Method for specifically degrading cancer suppressor protein p53 and oncoprotein KRAS, and composition therefor

Abstract

The present invention relates to specific degradation of cancer suppressor protein p53 and oncoprotein KRAS, and, more specifically, to a method in which a cell line expressing p53 and KRAS is treated with a complex of E3 ligase subunit SPOP167-374 and Rbx1delC and binders that specifically bind to p53 and KRAS, so that respective proteins are specifically degraded; and a composition used in the method. It has been identified that the method developed in the present invention allows p53 expressed in the nucleus to be degraded if a cell line is treated with Nb139_ SPOP167-374, and allows KRAS expressed in the cell membrane to be degraded if a cell line is treated with KRAS binder_ Rbx1delC.

Description

Method for specifically degrading tumor suppressor protein P53 and oncogenic protein KRAS and composition thereof

[0001] The present invention relates to the specific degradation of the tumor suppressor protein p53 and the oncogenic protein KRAS, and relates to a method for specifically degrading each protein by treating a cell line expressing p53 and KRAS with a complex of a binder that specifically binds to p53 and KRAS and an E3 ligase subunit, SPOP167-374 and Rbx1delC, and a composition used in the method.

[0002] Human cells are composed of diverse proteins. These numerous proteins function as signaling molecules that produce enzymes and hormones that initiate chemical reactions, thereby regulating cellular functions. Furthermore, they selectively degrade proteins within the cell to regulate their functions. The protein ubiquitination system is a mechanism that enables the degradation of specific proteins. Beyond this function, protein ubiquitination, a post-translational modification, is involved in diverse functions, including DNA repair, mRNA export, cell cycle regulation, protein-protein interactions in various signaling mechanisms, protein trafficking, and the formation of aggresomes, where ubiquitinated proteins tangle together to induce autophagy and removal.

[0003] Protein ubiquitination is mediated by ubiquitin. Ubiquitin is a regulatory protein composed of 76 amino acids. Ubiquitin is bound to substrate proteins through the sequential action of three enzymes: E1, E2, and E3. E1 is an active enzyme that binds to ubiquitin in the presence of ATP and catalyzes the acyl-adenylation of the ubiquitin C-terminus. Ubiquitin bound to E1 is transferred to the conjugating enzyme E2, forming a complex, which then binds to the ligase enzyme E3. The amine of the lysine group on the substrate undergoes a nucleophilic attack with the glycine group at the C-terminus of ubiquitin, forming a covalent bond, allowing ubiquitin to be transferred from E2 to the substrate protein.

[0004] This protein mechanism occurs repeatedly, whereby ubiquitin, composed of seven lysines, binds to other ubiquitins to form polyubiquitin chains. There are seven types of ubiquitin chains, and depending on the residue at which they bind, they are classified as K6, K11, K27, K29, K33, K48, and K63. Depending on the ubiquitin chain bound to the protein, they perform various functions.

[0005] Ubiquitin-Proteasom System (UPS)

[0006] Protein degradation within cells typically occurs through mechanisms involving the lysosome and the proteasome. A key difference between these two mechanisms is that lysosome degradation lacks selectivity and regulatory capacity, whereas proteasome degradation possesses both. Proteasomes exist in the nucleus and cytoplasm and degrade most proteins bound to ubiquitin chains.

[0007] The proteasome consists of two subunits: the 19S regulatory particle and the 20S core particle. The 19S regulatory particle recognizes proteins conjugated with ubiquitin chains and induces the degradation of the proteins into peptides by the 20S core particle.

[0008] E3 ligase

[0009] E3 ligases possess specificity, binding to both E2 and substrate proteins and determining which substrate proteins are degraded by the proteasome through ubiquitination. E3 ligases are mediators of enzymatic cascades, as they can confer high levels of specificity and selectivity for their target substrates in cells. The human proteome encodes over 600 E3s, which can be divided into three classes.

[0010] The largest class is the RING (Really Interesting New Gene / U-box) type E3 with about 600 members, followed by the HECT (Homologous to E6AP C-Terminus) type E3 with about 28 members, and the RBR (RING between RING) type E3 with about 14 members.

[0011] While RING E3 ligases act as scaffolds that bring E2 closer to its substrate and as allosteric activators of E2, HECT and RBR E3 ligases catalyze substrate ubiquitination in a two-step reaction. In the first step, activated Ub is accepted from E2 in a thiol exchange reaction on the catalytic cysteine, and in the second step, the Ub moiety is transferred to a lysine of the target substrate.

[0012] Regarding HECT-containing E3 ligases, at the C-terminus, all HECT E3s exhibit a catalytic HECT domain, consisting of a larger N-terminal lobe (N-lobe) containing the E2-binding domain and a C-terminal lobe (C-lobe) carrying the catalytic cysteine. The two lobes are connected by a flexible hinge region that allows the C-lobe to move to facilitate Ub transfer from E2 to E3.

[0013] 1) Rbx1

[0014] Rbx1 (RING-box protein 1) is a protein that constitutes the SCF complex, known as CRL (cullin-based RING ligase), and functions as an E3 ligase by binding to E2 and cullin. Rbx1 contains a RING motif that stabilizes its structure by binding to zinc ions, and H3, which consists of two large loops and an a-helix structure, binds to E2 and maintains the structure. H3 is located at the C terminus and plays an important role in Rbx1 function. In addition, cullin binds to the N terminus. (Zheng N, Schulman BA, Song L, et al. Nature. 2002;416(6882):703-9, Duda DM, Olszewski JL, Tron AE, et al. Mol Cell. 2012;47(3):371-82.)

[0015] 2) SPOP

[0016] SPOP (Speckle-type POZ protein) is a substrate-specific adaptor consisting of a MATH domain to which substrate proteins bind and a BTB domain to which cullin3 (Cul3) binds. The BTB domain contains a dimer-forming region that allows proteins with BTB domains to bind to each other, thereby conjugating E2-bound ubiquitin to substrate proteins. The MATH domain determines the specificity of substrate proteins and regulates the amount of proteins present in cells. Various substrate proteins of SPOP have a common motif that binds to SPOP (Zhuang M, Calabrese MF, Liu J, et al. Mol Cell. 2009;36(1):39-50.). Recent studies have revealed that changes in the MATH domain are a cause of prostate cancer (Gan W et al. 2015). The BTB domain includes a dimer domain (DD) that causes the dimer structure of SPOP and a 3-Box that binds to the N-terminus of Cul3, a subset (Zhuang M et al. 2009).

[0017] PROTACs, drugs recently being developed as a method for selectively removing target proteins, consist of a ligand-linker-E3 ubiquitin ligase ligand of the target protein. This structure is structurally complex, making it difficult to identify suitable ligand and linker combinations. Furthermore, the target proteins that can be degraded by PROTACs are limited, and their degradation efficiency is low. Furthermore, the drug can cause toxicity by affecting not only the problematic cells but also normal cells.

[0018] [Prior patent literature]

[0019] Republic of Korea Patent Publication No. 10-2017-0096878

[0020] The present invention has been conceived to solve the above problems and meet the above needs, and the purpose of the present invention is to provide a method for specifically decomposing the tumor suppressor protein p53.

[0021] Another object of the present invention is to provide a composition involved in a specific degradation method of the tumor suppressor protein p53.

[0022] Another object of the present invention is to provide a method for specifically degrading KRAS protein.

[0023] Another object of the present invention is to provide a composition involved in a method for specifically degrading KRAS protein.

[0024] In order to achieve the above purpose, the present invention provides a method for specifically degrading p53 protein in cells expressing p53 protein by treating the cells with a fusion of a nanobody recognizing p53 and residues 167-374 of SPOP (Speckle-type POZ protein), which is an E3 ligase subunit.

[0025] In one embodiment of the present invention, the fusion of the nanobody recognizing p 53 and residues 167-374 of SPOP (Speckle-type POZ protein), which is an E 3 ligase subunit, is preferably composed of the amino acid sequence of SEQ ID NO: 1, but all mutants that achieve the desired effect of the present invention through one or more substitutions, deletions, inversions, and translocation mutations in the amino acid sequence are also included in the scope of the present invention.

[0026] In addition, the present invention provides a composition for decomposing p53 protein, which comprises as an active ingredient a fusion of a nanobody and residues 167-374 of the SPOP protein, which is an E3 ligase subunit.

[0027] In addition, the present invention provides a method for specifically degrading a KRAS protein by treating a fusion of a RAS binder selected from the group consisting of NS1, K27, and DP among RAS binders that recognize the KRAS protein and Rbx1 (RING-box protein 1) with a C-terminal deletion to the KRAS protein.

[0028] In the present invention, the RAS binder is preferably a monobody, darpin, or RAF-ras binding domain, but is not limited thereto.

[0029] In one embodiment of the present invention, the fusion may be substituted for Rbx1 with a C-terminal deletion, Rbx2 (RING-box protein 2) with a C-terminal deletion, or APC11 (Anaphase-Promoting Complex 11) with a C-terminal deletion, but is not limited thereto.

[0030] To elaborate, existing technologies usually replace the substrate recognition site of E3 ligase with binders such as nanobodies, but in the case of the RAS binder fusion Rbx1delC of the present invention, it is not limited to the substrate recognition site of E3 ligase, and although Rbx1delC was used in the embodiment of the present invention, Rbx2 and APC11, which have similar functions, are also included in the scope of the present invention.

[0031] In one embodiment of the present invention, the fusion of one RAS binder selected from the group consisting of NS1, K27 and DP and Rbx1 (RING-box protein 1) with a C-terminal deletion is preferably composed of any one of the amino acid sequences of SEQ ID NOs: 5 to 7, but all mutants that achieve the desired effect of the present invention through one or more substitutions, deletions, inversions and translocation mutations in the amino acid sequence are also included in the scope of the present invention.

[0032] In addition, the present invention provides a composition for degrading KRAS protein, comprising as an active ingredient a fusion of one RAS binder selected from the group consisting of NS1, K27 and DP and Rbx1 (RING-box protein 1) with a C-terminal deletion.

[0033] The term "nanobody" of the present invention generally refers to a VHH, a humanized VHH, or generally a VHH that is sequence-optimized (e.g., optimized for chemical stability and / or solubility, maximum overlap with known human framework regions, and maximum expression) as defined in International Patent Application Publication No. WO08 / 020079 or WO09 / 138519. It is noted that the terms nanobody or nanobodies are registered trademarks of Ablynx and may therefore also be referred to as Nanobody® and / or Nanobodies®.

[0034] In one embodiment of the present invention, it is preferable that the nanobody is characterized by specifically recognizing p53, and in the embodiment of the present invention, Nb139 was used.

[0035] The present invention is described below.

[0036] The present invention relates to the specific degradation of the tumor suppressor protein p53 and the oncogenic protein KRAS, and relates to a method for specifically degrading each protein by treating a cell line expressing p53 and KRAS with a complex of a binder that specifically binds to p53 and KRAS and an E3 ligase subunit, SPOP167-374 and Rbx1delC, and a composition used in the method. It was confirmed that the method developed in the present invention degraded p53 expressed in the nucleus when Nb139_ SPOP167-374 was treated to the cell line, and degraded KRAS expressed in the cell membrane when KRAS binder_ Rbx1delC was treated to the cell line. Through this, it was confirmed that target proteins can be specifically degraded depending on the location of the protein within the cell, and it was confirmed through a xenograft mouse experiment that when Nb139_ SPOP167-374 is expressed, the proliferation of cancer cells related to p53 is also inhibited.

[0037] As can be seen through the present invention, it was confirmed that the method developed in the present invention degrades p53 expressed in the nucleus when Nb139_ SPOP167-374 was treated to a cell line, and degrades KRAS expressed in the cell membrane when KRAS binder_ Rbx1delC was treated to a cell line. Through this, it was confirmed that target proteins can be specifically degraded depending on the location of the protein within the cell, and it was confirmed through a xenograft mouse experiment that when Nb139_ SPOP167-374 is expressed, the proliferation of cancer cells related to p53 is also suppressed.

[0038] Figure 1 is a schematic diagram of the present invention;

[0039] Figure 2 shows FACS data obtained by transient transfection of various candidate proteins into 293tet-On-GFP cells. To determine whether the GFP-recognizing nanobody vhhGFP4 can degrade GFP expressed in 293teton by fusing E3 ligase subunits NSlimb, SPOP167-374, Rbx1WT, Rbx1ΔN, Rbx1ΔC, Rbx1ΔNΔ, and the GFP-non-recognizing nanobody vhhGFP4mut to Rbx1ΔC, the transient transfection was performed and analyzed by FACS. The above proteins are co-expressed with RFP670 by the Bi-TRE promoter when treated with doxycycline (1 μg / ml). After 24 hours of treatment with doxycycline (1 μg / ml), FACS analysis confirmed that vhhGFP4_Rbx1ΔC degrades GFP. However, it was confirmed that vhhGFP4_Rbx1WT, vhhGFP4_Rbx1ΔN, and vhhGFP4_Rbx1ΔNΔㅊC were not decomposed.

[0040] Figure 3 shows FACS data obtained by transient transfection of 293teton with Nb139-SPOP167-374, Rbx1delC and vhhGFP4-SPOP167-374, Rbx1delC. To determine whether the p53-recognizing nanobody Nb139 fused to the E3 ligase subunits SPOP167-374 and Rbx1delC could degrade endogenous p53 expressed in 293teton, immunostaining was performed after transient transfection and analyzed by FACS. In addition, vhhGFP4, a GFP-recognizing nanobody, was combined with SPOP167-374 and Rbx1delC and used as a negative control. p53 was immunostained using anti-p53 antibody (DO-1), and Nb139-SPOP167-374 and Nb139-Rbx1delC were expressed simultaneously with RFP670 by the Bi-TRE promoter when treated with doxycycline (1 μg / ml). As a result of checking using FACS 24 hours after treatment with doxycycline (1 μg / ml), it was confirmed that p53 was degraded in the case of Nb139-SPOP167-374. However, it was confirmed that p53 was not degraded in vhhGFP4-SPOP167-374, vhhGFP4-Rbx1delC, and Nb139-Rbx1delC.

[0041] Figure 4 shows Western data showing that FLAG-Nb139-SPOP167-374 degrades endogenous p53 in a HeLa stable cell line. The expression level of p53 was increased using 5-FU (1 μg / ml), and FLAG-Nb139-SPOP167-374 is expressed by the TRE promoter. After 24 hours of treatment with doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hours), it was confirmed in Lane 3 that FLAG-Nb139-SPOP167-374 was expressed and p53 was degraded.

[0042] Figure 5 shows Western data showing that FLAG-Nb139-Rbx1delC degrades endogenous p53 in a HeLa stable cell line. p53 expression levels were increased using 5-FU (1 μg / ml), and FLAG-Nb139-Rbx1delC is expressed by the TRE promoter. As a result of 24-hr treatment with Doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hr), unlike the result in (a), a slight degradation of p53 was confirmed in Lane 6, which was treated with MG132 and Doxycycline simultaneously.

[0043] Figure 6, left, shows FACS data analyzing a stable cell line created in Hela cells with Nb139-SPOP167-374. The p53 expression level was increased using 5-FU (1 μg / ml), and immunostaining was performed using an anti-p53 antibody. FLAG-Nb139-SPOP167-374 is expressed by the TRE promoter. After 24-hr treatment with doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hr), it was confirmed that the p53 level was reduced in MG132(-), 5-FU(+), and Dox(+).

[0044] The right side of the figure shows FACS data analyzing a stable cell line created in HeLa with Nb139-Rbx1delC. p53 expression levels were increased using 5-FU (1 μg / ml), and immunostaining was performed using an anti-p53 antibody. FLAG-Nb139-Rbx1delC is expressed by the TRE promoter. No significant changes in p53 levels were observed after 24-hour treatment with doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hours).

[0045] Figure 7 shows microscope data showing that FLAG-Nb139-SPOP167-374 degrades endogenous p53 in a HeLa stable cell line. The expression level of p53 was increased using 5-FU (1 μg / ml), and immunostaining was performed using an anti-p53 antibody. FLAG-Nb139-SPOP167-374 and TagRFP are expressed by the Bi-TRE promoter. After 24 hours of treatment with Doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hours), it was confirmed that the level of p53 was weakened in 5-FU(+), Dox(+), and MG132(-).

[0046] Figure 8 shows microscope data showing that FLAG-Nb139-Rbx1delC degrades endogenous p53 in a HeLa stable cell line. p53 expression levels were increased using 5-FU (1 μg / ml), and immunostaining was performed using an anti-p53 antibody. FLAG-Nb139-Rbx1delC and TagRFP are expressed by the Bi-TRE promoter. Treatment with doxycycline (1 μg / ml) and MG-132 (10 μM, 20 hr) did not show significant changes in p53 levels.

[0047] Figure 9 shows the degradation of p53 by p53 nanobody (Nb139)-SPOP167-374. A is a p53-recognizing nanobody, Nb139, and an E3 ligase adaptor protein, SPOP167-374. To confirm whether p53 can be degraded using SPOP167-374, an experiment was conducted using the SW480 stable cell line expressing Nb139-SPOP167-374 by a tetracycline-inducible promoter (TRE promoter). To induce the expression of Nb139-SPOP167-374 in the colon cancer cell line, SW480, doxycycline (1 μg / mL) was treated for 48 hours, and the result confirmed by Western blot showed that when Nb139-SPOP167-374 was expressed by treating with doxycycline, p53 degradation occurred effectively.

[0048] B To confirm whether the degradation of p53 by Nb139-SPOP167-374 occurs through the ubiquitin proteasome system (UPS), the expression of Nb139-SPOP167-374 was induced by treating the proteasome inhibitor MG132 (10 μM) for 4 hours and then treating with Doxycycline (1 mg / mL) for 20 hours. The results of Western blot analysis showed that p53 degradation was effectively induced in the MG132 (-) / Doxycycline (+) group, whereas p53 degradation was inhibited in the MG132 (+) / Doxycycline (+) group. This confirmed that p53 degradation by Nb139-SPOP167-374 occurs through the UPS.

[0049] Figure 10 shows the effect on the growth of target cells. To confirm whether the degradation of p53 by Nb139-SPOP167-374 in SW480, a colon cancer cell line with p53 mutation, could inhibit the colony formation ability of cells, stable cells (2X102 cells) were plated in a 6-well plate with or without medium containing Doxycycline (1μg / mL), and then Doxycycline (1μg / mL) was added every 72 hours. After 13 days, the colony formation ability was compared by counting the number of colonies after staining using Crystal Violet solution, and it was confirmed that the number of colonies was significantly reduced in the group treated with Doxycycline (1μg / mL). **p < 0.01

[0050] Figure 11 shows the effect on the invasion of target cells. Matrigel invasion chamber (Corning) was used to check whether the invasion ability of cells could also be inhibited. The constructed stable cell line was seeded at 1.5 X 10 in an 8 μm membrane pore insert. 5 Doxycycline (1 μg / mL) was added to the cells (-) and (+) groups, respectively. After 72 hours, non-invading cells were removed, and cells on the lower surface of the membrane were fixed and stained with Hematoxylin and Eosin Y. After staining, the number of stained cells was counted under a microscope, and when the invaded cells were counted and displayed in a graph, it was confirmed that the number of invaded cells was significantly reduced in the Doxycycline (+) group. **p < 0.01

[0051] Figure 12 shows the in vivo antitumor efficacy in xenograft mice. Nude mice were used to evaluate the in vivo antitumor efficacy of p53 degradation by Nb139-SPOP167-374. SW480 stable cells 3X106 expressing Nb139-SPOP167-374 by the TRE promoter were inoculated into the left flank of the mice. Nine days after inoculation, the mice were randomly assigned to an experimental group and a control group based on the tumor volume (approximately 50 mm3). The expression of Nb139-SPOP167-374 was induced in the experimental group by providing Doxycycline (2 μg / mL, 200 mg / kg) through water and food. The tumor size was measured with a digital caliper during the experimental period, and it was confirmed that the tumor volume was reduced in the group in which the expression of Nb139-SPOP167-374 was induced by Doxycycline compared to the control group.

[0052] Figure 13 shows antibody enzymes that degrade kRAS protein. In the figure, 1. 293Tet-On, 2-9. 293Tet-ON (GFP-kRAS, wild-type or G12V mutation), 3-9. Transient transfection was performed with AE (AntibodyEnzyme), and the AntibodyEnzyme (AE) was 3. iDAb-Rbx1△C 4. 12VC1-Rbx1△C 5. NS1-Rbx1△C, 6. K27-Rbx1△C, 7. DP-Rbx1△C, 8. K55-Rbx1△C, and 9. RAF1 (RBD-CRD)-Rbx1△C. As can be seen in Figure 10, the msfGFP-KRAS fusion protein was better degraded in Figure 5 (NS1-Rbx1delC), 6 (K27-Rbx1delC), and 7 (DP-Rbx1delC) than in other combinations.

[0053] Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

[0054] Example 1. Strains and culture conditions

[0055] 1) Escherichia coli strain and culture

[0056] In the present invention, Escherichia coli Top10F' (F'[lacIq Tn10(tetR)] mcrA ΔcrBC) φ80lacZΔM ΔlacX74 deoR nupG recA1 araD139 ΔgalU galK rpsL(StrR), Invitrogen) was used as a competent cell for amplifying the recombinant plasmid. This E. coli strain was cultured in LB medium (0.5% yeast extract, 1% Tryptone, 0.5% sodium chloride, Lennox) at 37°C and 225 rpm for 12 to 15 hours. Depending on the type of plasmid, antibiotics kanamycin (25 μg / ml) and carbenicillin (100 μg / ml) were added to the LB medium and cultured for drug selection.

[0057] 2) Animal cells and culture

[0058] 293TetOn cells were cultured in DMEM (containing 4.5 g / L glucose, sodium pyruvate, L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 5% CO2 and 37°C. 293T-CMV-msfGFP_WT KRAS cells and 293TetOn-CMV-msfGFP_KRAS_G12V cells used in the experiment were cultured (5% CO2, 37°C) in DMEM (containing 4.5 g / L glucose, L-glutamine, sodium pyruvate, [10% FBS, 10 mM HEPES, 1% penicillin / streptomycin]) supplemented with 2 μg / ml of puromycin to maintain msfGFP_WT KRAS and msfGFP_KRAS_G12V within the cells.

[0059] HeLa was cultured in DMEM (containing 4.5 g / L glucose, sodium pyruvate, and L-glutamine) medium supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 37°C and 5% CO2. Hela-Nb139_SPOP167-374 Cells and Hela-Nb139-Rbx1delC used in the experiment were cultured (5% CO2, 37℃) in a medium containing 10% FBS, 1% penicillin / streptomycin, 10 mM HEPES, and DMEM (containing 4.5 g / L glucose, sodium pyruvate, and L-glutamine) with 350 μg / ml of hygromycin used when creating the cells. SW480 was cultured in a medium containing RPMI1640 (containing 300 mg / L L-glutamine) with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 5% CO2 and 37℃. SW480-Nb139_SPOP167-374 Cells used in the experiment were cultured in a medium containing 350 μg / ml of hygromycin used when creating the cells. It was added to 10% FBS, 1% penicillin / streptomycin, 10 mM HEPES, RPMI1640 (containing 300 mg / L L-glutamine) medium at a concentration of 200 μg / ml and cultured (5% CO2, 37°C).

[0060] Example 2. Vector construction

[0061] 1) Construction of expression vectors for animal cells

[0062] The fluorescent protein (TagRFP) gene and E3 ubiquitin ligase were recombined into the TRE (tetracycline response element) promoter that is expressed in both directions in the plasmid containing pEM791-SRa-HygroB_rtTA3. The TRE promoter was used to enable expression only in the presence of doxycycline. The vector required for the experiment analyzed using FACS was recombined by amplifying the fluorescent protein genes RFP670 and TagRFP by PCR into the TRE promoter and treating with restriction enzymes (BstBⅠ / SalⅠ). The GFP-recognizing nanobody vhhGFP4 was flagged, and SPOP167-374 and Rbx1delC were recombined by treating with restriction enzymes (XhoⅠ / MluⅠ). Nb139, a nanobody that recognizes p53, was flagged and recombined with SPOP167-374 and Rbx1delC by treatment with restriction enzymes (XhoI / MluI).

[0063] To transform the animal cells used in the experiment, msfGFP fusion proteins (KRAS_WT, KRAS_G12V) synthesized with msfGFP and specific proteins were obtained through PCR, and the obtained PCR products were treated with restriction enzymes and recombined into the pPuro-CMV lentiviral vector. Flags were added to iDAb, 12VC1, NS1, K27, DP KRAS, K55, and RAF1_RBD_CRD, which are binders that can recognize KRAS, and Rbx1delC was recombined by treating it with restriction enzymes (XhoI / MluI).

[0064] Example 3. Transformation of recombinant vector

[0065] 1) Transformation of E. coli

[0066] During the cloning process, the mixture linking the vector and the inserted gene was mixed with Escherichia coli Top10F competent cells, left on ice for 5 minutes, and then heat-shocked at 42°C for 1 minute to allow the ligated DNA to enter the competent cells. After leaving on ice for 3 minutes, LB medium was added and cultured at 37°C, 225 rpm, 1 hour to allow the cells to recover. After that, the cells were cultured overnight (14-16 hours) on LB agar medium containing the necessary antibiotics.

[0067] 2) Transformation of animal cells

[0068] Animal cells cultured in a 10 cm plate were treated with 1.5 ml of 0.25% trypsin to recover the cells, and then centrifuged to remove trypsin and seeded in a 6-well plate at 7.6 x 10 5 Cells were inoculated at a concentration of 10 cells / well. After 24 hours, when the cells reached 60–70% confluence, a mixture of 2 μg of DNA, 8 μg of polyethylenimine (PEI), and 300 μl of Opti-MEM was added to each well after incubation at room temperature for 15 minutes. After culturing for 4 hours under conditions of 5% CO2 and 37°C, 2 μg of doxycycline was added.

[0069] Example 4. Cell line construction

[0070] (1) Construction of 293T-msfGFP_KRAS_WT and 293Tet-On-msfGFP_KRAS_G12V cell lines

[0071] To generate 293T-msfGFP_KRAS_WT and 293Tet-On-msfGFP_KRAS_G12V cell lines, cells were transfected with PEI and selected with medium containing 2 μg / ml of puromycin.

[0072] (2) Construction of Hela-Nb139_SPOP167-374 and Hela-Nb139_Rbx1delC cell lines

[0073] To establish Hela-Nb139_SPOP167-374 and Hela-Nb139_Rbx1delC cell lines, cells were transfected with PEI and selected with medium containing 350 μg / ml of hygromycin B.

[0074] (3) Construction of SW480-Nb139_SPOP167-374 cell line

[0075] To establish the SW480-Nb139_SPOP167-374 cell line, cells were transfected with PEI and selected with medium containing 200 μg / ml of hygromycin B.

[0076] Example 5. Fluorescence analysis using a flow cytometer

[0077] (1) Cell processing for flow cytometer analysis

[0078] Treat each well with 1 ml of 0.25% trypsin for 3 minutes, collect the cells, place them in 3 ml of complete medium, and centrifuge at 700 rpm for 3 minutes. Remove the supernatant, add 1 ml of 1X PBS (Phosphate-buffered saline), and suspend the cells. Place the suspended cells in a BD-Falcon FACS tube to prepare the sample.

[0079] (2) Flow cytometer analysis

[0080] (2-1) p53 immunofluorescence

[0081] Analysis of data obtained through FACS was performed using the FACScaliberTM System under the conditions of Alexa-488 (Excitation: 490 / Emission: 525) and RFP670 (Excitation: 643 / Emission: 670). Alexa-488 was excited using a 488 nm laser, and the emitted emission wavelength was confirmed with FL1 (530 / 30 bandpass filter). RFP670 was excited using a 635 nm laser, and the emitted emission wavelength was confirmed with FL4 (661 / 16 bandpass filter). Data were analyzed using the analysis program Kaluza (Backman Coulter).

[0082] (2-2) msfGFP_KRAS

[0083] Analysis of data obtained through FACS was performed using the FACScaliberTM System under the conditions of GFP (Excitation: 488 / Emission: 509) and TagRFP (Excitation: 555 / Emission: 584). GFP was excited using a 488 nm laser, and the emitted emission wavelength was confirmed with FL1 (530 / 30 bandpass filter). TagRFP was excited using a 488 nm laser, and the emitted emission wavelength was confirmed with FL2 (585 / 42 bandpass filter). Data were analyzed using the analysis program Kaluza (Backman Coulter).

[0084] Example 6. Fluorescence analysis using a fluorescence microscope

[0085] The fluorescence microscope used in the present invention was an Olympus IX71 (inverted microscope) equipped with a pE-2LED, and GFP, TagRFP, and Hoechst were observed. Images observed using the microscope were captured with an Andor Clara CCD camera and analyzed using the analysis program Metamorph (Molecular devices) software.

[0086] Example 7. Western Blot

[0087] 1) Cell lysate

[0088] The media in the plate was removed, 1.5 ml of ice-cold PBS was added to the plate to release the attached cells, and the released cells were transferred to a 1.5 ml microfuge tube. After removing the supernatant through centrifugation (3000 rpm, 5 min, 4℃), the cell pellet was washed using 1.5 ml ice-cold PBS. After centrifugation (3000 rpm, 1 min, 4℃), PBS was removed, and the cell pellet was suspended using 600 μl of lysis buffer and transferred to a 500 μl tube. Then, the cell lysate was incubated for 30 minutes in a cold-room tube rotator (end-over-end inversion possible / speed = 11), and the pellet was removed through centrifugation (12000 rpm, 15 min, 4℃) to obtain the supernatant. 50% GammaBind G sepharose bead slurry (50% GammaBind G sepharose: 10 μl Lysis buffer + 10 μl GammaBind G sepharose) was added to the sample obtained in this way and transferred to a cold-room tube rotator. After incubation for 1 hour, a sample excluding beads was obtained after brief centrifugation (12,000 rpm, 4℃).

[0089] 2) Immunoblotting

[0090] Sample buffer (1X SDS-PAGE loading buffer, 100 mM DTT) was added to the sample to denature it (95℃, 5 minutes). After spinning down, the sample was loaded on an SDS-PAGE gel and subjected to SDS-PAGE electrophoresis (60 mA, 90 minutes). The PVDF membrane was activated in methanol for 5 minutes and soaked in transfer buffer (25 mM Tris-Base / 192 mM Glycine / 20% Methanol) for more than 10 minutes. The proteins in the SDS-PAGE gel were transferred to the PVDF membrane under the condition of 100 V, 1 hour, and the membrane was blocked (RT, 1 hour) using 15 ml of blocking buffer (5% skim milk in TBST). The membrane was washed with 15 ml of TBST (50 mM pH 8.0 Tris-HCl, 140 mM NaCl, 0.05% Tween-20) to completely remove the milk. Then, the primary antibody (TBST containing 2% BSA (Sigma, A2153-100G)) was attached to the membrane in a cold room overnight (14-16 hours) and the membrane was washed with TBST. After that, the secondary antibody conjugated with HRP was added and incubated at room temperature for 1 hour and washed using TBST. ECL detection reagents (ECL: Peroxide = 1:1) were sprayed on the membrane to activate HRP and then photosensitized.

[0091] Example 8. Immunofluorescence

[0092] (1) Sample preparation method for FACS analysis

[0093] Remove the media from the plate, treat with 1 ml of 0.25% trypsin, and incubate for 3 minutes at 37°C. Add 500 μl of PFN (PBS + 10% FBS) to the trypsinized cells and transfer to a 1.5 ml microfuge tube. The supernatant was removed by centrifugation (1000 rpm, 4 minutes), and the cell pellet was resuspended in 100 μl of PFN. Add 100 μl of 4% PFA to the 1.5 ml of cells and fix for 30 minutes. After fixation, wash thoroughly with PFN. Then, treat with 400 μl of PFN containing 1% NP40 and permeabilize for 5 minutes. After permeabilization, wash thoroughly with PFN. Then, treat with 500 μl of PFN containing 2% normal goat serum and block for 30 minutes. After blocking, wash thoroughly with PFN. After washing, treat with 50 μl of primary antibody (in PFN with 2% normal goat serum) and incubate for 60 min. After primary antibody staining, wash thoroughly with PFN. After washing, treat with 50 μl of secondary antibody (in PFN with 2% normal goat serum) and incubate for 45 min. After secondary antibody staining, wash thoroughly with PBS. After washing, treat with 10 μl of mounting solution.

[0094] (2) Method for preparing samples for microscopic analysis

[0095] Remove the media from the plate, wash twice with 1 ml of PBS, and fix for 10 minutes with 1 ml of 4% PFA. After fixation, wash twice with 1 ml of PBS. Then, permeabilize for 5 minutes with 1 ml of PBS with 1% NP-40. After permeabilization, wash twice with 1 ml of PBS. Then, block for 30 minutes with 1 ml of PBS containing 1% BSA. After blocking, wash twice with 1 ml of PBS. After washing, treat with 200 μl of primary antibody (in blocking buffer) and incubate for 60 minutes. After primary antibody staining, wash twice with 1 ml of PBS. After washing, treat with 200 μl of secondary antibody (in blocking buffer) and incubate for 45 minutes. After secondary antibody staining, wash twice with 1 ml of PBS. After washing, treat with 1 ml of PBS containing 4 μg / ml Hoechst and incubate for 5 minutes. After nuclear staining, wash twice with 1 ml of PBS. Finally, mount using mounting solution.

[0096] Example 9. Xenograft mouse experiment

[0097] BALB / c-nude mice were used to evaluate the in vivo antitumor efficacy. SW480 stable cell line 3X10 6Cells were inoculated into the left flank of 6-week-old mice. Nine days after inoculation, mice were randomly assigned to experimental and control groups based on tumor volume (approximately 50 mm3). The expression of Nb139-SPOP167-374 was induced in the experimental group by providing doxycycline (2 μg / mL, 200 mg / kg) through water and food. During the experiment, tumor size was measured using a digital caliper and displayed graphically. Animals were euthanized when the tumor volume exceeded 1500 mm3 or when there was a rapid loss of body weight (20%).

Claims

1. A method for specifically degrading p53 protein by treating p53 protein with a fusion of a nanobody recognizing p53 and residues 167-374 of SPOP (Speckle-type POZ protein), an E3 ligase subunit.

2. In paragraph 1, A method for specifically degrading p53 protein, characterized in that the fusion product of a nanobody recognizing p53 and residues 167-374 of SPOP (Speckle-type POZ protein), an E3 ligase subunit, consists of the amino acid sequence of sequence number 1.

3. A composition for decomposing p53 protein, comprising as an active ingredient a fusion of a nanobody and residues 167-374 of the SPOP protein, which is an E3 ligase subunit.

4. A method for specifically degrading a KRAS protein by treating a fusion product of a RAS binder selected from the group consisting of NS1, K27, and DP among RAS binders that recognize the KRAS protein and Rbx1 (RING-box protein 1) with a C-terminal deletion to the KRAS protein.

5. A method for specifically degrading KRAS protein in claim 4, wherein the fusion is characterized in that Rbx1 with a C-terminal deletion is replaced with Rbx2 (RING-box protein 2) with a C-terminal deletion or APC11 (Anaphase-Promoting Complex 11) with a C-terminal deletion.

6. In the fourth paragraph, a method for specifically degrading a KRAS protein consisting of an amino acid sequence of any one of SEQ ID NOs: 5 to 7, wherein the fusion product of one RAS binder selected from the group consisting of NS1, K27 and DP and Rbx1 (RING-box protein 1) with a C-terminal deletion is used.

7. A composition for degrading KRAS protein, comprising as an active ingredient a fusion of one RAS binder selected from the group consisting of NS1, K27 and DP and Rbx1 (RING-box protein 1) with a C-terminal deletion.

8. A composition for degrading KRAS protein, characterized in that in paragraph 7, the fusion is a C-terminal deleted Rbx1 substituted with a C-terminal deleted Rbx2 (RING-box protein 2) or a C-terminal deleted APC11 (Anaphase-Promoting Complex 11).

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