KR20230105583A - Polynucleotide for treatment cancer encoding 5'-nucleotidase modified protein - Google Patents

Abstract

As a polynucleotide for novel cancer treatment, it is used in the form of being captured in liposomal nanoparticles that form a complex with a binder that binds to CD47, thereby maximizing the metabolic vulnerability of cancer cells and killing cancer cells. A polynucleotide for is disclosed. The present invention provides a polynucleotide for cancer treatment encoding the amino acid sequence represented by SEQ ID NO: 3.

Description

Polynucleotide for treatment cancer encoding 5'-nucleotidase modified protein {Polynucleotide for treatment cancer encoding 5'-nucleotidase modified protein}

The present invention relates to a polynucleotide for cancer treatment, and more particularly, to a polynucleotide for cancer treatment used in a form entrapped in a drug delivery material.

Cancer cells abuse signals used in normal mammalian biochemistry to prevent immune cells from destroying other cells, such as CD47. Interfering with these “don’t eat me” signals has had significant advantages in the development of effective cancer therapies that can target many types of cancer.

Immune cells, commonly called macrophages, detect cancer cells and then engulf and eat them. Researchers have discovered in recent years that proteins on the surface of cells send signals to macrophages not to eat or destroy them. This is useful for protecting the immune system from attacking normal cells, but cancer cells use these "don't eat me" signals to evade the immune system. Researchers have previously shown that the protein PD-L1 and the beta-2-microglobulin subunit of the major histocompatibility class 1 complex are utilized by cancer cells to protect themselves from immune cells. Antibodies that block CD47 are currently in clinical trials, and cancer therapies targeting the PD-L1 or PDL1 receptor are being used to treat patients.

This study showed that many cancers produce CD47 in abundance compared to normal cells and surrounding tissues. In a recent study, researchers showed that tumor-infiltrating macrophages can detect CD47 signals through a receptor called Sirpα. They also showed that if you mix a patient's cancer cells with macrophages in a dish and block the interaction of CD47 and Sirpα, the macrophages start eating the cancer cells. Finally, they transplanted human breast cancer cells into the mice. When the CD47 signal was blocked, the macrophages of the mouse's immune system attacked the cancer. Of particular note was the discovery that blocking the CD47 signal had a significant effect on blood cancer and triple-negative breast cancer.

[Prior patent literature]

- Korean Patent Publication No. 2006-0121150 (2006.11.28.)

The present invention is a novel polynucleotide for cancer treatment, which is used in the form of being encapsulated in liposome-type nanoparticles that form a complex with a binder that binds to CD47, thereby maximizing the metabolic vulnerability of cancer cells to kill cancer cells. It is intended to provide polynucleotides for cancer treatment.

In order to solve the above problems, the present invention provides a polynucleotide for cancer treatment encoding the amino acid sequence represented by SEQ ID NO: 3.

In addition, the polynucleotide is characterized by comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 41.

In addition, the polynucleotide provides a polynucleotide for cancer treatment, characterized in that the mRNA.

In addition, the mRNA provides a polynucleotide for cancer treatment characterized in that it enters cancer cells and inhibits nucleic acid metabolism.

In addition, the nucleic acid metabolism provides a polynucleotide for cancer treatment, characterized in that dTTP biosynthetic metabolism.

In addition, the polynucleotide provides a polynucleotide for cancer treatment, characterized in that it further comprises a 5'-UTR represented by SEQ ID NO: 19 and a 3'-UTR represented by SEQ ID NO: 20.

In addition, the polynucleotide provides a polynucleotide for cancer treatment, characterized in that it further comprises a nucleic acid sequence encoding a nuclear localization signal (NLS) represented by SEQ ID NO: 22.

In addition, the polynucleotide provides a polynucleotide for cancer treatment, characterized in that it further comprises a nucleic acid sequence encoding a mitochondrial localization signal (MLS) represented by SEQ ID NO: 23.

In addition, it provides a polynucleotide for the treatment of cancer, characterized in that the cancer is colon cancer or breast cancer.

The present invention is a polynucleotide for cancer treatment having a mechanism of killing cancer cells by maximizing the metabolic vulnerability of cancer cells when used in the form of being captured in liposomal nanoparticles that form a complex with a binder that binds to CD47, It is possible to provide a polynucleotide for cancer treatment in which the gene sequence is optimized to minimize the site acting as a non-specific microRNA (microRNA) when delivered into human cells and to maximize expression.

1 is a diagram showing a comparison of the amino acid sequences of Sirpα, SV1 and SV4. Bold letters indicate residues that are not partially conserved. Sequence alignment was done with ClustalW, and images were created with the BioEdit sequence alignment editing program.
Figure 2 is a diagram explaining mutation of SV1 for correct directionality. In FIG. 2A, the SV domain (green ribbon) is bound to the CD47 (red sphere) domain, and in the model of FIG. show that it is mutated for 2B shows the analysis results using mass spectrometry analysis of DSPE-conjugated SV1 and SV4.
Figure 3 is a diagram showing the conserved motif of T001 and human NT5M. As a sequence alignment of T001 and human NT5M, it was aligned through ClustalW to confirm the sequence similarity of human NT5M and T001. The Swiss-Prot/TrEMBL accession numbers of the sequences used for alignment are human NT5M and T001. Red fields represent fully conserved amino acid residues, and red letters in white fields represent partially conserved amino acid residues with similar biochemical functions. Sequence alignment was done with ClustalW, and images were created with the ESPript server.
4 is a model showing a comparison of structures of T001 and NT5M. Cytoplasmic T001 (CT) and dTMP-bound human NT5M (yellow) are shown superimposed, nitrogen is shown in white and oxygen is shown in red.
5 is a schematic diagram showing candidate structures for UTR screening for optimal expression of T001.
6a to 6m are photographs and graphs showing FACS analysis results in UTR screening for optimal expression of T001. The assay conditions were as follows: GFP fluorescence, HCT-116, 6well (5x10 ⁵ cells/well), 24h mRNA transfection, 10% FBS, 2mM Gln MEM media.
7 is a schematic diagram showing the N-terminal and C-terminal sequences of mStrawberry. The estimated import signal is located as shown in FIG. 7 .
8 is a photograph showing the intracellular action site of mStrawberry-NLS and mStrawberry-MLS revealed by fluorescence microscopy. White arrows indicate the location of the nucleus, and black arrows indicate the location of the mitochondria.
9 is a diagram showing the mode of action (MOA) and pathway of target metabolism in cancer cells.
10 is a photograph showing the results of a live and dead assay after mRNA transfection in MCF7 cell line. Cell viability of MCF7 cells was observed after treatment with 5 μg/well of mRNA using a fluorescence microscope. Cell viability was effectively reduced at 24 hours post-transfection, and this effect was either time dependent or dose dependent.
11 is a graph showing the results of MTT analysis after mRNA transfection in MCF7 cell line.
12a to 12d are graphs showing apoptosis analysis results according to Annexin V staining after mRNA transfection in MCF7 cell line. The early apoptotic portion (lower right quadrant) increased continuously after mRNA transfection, and the late apoptotic portion (upper right quadrant) also increased.
13 is a graph showing comparison results of cytotoxicity and cell growth inhibition according to T001 and NT5M transfection.
14a to 14c are graphs showing apoptosis results induced by CT and NT5M transfection.
15a to 15c are graphs showing cell cycle arrest by T001 transfection.
16 is a graph showing the ratio of apoptosis-inducing cells according to the concentration in the colorectal cancer cell line HCT-116.
17a to 17c are graphs showing the effect of T001 offset by siRNA treatment of T001.
18 is a graph showing the ratio of apoptosis-inducing cells according to the concentration in triple-negative breast cancer (TNBC).
19 is a photograph showing the results of Western blot analysis of DNA damage markers after CT treatment for triple-negative breast cancer.
20 is a schematic diagram showing an anticancer agent using a SV4 binder and a drug T001.
21 is a schematic diagram schematically showing the composition of T001 mRNA.
22 is a schematic diagram and photographs explaining the results of in vitro analysis of carboxy fluorescein-DSPE complexed with an immuno-liposome (iLP) containing NLS-mStrawberry mRNA. The location of each translated mStrawberry protein and mRNA was confirmed by nuclear transfection through fluorescence detection in the MCF7 cell line. A is 2.5 μg of NLS-mStrawberry mRNA and B is 5 μg of NLS-mStrawberry mRNA.
23 is a photograph showing the results of CD47 masking assay in vitro and in vivo .
24 is a photograph showing the distribution of MCF7 xenograft mice after intravenous (IV) injection of SV4-conjugated iLP-NIR RFP mRNA. A is a xenograft mouse, B is 1 hour after injection, C is 3 hours after injection, D is 6 hours after injection, and E is the cut cancer tissue.
25 is a graph and a tumor photograph showing tumor volume by period after iLPD intravenous injection in vivo .
26 is a graph showing the results of toxicity test of mouse organs by iLPD treatment.
27 is a schematic diagram showing the mechanism of anticancer agents using the SV4 binder according to the present invention.
28 is a diagram showing a comparison of DNA sequences of SIRPα and SV1.
29 is a diagram explaining the conjugation process between DSPE-PEG ₂₀₀₀ -NHS and SV4 protein.

Hereinafter, the present invention will be described in detail through examples. Prior to this, the terms or words used in this specification and claims should not be construed as being limited to the usual or dictionary meaning, and the inventor appropriately uses the concept of the term in order to explain his/her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical spirit of the present invention. Therefore, since the configurations of the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, various equivalents and modifications that can replace them at the time of this application It should be understood that there may be

The terms used herein can be understood as follows.

The term “CD47” herein is not particularly limited, and may be derived from any animal, preferably a mammal, and more preferably human CD47. The amino acid sequence and nucleotide sequence of human CD47 are already known (J. Cell. Biol., 123, 485-496, (1993), Journal of Cell Science, 108, 3419-3425, (1995), GenBank: Z25521). .

As used herein, the term “binder” refers to a protein that binds to a receptor, in particular CD47, wherein the binder binds to CD47, particularly in cancer cells, enabling recognition and/or interaction with a cell-transmitter.

As used herein, the term "conjugated" or "conjugate" refers to a chemical compound formed by combining two or more compounds with one or more chemical bonds or linkers. In one embodiment of the invention, the binder and the liposome form a conjugate.

As used herein, the term “PEGylation” refers to a technique for conjugating polyethylene glycol (PEG) to a target material to increase stability, and in the present invention, a pegylated phospholipid may be used, for example, DSPE-PEG ₂₀₀₀ or the like may be used. can DSPE-PEG ₂₀₀₀ means DSPE attached with PEG having a number average molecular weight of about 2000.

As used herein, the term “polynucleotide” generally refers to deoxyribonucleotides or polymers of ribonucleotides, which may be RNA or DNA, or modified RNA or DNA, in single-stranded or double-stranded form. In one embodiment of the present invention, the polynucleotide is a synthesized single-chain "mRNA".

As used herein, the term "5'-Untranslated Region (5'-UTR)" is commonly understood as a specific portion of mRNA, and is located 5' of the protein coding region (ie, open reading frame (ORF)) of mRNA. Typically, the 5'-UTR begins at the transcription start region and ends one nucleotide before the initiation codon of the open reading frame.

As used herein, the term "3'-Untranslated Region (3'-UTR)" is a part of mRNA that is usually located between the open reading frame (ORF) of mRNA and the polyA sequence. The 3'-UTR of mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is usually encoded by a gene that is transcribed into each mRNA during gene expression.

The term “Kozak sequence” herein refers to a translational initiation enhancer element that improves expression of a gene or open reading frame (ORF), which in eukaryotes is located in the 5'-UTR but at position -3 of the initiation codon is an A or It is located in a sequence containing G and may further include a sequence (eg, -GTG-) containing G at position +1 of the initiation codon.

As used herein, the terms “nuclear localization signal (NLS)” and “mitochondrial localization signal (MLS)” are amino acid sequences that serve to transport specific substances such as proteins or nucleic acids into the cell nucleus and mitochondria, respectively. means

As used herein, the term “transfection” refers to a process in which an extracellular polynucleotide enters a host cell, particularly a cancer cell, with or without an accompanying substance. “Transfected cell” may mean, for example, a cell having an extracellular mRNA by introducing an extracellular mRNA into the cell.

The present invention discloses a polynucleotide for cancer treatment encoding the amino acid sequence represented by SEQ ID NO: 3.

According to one embodiment, the polynucleotide for cancer treatment according to the present invention is used in the form of being encapsulated in liposomal nanoparticles that form a complex with a binder that binds to CD47, thereby maximizing the metabolic vulnerability of cancer cells. As a polynucleotide for cancer treatment having a mechanism of killing, preferably, the gene sequence is optimized to minimize the site acting as a non-specific microRNA (microRNA) and maximize expression when delivered into human cells. As the polynucleotide whose gene sequence is optimized, it can be selected from the nucleotide sequences represented by SEQ ID NO: 39 to SEQ ID NO: 41, and preferably, the nucleotide sequence represented by SEQ ID NO: 39 can be selected. SEQ ID NO: 38 represents a wild type sequence.

In one embodiment of the present invention, the binder binds to CD47 overexpressed in cancer cells, and includes the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

In the present invention, the binder comprising the amino acid sequence represented by SEQ ID NO: 1 was named 'SV1', and the binder comprising the amino acid sequence represented by SEQ ID NO: 2 was named 'SV4'.

First, Sirpα-variant-version1 (SV1) was selected from a mutant library derived from pure Sirpα (see amino acid sequence (SEQ ID NO: 24)), which is the soluble domain of the original CD47 ligand. The selection process for the SV1 mutation is as follows.

Weiskopf K et al. (Weiskopf, K., et al. (2013). "Engineered SIRPalpha variants as immunotherapeutic adjuvants to anticancer antibodies." Science (New York, N.Y.) 341 (6141): 88-91.), the gene was obtained by synthesizing a single 14-kD binding domain of human SIRPa. Amino acids changed from wild-type SIRPα are as follows. Valine at the 6th position is converted to isoleucine, valine located at the 27th position to isoleucine, isoleucine located at the 31st position to phenylalanine, Glutamate at position valine, lysine at position 53 to arginine, glutamate at position 54 to glutamine, position at position 56 Histidine was replaced with proline, serine at position 66 with threonine, and valine at position 92 was replaced with isoleucine. For protein expression in E. coli, SV1 was prepared by modifying the existing nucleic acid sequence through codon optimization, and FIG. 28 shows a comparison between the SIRPα sequence (SEQ ID NO: 25) and the DNA sequence (SEQ ID NO: 26) of SV1.

On the other hand, SV1-(C)CRM197, which is a protein in which CRM197 protein is added to the C-terminus of SV1, CRM197(N)-SV1 in which CRM197 protein is added to the N-terminus, and SV1 protein are prepared to make SV1 N-terminus and CRM197 protein. - The effect of adding the protein at the end was confirmed through SPR (Surface Plasmon Resonance) analysis. Each sample was analyzed through binding kinetics of SPR using a Biacore X100 instrument. At this time, as a chip for analysis, a chip in which recombinant CD47 protein was conjugated to the surface of Protein G to 500 RU was used, and HBS-P was analyzed at a flow rate of 5 μl/min. Each sensogram result was analyzed with BiaEvaluation software using a 1:1 binding model and global fitting, and the results are shown in Table 1 below.

Analyte K _D (nM) SV1 protein 0.877 SV1-CRM197 protein 0.894 CRM197-SV1 protein 19.9

Referring to Table 1, in the results of SPR analysis, the _KD value of SV1-CRM197 with CRM197 inserted at the C-terminus did not change significantly compared to SV1, but in the case of CRM197-SV1 with CRM197 inserted at the N-terminus, affinity was It was confirmed that the _KD value increased about 2 times to 19.9 nM, and the affinity decreased. Therefore, it was confirmed that when a protein was added at the N-terminus of SV1, the affinity for CD47 was reduced compared to SV1 due to steric hindrance caused by preventing SV from correctly binding to CD47.

Hereinafter, specific examples of the SV4 binder related to the present invention and the liposome conjugated with the binder will be described in detail, and then the T001 drug encapsulated in the binder-conjugated liposome according to the present invention, and the anticancer agent using the SV4 binder and the T001 drug The present invention will be described in detail with reference to specific examples.

SV4-conjugated liposomes

Drug delivery systems based on lipid nanoparticles have been widely used with significant advantages. Lipid nanoparticles have high drug capacity, high stability, and high specificity, and the release point can be controlled. Because of the genetic material used as a drug, cationic liposomes have been used to deliver drugs to target cancers. The positively charged liposomes attract the genetic drug and form a spherical complex that covers the drug for protection until it meets its target.

In the present invention, to form cationic liposomes, pro-liposomes were prepared using one of cationic phospholipids, DOTAP and cholesterol. In addition, PEG ₁₀₀₀ -DSPE was additionally added to pro-liposomes to prepare pegylated liposomes to increase serum stability during drug delivery to the target through blood vessels. In addition to pegylated liposomes, DSPE-PEG ₂₀₀₀ -SV4 was prepared by conjugating NHS activated DSPE-PEG ₂₀₀₀ with SV4 protein. As a final step to prepare SV4 conjugated liposomes, mRNA encapsulated pro-liposomes were mixed with DSPE-PEG ₂₀₀₀ -SV4. The specific SV4-conjugated liposome manufacturing process is as follows.

Liposomes were prepared by dry-film method. Cationic liposomes composed of DOTAP (Avanti Polar Lipids) and cholesterol (Sigma) (1:1 molar ratio, 10 mM) and PEG-DSPE1000 (1 mM; Avanti Polar Lipids) were added. Cationic liposomes were dissolved in chloroform and methanol (2:1 (v/v)) in a round bottom glass flask. Lipid drying was performed under vacuum on a rotary evaporator at 50 °C. The lipid film was freeze-dried overnight to completely remove chloroform and methanol. After evaporation, the lipid film was rehydrated with nuclease free water at 50 °C for up to 1 hour. The hydrated lipid membrane was sonicated to form unilamellar vesicles. Finally, lipids were extruded with a mini extruder (Avanti Polar Lipids) using a membrane with a 100 nm pore.

Conjugation of DSPE-PEG ₂₀₀₀ -NHS with SV4 protein was prepared as follows (see Figure 29). DSPE-PEG ₂₀₀₀ -NHS was prepared by dry-film method. DSPE-PEG ₂₀₀₀ -NHS dissolved in chloroform was evaporated in a round bottom glass flask. Lipid drying was performed by rotary evaporator at 30° C. under vacuum for 1 hour. After evaporation, the lipid film was rehydrated with SV4 protein dissolved in nuclease-free water for 1 hour at 30 °C. A 10,000 MWCO (Thermo Scientific) dialysis cassette was run overnight in PBS pH 6.8 to remove residual unconjugated DSPE-PEG ₂₀₀₀ -NHS.

Liposomes containing encapsulated drug and binding ligand were prepared as described above. 3 mg of cationic liposome, 25 μg of protamine, and diethylpyrocarbonate water were mixed to obtain solution A, and 50 μg of mRNA and diethylpyrocarbonate water were mixed to obtain solution B. Solutions A and B were equalized in volume with diethyl pyrocarbonate water and incubated for 30 minutes. Thereafter, mixing and incubation for 30 minutes formed liposomes with the encapsulated drug. For the binding of the DSPE-PEG ₂₀₀₀ -NHS conjugate SV4 ligand, liposomes (1:100 molar ratio) containing the encapsulated drug were mixed at 50°C for 15 minutes.

SV1 as a conjugation target

By modifying the sequence of Sirpα present in nature, SV1 with improved binding ability to CD47 was selected through mutation, and SV4, which can be inserted in the correct direction and reacted when preparing a liposome formulation, was secured through mutation (FIG. 1). reference). SV4 mutant construction was performed in the following way. In the SV1 sequence obtained above, 11th and 104th lysines were substituted with leucine for binding in the correct direction with CD47. For substitution, using a plasmid in which the SV1 gene was inserted into the pET28a vector, primers (see Table 2 below) were prepared for point-mutation to sequences corresponding to the 11th and 104th positions, and Quickchange II site-directed According to the method of the mutagenesis kit (Agilent), mutant genes each or simultaneously substituted were prepared.

division order sequence number Leu 11 mutagenesis (KKtoLL11_F1) Forward GAT TAT TCA GCC GGA CCT GTC CGT AAG CGT TGC 27 (KKtoLL11_R1) Reverse GCA ACG CTT ACG GAC AGG TCC GGC TGA ATA ATC 28 Leu 104 mutagenesis (KKtoLL104_F2) Forward CTG ACA CGG AGT TTC TGT CTG GCG CAG 29 (KKtoLL104_R2) Reverse CTG CGC CAG ACA GAA ACT CCG TGT CAG 30

SV1 protein has six lysine residues in addition to the N-terminal amino group (see FIG. 2A). In order to deliver a drug through binding to CD47 by linking SV1 with liposome through conjugation, it is necessary to improve the binding force. Among the residues that can act in the chemical reaction linking DSPE through NHS conjugation, residues that can act in the correct orientation without interfering with CD47 binding are selected, and residues other than these residues are selected as Leucine. can be modified without loss of binding activity. As shown in FIG. 2, DSPE-conjugated SV4 was prepared through simple coupling by reducing the number of residues binding to DSPE through substitution.

On the other hand, in order to confirm the Lysine substitution effect for the correct orientation of SV4, a DSPE-conjugated form and a form inserted into liposomes were prepared for comparison with SV1, respectively, and affinity with CD47 was measured by SPR ( Surface Plasmon Resonance) was analyzed. For analysis, after splicing CD47 using CM5 Chip, compare _KD values by measuring association and dissociation of SV1, DSPE-SV1, LPD-SV1, SV4, DSPE-SV4 and LPD-SV4 did Specifically, the recombinant CD47 protein was conjugated to the surface of the CM5 chip with a target SPR reaction of 250 RU through the EDC-NHS reaction, and HBS-P was used at a flow rate of 30 μl/min for 3 minutes of association and 10 minutes of dissociation The sensogram obtained through was analyzed with BiaEvaluation software using a 1:1 binding model and global fitting. 10 mM glycine pH 2.0 was used for the SV1 and SV4 proteins, and 2.0 M MgCl ₂ was used for the sample with attached fatty acids as a dissociation buffer according to the characteristics of the sample to be analyzed.

In the results of SPR analysis, the _KD value of SV1 was significantly improved from 280 nM to 0.87 nM compared to Sirpα (wt) (see Table 1). The affinity of SV1 was greatly improved, but when SV1-DSPE was prepared through the NHS conjugation reaction, the affinity decreased due to the increase in _KD value from 0.87 nM to 2.67 nM. Also, in the case of SV1-iLP inserted into liposomes, the affinity was further decreased and the _KD value increased to 10.9 nM. In contrast, in the case of SV4, in which two lysines, which are NHS reactive residues, were substituted with leucine, the affinity of the protein itself was slightly reduced, but due to the correct orientation, it was conjugated with DSPE and even when inserted into liposomes, it was within the error range without a significant decrease. It was confirmed that the binding activity was maintained (see Table 3).

Analyte k _a (M ^-1 s ^-1 ) k _d ( s ^-1 ) K _D (nM) Sirpα 6.10×10 ⁵ 3.70×10 ^-1 290.0 SV1 3.53×10 ⁵ 3.08×10 ^-4 0.87 SV1-DSPE 7.34×10 ⁴ 1.96×10 ^-4 2.67 SV1-iLP 1.73×10 ⁴ 1.89×10 ^-4 10.9 SV4 1.64×10 ⁵ 2.75×10 ^-4 1.67 SV4-DSPE 2.27E×10 ⁵ 2.56×10 ^-4 1.13 SV4-iLP 3.12E×10 ⁵ 3.95×10 ^-4 1.27

Binder-conjugated liposomal entrapment drugs

Hereinafter, embodiments of the drug encapsulated in the binder (SV4)-conjugated liposome will be described.

Thymidylate 5' derived from Bacteriophage PBS2, which has activity similar to that of human 5'-nucleotidase, but has exceptionally high specificity only for dTMP and dUMP without specificity for other nucleic acids. -Phosphohydrolase (Thymidylate 5'-phosphohydrolase) was selected as a drug candidate with a mechanism to kill cancer cells by maximizing the metabolic vulnerability of cancer cells, and when delivered into human cells, non-specific microRNA (microRNA) ), the amino acid sequence represented by SEQ ID NO: 3 and the polynucleotide encoding it (SEQ ID NO: 39) were named 'T001' by optimizing the gene sequence to maximize expression and minimize the site acting as Nucleotides (SEQ ID NO: 40 and SEQ ID NO: 41) were named 'T002' and 'T007', respectively. The sequence optimization process of T001 is as follows.

To optimize codons for mammalian cell expression in the DNA sequence, natural codons were replaced with the following optimal codons: alanine (GCC), arginine (CGC), asparagine (AAC), aspartic acid (GAC). ), cysteine (TGC), glutamic acid (GAG), glutamine (CAG), glycine (GGC), histidine (CAC), isoleucine (ATC), leucine (CTG), lysine (AAG), methionine (ATG), phenylalanine ( TTC), proline (CCC), serine (TCC), threonine (ACC), tryptophan (TGG), tyrosine (TAC), and valine (GTG). Runs of Cs and Gs were avoided to simplify PCR conditions as well as oligonucleotide synthesis.

Similar to T001, enzymes involved in nucleic acid metabolism in human cells are 5'-nucleotidases, and three types of enzymes are known according to the location of action: NT5C (cytoplasm), NT5M (mitochondria), and NT5E (extracellular membrane). These three enzymes are known to have different preferred substrate nucleic acid species due to structural differences in activity and structural differences in hydrolyzing the phosphate group of NMP or dNMP as well as the site of action, and most of them have broad specificity for NMP or dNMP. Among them, NT5M was presumed to be generally similar in structure to T001 compared to T001 in terms of amino acid sequence (see FIG. 3). In particular, despite the difference in sequence, the sequence of the action site was conserved and it was identified as belonging to the HAD superfamily (haloalkanoic acid dehalogenase superfamily).

Despite structural homology, the structural difference between NT5M and T001 is that T001 has a unique binding loop structure that does not exist in NT5M, and this is the biggest characteristic difference (see FIG. 4).

The binding loop is closely related to the substrate binding sites of NT5M and T001, and is presumed to be related to the specificity of the substrate. Until now, little is known about the function of the binding loop, but in the present invention, it was partially confirmed that the binding loop has higher affinity for dTMP than NT5M and high dTMP resolution (see Table 4).

5' NT Alias Preferred substrate (km) References PBS2 TMP phosphohydrolase T001 dTMP (0.01 mM) dGMP (0.7 mM)
dUMP (0.8 mM) Methods Enzymol. 51:285-290 (1978);
Also In house data Human mitochondrial 5'doxyribonucleotidase NT5M (hdNT-2) dGMP (0.09 mM)
dUMP (0.16 mM)
dTMP (0.3 mM) Biochem. 46:13809-13818 (2007)
Biochemical Pharmacol. 66: 471-479 (2003) Human cytosolic 5'deoxyribonucleotidase hdNT-1 dUMP (1.5 mM)dTMP (1.5 mM)
dAMP (3.0 mM)
dGMP (3.3 mM) J. Biol. Chem. 265:6589-6595 (1990)
Biochemical Pharmacol. 66: 471-479 (2003) Murine cytosolic 5'deoxyribnucleotidase mdNT-1 dUMP (0.8 mM) dAMP (1.0 mM)
dGMP (1.2 mM)
dTMP (1.4 mM) J. Biol. Chem. 275:5409-5415 (1990)
Biochemical Pharmacol. 66: 471-479 (2003)

As shown in Table 4, it is known to have the highest affinity for dTMP among other 5'-deoxyribonucleotidase known to date. According to what has been reported so far, it can be seen that other enzymes show a relatively broad substrate specificity as well as low affinity for dTMP compared to T001 presented in the present invention. In particular, even when compared with the structurally similar human NT5M, the spectrum for the substrate is different, and the affinity for dTMP is also about 30 times lower according to Table 4. This difference is presumed to be due to the presence of a binding loop in T001 that does not exist in NT5M.

T001 mRNA drug - localization and mRNA structure

The form of the final drug of T001 is in the form of mRNA, and UTR (untranslated region) and Kozak sequence were optimized by optimizing each component required for expression. To this end, expression efficiency was determined by selecting a candidate structure as shown in FIG. 5 using GFP (Green Fluorescent Protein) as a reporter gene. Sequence information of each used UTR is shown in Table 5 below.

No 5′-UTR 5'-UTR source 3′-UTR 3'-UTR source Ref One TTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTA
(SEQ ID NO: 4) Chimeric introns ACGCGTCGAGCATGCATCTAGGGCGGCCAATTCCGCCCCTCTCCCCCCCCCCTCTCCCTCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCAAAGGAATGCAA GGTCTGTTGA
(SEQ ID NO: 5) IRES - 2 (SEQ ID NO: 6) Chimeric introns - IRES - 3 (SEQ ID NO: 7) Chimeric introns GCATCACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTAGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAATGCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCTCTTTTCAGAGCCACCAGAATT (SEQ ID NO: 8) Albumin Andreas Thess (2015) 4 (SEQ ID NO: 9) Chimeric introns GCATCACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTAGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAATGCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCTCTTTTCAGAGCCACCAGAATT (SEQ ID NO: 10) Albumin Andreas Thess (2015) 5 GGGTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATTCAAGCGCCACC (SEQ ID NO: 11) Andreas Thess (2015) GCATCACATTTAAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCTAGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAATGCATCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAAGGCTCTTTTCAGAGCCACCAGAATT (SEQ ID NO: 12) Albumin Andreas Thess (2015) 6 CTTCCTACTCAGGCTTTATACAAAGACCAAGAGGTACAGGTGCAAGGGAGAGAAGAAGAGTAAGAAGAAATATAAGGCCACC (SEQ ID NO: 13) Mus musculus_alpha-globin (Hbat1) GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID NO: 14) Mus musculus hemoglobin alpha (Hba-a1) UTRdb 7 AGTAAGAAGAAATATAAGGCCACC (SEQ ID NO: 15) Rhinatrema bivittatum thiamin pyrophosphokinase 1 GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID NO: 16) Mus musculus hemoglobin alpha (Hba-a1) UTRdb 8 GAGACTGCCACC (SEQ ID NO: 17) Zeljka Trepotec (2019) GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID NO: 18) Mus musculus hemoglobin alpha (Hba-a1) UTRdb 9 GAGACTGCCAAG (SEQ ID NO: 19) Zeljka Trepotec (2019) GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID NO: 20) Mus musculus hemoglobin alpha (Hba-a1) UTRdb 10 - - GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG
(SEQ ID NO: 21) Mus musculus hemoglobin alpha (Hba-a1) UTRdb

Expression efficiency for each UTR was analyzed by FACS through the degree of GFP fluorescence. The FACS analysis method is as follows.

5 × 10 ⁵ cells per each cell of a 6-well cell culture plate were cultured for 24 hours in a cell incubator at 37°C and 5% CO ₂ , and then adhered to the bottom of the plate. EGFP expressing green fluorescence having each UTR structure mRNA was transfected using lipofectamine messengerMAX reagent. First, mix 125 μl of OPTI-MEM medium and 3.5 μl of reagent in a microtube and incubate for 10 minutes at room temperature. In another microtube, add 125 μl of medium and 1.25 μg of mRNA having each UTR structure, and mix the mRNA diluted medium with the above reagent. After mixing and further incubating for 5 minutes, it was administered to the cells of each sphere. After 4 hours, the cells in each sphere were lightly washed with phosphate buffered saline, replaced with cell culture medium, and then further cultured for 20 hours. The green fluorescent protein expression level of EGFP mRNA of each UTR was compared and analyzed by fluorescence microscopy and flow cytometry. . As a control, EGFP mRNA purchased from Trilink Biotechnology was used. First, the intensity of green fluorescence of the cultured cells was compared and analyzed as an image through a fluorescence microscope, and then the cells treated with EGFP mRNA with each UTR, including the control group, were detached from the bottom of the plate with trypsin enzyme, collected in a microcentrifuge tube, and phosphorus acid It was diluted in buffered physiological saline. Through the cell flow cytometer recovered in this way, the distribution of the cell population expressing green fluorescence compared to the control group was compared into three levels, strong, medium, and weak, according to intensity.

As a result of FACS analysis, the final expression form in the form of a shortened UTR was confirmed as UTR No. 9 (see FIGS. 6a to 6m).

In order to diversify the intracellular action site based on UTR No. 9, the location signal sequence (NLS: SEQ ID NO: 22, MLS: SEQ ID NO: 23) as shown in FIG. After addition, the reporter gene mStrawberry was expressed according to the positioning signal sequence, and the position of the protein was confirmed by fluorescence (see FIG. 8). The specific process of the fluorescence localization test (mRNA transfection: lipofectamine) according to the localization signal sequence is as follows.

One cover glass for observation under a confocal microscope is placed in each sphere of the 6-hole cell culture plate, and 5×10 ⁵ cells are cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ , and adhered to the cover glass. mRNAs linked with each localization signal sequence and the mStrawberry reporter gene sequence expressing red fluorescent protein were transfected using lipofectamine messengerMAX reagent. First, mix 125 μl of OPTI-MEM medium and 3.5 μl of reagent in a microtube, incubate at room temperature for 10 minutes, and in another microtube, add 125 μl of medium and 1.25 μg of reporter mRNA having each positional signal sequence, and mix the mRNA diluted medium. It was added to the above reagent, mixed, and after additional incubation for 5 minutes, it was administered to the cells of each sphere. After 4 hours, the cells in each sphere were lightly washed with phosphate-buffered saline, replaced with cell culture medium, and then cultured for 20 hours, and samples for confocal microscopy were prepared. For sample preparation, 4% paraformaldehyde reagent was added, incubated for 10 minutes to fix the cells, washed with phosphate-buffered saline, and then put 20 μl of preservation solution on a slide glass and put a fixed and washed cover glass on top of it. After completion, it was confirmed that the localization signal sequence worked well with red fluorescence of a confocal microscope, and that the mStrawberry protein was located in the cell nucleus and mitochondria.

T001 mRNA synthesis was performed as follows.

Template DNA preparation: The vector for IVT (In Vitro Transcribed) mRNA synthesis was modified from the pIRES vector. Briefly, the 5'UTR-T001-3'UTR cassette was cloned into the MCS of pIRES vector. To generate the IVT template, the plasmid was treated with SacI/HpaI enzymes to form a linear strand, and the 1.5 kb linear strand containing the T7 promoter and the T001 cassette was column-purified as a template for PCR. used A forward primer (gtgcttctgacacaacagtctcgaacttaagc; SEQ ID NO: 36) and a reverse primer (gaaGCGGCCGCCTTCCTACTCAGGCTTTATTC; SEQ ID NO: 37) were used in the PCR reaction, and all PCR reactions were performed using Pfu polymerase at 95° C. for 1 min, 61° C. for 1 min; PCR was performed at 72° C. for 3 minutes, for a total of 30 cycles. PCR products were run on an agarose gel and extracted using the Qiagen cleanup kit before further processing.

IVT mNRA synthesis: After PCR, genetic information is transcribed from DNA to mRNA in vitro using the HiScribe™ T7 ARCA mRNA kit (New England Biolabs, Cot. #. E2065). 20 μl of IVT reaction mixture was prepared by adding 10 μl of NTP/cap analog mixture, 1 μg of template DNA, and 2 μl of 1×T7 RNA polymerase mixture to the reaction solution. The IVT reaction mixture was incubated at 37°C for 30 minutes. To remove template DNA, 1 μl DNase was added to the IVT reaction mixture and incubated at 37° C. for 15 minutes. For poly (A) tailing, 20 μl of IVT reaction mixture was prepared by adding 5 μl 10× poly (A) polymerase reaction buffer, 5 μl poly (A) polymerase and 20 μl nuclease-free water to a 40 minutes at 37°C. Then, after purification using the RNeasy Mini Kit (Qiagen, Hilden, Germany), the synthesized mRNA was purified by elution from the spin column membrane with 89 μl of nuclease-free water. Subsequently, 1 μl of Antarctic phosphatase was treated at 37 ° C for 30 minutes to remove 5'-phosphate, and then purified again using the RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by spin column with 50 μl nuclease-free water. The mRNA synthesized by elution from the membrane was recovered. For the next experiment, after adjusting the final concentration to 500 ng/μl, it was aliquoted and stored at -80°C.

Target metabolism in cancer cells

Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are the main types of cancer treatment, but their use is limited due to off-target effects and toxicity. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to alternative dTMP production pathways by restoring thymidine from the tumor milieu and may modulate resistance to TS-targeting drugs. Since there was a report that downregulation of TKs with siRNA increased the capacity of TS siRNA to detect tumor cells compared to existing TS protein-targeting drugs (5FUdR and pemetrexed), the present invention focused on dTTP biosynthetic metabolism.

Complex downregulation of these enzymes is an attractive strategy to enhance TS-targeted anti-cancer therapy, but normal cytotoxicity and resistance to cancer drugs can be problematic. An alternative to avoiding these deficiencies is to obtain a high dTMP specific hydrolase, T001. T001 can hydrolyze dTMP to thymidine without hydrolyzing other dNMP (deoxynucleotide monophosphate). An imbalanced nucleotide pool in cells can be caused by dTTP deficiency, resulting in severe damage to cell growth and proliferation. Considering the substrate selectivity and activity of T001, we hypothesized that (1) an imbalanced nucleotide pool induces human tumor cells to accumulate damage, (2) overexpression of T001 may cause an imbalanced nucleotide pool, and , (3) imbalanced nucleotide pools can lead to cell death by excessive repair frequencies.

Reducing the number of viable cells through transfection of T001 mRNAs into cancer cells

First, in order to evaluate tumor cell growth after transfection with each T001 mRNA, the number of cells in each group was estimated, and the growth of cultured cells was analyzed by a live and dead assay (see FIG. 10). A live and dead assay was performed as follows.

5×10 ⁵ cells are put into each cell of a 6-hole cell culture plate and cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ , and adhered to the bottom. After 24 hours, each mRNA version was transfected using lipofectamine and cultured for an additional 24 hours. The cells of each experimental group were collected, and 2 μM calcein AM and 4 μM EthD-1, which are viability assay reagents, were treated on the recovered cells, reacted for 30 minutes, and then the cells were examined under a fluorescence microscope. Calcein AM enters cells and shows green fluorescence after being degraded by enzymes of living cells, and EthD-1 enters cells of dead cells and stains the nucleus to show red fluorescence.

As shown in Figure 10, viable cells at 24 hours after transfection were significantly reduced compared to the control group, and there was no difference between the mRNA versions (NT, MT and CT).

Inhibition of viable cell proliferation through transfection of T001 mRNAs into cancer cells

MTT assay was performed to quantify cell viability. MTT analysis method is as follows.

Put 10,000 cells per cell in a 96-hole cell culture plate and incubate for 24 hours in a cell incubator at 37° C., 5% CO ₂ conditions to attach to the bottom. After 24 hours, lipofectamine reagents were prepared for each mRNA version and concentration for transfection, and cell activity assays were performed for each time after further culturing for 24 hours, 48 hours, and 72 hours. 10 μl of EZ-Cytox, a cell activity assay reagent, is administered to each sample for each time period, and after 2 hours of reaction, the absorbance value at a wavelength of 450 nm is measured using a plate reader. Cell viability was analyzed by treatment time and concentration by comparing the values of the mRNA treatment group for each version compared to the control group.

As a result of the MTT assay, compared to the control group, the proliferation of cells transfected with T001 mRNA was significantly inhibited at 24 hours after transfection, and the inhibitory effect was maintained up to 72 hours, resulting in a continuous decrease in the viability of each cell. appeared to be Cell viability was dose-dependent (see FIG. 11).

Induction of apoptosis through transfection of T001 mRNAs into cancer cells

According to the above experimental results, flow cytometric analysis was performed after Annexin V staining to investigate whether T001 induces apoptosis to reduce cell number or inhibit cell proliferation. The specific analysis method is as follows.

5 × 10 ⁵ cells per each cell of a 6-well cell culture plate were cultured for 24 hours in a cell incubator at 37 ° C and 5% CO ₂ to attach to the bottom of the plate, and mRNA for each version was prepared using lipofectamine messengerMAX reagent. transfected. First, mix 125 μl of OPTI-MEM medium and 3.5 μl of reagent in a microtube and incubate for 10 minutes at room temperature. In another microtube, add 125 μl of medium and 1.25 μg of mRNA having each UTR structure, and mix the mRNA diluted medium with the above reagent. After mixing and further incubating for 5 minutes, it was administered to the cells of each sphere. After 4 hours, the cells in each sphere were lightly washed with phosphate-buffered saline, replaced with cell culture medium, and then further cultured for 20 hours, then detached from the bottom of the plate with trypsin enzyme, collected in a microcentrifuge tube, and diluted in phosphate-buffered saline. . 3 μl of Annexin V staining reagent and Propidium Iodide staining reagent were added to each microcentrifuge tube, reacted at room temperature for 15 minutes, and the degree of apoptosis induced cell group by version of T001 was compared through flow cytometry. As a control, lipofectamine messengerMAX reagent without mRNA was used. Annexin V reagent stains the cell membrane of cells undergoing apoptosis, and Propidium Iodide stains the intracellular nucleus of dead cells. Therefore, on the flow cytometry graph, cells that do not undergo apoptosis are distributed in the third quadrant, and the position of the cell population moves from the fourth quadrant to the first quadrant according to the degree of apoptosis.

As a result of the analysis, the apoptosis rates in all versions were similar to each other. The apoptosis rate of the control group was 3.75%, and the premature apoptosis rates of cells transfected with NT, MT, and CT were 21.59%, 25.11%, and 24.65%, respectively. The death rates of cells transferred with NT, MT, and CT were 9.85%, 8.42%, and 11.47%, respectively (see FIGS. 12A to 12D). Apoptosis rates increased in a dose-dependent manner, with slight differences between each T001 version.

Comparison of cytotoxicity of T001 and NT5M against colorectal cancer cells (HCT-116)

T001 is more specific for nucleic acid T due to other structural differences from NT5M. Therefore, the imbalance of nucleic acid due to the loss of dTTP rather than the overall loss of nucleic acid can have a greater effect on the metabolism of cancer cells, thereby inhibiting the growth of cancer cells. To prove this, the mature form of human NT5M and the non-mature form containing introns were compared with cytoplasmic T001 (CT) in terms of cell viability and cell growth inhibitory effect. Depending on the substrate specificity, the effect on cells was investigated. Each mRNA was transfected into the colorectal cancer cell line HCT-116, and 24 hours later, Trypan blue assay was performed, and the results of HCT-116 cell viability and cell growth inhibition are shown in FIG. 13 . The specific test method is as follows.

HCT-116 cells were transfected with each of 1.25 μg of non-mature form NT5M, mature form NT5M, and CT mRNA. After 24 hours, trypsin was treated and the cells were harvested using a centrifuge. After resuspension of the collected cells with 1× DPBS, a certain amount of cells was mixed with trypan blue reagent at a ratio of 1:1. After 2 minutes of incubation at room temperature (RT), the number of cells was counted using a hemocytometer. After obtaining the number of cells stained and unstained with trypan blue reagent to obtain the number of viable cells and non-viable cells, divide by the total number of cells to obtain viability. did Each experiment was repeated three or more times to evaluate significance.

As shown in FIG. 13, in the case of T001 having high affinity and activity for dTMP, the effect on cells was greater than that of human NT5M having similar activity.

In particular, it was confirmed that the toxicity shown in the cells was mainly caused by the induction of apoptosis (see FIGS. 14a to 14c). HCT-116 cells were transfected with 1.25 μg each of immature NT5M, mature NT5M, and CT mRNA, and the degree of apoptosis was measured and analyzed after culturing for 24 hours. ), it was confirmed that the CT mRNA increased by about 25% compared to the control group, whereas the Sub G1 phase increased by about 12% compared to the control group. Apoptosis caused by expression of T001 is believed to originate from changes in the cell cycle caused by depletion of intracellular nucleic acids, and selective deficiency of specific nucleic acids and dTTP among substrates leads to arrest of the cell cycle, intensifying this process. It is presumed that apoptosis will eventually be induced if this occurs (see FIGS. 15a to 15c).

The apoptosis-inducing effect shown in the above results showed a concentration-dependent tendency according to the treatment concentration of CT in colon cancer cell lines, indicating that the activity of CT was directly involved in apoptosis (see FIG. 16).

In order to confirm that the apoptosis of these cancer cells is the effect of T001, T001 was knocked down using siRNA against CT, and then the change in the degree of apoptosis was confirmed. Two types of siRNA targeting T001 were prepared and transfected into HCT-116 cells, and then CT mRNA was transfected. After 24 hours, the degree of apoptosis was confirmed by Annexin V/PI staining. The specific test method is as follows.

Two types of siRNA targeting T001 were prepared and transfected using RNAiMAX reagent, and CT mRNA was transfected 1 hour later. After 24 hours, cells were collected and stained using FITC Annexin V Apoptosis Detection Kit I. After incubation at room temperature for 20 minutes, the degree of fluorescence was analyzed using flow cytometry. 10,000 cells were analyzed for each sample. The sequence of the siRNA used is as follows.

[siT001-1]

sense (SEQ ID NO: 32): CGAGAAGAAGUCAGAUUACAUCAAG

antisense (SEQ ID NO: 33): CUUGAUGUAAUCUGACUUCUUCUCG

[siT001-2]

sense (SEQ ID NO: 34): CGCAAAUUCAUUGAAACCUUCCUGA

antisense (SEQ ID NO: 35): UCAGGAAGGUUUCAAUGAAUUUGCG

As a result of the analysis, it was confirmed that apoptosis was reduced in the group transfected with CT mRNA after transfection with siRNA compared to the group transfected with CT mRNA alone (see FIGS. 17A to 17C ).

Concentration-dependent inhibitory effect of T001 in triple-negative carcinoma

It is thought that T001 can induce cancer cell death in carcinomas other than colorectal cancer based on its mechanism of action, and the same test was performed on triple-negative breast cancer (TNBC) to confirm its ability to induce apoptosis. The specific test method is as follows.

MDA-MB-231 and MDA-MB-468 cells were each seeded at 5×10 ⁵ cells/well, and 0, 0.625, 1.25, and 2.5 μg of CT mRNA were respectively transfected. After 24 hours, cells were collected and stained using FITC Annexin V Apoptosis Detection Kit I. After incubation at room temperature for 20 minutes, the degree of fluorescence was analyzed using a flow cytometer. 10,000 cells were analyzed for each sample. Significance was evaluated through three repeated experiments.

After treatment with cytoplasmic T001 (CT) mRNA for each concentration of two types of triple-negative breast cancer, FACS analysis was performed on cells cultured for 24 hours through Annexin V/PI staining. It was also confirmed that the proportion of apoptotic cells increased (see FIG. 18).

To analyze the protein level of the cause of apoptosis, 1.25 μg of CT mRNA was transfected into TNBC cell lines MDA-MB-231 and MDA-MB-468, and Western blotting was performed 18 hours later. . The specific analysis method is as follows.

After transfection with CT mRNA, cells were collected 18 hours later. Cells were lysed on ice for 30 minutes using RIPA buffer (+ protease inhibitor, phosphatase inhibitor), and proteins were separated using a 4° C. centrifuge for 15 minutes. Separated proteins were quantified and 10 to 20 μg of protein was loaded on an acrylamide gel. After transferring the gel to a PVDF membrane, it was blocked for 1 hour at RT with 5% skim milk. Each 1' antibody was added and stored overnight at 4°C. After washing with 0.1% TBST, the 2' antibody was added and incubated for 1 hour at RT. After washing with 0.1% TBST, proteins were detected using ECL solution. Protein expression was analyzed using ChemiDoc XRS+.

As a result of the analysis, as shown in FIG. 19 , it was confirmed that cleavage of PARP, which is an apoptosis marker, and increased expression of gamma-H2AX, which is a DNA damage marker, were confirmed. It was confirmed that DNA damage occurred due to dTTP deficiency during CT mRNA treatment, and it was confirmed that apoptosis was induced through this.

Anticancer drug using SV4 binder and T001 drug

Hereinafter, an embodiment of an anticancer agent using the SV4 binder and the drug T001 will be described.

Anticancer drugs using SV4 binder and T001 drug primarily detect and bind to CD47, which is overexpressed on the surface of cancer cells, and secondarily, mRNA-type nucleic acid metabolism inhibitors that enter cancer cells detect and inhibit excessive nucleic acid synthesis metabolism in cancer cells, thereby inhibiting cancer cells It is a 4th-generation targeted metabolic cancer drug that innovatively reduces side effects of normal cells by targeting immune evasion and metabolic vulnerability. In general, cancer cell-specific surface proteins are often distributed even in normal cells, so when a target protein that recognizes a specific surface protein is combined with a toxic substance, damage to normal cells is inevitable. However, recognition alone does not cause toxicity, and when targeting the metabolism of cancer cells that attempt to synthesize nucleic acids excessively, the recognition rate for cancer cells is high, and damage to normal cells can be reduced. In other words, most normal cells that do not amplify nucleic acid for growth, even if CD47 is recognized and T001 mRNA is introduced into the cell, suffer little damage because they require little nucleic acid. Nucleic acid imbalance caused by deficiency causes great damage. Therefore, in the process of recognizing high-expressed CD47 in cancer cells, even if cells with relatively high CD47 levels are targeted among some normal cells, they cause more damage to cancer cells through the target of nucleic acid metabolism introduced into the inside, thereby increasing the recognition rate of cancer cells and damaging normal cells. can reduce

As shown in Figure 20, the composition of the anticancer agent using the SV4 binder is an mRNA that has a CD47 recognition protein on the outside and an mRNA-type nucleic acid metabolism inhibitor on the inside and a location signal (nuclear, cytoplasmic, or mitochondrial) in different combinations depending on the type of carcinoma. It is in the form of liposome-type nanoparticles encapsulated. 27 schematically shows the mechanism of the anticancer agent using the SV4 binder according to the present invention.

anticancer component

(1) CD47 binder (SV4)

A high-affinity CD47 complex modified from a protein derived from Sirpα and SV4 modified to be positioned outside the liposome in the correct direction upon conjugation with DSPE were used as CD47 binders, and the estimated structure of SV4 is shown in FIG. 1A. was As a result of comparing the K _D values of each of the samples combined with DSPE and the samples inserted into liposomes, it can be seen that the liposome form was improved by sequence mutation (see Table 1 above).

(2) Components of liposome-based drug delivery system

As a material used for smooth drug delivery to the target by positively charging the liposome, the structures of cationic phospholipid (DSPE-PEG ₁₀₀₀ ), DOTAP and cholesterol are shown in Chemical Formulas 1 to 3, respectively.

[Formula 1]

[Formula 2]

[Formula 3]

(3) Medication

As described above, the structure of T001 mRNA is schematically shown in FIG. 21 as an mRNA capable of expression in the nucleus, cytoplasm, and mitochondria.

Positioning of translated proteins by mStrrawberry mRNA fused with positional signal

To confirm that localization signals worked well, mRNA was transfected into MCF7 with nuclear and mitochondrial localization signals. mStrawberry fluorescence was detected to confirm successful transfection and localization of each mRNA (see FIG. 22). The specific test method is as follows.

5 × 10 ⁵ cells per each cell of a 6-hole cell culture plate are cultured for 24 hours in a cell incubator at 37° C. and 5% CO ₂ , and adhered to the bottom of the plate. In liposomes constructed using carboxyfluorescein conjugated DSPE, mStrawberry mRNA producing red fluorescent protein linked to intracellular cytoplasmic localization signals synthesized in the laboratory was captured and cultured for 24 hours in MCF-7 cells. treated and transfected. After 24 hours of additional incubation, confocal laser fluorescence microscopy confirmed the location on cells of green fluorescence where carboxyfluorescein of liposomes was generated with green wavelengths, and mRNA transfected with red wavelengths produced mStawberry protein and was located in the cytoplasm. Confirmed.

Confirmation of CD47-mediated drug delivery

To confirm whether drug delivery occurs through CD47, MCF-7 cells were treated with SV4-conjugated iLP, in which epirubicin was captured. At this time, to confirm CD47 mediation, the experimental group treated with CD47 antibody (polyclonal) and the experimental group not treated were compared. The specific CD47 masking assay method is as follows.

5 × 10 ⁵ cells per each sphere of a 6-hole cell culture plate were cultured for 24 hours in a cell incubator at 37 ° C and 5% CO ₂ conditions, and then attached to the bottom, and only one cell sample had a final concentration of 5 μg / ml. CD47 antibody is added and reacted for 4 hours to block CD47 on the cell surface. Epirubicin was collected here, and SV4-conjugated liposomes (iLP) were treated with iLP so that the final concentration of epirubicin was 10 μM for each cell, and cultured for 24 hours. After 24 hours, the degree of fluorescence of epirubicin, which was introduced into the cells and exhibited red fluorescence in the cell nucleus, was compared and analyzed using a fluorescence microscope. At this time, as a control, a cell sample directly treated with epirubicin was used (see FIG. 23A). 100 μg of SV4 protein was first administered to MCF7-xenotransplanted mice to saturate them, and the same volume of phosphate-buffered saline was administered to the mice in a state in which CD47 was blocked in cancer cells in the mouse, so that CD47 in cancer cells was not blocked. Here, 100 μl (mRNA 5 μg/Liposome 0.5 mg) of liposome containing T001 mRNA into which SV4-DSPE and Cyanine 5.5-DSPE, which exhibit red fluorescence in the near-infrared region are simultaneously inserted, is injected into the mouse body through intravascular injection to detect CD47 The accessibility to the target through the medium was confirmed.

As a result of the experiment, as shown in FIG. 23A, fluorescence of epirubicin was not observed in the treatment group in which CD47 was blocked, but fluorescence of epirubicin was shown in the experimental group in which CD47 was not blocked.

In addition, in the MCF7-xenotransplantation mouse model experiment to confirm the CD47-mediated drug delivery ability in vivo , overall noise was observed due to CD47 present in blood cells, but it was higher in unblocked mice than in SV4-blocked mice. Stronger drug delivery was confirmed in cancer cells (see Fig. 23B).

In the MCF7 xenograft mouse model, SV4-conjugated iLP containing mRNA of Near Infrared Red Fluorescence Protein (NIR RFP) was intravenously injected, and fluorescence images were analyzed over time, and the results were analyzed. 24. The specific test method is as follows.

MCF7, a human breast cancer cell line, was cultured and xenotransplanted into nude mice. SV4-iLP, in which NIR-RFP (Near Infrared-Red Fluorescence Protein) mRNA was captured, was injected intravenously, adjusted so that 5 μg mRNA was administered per 25 g mouse, and then injected using an in vivo optical imaging system. The degree of fluorescence of the sample was tracked.

Referring to FIG. 24, although mRNA delivery occurs through CD47 in some blood cells, a phenomenon in which expression is accumulated in cancer cells at a relatively high level was observed.

MCF7 xenograft mouse IV cancer growth control efficacy test (28 days) was performed, and specifically MCF7 was cultured and injected subcutaneously into nude mice. After randomly classifying nude mice with tumors, PBS, Liposome, Liposome-NT, and Liposome-MT (Lipsome 20 mpk, 10 μg mRNA/mg liposome) were treated intravenously (IV), respectively. Each mRNA was treated with 5 μg, a total of 5 times for 28 days at 3-day intervals. The volume of the tumor was examined at 3-day intervals and displayed in a graph. On day 28, nude mice were sacrificed, tumors were separated and the volume was measured, and the results are shown in FIG. 25 .

Referring to FIG. 25 , tumor growth rates in the iLPD-NT and iLPD-MT groups were significantly decreased compared to the two control groups (P = 0.03). There was also a slight difference between the PBS and Void Liposome injection samples.

In order to evaluate the toxic effect of mRNA in the body ( in vivo ), the body weight of mice was measured during the experimental period. In all groups, the body weight of mice increased slightly throughout the entire experimental period, and the change in body weight after sample injection compared to the control group was It turned out not to be large. As shown in FIG. 25, the final size of the tumor tissue collected from the sacrificed mouse after completion of the experiment was compared and shown.

After completion of the experiment, hematological and histological examinations were performed with blood and tissues collected from the sacrificed mice, and the results are shown in FIG. 26 . Referring to FIG. 26, except for the number of WBCs in the blood and liver of mice treated with samples compared to the control group, no significant toxic effects were observed.

Preferred embodiments of the present invention described above have been disclosed to solve the technical problems, and those skilled in the art will be able to make various modifications, changes, additions, etc. within the spirit and scope of the present invention. , such modifications and changes should be regarded as belonging to the scope of the following claims.

<110> BPGene Co., Ltd. <120> Polynucleotide for treatment cancer encoding 5'-nucleotidase modified protein <130> NP21-09070 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> < 223> CD47 binder SV1 <400> 1 Met Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Lys Ser Val Ser Val 1 5 10 15 Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe 20 25 30 Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val 35 40 45 Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val 50 55 60 Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser 65 70 75 80 Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg 85 90 95 Lys Gly Ser Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> CD47 binder SV4 <400> 2 Met Glu Glu Glu Leu Gln Ile Ile Gln Pro Asp Leu Ser Val Ser Val 1 5 10 15 Ala Ala Gly Glu Ser Ala Ile Leu His Cys Thr Ile Thr Ser Leu Phe 20 25 30 Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Val 35 40 45 Leu Ile Tyr Asn Gln Arg Gln Gly Pro Phe Pro Arg Val Thr Thr Val 50 55 60 Ser Glu Thr Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser 65 70 75 80 Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Ile Lys Phe Arg 85 90 95 Lys Gly Ser Pro Asp Thr Glu Phe Leu Ser Gly Ala Gly Thr Glu Leu 100 105 110 Ser Val Arg Ala Lys Pro Ser 115 <210> 3 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> T001 < 400>3 Met Phe Ser Ile Lys Glu Pro Phe Ser Ile Val Thr Asp Cys Asp Glu 1 5 10 15 Val Leu Thr Asp Ile Ser Pro Leu Trp Val His Lys Ile Gln Gln Asn 20 25 30 Ala Asp Tyr Phe Gly Lys Tyr Phe Asp Leu Ser Lys Leu Glu Gly Leu 35 40 45 Glu Phe Gly Thr Phe Glu His Tyr Gln Thr Val Leu Ser Arg Pro Glu 50 55 60 Phe His Leu Asn Lys Trp Leu Arg Lys Glu Asn Leu Val Leu Ser Asp 65 70 75 80 Glu Glu Glu Lys Glu Leu Phe Glu Arg Phe Tyr Ser Leu Tyr Asp Asn 85 90 95 Asp Glu Phe Tyr Glu Asp Cys Met Pro Thr Lys Met Cys Glu Gly Ile 100 105 110 Tyr Lys Leu Ser Leu Gln Lys Phe Val Asp Lys Ile Tyr Val Val Thr 115 120 125 Arg Thr Ser Glu Gly Thr Lys Glu Gly Lys Arg Lys Phe Ile Glu Thr 130 135 140 Phe Leu Asn Ser Asn Lys Val Glu Ile Ile Phe Val Gly Lys Asn Glu 145 150 155 160 Lys Lys Ser Asp Tyr Ile Lys Asn Leu Lys Asn Val Lys Met Ile Val 165 170 175 Glu Asp Glu Leu Ser Asn Ile Asn Asp Ile Val Glu Asn Cys Asn Asp 180 185 190 Gly Phe Glu Glu Val Asp Thr Tyr Ile Pro Ser Thr Gly Tyr Asn Asn 195 200 205 Lys Asp Ile Asp Gly Phe Asn Glu Asn Leu Met Lys Lys Gly Phe Asn 210 215 220 Ala Val Pro Tyr Val Ile Ile Glu Arg Pro Glu Thr Glu Glu Lys Ala 225 230 235 240 Val <210> 4 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 4 ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca 60 atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg 120 tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt tcaatta 177 <210> 5 <211> 247 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 5 acgcgtcgag catgcatcta gggcggccaa ttccgcccct ctcccccccc cccctctccc 60 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccgg tg tgcgtttgtc 120 tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 180 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 240 ctgttga 247 <210> 6 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 6 ttggtcgtga ggcactgggc aggta agtat caaggttaca agacaggttt aaggagacca 60 atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg 120 tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt tcaatta 177 <210> 7 <211> 177 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 7 ttggt cgtga ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca 60 atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg 120 tcttactgac atccactttg cctttctctc cacaggtgtc cactcccagt tcaatta 177 <210> 8 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 3 '-UTR sequence <400> 8 gcatcacatt taaaagcatc tcagcctacc atgagaataa gagaaagaaa atgaagatca 60 atagcttatt catctctttt tctttttcgt tggtgtaaag ccaacaccct gtctaaaaaa 120 cataaatttc tttaatcatt ttgcctcttt tctctgtgct tcaattaata aaaaatggaa 180 agaacctaga tctaaaaaaa a aaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240 aaaaaaaaaa aaaaaaatgc atcccccccc cccccccccc cccccccccc cccaaaggct 300 cttttcagag ccaccagaat t 321 <210> 9 <211> 1 77 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 9 ttggtcgtga ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca 60 atagaaactg ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg 120 tcttactgac atccacttt g cctttctctc cacaggtgtc cactcccagt tcaatta 177 <210> 10 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 10 gcatcacatt taaaagcatc tcagcctacc atgagaataa gagaaagaaa atgaagatca 60 atagcttatt catctctttt tctttttcgt tggtgtaaag ccaacaccct gtctaaaaaaa 120 cataaattt c tttaatcatt ttgcctcttt tctctgtgct tcaattaata aaaaatggaa 180 agaacctaga tctaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240 aaaaaaaaaa aaaaaaatgc atcccccccc cccccccccc cccccccccc cccaaaggct 300 cttttcagag ccaccagaat t 321 <210> 11 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 11 gggtcccgca gtcggcgtcc agcggct ctg cttgttcgtg tgtgtgtcgt tgcaggcctt 60 attcaagcgc cacc 74 <210> 12 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 12 gcatcacatt taaaagcatc tcagcctacc atgagaataa gagaaagaaa atgaagatca 60 atagcttatt catctctttt tctttttcgt tggtg taaag ccaacaccct gtctaaaaaa 120 cataaatttc tttaatcatt ttgcctcttt tctctgtgct tcaattaata aaaaatggaa 180 agaacctaga tctaaaaaaa aaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240 aaaaaaaaaa aaaaaatgc atcccccccc cccccccccc cccccccccc cc caaaggct 300 cttttcagag ccaccagaat t 321 <210> 13 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 13 cttcctactc aggctttata caaagaccaa gaggtacagg tgcaagggag agaagaagag 60 taagaagaaa tataagagcc acc 83 <210> 14 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 14 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 93 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 1 5 agtaagaaga aatataagag ccacc 25 <210 > 16 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 16 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 93 <21 0> 17 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 17 gagactgcca cc 12 <210> 18 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 18 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 93 <210> 19 <211> 12 <212> DNA <213> Artificial Sequence <220> <223 > 5'-UTR sequence < 400> 19 gagactgcca ag 12 <210> 20 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> 3'-UTR sequence <400> 20 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttgg tct ttgaataaag cctgagtagg aag 93 <210> 21 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR sequence <400> 21 gctgccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac 60 ctcttggtct ttgaataaag cctgagtagg aag 9 3 <210> 22 <211 > 18 <212> PRT <213> Artificial Sequence <220> <223> Nucleoplasmin NLS sequence <400> 22 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 Leu Asp <210> 23 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Poxvirus MLS sequence <400> 23 Lys Ile Ser Val Tyr Leu Thr Ala Ala Val Val Gly Phe Val Ala Tyr 1 5 10 15 Gly Ile Leu Lys Trp Tyr Arg Gly Thr 20 25 <210> 24 <211> 503 <212> PRT <213> Unknown <220> <223> Human Sirpa <400> 24 Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys 1 5 10 15 Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu 20 25 30 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly 35 40 45 Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly 50 55 60 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr 65 70 75 80 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser 85 90 95 Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr 100 105 110 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 115 120 125 Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg 130 135 140 Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr 145 150 155 160 Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro 165 170 175 Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp 180 185 190 Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile 195 200 205 His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln 210 215 220 Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg 225 230 235 240 Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Thr Leu Glu 245 250 255 Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys 260 265 270 Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu 275 280 285 Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn 290 295 300 Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser 305 310 315 320 Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly 325 330 335 Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His Pro 340 345 350 Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn Glu 355 360 365 Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val Ala 370 375 380 Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys Ala 385 390 395 400 Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn Ala 405 410 415 Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn 420 425 430 Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn 435 440 445 His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser Glu 450 455 460 Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg Thr 465 470 475 480 Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr Ala 485 490 495 Ser Val Gln Val Pro Arg Lys 500 <210> 25 <211> 357 <212> DNA <213> Unknown <220> <223> Human Sirpa DNA sequence <400> 25 ggtgaggagg agctgcaggt gattcagcct gacaagtccg tatcagttgc agctggagag 60 tcggccattc tgcactgcac tgtgacctcc ctgatccctg tggggcccat ccagtggttc 120 agagg agctg gaccagcccg ggaattaatc tacaatcaaa aagaaggcca cttcccccgg 180 gtaacaactg tttcagagtc cacaaagaga gaaaacatgg acttttccat cagcatcagt 240 aacatcaccc cagcagatgc cggcacctac tactgtgtga agttccggaa agggagccct 300 gacacggagt ttaagtctgg agcaggcact gagctgtctg tgcgtgccaa accctct 357 <210> 26 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> SV1 DNA sequence <400> 26 gaagag gaac tgcagattat tcagccggac aagtccgtaa gcgttgcagc tggcgagagc 60 gccattctgc actgcactat tacctccctg tttccggtgg gcccgatcca gtggttccgt 120 ggcgctggcc cagcccgtgt gctgatctac aatcaacgtc agggcccgtt cccgcgtgta 180 accactgtta gcgagaccac caagcgtgaa aacatggact tttccatcag catcagcaac 240 atcaccccag cagat gccgg cacctactac tgtattaagt tccgtaaagg cagccctgac 300 acggagttta agtctggcgc aggcactgag ctgtctgtgc gtgccaaacc gtct 354 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> KKtoLL11_F1 Primer <400> 27 gattattcag ccggacctgt ccgtaagcgt tgc 33 <210> 28 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> KKtoLL11_R1 Primer <400> 28 gcaacgctta cgg acaggtc cggctgaata atc 33 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> KKtoLL104_F2 Primer <400> 29 ctgacacgga gtttctgtct ggcgcag 27 <210> 30 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> KKtoLL104_R2 Primer <400> 30 ctgcgccaga cagaaactcc gtgtcag 27 <210> 31 <211> 198 <212> PRT <213> Unknown <220> <223> NT5M <400> 31 Met Gly Gly Arg Ala Leu Arg Val Leu Val Asp Met Asp Gly Val Leu 1 5 10 15 Ala Asp Phe Glu Gly Gly Phe Leu Arg Lys Phe Arg Ala Arg Phe Pro 20 25 30 Asp Gln Pro Phe Ile Ala Leu Glu Asp Arg Arg Gly Phe Trp Val Ser 35 40 45 Glu Gln Tyr Gly Arg Leu Arg Pro Gly Leu Ser Glu Lys Ala Ile Ser 50 55 60 Ile Trp Glu Ser Lys Asn Phe Phe Phe Glu Leu Glu Pro Leu Pro Gly 65 70 75 80 Ala Val Glu Ala Val Lys Glu Met Ala Ser Leu Gln Asn Thr Asp Val 85 90 95 Phe Ile Cys Thr Ser Pro Ile Lys Met Phe Lys Tyr Cys Pro Tyr Glu 100 105 110 Lys Tyr Ala Trp Val Glu Lys Tyr Phe Gly Pro Asp Phe Leu Glu Gln 115 120 125 Ile Val Leu Thr Arg Asp Lys Thr Val Val Ser Ala Asp Leu Leu Ile 130 135 140 Asp Asp Arg Pro Asp Ile Thr Gly Ala Glu Pro Thr Pro Ser Trp Glu 145 150 155 160 His Val Leu Phe Thr Ala Cys His Asn Gln His Leu Gln Leu Gln Pro 165 170 175 Pro Arg Arg Arg Leu His Ser Trp Ala Asp Asp Trp Lys Ala Ile Leu 180 185 190 Asp Ser Lys Arg Pro Cys 195 <210> 32 <211> 25 <212> RNA <213> Artificial Sequence < 220> <223> siT001-1 sense <400> 32 cgagaagaag ucagauuaca ucaag 25 <210> 33 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siT001-1 antisense <400> 33 cuugauguaa ucugacuucu ucucg 25 <210> 34 <211> 25 <212> RNA <213> Artificial Sequence <220> <223> siT001-2 sense <400> 34 cgcaaauuca uugaaaccuu ccuga 25 <210> 35 <211> 25 <212> RNA < 213> Artificial Sequence <220> <223> siT001-2 antisense <400> 35 ucaggaaggu uucaaugaau uugcg 25 <210> 36 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for PCR < 400> 36 gtgcttctga cacaacagtc tcgaacttaa gc 32 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for PCR <400> 37 gaagcggccg ccttcctact caggctttat tc 32 <210> 38 <2 11 > 726 <212> DNA <213> Artificial Sequence <220> <223> 5'-nucleotidase variant, wild type <400> 38 atgttttcta ttaaagaacc attttcaatt gttacagact gcgatgaggt attaactgac 60 attagtcctt tatgggttca taagattcag caaaatgctg attattttgg a aaatacttt 120 gatttaagta aactagaagg attggaattt ggtacatttg aacattatca aacagtacta 180 tcacgaccag aatttcattt aaataaatgg ctaagaaaag agaatcttgt attatcagat 240 gaagaagaaa aagaattattt tgaaagattt tattcgttat atgataatga tgaattttat 300 gaagattgta tgccaactaa aatgtgtgaa ggaatttata aattatcatt acaaaaattc 360 g tagataaaa tctatgttgt aacaagaaca agtgaaggaa ccaaagaagg aaaaagaaaa 420 tttattgaaa ctttcttaaa ttctaataaa gtagagatta tttttgttgg gaaaaatgaa 480 aagaaatcag attatattaa gaatctaaag aatgtaaaaa tgattgtaga aga tgaatta 540 tcaaatatta atgatattgt agaaaattgt aatgatggtt ttgaagaagt agatacttat 600 attccatcaa ctggttataa caataaagat attgatggtt ttaatgaaaa tctaatgaaa 660 aaaggattta atgccgttcc atatgtaatt atagaaagac cagaaacaga agagaaagca 720 gtataa 726 <210> 39 <211> 726 <212> DNA <213> Artificial Sequence <220> <2 23> 5'-nucleotidase variant, T001 <400> 39 atgttctcca tcaaagaacc attctccatc gtgaccgact gcgatgaggt gctgaccgac 60 attagccctc tgtgggtgca taagattcag cagaacgctg attacttcgg caaatacttt 120 gatctgagca aactggaagg cctggaattt ggtacgtttg aacattatca gacggtgctg 180 tcacgcccag agttccatct gaacaaatgg ctgcgcaaag agaacctggt gctgtcagat 240 gaagaagaga aagaactgtt tgaacgcttc tattcgctgt atgataatga tgagttct ac 300 gaagattgta tgccaaccaa aatgtgcgaa ggcatctaca aactgtcact gcagaaattc 360 gtggataaaa tctatgtggt aacccgcacc agtgaaggca ccaaagaagg caacgcaaa 420 ttcattgaaa ccttcctgaa ctctaacaaa gtggagatta tcttcgt tgg caagaacgag 480 aagaagtcag attacatcaa gaacctgaag aatgtgaaaa tgattgtgga agatgaactg 540 tccaaca acgatattgt ggagaactgt aacgatggct ttgaagaagt ggatacctac 600 attccgtcaa ctggttacaa caacaaagat attgatggct tcaacgaaaa cctgatgaaa 660 aaaggcttca acgccgttcc atacgtgatt atcgaacgcc cagaaaccga agagaaagca 720 gtgtaa 726 <210> 40 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> 5'-nucleotidase variant , T002 <400> 40 atgttctcca tcaaggagcc gttctccatc gtgaccgact gcgacgaggt gctgaccgac 60 atcagcccgc tgtgggtgca caagatccag cagaacgccg actacttcgg caagtacttc 120 gacctgagca agctggaggg cctggagttc ggcacgt tcg agcactacca gacggtgctg 180 tcgcgcccgg agttccacct gaacaagtgg ctgcggaaag aaaacctcgt cctctccgac 240 gaggaggaga aggagctgtt cgagcgcttc tactcgctgt acgacaacga cgagttctac 300 gaggactgca tgccgaccaa gat gtgcgag ggcatctaca agctgtcgct gcagaagttc 360 gtggacaaga tctacgtggt cacccgcacg agcgagggca ccaaggaggg caagcgcaag 420 ttcatcgaga ccttcctgaa ctcgaacaag gtggagatca tcttcgtcgg caagaacgag 480 aagaagtcag actacatcaa gaacctgaag aacgtgaaga tgatcgtgga ggatgagctg 540 tccaacat ca acgacatcgt ggagaactgc aacgacggct tcgaggaggt ggacacctac 600 atcccgtcaa ctggttacaa caacaaggat atcgacggct tcaacgagaa cctgatgaag 660 aagggcttca acgccgtgcc atacgtgatc atcgagcgcc cagagaccga ggagaaggca 720 gtgtaa 7 26 <210> 41 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> 5'-nucleotidase variant, T007 <400> 41 atgttcagca tcaaggagcc cttctccatc gtgaccgact gcgacgaagt cctcaccgac 60 atcagccccc tgtgggtgca caagatccag cagaacgccg actacttcgg ga aatacttc 120 gacctgagca agctggaggg cctggagttc gggaccttcg agcactacca gaccgtgctc 180 tcccgccccg aattccacct gaacaagtgg ctccgcaagg agaacctggt cctgtccgac 240 gaggaggaga aggagctgtt cgagcgcttc tacagcctgt acgacaacga cgagttctac 300 gaggactgca tgcccactaa gatgtgcgag ggcatctaca agctgagcct gcagaagttc 360 gtggataaga tctatgtcgt gaccc gcacc agcgagggca ccaaggaggg caagcgcaag 420 ttcatcgaga ctttcctcaa cagcaacaag gtggagatca tcttcgtcgg caagaacgaa 480 aaaaagagcg actacatcaa gaacctgaag aacgtgaaga tgatcgtgga ggacgagctg 540 tccaaca acga catcgt ggagaactgc aacgacggct tcgaggaggt ggacacctac 600 atccccagca ctgggtacaa caacaaggac atcgacggct tcaacgagaa cctgatgaag 660 aagggcttca acgccgtgcc ctacgtgatc atcgagcgcc ccgagaccga ggagaaggcc 720gtgtaa 726

Claims (13)

A polynucleotide for cancer treatment encoding the amino acid sequence represented by SEQ ID NO: 3. According to claim 1,
The polynucleotide characterized in that it comprises a base sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 41. According to claim 1,
The polynucleotide for the treatment of cancer, characterized in that the polynucleotide is mRNA. According to claim 3,
The polynucleotide for cancer treatment, characterized in that the mRNA enters cancer cells and inhibits nucleic acid metabolism. According to claim 4,
The nucleic acid metabolism is a polynucleotide for cancer treatment, characterized in that dTTP biosynthetic metabolism. According to claim 1,
The polynucleotide for the treatment of cancer, characterized in that the polynucleotide further comprises a 5'-UTR represented by SEQ ID NO: 19 and a 3'-UTR represented by SEQ ID NO: 20. According to claim 1,
The polynucleotide for the treatment of cancer, characterized in that the polynucleotide further comprises a nucleic acid sequence encoding a nuclear localization signal (NLS) represented by SEQ ID NO: 22. According to claim 1,
The polynucleotide for the treatment of cancer, characterized in that the polynucleotide further comprises a nucleic acid sequence encoding the mitochondrial localization signal (MLS) represented by SEQ ID NO: 23. According to claim 1,
The polynucleotide for cancer treatment, characterized in that the cancer is colon cancer or breast cancer. A polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 3;
liposomes encapsulating the polynucleotide; and
a binder conjugated to the liposome and binding to CD47 overexpressed in cancer cells, comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
Complex for cancer treatment comprising a. According to claim 10,
The binder is DSPE (1,2-Distearoyl-sn-glycero-3-phosphorylethanolamine)-conjugated complex for cancer treatment, characterized in that. According to claim 10,
The liposome is a complex for cancer treatment, characterized in that positively charged using at least one material selected from the group consisting of cationic phospholipids, DOTAP and cholesterol. According to claim 1,
The liposome is a complex for cancer treatment, characterized in that pegylated.

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