds to the tumor-associated antigen may be present in the outer membrane of the mitochondria at an appropriate ratio. For example, the ratio of the enzyme or fragment thereof and the antibody or fragment thereof based on the weight ratio may be about 1:10 to about 10:1, about 1:9 to about 9:1, about 1:8 to about 8:1, about 1:7 to about 7:1, about 1:6 to about 6:1, about 1:5 to about 5:1, about 1:4 to about 4:1, about 1:3 to about 3:1, about 1:2 to about 2:1, or about 1:1. Preferably, it may be about 1:5.

[0163] Method for producing fusion proteins

[0164] Another aspect of the present invention provides a method for producing a fusion protein, comprising isolating a fusion protein from a cell into which a vector encoding the fusion protein, which comprises a mitochondrial outer membrane targeting protein and a prodrug converting enzyme, has been introduced. The fusion protein is the same as described above. The cell may be a prokaryotic cell or a eukaryotic cell.

[0165]

[0166] Hereinafter, preferred embodiments are presented to help understand the present invention.

[0167] However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[0168] Example 1. Production of a yeast-derived cytosine deaminase expression vector capable of fusion to the mitochondrial outer membrane.

[0169] Example 1.1. Construction of a yeast-derived cytosine deaminase expression vector.

[0170] In order to express yeast-derived cytosine deaminase as a recombinant protein, gene synthesis was requested from Bionics Co., Ltd., and the expression vector loaded with the yeast-derived cytosine deaminase gene was named pUC57-yCD, and the base sequence of yeast-derived cytosine deaminase is the same as the base sequence of SEQ ID NO: 3.

[0171] Example 1.2. Construction of a yeast-derived cytosine deaminase expression vector capable of fusion to the mitochondrial outer membrane.

[0172] In order to produce a yeast-derived cytosine deaminase protein in a fused form with TOM7 that binds to the mitochondrial outer membrane, an expression vector capable of expressing a yeast-derived cytosine deaminase in a fused form with TOM7 was produced.

[0173] Primer sequences: RyCD5'- AAA AAA GAA TTC ATG GTG ACA GGG GGA ATG GCA AGC -3'Sequence number 4yCD-myc-TOM7-F5'-GGT TTG AAG ATA TTG GTG AGG AAC AAA AAC TCA TCT CAG AAG AGG ATC TGG TGA AGC TGA GCA AAG AGG CCA AG -3'Sequence number 5yCD-myc-TOM7-R5'-CTT GGC CTC TTT GCT CAG CTT CAC CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CTC ACC AAT ATC TTC AAA CC -3'Sequence number 6XTOM7(T)5'- AAA AAA CTC GAG TTA TCC CCA AAG TAG GCT CAA AAC -3'Sequence number 7

[0174] Using the plasmid pUC57-yCD obtained by the method of Example 1.1 as a template, 0.2 pmol primer (RyCD) and 0.2 pmol primer (yCD-myc-TOM7-R) were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase, and then 25 amplification reactions were performed at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute in a polymerase chain reaction apparatus to obtain the gene yCD-myc. The DNA fragment amplified by the above method was named N-yCD-myc.

[0175] Plasmid pT-TOM7 obtained by the method disclosed in Korean Patent Publication No. 2019-0124656 was used as a template, and 0.2 pmol primer (yCD-myc-TOM7-F) and 0.2 pmol primer (XTOM7(T)) were added, 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase were mixed, and then 25 amplification reactions were performed at 95°C for 40 seconds, 58°C for 30 seconds, and 72°C for 1 minute in a polymerase chain reaction apparatus to obtain the gene myc-TOM7. The DNA fragment amplified through the above method was named C-myc-TOM7.

[0176] Using the amplified DNA fragments N-yCD-myc and C-myc-TOM7 as templates, 0.2 pmol primer (RyCD), 0.2 pmol primer (XTOM7(T)) were mixed with 0.2 nM dNTP, 1x AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase, and then 25 amplification reactions were performed in a polymerase chain reaction apparatus at 95°C for 40 sec, 58°C for 30 sec, and 72°C for 1 min to obtain the yeast-derived cytosine deaminase gene yCD-myc-TOM7 (SEQ ID NO: 26) fused with the amplified TOM7.

[0177] The amplified yCD-myc-TOM7 gene was digested with restriction enzymes EcoRI and XhoI, and DNA fragments were obtained by 2% agarose gel electrophoresis. The fragments were then inserted into pcDNA3.1 vector (Invitrogen, USA) digested with restriction enzymes EcoRI and XhoI using T4 DNA ligase to produce plasmid pCMV-yCD-myc-TOM7 (Fig. 1).

[0178] Example 2. Transformation and expression of yeast-derived cytosine deaminase gene into human fetal kidney cells.

[0179] To introduce pCMV-yCD-myc-TOM7, a cytosine deaminase expression vector produced in the same manner as Example 1, into a human fetal kidney cell line (HEK293), Lipofectamine 2000 (Thermo Scintific, 11668019) was used and the experimental method provided by the manufacturer was referenced.

[0180] HEK293 cells were cultured in DMEM (hyclone, SH30243) culture medium containing 10% fetal bovine serum (FBS) (Gibco, 12483-020) and penicillin-streptomycin (Hyclone, SV30010) at 37°C in 5% CO2.

[0181] For plasmid introduction, HEK293 cells were seeded at 1X10 in 6-well plates. 6 The cells were seeded at 10 cells / well and cultured for 24 hours. 2.5 μg of plasmid and 12.5 μl of Lipofectamine 2000 were added to Opti-MEM (Gibco, 31985-070) and mixed to form a DNA-Lipid complex, which was then treated to introduce the plasmid into HEK293 cells. After 24 hours, the cells were washed with PBS, replaced with fresh culture medium, and cultured for an additional 24 hours.

[0182] To confirm the expression of the introduced gene in HEK293 cells, cells were lysed using cell lysis buffer (Cell Signaling Technologies, 9803), proteins were extracted, and Western blotting was performed. To confirm the expressed yeast-derived cytosine deaminase, anti-myc antibody (Roche, 11667149001) was used as the primary antibody, and anti-mouse IgG HRP (Abcam, 6789) was used as the secondary antibody.

[0183] As a result, as shown in Fig. 2, it was confirmed that yeast-derived cytosine deaminase was expressed in HEK293 cells.

[0184] Example 3. Measurement of yeast-derived cytosine deaminase enzyme activity

[0185] In order to measure the enzyme activity of yeast-derived cytosine deaminase expressed from HEK293 cells produced in Example 2, the amount of 5-FU converted from 5-FC was measured using the absorbance at 314 nm.

[0186] Specifically, HEK293 or cytosine deaminase-expressing HEK293 (HEK293-yCD) cells were seeded at 1X10 in 6-well plates. 6 Cells were seeded at 10 cells / well and cultured for 24 hours. After 24 hours of culture, cells were harvested by treatment with 0.05% trypsin-EDTA (gibco, 25300) and washed twice with PBS. Afterwards, the cells were resuspended in PBS (pH 7.4) and disrupted using an ultrasonic disruptor. The disrupted cells were centrifuged at 2,000 x g for 10 minutes. The separated supernatant was collected and transferred to a new microtube, treated with 5 mM 5-FC (sigma, F7129), and incubated at 37°C.

[0187] The reaction solution was taken at intervals of 1 hour, 2 hours, 4 hours, and 16 hours, and 200 μl of the reaction solution was dispensed into a 96-well plate, and the absorbance was measured at 314 nm using an ELISA reader. At this time, it was confirmed that only 5-FU was measured and not 5-FC at an absorbance of 314 nm, and the amount of 5-FU obtained from the reaction solution was quantified using a standard curve for each concentration of 5-FU (sigma, F6627).

[0188] Figure 3 shows the results of a standard curve for 5-FU concentration, and Figure 3 B shows the results of a graph showing the amount of detected 5-FU over time. 5-FU was not detected in the reaction solution obtained from HEK293 cells, but 12.3 mM of 5-FU was detected in the reaction solution obtained from HEK293 cells expressing cytosine deaminase, indicating that 5-FC was converted to 5-FU by yeast-derived cytosine deaminase.

[0189] Example 4. Measurement of cytotoxicity by 5-FC, a prodrug

[0190] HEK293 cell line produced in Example 1 or HEK293 (HEK293-yCD) cells expressing cytosine deaminase were seeded at 5,000 cells / well in a 96-well plate and cultured for 24 hours. After 24 hours of culture, the prodrug 5-FC (sigma, F7129) was treated at a concentration of 500 μg / mL and cultured for 120 hours. The death degree of HEK293 cells cultured for 120 hours was observed under an optical microscope, and cell proliferation was measured using the WST-1 (Roche, 11644807001) assay.

[0191] As a result, as shown in Figures 5 and 6, in the 5-FC untreated group, no cell proliferation inhibitory effect was observed in either HEK293 or HEK293 (HEK293-yCD) cells expressing cytosine deaminase. On the other hand, in the 5-FC treated group, it was confirmed that cell growth was inhibited by 90% in HEK293-yCD cells compared to HEK293 cells.

[0192] Example 5. Production of a cell line containing stabilized yeast-derived cytosine deaminase.

[0193] In order to produce a cell line in which cytosine deaminase is continuously expressed in the HEK293 (HEK 293-yCD) cells produced in Example 2 above, 1,000 ㎍ / mL of the antibiotic G418 (Promega, V7983) was administered to select cells with resistance, thereby producing a transformed cell line (stable cell line).

[0194] Specifically, the HEK293 (HEK 293-yCD) cells expressing the cytosine deaminase selected in Example 2 were cultured in a 100 mm dish plate at a cell density of approximately 80%, and then the culture medium containing 1,000 μg / mL of G418 (Promega, V7983) was replaced every other day for two weeks to induce the death of cells that were not introduced with the plasmid. After two weeks of G418 treatment, 80-90% of the cells were dead, and when colonies formed by the surviving cells growing and dividing were observed, each colony was transferred to a 24-well plate using 0.05% trypsin-EDTA (gibco, 25300) and cultured. During the subsequent culture process, the culture medium containing 1,000 μg / mL of G418 was always used.

[0195] When the selected colonies grew to a cell density of 60% or greater in a 24-well plate, colonies with high protein expression were selectively isolated through Western blotting. The selected colonies were scaled up and cultured.

[0196] As a result, as shown in Fig. 7, HEK293-CD#5, HEK293-CD#6, and HEK293-CD#7 colonies with high protein expression levels were scaled up and cultured, and subsequent experiments were conducted using the HEK293-CD#5 colony.

[0197] Example 6. Confirmation of the intracellular expression location of yeast-derived cytosine deaminase and MT yCD manufacturing

[0198] To confirm whether cytosine deaminase is expressed in the mitochondria of HEK293 (HEK293-yCD) cells in HEK293-CD#5 produced in Example 5 above, Western blot and cell immunochemical staining were performed.

[0199] Western blot was performed using the following method. Specifically, HEK293-CD#5 cells expressing cytosine deaminase were cultured at 1X10 7 After harvesting cells and washing them twice with PBS, the cells were suspended in 300 ㎕ of SHE (Sucrose 250 mM, HEPES 20 mM (pH 7.4), EGTA 2 mM) buffer and disrupted using an ultrasonic disruptor. The disrupted cells were centrifuged at 2,000Хg for 10 minutes. The separated supernatant was obtained and transferred to a new microtube, centrifuged at 12,000Хg for 15 minutes to separate the cytoplasm from the supernatant, and the precipitated portion was separated into mitochondria.

[0200] The above fractions were each subjected to electrophoresis and Western blotting was performed. To identify expressed cytosine deaminase, mitochondrial marker, and cytoplasmic marker proteins, anti-myc antibody (Roche, 11667149001), anti-COX4 antibody (abcam, 33985), and anti-β-tubulin antibody (Thermo, MA5-16308) were used as primary antibodies, respectively. Anti-mouse IgG HRP (abcam, 6789) or anti-rabbit IgG HRP (abcam, 6721) was used as secondary antibodies.

[0201] As a result, as shown in Figure 8, cytosine deaminase was confirmed to exist in the mitochondrial fraction together with COX4, which was used as a mitochondrial marker. In addition, it was confirmed that it was not present in the fraction containing β-tubulin, a cytoplasmic marker.

[0202] Cell immunochemical staining was performed using the following method. Specifically, HEK293 cells and HEK293-CD#5 cell lines were seeded in 24-well plates at a density of 5 x 10 4 Cells were seeded at 10 cells / well and cultured for 24 hours. After 24 hours of culture, the cells were washed twice with PBS and fixed with 4% formaldehyde (Thermo, 28908). After removing the formaldehyde, anti-myc antibody (Roche, 11667149001) and anti-MTCO2 antibody (abcam, 79393) were diluted 1:1000 in PBS containing 0.1% bovine serum albumin (BSA), 0.1% TritonX-100, and 1% goat serum (life technologies, 500622) and treated to the cells, and incubated at 4°C for 18 hours.

[0203] After the reaction was completed, the primary antibody was removed by washing four times with PBS containing 0.1% BSA, and then the cells were treated with Alexa Fluor®488-labeled anti-mouse IgG antibody (Invitrogen, 11001) and anti-rabbit IgG antibody (Invitrogen, 21428) diluted 1:1000 in PBS containing 0.1% BSA, and reacted at room temperature for approximately 1 hour. After the reaction was completed, the cells were washed four times each with PBS containing 0.1% BSA and distilled water, and the nuclei were stained using mounting media containing DAPI (VECTASHIELD, H-1200), and the cells were observed using a confocal microscope.

[0204] As a result, as shown in Fig. 9, it was confirmed that the positions of the red fluorescence labeling MTCO-2, a mitochondrial positive marker, and the green fluorescence labeling cytosine deaminase were identical.

[0205] Mitochondria obtained from the mitochondrial fractionation method in the above HEK293 cells and HEK293-CD#5 cells were designated as MT or MT, respectively. yCDIt was named as. At this time, MT is a mitochondria obtained from control HEK293 cells in which yeast-derived cytosine deaminase is not expressed, and MT yCD It is a type of mitochondrion isolated from HEK293-CD#5 cell line, in which cytosine deaminase is fused to the mitochondrial outer membrane.

[0206] Example 7. Evaluation of anticancer activity of mitochondria containing yeast-derived cytosine deaminase against cancer cells.

[0207] MT or MT obtained by the same method as in Example 6 above yCD After treating human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2), the morphological changes of cancer cells were observed using an optical microscope by treating with prodrug 5-FC, and cancer cell death and cell proliferation were measured using the WST-1 assay.

[0208] AsPc-1, Capan-1, and Capan-2 cells were cultured in RPMI-1640 (welgene, LM011-51) containing 10% FBS and penicillin-streptomycin. MIA Paca-2 cells were cultured in DMEM (hyclone, SH30243) containing 10% FBS and penicillin-streptomycin.

[0209] Specifically, each human pancreatic cancer cell line (AsPc-1, Capan-1, Capan-2, and MIA Paca-2) was seeded at 5,000 cells / well in a 96-well plate and cultured for 24 hours. After 24 hours of culture, MT or MT obtained in the same manner as in Example 6 was cultured. yCDEach well was treated with 0 ㎍, 0.5 ㎍, 1 ㎍, or 2 ㎍ and incubated for 2 hours. After 2 hours, the cells were washed once with culture medium to remove mitochondria that were not transferred to the cancer cell line. Afterwards, the prodrug 5-FC was treated at a concentration of 0 ㎍ / mL, 20 ㎍ / mL, 100 ㎍ / mL, or 500 ㎍ / mL and incubated for 120 hours.

[0210] As a result, as shown in Figures 10 to 12, in the 5-FC untreated group, MT or MT yCD No inhibitory effect on cell proliferation was observed by treatment. On the other hand, in the 5-FC treatment group, MT yCD It was confirmed that cell growth was inhibited by about 60% to 90% or more compared to the MT treatment group by treatment. In addition, MT yCD It can be seen that the cell death rate increases in a concentration-dependent manner as the treatment concentration or the treatment concentration of 5-FC increases.

[0211] Example 8. MT through Western blot analysis yCD Confirmation of natural apoptosis of cancer cells by treatment

[0212] MT or MT obtained by the same method as in Example 6 above yCD After treatment with human pancreatic cancer cell lines, prodrug 5-FC was treated and Western blot was performed using antibodies against apoptosis markers.

[0213] Specifically, human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2 and MIA Paca-2) were cultured in 6-well plates at 5X10 5 Each well was inoculated with cells and cultured for 24 hours. After 24 hours of culture, MT or MT obtained in the same manner as in Example 6 yCD Each well was treated with 50 μg of the mitochondria and incubated for 2 hours. After 2 hours, the cells were washed once with culture medium to remove mitochondria that were not transferred to the cancer cell line.

[0214] After treatment with the prodrug 5-FC at a concentration of 100 μg / mL and incubation for 96 hours, cells were lysed using cell lysis buffer (Cell Signaling Technologies, 9803), and proteins were extracted. The extracted proteins were quantified using the BCA assay (Thermo Scientific, 23225). For each sample, an equal amount of protein was electrophoresed, followed by Western blotting.

[0215] Primary antibodies used were anti-phospho-p53 antibody (ABclonal, AP0762), anti-cleaved caspase 3 antibody (Cell signaling, 9664), and anti-β-tubulin antibody (Thermo MA5-16308). Secondary antibodies used were anti-mouse IgG HRP (abcam, 6789) or anti-rabbit IgG HRP (abcam, 6721). Protein amounts were corrected for β-tubulin protein expression.

[0216] As a result, as shown in Fig. 13, MT yCD In the 5-FC treatment group, cleaved caspase-3 and phosphorylated p53, which are observed during apoptosis, were observed only in the 5-FC treatment group. In addition, MT or MT yCD Or, in the 5-FC only treatment group and the 5-FC treatment group, no expression of apoptosis markers was observed similar to the control group (untreated).

Claims

1. A fusion protein comprising a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein.

2. In paragraph 1, A fusion protein wherein the prodrug converting enzyme is an enzyme having an activity of converting a non-toxic prodrug into a toxic drug.

3. In paragraph 3, A fusion protein, wherein the prodrug converting enzyme is any one selected from the group consisting of cytosine deaminase (CD), carboxyl esterase (CE), simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine-nucleoside-phosphorylase (PNP), and horseradish peroxidase (HRP).

4. In paragraph 1, A fusion protein wherein the mitochondrial outer membrane targeting protein is any one selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

5. In paragraph 1, A fusion protein in which the mitochondrial outer membrane targeting protein and a prodrug converting enzyme or a fragment thereof are linked from the N-terminus to the C-terminus, when the mitochondrial outer membrane targeting protein is TOM20, TOM70 or OM45.

6. In paragraph 1, A fusion protein in which a prodrug converting enzyme or a fragment thereof and a mitochondrial targeting protein are linked from the N-terminus to the C-terminus, wherein the mitochondrial outer membrane targeting protein is any one selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

7. In paragraph 1, A fusion protein, wherein a prodrug converting enzyme or a fragment thereof and a mitochondrial outer membrane targeting protein are connected by a linker.

8. A polynucleotide encoding a fusion protein according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide of paragraph 8.

10. A transformed cell into which the vector of clause 9 has been introduced.

11. Modified mitochondria in which prodrug converting enzyme or a fragment thereof is present in the outer mitochondrial membrane.

12. In paragraph 11, Modified mitochondria wherein an antibody or fragment thereof that specifically binds to a tumor-associated antigen is further present in the mitochondrial outer membrane.

13. In paragraph 11, A modified mitochondrion, wherein the prodrug converting enzyme is any one selected from the group consisting of cytosine deaminase (CD), carboxyl esterase (CE), simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine-nucleoside-phosphorylase (PNP), and horseradish peroxidase (HRP).

14. In paragraph 12, Modified mitochondria, wherein the tumor-related antigen is any one selected from the group consisting of CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), mucin 1 cell surface associated (MUC-1), prostatic acid phosphatase (PAP), prostate specific antigen (PSA), survivin, tyrosine related protein 1 (tyrp1), tyrosine related protein 1 (tyrp2), Brachyury, Mesothelin, Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), ERBB2, Wilms tumor protein (WT1), FAP, EpCAM, PD-L1, ACPP, CPT1A, IFNG, CD274, FOLR1, EPCAM, ICAM2, NCAM1, LRRC4, UNC5H2 LILRB2, CEACAM, Nectin-3, and combinations thereof.

15. In paragraph 12, Modified mitochondria, wherein the fragment of the above antibody is any one selected from the group consisting of Fab, Fab', scFv and F(ab)2.

16. A pharmaceutical composition for preventing and treating cancer, comprising the modified mitochondria of claim 11 or 12 as an active ingredient.

17. In paragraph 16, A pharmaceutical composition for preventing and treating cancer, wherein the cancer is any one selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, glioma, stomach cancer, liver cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myeloid leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.

18. Use of the modified mitochondria of claim 11 for preparing a pharmaceutical composition for treating cancer.

19. A method for treating cancer comprising administering to a subject the modified mitochondria of paragraph 11.

20. In paragraph 19, A method for treating cancer, comprising administering the modified mitochondria in combination with an anticancer prodrug.

21. In paragraph 20, A method for treating cancer, wherein the anticancer prodrug is any one selected from the group consisting of 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazaphosphorine, 6-methylpurine deoxyriboside, and indole-3-acetic acid (IAA).

22. A kit for treating cancer comprising the modified mitochondria and prodrug of Article 11.

23. In paragraph 22, A kit for treating cancer, wherein the prodrug is any one selected from the group consisting of 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazaphosphorine, 6-methylpurine deoxyriboside, and indole-3-acetic acid (IAA).

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