Methods of specifically degrading g12v mutant proteins of krass and compositions thereof

Abstract

The present invention relates to a method for specifically degrading KRAS mutant (G12V) by treating a cell line expressing KRAS mutant (G12V) with a complex of a RAS binder that specifically binds to KRAS mutant (G12V) and an E3 ligase (E3 ligase) NEDD4 HECT, and a composition for use in the method. It can be confirmed that the method developed in the present invention, when treating a cell line expressing KRAS wild type, KRAS wild type is not degraded, in a KRAS mutant (G12V) cell line, the protein level is decreased, the ERK activity is inhibited, and the proliferation, penetration of cancer cells related to KRAS mutant (G12V) is inhibited.

Description

Technical Field

[0001] This invention relates to a method for specifically degrading KRAS mutation (G12V) by treating a cell line expressing KRAS mutation (G12V) with a complex of a RAS conjugate that specifically binds to KRAS mutation (G12V) and an E3 ligase (NEDD4HECT), and to the composition used in the method. Background Technology

[0002] Human cells are composed of various proteins. Many proteins, acting as enzymes that trigger chemical reactions and signaling molecules that produce hormones, play a regulatory role within the cell, modulating their function by selectively degrading proteins present in the cell. Therefore, the protein ubiquitination system is a mechanism that enables the degradation of specific proteins.

[0003] Beyond these functions, protein ubiquitination, as a post-translational modification, participates in various functions such as DNA repair, mRNA export, cell cycle regulation, protein-protein interactions in various signal transduction mechanisms, protein transport, and the formation of aggregates from ubiquitinated proteins that trigger autophagy for removal.

[0004] Protein ubiquitination is induced by ubiquitin. Ubiquitin is a regulatory protein composed of 76 amino acids, which undergoes ubiquitination by binding to the substrate protein 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, thereby promoting the acyl-adenosine conversion of the C-terminus of ubiquitin. The ubiquitin bound to E1 moves to E2, which acts as a conjugating enzyme, to form a complex, and then binds to E3, which acts as a ligase enzyme. Furthermore, the amine of the lysine group present in the substrate launches a nucleophilic attack on the glycine at the C-terminus of ubiquitin, forming a covalent bond, thereby causing ubiquitin to move from E2 to the substrate protein.

[0005] These protein mechanisms occur repeatedly, causing ubiquitin, composed of seven lysine residues, to bind with other ubiquitins to form polyubiquitin chains. There are seven types of ubiquitin chains, classified as K6, K11, K27, K29, K33, K48, and K63 based on their position on the protein chain. Furthermore, different ubiquitin chains perform various functions depending on which residue they bind to.

[0006] Ubiquitin-Proteasome System (UPS) Typically, intracellular protein degradation includes mechanisms mediated by lysosomes and proteasomes. The significant difference between these two mechanisms is that lysosomal-mediated mechanisms lack selectivity or regulatory capacity, while proteasome-mediated mechanisms possess selectivity or regulatory capacity.

[0007] The proteasome is located in the cell nucleus and cytoplasm and degrades most proteins that are bound to ubiquitin chains.

[0008] The proteasome consists of two subunits: a 19S regulatory particle and a 20S core particle. The 19S regulatory particle recognizes proteins that bind to the ubiquitin chain and induces the 20S core particle to degrade the protein into peptides.

[0009] E3 ligase E3 ligases are specific, binding simultaneously to both E2 proteins and substrate proteins and identifying substrate proteins to be degraded by the proteasome through ubiquitination. E3 ligases act as mediators of enzyme cascades because they can confer high levels of specificity and selectivity to cellular target substrates. The human proteome encodes more than 600 E3 ligases that can be classified into three classes.

[0010] The largest category 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, consisting of about 28 members, and the RBR (RING between RING) type E3, consisting of about 14 members.

[0011] RING E3 ligases act as a scaffold to bring E2 closer to the substrate and as an allosteric activator of E2, while HECT and RBR E3 ligases catalyze substrate ubiquitination through a two-step reaction. In the first step, activated Ub is accepted from E2 via a thiol exchange reaction targeting cysteine. In the second step, the Ub moiety is transferred to the lysine residue of the target substrate.

[0012] Examining HECT-containing E3 ligases, at the C-terminus, all HECT E3 ligases exhibit a catalytic HECT domain, consisting of a larger N-lobe containing an E2-binding domain and a C-lobe carrying a catalytic cysteine ​​residue. These two lobes are connected by a flexible hinge region, allowing the C-lobe to move and facilitate the transfer of Ub from E2 to E3.

[0013] In recent years, PROTACs, drugs developed as a selective removal method for target proteins, consist of a target protein ligand-linker-E3 ubiquitin ligase ligand. These structures are complex, and finding suitable ligand-linker combinations is difficult. Furthermore, the number of target proteins that can be induced to degrade by PROTACs is limited, leading to decreased degradation efficiency. Additionally, these drugs may affect not only problematic cells but also normal cells, potentially causing toxicity.

[0014] Existing patent literature Korean Patent Publication No. 10-2017-0096878 Summary of the Invention

[0015] Technical issues The present invention is proposed to solve the above-mentioned objectives and in accordance with the above-mentioned needs. The objective of the present invention is to provide a method for specifically degrading the G12V mutant of KRAS protein.

[0016] Another object of the present invention is to provide a composition for a method of specifically degrading a G12V mutant of KRAS protein.

[0017] Technical solution To achieve the above objectives, the present invention provides a method for specifically degrading the G12V mutant protein of KRAS. This method specifically degrades the G12V mutant protein of KRAS by treating it with a fusion of a RAS binder that recognizes KRAS and a partially deleted neural precursor cell expressing a developmentally down-regulated 4-like (NEDD4L) E6-AP carboxyl terminus (HECT) domain.

[0018] In this invention, preferably, the RAS binder is a monobody, darpin, or RAF-ras binding domain, but is not limited thereto.

[0019] In one embodiment of the present invention, preferably, the RAS conjugate that identifies kRAS is a RAS conjugate selected from the group consisting of iDAb, 12VC1 and K55, but is not limited thereto.

[0020] In another embodiment of the present invention, preferably, the fusion of the RAS conjugate that recognizes kRAS and the partially deleted HECT domain of the NEDD4L that recognizes the substrate is one of SEQ ID NO: 1 to 3, but all mutants that achieve the effect targeted by the present invention by introducing more than one substitution, deletion, inversion and translocation mutation in the amino acid sequence are also included within the scope of the present invention.

[0021] Furthermore, the present invention provides a composition for degrading G12V mutant protein of KRAS, the composition comprising, as an active ingredient, a fusion of a RAS conjugate that recognizes kRAS and a partially deleted E6-AP carboxyl terminus (HECT) domain that recognizes a substrate in neural precursor cells expressing developmentally down-regulated 4-like (NEDD4L).

[0022] In one embodiment of the present invention, preferably, the RAS conjugate that identifies kRAS is a RAS conjugate selected from the group consisting of iDAb, 12VC1 and K55, but is not limited thereto.

[0023] In another embodiment of the invention, preferably, the fusion of the RAS conjugate that recognizes kRAS and the partially deleted HECT domain of the NEDD4L that recognizes the substrate is one of SEQ ID NO: 1 to 3, but all mutants that achieve the effect targeted by the invention by introducing one or more substitution, deletion, inversion and translocation mutations into the amino acid sequence are also included in the scope of the invention.

[0024] The present invention will now be described.

[0025] This invention relates to the specific degradation of the oncogenic protein KRAS mutation (G12V), specifically a method for treating cell lines expressing KRAS mutation (G12V) with a complex of a RAS conjugate specifically binding to KRAS mutation (G12V) and an E3 ligase (NEDD4HECT), as well as the composition used in this method. It has been confirmed that the method developed in this invention does not degrade KRAS wild-type cells when treating KRAS wild-type cells; in KRAS mutation (G12V) cell lines, protein levels decrease, ERK activity is inhibited, and the proliferation and penetration of cancer cells associated with KRAS mutation (G12V) are suppressed.

[0026] The effects of the invention As can be seen from the present invention, when the method developed in the present invention is used to treat cell lines expressing KRAS wild-type, KRAS wild-type is not degraded, while in KRAS mutant (G12V) cell lines, protein levels decrease, ERK activity is inhibited, and the proliferation and infiltration of cancer cells associated with KRAS mutant (G12V) are inhibited. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the present invention.

[0028] Figure 2 The diagrams illustrating the antibody enzymes that degrade the kRAS mutant protein show this effect in numbers 3, 4, and 8.

[0029] Figure 3Part (a) shows a diagram illustrating KRAS G12V degradation induced by 12VC1-NEDDHECT. To confirm whether the KRAS protein could be degraded using the monoclonal antibody 12VC1, which has high affinity for KRAS G12V and G12C, and NEDDHECT, which acts as a HECT E3 ubiquitin ligase, experiments were conducted using a stable SW480 cell line expressing 12VC1-NEDDHECT induced by TRE. To induce 12VC1-NEDDHECT expression in the colon cancer cell line SW480 (KRASG12V), treatment with doxycycline (1 μg / mL) for 20 hours and confirmation by Western blot showed that KRAS protein was effectively degraded when treated with doxycycline and expressing 12VC1-NEDDHECT. Furthermore, it was confirmed that when KRAS protein degradation occurs, pERK, which is a downstream signal of KRAS, also decreases (Lane 1,2). Furthermore, to confirm whether KRAS G12V degradation induced by 12VC1-NEDDHECT occurs via UPS, treatment with MG132 (10 mM), a proteasome inhibitor, for 6 hours was followed by treatment with doxycycline (1 μg / mL) for 20 hours. Western blot analysis confirmed that KRAS protein degradation occurred effectively under MG132(-) / doxycycline(+) ​​conditions, while degradation was inhibited under MG132(+) / doxycycline(+) ​​conditions. Therefore, it can be confirmed that KRAS protein degradation induced by 12VC1-NEDDHECT occurs via UPS (Lane 3,4).

[0030] Figure 3Part (b) shows that, to confirm the effectiveness of the monoclonal antibody 12VC1, selective for KRAS G12V and G12C, and NEDDHECT, as a HECT E3 ubiquitin ligase, in KRAS WT cells, experiments were conducted using the HT29 stable cell line, which expressed 12VC1-NEDDHECT via TRE induction. To induce 12VC1-NEDDHECT expression in HT29, a KRAS WT colorectal cancer cell line, treatment with doxycycline (1 μg / mL) for 20 hours and confirmation by Western blot confirmed that even with 12VC-NEDDHECT expression, KRAS protein degradation did not occur in KRAS WT cells.

[0031] Figure 4 To illustrate the effects on target cell growth (proliferation), and to confirm whether KRAS (G12V) degradation induced by 12VC1-NEDDHECT could inhibit cell growth in the KRAS-mutant colorectal cancer cell line SW480, stable cells (5 x 10⁻⁶ cells) were constructed. 3 Cells were divided into two groups: one in medium containing doxycycline (1 mg / mL) and the other in medium without doxycycline. Cells were seeded in 96-well plates, and viability was confirmed using CCK-8 (Abcam) after 24, 48, and 72 hours. When absorbance was measured at 460 nm after 1 hour and 30 minutes of CCK-8 treatment and plotted graphically, it was confirmed that the absorbance significantly decreased after 72 hours in the doxycycline (+) group. ***p≤0.01 Figure 5 To illustrate the effect on target cell growth (colony formation), and to confirm whether KRAS degradation induced by 12VC1-NEDDHECT could inhibit the cells' colony formation ability, constructed SW480 stable cells (2 x 10⁻⁶ cells) were used. 2Cells were divided into two groups: one with doxycycline (1 mg / mL) and the other without, and seeded into 6-well plates. Doxycycline (1 μg / mL) was added every 72 hours. After 13 days, colony counts were performed using crystal violet solution, and colony-forming ability was compared. The results confirmed that the colony count in the doxycycline (1 μg / mL) treatment group was significantly reduced. **p<0.01 Figure 6 To illustrate the effect on invasion of target cells, To confirm whether cell invasion could still be suppressed, a Matrigel-based invasion chamber (Corning) was used. Stable cells (1.5 x 10⁻⁶ cells) were then introduced into the chamber. 5 Cells were divided into doxycycline (1 mg / mL) (-) and (+) samples and added to 8 mm pore matrigel inserts. After 72 hours, non-invading cells were removed, and the 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 determined under a microscope. When the number of invading cells was counted and plotted, it was confirmed that the number of invading cells was significantly reduced in the doxycycline (+) sample. **p<0.01 Figure 7 A graph illustrating the antitumor efficacy of xenograft mice in vivo. To evaluate the in vivo antitumor efficacy of KRAS (G12V) degradation induced by 12VC1-NEDDHECT, nude mice were used. 3 x 10 6 SW480 stable cells expressing 12VC1-NEDDHECT via the TRE promoter were seeded into the left ventral region of mice. Eight days after seeding, tumor volume (approximately 70 mm) was used to determine the tumor size. 3Students were randomly divided into an experimental group and a control group. The experimental group was given doxycycline (650 mg / kg) via food to induce the expression of 12VC1-NEDDHECT. During the experiment, the size of the tumor was measured using digital calipers, and it was confirmed that the tumor volume in the group whose 12VC1-NEDDHECT expression was induced by doxycycline was significantly reduced compared with the control group. Detailed Implementation

[0032] The present invention will now be described in more detail through non-limiting embodiments. However, the following embodiments are described in order to illustrate the intent of the invention, and the scope of the invention should not be construed as limited to the following embodiments.

[0033] Example 1. Strains and Culture Conditions 1) Escherichia coli strains and culture In this invention, Escherichia coli (E. coli) is used. Escherichia coli The *E. coli* strain was used as the competent cells for amplifying the recombinant plasmid. The strain was cultured in LB medium (0.5% yeast extract, 1% tryptone, 0.5% sodium chloride, Lennox) at 37°C and 225 rpm for 12–15 hours. To perform drug selection based on plasmid type, kanamycin (25 μg / ml) and carbenicillin (100 μg / ml), both antibiotics, were added to LB medium for culture.

[0034] 2) Animal cells and culture 293TetOn cells were cultured in DMEM medium (containing 4.5 g / L glucose, sodium pyruvate, and L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 5% CO2 and 37°C. The 293T-CMV-msfGFP_WT KRAS cells and 293TetOn-CMV-msfGFP_KRAS_G12V cells used in the experiment were cultured in DMEM medium (containing 4.5 g / L glucose, L-glutamine, sodium pyruvate, [10% FBS, 10 mM HEPES, 1% penicillin / streptomycin]) supplemented with 2 μg / ml puromycin (5% CO2, 37℃) to maintain the msfGFP_WT KRAS and msfGFP_KRAS_G12V cells within the cells.

[0035] SW480 cells were cultured in RPMI 1640 medium (containing 300 mg / L L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 5% CO2 and 37°C. The SW480-Nb139_SPOP cells used in the experiment were... 167-374 Cells were cultured in 10% FBS, 1% penicillin / streptomycin, 10mM HEPES, and RPMI 1640 (containing 300mg / L L-glutamine) medium supplemented with 200μg / ml of the hygromycin used in cell construction (5% CO2, 37°C).

[0036] HT-29 cells were cultured in RPMI 1640 medium (containing 300 mg / L L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 5% CO2 and 37°C. The HT-29-12VC1_NEDD cells used in the experiment were... HECTCells were cultured in 10% FBS, 1% penicillin / streptomycin, 10mM HEPES, and RPMI 1640 (containing 300mg / L L-glutamine) medium supplemented with 40μg / ml of puromycin used in cell construction (5% CO2, 37°C).

[0037] Example 2. Constructing a carrier 1) Constructing expression vectors for animal cells In a plasmid containing pEM791-SRa-HygroB_rtTA3, the fluorescent protein (TagRFP) gene and E3 ubiquitin ligase were recombined into a bidirectional tetracycline response element (TRE) promoter. The TRE promoter was used to ensure that expression only occurred in the presence of doxycycline.

[0038] The vector required for the FACS analysis is the recombinant vector into the TRE promoter after the fluorescent protein genes RFP670 and TagRFP are amplified by PCR and treated with restriction endonucleases (BstBI / SalBI).

[0039] Tags were added to iDAb, 12VC1, NS1, K27, DPKRAS, K55, and RAF1_RBD_CRD, which are KRAS-recognizing binders, and NEDD4 was treated with restriction endonucleases (XhoⅠ / MluⅠ). HECT Recombination was then performed. In order to transform the animal cells used in the experiment, msfGFP fusion proteins (KRAS_WT, KRAS_G12V) synthesized by PCR were obtained by combining msfGFP with specific proteins. The PCR products obtained were then treated with restriction endonucleases and recombined into the pPuro-CMV lentiviral vector.

[0040] 2) Constructing a vector for producing lentivirus. Flag-12VC1-NEDD HECTRecombinantly, after PCR amplification and treatment with restriction endonucleases (MfeⅠ / NheⅠ), was incorporated into the tetracycline response element (TRE) promoter of the pCW57.1-DUX4-CA_EcoRI_NheRI_IRES2_mCherry plasmid. The TRE promoter was used to ensure expression only in the presence of doxycycline.

[0041] Example 3. Transformation of recombinant vectors 1) Transformation of Escherichia coli During the cloning process, the vector is ligated with the mixture containing the inserted gene and E. coli cells serving as competent cells. Escherichia coli Mix Top10F, then incubate on ice for 5 minutes, followed by heat shock at 42°C for 1 minute to allow the ligated DNA to enter competent cells. Incubate on ice for 3 minutes, then add LB medium and incubate at 37°C and 225 rpm for 1 hour to allow the cells to recover.

[0042] Then, incubate overnight (14–16 hours) in LB agar medium containing the desired antibiotics.

[0043] 2) Transformation of animal cells Animal cells cultured in 10 cm plates were treated with 1.5 ml of 0.25% trypsin to recover the cells, followed by centrifugation to remove the trypsin. The cells were then separated at 7.6 x 10⁻⁶ cm⁻¹. 5 A concentration of 1 cell / well was seeded in a 6-well plate.

[0044] Twenty-four hours later, when the cells had grown to 60-70% confluence, a mixture of 2 μg DNA, 8 μg polyethyleneimine (PEI), and 300 μl Opti-MEM was added to each well after being allowed to stand at room temperature for 15 minutes. The cells were then incubated at 37°C with 5% CO2 for 4 hours, followed by the addition of 2 μg doxycycline.

[0045] Example 4. Fluorescence analysis using flow cytometer (1) Cell processing for flow cytometer analysis Treat each well with 1 ml of 0.25% trypsin for 3 minutes, recover the cells and transfer them to 3 ml of complete culture medium. Centrifuge at 700 rpm for 3 minutes. Then, remove the supernatant, add 1 ml of 1X phosphate-buffered saline (PBS) to resuspend the cells, and then transfer the resuspended cells into BD-FalconFACS tubes to prepare samples.

[0046] (2) Flow cytometer analysis Using FACScaliber TM The system analyzed data obtained via FACS under GFP (excitation: 488 / emission: 509) and TagRFP (excitation: 555 / emission: 584) conditions. GFP was induced into the excitation state using a 488 nm laser, and the emission wavelength was confirmed using FL1 (530 / 30 bandpass filter). TagRFP was also induced into the excitation state using a 488 nm laser, and the emission wavelength was confirmed using FL2 (585 / 42 bandpass filter). The data were analyzed using the Kaluza analysis program (Backman Coulter).

[0047] Example 5. Construction of cell lines (1) Constructing 293T-msfGFP_KRAS_WT and 293Tet-On-msfGFP_KRAS_G12V cell lines To construct the 293T-msfGFP_KRAS_WT and 293Tet-On-msfGFP_KRAS_G12V cell lines, cells were transformed with PEI and selected using a medium containing 2 μg / ml puromycin.

[0048] (2) Construct SW480-12VC1-NEDD using lentivirus. HECT HT-29-12VC1-NEDD HECT cell lines (2-1) Production of lentiviruses 3x10 6 HEK293T cells were seeded in 100 mm culture dishes. After 24 hours, they were transformed with 12 μg of pCW57.1-DUX4-CA_FLAG_12VC1_NEDDHECT_IRES2_mCherry vector, 8.5 μg of psPAX2 (lentiviral packaging vector), 2.8 μg of pMD2.G (envelope vector), and 68.5 μg of PEI. Forty-eight hours after transformation, the supernatant was collected, filtered through a 0.45 μm filter, and concentrated with 3 ml of Lenti-X concentrate for 24 hours. The concentrated mixture was centrifuged at 1500 x g at 4 °C for 45 minutes and then dispersed with 1 ml of culture medium.

[0049] (2-2) Virus transduction and protein expression Cells were added to 1.5 ml of medium containing 10 μg / ml polybrene and transduced with lentivirus in 6-well plates for 48 hours. Transduced cells were then selected using 40 μg / ml puromycin. FLAG_12VC1_NEDDHECT was expressed via the TRE promoter upon treatment with 1 μg / ml doxycycline.

[0050] Example 6. Measurement of target cell growth (cell proliferation) To determine the growth (proliferation) of target cells, the constructed SW480 stable cell line was divided into a medium containing doxycycline (1 μg / mL) and a medium without doxycycline, at a concentration of 5 x 10⁻⁶ cells / mL. 3 Cells were seeded in 96-well plates (cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin). At 24, 48, and 72 hours post-seeding, CCK-8 (Abcam) was added to the live cells, and the absorbance was measured at 460 nm using a microplate reader 1 hour and 3 minutes later.

[0051] Example 7. Measurement of target cell colonies To determine target cell colony formation, the constructed stable SW480 cell line was divided into a medium containing doxycycline (1 μg / mL) and a medium without doxycycline, at a concentration of 2 x 10⁻⁶ cells / mL. 2 Cells were seeded in 6-well plates. Doxycycline (1 μg / mL) was added every 72 hours. After 12-13 days, the cells were washed with PBS, fixed with methanol, stained with crystal violet, and counted.

[0052] Example 8. Determining the invasive ability of cells To determine the invasive ability of target cells, SW480 stable cell lines were sputtered at 1.5 x 10⁻⁶ cells / year. 2 Cells were seeded into inserts in Matrigel invasion chambers (Corning), separately supplemented with doxycycline (1 mg / mL) in both (-) and (+) forms. After 72 hours, non-invading cells were removed, and cells on the lower membrane surface were fixed and stained with hematoxylin and eosin Y.

[0053] Example 9. Xenotransplantation mouse experiment To evaluate in vivo antitumor efficacy, BALB / c-nude mice were used. The SW480 stable cell line (12VC1-NEDDHECT) was cultured at 3 x 10⁻⁶ cells / mL. 6 Cells were inoculated into the left ventral region of 6-week-old mice. Eight days after inoculation, tumor volume (approximately 70 mm) was used to determine the tumor size. 3 Patients were randomly assigned to an experimental group and a control group. The experimental group received doxycycline (650 mg / kg) via food, which induced the expression of 12VC1-NEDDHECT. During the experiment, tumor size was measured using digital calipers and plotted graphically.

Claims

1. A method for specifically degrading the G12V mutant protein of KRAS, characterized in that, The G12V mutant protein of KRAS was specifically degraded by treating the G12V mutant protein of KRAS with a fusion of the E6-AP C-terminal homologous HECT domain of four NEDD4L-like proteins that downregulated development by expressing a RAS conjugate that recognizes KRAS and a partially deleted recognition substrate in neural progenitor cells.

2. The method for specifically degrading the G12V mutant protein of KRAS according to claim 1, characterized in that, The RAS conjugate that identifies kRAS is one RAS conjugate selected from the group consisting of iDAb, 12VC1, and K55.

3. The method for specifically degrading the G12V mutant protein of KRAS according to claim 1, characterized in that, The fusion of the RAS conjugate that recognizes kRAS and the partially deleted HECT domain of the NEDD4L that recognizes the substrate is one of SEQ ID NO: 1 to 3.

4. A composition for degrading the G12V mutant protein of KRAS, characterized in that, The active ingredient is a fusion of the E6-AP C-terminal homologous HECT domain of four NEDD4L-like cells that downregulate the expression of a RAS conjugate that recognizes kRAS and a partially deleted substrate recognition component.

5. The composition for degrading the G12V mutant protein of KRAS according to claim 4, characterized in that, The RAS conjugate that identifies kRAS is one RAS conjugate selected from the group consisting of iDAb, 12VC1, and K55.

6. The composition for degrading the G12V mutant protein of KRAS according to claim 4, characterized in that, The fusion of the RAS conjugate that recognizes kRAS and the partially deleted HECT domain of the NEDD4L that recognizes the substrate is one of SEQ ID NO: 1 to 3.

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