Method for specifically degrading g12v mutant protein of KRAS and composition therefor

Abstract

The present invention relates to a method for specifically degrading a KRAS mutant (G12V) by treating a cell line expressing the KRAS mutant (G12V) with a complex of a RAS binder binding specifically to the KRAS mutant (G12V) and an E3 ligase NEDD4HECT, and a composition used in the method. When the method developed in the present invention is applied to a cell line expressing a wild-type KRAS, the wild-type KRAS is not degraded. In a KRAS mutant (G12V) cell line, it was confirmed that while a protein level is reduced, ERK activity is inhibited and proliferation and penetration of cancer cells related to the KRAS mutant (G12V) are inhibited.

Description

Method for specific degradation of G12V mutant protein of KRAS and composition thereof

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

[0002] Human cells are composed of diverse proteins. These numerous proteins regulate the cell's internal functions as signaling molecules that produce enzymes and hormones that initiate chemical reactions. They also selectively degrade proteins within the cell, thereby regulating their functions. The protein ubiquitination system is a mechanism that enables the degradation of specific proteins.

[0003] In addition to these functions, protein ubiquitination is a post-translational modification that is involved in various functions such as DNA repair, mRNA export, cell cycle regulation, protein-protein interactions in various signal transduction mechanisms, protein trafficking, and ubiquitinated proteins tangle together to form aggresomes, which induce autophagy and are then removed.

[0004] 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.

[0005] 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.

[0006] Ubiquitin-Proteasom System (UPS)

[0007] Representative mechanisms for protein degradation within cells exist, involving lysosomes and proteasomes. These two mechanisms differ significantly: lysosomes lack selectivity or regulatory capacity, while proteasomes possess both.

[0008] Proteasomes exist in the nucleus and cytoplasm and degrade most proteins linked to ubiquitin chains.

[0009] 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.

[0010] E3 ligase

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] [Prior patent literature]

[0017] Republic of Korea Patent Publication No. 10-2017-0096878

[0018] 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 a G12V mutant of KRAS protein.

[0019] Another object of the present invention is to provide a composition involved in a method for specifically degrading a G12V mutant of KRAS protein.

[0020] In order to achieve the above purpose, the present invention provides a method for specifically degrading a G12V mutant protein of KRAS by treating a fusion of a RAS binder that recognizes kRAS and a HECT (homology to E6-AP carboxyl terminus) domain of NEDD4L (Neural precursor cell expressed developmentally down-regulated 4-like) in which a portion that recognizes a substrate is removed to the G12V mutant protein of KRAS.

[0021] In the present invention, the RAS binder is preferably a monobody, darpin, or RAF-ras binding domain, but is not limited thereto.

[0022] In one embodiment of the present invention, the RAS binder recognizing the kRAS is preferably, but is not limited to, one RAS binder selected from the group consisting of iDAb, 12VC1, and K55.

[0023] In another embodiment of the present invention, the fusion of the RAS binder recognizing kRAS and the HECT domain of NEDD4L with the substrate-recognizing portion removed is preferably one of SEQ ID NOs. 1 to 3, 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.

[0024] In addition, the present invention provides a composition for degrading a G12V mutant protein of KRAS, which comprises as an active ingredient a fusion of a RAS binder that recognizes kRAS and a HECT (homology to E6-AP carboxyl terminus) domain of NEDD4L (Neural precursor cell expressed developmentally down-regulated 4-like) in which a portion that recognizes a substrate is removed.

[0025] In one embodiment of the present invention, the RAS binder recognizing the kRAS is preferably, but is not limited to, one RAS binder selected from the group consisting of iDAb, 12VC1, and K55.

[0026] In another embodiment of the present invention, the fusion of the RAS binder recognizing kRAS and the HECT domain of NEDD4L with the substrate-recognizing portion removed is preferably one of SEQ ID NOs. 1 to 3, 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.

[0027] The present invention is described below.

[0028] The present invention relates to the specific degradation of the oncogenic protein KRAS mutant (G12V), and 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 NEDD4HECT, and a composition used in the method. When the method developed in the present invention is treated to a cell line expressing KRAS wild type, KRAS wild type is not degraded, and it can be confirmed that ERK activity is suppressed while the protein level decreases in the KRAS mutant (G12V) cell line, and proliferation and invasion of cancer cells related to KRAS mutant (G12V) are suppressed.

[0029] As can be seen through the present invention, when the method developed in the present invention is applied to a cell line expressing KRAS wild type, KRAS wild type is not degraded, and in a KRAS mutant (G12V) cell line, protein levels are reduced while ERK activity is suppressed, and proliferation and invasion of cancer cells related to KRAS mutant (G12V) are suppressed.

[0030] Figure 1 is a schematic diagram of the present invention;

[0031] Figure 2 is a diagram showing antibody enzymes that decompose kRAS mutant proteins, and this effect is observed in numbers 3, 4, and 8.

[0032] Figure 3(a) is a diagram showing the degradation of KRAS G12V by 12VC1-NEDDHECT. In order to confirm whether KRAS protein can be degraded using 12VC1, a monobody with high affinity for KRAS G12V and G12C, and NEDDHECT, a HECT E3 ubiquitin ligase, an experiment was conducted using the SW480 stable cell line expressing 12VC1-NEDDHECT by TRE. To induce the expression of 12VC1-NEDDHECT in the colon cancer cell line SW480 (KRASG12V), Doxycycline (1 μg / mL) was treated for 20 hours, and the result confirmed by Western blot showed that when 12VC1-NEDDHECT was expressed by treating with Doxycycline, KRAS protein was effectively degraded. In addition, it was confirmed that when KRAS protein degradation occurs, pERK, a downstream signal of KRAS, also decreases (Lane 1,2).

[0033] In addition, to confirm whether the degradation of KRAS G12V by 12VC1-NEDDHECT occurs through the UPS, the cells were treated with the proteasome inhibitor MG132 (10 mM) for 6 hours, followed by Doxycycline (1 μg / mL) for 20 hours. The results were confirmed through Western blot. MG132 (-) / Doxycycline (+) effectively degraded KRAS protein, whereas MG132 (+) / Doxycycline (+) inhibited KRAS degraded protein. This confirmed that KRAS protein degradation by 12VC1-NEDDHECT occurs through the UPS. (Lane 3,4).

[0034] Figure 3b shows an experiment conducted using HT29 stable cell line expressing 12VC1-NEDDHECT by TRE to confirm the effect of 12VC1, a monobody selective for KRAS G12V, G12C, and NEDDHECT, a HECT E3 ubiquitin ligase, on KRAS WT cells. To induce 12VC1-NEDDHECT expression in HT29, a KRAS WT colon cancer cell line, doxycycline (1 μg / mL) was treated for 20 hours, and the result confirmed by Western blot showed that KRAS protein degradation did not occur in KRAS WT cells even when 12VC-NEDDHECT was expressed.

[0035] Figure 4 is a drawing showing the effect on the growth (proliferation) of target cells. Stable cells (5X10) were constructed to confirm whether the degradation of KRAS (G12V) by 12VC1-NEDDHECT in SW480, a colon cancer cell line with KRAS mutation, can inhibit cell growth. 3 Cells) were plated in 96-well plates with or without Doxycycline (1 mg / mL) and viable cells were identified 24, 48, and 72 hours after plating using CCK-8 (Abcam). The absorbance was measured at 460 nm 1 hour and 30 minutes after CCK-8 treatment, and when this was graphed, it was confirmed that the value after 72 hours was significantly reduced in Doxycycline (+). ***p ≤ 0.01

[0036] Figure 5 is a drawing showing the effect on the growth of target cells (Colony formation). SW480 Stable cells (2X10) were constructed to confirm whether the degradation of KRAS by 12VC1-NEDDHECT can inhibit the colony formation ability of cells. 2Cells were divided into 6-well plates, and doxycycline (1 μg / mL) was added every 72 hours after plating. After 13 days, the colonies were counted after staining with Crystal Violet solution and the colony forming ability was compared. As a result, it was confirmed that the number of colonies in the group treated with doxycycline (1 μg / mL) was significantly reduced. **p < 0.01

[0037] Figure 6 is a diagram showing the effect on invasion of target cells.

[0038] To determine whether the cell invasion ability could also be inhibited, a Matrigel invasion chamber (Corning) was used. The constructed stable cells (1.5 X 10 5 Cells) were divided into (-) and (+) groups and added with Doxycycline (1 mg / mL) to 8 mm pore matrigel inserts. 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 (+) sample. **p < 0.01

[0039] Figure 7 is a diagram showing the in vivo antitumor efficacy in xenograft mice.

[0040] To evaluate the in vivo antitumor efficacy of KRAS(G12V) degradation by 12VC1-NEDDHECT, nude mice were used. 3X10 SW480 stable cells expressing 12VC1-NEDDHECT by the TRE promoter6 was inoculated into the left flank of the mouse. Based on the tumor volume (approximately 70 mm ) 8 days after inoculation 3 ) were randomly assigned to an experimental group and a control group, and the expression of 12VC1-NEDDHECT was induced in the experimental group by providing Doxycycline (650 mg / kg) through food. During the experimental period, the tumor size was measured with a digital caliper, and it was confirmed that the tumor volume in the group in which the expression of 12VC1-NEDDHECT was induced through Doxycycline was significantly reduced compared to the control group.

[0041] 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.

[0042] Example 1. Strains and culture conditions

[0043] 1) Escherichia coli strain and culture

[0044] In the present invention, Escherichia coliTop10F' (F'[lacIq Tn10(tetR)] mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR nupG recA1 araD139 Δ(ara-leu)7697 galU 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.

[0045] 2) Animal cells and culture

[0046] 293TetOn cells were cultured in DMEM (containing 4.5 g / L glucose, sodium pyruvate, and L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 37°C and 5% CO2. 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]) medium with puromycin added at a concentration of 2 μg / ml to maintain msfGFP_WT KRAS and msfGFP_KRAS_G12V within the cells.

[0047] SW480 was cultured in RPMI1640 medium (containing 300 mg / L L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 37°C and 5% CO2. SW480-Nb139_SPOP used in the experiment 167-374 Cells were cultured (5% CO2, 37°C) in a medium containing 10% FBS, 1% penicillin / streptomycin, 10 mM HEPES, and RPMI1640 (containing 300 mg / L L-glutamine) at a concentration of 200 μg / ml of hygromycin used to create the cells.

[0048] HT-29 was cultured in RPMI1640 medium (containing 300 mg / L L-glutamine) supplemented with 10% FBS, 1% penicillin / streptomycin, and 10 mM HEPES at 37°C and 5% CO2. HT-29-12VC1_NEDD used in the experiment HECTCells were cultured (5% CO2, 37°C) in a medium containing 10% FBS, 1% penicillin / streptomycin, 10 mM HEPES, and RPMI1640 (containing 300 mg / L L-glutamine) at a concentration of 40 μg / ml of puromycin, which was used to create the cells.

[0049] Example 2. Vector construction

[0050] 1) Construction of expression vectors for animal cells

[0051] The fluorescent protein (TagRFP) gene and E3 ubiquitin ligase were recombined into the bilaterally expressed tetracycline response element (TRE) promoter in the plasmid containing pEM791-SRa-HygroB_rtTA3. The TRE promoter was used to ensure expression only in the presence of doxycycline.

[0052] The vector required for the experiment analyzed using FACS was amplified by PCR with the fluorescent protein genes RFP670 and TagRFP in the TRE promoter and recombined by treatment with restriction enzymes (BstBI / SalI).

[0053] Flag iDAb, 12VC1, NS1, K27, DP KRAS, K55, RAF1_RBD_CRD, which are binders that can recognize KRAS, and NEDD4 HECT was recombined by restriction enzyme (XhoⅠ / MluⅠ) treatment. In order to transform animal cells used in the experiment, msfGFP and a specific protein-synthesized msfGFP fusion protein (KRAS_WT, KRAS_G12V) were obtained through PCR, and the obtained PCR product was treated with restriction enzymes and recombined into the pPuro-CMV lentiviral vector.

[0054] 2) Construction of vectors for lentivirus production

[0055] Flag-12VC1-NEDD was cloned into the TRE (tetracycline response element) promoter of pCW57.1-DUX4-CA_EcoR I_Nhe I_IRES2_mCherry plasmid by PCR. HECT After amplification, it was recombined by treatment with restriction enzymes (MfeⅠ / NheⅠ). The TRE promoter was used to enable expression only in the presence of doxycycline.

[0056] Example 3. Transformation of recombinant vector

[0057] 1) Transformation of E. coli

[0058] During the cloning process, the mixture linking the vector and the inserted gene was mixed with competent cells (Escherichia coliTop10F), 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, and 1 hour to allow the cells to recover.

[0059] Afterwards, it was cultured overnight (14-16 hours) on LB agar medium containing the necessary antibiotics.

[0060] 2) Transformation of animal cells

[0061] 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 Inoculated at a concentration of 10 cells / well.

[0062] After 24 hours, when the cells were 60-70% confluent, a mixture of 2 μg of DNA, 8 μg of polyethylenimine (PEI), and 300 μl of Opti-MEM per well was added 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.

[0063] Example 4. Fluorescence analysis using a flow cytometer

[0064] (1) Cell processing for flow cytometer analysis

[0065] 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.

[0066] (2) Flow cytometer analysis

[0067] Analysis of data obtained through FACS is performed using FACScaliber TMThe system was used to analyze GFP (Excitation: 488 / Emission: 509) and TagRFP (Excitation: 555 / Emission: 584) conditions. 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).

[0068] Example 5. Cell line construction

[0069] (1) Construction of 293T-msfGFP_KRAS_WT and 293Tet-On-msfGFP_KRAS_G12V cell lines

[0070] 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.

[0071] (2) SW480-12VC1-NEDD using lentivirus HECT ,HT-29-12VC1-NEDD HECT Cell line construction

[0072] (2-1) Lentivirus production

[0073] 3 x 10 6HEK293T cells were seeded in 100 mm dishes and transfected 24 hours later 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. 48 hours after transfection, the supernatant was collected, filtered using a 0.45 μm filter, and 3 ml of Lenti-X concentrator was added to concentrate for 24 hours. The concentrated mixture was centrifuged at 1,500 x g for 45 minutes at 4°C and resuspended in 1 ml of medium.

[0074] (2-2) Virus transduction and protein expression

[0075] Cells were transduced with lentivirus in 6-well plates in 1.5 ml of medium containing 10 μg / ml polybrene for 48 h. Transduced cells were selected with 40 μg / ml puromycin. FLAG_12VC1_NEDDHECT is expressed by the TRE promoter upon treatment with 1 μg / ml doxycycline.

[0076] Example 6. Measurement of target cell proliferation

[0077] To measure growth (proliferation) in target cells, 5X10 of the SW480 stable cell line was established in a 96-well plate. 3Cells were plated in medium containing or without doxycycline (1 μg / mL) (cultured in RPMI1640 medium containing 10% FBS and 1% Penicillin / streptomycin). After 24, 48, and 72 hours of plating, living cells were incubated with CCK-8 (Abcam), and the absorbance was measured at 460 nm using a microplate reader after 1 hour and 30 minutes.

[0078] Example 7. Measurement of target cell colonies

[0079] 2X10 SW480 stable cell lines constructed to measure colony formation in target cells 2 Cells were plated in 6-well plates divided into medium containing or without doxycycline (1 μg / mL). Doxycycline (1 μg / mL) was added every 72 hours, and after 12–13 days, the cells were washed with PBS, fixed with methanol, stained with crystal violet solution, and counted.

[0080] Example 8. Measurement of cell invasive ability

[0081] To measure the invasion ability in target cells, 1.5X10 of SW480 Stable cell line 2 Cells were divided into (-) and (+) groups and added with doxycycline (1 mg / mL) to Matrigel invasion chamber (Corning) inserts. After 72 hours, non-invading cells were removed, and cells on the surface beneath the membrane were fixed and stained with Hematoxylin and Eosin Y.

[0082] Example 9. Xenograft mouse experiment

[0083] To evaluate the in vivo antitumor efficacy, BALB / c-nude mice were used. SW480 stable cell line (12VC1-NEDDHECT) 3X10 6 Cells were inoculated into the left flank of 6-week-old mice. Based on the tumor volume (approximately 70 mm) 8 days after inoculation 3 ) were randomly assigned to an experimental group and a control group, and the expression of 12VC1-NEDDHECT was induced in the experimental group by providing Doxycycline (650 mg / kg) through food. During the experimental period, the tumor size was measured using a digital caliper and presented in a graph.

Claims

1. A method for specifically degrading a G12V mutant protein of KRAS by treating a fusion of a RAS binder that recognizes kRAS and a HECT (homology to E6-AP carboxyl terminus) domain of NEDD4L (Neural precursor cell expressed developmentally down-regulated 4-like) with a portion that recognizes a substrate removed to a G12V mutant protein of KRAS.

2. A method for specifically degrading a G12V mutant protein of KRAS, characterized in that the RAS binder recognizing the kRAS in the first paragraph is one RAS binder selected from the group consisting of iDAb, 12VC1, and K55.

3. A method for specifically degrading a G12V mutant protein of KRAS, characterized in that the fusion product of the RAS binder that recognizes kRAS and the HECT domain of NEDD4L with the substrate-recognizing portion removed in the first paragraph is one of SEQ ID NOs. 1 to 3. A composition for degrading a G12V mutant protein of KRAS, comprising as an active ingredient a fusion of a RAS binder that recognizes 4.kRAS and a HECT (homology to E6-AP carboxyl terminus) domain of NEDD4L (Neural precursor cell expressed developmentally down-regulated 4-like) with a portion that recognizes a substrate removed.

5. A composition for degrading a G12V mutant protein of KRAS, characterized in that the RAS binder recognizing the kRAS in the fourth paragraph is one RAS binder selected from the group consisting of iDAb, 12VC1, and K55.

6. A composition for degrading a G12V mutant protein of KRAS, characterized in that the fusion product of the RAS binder that recognizes kRAS and the HECT domain of NEDD4L with the substrate-recognizing portion removed in the fourth paragraph is one of SEQ ID NOs. 1 to 3.

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