Fusion protein

Abstract

The present invention addresses the problem of providing a substance having a stronger Ras inhibitory effect and / or a stronger anticancer effect. The problem is solved by a fusion protein comprising a Ras-cleaving / modifying enzyme domain and a Ras-binding domain.

Description

Fusion proteins

[0001] The present invention relates to fusion proteins and the like.

[0002] Ras, a protein produced by an oncogene, is known to be activated and become mutated in many cancers, including pancreatic and colon cancers. This mutated Ras activates downstream signaling pathways, the MEK / ERK signaling pathway and the AKT / mTOR signaling pathway, promoting the development, maintenance, and proliferation of cancer. For this reason, a worldwide search is underway for substances that target mutated Ras and inhibit its function as a method for developing cancer therapeutics.

[0003] In recent years, small molecule inhibitors targeting K-Ras G12C, a type of mutant Ras, have been developed and have demonstrated anti-cancer activity in clinical trials. These small molecule inhibitors inhibit Ras function by forming covalent bonds with the mutated cysteine ​​residue present in K-Ras G12C. However, K-Ras G12C accounts for only about 15% of all mutant Ras, and developing inhibitors for other mutant Ras has been extremely difficult.

[0004] Non-Patent Document 1 reports that administration of the Ras-cleaving enzyme RRSP (Ras / Rap1A-specific protease) to mice transplanted with human cancer cells resulted in tumor regression. However, this report used diphtheria toxin (which has the property of penetrating only cells expressing the human HGF receptor) for intracellular delivery, resulting in the specific delivery of RRSP to human cancer cells. This experimental system, therefore, does not allow for evaluation of toxicity to normal mouse tissues, making it difficult to extrapolate to humans, particularly in terms of safety. Therefore, this technique cannot be directly applied to humans.

[0005] In addition to RRSP, various enzymes that inactivate Ras by cleaving or modifying it have been reported (Non-Patent Documents 2 and 3). However, when Ras cleaving / modifying enzymes are used alone, their Ras inhibitory and / or anticancer effects are limited.

[0006] Vidimar, Vania, et al. "Proteolytic pan-RAS cleavage leads to tumor regression in patient-derived pancreatic cancer xenografts." Molecular cancer therapeutics 21.5 (2022): 810-820.HENRIKSSON, ML, et al. (2000). "Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras." Biochemical Journal 347(1): 217-222Schorch, B., et al. (2018). "Targeting oncogenic Ras by the Clostridium perfringens toxin TpeL." Oncotarget 9(23): 16489

[0007] An object of the present invention is to provide a substance with a higher Ras inhibitory effect and / or anti-cancer effect.

[0008] In view of the above problems, the present inventors have conducted extensive research and found that a fusion protein containing a Ras cleavage / modification enzyme domain and a Ras binding domain can solve the above problems. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0009] Item 1. A fusion protein comprising a Ras cleavage / modification enzyme domain and a Ras binding domain.

[0010] Item 2. The fusion protein according to Item 1, wherein the Ras cleavage / modification enzyme is an enzyme that cleaves or modifies a Ras switch region.

[0011] Item 3. The fusion protein according to Item 1 or 2, wherein the Ras cleavage / modification enzyme domain is a domain comprising an amino acid sequence A1 set forth in any one of SEQ ID NOs: 1 to 3, or an amino acid sequence A2 having 70% or more identity to the amino acid sequence A1.

[0012] Item 4. The fusion protein according to any one of Items 1 to 3, wherein the Ras-binding domain comprises a Ras-binding domain of a natural Ras-binding protein, an artificial antibody against Ras, or an antibody against Ras.

[0013] Item 5. The fusion protein according to any one of Items 1 to 4, wherein the Ras-binding domain is a domain comprising an amino acid sequence B1 set forth in any one of SEQ ID NOs: 4 to 14, or an amino acid sequence B2 having 70% or more identity to the amino acid sequence B1.

[0014] Item 6. The fusion protein according to any one of Items 1 to 5, further comprising a cell membrane-penetrating peptide region.

[0015] Item 7. The fusion protein according to Item 6, wherein the cell membrane-penetrating peptide region is a region comprising an amino acid sequence C1 set forth in any one of SEQ ID NOs: 15 to 29, or an amino acid sequence C2 in which one or more amino acids have been mutated relative to the amino acid sequence C1.

[0016] Item 8. The fusion protein according to any one of Items 1 to 7, wherein a linker is contained between the Ras cleavage / modification enzyme domain and the Ras binding domain, between the Ras cleavage / modification enzyme domain and the cell membrane-permeable peptide region, or between the Ras binding domain and the cell membrane-permeable peptide region.

[0017] Item 9. The fusion protein according to any one of Items 1 to 8, wherein the Ras cleavage / modification enzyme domain and the Ras binding domain are arranged in this order from the N-terminus.

[0018] Item 10. The fusion protein according to any one of Items 1 to 9, wherein the Ras cleavage / modification enzyme domain is located at the N-terminus.

[0019] Item 11. A polynucleotide comprising a coding sequence for the fusion protein according to any one of Items 1 to 10.

[0020] Item 12. The polynucleotide according to Item 11, which is an expression plasmid or mRNA.

[0021] Item 13. A Ras inhibitor comprising at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein.

[0022] Item 13A: A method for inhibiting Ras, comprising administering to a subject at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein.

[0023] Item 13B: At least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein, for use as a Ras inhibitor.

[0024] Item 13C: Use of at least one selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein, for the production of a Ras inhibitor.

[0025] Item 13D: Use of at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein, for inhibiting Ras.

[0026] Item 14. An anticancer agent comprising at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein.

[0027] Item 14A: A method for preventing or treating cancer, comprising administering to a subject at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein.

[0028] Item 14B: At least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein, for use as an anticancer agent.

[0029] Item 14C: Use of at least one selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein, for the production of an anticancer agent.

[0030] Item 14D: Use of at least one member selected from the group consisting of the fusion protein according to any one of Items 1 to 10 and a polynucleotide comprising a coding sequence for the fusion protein for the prevention or treatment of cancer.

[0031] According to the present invention, a substance having a higher Ras inhibitory effect and / or anticancer effect can be provided.

[0032] 1 shows the results of Western blot for Test Example 1. The upper part of the photograph shows the protein encoded by the introduced vector. Ctrl indicates an empty vector. The left side of the photograph shows the target of the primary antibody. 1 shows the results of Western blot for Test Example 1. The upper part of the photograph shows the protein encoded by the introduced vector. Ctrl indicates an empty vector. The right side of the photograph shows the target of the primary antibody. 1 shows the results of a BRET test for Test Example 2. The horizontal axis shows the protein encoded by the introduced vector. 1 shows the results of the in vivo antitumor assay for Test Example 5, where mice transplanted with Colon-26 cells were used. Graph A shows the change in tumor volume over time, and Graph B shows the tumor volume 16 days after cancer cell transplantation. The right side of graph A and the horizontal axis of graph B show the administered fusion protein (Test Example 3) (Vehicle is a solvent control). The arrow above graph A indicates the day of administration of the fusion protein or solvent control. 1 shows the results of the in vivo antitumor assay for Test Example 5, where mice transplanted with cancer cells (shown above each graph) were used. The vertical line above each graph indicates the day of administration of the fusion protein (RRSP-v1-TAT) or the vehicle control. The black curve (the curve located on the upper side at the time point on the far right of the horizontal axis) represents the vehicle control administration group, and the red curve (the curve located on the lower side at the time point on the far right of the horizontal axis) represents the fusion protein (RRSP-v1-TAT) administration group.

[0033] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0034] As used herein, "identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0035] As used herein, amino acid mutation specifically refers to amino acid deletion, substitution, insertion, or addition.

[0036] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0037] As used herein, polynucleotides such as DNA and RNA may be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also preferably used are BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide.

[0038] As used herein, the term "coding sequence" refers to a base sequence that codes for the amino acid sequence of a protein, and is not particularly limited thereto.

[0039] In this specification, the position of an amino acid in an amino acid sequence may be indicated by the single-letter amino acid code plus the amino acid number counted from the N-terminal amino acid. For example, "R20" indicates arginine, the 20th amino acid from the N-terminus. For example, "X20" indicates the 20th amino acid from the N-terminus (X is any amino acid). When an amino acid mutation is indicated, the mutated amino acid is indicated to the right of the amino acid number. For example, "R20E" indicates that arginine, the 20th amino acid from the N-terminus, has been mutated to glutamic acid.

[0040] 2. Fusion Protein In one aspect, the present invention relates to a fusion protein (sometimes referred to herein as the "fusion protein of the present invention") comprising a Ras cleavage / modification enzyme domain and a Ras binding domain, which is described below.

[0041] Ras is a small GTP-binding protein involved in cell proliferation and other phenomena. Examples of Ras include K-Ras, N-Ras, and H-Ras. Ras contains two regions (switch regions: switch I and switch II) that are important for binding and recognizing various target proteins, including Raf.

[0042] The Ras targeted by the present invention includes not only wild-type Ras but also mutant Ras. Examples of mutant Ras include cancer mutant Ras (mutant Ras that activates cell proliferation signals). More specifically, in the case of human K-Ras, examples include G12S, G12R, G12C, G12D, G12A, G12V, G13D, A59T, Q61L, Q61R, Q61H, Q61H, K117N, K117N, A146T, A146V, etc., and in the case of N-Ras, examples include G12S, G12R, G12C, G12D, G12A, G12V, G13R, G13D, G13V, A59T, Q61K, Q61R, Q61L, Q61H, Q61H, K117N, K117N, A146T, etc.

[0043] The species of origin of Ras targeted by the present invention is not particularly limited and includes various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, deer, etc. The species of origin of the cells to be contacted with the fusion protein of the present invention is preferably the same as that of the cells, but may be heterologous (e.g., heterologous within a mammal).

[0044] The amino acid sequences of Ras derived from various species are known. For example, human K-Ras is a protein consisting of the amino acid sequence shown in SEQ ID NO: 48 (K-Ras4A) or SEQ ID NO: 49 (K-Ras4B), human N-Ras is a protein consisting of the amino acid sequence shown in SEQ ID NO: 50, and human H-Ras is a protein consisting of the amino acid sequence shown in SEQ ID NO: 51.

[0045] The switch region I is the region from the 30th to the 41st amino acids in human Ras.

[0046] The switch region II is the region from the 60th to the 76th amino acids in human Ras.

[0047] The switch regions in other Ras correspond to the above-mentioned human switch regions, i.e., regions that correspond to the switch regions in human wild-type Ras when the amino acid sequences of the other Ras are compared (e.g., by BLAST using default parameters).

[0048] The Ras targeted by the present invention may have a mutation that can occur naturally in an organism.

[0049] When Ras is a mutant Ras, there are no particular limitations as long as it has the ability to activate cell proliferation. The presence or absence of the ability to activate cell proliferation can be determined, for example, by examining whether cell proliferation is enhanced by overexpressing the mutant Ras in cells.

[0050] Preferred examples of Ras targeted by the present invention include proteins consisting of the amino acid sequence X1 set forth in any one of SEQ ID NOS: 48 to 51, or the amino acid sequence X2 having 70% or more (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) identity to the amino acid sequence X1. The amino acid sequence X2 can be an amino acid sequence obtained by mutating one or more amino acids (for example, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1) from the amino acid sequence X1.

[0051] The Ras cleavage / modification enzyme domain is a domain consisting of a Ras cleavage / modification enzyme. The Ras cleavage / modification enzyme is an enzyme that can attenuate the cell proliferation activation ability of Ras by cleaving Ras and modifying amino acid residues of Ras, and is not particularly limited thereto. Cleavage is typically cleavage by degradation of peptide bonds. Examples of modifications of amino acid residues include ADP-ribosylation, glycosylation, deamidation, etc., and in one embodiment of the present invention, the modification is preferably ADP-ribosylation.

[0052] The Ras cleavage / modification enzyme is preferably a cleavage enzyme from the viewpoint of more effectively attenuating the cell proliferation activation ability of Ras, and from the same viewpoint, the Ras cleavage / modification enzyme is preferably an enzyme that cleaves or modifies the Ras switch region (particularly switch region I).

[0053] Various Ras cleavage / modification enzymes are known, for example, as bacterial toxins. Because Ras is a factor involved in cell proliferation, bacterial evolution has evolved toxins that inhibit this factor in order to attack the host. Various bacterial toxin cleavage / modification enzymes for small G proteins such as Ras have been reported, for example, in Reference 1 (Ras Superfamily Small G Proteins: Biology and Mechanisms 1: General Features, Signaling, Alfred Wittinghofer Editor, 2014 / 10 / 2) and Reference 2 (Nat Commun. 2015; 6: 7396. Published online 2015 Jun 8. doi: 10.1038 / ncomms8396). Specific examples of bacterial Ras cleavage / modification enzymes include RRSP (SEQ ID NO: 1), TpeL (SEQ ID NO: 2), and ExoS (SEQ ID NO: 3). Among these, RRSP and ExoS are preferred, with RRSP being particularly preferred, from the perspective of the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention.

[0054] The Ras cleavage / modification enzyme may be mutated as long as it retains the Ras cleavage / modification ability. The presence or absence of Ras cleavage / modification ability can be determined by expressing the fusion protein of the present invention using the mutant in cells and examining whether the amount of Ras cleavage / modification product increases, whether the amount of Ras detected decreases, or whether the Ras downstream signal is attenuated. Specifically, this can be determined, for example, according to the method described in Test Example 1 below.

[0055] For example, in the case of RRSP, its domain structure has been analyzed, and based on information from previous reports (e.g., J. Biol. Chem. (2018) 293(47) 18110-18122), it is possible to identify mutations that do not impair Ras cleavage / modification ability.

[0056] Preferred examples of the Ras cleavage / modification enzyme domain include a domain comprising the amino acid sequence A1 set forth in any one of SEQ ID NOS: 1 to 3, or an amino acid sequence A2 having 70% or more identity (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) to the amino acid sequence A1. The amino acid sequence A2 can be an amino acid sequence obtained by mutating one or more amino acids (e.g., 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1) from the amino acid sequence A1.

[0057] Fusion proteins of the invention can contain one or more (e.g., 1-3, 1-2, or 1) Ras cleavage / modification enzyme domains. Preferably, fusion proteins of the invention contain one Ras cleavage / modification enzyme domain.

[0058] The Ras-binding domain is not particularly limited as long as it has binding ability to Ras. With regard to binding ability, the Ras-binding domain itself has a dissociation constant for binding to at least one type of Ras of, for example, 2 μM or less, preferably 1 μM or less, more preferably 500 nM or less, even more preferably 200 nM or less, even more preferably 100 nM or less, particularly preferably 50 nM or less, especially more preferably 20 nM or less, and particularly preferably 10 nM or less.

[0059] The number of amino acid residues in the Ras-binding domain is not particularly limited, but from the viewpoints of cell membrane permeability, binding to Ras, etc., it is, for example, 30 to 200, preferably 50 to 150, more preferably 60 to 120, and particularly preferably 60 to 100.

[0060] In one embodiment of the present invention, the Ras binding domain preferably satisfies either one or both of the following two conditions: 1) It has a three-dimensional structure similar to that of ubiquitin, i.e., five β-sheet structures and two α-helices or three 101) Having a helix structure. It is known that adding a cell membrane-penetrating peptide to ubiquitin allows it to penetrate cancer cell membranes (Inomata, K., et al. (2009). "High-resolution multi-dimensional NMR spectroscopy of proteins in human cells." Nature 458(7234): 106.). Based on this fact, it is likely that peptide chains with a structure similar to ubiquitin will be able to penetrate cell membranes by adding a cell membrane-penetrating peptide. Ubiquitin itself does not bind to Ras. 2) Having the ability to bind to the β2 peripheral region of Ras. This region is essential for Ras to activate downstream signals such as MEK / ERK and AKT / mTOR. Therefore, peptide chains that bind to this region are more likely to exert competitive inhibitory effects by binding to Ras.

[0061] More specifically, the Ras-binding domain includes, for example, a natural Ras-binding protein, an artificial antibody against Ras, or a Ras-binding domain possessed by an antibody against Ras.

[0062] Natural Ras-binding proteins are Ras-binding proteins encoded by the cellular genome. Various reports have been published on Ras interaction analysis, and information on natural Ras-binding proteins can be obtained based on these reports. Examples of natural Ras-binding proteins include cRaf, RalGDS, Afadin, PLCε1, RASSF5, and RIN1. These natural Ras-binding proteins can be used as Ras-binding domains as they are, or deleted regions that do not impair Ras-binding ability can be used as Ras-binding domains. Ras-binding domains derived from natural Ras-binding proteins may contain mutations as long as they retain Ras-binding ability. The presence or absence of Ras-binding ability can be measured and determined, for example, by the BRET method according to Test Example 2. Mutations can also be used to improve binding ability.

[0063] Ras-binding domains obtained by deleting or mutating natural Ras-binding proteins are known, and examples thereof include domains consisting of the amino acid sequences shown in any of SEQ ID NOS: 4 to 11.

[0064] Antibodies to Ras include not only immunoglobulins but also fragments thereof that have Ras-binding activity, such as IgG (e.g., IgG1, IgG2, IgG3, and IgG4), IgA (e.g., IgA1 and IgA2), IgD, IgE, IgM, Fab, F(ab')2, minibodies, scFv-Fc, Fv, scFv, diabodies, triabodies, and tetrabodies.

[0065] Antibodies against Ras can be obtained by immunizing an animal (e.g., a mammal such as a mouse, rabbit, or camel) with Ras or a peptide thereof (e.g., 8 or more amino acid residues, 12 or more amino acid residues, or 15 or more amino acid residues) according to or in accordance with known methods, and then obtaining the antibodies from the serum of the resulting animal (polyclonal antibodies), or from antibody-producing cells of the resulting animal (monoclonal antibodies).

[0066] An artificial antibody is a molecule having a variable region capable of recognizing an antigen and a scaffold region that displays the variable region on its surface, and is a substance / molecule that can be used to screen for those that have binding affinity to a target antigen, and is not particularly limited insofar as such. Examples of artificial antibodies include monobodies, DARPins, affibodies, anticalins, FN3, and aPP (avian pancreatic polypeptide)-based miniproteins.

[0067] Artificial antibodies against Ras are known, and examples thereof include proteins consisting of the amino acid sequence shown in any one of SEQ ID NOS: 12 to 14.

[0068] Specific preferred examples of the Ras-binding domain include a domain comprising the amino acid sequence B1 set forth in any one of SEQ ID NOS: 4 to 14 (among these, from the viewpoint of the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention, preferably SEQ ID NOS: 10, 14, and 11, more preferably SEQ ID NOS: 10 and 14, and particularly preferably SEQ ID NOS: 10), or an amino acid sequence B2 having 70% or more identity (preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and still more preferably 99% or more) to the amino acid sequence B1. The amino acid sequence B2 can be an amino acid sequence obtained by mutating one or more amino acids (e.g., 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1) from the amino acid sequence B1.

[0069] Fusion proteins of the invention can contain one or more (e.g., 1-3, 1-2, or 1) Ras-binding domains. Preferably, fusion proteins of the invention contain one Ras-binding domain.

[0070] The fusion protein of the present invention preferably includes a region (core region A) in which the Ras cleavage / modification enzyme domain and the Ras binding domain are linked via another sequence (preferably a linker) (i.e., the Ras cleavage / modification enzyme domain, the other sequence, and the Ras binding domain are directly linked in this order), or a region (core region B) in which the Ras cleavage / modification enzyme domain and the Ras binding domain are directly linked (collectively referred to as the "core region"). This further enhances the Ras inhibitory and / or anticancer activity of the fusion protein of the present invention. From the viewpoint of said activity, the core region is preferably core region A. From the viewpoint of said activity, it is preferable that the Ras cleavage / modification enzyme domain be located on the N-terminus of the core region. From the viewpoint of said activity, it is also preferable that the Ras cleavage / modification enzyme domain be located on the N-terminus of the fusion protein of the present invention (i.e., no other sequence is present on the N-terminus of the Ras cleavage / modification enzyme domain).

[0071] The linker is not particularly limited as long as it is a flexibly movable linker. Examples of linkers include peptide linkers such as a linker composed of glycine and serine (GS linker), an α-helix linker (e.g., EAAAK (SEQ ID NO: 52) or a repeat sequence thereof), and a protein tag (e.g., HA tag, FLAG tag, His tag, etc.) linker. Examples of structural units of a GS linker include GGGGS (SEQ ID NO: 53), GSG, SAGG (SEQ ID NO: 54), GGGS (SEQ ID NO: 55), and GGSG (SEQ ID NO: 56). The linker may be a single type or a combination of two or more types. The number of amino acid residues in the peptide linker is, for example, 1 to 100, preferably 2 to 50, more preferably 3 to 30, even more preferably 5 to 25, even more preferably 8 to 20, and particularly preferably 10 to 20.

[0072] The fusion protein of the present invention preferably further comprises a cell membrane-penetrating peptide region, which can further enhance the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention.

[0073] The cell membrane-permeable peptide region is a region consisting of the amino acid sequence of a cell membrane-permeable peptide. The cell membrane-permeable peptide is a peptide that has cell membrane permeability, and is not particularly limited insofar as this is the case. Specifically, it refers to any peptide that, when administered in the presence of a desired drug, can improve the permeability of the drug into cells (e.g., increase the amount of permeation) compared to when administered in the absence of the cell membrane-permeable peptide.

[0074] Specific examples of cell membrane-permeable peptides include TAT, Pen, ZF5.3, GET, MAP, M918, PenArg, S19TAT, PTD4, TP10, CPP2, 10HPen, R8, CPP44, CPP12, etc. These cell membrane-permeable peptides may be mutated as long as they have cell membrane permeability.

[0075] A preferred example of the cell membrane-permeable peptide region is a region comprising the amino acid sequence C1 shown in any of SEQ ID NOS: 15 to 29 (among these, SEQ ID NOS: 15 is preferred in terms of the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention), or the amino acid sequence C2 obtained by mutating one or more amino acids (e.g., 1 to 5, 1 to 3, 1 to 2, or 1) in the amino acid sequence C1. The amino acid sequence C2 is preferably an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence C1.

[0076] The fusion protein of the present invention can comprise one or more (e.g., 1 to 3, 1 to 2, or 1) cell membrane-penetrating peptide regions. Preferably, the fusion protein of the present invention comprises one cell membrane-penetrating peptide region.

[0077] When the fusion protein of the present invention contains a cell membrane-penetrating peptide domain, it is preferable that the cell membrane-penetrating peptide domain is located at a terminal end, particularly at the C-terminus, which can further enhance the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention.

[0078] When the fusion protein of the present invention contains a cell membrane-penetrating peptide domain, the cell membrane-penetrating peptide domain is preferably linked to another sequence (e.g., a core domain) via a linker. The linker is as described above. This can further enhance the Ras inhibitory activity and / or anticancer activity of the fusion protein of the present invention.

[0079] The number of amino acid residues of the fusion protein of the present invention is, for example, more than 300, preferably 350 or more, more preferably 400 or more, and even more preferably 500 or more, and is, for example, 1000 or less, preferably 800 or less, more preferably 700 or less, and even more preferably 650 or less.

[0080] In a particularly preferred embodiment of the present invention, the fusion protein of the present invention comprises a core region, a linker, and a cell membrane-penetrating peptide region arranged from the N-terminus. In this embodiment, it is preferred that the core region is at the N-terminus and the cell membrane-penetrating peptide region is at the C-terminus.

[0081] The fusion protein of the present invention may be chemically modified as long as the Ras inhibitory activity and / or anticancer activity is not significantly inhibited.

[0082] The fusion protein of the present invention has a C-terminus containing a carboxyl group (-COOH), a carboxylate group (-COO － ), amide (-CONH2) or ester (-COOR).

[0083] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group 7－14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0084] The fusion protein of the present invention may have amidated or esterified carboxyl groups (or carboxylates) other than those at the C-terminus. In this case, the esters used may be, for example, the C-terminal esters described above.

[0085] Furthermore, in the fusion protein of the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1－6 C such as alkanoyl group 1－6 and those protected with an acyl group or the like.

[0086] The fusion protein of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is pharmaceutically acceptable, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0087] The fusion protein of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0088] The fusion protein of the present invention can be easily produced according to its amino acid sequence using known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0089] The fusion protein of the present invention may be purified after synthesis. For example, the fusion protein of the present invention is extracted from bacterial cells collected by centrifugation, filtration, or the like from the culture. The first step of extraction involves disrupting the bacterial cells using enzyme digestion, osmotic disruption, rapid pressure / pressure / vacuum application, ultrasound, various homogenizers, and the like. The disrupted bacterial cells can then be fractionated using physical methods such as low-speed centrifugation, ultracentrifugation, filtration, molecular sieving, and membrane concentration; chemical methods such as the use of precipitants, solubilizers, adsorbents, and dispersants; and physicochemical methods such as electrophoresis, column chromatography, support, dialysis, and salting out, in combination. When applying these methods, physicochemical conditions such as temperature, pressure, pH, and ionic strength can be appropriately adjusted.

[0090] 3. Polynucleotides and Cells In one aspect, the present invention relates to a polynucleotide (sometimes referred to herein as a "polynucleotide of the present invention") comprising a coding sequence for the fusion protein of the present invention, and a cell (sometimes referred to herein as a "cell of the present invention") comprising the polynucleotide of the present invention. These are described below. For matters not explained below, the description in Section 2 above is incorporated by reference.

[0091] The coding sequence of the fusion protein of the present invention is not particularly limited, as long as it is a polynucleotide consisting of a base sequence that encodes the fusion protein of the present invention.

[0092] In one embodiment, the polynucleotide of the present invention comprises an expression cassette for the fusion protein of the present invention.

[0093] The expression cassette for the fusion protein of the present invention is not particularly limited as long as it is a polynucleotide that can express the fusion protein of the present invention in cells. Typical examples of the expression cassette for the fusion protein of the present invention include a promoter and a polynucleotide comprising a coding sequence for the fusion protein of the present invention placed under the control of the promoter.

[0094] The promoter contained in the expression cassette of the fusion protein of the present invention is not particularly limited and can be selected appropriately depending on the target cell. For example, various Pol II promoters can be used. Pol II promoters are not particularly limited, but examples include the CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, and CAG promoter. Other examples of promoters include tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, alcohol (e.g., methanol)-inducible promoter, arabinose-inducible promoter, cold shock promoter, and tetracycline-inducible promoter.

[0095] The polynucleotide of the present invention may contain other elements, as necessary (for example, a multiple cloning site (MCS), a drug resistance gene, an origin of replication, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein (e.g., a fluorescent protein), a drug resistance gene coding sequence, etc.).

[0096] The polynucleotide of the present invention may be in the form of a vector. An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include M13 phage or its variants, λ phage or its variants, and pBR322 or its variants (pB325, pAT153, pUC8, etc.); examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, and pPICZα A; examples of vectors using insect cells as a host include pAc and pVL; and examples of vectors using mammalian cells as a host include pcDNA, pCDM8, and pMT2PC.

[0097] In a preferred embodiment, the polynucleotide of the present invention is an expression plasmid or mRNA, which allows the fusion protein of the present invention to be more efficiently expressed in the target organism.

[0098] The cells of the present invention are not particularly limited as long as they contain the polynucleotide of the present invention. Examples of cells include Escherichia coli such as Escherichia coli K12, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, the Sf cell line derived from Spodoptera frugiperda or the HighFive cell line derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells such as COS7 cells. Preferred animal cells include cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0099] In one embodiment, the cell of the present invention expresses the fusion protein of the present invention.

[0100] In one embodiment, the cell of the present invention can be a cell in which the polynucleotide of the present invention has been integrated into the chromosomal DNA (a stable expression cell line).

[0101] 4. Uses In one aspect, the present invention relates to a pharmaceutical (e.g., pharmaceutical composition), reagent, etc., containing at least one selected from the group consisting of the fusion protein of the present invention and a polynucleotide comprising a coding sequence for the fusion protein (hereinafter, sometimes referred to as an "active ingredient"). More specifically, the active ingredient can be used as a Ras inhibitor, an anticancer agent, etc.

[0102] There are no particular limitations on the cancers to which the present invention can be applied, but specific examples include solid cancers such as lung cancer, pancreatic cancer, esophageal cancer, colorectal cancer, colon cancer, stomach cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer, gallbladder cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, brain tumors, glioma, glioblastoma, hepatoblastoma, glioblastoma multiforme, and peritoneal dissemination; and blood cancers such as leukemia and malignant lymphoma.

[0103] The cancer to which the present invention is applied may be either a Ras wild-type cancer or a Ras mutant cancer, and the type of mutation in the Ras mutant cancer is not particularly limited.

[0104] The target organisms of the active ingredient of the present invention are not particularly limited, and examples include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits.

[0105] The content of the active ingredient in the medicament of the present invention can be appropriately determined taking into consideration the type of target disease, the desired therapeutic effect, the administration method, the treatment period, the age and body weight of the patient, etc. For example, the content of the active ingredient in the medicament of the present invention can be about 0.0001 to 100 parts by weight, assuming that the total amount of the medicament of the present invention is 100 parts by weight.

[0106] The dosage form of the pharmaceutical of the present invention is not particularly limited as long as the desired effect is obtained, and can be administered to mammals, including humans, by either oral administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Parenteral administration is preferred, and intravenous injection is more preferred. Dosage forms for oral and parenteral administration and their preparation methods are well known to those skilled in the art, and can be prepared by standard methods such as mixing the active ingredient with a pharmaceutically acceptable carrier.

[0107] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, ophthalmic preparations, eye ointments, nasal drops, ear drops, liposomes, and nanolipid particles. For example, injectable preparations are prepared by dissolving the active ingredient in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. Pharmaceuticals can also be freeze-dried for immediate preparation.

[0108] The medicament of the present invention may further contain other drugs that are effective in treating or preventing diseases. The medicament of the present invention may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as needed.

[0109] Carriers used in formulating the pharmaceutical of the present invention include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like that are commonly used in the art.

[0110] The dosage of the medicament of the present invention can be determined by a clinician based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether the medicament is administered as part of a combination of other drugs. The dosage of the medicament of the present invention can be, for example, approximately 1 μg / kg (body weight) to 10 g / kg (body weight) per day. The administration schedule of the medicament of the present invention can also be determined taking into account factors similar to those for the dosage. For example, the above-mentioned daily dosage can be administered once every day to once a month.

[0111] The reagent of the present invention may be in the form of a composition containing an active ingredient. The composition may contain other ingredients as needed. Examples of other ingredients include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0112] The reagent of the present invention may be in the form of a kit containing an active ingredient. The kit may also include instruments, reagents, etc. Examples of such instruments and reagents include test tubes, microtiter plates, purification columns, labeled antibodies, and standard samples (positive and negative controls).

[0113] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0114] Reference Example 1. Preparation of Ras cleavage / modification enzyme domain, Ras binding domain, and cell membrane-permeable peptide region The amino acid sequences of specific examples of Ras cleavage / modification enzyme domains are shown in Table 1. RRSP (Ras / Rap1A-specific protease) is an enzyme that cleaves Ras between the 31st and 32nd amino acid sequences, inactivating it. TpeL is an enzyme that glycosylates the 35th tyrosine residue of Ras, inactivating it. ExoS is an enzyme that ADP-ribosylates residues such as the 41st arginine residue of Ras, inactivating it.

[0115]

[0116] Specific examples of Ras binding domains are shown in Table 2.

[0117]

[0118] Specific examples of cell membrane-permeable peptide regions are shown in Table 3.

[0119]

[0120] In the following test examples, the names of the domains / areas are shown as the names listed in Tables 1 to 3 or their abbreviations.

[0121] Test Example 1. Intracellular expression of a fusion protein of a Ras cleavage / modification enzyme domain and a Ras binding domain 1. A fusion protein of a Ras cleavage / modification enzyme domain and a Ras binding domain was forced to be expressed in cells to examine whether its Ras inhibitory activity was improved. Specifically, this was done as follows.

[0122] DNA encoding the following five proteins was outsourced to Eurofins Genomics or Integrated DNA Technologies for synthesis, and inserted into the pcDNA3.1 vector (Thermofisher) with a 2xHA tag added to the C-terminus.

[0123] For the assay, PANC-1 cells were seeded in 24-well plates containing EMEM medium supplemented with 10% FBS, allowed to adhere overnight, and transfected with plasmid vectors using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific #L3000001). After incubation for 1–6 days, cells were washed once with chilled PBS, lysed with 1% SDS, and sonicated using an ultrasonic processor. Protein concentrations of cell lysates were measured using the DC protein assay (Biorad #5000112JA). After uniform protein concentrations across all samples, lysates were added to 5x sample buffer [0.25 M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 0.5 M DTT, and 50% glycerol] and boiled for 5 minutes. Proteins were separated using 12.5 or 15% SuperCep Acylamide gels (Wako #196-14981 or #190-15001) and transferred to Immobileon-P membranes (Millipore #IPVH00010). The membranes were blocked with PVDF Blocking reagent (TOYOBO #NYPBR01), probed with primary antibodies, and reacted with secondary antibodies. Proteins were stained with Luminata Forte Western HRP substrate (Millipore #WBLUF0100) and detected using an ImageQuant LAS4000.

[0124] The results are shown in Figures 1 and 2. It was shown that the fusion of the RAS cleavage / modification enzyme domain with the Ras binding domain strongly enhanced the cleavage / modification of RAS and the suppression of downstream signals of RAS (p-ERK: ERK phosphorylation) (see boxes in Figures 1 and 2). It is known that ExoS has the ability to modify RAS, which prevents the binding of anti-RAS antibodies in immunoblots, resulting in the apparent faintness of the RAS band.

[0125] Test Example 2. Intracellular expression of a fusion protein of a Ras cleavage / modification enzyme domain and a Ras binding domain. We investigated whether the fusion of a Ras cleavage / modification enzyme domain with a Ras binding domain improves binding to Ras. Specifically, we performed the following procedure.

[0126] DNA encoding KRAS-G12V was inserted into the pFN31K Nluc vector (Promega). DNA encoding the following 10 proteins was inserted into the pFN21A Halotag Vector (Promega): RRSP, RRSP-v1, ExoS, ExoS-v1, TpeL (SEQ ID NO: 2), TpeL-v1 (a fusion protein in which v1 is located at the C-terminus of TpeL via a linker (GSSGGGGSGGGGSSMG: SEQ ID NO: 30)), cRaf (SEQ ID NO: 4), v1, RRSP* (SEQ ID NO: 47: an RRSP mutant with inactivated RAS cleavage activity), or RRSP*-v1.

[0127] For the assay, MiaPaCa-2 cells were seeded in 24-well plates containing EMEM medium supplemented with 10% FBS, allowed to adhere overnight, and transfected with plasmid vectors using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific #L3000001). After overnight incubation, cells were detached by trypsinization, labeled with HaloTag® NanoBRET™ 618 ligand (Promega #980A) as needed, and replated into 96-well CELLSTAR plates (Greiner #655083). After overnight incubation, the medium was replaced with Opti-MEM medium supplemented with 4% FBS, incubated for 1 hour, and then NanoBRET™ Nano-Glo® substrate (Promega, #N157A) was added. Emission at 480 and 620 nm was immediately measured by a SpectraMax iD5 (Molecular Devices), and the BRET ratio was calculated according to the manufacturer's protocol.

[0128] The results are shown in Figure 3. A higher BRET ratio indicates a stronger interaction. The fusion of the RAS cleavage / modification enzyme domain with the Ras-binding domain demonstrated improved binding to Ras. Similar results were also obtained when TpeL was used as the Ras cleavage / modification enzyme domain.

[0129] Test Example 3. Preparation of a fusion protein of a Ras cleavage / modification enzyme domain, a Ras binding domain, and an intracellular penetrating peptide domain A fusion protein of a Ras cleavage / modification enzyme domain, a Ras binding domain, and an intracellular penetrating peptide domain was prepared. The domain structure of the fusion protein prepared in this test example is shown in Table 4. In Table 4, Domain 1 is at the N-terminus, and the sequences are Linker 1, Domain 2, Linker 2, Domain 3, Linker 3, and Domain 4. In Table 4, "-" indicates that the domain / linker is not present.

[0130]

[0131] Each domain / region was inserted into the pRSET A vector (Thermo Fisher Scientific #V35120), which encodes various cell-penetrating peptides established in a previous study (Cell Chemical Biology, 28 (2021) 1581-1589. e1586.), to prepare plasmids encoding the fusion proteins listed in Table 4. Plasmids were transformed into ECOS Competent E. coli BL21 using the manufacturer's protocol (NIPPON GENE #314-06533). The next day, a single colony was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and pre-cultured at 37°C with shaking at 180-240 rpm until mid-logarithmic phase. The pre-cultured E. coli was then inoculated into 1.25 L of LB medium containing 50 μg / mL ampicillin and pre-cultured until mid-logarithmic phase with shaking under the same conditions. Protein expression was then induced with 0.1 mM IPTG and shaking at 16°C for 24 hours. The E. coli cells were harvested by centrifugation at 7,000 g for 12 minutes, resuspended in buffer A (50 mM Tris-HCl, pH 8, 300 mM NaCl), and sonicated on ice using a VP-300N ultrasonic processor (TAITEC, #0075955-). The disrupted E. coli cells were centrifuged twice (at 3,430 g for 25 minutes and at 30,000 g for 25 minutes), and the supernatant fraction was collected.

[0132] The supernatant fraction was purified using immobilized metal affinity chromatography (IMAC) using standard procedures. Briefly, Ni Sepharose 6 Fast Flow resin (Cytiva) was loaded onto a column, washed with deionized water, and then charged with 0.1 M NiSO4. The resin was washed again with deionized water, and the supernatant fraction was loaded onto the column and rotated at 4 °C for 30 min. The resin was washed three times with buffer A supplemented with 20 mM imidazole and 1% Triton X-114, three times with buffer A supplemented with 20 mM imidazole and 1.7 M NaCl, and twice with buffer A supplemented with 20 mM imidazole. Finally, the protein was eluted by applying buffer A supplemented with 500 mM imidazole.

[0133] Example 4: In Vitro Cell Viability Assay. PANC-1 cells were resuspended in RPMI 1640 medium containing 10% FBS and seeded into a Nunclon Delta-treated 96-well plate (Thermo Fisher Scientific #167425). The next day, the cells were treated with medium containing various concentrations of the fusion protein (Example 3). After 6 days of incubation, cell viability was measured using Cell Counting Kit-8 (Wako #343-07623) according to the manufacturer's protocol. The IC50 was calculated by fitting the dose-response curve to a sigmoidal function.

[0134] The results are shown in Table 4. The fusion protein of the Ras cleavage / modification enzyme domain, the Ras-binding domain, and the cell-penetrating peptide domain showed a lower IC50 than when a domain that does not bind to Ras was used instead of the Ras-binding domain, or when the Ras-binding domain was omitted. Similar results were also obtained when ExoS was used as the Ras cleavage / modification enzyme domain.

[0135] Test Example 5: In Vivo Antitumor Assay. Colon-26, CT-26, MC-38, MB49, 4T1, or B16F1 cancer cells were implanted into the right flank of 6- to 8-week-old female BALB / c mice. After tumor establishment, mice were randomly assigned (n = 6 per group) and administered vehicle control or fusion protein (Test Example 3) at a dose of 50 mg / kg body weight (per administration) via the tail vein. The administered fusion protein was dissolved in 100 μL of D-PBS(-) at a concentration of 10 mg / mL. Tumor volume was calculated by 0.5 × width × length.

[0136] The results are shown in Figures 4 and 5. It was demonstrated that the compound exerts an anticancer effect against non-G12C mutant Ras and wild-type Ras tumors, which had previously been difficult to demonstrate.

Claims

1. A fusion protein comprising a Ras cleavage / modification enzyme domain and a Ras binding domain.

2. The fusion protein of claim 1, wherein the Ras cleavage / modification enzyme is an enzyme that cleaves or modifies a Ras switch region.

3. The fusion protein described in claim 1, wherein the Ras cleavage / modification enzyme domain is a domain containing the amino acid sequence A1 shown in any one of SEQ ID NOs: 1 to 3, or the amino acid sequence A2 having 70% or more identity to the amino acid sequence A1.

4. The fusion protein of claim 1, wherein the Ras-binding domain comprises a Ras-binding domain carried by a natural Ras-binding protein, an artificial antibody against Ras, or an antibody against Ras.

5. The fusion protein described in claim 1, wherein the Ras binding domain is a domain comprising the amino acid sequence B1 shown in any one of SEQ ID NOs: 4 to 14, or the amino acid sequence B2 having 70% or more identity to the amino acid sequence B1.

6. The fusion protein of claim 1, further comprising a cell membrane-permeable peptide region.

7. The fusion protein according to claim 6, wherein the cell membrane-permeable peptide region is a region comprising the amino acid sequence C1 set forth in any one of SEQ ID NOs: 15 to 29, or the amino acid sequence C2 in which one or more amino acids have been mutated relative to the amino acid sequence C1.

8. The fusion protein of claim 1, comprising a linker between the Ras cleavage / modification enzyme domain and the Ras binding domain, between the Ras cleavage / modification enzyme domain and the cell membrane-permeable peptide region, or between the Ras binding domain and the cell membrane-permeable peptide region.

9. The fusion protein according to claim 1, in which a Ras cleavage / modification enzyme domain and a Ras binding domain are arranged in this order from the N-terminus.

10. The fusion protein of claim 1, wherein the Ras cleavage / modification enzyme domain is located at the N-terminus.

11. A polynucleotide comprising a coding sequence for the fusion protein of any one of claims 1 to 10.

12. The polynucleotide of claim 11, which is an expression plasmid or mRNA.

13. A Ras inhibitor comprising at least one member selected from the group consisting of the fusion protein according to any one of claims 1 to 10 and a polynucleotide comprising a coding sequence for said fusion protein.

14. An anti-cancer agent comprising at least one selected from the group consisting of the fusion protein according to any one of claims 1 to 10 and a polynucleotide comprising a coding sequence for said fusion protein.

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