Cancer targeting peptide, transport carrier, and pharmaceutical composition

Abstract

The present invention provides a peptide that specifically binds to cells constituting cancer, and a pharmaceutical composition containing the peptide. The present invention is a cancer targeting peptide containing a peptide that binds to cancer cells and that comprises one or more amino acid sequences, each identical to a partial region of a human protein selected from the group consisting of extracellular matrix-constituting proteins, transcriptional regulators, nuclear regulators, signaling pathway proteins, and RNA regulatory proteins. The partial region comprises 5-7 amino acid residues.

Description

Cancer targeting peptide, transport carrier, and pharmaceutical composition

[0001] The present invention relates to a cancer-targeting peptide, a transport carrier using the peptide, and a pharmaceutical composition.

[0002] Techniques for targeting specific organs are expected to be applied to, for example, drug delivery systems (DDS) for delivering drugs. For example, transport efficiency can be improved by modifying a transport carrier targeted to a specific tissue with a peptide that specifically binds to cells constituting the tissue. Examples of peptides targeting specific tissues or cells include a neovascularization-specific peptide described in Patent Document 1, a cardiac-specific peptide described in Patent Document 2, and a spinal cord tissue-specific peptide described in Patent Document 3. Furthermore, Patent Document 4 describes a peptide that can penetrate the blood-brain barrier and is used to modify a transport carrier targeted to the brain.

[0003] International Publication No. 2000 / 023476 U.S. Patent No. 6,303,573 Japanese Patent No. 6479331 Japanese Patent No. 7378046

[0004] An object of the present invention is to provide a peptide that specifically binds to cancer cells, and a pharmaceutical composition containing the peptide.

[0005] The present invention includes the following aspects: [1] A cancer targeting peptide comprising a peptide that binds to cancer cells, the peptide consisting of an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues in a human protein, and the human protein being one or more proteins selected from the group consisting of extracellular matrix constituent proteins, transcriptional regulators, nuclear regulators, signal transduction pathway proteins, and RNA regulatory proteins. [2] The cancer targeting peptide of [1] above, wherein the human protein is an extracellular matrix constituent protein and is a protein that constitutes a collagen cluster or a protein involved in fibrin lamin regulation. [3] The cancer targeting peptide of [2] above, wherein the protein that constitutes a collagen cluster is COL4A3, COL4A4, COL6A6, COL4A1, COL6A3, COL13A1, COL16A1, COL5A2, or COL23A1, and the protein involved in fibrin lamin regulation is EMILIN1, LAMA4, ADAMTS2, or FBLN2. [4] The cancer target peptide of [1], wherein the human protein is the transcription factor, a protein involved in transcription regulation, a TBP-related factor, or a transcription mediator. [5] The cancer target peptide of [4], wherein the protein involved in transcription regulation is CCNK, AFF3, or AFF4, the TBP-related factor is TAF1, TAF1L, TAF6, or TAF2, and the transcription mediator is FBXL19, MED12, or MED13L. [6] The cancer target peptide of [1], wherein the human protein is the nuclear regulatory factor, a protein involved in repair regulation, or a protein involved in histone modification. [7] The cancer target peptide according to [6], wherein the protein involved in repair control is SMARCAL1, ERCC1, MSH6, E2F7, ATRX, CHAF1B, TICRR, TCHP, or TOP2A, and the protein involved in histone modification is KMT2C, MTA1, MTA2, SETD7, UBE2A, UBE2B, MPND, MYSM1, BTAF1, USP22, or USP27X.[8] The cancer targeting peptide of [1], wherein the human protein is the signal transduction pathway protein, and is a protein involved in complement / thrombosis formation, a protein involved in signal response, or a protein involved in fatty acid regulation. [9] The cancer targeting peptide of [8], wherein the protein involved in complement / thrombosis formation is C3, F5, APOH, or FGB, the protein involved in signal response is ATG7, PIK3C3, INPP4A, PIK3CD, PLCG2, ANGPT1, ANGPT2, ENG, LEP, JAK2, CD4, PXDN, IFIT1, AZI2, or TLR1, and the protein involved in fatty acid regulation is CYP2E1, CYP2J2, PLIN2, PDK4, or ACACA.

[10] The cancer targeting peptide of [1], wherein the human protein is the RNA regulatory protein, and is a protein involved in ribosomes.

[11] The cancer targeting peptide of

[10] above, wherein the ribosome-related protein is TARDBP, MATR3-2, DHX38, CHERP, NUP107, EIF3B, or DDX42.

[12] The cancer targeting peptide of [1] above, wherein the peptide that binds to cancer cells consists of any of the amino acid sequences of SEQ ID NOs: 1 to 59.

[13] The cancer targeting peptide of any of [1] to

[12] above, which targets one or more cancers selected from the group consisting of pancreatic cancer and liver cancer.

[14] A transport carrier for cancer-specific transport, comprising the cancer targeting peptide of any of [1] to

[13] above.

[15] A pharmaceutical composition comprising the cancer targeting peptide of any of [1] to

[13] above.

[16] The pharmaceutical composition of

[15] above, which is used for cancer treatment.

[0006] According to the present invention, it is possible to provide a cancer targeting peptide having excellent selectivity for cancer, and a pharmaceutical composition containing the peptide.

[0007] 1 is a diagram in which the number of pancreatic cancer reads is plotted on the vertical axis and the number of liver cancer reads is plotted on the horizontal axis for 883 peptides that bind to both liver cancer and pancreatic cancer in Example 1.

[0008] Hereinafter, embodiments of the present invention will be specifically described.

[0009] In the present invention and the present specification, "X １ ~X ２ (X １ and X ２ is X １ <X ２ "X" is a real number that satisfies １ More than X ２ means "below."

[0010] In the present invention and this specification, the term "polypeptide" refers to a polymer of 25 or more amino acids bonded together by peptide bonds, and the term "peptide" refers to a polymer of 2 to 24 amino acids bonded together by peptide bonds.

[0011] In the present invention and this specification, "targeting (specific cells or tissues)" means targeting specific cells or tissues as the binding target. For example, a "(specific cell or tissue) targeting peptide" means a peptide that binds to a specific cell or tissue. A "(specific cell or tissue) targeted transport carrier" means a transport carrier for transporting a substance to a specific cell or tissue.

[0012] In the present invention and this specification, the term "specifically bind (to a specific cell or tissue)" means that the affinity (strength of binding) for a specific cell or tissue is higher than the affinity for the majority of other cells or tissues.

[0013] In the present invention and this specification, a "peptide consisting of the amino acid sequence of SEQ ID NO: n (n is a natural number)" may be referred to as "peptide n."

[0014] <Cancer Targeting Peptide> The cancer targeting peptide of this embodiment binds to cancer cells and comprises a peptide consisting of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues in a human protein. Hereinafter, the "peptide that binds to at least one type of cancer cell" may be referred to as a "cancer-specific binding peptide." Furthermore, the region of the "peptide that binds to at least one type of cancer cell" in the cancer targeting peptide of this embodiment may be referred to as a "cancer-specific binding peptide portion." In other words, the cancer targeting peptide of this embodiment comprises at least one cancer-specific binding peptide.

[0015] The cancer-targeting peptide of this embodiment contains a cancer-specific binding peptide portion, which allows it to specifically bind to cancer cells and tissues containing cancer cells, thereby functioning as a cancer-targeting peptide. Furthermore, the cancer-specific binding peptide portion has the same amino acid sequence as a partial region of a human protein, and therefore has low immunogenicity. Therefore, the cancer-targeting peptide of this embodiment is expected to be relatively safe for administration to humans.

[0016] The cancer-specific binding peptide portion in the cancer-targeting peptide of this embodiment has the same amino acid sequence as a partial region of one or more proteins selected from the group consisting of extracellular matrix constituent proteins, transcriptional regulators, nuclear regulatory factors, signal transduction pathway proteins, and RNA regulatory proteins. Extracellular matrix constituent proteins, transcriptional regulators, nuclear regulatory factors, signal transduction pathway proteins, and RNA regulatory proteins are all proteins involved in cell proliferation and carcinogenesis, and the cancer-targeting peptide of this embodiment is capable of binding to these proteins at the cancer-specific binding peptide portion, suggesting that it has high affinity for cancer cells.

[0017] The cancer-specific binding peptide portion of the cancer-targeting peptide of this embodiment preferably has an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a protein constituting a collagen cluster or a protein involved in fibrinlamin regulation, particularly among extracellular matrix-constituting proteins. Proteins constituting collagen clusters include COL4A3, COL4A4, COL6A6, COL4A1, COL6A3, COL13A1, COL16A1, COL5A2, and COL23A1. Proteins involved in fibrinlamin regulation include EMILIN1, LAMA4, ADAMTS2, and FBLN2. The cancer-specific binding peptide portion of the cancer-targeting peptide of this embodiment preferably has an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a protein involved in fibrinlamin regulation, and more preferably has an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of EMILIN1 or LAMA4.

[0018] Examples of cancer-specific binding peptide moieties in the cancer targeting peptides of this embodiment include the peptides listed in Table 1, with peptide 1 and peptide 2 being particularly preferred due to their higher affinity for cancer cells. In the table, the "human protein" column indicates the name of the human protein that has each amino acid sequence as a partial region and the registration number (STRING_ID) in the protein-protein interaction (PPI) database "STRING." The same applies to the following tables.

[0019]

[0020] The cancer-specific binding peptide portion in the cancer-targeting peptide of this embodiment preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a transcriptional regulatory factor, particularly a protein involved in transcriptional regulation, a TBP-related factor, or a transcription mediator. Proteins involved in transcriptional regulation include CCNK, AFF3, and AFF4. TBP-related factors include TAF1, TAF1L, TAF6, and TAF2. Transcriptional mediators include FBXL19, MED12, and MED13L. The cancer-specific binding peptide portion in the cancer-targeting peptide of this embodiment preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a protein involved in transcriptional regulation or a TBP-related factor, and more preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of CCNK or TAF1.

[0021] Examples of the cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment include the peptides listed in Table 2, and peptide 9 and peptide 10 are particularly preferred because of their higher affinity for cancer cells.

[0022]

[0023] The cancer-specific binding peptide portion of the cancer targeting peptide of this embodiment preferably has an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a protein involved in repair control or a protein involved in histone modification, particularly among nuclear regulatory factors. Proteins involved in repair control include SMARCAL1, ERCC1, MSH6, E2F7, ATRX, CHAF1B, TICRR, TCPH, and TOP2A. Proteins involved in histone modification include KMT2C, MTA1, MTA2, SETD7, UBE2A, UBE2B, MPND, MYSM1, BTAF1, USP22, and USP27X. The cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of SMARCAL1, ATRX, TOP2A, KMT2C, UBE2A, or BTAF1, more preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of SMARCAL1, TOP2A, KMT2C, or BTAF1, and even more preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of KMT2C.

[0024] Examples of the cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment include the peptides listed in Table 3. In particular, peptides 15 to 20 are preferred, peptides 15 to 18 are more preferred, and peptide 15 is particularly preferred, because of their higher affinity for cancer cells.

[0025]

[0026] The cancer-specific binding peptide portion of the cancer targeting peptide of this embodiment preferably has an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of a signal transduction pathway protein, particularly a protein involved in complement / thrombus formation, a protein involved in signal response, or a protein involved in fatty acid regulation. Proteins involved in complement / thrombus formation include C3, F5, APOH, and FGB. Proteins involved in signal response include ATG7, PIK3C3, INPP4A, PIK3CD, PLCG2, ANGPT1, ANGPT2, ENG, LEP, JAK2, CD4, PXDN, IFIT1, AZI2, and TLR1. Proteins involved in fatty acid regulation include CYP2E1, CYP2J2, PLIN2, PDK4, and ACACA. The cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of C3, APOH, PIK3CD, ANGPT2, LEP, or PDK4, more preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of C3, APOH, PIK3CD, ANGPT2, or LEP, even more preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of APOH, PIK3CD, or ANGPT2, and particularly preferably consists of the same amino acid sequence as a partial region consisting of 5 to 7 amino acid residues of ANGPT2.

[0027] Examples of the cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment include the peptides listed in Table 4. In particular, because of their higher affinity for cancer cells, peptides 31 to 36 are preferred, peptides 31 to 35 are more preferred, peptides 31 to 33 are even more preferred, and peptide 31 is particularly preferred.

[0028]

[0029] The cancer-specific binding peptide portion in the cancer-targeting peptide of this embodiment preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of an RNA regulatory protein, particularly a protein involved in ribosomes. Examples of proteins involved in ribosomes include TARDBP, MATR3-2, DHX38, CHERP, NUP107, EIF3B, and DDX42. The cancer-specific binding peptide portion in the cancer-targeting peptide of this embodiment preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of CHERP, NUP107, or EIF3B, more preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of CHERP or NUP107, and even more preferably comprises an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues of CHERP.

[0030] Examples of the cancer-specific binding peptide portion in the cancer targeting peptide of this embodiment include the peptides listed in Table 5. In particular, peptides 53 to 55 are preferred, peptides 53 or 54 are more preferred, and peptide 53 is even more preferred, in terms of their higher affinity for cancer cells.

[0031]

[0032] As shown in Example 1 below, all of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 59 bind to cancer cells but not to normal cells. Here, "peptides that do not bind to normal cells" refer to "peptides that do not bind to tissues in the body when administered intravenously and remain in the serum." The cancer targeting peptide of this embodiment has a peptide portion that binds to cancer cells but not to normal cells, and therefore selectively binds to cancer cells when administered to the body. Therefore, when used as a transport carrier for drugs that target cancer cells, such as anticancer drugs, the effect on normal cells can be kept extremely low.

[0033] The cancer targeted by the cancer targeting peptide of this embodiment is not particularly limited and may be a solid cancer or a hematopoietic tumor. Examples of solid cancers include brain tumors, thyroid cancer, lung cancer, breast cancer, laryngeal cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, liver cancer, spleen cancer, colon cancer, kidney cancer, bladder cancer, prostate cancer, uterine cancer, ovarian cancer, testicular cancer, skin cancer, osteosarcoma, and chondrosarcoma. Examples of hematopoietic tumors include leukemia, malignant lymphoma, and multiple myeloma. The cancer targeted by the cancer targeting peptide of this embodiment is preferably a solid cancer, more preferably pancreatic cancer, liver cancer, gastric cancer, or colon cancer, and particularly preferably pancreatic cancer or liver cancer.

[0034] The cancer targeted by the cancer targeting peptide of this embodiment may be a primary cancer or a metastatic cancer. The stage of the cancer targeted by the cancer targeting peptide of this embodiment is not particularly limited, and may be any cancer from stage 0 to stage IV.

[0035] The cancer targeting peptide of this embodiment may be a peptide consisting solely of a cancer-specific binding peptide (a peptide consisting solely of the amino acid sequence of any one of SEQ ID NOS: 1 to 59), or may contain other amino acid sequence moieties. The other amino acid sequence moieties are not particularly limited, as long as they do not impair the specific binding ability of the cancer-specific binding peptide to cancer cells. Examples of the other amino acid sequence moieties include various signal peptides and various tag peptides. These signal peptides and tag peptides can be appropriately selected from various signal peptides and tag peptides commonly used in the production of recombinant proteins. Examples of the tag peptides include His tags, HA (hemagglutinin) tags, Myc tags, and Flag tags. The other amino acid sequence moieties preferably include linkers for binding to other substances or amino acid residues that contribute to binding to other substances. Examples of amino acid residues that contribute to binding to other substances include cysteine ​​residues.

[0036] The cancer targeting peptide of this embodiment may be a linear or cyclic peptide. Cyclization of the peptide is expected to improve hydrolysis resistance and in vivo stability.

[0037] The cancer targeting peptide of this embodiment may be modified in various ways, such as acetylation of the N-terminus or amidation of the C-terminus, as long as the modification does not impair its ability to specifically bind to cancer cells.

[0038] The cancer targeting peptide of this embodiment may be a peptide consisting only of L-amino acids, a peptide containing both L-amino acids and D-amino acids, or a peptide consisting only of D-amino acids. Furthermore, some or all of the amino acids constituting the cancer targeting peptide of this embodiment may be replaced with artificial amino acids.

[0039] The cancer targeting peptide of this embodiment can be easily synthesized by commonly used peptide synthesis techniques, and can also be easily produced by using commonly used expression systems such as Escherichia coli.

[0040] The cancer targeting peptide of this embodiment may be bound to a substance other than a peptide or polypeptide. The substance can be appropriately selected from various labeling substances used to label peptides. Examples of the labeling substance include low molecular weight compounds such as biotin, fluorescent substances, etc.

[0041] When the cancer-targeting peptide of this embodiment is a substance in which a peptide or polypeptide containing a cancer-specific-binding peptide moiety is linked to a labeling substance, the two substances may be linked directly or indirectly via a linker. The linker is not particularly limited as long as it is a divalent or higher linking group that does not impair the specific binding ability of the cancer-specific-binding peptide moiety to cancer. Examples of the linking group include hydrocarbon groups, -NH-, -O-, -CO-, -S-, -SO-, divalent or higher aromatic ring groups, maleimide groups, and combinations of two or more of these. Linking of a peptide or polypeptide containing a cancer-specific-binding peptide moiety to a labeling substance can be carried out using chemical reactions commonly used in the synthesis of chemical substances.

[0042] By contacting the cancer targeting peptide of this embodiment with cancer cells or cancer tissue containing cancer cells, the cancer targeting peptide can bind to cells constituting the cancer cells or cancer tissue. The method for contacting the cancer targeting peptide of this embodiment with cancer is not particularly limited. For example, when contacting the cancer targeting peptide of this embodiment with cancer in vitro or ex vivo, an appropriate amount of the peptide can be contacted with a subject containing cancer cells or cancer tissue, and then incubated as needed. Furthermore, when targeting the cancer targeting peptide of this embodiment in vivo, it can be achieved by direct injection into the cancer, intravenous, subcutaneous, intramuscular, or peritoneal injection, as well as oral administration, inhalation administration, transmucosal administration, and the like. In particular, peptides consisting of any of the amino acid sequences represented by SEQ ID NOs: 1 to 59 do not bind to normal cells when administered intravenously. Therefore, the cancer targeting peptide of this embodiment containing these peptides as an active ingredient suppresses the effect on normal cells even when administered systemically via intravenous injection.

[0043] The cancer targeting peptide of this embodiment specifically binds to cancer and cancer-constituting cells, and is therefore useful for the observation and diagnosis of cancer tissue. The peptide can be used for a variety of applications, for example, in the pharmaceutical field, such as molecular imaging of cancer tissue, reduction or treatment, and as a tool for evaluating the effectiveness of cancer treatment. Furthermore, when the cancer targeting peptide of this embodiment is a peptide linked to a labeling substance such as a fluorescent substance, the cancer targeting peptide can be used to label cancer and cancer-constituting cells, and is useful for the observation and diagnosis of cancer.

[0044] The cancer and cancer-constituting cells targeted by the cancer targeting peptide of this embodiment are not particularly limited in biological species. Examples of animals include vertebrates such as mammals, birds, reptiles, amphibians, and fish. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, and humans.

[0045] <Cancer-targeting transport carrier> The transport carrier of this embodiment is a transport carrier for specifically transporting cancer (cancer-targeting transport carrier) and includes the cancer-targeting peptide of this embodiment, i.e., a peptide having a cancer-specific binding peptide moiety. The transport carrier of this embodiment utilizes the specific binding ability to cancer of the cancer-specific binding peptide moiety, and is therefore suitable for use in delivering a substance to be delivered to cancer tissue. The transport carrier of this embodiment can transport the substance to cancer tissue more efficiently, thereby allowing the beneficial effects of the substance to be delivered to be efficiently exerted in the cancer tissue. Furthermore, the transport carrier of this embodiment can reduce or avoid side effects on normal tissues and normal cells.

[0046] The transport carrier of this embodiment has a carrier functional moiety for holding a substance to be delivered, in addition to a moiety derived from the cancer targeting peptide of this embodiment (cancer targeting peptide moiety). Examples of the carrier moiety include known DDS carriers and their constituent components, such as polymers constituting polymeric micelles and lipids constituting liposomes. The carrier moiety may also be a protein, such as serum albumin.

[0047] Examples of polymers constituting polymeric micelles include cationic polymers such as DEAE-dextran, Dendrimer, Polybrene (registered trademark) (CAS: 28728-55-4), Polyethyleneimine (PEI), and various derivatives thereof. Calcium phosphate or cationic lipids can also be used in place of the cationic polymers. Examples of lipids constituting liposomes include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and modified forms thereof; sphingophospholipids such as sphingomyelin, sphingoethanolamine, and modified forms thereof; cholesterol and modified forms thereof; and the like.

[0048] The transport carrier of this embodiment may be a complex in which the cancer targeting peptide moiety and the carrier functional moiety are directly linked, or indirectly linked via a linker. The linker is not particularly limited as long as it is a divalent or higher linking group that does not impair the specific binding ability of the cancer targeting peptide moiety to cancer. Examples of such linking groups include hydrocarbon groups, -NH-, -O-, -CO-, -S-, -SO-, divalent or higher aromatic ring groups, maleimide groups, and combinations of two or more of these. Linking between the cancer targeting peptide moiety and the carrier functional moiety, and linking between the cancer targeting peptide moiety, the carrier functional moiety, and the linker can be performed using chemical reactions commonly used in the synthesis of chemical substances.

[0049] The transport carrier of this embodiment may be directly or indirectly linked to the substance to be transported, or may not be linked. When the surface of the substance to be transported is anionic, the transport carrier of this embodiment and the substance to be transported can form a complex by electrostatic interaction with the cationic polymer moiety in the transport carrier simply by mixing them in an appropriate solvent. The complex formed between the transport carrier and the substance to be transported can be used to specifically transport the substance to cancer.

[0050] Examples of substances that can be transported by the transport carrier of this embodiment include nucleic acids, peptides, proteins (e.g., protein-based drugs such as antibodies, antibody fragments, antagonists, and agonists), lipids, peptide lipids, sugars, low-molecular-weight compounds, and other synthetic or natural compounds. These substances can be used alone or in combination of two or more. The size of the substance to be transported is not particularly limited, as long as it is large enough to be introduced into cancer cells using the transport carrier of this embodiment.

[0051] Among the substances to be transported by the transport carrier of this embodiment, nucleic acids are particularly preferred. Examples of nucleic acids include DNA such as plasmid DNA, cDNA, and antisense DNA, and RNA such as siRNA, shRNA, miRNA, antisense RNA, mRNA, tRNA, and rRNA. Examples of such nucleic acids include nucleic acids that serve as active ingredients in gene therapy and mRNA vaccine therapy.

[0052] <Pharmaceutical Composition> The pharmaceutical composition of this embodiment includes the cancer targeting peptide of this embodiment or the transport carrier of this embodiment. For example, the transport carrier of this embodiment, which includes a therapeutic agent or detection agent targeted to cancer tissue as the transported substance, is suitable as an active ingredient of a pharmaceutical composition targeting cancer. For example, by using a drug that acts as a therapeutic agent among cancer targeting substances as the transported substance and the transport carrier of this embodiment, the therapeutic agent can be efficiently transported to the cancer, and improved therapeutic effects can be expected. Furthermore, by using a detection agent for detecting cancer as the transported substance and the transport carrier of this embodiment, cancer cells and cancer tissue can be efficiently detected. Similarly, among the cancer targeting peptides of this embodiment, peptides in which a therapeutic agent or detection agent targeted to cancer tissue is directly or indirectly linked to a cancer-specific binding peptide moiety are also suitable as active ingredients of pharmaceutical compositions targeting cancer.

[0053] The content of the substance to be delivered in the pharmaceutical composition of this embodiment is not limited as long as the useful effect of the substance to be delivered is exerted in cancer cells, and can be appropriately determined by a person skilled in the art depending on the form of the pharmaceutical composition, the manner of use, the type and progression of cancer tissue, weight, etc.

[0054] The pharmaceutical composition of this embodiment may consist solely of the cancer targeting peptide of this embodiment or the transport carrier of this embodiment, or may contain a pharmaceutically acceptable carrier, if necessary. The carrier is not limited as long as the effects of the present invention are obtained, and examples thereof include purified water, buffer solution, physiological saline, RNase-free water, DNase-free water, protease-free water, aqueous glucose solution, isotonicity agent, excipient, binder, lubricant, disintegrant, fluidizing agent, diluent, thickener, stabilizer, buffer, preservative, antioxidant, flavoring agent, colorant, etc., and the carrier may be appropriately selected by those skilled in the art depending on the form of use.

[0055] The dosage form of the pharmaceutical composition of this embodiment is not particularly limited and can be appropriately selected from commonly used dosage forms, taking into consideration the administration method, etc. Examples of such dosage forms include tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (pulverized drugs), granules, fine granules, liquids, suspensions, emulsions, pastes, syrups, and injections (including those prepared as liquids by mixing with distilled water or infusions such as amino acid infusions or electrolyte infusions at the time of use).

[0056] The method of administration of the pharmaceutical composition of this embodiment is not particularly limited, and can be, for example, intra-arterial administration, intravenous administration, oral administration, rectal administration, enteral administration, transdermal administration, oral administration, etc. The pharmaceutical composition of this embodiment is administered to an animal that has developed or is likely to develop cancer, and is preferably administered to a vertebrate having cancer cells, more preferably to a mammal having cancer cells, and particularly preferably to a primate cancer patient, including a human.

[0057] The amount of the cancer targeting peptide of this embodiment or the transport carrier of this embodiment contained in the pharmaceutical composition of this embodiment is not particularly limited. The pharmaceutical composition of this embodiment is preferably adjusted so that the cancer targeting peptide and the transport carrier can deliver a sufficient amount of the target substance to cancer tissue. The dosage of the pharmaceutical composition of this embodiment can be determined appropriately depending on various conditions such as the patient's weight, age, sex, symptoms, administration route, and dosage form.

[0058] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0059] [Experimental Example 1] A peptide that does not bind to normal cells but specifically binds to cancer cells was identified.

[0060] (1) Recovery of peptides that do not bind to normal mouse cells. １１ The phage library of 1000 cfu was administered via the tail vein (first administration).ＴＭ The phage library was constructed using a "Peptide Library Kit" (New England Biolabs) that expressed random peptides consisting of seven amino acids on the phage surface.

[0061] Five minutes after administration of the phage library, the mice were anesthetized, cardiac blood was collected, and serum was separated and collected from the collected blood. Next, the phages contained in the collected serum were propagated. Specifically, the phage were propagated by infecting Escherichia coli with the serum, adding it to top agar, and inoculating it onto an LB plate and culturing it overnight at 37°C. After confirming the phage plaques, the phage were cultured in SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 8 mM MgSO ４ The phages were recovered and propagated using 0.01% gelatin.

[0062] The amplified phages were administered to a new mouse (second administration), and five minutes after administration, the mouse was anesthetized, blood was collected from the heart, and serum was separated and recovered from the collected blood. The phages contained in the collected serum were amplified, and the amplified phages were administered to a new mouse (third administration), and five minutes after administration, the mouse was anesthetized, and blood was collected from the heart. Serum was separated and recovered from the collected blood, and the phages contained in the collected serum were amplified. The amplified phages contained in the serum and the administration of the phages to the mouse were performed in the same manner as the first administration.

[0063] (2) Recovery of peptides that do not bind to normal monkey cells. 4 x 10 phages obtained in (1) above were collected. １３ cfu was intravenously administered to a cynomolgus monkey, and 5 minutes later, the monkey was anesthetized, blood was collected from the heart, and serum was separated and recovered from the collected blood. The proliferation of phages contained in the serum was carried out in the same manner as in (1) above.

[0064] (3) Recovery of peptides that bind to cancer cells using pancreatic cancer-bearing mice Peptides that bind to pancreatic cancer cells were recovered using xenograft model mice bearing the human pancreatic ductal carcinoma cell line MIA PaCa-2 (obtained from RIKEN, National Research and Development Agency).

[0065] Xenograft model mice were prepared by inoculating 1×10 MIA PaCa-2 cells onto the dorsal surface of NOD / Scid mice. ６ The cells were prepared by subcutaneously injecting 100 μL of PBS / Matrigel (volume ratio 1 / 1).

[0066] Three to four weeks after the cancer cell transplantation, the mice were treated with 2 × 10 phage obtained in (2) above. １１ cfu (1 x 10 ９ cfu / μL × 200 μL) was administered via the tail vein (first administration). Five minutes after phage administration, the mice were anesthetized, cardiac blood was collected, and then perfused and bled with phosphate-buffered saline (PBS). The tumor site formed on the dorsal surface of the mouse was then collected. The collected tumor site was then homogenized to obtain a phage solution. The obtained phage solution was propagated in the same manner as in (1) above. The phage solution obtained after proliferation was administered to a new xenograft model mouse (second administration). Five minutes after administration, the mouse was anesthetized, cardiac blood was collected, and the tumor site was then collected. The phage solution was obtained from the tumor site and propagated. The phage solution obtained after proliferation was administered to a new xenograft model mouse (third administration). Five minutes after administration, the mouse was anesthetized, cardiac blood was collected, and the tumor site was then collected. The obtained tumor site was homogenized to obtain a phage solution, which was then propagated. Administration of the phage solution to the xenograft model, collection of the tumor site from the xenograft model, preparation of the phage solution, and proliferation of the phage solution were carried out in the same manner as in the first round.

[0067] (4) Recovery of peptides that bind to cancer cells using liver cancer-bearing mice Peptides that bind to liver cancer cells were recovered in the same manner as in (3) above, except that xenograft model mice bearing human liver cancer cells HuH-7 (obtained from RIKEN, National Research and Development Agency) were used as xenograft model mice to which phages were administered, instead of xenograft model mice subcutaneously injected with MIA PaCa-2.

[0068] Xenograft model mice were prepared by inoculating 1 × 10 HuH-7 cells onto the dorsal surface of NOD / Scid mice. ６The cells were prepared by subcutaneously injecting 100 μL of PBS / Matrigel (volume ratio 1 / 1).

[0069] (5) Analysis by Next-Generation Sequencing: PCR amplification was performed on the phage solution obtained after the third round of amplification in (3) above, and the PCR product was electrophoresed, gel extracted, and purified to prepare a sample for next-generation sequencing (pancreatic cancer-binding peptide sample). The base sequence of the prepared sample for next-generation sequencing was then analyzed using a next-generation sequencer. Based on the results of next-generation sequencing, the amino acid sequences of a group of peptides (pancreatic cancer-binding peptide group) that bind to human pancreatic ductal carcinoma cells but not to normal mouse or monkey cells were analyzed.

[0070] Similarly, PCR amplification was performed on the phage solution obtained after the third round of amplification in (4) above to prepare samples for next-generation sequencing (liver cancer-binding peptide samples), and the base sequences of the prepared samples for next-generation sequencing were analyzed. Based on the results of next-generation sequencing, the amino acid sequences of a group of peptides that do not bind to normal mouse or monkey cells but have binding activity to human liver cancer cells (liver cancer-binding peptide group) were analyzed.

[0071] Next-generation sequencing identified 883 peptides that were present in both the pancreatic cancer-binding peptide group and the liver cancer-binding peptide group, i.e., peptides that bind to both human pancreatic cancer cells and human liver cancer cells. Furthermore, peptides with amino acid sequences that had a high number of reads in the pancreatic cancer-binding peptide group tended to also have a high number of reads in the liver cancer-binding peptide group. Figure 1 shows the relationship between the number of pancreatic cancer reads and the number of liver cancer reads for the 883 peptides that bind to both liver cancer and pancreatic cancer.

[0072] In addition, each sample with a large number of "reads" read in next-generation sequencing analysis contained a large number of the peptide, indicating a high affinity for cancer cells. Hereinafter, "pancreatic cancer reads" refers to the number of reads obtained as a result of next-generation sequencing of pancreatic cancer-binding peptide samples, and "liver cancer reads" refers to the number of reads obtained as a result of next-generation sequencing of liver cancer-binding peptide samples.

[0073] The amino acid sequences of the 883 peptides were searched (BlastP search) against amino acid sequence data of human proteins registered in a protein amino acid sequence database (Swiss-Prot) to determine whether there were any human proteins with partial regions matching these amino acid sequences. For the database search, human proteins matching 5 to 7 amino acid residues out of the total 7 amino acid residues of each amino acid sequence were selected. As a result, 146 amino acid sequences were identical to partial regions consisting of 5 to 7 amino acid residues of 279 human proteins. The majority of the remaining amino acid sequences matched partial regions of bacterial and viral proteins. These 146 peptides did not bind to normal cells but bound to cancer cells, and were designated "cancer-specific binding peptide group A," as they consisted of amino acid sequences identical to partial regions consisting of 5 to 7 amino acid residues of human proteins.

[0074] When the 279 human proteins that matched each of the 146 peptides included in cancer-specific binding peptide group A were searched against the PPI database "STRING," a total of 7,239 human proteins were found. Using information on whether the 279 human protein groups that matched each of the 146 peptides included in cancer-specific binding peptide group A interacted with the 7,239 human proteins, the Dice distance (defined as [Dice distance] = 1 - [Dice coefficient]) between these 279 proteins in the human protein group was calculated. Clustering was performed using the Ward method, a type of hierarchical clustering, and the proteins were classified into five groups. These five groups were ultimately found to be extracellular matrix component proteins, transcriptional regulators, nuclear regulators, signal transduction pathway proteins, and RNA regulatory proteins. The Dice coefficient between two groups is the ratio of the number of common proteins to the average number of proteins in the two groups. The clustering method is not limited to the Ward method, but may be a centroid method, a Kmeans method, DBSCAN, or the like.

[0075] Among the 279 human proteins, proteins classified as extracellular matrix constituent proteins include COL4A3, COL4A4, COL6A6, COL4A1, COL6A3, COL13A1, COL16A1, COL5A2, and COL23A1, which are proteins that constitute collagen clusters, and EMILIN1, LAMA4, ADAMTS2, and FBLN2, which are proteins involved in fibrin lamin regulation. Table 6 shows peptides that match partial regions of these human proteins, along with the number of pancreatic cancer and liver cancer reads for those peptides. Peptides 1 to 8 all had similar numbers of pancreatic cancer and liver cancer reads, confirming their usefulness as cancer targeting peptides targeting pancreatic or liver cancer. In particular, peptides 1 and 2 had over 100 reads and were confirmed to bind with very high affinity to both pancreatic and liver cancer. Peptides with high affinity for both pancreatic cancer and liver cancer are expected to also have high affinity for cancer cells derived from other tissues, making them useful as cancer-targeting peptides for various types of cancer. In other words, peptides 1 and 2 were confirmed to have very high affinity for a wide range of cancer cells, regardless of cancer type.

[0076]

[0077] Among the 279 human proteins, proteins classified as transcription factors included CCNK, AFF3, and AFF4, proteins involved in transcriptional regulation; TAF1, TAF1L, TAF6, and TAF2, which are TBP-associated factors; and FBXL19, MED12, and MED13L, which are transcriptional mediators. Table 7 shows peptides matching partial regions of these human proteins, along with their pancreatic cancer and liver cancer read counts. Peptides 9 to 14 all had similar pancreatic cancer and liver cancer read counts, confirming their usefulness as cancer targeting peptides targeting pancreatic or liver cancer. In particular, peptides 9 and 10 had over 100 read counts and were confirmed to have high affinity for a wide range of cancer cells, regardless of cancer type.

[0078]

[0079] Among the 279 human proteins, proteins classified as nuclear regulatory factors included SMARCAL1, ERCC1, MSH6, E2F7, ATRX, CHAF1B, TICRR, TCPH, and TOP2A, which are proteins involved in repair control, and KMT2C, MTA1, MTA2, SETD7, UBE2A, UBE2B, MPND, MYSM1, BTAF1, USP22, and USP27X, which are proteins involved in histone modification. Table 8 shows peptides matching partial regions of these human proteins and the number of pancreatic cancer reads and liver cancer reads for these peptides. Peptides 15 to 30 all had similar numbers of pancreatic cancer reads and liver cancer reads, confirming their usefulness as cancer targeting peptides targeting pancreatic or liver cancer. In particular, peptides 15 to 18 were confirmed to have high affinity for cancer cells, with both the number of pancreatic cancer reads and the number of liver cancer reads being 20 or more. Peptides 19 and 20 also had the number of pancreatic cancer reads being 10 or more, and were found to be particularly suitable as peptides targeting pancreatic cancer.

[0080]

[0081] Among the 279 human proteins, proteins classified as signal transduction pathway proteins included proteins involved in complement and thrombosis, such as C3, F5, APOH, and FGB; proteins involved in signal response, such as ATG7, PIK3C3, INPP4A, PIK3CD, PLCG2, ANGPT1, ANGPT2, ENG, LEP, JAK2, CD4, PXDN, IFIT1, AZI2, and TLR1; and proteins involved in fatty acid regulation, such as CYP2E1, CYP2J2, PLIN2, PDK4, and ACACA. Table 9 shows peptides matching partial regions of these human proteins and the number of pancreatic cancer and liver cancer reads for these peptides. Peptides 31 to 50 all had similar numbers of pancreatic cancer and liver cancer reads, confirming their usefulness as cancer targeting peptides targeting pancreatic or liver cancer. Peptides 31 to 52 had a pancreatic cancer lead count of 10 or more and a liver cancer lead count of 10 or more, confirming their high affinity for cancer cells. In particular, peptides 31 to 33 had a pancreatic cancer lead count of 100 or more and a liver cancer lead count of 100 or more, confirming their high affinity for a wide range of cancer cells regardless of cancer type. Peptide 36 also had a pancreatic cancer lead count of 10 or more, and was found to be particularly suitable as a peptide targeting pancreatic cancer.

[0082]

[0083] Among the 279 human proteins, proteins classified as RNA regulatory proteins included TARDBP, MATR3-2, DHX38, CHERP, NUP107, EIF3B, and DDX42A, which are proteins involved in ribosomes. Table 10 shows peptides matching partial regions of these human proteins, along with the number of pancreatic cancer reads and liver cancer reads for the peptides. Peptides 53-59 all had similar numbers of pancreatic cancer reads and liver cancer reads, confirming their usefulness as cancer targeting peptides targeting pancreatic or liver cancer. Peptides 53-55 had both 40 or more pancreatic cancer reads and liver cancer reads, confirming their high affinity for cancer cells. In particular, peptides 53 and 54 had both 100 or more pancreatic cancer reads and liver cancer reads, confirming their high affinity for a wide range of cancer cells, regardless of cancer type.

[0084]

[0085] The cancer targeting peptide, transport carrier, and pharmaceutical composition containing the same of this embodiment are very useful for molecular imaging of cancer tissue, observation of cancer pathology, anti-cancer treatment using delivered substances, etc. Furthermore, by further clarifying the state of cancer tissue using the present invention, it will lead to further progress in disease diagnosis, prevention, and treatment methods, as well as personalized medicine, drug discovery, and elucidation of the causes of disease.

Claims

1. A cancer targeting peptide comprising a peptide that binds to cancer cells, said peptide consisting of an amino acid sequence identical to a partial region consisting of 5 to 7 amino acid residues in a human protein, said human protein being one or more proteins selected from the group consisting of extracellular matrix constituent proteins, transcriptional regulatory factors, nuclear regulatory factors, signal transduction pathway proteins, and RNA regulatory proteins.

2. The cancer targeting peptide according to claim 1, wherein the human protein is an extracellular matrix constituent protein, which is a protein that constitutes collagen clusters or a protein involved in fibrin lamin regulation.

3. The cancer targeting peptide according to claim 2, wherein the protein constituting the collagen cluster is COL4A3, COL4A4, COL6A6, COL4A1, COL6A3, COL13A1, COL16A1, COL5A2, or COL23A1, and the protein involved in fibrin lamin regulation is EMILIN1, LAMA4, ADAMTS2, or FBLN2.

4. The cancer targeting peptide according to claim 1, wherein the human protein is the transcriptional regulatory factor, a protein involved in transcriptional regulation, a TBP-associated factor, or a transcriptional mediator.

5. The cancer targeting peptide according to claim 4, wherein the protein involved in transcriptional regulation is CCNK, AFF3, or AFF4, the TBP-associated factor is TAF1, TAF1L, TAF6, or TAF2, and the transcription mediator is FBXL19, MED12, or MED13L.

6. The cancer targeting peptide according to claim 1, wherein the human protein is a nuclear regulatory factor that is involved in repair control or histone modification.

7. The cancer targeting peptide of claim 6, wherein the protein involved in repair control is SMARCAL1, ERCC1, MSH6, E2F7, ATRX, CHAF1B, TICRR, TCHP, or TOP2A, and the protein involved in histone modification is KMT2C, MTA1, MTA2, SETD7, UBE2A, UBE2B, MPND, MYSM1, BTAF1, USP22, or USP27X.

8. The cancer targeting peptide of claim 1, wherein the human protein is a signal transduction pathway protein, and is a protein involved in complement / thrombosis formation, a protein involved in signal response, or a protein involved in fatty acid regulation.

9. The cancer targeting peptide of claim 8, wherein the protein involved in complement / thrombus formation is C3, F5, APOH, or FGB; the protein involved in signal response is ATG7, PIK3C3, INPP4A, PIK3CD, PLCG2, ANGPT1, ANGPT2, ENG, LEP, JAK2, CD4, PXDN, IFIT1, AZI2, or TLR1; and the protein involved in fatty acid regulation is CYP2E1, CYP2J2, PLIN2, PDK4, or ACACA.

10. The cancer targeting peptide of claim 1, wherein the human protein is an RNA regulatory protein and is a protein involved in ribosomes.

11. The cancer targeting peptide of claim 10, wherein the ribosome-associated protein is TARDBP, MATR3-2, DHX38, CHERP, NUP107, EIF3B, or DDX42.

12. The cancer targeting peptide of claim 1, wherein the peptide that binds to cancer cells consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 59.

13. The cancer targeting peptide of claim 1, which targets one or more cancers selected from the group consisting of pancreatic cancer and liver cancer.

14. A transport carrier for specifically transporting cancer, comprising the cancer targeting peptide according to any one of claims 1 to 13.

15. A pharmaceutical composition comprising the cancer targeting peptide according to any one of claims 1 to 13.

16. The pharmaceutical composition according to claim 15, which is used in the treatment of cancer.

Images