Cancer-targeting peptide, transport carrier, and pharmaceutical composition

Abstract

The present invention provides: a cancer-targeting peptide which comprises a peptide comprising an amino acid sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 47; the cancer-targeting peptide which targets at least one type of cancer selected from the group consisting of pancreatic cancer and liver cancer; a transport carrier which is intended to be used for transporting in a cancer-specific manner and comprises any one of the cancer-targeting peptides; a pharmaceutical composition which contains any one of the cancer-targeting peptides; and the pharmaceutical composition which is intended to be used for cancer treatment.

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. This application claims priority to Japanese Patent Application No. 2024-150267, filed on August 30, 2024, the contents of which are incorporated herein by reference.

[0002] The technology for targeting specific organs is 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. As peptides targeting specific tissues or cells, for example, Patent Document 1 describes a peptide specific to neovascularization, Patent Document 2 describes a cardiac-specific peptide, and Patent Document 3 describes a spinal cord tissue-specific peptide. 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 consisting of the amino acid sequence of any one of SEQ ID NOS: 1 to 47. [2] The cancer targeting peptide of [1] above, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOS: 1 to 28. [3] The cancer targeting peptide of [1] above, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOS: 1 to 13. [4] The cancer targeting peptide of [1] above, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOS: 1 to 10. [5] The cancer targeting peptide of [1] above, wherein the peptide consists of the amino acid sequence of any one of SEQ ID NOS: 1 to 6. [6] The cancer targeting peptide of any one of [1] to [5] above, which targets one or more cancers selected from the group consisting of pancreatic cancer and liver cancer. [7] A transport carrier for cancer-specific transport, comprising the cancer targeting peptide of any one of [1] to [6] above. [8] A pharmaceutical composition comprising the cancer targeting peptide of any one of [1] to [6] above. [9] The pharmaceutical composition of [8] 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 on the horizontal axis for 883 peptides that bind to both liver cancer and pancreatic cancer in Example 1. In Example 1, this is an image of cancer tissue and pancreatic tissue sections from a xenograft model mouse carrying pancreatic cancer cells, fluorescently stained with P2 peptide, which strongly binds to pancreatic cancer. In Example 1, this is an image of cancer tissue and liver tissue sections from a xenograft model mouse carrying pancreatic cancer cells, fluorescently stained with P1 peptide, P3 peptide, or P4 peptide, which strongly binds to pancreatic cancer. In Example 1, this is an image of cancer tissue and liver tissue sections from a xenograft model mouse carrying liver cancer cells, fluorescently stained with P7 peptide, which strongly binds to liver cancer.

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

[0009] In the present invention and the present specification, "X １ ~X２ (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 includes a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 47. All of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 47 are peptides that bind to at least one type of cancer cell. By including any of the amino acid sequences of SEQ ID NOs: 1 to 47, the cancer targeting peptide of this embodiment can specifically bind to cancer cells or tissues containing such cells, and functions as a cancer-targeting peptide.

[0015]

[0016]

[0017] The cancer targeting peptide of this embodiment may be any peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 47; however, in terms of higher affinity for cancer cells, it is preferred that the cancer targeting peptide comprises a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 28; it is more preferred that the cancer targeting peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 23; it is even more preferred that the cancer targeting peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 1 ...0; and it is particularly preferred that the cancer targeting peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 6.

[0018] In the present specification, a region of the cancer targeting peptide of this embodiment consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 47 may be referred to as a "cancer-specific binding peptide portion." A "peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 47" may be referred to as a "cancer-specific binding peptide." That is, the cancer targeting peptide of this embodiment contains at least one cancer-specific binding peptide. As shown in Example 1 below, all of the peptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 47 bind to cancer cells but not to normal cells. Here, a "peptide that does not bind to normal cells" refers to a "peptide that does not bind to tissues in the body when administered intravenously and remains in the serum." The cancer targeting peptide of this embodiment has a peptide portion that binds to cancer cells but not to normal cells. Therefore, when administered to the body, it selectively binds to cancer cells. 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.

[0019] 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.

[0020] 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.

[0021] 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 47), 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.

[0022] 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.

[0023] 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.

[0024] 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. Some or all of the amino acids constituting the cancer targeting peptide of this embodiment may also be replaced with artificial amino acids.

[0025] 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.

[0026] 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.

[0027] 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 a hydrocarbon group, -NH-, -O-, -CO-, -S-, -SO-, a divalent or higher aromatic ring group, a maleimide group, and combinations of two or more of these. The linking of the peptide or polypeptide containing a cancer-specific-binding peptide moiety to the labeling substance can be carried out using chemical reactions commonly used in the synthesis of chemical substances.

[0028] 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, followed by incubation as needed. 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 47 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.

[0029] 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 various purposes, for example, in the pharmaceutical field, such as molecular imaging of cancer tissue, alleviation or treatment, and as a tool for evaluating the effectiveness of cancer treatment. 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.

[0030] 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.

[0031] <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 cancer-specific binding ability of the amino acid sequence moiety represented by any one of SEQ ID NOS: 1 to 47, 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. The transport carrier of this embodiment can reduce or avoid side effects on normal tissues and normal cells.

[0032] 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.

[0033] 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.

[0034] 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 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 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.

[0035] 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 be mixed in an appropriate solvent to form a complex through electrostatic interaction with the cationic polymer moiety in the transport carrier. The complex formed between the transport carrier and the substance to be transported can be used to specifically transport the substance to cancer.

[0036] 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.

[0037] The substance to be transported by the transport carrier of this embodiment is preferably a nucleic acid. 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 the nucleic acid include nucleic acids that serve as active ingredients in gene therapy and mRNA vaccine therapy.

[0038] <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. 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.

[0039] 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.

[0040] 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.

[0041] 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 the dosage form 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).

[0042] 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.

[0043] 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 a sufficient amount of the target substance can be delivered to cancer tissue by the cancer targeting peptide and the transport carrier. 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.

[0044] 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.

[0045] Example 1 A peptide that specifically binds to cancer cells but not to normal cells was identified.

[0046] (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.

[0047] 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.

[0048] 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.

[0049] (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.

[0050] (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).

[0051] 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).

[0052] 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 time.

[0053] (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 the human liver cancer cell line HuH-7 (obtained from RIKEN, National Research and Development Agency) were used as the xenograft model mice to which phages were administered, instead of xenograft model mice subcutaneously injected with MIA PaCa-2.

[0054] 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).

[0055] (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 cancer cells but not to normal mouse or monkey cells were analyzed.

[0056] 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.

[0057] Next-generation sequencing identified 883 peptides contained 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. Of these, the amino acid sequences of 114 peptides, including those with 5 or more reads in the pancreatic cancer-binding peptide group and those with 5 or more reads in the liver cancer-binding peptide group, are shown in Tables 3 to 6. Note that each sample with a high "number of reads" in next-generation sequencing analysis contained a large number of these peptides, indicating a high affinity for cancer cells. Hereinafter, "number of pancreatic cancer reads" refers to the number of reads obtained as a result of next-generation sequencing of a pancreatic cancer-binding peptide sample, and "number of liver cancer reads" refers to the number of reads obtained as a result of next-generation sequencing of a liver cancer-binding peptide sample.

[0058]

[0059]

[0060]

[0061]

[0062] As shown in Tables 3 to 6, 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. In particular, all of the peptides ranked in the top 10 for number of pancreatic cancer reads (peptides consisting of the amino acid sequences of SEQ ID NOS: 1 to 10) were among the top 13 for number of liver cancer reads. Figure 1 shows the relationship between the number of pancreatic cancer reads and the number of liver cancer reads for 883 peptides that bind to both liver cancer and pancreatic cancer. As shown in Figure 1, a high correlation was observed between the two.

[0063] Among the peptides listed in Tables 3 to 6, peptides consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 47 had at least one of a pancreatic cancer read number and a liver cancer read number of 10 or more, and were confirmed to bind with high affinity to either pancreatic cancer or liver cancer. Peptides consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 28 had both a pancreatic cancer read number and a liver cancer read number of 10 or more, and were confirmed to bind with high affinity to both pancreatic cancer and liver cancer. In particular, peptides consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 23 had both a pancreatic cancer read number and a liver cancer read number of 17 or more, and were confirmed to bind with very high affinity to both pancreatic cancer and liver cancer. Peptides with high affinity to both pancreatic cancer and liver cancer are expected to also have high affinity for cancer cells derived from other tissues, and are useful as cancer targeting peptides that target various types of cancer.

[0064] (6) Immunostaining of pancreatic cancer tissue sections. The peptides with the amino acid sequences that had the highest number of reads in the pancreatic cancer-binding peptide group in (5) above were used to confirm whether they could detect pancreatic cancer cells by immunostaining. Biotinylated peptides were used, modified at the N-terminus with biotin: the peptide with the amino acid sequence (SEQ ID NO: 1) with the highest number of reads in the pancreatic cancer-binding peptide group (hereinafter referred to as "P1 peptide"), the peptide with the amino acid sequence (SEQ ID NO: 2) with the second highest number of reads in the pancreatic cancer-binding peptide group (hereinafter referred to as "P2 peptide"), the peptide with the amino acid sequence (SEQ ID NO: 3) with the third highest number of reads in the pancreatic cancer-binding peptide group (hereinafter referred to as "P3 peptide"), and the peptide with the amino acid sequence (SEQ ID NO: 4) with the fourth highest number of reads in the pancreatic cancer-binding peptide group (hereinafter referred to as "P4 peptide").

[0065] Specifically, cancer tissue and pancreatic tissue were excised from xenograft model mice carrying MIA PaCa-2 cells prepared as described in (3) above, 3 to 4 weeks after cancer cell transplantation, embedded in paraffin, and tissue sections were prepared. The obtained tissue sections were immersed in a solution containing a biotinylated peptide, washed, and then fluorescently stained by immersion in a solution containing streptavidin labeled with the fluorescent dye FITC and the nuclear stain DAPI. The stained tissue sections were observed using a confocal microscope SP8 (Leica) and images were taken at 400x or 1000x magnification.

[0066] Figure 2 shows fluorescent staining images using biotinylated P2 peptide. In the figure, "Pancreas" indicates the results for pancreatic tissue sections, and "tumor" indicates the results for cancer tissue sections. "P2" indicates the results for cells treated with biotinylated P2 peptide, and "No treatment" indicates the results for cells not treated with biotinylated peptide. In each figure, the upper row is a nuclear staining image, and the lower row is an FITC image.

[0067] Figure 3 shows fluorescent staining images of cancer tissue sections using biotinylated P1 peptide, biotinylated P3 peptide, and biotinylated P4 peptide. In the figure, "P1," "P3," and "P4" represent the results of cells treated with biotinylated P1 peptide, biotinylated P3 peptide, and biotinylated P4 peptide, respectively. In the figure, "x400" and "x1000" represent images taken at 400x and 1000x magnification, respectively. In each figure, the upper row is a nuclear staining image, and the lower row is an FITC image.

[0068] As shown in Figure 2, in the fluorescent image stained with P2 peptide, FITC fluorescence was observed only in the cancer tissue section. FITC fluorescence was mainly detected on the cell surface of cancer cells. These results confirmed that P2 peptide does not bind to normal pancreatic cells, but only to cancer cells.

[0069] As shown in Figure 3, the fluorescent images stained with the P1 peptide, P3 peptide, and P4 peptide confirmed binding to the cell surface in the cancer tissue section, similar to the fluorescent image stained with the P2 peptide.

[0070] (7) Immunostaining of Liver Cancer Tissue Sections Whether the peptides with the amino acid sequence that had the most reads in the liver cancer-binding peptide group in (5) above can detect liver cancer cells was confirmed by immunostaining. A biotinylated peptide in which the N-terminus of the peptide with the amino acid sequence (SEQ ID NO: 7) that had the sixth most reads in the liver cancer-binding peptide group (hereinafter sometimes referred to as "P7 peptide") was modified with biotin was used.

[0071] Specifically, from xenograft model mice carrying HuH-7 cells prepared as described in (4) above, cancer tissue and liver tissue were excised 3 to 4 weeks after cancer cell transplantation, embedded in paraffin, and tissue sections were prepared. The obtained tissue sections were immersed in a solution containing a biotinylated peptide, washed, and then fluorescently stained by immersion in a solution containing streptavidin labeled with the fluorescent dye FITC and the nuclear stain DAPI. The stained tissue sections were observed using a confocal microscope SP8 (Leica) and images were taken at 400x or 1000x magnification.

[0072] Figure 4 shows fluorescent staining images using biotinylated P7 peptide. In the figure, "Liver" indicates the results for liver tissue sections, and "tumor" indicates the results for cancer tissue sections. In the figure, "x400" and "x1000" indicate images taken at 400x and 1000x magnifications, respectively. In each figure, the upper row is a nuclear staining image, and the lower row is an FITC image.

[0073] As shown in Figure 4, in the fluorescent image stained with P7 peptide, FITC fluorescence was observed only in the cancer tissue section. FITC fluorescence was mainly detected on the cell surface of cancer cells. These results confirmed that P7 peptide does not bind to normal liver cells, but only to cancer cells.

[0074] The cancer targeting peptide, transport carrier, and pharmaceutical composition containing the same of this embodiment are extremely useful for molecular imaging of cancer tissue, observation of cancer pathology, anti-cancer treatment using delivered substances, etc. Further elucidation of the state of cancer tissue through the present invention will lead to further advances in disease diagnosis methods, prevention methods, treatment methods, personalized medicine, drug discovery, elucidation of the causes of disease, etc.

Claims

1. A cancer targeting peptide comprising a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 47.

2. The cancer targeting peptide of claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 28.

3. The cancer targeting peptide according to claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 13.

4. The cancer targeting peptide of claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 10.

5. The cancer targeting peptide of claim 1, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 6.

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

7. A transport carrier for specifically transporting cancer, comprising the cancer targeting peptide according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the cancer targeting peptide according to any one of claims 1 to 6.

9. The pharmaceutical composition according to claim 8, which is used for the treatment of cancer.

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