Modified mitochondria comprising prodrug invertase and uses thereof

Abstract

The present invention relates to a modified mitochondrial comprising, on the outer membrane of the mitochondrial, an antibody that binds to a cancer cell-specific protein and a cytosine deaminase. It is proved that when the mitochondria and 5-FC are jointly used for treating a pancreatic cancer cell line, the cytotoxicity induced by 5-FC treatment is remarkably enhanced. The effect is proved as follows: 5-FC is converted into 5-FU by cytosine deaminase expressed on a mitochondrial outer membrane, so that pancreatic cancer cells are induced to generate apoptosis. Therefore, the modified mitochondria according to the present invention can be effectively used in anticancer therapy using 5-FC as an anticancer agent.

Description

Technical Field

[0001] The present invention provides a modified mitochondria wherein a prodrug converting enzyme is located on the outer mitochondrial membrane, and a pharmaceutical composition for the prevention and treatment of cancer, the active ingredient of which includes the modified mitochondria. Background Technology

[0002] Prodrug-converting enzymes are enzymes that convert harmless or low-toxic prodrugs into highly cytotoxic drugs. Furthermore, upon administration of a prodrug, these enzymes can induce apoptosis in surrounding cells. Cytosine deaminase (CD), one such prodrug-converting enzyme, is known to convert the non-toxic prodrug 5-fluorocytosine (5-FC) in mammalian cells into the highly cytotoxic drug 5-fluorouracil (5-FU).

[0003] 5-FU is known to induce apoptosis by inhibiting protein and DNA synthesis. Intracellularly, 5-FU is converted to 5-FUTP by cellular enzymes, which inhibits protein synthesis by suppressing mRNA biosynthesis. Furthermore, 5-FU can also be converted to 5-fluoro-dUTP (5-FdUTP) by cellular enzymes, which induces dTTP depletion and inhibits DNA replication by inhibiting thymidine synthase activity.

[0004] The most common way to use cytosine deaminase as an anticancer agent is through gene therapy using viral genes as vectors. However, the use of viruses as delivery vectors is severely limited due to safety concerns. Another approach is to express the apoptosis gene cytosine deaminase in mesenchymal stem cells with homing effects, and then inject these mesenchymal stem cells. In this case, 5-FC is converted into 5-FU within the mesenchymal stem cells and secreted extracellularly, thereby producing a bystander effect that kills neighboring cancer cells (Korean Patent Application Publication No. 10-2007-0036289).

[0005] However, mesenchymal stem cells have a very weak homing effect on cancer cells, making it difficult to overcome the potential problem of insufficient cytotoxicity. Invention Details

[0007] Technical issues

[0008] The present invention aims to provide a substance that, by coupling a prodrug-converting enzyme to an antibody capable of binding to cancer cell-specific proteins on the outer mitochondrial membrane, transports the prodrug-converting enzyme to the vicinity of cancer cells in a cancer cell-specific manner, and by administering the prodrug, activates an anticancer agent in a cancer cell-specific manner; and to provide a composition for treating cancer using said substance; and to provide a method for treating cancer using said substance.

[0009] Technical solutions to the problem

[0010] To address the aforementioned problems, in one aspect of the present invention, a fusion protein is provided, the fusion protein comprising a prodrug convertase or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0011] In another aspect of the invention, a polynucleotide encoding the fusion protein, a vector loaded with the polynucleotide, and a transformed cell introduced into the vector are provided.

[0012] In another aspect of the invention, a modified mitochondria is provided, wherein a prodrug convertase or a fragment thereof is located on the outer mitochondrial membrane.

[0013] In another aspect of the invention, a modified mitochondria is provided, wherein a prodrug converting enzyme or a fragment thereof, and an antibody or a fragment thereof capable of specifically binding to tumor-associated antigens are located on the outer mitochondrial membrane.

[0014] In another aspect of the invention, a pharmaceutical composition for the prevention and treatment of cancer is provided, the pharmaceutical composition comprising the modified mitochondria as an active ingredient.

[0015] In another aspect of the invention, the use of the modified mitochondria in the preparation of a medicament for treating cancer is provided.

[0016] In another aspect of the invention, a method for treating cancer is provided, the method comprising administering the modified mitochondria to a subject.

[0017] In another aspect of the invention, a kit for treating cancer is provided, the kit comprising the modified mitochondria and a prodrug.

[0018] Invention Effects

[0019] When pancreatic cancer cells are treated with the modified mitochondria of the present invention in combination with 5-FC, the outer membrane of the modified mitochondria expresses antibodies capable of binding cancer cell-specific proteins and the prodrug-converting enzyme cytosine deaminase, exhibiting higher cytotoxicity compared to the group treated with unmodified mitochondria. Based on the above results, the modified mitochondria of the present invention containing the prodrug-converting enzyme can be effectively used in anticancer therapy using 5-FC as an anticancer agent. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the expression vector (pCMV-yCD-myc-TOM7), which is used to express a yeast-derived cytosine deaminase fragment fused with mitochondria.

[0021] Figure 2This figure shows the results of Western blotting detection of yeast-derived cytosine deaminase fragment expression after pCMV-yCD-myc-TOM7 was transfected into the human embryonic kidney cell line (HEK293). Lane 1 represents HEK293 cells, Lane 2 represents cells transformed with the empty vector (HEK293-EV), and Lane 3 represents cells transfected with a vector carrying the gene encoding yeast-derived cytosine deaminase (HEK293-CD).

[0022] Figure 3 To measure the absorbance of 5-FC (5-fluorocytosine) and 5-FU (5-fluorouracil) at 314 nm and to plot standard curves with absorbance at each concentration.

[0023] Figure 4 The figure shows the results of detecting the enzyme activity of cytosine deaminase expressed in HEK293-CD cells in converting 5-FC to 5-FU.

[0024] Figure 5 The image shows the morphological changes of HEK293 cells and HEK293-CD cells transformed with a vector encoding the cytosine deaminase gene after treatment with 5-FC (a precursor to the anticancer drug 5-FU) under an optical microscope.

[0025] Figure 6 The figure shows the cytotoxicity assay results of HEK293 cells and HEK293-CD cells transformed with a vector encoding the cytosine deaminase gene after treatment with 5-FC (a precursor to the anticancer drug 5-FU).

[0026] Figure 7 Western blotting results of HEK293-CD stable cell lines for screening yeast-derived cytosine deaminase (yCD-myc-TOM7).

[0027] Figure 8 This is a Western blotting result showing the co-localization of yeast-derived cytosine deaminase (yCD-myc-TOM7) expressed in the HEK293-CD cell line with mitochondria.

[0028] Figure 9 A confocal microscope image showing the intracellular localization of yeast-derived cytosine deaminase (yCD-myc-TOM7) expressed in the HEK293-CD cell line.

[0029] Figure 10 To modify mitochondria (MT) yCD-myc-TOM7Mitochondria (MT) were injected into human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2), followed by treatment with 5-FC. The results of morphological changes and apoptosis of cancer cells were observed under a light microscope. Among them, MT refers to mitochondria isolated from HEK293 cells without being coupled with yeast-derived cytosine deaminase.

[0030] Figure 11 To use MT or MT yCD-myc-TOM7 The results of cytotoxicity assays after injecting different concentrations of the drug into human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2) and treating them with 5-FC are shown in the figure.

[0031] Figure 12 To use MT or MT yCD-myc-TOM7 The results of cytotoxicity assays after injection into human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIAPaca-2) and subsequent treatment with different concentrations of 5-FC are shown in the figure.

[0032] Figure 13 To use MT or MT yCD-myc-TOM7 The cells were injected into human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIAPaca-2), and then treated with 5-FC. The induced apoptosis was confirmed by Western blotting.

[0033] Detailed Implementation Plan

[0034] The present invention will now be described in detail.

[0035] Fusion protein

[0036] In one aspect of the invention, a fusion protein is provided, the fusion protein comprising a prodrug convertase or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0037] In this article, the term "prodrug" refers to a drug that has little or no pharmacological activity, but is converted into a pharmacologically active metabolite in vivo through enzymatic or chemical changes.

[0038] As used in this article, the term "prodrug-converting enzyme" refers to an enzyme capable of converting a non-toxic prodrug into a toxic drug. Prodrug-converting enzymes are commonly used in enzyme / prodrug therapy.

[0039] In this invention, the prodrug-converting enzyme can be any one selected from the group consisting of cytosine deaminase (CD), carboxylesterase (CE), herpes simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine nucleoside phosphorylase (PNP), and horseradish peroxidase (HRP), but is not limited to the above enzymes as long as it has the function of converting prodrugs into toxic drugs.

[0040] In this invention, the prodrug-converting enzyme includes all its natural forms, variants, and fragments. The term "natural form" generally refers to a polypeptide containing the amino acid sequence of each natural prodrug-converting enzyme. The amino acid sequence of the natural prodrug-converting enzyme generally refers to the amino acid sequence present in each naturally derived prodrug-converting enzyme. Furthermore, provided that the prodrug-converting enzyme has the same activity as the natural prodrug-converting enzyme, or the gene encoding the enzyme is located at the same chromosomal position, the sequence of the prodrug-converting enzyme may include the addition, deletion, or substitution of one or more amino acids in the protein.

[0041] Specifically, the prodrug converting enzyme of the present invention may have 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% sequence identity with the amino acid sequence of each natural enzyme.

[0042] As used herein, the term "prodrug-converting enzyme fragment" refers to a truncated fragment of the enzyme's N-terminus, C-terminus, or a portion thereof, and possesses enzymatic activity for converting a prodrug into a toxic drug. The activity of the prodrug-converting enzyme can be determined using methods known to those skilled in the art. In this case, the prodrug-converting enzyme may include variants thereof.

[0043] As used herein, the term "variant" refers to a form in which some amino acids of the aforementioned prodrug-converting enzyme or its fragments have been replaced, and which possesses the function of activating the prodrug into a toxic anticancer agent. In other words, a variant of the prodrug-converting enzyme may have a different sequence from the natural prodrug-converting enzyme, but still retain the function of activating the prodrug. The enzyme activity for activating the prodrug can be determined using methods known to those skilled in the art.

[0044] In one specific embodiment of the present invention, the prodrug converting enzyme may be cytosine deaminase. Cytosine deaminase (CD) is an enzyme that catalyzes the reaction of cytosine as a substrate, by hydrolyzing its amino group to generate uracil. The cytosine deaminase uses the non-toxic prodrug 5-fluorocytosine (5-FC) as a reactant, and generates 5-fluorouracil (5-FU) through a deamination reaction. The generated 5-FU inhibits DNA synthesis, thereby exerting specific toxicity on rapidly dividing cancer cells. The cytosine deaminase of the present invention may be derived from yeast (SEQ ID NO: 1) or bacteria (SEQ ID NO: 2). Preferably, it is a cytosine deaminase derived from yeast.

[0045] Information regarding cytosine deaminases can be obtained from well-known databases such as the GenBank database of the National Institutes of Health (NIH). The cytosine deaminase may specifically be an enzyme having the amino acid sequence shown in SEQ ID NO: 1, but is not limited thereto. Furthermore, the cytosine deaminase of the present invention 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%, about 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the cytosine deaminase may have the amino acid sequence shown in 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%, about 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2. Furthermore, the cytosine deaminase of the present invention includes variants thereof and fragments thereof. The variants and fragments are as defined above.

[0046] As used herein, the term "membrane protein" refers to a protein that constitutes a part of or interacts with a biological membrane. In this context, membrane proteins include both integrated membrane proteins that constitute a part of or are permanently attached to a membrane, and peripheral membrane proteins that are temporarily attached to the phospholipid bilayer or other integrated membrane proteins.

[0047] As used herein, the term "mitochondrial outer membrane protein" refers to a protein capable of localizing to the outer mitochondrial membrane. The term "mitochondrial outer membrane targeting protein" refers to a protein containing the N-terminus or C-terminus of a protein present in the outer mitochondrial membrane and having an amino acid sequence that allows it to specifically localize to the outer mitochondrial membrane. In this case, the mitochondrial outer membrane targeting protein enables the fusion protein described herein to attach to the outer mitochondrial membrane. Furthermore, the mitochondrial outer membrane targeting protein prevents the fusion protein described herein from entering the mitochondria.

[0048] The mitochondrial outer membrane targeting protein can be selected from proteins present in eukaryotic mitochondria. For example, it can be selected from proteins present in the outer membrane of yeast, animal cells, or human cells.

[0049] In one embodiment, the mitochondrial outer membrane targeting protein may be a protein comprising the N-terminus or C-terminus of any protein selected from the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

[0050] Specifically, when the mitochondrial outer membrane targeting protein is derived from any one of the group consisting of TOM20, TOM70, and OM45, it may include the N-terminal region of TOM20, TOM70, or OM45. In one embodiment, the mitochondrial outer membrane targeting protein may be yeast-derived TOM70 as shown in SEQ ID NO: 8 or human-derived TOM70 as shown in SEQ ID NO: 9. In another embodiment, it may be yeast-derived TOM20 as shown in SEQ ID NO: 10 or human-derived TOM20 as shown in SEQ ID NO: 11. In yet another embodiment, it may be yeast-derived OM45 as shown in SEQ ID NO: 12.

[0051] Furthermore, when the mitochondrial outer membrane targeting protein is derived from any one of the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, it may include the C-terminal region of any one of the proteins selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In one embodiment, the mitochondrial outer membrane targeting protein may be yeast-derived TOM5 as shown in SEQ ID NO: 13 or human-derived TOM5 as shown in SEQ ID NO: 14. In another embodiment, it may be yeast-derived TOM7 as shown in SEQ ID NO: 15 or human-derived TOM7 as shown in SEQ ID NO: 16. In another embodiment, it may be yeast-derived TOM22 as shown in SEQ ID NO: 17 or human-derived TOM22 as shown in SEQ ID NO: 18. In another embodiment, it may be yeast-derived Fis1 as shown in SEQ ID NO: 19 or human-derived Fis1 as shown in SEQ ID NO: 20. In another embodiment, it may be the human Bcl-2 alpha shown in SEQ ID NO: 21. In another embodiment, it may be the yeast-derived VAMP1 shown in SEQ ID NO: 22 or the human VAMP1 shown in SEQ ID NO: 23.

[0052] The prodrug-converting enzyme or a fragment thereof can bind to the outer mitochondrial membrane via the outer mitochondrial membrane targeting protein. The outer mitochondrial membrane targeting protein and the prodrug-converting enzyme can be linked in a direction from the N-terminus to the C-terminus.

[0053] The fusion proteins can be linked in the following order: <Structure 1> N-terminus - mitochondrial outer membrane targeting protein - prodrug convertase or fragment thereof - C-terminus; or

[0054] <Structure 2>

[0055] N-terminus - prodrug convertase or fragment thereof - mitochondrial outer membrane targeting protein - C-terminus.

[0056] In structural formula 1, the mitochondrial outer membrane targeting protein can be the N-terminal sequence of any protein selected from the group consisting of TOM20, TOM70 and OM45.

[0057] In structural formula 2, the mitochondrial outer membrane targeting protein can be the C-terminal sequence of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X, and VAMP1B. In this case, the prodrug convertase or a fragment thereof is the same as defined above.

[0058] Furthermore, 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 consist 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 consist of an amino acid sequence appropriately selected from 20 amino acids, preferably composed of glycine and / or serine. In one embodiment, the linker may be a sequence of 5 to 50 amino acids composed of glycine and serine. In one embodiment, the linker may be (G4S)n (SEQ ID NO: 24), where n is an integer from 1 to 10, and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0059] The fusion proteins can also be linked in the following order: <Structure 3> N-terminus - mitochondrial outer membrane targeting protein - linker - prodrug convertase or fragment thereof - C-terminus; or

[0060] <Structure 4>

[0061] N-terminus - prodrug convertase or fragment thereof - linker - mitochondrial outer membrane target protein - C-terminus.

[0062] In structural formula 3, the mitochondrial outer membrane targeting protein can be the N-terminal sequence of any protein selected from the group consisting of TOM20, TOM70 and OM45.

[0063] In structural formula 4, the mitochondrial outer membrane targeting protein can be the C-terminal sequence of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-X and VAMP1B.

[0064] In another aspect of the invention, a polynucleotide encoding a fusion protein is provided, the fusion protein comprising a prodrug convertase or a fragment thereof and a mitochondrial outer membrane targeting protein. The prodrug convertase or a fragment thereof, the mitochondrial outer membrane targeting protein, and the fusion protein are as defined above.

[0065] If the polynucleotides encode the same polypeptide, one or more bases can be mutated by substitution, deletion, insertion, or a combination thereof. When preparing polynucleotide sequences by chemical synthesis, synthetic methods known in the art can be used, such as those described by Engels and Uhlmann in Angew Chem Int Ed Engl (37:73-127, 1988). Examples of such methods include the triester method, the phosphite method, the phosphoramide method, the H-phosphate method, PCR and other self-priming methods, and solid-phase oligonucleotide synthesis methods.

[0066] In one embodiment, the polynucleotide may include 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% sequence identity with the nucleotide sequence encoding the polynucleotide of the fusion protein, wherein the fusion protein comprises a prodrug convertase or a fragment thereof and a mitochondrial outer membrane targeting protein.

[0067] In another aspect of the invention, a vector loaded with the polynucleotide encoding the fusion protein is provided. The fusion protein is as defined above.

[0068] As used herein, the term "vector" refers to a vector capable of being introduced into a host cell and undergoing recombination and insertion within the host cell's genome. Alternatively, the vector can also be understood as a nucleic acid vector containing a nucleotide sequence capable of autonomous replication as an episome. Examples of vectors include linear nucleic acids, plasmids, phage particles, granules, RNA vectors, viral vectors, small chromosomes, and analogues thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0069] Specifically, the vector can be plasmid DNA, bacteriophage DNA, etc., and can be commercially developed plasmids (e.g., pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from *Escherichia coli* (e.g., pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from *Bacillus subtilis* (e.g., pUB110, pTP5, etc.), plasmids derived from yeast (e.g., YEp13, YEp24, YCp50, etc.), bacteriophage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal viral vectors (e.g., retroviruses, adenoviruses, vaccinia viruses, etc.), or insect viral vectors (e.g., baculoviruses, etc.). Since the vector exhibits different protein expression levels and patterns depending on the host cell, it is preferable to select the host cell most suitable for the purpose.

[0070] Furthermore, the plasmid may include selectable markers, such as antibiotic resistance genes, and the host cell carrying the plasmid may be cultured under selectable conditions.

[0071] As used herein, the term "gene expression" or "expression" refers to the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of a fusion protein product or fragment thereof. Useful expression vectors may be RcCMV (Invitrogen, Carlsbad) or variants thereof. These expression vectors may include a human CMV (cytomegalovirus) promoter to facilitate sustained transcription of the target gene in mammalian cells and include a bovine growth hormone polyadenylation signaling sequence to enhance posttranscriptional RNA homeostasis.

[0072] In another aspect of the invention, a transformed cell is provided, wherein a vector loaded with a polynucleotide encoding the fusion protein is introduced.

[0073] As used herein, the term "transformed cell" refers to a prokaryotic or eukaryotic cell capable of receiving a recombinant expression vector. The transformed cell can be constructed by introducing the vector into a host cell and transforming it. Furthermore, the polynucleotides contained in the vector can be expressed to produce the fusion protein of this invention.

[0074] Transformation can be performed using various methods. The specific method is not particularly limited as long as it produces the fusion protein of this invention. Specifically, transformation methods may include CaCl2 precipitation, the Hanahan method (using a reducing agent such as dimethyl sulfoxide (DMSO) to improve efficiency in CaCl2 precipitation), electroporation, calcium phosphate precipitation, protoplast fusion, stirring with silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate-mediated transformation, liposome-mediated transformation, or drying / inhibition-mediated transformation. Furthermore, the target substance can be delivered into cells via viral particles through infection. Additionally, the vector can be introduced into host cells through methods such as gene bombardment.

[0075] Furthermore, the host cell used to construct the transformed cells is not particularly limited, as long as it can produce the fusion protein of the present invention. Specifically, the host cell can include, but is not limited to, prokaryotic cells, eukaryotic cells, and cells derived from mammals, plants, insects, fungi, or other cell sources. For example, *Escherichia coli* can be used as a prokaryotic cell. For example, yeast can be used. In addition, for mammalian cells, CHO cells, F2N cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, SP2 / 0 cells, human lymphoblastoid cells, NSO cells, HT-1080 cells, PERC.6 cells, HEK293 cells, HEK293T cells, etc., can be used, but are not limited to these. Any cell known to those skilled in the art as a mammalian host cell can be used.

[0076] As described above, in order to optimize the properties of fusion proteins as therapeutic agents or for other uses, the glycan patterns (e.g., sialylation, fucosylation, glycosylation) of the fusion protein can be modulated by employing methods known to those skilled in the art to regulate glycosylation-related genes present in the host cell.

[0077] Modified mitochondria

[0078] In another aspect of the invention, a modified mitochondria is provided, wherein a prodrug convertase or a fragment thereof is located on the outer mitochondrial membrane.

[0079] In this article, the term "mitochondria" refers to organelles in eukaryotic cells that participate in the synthesis and regulation of adenosine triphosphate (ATP), the intracellular energy source. Mitochondria are involved in a variety of metabolic pathways in the body, such as cell signaling, cell differentiation, cell death, and the regulation of the cell cycle and cell growth.

[0080] Therefore, reports indicate that mitochondrial dysfunction or impairment caused by genetic, environmental, or unknown reasons is associated with the development of a variety of 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 or Alzheimer's disease, as well as the occurrence and metastasis of various cancers.

[0081] The aforementioned mitochondria can originate from mammals or humans. Specifically, they can be isolated from cells or tissues, or from blood cells or platelets. Furthermore, they can be normal mitochondria derived from cells possessing normal mitochondrial biological activity. In this case, the mitochondria can be isolated and used after tissue or cell concentration and disruption, or they can be isolated and disrupted from thawed, cryopreserved tissue or cell samples. Additionally, they can be isolated from thawed, cryopreserved samples of cells or tissues cultured in vitro. For example, the mitochondria can originate from somatic cells, germ cells, or stem cells.

[0082] Specifically, the somatic cells can be muscle cells, liver cells, nerve cells, fibroblasts, epithelial cells, adipocytes, bone cells, leukocytes, lymphocytes, platelets, or mucosal cells.

[0083] Furthermore, the stem cells are undifferentiated cells capable of differentiating into various tissue cells, and can be selected from any of the following groups: mesenchymal stem cells, adult stem cells, dedifferentiated 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 can be derived from any of the following groups: umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, synovial fluid, testis, amnion, and placenta.

[0084] Furthermore, the mitochondria can be derived from the individual, an allogeneic individual, or a heterogeneous individual. Specifically, autologous mitochondria refer to mitochondria derived from the tissue or cells of the same donor. Allogeneic mitochondria refer to mitochondria derived from an individual belonging to the same species as the donor but with a different allele genotype. Heterogeneous mitochondria refer to mitochondria derived from an individual belonging to a different species than the subject.

[0085] Furthermore, the mitochondria can be mitochondria isolated from cells. Additionally, the mitochondria can be intact mitochondria with mitochondrial activity.

[0086] Meanwhile, mitochondria can be isolated from specific cells using a variety of modified methods, including various well-known methods, such as using specific buffer solutions or methods utilizing potential differences and magnetic fields.

[0087] To maintain mitochondrial activity, mitochondria can be isolated by disrupting tissue or cells and centrifuging. In one embodiment, mitochondrial isolation can be achieved by culturing cells and performing a first centrifugation on a composition containing the cells to form a precipitate; resuspending the precipitate in a buffer and homogenizing it; performing a second centrifugation on the homogenate to obtain a supernatant; and finally performing a third centrifugation on the supernatant to purify the mitochondria. In this case, to maintain cell activity, it is preferable to set the second centrifugation time shorter than the first and third centrifugation times, and the centrifugation speed can be gradually increased from the first to the third centrifugation.

[0088] Specifically, the first to third centrifugations can be performed at a temperature of about 0°C to about 10°C, preferably at a temperature of about 3°C ​​to about 5°C. Furthermore, the centrifugation time can be from about 1 minute to 50 minutes, and can be adjusted appropriately according to the number of centrifugations and the sample content.

[0089] Furthermore, the first centrifugation can be performed at speeds of approximately 100 ×g to approximately 1,000 ×g, approximately 200 ×g to approximately 700 ×g, or approximately 300 ×g to approximately 450 ×g. The second centrifugation can be performed at speeds of approximately 1 ×g to approximately 2,000 ×g, approximately 25 ×g to approximately 1,800 ×g, or approximately 500 ×g to approximately 1,600 ×g. The third centrifugation can be performed at speeds of approximately 100 ×g to approximately 20,000 ×g, approximately 500 ×g to approximately 18,000 ×g, or approximately 800 ×g to approximately 15,000 ×g.

[0090] Furthermore, pharmaceutically acceptable sugars can be used to stabilize the obtained mitochondria. Specifically, the sugars can be sucrose, mannitol, trehalose, etc., but are not limited to these. In addition, pharmaceutically acceptable Tris, HEPES, phosphates, etc., can be used as pH buffers, but are not limited to these.

[0091] Meanwhile, after obtaining mitochondria, additives such as chelating agents and antioxidants can be used to remove the damage caused by ion efflux and inhibit oxidative stress. The use of these additives is unrestricted and includes reagents known in the art. For example, they can be EDTA, EGTA, citrate, glycine, taurine, ATP, etc., but are not limited to these.

[0092] In addition, to maintain mitochondrial activity, mitochondria can be isolated by thawing, disrupting, and centrifuging frozen cells or tissues. Methods for obtaining mitochondria may include freezing cells or tissues, thawing said cells or tissues, and disrupting the thawed cells or tissues.

[0093] As used herein, the term "modified mitochondria" refers to mitochondria with foreign proteins bound to their outer mitochondrial membrane. Specifically, the foreign protein may be a recombinant fusion protein including an outer mitochondrial membrane targeting protein and / or a target protein. The outer mitochondrial membrane targeting protein is the same as defined above.

[0094] In this case, the target protein can be any of the proteins selected from those that are prodrug-converting enzymes active both intracellularly and extracellularly, and proteins capable of binding to receptors or ligands present on the cell membrane. The fusion protein comprising a prodrug-converting enzyme and a mitochondrial outer membrane targeting protein is the same as defined above.

[0095] In this invention, the target protein can be a protein capable of binding to receptors or ligands present on the cell membrane. In this case, the protein capable of binding to receptors or ligands present on the cell membrane can be an antibody or a fragment thereof capable of specifically binding to tumor-associated antigens.

[0096] The tumor-associated antigen may be any one selected from CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), cell surface-associated mucin 1 (MUC-1), prostate acid phosphatase (PAP), prostate-specific antigen (PSA), Survivin, tyrosinase-associated protein 1 (tyrp1), tyrosinase-associated protein 2 (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, but is not limited to any one of these as long as it is a protein specifically present on the surface of tumor cells.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of generating an immune response to a specific antigen, and is a protein molecule capable of specifically recognizing said antigen. In this context, an antibody fragment refers to a fragment having the same antigen-determining region (CDR) as the antibody and possessing antigen-binding activity. Specifically, the antibody fragment may be Fab, scFv, F(ab')2, or a combination thereof.

[0098] If the target protein is an antibody or fragment thereof capable of binding to tumor-associated antigens, then the target protein may bind to a mitochondrial outer membrane targeting protein comprising the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B, and may be linked in the following order: <Structure 5> N-terminus - Target protein - A mitochondrial outer membrane target protein containing the C-terminal region of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B.

[0099] In this case, the target protein can be an antibody or a fragment thereof capable of binding to tumor-associated antigens.

[0100] Furthermore, the exogenous protein may further include a linker between the target protein and the C-terminal region of the mitochondrial outer membrane targeting protein, wherein the C-terminal region is selected from any one of the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x, and VAMP1B. In this case, the linker is the same as defined above and can be connected in the following order: <Structure 6> N-terminus - target protein - linker - contains the C-terminal region of a mitochondrial outer membrane target protein - C-terminus of any protein selected from the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

[0101] In this case, the target protein may be an antibody or a fragment thereof capable of binding to tumor-associated antigens.

[0102] Modified mitochondria that bind to the aforementioned target proteins can be easily introduced into specific targets, thereby effectively allowing mitochondria to enter specific cells.

[0103] Therefore, in another aspect of the invention, a modified mitochondria is provided, wherein a prodrug-converting enzyme or a fragment thereof is present on the mitochondrial outer membrane, and an antibody or a fragment thereof capable of specifically binding to a tumor-associated antigen. In this case, the prodrug-converting enzyme, the tumor-associated antigen, the antibody or a fragment thereof, and the modified mitochondria are the same as defined above.

[0104] Pharmaceutical compositions with modified mitochondria as the active ingredient

[0105] In another aspect of the invention, a pharmaceutical composition for the prevention and treatment of cancer is provided, the pharmaceutical composition comprising the modified mitochondria as an active ingredient. In this case, the pharmaceutical composition can be used for the prevention or treatment of cancer.

[0106] As used herein, the term "cancer" refers to a disease in which normal tissue cells proliferate uncontrollably for some reason and continue to develop rapidly regardless of the body's living conditions or the state of surrounding tissues. In this invention, the cancer may be any one of the following groups, but is not limited to: breast cancer, lung cancer, pancreatic cancer, glioma, gastric cancer, liver cancer, colorectal 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.

[0107] For the pharmaceutical composition, the concentration of the mitochondria can be from about 0.1 µg / mL to about 500 µg / mL, from about 0.2 µg / mL to about 450 µg / mL, or from about 0.5 µg / mL to about 400 µg / mL, but is not limited thereto. When the mitochondria are within the above concentration range, it is beneficial to adjust the dosage during administration and to help improve patient symptoms. In this case, the dosage of the mitochondria can be determined by quantifying the membrane proteins of the isolated mitochondria. Specifically, the isolated mitochondria can be quantified by the Bradford protein assay. Specifically, the isolated mitochondria can be quantified by the Bradford protein assay [paper by James D. McCully (JVis Exp. 2014;(91):51682)].

[0108] Furthermore, for the pharmaceutical composition, the concentration of the prodrug-converting enzyme binding to mitochondria can be from 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, but is not limited thereto. When the prodrug-converting enzyme is within the above concentration range, it is beneficial for dose adjustment during administration and helps improve patient symptoms. The prodrug-converting enzyme is the same as defined above.

[0109] Furthermore, in the pharmaceutical composition, the concentration of the antibody or fragment thereof capable of binding to tumor-associated antigens and delivering them to specific cells via mitochondria may be from about 0.1 µg / mL to about 500 µg / mL, from about 0.2 µg / mL to about 450 µg / mL, or from about 0.5 µg / mL to about 400 µg / mL, but is not limited thereto. When the antibody or fragment thereof is within the above concentration range, it is advantageous to adjust the dosage during administration and helps to improve patient symptoms.

[0110] For the pharmaceutical composition, when the modified mitochondria simultaneously comprise a prodrug-converting enzyme as an exogenous protein and an antibody or fragment thereof capable of binding tumor-associated antigens, the prodrug-converting enzyme and the antibody may be present on the outer mitochondrial membrane in an appropriate ratio. For example, by weight, the ratio of the prodrug-converting enzyme to the antibody or fragment thereof 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 is about 1:5. In this case, the prodrug-converting enzyme may be a prodrug-converting enzyme or a fragment thereof.

[0111] As used herein, the term "prevention" refers to any action that inhibits or delays the occurrence of cancer by administering the pharmaceutical composition described herein. Furthermore, "treatment" refers to any action that delays or beneficially alters the symptoms of cancer by administering the pharmaceutical composition described herein.

[0112] As used herein, the term "efficacy" can be determined by one or more parameters, such as survival rate or disease-free survival over a 1-year, 5-year, or 10-year period. Furthermore, these parameters may also include the suppression of the size of at least one tumor in the subject's body.

[0113] The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art. In the pharmaceutical compositions of the present invention for the prevention or treatment of cancer, the active ingredient may be added in any amount (effective amount) as long as it exhibits anticancer activity, depending on the intended use, formulation, combination purpose, etc. In this case, "effective amount" refers to the amount of active ingredient capable of inducing an anticancer effect. The effective amount can be determined experimentally within the conventional capabilities of those skilled in the art.

[0114] The pharmaceutical compositions of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any material suitable for delivery to a patient and non-toxic. The carrier may include distilled water, alcohols, lipids, waxes, and inert solids. Furthermore, the pharmaceutical compositions may also include pharmaceutically acceptable excipients (e.g., buffers, dispersants).

[0115] Specifically, the pharmaceutical composition may be prepared as a parenteral formulation by adding a pharmaceutically acceptable carrier in addition to the active ingredient, according to conventional methods known in the art, depending on the route of administration. In this case, "pharmaceuticalally acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity exceeding that tolerated by the user (prescribing) the drug.

[0116] When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be prepared as an injection, transdermal formulation, or nasal inhalation formulation according to methods known in the art, together with a suitable carrier. When preparing as an injection, sterile water, ethanol, glycerol, or propylene glycol, or mixtures thereof, can be selected as a suitable carrier. Preferably, Ringer's solution, PBS (phosphate-buffered saline) containing triethanolamine, sterile water for injection, or 5% glucose isotonic solution can be used.

[0117] The pharmaceutical compositions of the present invention can be used as injectable formulations. Therefore, the pharmaceutical compositions of the present invention can be prepared as highly physically or chemically stable injectable formulations by adjusting the pH using an acidic aqueous solution or a buffer solution such as a phosphate buffer to ensure product stability when dispensed as an injectable formulation according to the prescription.

[0118] Specifically, the pharmaceutical composition of the present invention may include water for injection.

[0119] The water for injection refers to distilled water used to dissolve solid injections or to dilute water-soluble injections. The water for injection 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, lactated Ringer's solution, or a phosphate buffer or sodium dihydrogen phosphate-citrate buffer with a pH of approximately 3.5 to 7.5.

[0120] The pharmaceutical compositions of the present invention may further include stabilizers or solubilizers. For example, the stabilizer may be metabisulfite or ethylenediaminetetraacetic acid (EDTA), and the solubilizer may be hydrochloric acid, acetic acid, potassium hydroxide phosphate, potassium bicarbonate, potassium carbonate, or Tris. In one embodiment, the pharmaceutical composition may include a mixed preservation solution, such as a TTG (trehalose-tris(hydroxymethyl)aminomethane-glycine) solution, which is conventionally used in pharmaceutically acceptable formulations.

[0121] Specifically, in addition to modified mitochondria, the pharmaceutical composition of the present invention may also contain an injectable liquid composition. In this case, the modified mitochondria are the same as defined above. The pharmaceutical composition of the present invention contains an injectable liquid composition, thereby forming a composition for the prevention or treatment of diseases related to mitochondrial function. When administered to mitochondria by injection, this composition can inhibit thrombosis caused by mitochondrial aggregation, thrombocytopenia, and other factors, and can maintain and / or enhance mitochondrial stability, while also stably maintaining mitochondrial activity.

[0122] In this case, the liquid composition may include glycine, sugars, and buffers.

[0123] The concentration of glycine in the injectable liquid composition may be, but is not limited to, about 15 mM or more. Specifically, it may be 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. Furthermore, the glycine may be used with at least one amino acid selected from, but not limited to, the group consisting of, histidine, isoleucine, leucine, lysine acetate, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, proline, serine, and tyrosine.

[0124] Furthermore, the sugar contained in the injectable liquid composition may be at least one selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, glucose, fructose, mannose, maltose, lactose, isomaltose, dextran, and dextrin, but is not limited thereto. In particular, the sugar may be trehalose, mannitol, or sucrose. Preferably, the sugar is trehalose.

[0125] The buffer contained in the injectable liquid composition may be selected from, but is not limited to, Tris buffer, HEPES buffer (hydroxyethylpiperazine ethanesulfonic acid), MOPS buffer (3-(N-morpholino)propanesulfonic acid), and buffers containing acetate or phosphate. Preferably, the buffer is injectable grade Tris buffer.

[0126] In this case, the pH of the buffer solution may be from about 7.0 to about 7.8, from about 7.2 to about 7.6, or from about 7.3 to about 7.5, but is not limited thereto.

[0127] Furthermore, the concentration of the buffer in the injectable liquid composition may be from about 5 mM to about 50 mM, from about 8 mM to about 40 mM, from about 10 mM to about 35 mM, from about 13 mM to about 30 mM, or from about 15 mM to about 25 mM, but is not limited thereto.

[0128] The osmotic pressure of the above-described injectable liquid composition can be 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 osmotic pressure range is advantageous for long-term storage at temperatures from 2°C to 8°C or higher, while also making the composition suitable for parenteral administration, such as intravenous injection, intramuscular injection, or subcutaneous injection, without causing adverse reactions in the subject.

[0129] As used herein, the term "osmotic pressure" refers to the number of moles of solute per unit mass of solvent that contribute to the osmotic pressure of a solution. The osmotic pressure can be determined by measuring the freezing point depression of a sample using an osmometer.

[0130] Furthermore, the liquid composition for injection may further include a chelating agent.

[0131] The chelating agent may be at least one selected from, but is not limited to, injectable EGTA (ethylene glycol tetraacetic acid), EDTA (ethylenediaminetetraacetic acid), and BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. The chelating agent can inhibit damage caused by ion efflux after obtaining the mitochondria contained in the injectable liquid composition.

[0132] In addition, the pharmaceutical composition may also include antioxidants, ATP, magnesium, and other additives that help maintain mitochondrial function and activity.

[0133] The pharmaceutical compositions of the present invention can be stored in containers selected from the group consisting of vials, cartridges, syringes, and autoinjectors. Furthermore, the containers storing the pharmaceutical compositions can be stored at room temperature, at a refrigerated temperature of 2°C to 8°C, or at a temperature of 25°C to 40°C until administered to a subject requiring treatment.

[0134] The subject can be a mammal, such as a human, dog, cow, horse, pig, sheep, goat, cat, mouse, rabbit, or rat, but is not limited thereto, and is preferably a human.

[0135] Simultaneously, the pharmaceutical compositions of the present invention are administered at pharmaceutically effective amounts. The terms "therapeuticly effective amount" or "pharmaceutically effective amount" refer to an effective amount of a compound or composition sufficient to prevent or treat a target disease, a dose sufficient to treat the disease with a reasonable benefit / risk ratio in medical treatment without causing side effects. The level of the effective amount can be determined based on factors including the patient's health status, disease type and severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration, excretion rate, duration of treatment, drugs used in combination or concurrently, and other factors known in the medical field. In one embodiment, the therapeutically effective amount refers to a drug amount capable of effectively treating cancer.

[0136] As used herein, the term "administration" refers to the introduction of a predetermined substance into a subject by an appropriate method. The route of administration of the composition can be any conventional route, as long as it reaches the target tissue. The administration method can be intraperitoneal injection, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, local administration, intranasal administration, or rectal administration, but is not limited thereto. Preferred doses of the pharmaceutical composition of the present invention can be, but are not limited to, mitochondrial doses of 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 based on the subject's body weight per administration. In other words, from the perspective of cellular activity, it is preferable to administer modified mitochondria containing an anticancer agent according to the above-mentioned dose range and the body weight of a subject with cancerous tissue. Furthermore, the pharmaceutical composition can be administered 1 to 10 times, 3 to 8 times, or 5 to 6 times, preferably 5 times. In this case, the dosing interval can be 1 to 7 days or 2 to 5 days, preferably 3 days. The above dosages should not limit the scope of the invention in any way.

[0137] In this case, the pharmaceutical composition can be administered parenterally using an 18 G to 32 G needle in a volume of less than 5 mL, less than 3 mL, or less than 2 mL, but is not limited thereto.

[0138] The term "subject" refers to an individual to whom the (prescription) composition of the present invention may be administered, and may be a subject suffering from cancer. Furthermore, the subject may be a mammal, including humans, such as rats, mice, or livestock, but preferably humans. In addition to modified mitochondria, the compositions of the present invention may further comprise any compound or natural extract that has been proven safe and is known to have anticancer activity to enhance or improve the anticancer activity. In this case, the pharmaceutical composition and the compound or natural extract with anticancer activity may be administered simultaneously or sequentially.

[0139] Uses of mitochondrial modification

[0140] In another aspect of the invention, the use of modified mitochondria in the preparation of pharmaceutical compositions for treating cancer is provided.

[0141] In this context, the cancer, the treatment, and the modified mitochondria are as defined above. The modified mitochondria can serve as a carrier for delivering exogenous proteins. Specifically, the modified mitochondria can serve as a carrier for delivering a target protein capable of functioning within the cell, wherein the target protein is a prodrug-converting enzyme or a fragment thereof. Furthermore, by introducing an antibody or a fragment thereof capable of binding tumor-associated antigens into the outer mitochondrial membrane, the modified mitochondria can effectively target tumor cells, thereby delivering the target protein, which is a prodrug-converting enzyme, into the cell.

[0142] Therefore, the modified mitochondria can be used as an efficient protein delivery system. In this case, the prodrug convertase or a fragment thereof is the same as defined above.

[0143] Methods of treating cancer using modified mitochondria

[0144] In another aspect of the invention, a method for treating cancer is provided, comprising administering modified mitochondria to a subject.

[0145] In this case, the modified mitochondria can be administered in combination with an anticancer prodrug. The anticancer prodrug can be any one selected from the group consisting of 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazolidinyl compounds, 6-methylpurine deoxynucleoside, and indole-3-acetic acid (IAA). However, any compound that can be converted from a non-toxic compound to a toxic anticancer agent by a prodrug-converting enzyme is also included.

[0146] Furthermore, when applying modified mitochondria comprising one or more prodrug-converting enzymes or fragments thereof, one or more prodrugs may be applied in combination, depending on the combination of the prodrug-converting enzymes. In this case, the modified mitochondria may comprise a single prodrug-converting enzyme, or may be modified mitochondria comprising one or more prodrug-converting enzymes.

[0147] The modified mitochondria, the cancer, and the treatment are the same as defined above.

[0148] The subject may be a person suffering from cancer. Furthermore, the subject may be a mammal, preferably a human.

[0149] Kits for modified mitochondria used in cancer prevention and treatment

[0150] In another aspect of the invention, a kit for the prevention and treatment of cancer is provided, comprising modified mitochondria and a prodrug. In this case, the modified mitochondria, the prodrug, the cancer, the prevention, and the treatment are as defined above.

[0151] Furthermore, when the kit contains mitochondria modified with one or more prodrug-converting enzymes or fragments thereof, one or more prodrugs may be used in combination, depending on the prodrug-converting enzyme or fragment thereof used. In this case, the modified mitochondria may each comprise a prodrug-converting enzyme or fragment thereof, or may be mitochondria comprising one or more prodrug-converting enzymes or fragments thereof.

[0152] Methods for preparing modified mitochondria

[0153] In another aspect of the invention, a method for preparing modified mitochondria is provided, wherein the outer mitochondrial membrane of the modified mitochondria expresses a prodrug-converting enzyme or a fragment thereof, the method comprising the steps of: expressing the prodrug-converting enzyme or a fragment thereof on the outer mitochondrial membrane within a cell; and isolating the mitochondria. In this case, the prodrug-converting enzyme and the modified mitochondria are as defined above.

[0154] Furthermore, in another aspect of the present invention, a method for preparing modified mitochondria is provided, wherein the outer mitochondrial membrane of the modified mitochondria expresses a prodrug-converting enzyme or a fragment thereof, and an antibody or a fragment thereof capable of specifically binding to a tumor-associated antigen. The method includes the steps of: expressing a prodrug-converting enzyme or a fragment thereof, and an antibody or a fragment thereof capable of specifically binding to a tumor-associated antigen, on the outer mitochondrial membrane within a cell; and isolating the mitochondria. In this case, the prodrug-converting enzyme, the tumor-associated antigen, the antibody, and the modified mitochondria are the same as defined above.

[0155] In this case, the prodrug-converting enzyme or a fragment thereof, and the antibody or a fragment thereof capable of specifically binding to tumor-associated antigens, can be present on the outer mitochondrial membrane in an appropriate ratio. For example, by weight, the ratio of the enzyme or a fragment thereof to the antibody or a fragment thereof can 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 is about 1:5.

[0156] Preparation methods of fusion proteins

[0157] In another aspect of the invention, a method for preparing a fusion protein is provided, wherein the method includes isolating the fusion protein from a cell into which a vector encoding the fusion protein has been introduced, the fusion protein comprising a mitochondrial outer membrane targeting protein and a prodrug convertase. The fusion protein is as defined above. In this case, the cell may be a prokaryotic cell or a eukaryotic cell.

[0158] The present invention will be described below through preferred embodiments to facilitate understanding of the invention.

[0159] However, the following embodiments are only used to help understand the present invention, and the content of the present invention is not limited to the following embodiments.

[0160] Example 1: Construction of an expression vector encoding a yeast-derived cytosine deaminase that can fuse to the outer mitochondrial membrane

[0161] Example 1.1 Construction of yeast-derived cytosine deaminase expression vector

[0162] To express yeast-derived cytosine deaminase as a recombinant protein, gene synthesis was commissioned to BIONICS. The obtained yeast-derived cytosine deaminase gene was loaded into an expression vector and named pUC57-yCD. The nucleotide sequence of the yeast-derived cytosine deaminase is identical to that of SEQ ID NO: 3.

[0163] Example 1.2 Construction of an expression vector encoding a yeast-derived cytosine deaminase that can fuse to the outer mitochondrial membrane

[0164] To prepare a yeast-derived cytosine deaminase protein fused with the mitochondrial outer membrane binding protein TOM7, an expression vector capable of expressing the fusion protein was constructed.

[0165] Table 1

[0166] Using the plasmid pUC57-yCD obtained by the method in Example 1.1 above as a template, 0.2 pmol primers (RyCD) and 0.2 pmol primers (yCD-myc-TOM7-R) were added, along with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The mixture was subjected to 25 cycles of amplification reaction in a polymerase chain reaction (PCR) instrument: 95 ℃ for 40 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute, thereby obtaining the yCD-myc gene. The DNA fragment amplified by the above method was named N-yCD-myc.

[0167] Using plasmid pT-TOM7 obtained by the method disclosed in Korean Patent Application Publication No. 2019-0124656 as a template, 0.2 pmol primers (yCD-myc-TOM7-F) and 0.2 pmol primers (XTOM7(T)) were added, along with 0.2 nM dNTPs, 1×AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The mixture was subjected to 25 cycles of amplification reaction in a polymerase chain reaction (PCR) instrument: 95 °C for 40 seconds, 58 °C for 30 seconds, and 72 °C for 1 minute, thereby obtaining the myc-TOM7 gene. The DNA fragment amplified by the above method was named C-myc-TOM7.

[0168] The amplified DNA fragments N-yCD-myc and C-myc-TOM7 were used as templates. 0.2 pmol primers (RyCD) and 0.2 pmol primers (XTOM7(T)) were added, and the mixture was combined with 0.2 nM dNTPs, 1× AccuPrime Taq DNA polymerase reaction buffer (Invitrogen, USA), and 1 unit of AccuPrime Taq DNA polymerase. The amplification reaction was carried out in a polymerase chain reaction instrument for 25 cycles: 95 ℃ for 40 seconds, 58 ℃ for 30 seconds, and 72 ℃ for 1 minute, thereby obtaining the yeast cytosine deaminase gene yCD-myc-TOM7 (SEQ ID NO: 26) fused with the amplified TOM7.

[0169] The amplified yCD-myc-TOM7 gene was digested with restriction endonucleases EcoRI and XhoI, and the DNA fragments were separated by electrophoresis on a 2% agarose gel. Subsequently, the DNA fragments were inserted into the pcDNA3.1 vector (Invitrogen, USA), which had also been digested with EcoRI and XhoI, using T4 DNA ligase to construct the plasmid pCMV-yCD-myc-TOM7. Figure 1 ).

[0170] Example 2: Confirmation of the introduction and expression of yeast-derived cytosine deaminase gene in human embryonic kidney cells

[0171] To introduce the expression vector pCMV-yCD-myc-TOM7 encoding cytosine deaminase constructed in Example 1 into the human embryonic kidney cell line (HEK293), Lipofectamine 2000 (Thermo Scientific, 11668019) was used, and the procedure was performed according to the manufacturer's instructions.

[0172] HEK293 cells were cultured in DMEM (Hyclone, SH30243) medium containing 10% fetal bovine serum (FBS) (Gibco, 12483-020) and penicillin-streptomycin (Hyclone, SV30010) at 37 °C and 5% CO2.

[0173] When performing plasmid introduction, HEK293 cells were loaded at a rate of 1 × 10⁻⁶. 6Cells were seeded at a density of 10 cells / well in 6-well plates 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. HEK293 cells were then treated with this complex to introduce the plasmid. After 24 hours, the cells were washed with PBS, replaced with fresh culture medium, and cultured for another 24 hours.

[0174] To verify the expression of the introduced gene in HEK293 cells, cells were lysed using cell lysis buffer (Cell Signaling Technologies, 9803), proteins were extracted, and then analyzed by Western blot. To confirm the expression of 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.

[0175] The results are as follows Figure 2 As shown, yeast-derived cytosine deaminase was confirmed to be expressed in HEK293 cells.

[0176] Example 3: Enzyme activity assay of yeast-derived cytosine deaminase

[0177] To determine the enzyme activity of yeast-derived cytosine deaminase expressed in HEK293 cells constructed in Example 2 above, the amount of 5-FU generated from the conversion of 5-FC was detected by measuring the absorbance at a wavelength of 314 nm.

[0178] Specifically, HEK293 cells or HEK293 cells expressing cytosine deaminase (HEK293-yCD) were used at a concentration of 1 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours. After 24 hours of culture, cells were collected by treating with 0.05% trypsin-EDTA (Gibco, 25300) and washed twice with PBS. The cells were then resuspended in PBS (pH 7.4) and sonicated. The lysed cells were centrifuged at 2,000 × g for 10 minutes, and the supernatant was collected and transferred to a new microcentrifuge tube. 5 mM 5-FC (Sigma, F7129) was then added, and the reaction was carried out at 37 °C.

[0179] Samples were taken at 1, 2, 4, and 16 hours, with 200 μL of reaction solution added to each well of a 96-well plate. The absorbance was then measured at 314 nm using an ELISA reader. Under these conditions, it was confirmed that 5-FC showed no absorbance signal at 314 nm, and only 5-FU was detected. Furthermore, standard curves were plotted using different concentrations of 5-FU (Sigma, F6627) to quantify the 5-FU generated in the reaction solution.

[0180] Figure 3 Standard curves for different concentrations of 5-FU are shown. Figure 3 B shows the curve of 5-FU content change over time. 5-FU was not detected in the reaction solution derived from HEK293 cells, but 12.3 mM of 5-FU was detected in the reaction solution derived from HEK293 cells expressing cytosine deaminase, indicating that 5-FC was converted to 5-FU by yeast-derived cytosine deaminase.

[0181] Example 4 Cytotoxicity assay of prodrug 5-FC

[0182] HEK293 cells constructed in Example 1 or HEK293 cells expressing cytosine deaminase (HEK293-yCD) were seeded at a density of 5,000 cells / well in 96-well plates and cultured for 24 hours. After 24 hours of culture, the cells were treated with the prodrug 5-FC (Sigma, F7129) at a concentration of 500 μg / mL, and cultured for another 120 hours. After 120 hours of culture, apoptosis of HEK293 cells was observed under an optical microscope, and cell proliferation was measured using the WST-1 method (Roche, 11644807001).

[0183] The results are as follows Figure 5 and Figure 6 As shown, in the untreated 5-FC group, no cell proliferation inhibition was observed in either HEK293 cells or HEK293 cells expressing cytosine deaminase (HEK293-yCD). Conversely, in the 5-FC treated group, the cell growth of HEK293-yCD cells was inhibited by approximately 90% compared to HEK293 cells.

[0184] Example 5: Construction of a stable cell line expressing yeast-derived cytosine deaminase

[0185] To obtain a cell line that continuously expresses cytosine deaminase from the HEK293 cells (HEK293-yCD) expressing cytosine deaminase constructed in Example 2 above, 1,000 µg / mL of G418 (Promega, V7983) antibiotic was added for screening, and drug-resistant cells were selected to construct a transformed cell line (stable cell line).

[0186] Specifically, HEK293 (HEK293-yCD) cells expressing cytosine deaminase, selected in Example 2, were cultured in 100 mm culture dishes to approximately 80% cell density. Subsequently, the medium containing 1,000 μg / mL G418 (Promega, V7983) was changed every two days for two weeks to induce apoptosis in cells without plasmid infusion. After two weeks of G418 treatment, approximately 80–90% of the cells died. When clones formed from the growth and division of surviving cells were observed, each clone was transferred to a 24-well plate using 0.05% trypsin-EDTA (Gibco, 25300) for further culture. Throughout subsequent culture, medium containing 1,000 μg / mL G418 was used.

[0187] When the selected clones reached a cell density of over 60% in 24-well plates, Western blot analysis was used to screen clones with higher protein expression levels. The selected clones were then expanded into larger-scale cultures.

[0188] The results are as follows Figure 7 As shown, clones HEK293-CD#5, HEK293-CD#6, and HEK293-CD#7 with high protein expression levels were expanded and cultured, with the HEK293-CD#5 clone used for subsequent experiments.

[0189] Example 6 Verification of intracellular expression localization of yeast-derived cytosine deaminase and preparation of MTYCD

[0190] To verify whether cytosine deaminase is expressed in mitochondria in HEK293-CD#5 cells constructed in Example 5 above, Western blot analysis and immunocytochemical staining were performed on HEK293 (HEK293-yCD) cells.

[0191] Western blot analysis was performed using the following method. Specifically, 1 × 10⁻⁶ samples were collected. 7 HEK293-CD#5 cells expressing cytosine deaminase were washed twice with PBS, resuspended in 300 µL SHE buffer (250 mM sucrose, 20 mM HEPES (pH 7.4), 2 mM EGTA), and sonicated. The disrupted cells were centrifuged at 2,000 × g for 10 minutes, and the supernatant was collected and transferred to a new microcentrifuge tube. The tube was then centrifuged at 12,000 × g for 15 minutes to separate the cytoplasm and mitochondrial precipitate, which was used to isolate the mitochondria.

[0192] Each of the above components was subjected to electrophoresis and Western blot analysis. To confirm the expression of cytosine deaminase, mitochondrial marker proteins, 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. Secondary antibodies were anti-mouse IgG HRP (abcam, 6789) or anti-rabbit IgG HRP (abcam, 6721).

[0193] The results are as follows Figure 8 As shown, cytosine deaminase was confirmed to exist in the mitochondrial fraction along with COX4, a mitochondrial marker protein. Furthermore, it was confirmed that it was not present in the fraction containing the cytoplasmic marker protein β-tubulin.

[0194] Immunocytochemical staining was performed using the following method. Specifically, HEK293 cells and HEK293-CD#5 cells were stained at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 cells / well in 24-well plates 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 used as primary antibodies, diluted 1:1000 in PBS containing 0.1% bovine serum albumin (BSA), 0.1% Triton X-100, and 1% goat serum (Life Technologies, 500622). The cells were treated with this mixture and incubated at 4 °C for 18 hours.

[0195] After the reaction, the cells were washed four times with PBS containing 0.1% BSA to remove the primary antibody. Subsequently, Alexa Fluor® 488-labeled anti-mouse IgG antibody (Invitrogen, 11001) and anti-rabbit IgG antibody (Invitrogen, 21428) were diluted 1:1000 in PBS containing 0.1% BSA, and the cells were treated with this mixture at room temperature for approximately 1 hour. After the reaction, the cells were washed four times each with PBS containing 0.1% BSA and distilled water, and the nuclei were stained using mounting medium containing DAPI (VECTASHIELD, H-1200) and observed under a confocal microscope.

[0196] The results are as follows Figure 9 As shown, the localization of the red fluorescently labeled mitochondrial positive marker MTCO-2 is consistent with the localization of the green fluorescently labeled cytosine deaminase.

[0197] Mitochondria obtained from HEK293 cells and HEK293-CD#5 cells using a mitochondrial fractionation method were named MT and MT, respectively. yCD MT refers to the mitochondria of control HEK293 cells that do not express yeast-derived cytosine deaminase. yCD It refers to mitochondria derived from the HEK293-CD#5 cell line, in which cytosine deaminase is fused and expressed on the outer mitochondrial membrane.

[0198] Example 7 Evaluation of the anticancer activity of mitochondria containing yeast-derived cytosine deaminase against cancer cells

[0199] The MT or MT obtained using the same method as in Example 6 above. yCD Human pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2) were treated with the prodrug 5-FC. Morphological changes in the cancer cells were then observed under a light microscope, and apoptosis and cell proliferation were determined using the WST-1 assay.

[0200] AsPc-1, Capan-1, and Capan-2 cells were cultured in RPMI-1640 (Welgene, LM011-51) medium containing 10% FBS and penicillin-streptomycin. MIA Paca-2 cells were cultured in DMEM (Hyclone, SH30243) medium containing 10% FBS and penicillin-streptomycin.

[0201] Specifically, pancreatic cancer cell lines (AsPc-1, Capan-1, Capan-2, and MIA Paca-2) were seeded at a density of 5,000 cells / well in 96-well plates and cultured for 24 hours. After 24 hours of culture, 0 μg, 0.5 μg, 1 μg, or 2 μg of MT or MT obtained in the same manner as in Example 6 were added to each well. yCD The reaction was carried out for 2 hours. After 2 hours, the cells were washed once with culture medium to remove mitochondria that had not metastasized to the cancer cells. Then, the prodrug 5-FC was added to achieve final concentrations of 0 µg / mL, 20 µg / mL, 100 µg / mL, or 500 µg / mL, and the cells were cultured for another 120 hours.

[0202] The results are as follows Figures 10 to 12 As shown, in the untreated 5-FC group, MT or MT yCD No cell proliferation inhibition was observed in any of the treatments. Conversely, in the 5-FC treatment group, compared to the MT treatment group, MT... yCD Treatment can inhibit cell growth by approximately 60% to over 90%. Furthermore, it can be seen that with MT...yCD Increasing the treatment concentration or the concentration of 5-FC treatment led to a concentration-dependent increase in cell death rate.

[0203] Example 8: Confirmation of MT by Western blot yCD Treatment of induced cancer cell apoptosis

[0204] The MT or MT obtained using the same method as in Example 6 above. yCD Human pancreatic cancer cell lines were treated. Subsequently, the cells were treated with the prodrug 5-FC, and Western blot analysis was performed using antibodies targeting apoptosis markers.

[0205] Specifically, pancreatic cancer cell lines from various individuals (AsPc-1, Capan-1, Capan-2, and MIA Paca-2) were divided into groups of 5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 24 hours. After 24 hours of culture, 50 µg of MT or MT obtained in the same manner as in Example 6 was added to each well. yCD The reaction was carried out for 2 hours. After 2 hours, the cells were washed once with culture medium to remove mitochondria that had not metastasized to the cancer cells.

[0206] The prodrug 5-FC was then added to a final concentration of 100 μg / mL, and the cells were cultured for another 96 hours. Cells were then lysed using cell lysis buffer (Cell Signaling Technologies, 9803) to extract proteins. The extracted proteins were quantified using the BCA method (Thermo Scientific, 23225). Equal volumes of protein from each sample were subjected to electrophoresis and Western blot analysis.

[0207] Anti-phosphorylated p53 antibody (ABclonal, AP0762), anti-cleaved caspase-3 antibody (CellSignaling, 9664), and anti-β-tubulin antibody (Thermo, MA5-16308) were used as primary antibodies. Secondary antibodies were anti-mouse IgG HRP (abcam, 6789) or anti-rabbit IgG HRP (abcam, 6721). Protein expression levels were corrected for β-tubulin protein expression levels.

[0208] The results are as follows Figure 13 As shown, only in MT yCD Expression of apoptosis-related proteins cleaved caspase-3 and phosphorylated p53 was observed in the group treated with 5-FC. Furthermore, in the group treated with MT alone, MT... yCDNo expression of apoptosis markers was observed in any of the 5-FC groups; in the 5-FC-only treatment groups, the results were similar to those in the control group (untreated group).

Claims

1. A fusion protein, characterized in that, The fusion protein includes a prodrug convertase or a fragment thereof, and a mitochondrial outer membrane targeting protein.

2. The fusion protein as described in claim 1, characterized in that, The prodrug-converting enzyme is an enzyme capable of converting a non-toxic prodrug into a toxic drug.

3. The fusion protein as described in claim 3, characterized in that, The prodrug-converting enzyme is selected from any one of the following groups: cytosine deaminase (CD), carboxylesterase (CE), herpes simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine nucleoside phosphorylase (PNP), and horseradish peroxidase (HRP).

4. The fusion protein as described in claim 1, characterized in that, The mitochondrial outer membrane targeting protein is selected from any one of the group consisting of TOM20, TOM70, OM45, TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B.

5. The fusion protein as described in claim 1, characterized in that, When the mitochondrial outer membrane targeting protein is TOM20, TOM70, or OM45, the mitochondrial outer membrane targeting protein is linked to the prodrug convertase or a fragment thereof in order from the N-terminus to the C-terminus.

6. The fusion protein as described in claim 1, characterized in that, When the mitochondrial outer membrane targeting protein is selected from any one of the group consisting of TOM5, TOM6, TOM7, TOM22, Fis1, Bcl-2, Bcl-x and VAMP1B, the prodrug convertase or a fragment thereof is linked to the mitochondrial outer membrane targeting protein in the order from N-terminus to C-terminus.

7. The fusion protein as described in claim 1, characterized in that, The prodrug converting enzyme or a fragment thereof is linked to the mitochondrial outer membrane targeting protein via a linker.

8. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein as described in any one of claims 1-7.

9. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 8.

10. A transformed cell, characterized in that, The transformed cells are introduced into the vector as described in claim 9.

11. A modified mitochondria, characterized in that, Prodrug-converting enzymes or fragments thereof are present on the outer mitochondrial membrane.

12. The modified mitochondria as described in claim 11, characterized in that, The outer mitochondrial membrane further contains antibodies or fragments thereof that specifically bind to tumor-associated antigens.

13. The modified mitochondria as described in claim 11, characterized in that, The prodrug-converting enzyme is selected from any one of the following groups: cytosine deaminase (CD), carboxylesterase (CE), herpes simplex virus thymidine kinase (HSV-TK), cytochrome P450 (CYP450), purine nucleoside phosphorylase (PNP), and horseradish peroxidase (HRP).

14. The modified mitochondria as described in claim 12, characterized in that, The tumor-associated antigen is selected from any one of the following groups: CD19, CD20, melanoma antigen E (MAGE), NY-ESO-1, carcinoembryonic antigen (CEA), cell surface-associated mucin 1 (MUC-1), prostate acid phosphatase (PAP), prostate-specific antigen (PSA), Survivin, tyrosine-associated protein 1 (tyrp1), tyrosine-associated protein 2 (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. The modified mitochondria as described in claim 12, characterized in that, The antibody fragment is selected from any one of the group consisting of Fab, Fab', scFv and F(ab)2.

16. A pharmaceutical composition for the prevention and treatment of cancer, characterized in that, The pharmaceutical composition comprises modified mitochondria as described in claim 11 or 12 as an active ingredient.

17. The pharmaceutical composition for the prevention and treatment of cancer as described in claim 16, characterized in that, The cancer is selected from any one of the following groups: breast cancer, lung cancer, pancreatic cancer, glioma, stomach cancer, liver cancer, colorectal 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 as described in claim 11 in the preparation of a medicament for treating cancer.

19. A method for treating cancer, characterized in that, The method includes administering the modified mitochondria as described in claim 11 to a subject.

20. The method for treating cancer as described in claim 19, characterized in that, The modified mitochondria are administered in combination with an anticancer prodrug.

21. The method for treating cancer as described in claim 20, characterized in that, The anticancer prodrug is selected from any one of the following groups: 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazolidinyl compounds, 6-methylpurine deoxynucleoside, and indole-3-acetic acid (IAA).

22. A reagent kit for treating cancer, characterized in that, Includes the modified mitochondria and prodrug as described in claim 11.

23. The reagent kit for treating cancer as described in claim 22, characterized in that, The prodrug is selected from any one of the group consisting of 5-fluorocytosine (5-FC), irinotecan, ganciclovir, oxazolidinyl compounds, 6-methylpurine deoxynucleoside, and indole-3-acetic acid (IAA).

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