D-type peptides and uses thereof

Abstract

The present invention provides a peptide comprising an amino acid sequence of any one of formulae (I) to (V), in which each amino acid symbol having a symbol [D] in the front represents the D-type of the amino acid. (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D] Q [D] (X2) [D] A [D] T [D] (X5) [D] L [D] K in which X2 represents Y or L and X5 represents N, K or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in which X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) an amino acid sequence having an insertion, substitution, deletion of one or more amino acids or a combination thereof in any one of the amino acid sequences (I)-(III); and (V) a reverse reversal amino acid sequence of any one of the amino acid sequences (I)-(IV).

Description

Technical Field

[0001] This invention relates to peptides containing specific amino acids that bind to Annexin A1, and conjugates containing the peptide with one or more components. Furthermore, this invention relates to compositions containing the peptide or conjugate and their uses. Background Technology

[0002] In recent years, antibody-drug conjugates (ADCs), which combine highly cytotoxic but side-effect-prone anticancer agents with antibodies targeting molecules on the surface of cancer cells, have attracted considerable attention in the field of anticancer therapy. ADCs are compounds that combine low-molecular-weight compounds with high cytotoxic effects with antibodies, enabling their release at the target site. They are expected to be next-generation antibody drugs. Because antibodies bind with antigens present at the target site with strong affinity, ADCs can selectively deliver the bound low-molecular-weight compound to the target site. Therefore, they can simultaneously leverage the characteristics of both antibodies and low-molecular-weight anticancer agents. Antibodies have very high affinity for antigens and long half-lives, but can only target antigens present on the cell surface. On the other hand, low-molecular-weight anticancer agents are selective, have short in vivo residence time, but high permeability and can also target intracellular proteins. ADCs, which combine low-molecular-weight compounds with long-lived antibodies to sustainably release low-molecular-weight anticancer agents at the target site and thus target intracellular proteins, are also a very suitable form for drug delivery systems (DDS).

[0003] However, the soaring research and development costs of antibody drugs based on antibody-drug conjugates (ADCs) are placing an increasing financial burden on patients. There are concerns that this trend will worsen further due to advancements in healthcare, such as genetically based diagnostic systems. Furthermore, the impact of these high drug prices on national healthcare costs is immeasurable. Moreover, the exorbitant cost of antibody drugs will make them unaffordable for less developed countries, leading to further widening healthcare disparities worldwide. To address these issues from a longer-term perspective, in addition to pursuing superior drug candidates, it is necessary to explore the possibility of inexpensive biopharmaceuticals such as short-chain peptides.

[0004] Similar to the aforementioned ADCs, peptide-drug conjugates (PDCs) are known. Regarding PDCs, recent years have seen reports of peptides using glycan-mimicking structures successfully inhibiting glycan-dependent cancer metastasis for the first time globally (Non-Patent Literature 1). Furthermore, in investigating the interactions of these glycan-mimicking peptide groups with vascular endothelial receptors, the binding of a peptide called IF7 to Annexin A1 (ANXA1) has been reported (Non-Patent Literature 2). Similarly, a peptide called dTIT7 is known to bind to ANXA1, and it is known to be used as a PDC for the anticancer agent DDS (Patent Literature 1).

[0005] Among the known tumor angiogenesis-specific biomarkers, ANXA1 is clearly highly specific. It is expressed intracellularly in normal cells, but strongly expressed on the luminal surface of blood vessels in tumor neovascular endothelial cells (Non-Patent Literature 3). In addition, it has been reported that ANXA1 expression is elevated in multiple cancer types and is associated with prognosis (Non-Patent Literature 4).

[0006] As peptides targeting ANXA1, IF7 and dTIT7 are known, but even when attempting to treat cancer using PDCs containing these peptides, there is a risk of not achieving sufficient efficacy due to their poor solubility and low stability in vivo. Therefore, there has been a strong desire to develop peptides suitable for targeting ANXA1 in PDCs.

[0007] Existing technical documents Patent documents Patent Document 1: International Publication No. 2018 / 034356 Non-patent literature Non-patent literature 1: Fukuda et al., Cancer Res., 60:450-6, 2000 Non-patent literature 2: Hatakeyama et al., Proc. Natl. Acad. Sci. USA, 108: 19587-92, 2011 Non-patent literature 3: Oh et al., Nature, 429: 629-35, 2004 Non-patent literature 4: Lauren K et al., Immunology, 166(1):2-16, 2022. Summary of the Invention

[0008] The problem that the invention aims to solve Therefore, the objective of this invention is to provide peptides containing specific amino acids that bind to Annexin A1, and conjugates containing the peptide and one or more other components. Furthermore, the objective of this invention is also to provide compositions containing the peptide or conjugates and their uses.

[0009] Methods for solving problems The inventors devised a method different from dTIT7, namely, screening only for D-type peptide sequences that bind to the L-type peptide sequence of the N-terminus 15 residues of Annexin A1 within 5 minutes under biological conditions at 37°C. Therefore, a mirror-image T7 phage library screening was performed to attempt the identification of D-type peptides that selectively bind to the N-terminus of ANXA1. As a result, novel peptides with specific 7-residue D-type amino acids exhibiting high affinity for ANXA1 were successfully obtained. Furthermore, the obtained peptides possess excellent solubility. Surprisingly, the obtained peptides showed tumor-specific aggregation in bladder cancer, prostate cancer, and head and neck cancer models 24 hours after administration to mice. Based on these results, the inventors believe that the obtained peptides are suitable for DDS (Distributed Drug Components) of boron compounds and anticancer agents.

[0010] The inventors have previously confirmed that IF7- formed by combining boron compounds with IF7... 10 Compound B accumulates in tumor tissue, thus exhibiting the therapeutic effects of boron neutron capture therapy (BNCT), a heavy ion beam therapy that has been approved in recent years to show specific cell damage to tumors (International Publication No. 2019 / 244954, Yoneyama et al., BMC cancer, 21:72, 2021). However, IF7 suffers from poor solubility and low stability in blood, and novel peptides for PDC have been explored as a fundamental solution. Therefore, the feasibility of using a readily soluble peptide in BNCT was investigated. In-depth research revealed that in BNCT using this obtained peptide, conjugates (peptide-drug conjugates) formed by combining boron compounds with this peptide exhibited excellent tumor aggregation and anti-tumor effects. Based on these insights, further repeated studies resulted in the formulation of the present invention: a peptide containing a specific amino acid that binds to Annexin A1, and conjugates containing this peptide with one or more other components. In addition, compositions containing the peptide or conjugate of the present invention and their uses (e.g., for neutron capture therapy, for cancer chemotherapy, for cancer examination, etc.) have also been completed.

[0011] That is, the present invention is as follows.

[0012] [1] A peptide comprising any of the amino acid sequences in formulas (I) to (V) below, wherein each amino acid preceding the symbol [D] in the following sequences indicates the D-type of that amino acid: (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S, in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in this sequence, X2 represents Y or L, and X5 represents N, K or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in this sequence, X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions or deletions or combinations thereof in any of the amino acid sequences in (I) to (III) above; (V) The reverse amino acid sequence of any of the amino acid sequences in (I) to (IV) above.

[0013] [2] The peptide according to [1] comprises any of the following amino acid sequences (I') to (V), in which the symbol [D] has the same meaning as described above: (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S, in which X1 represents H or Q, and X2 represents P or S; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in this sequence, X2 represents Y or L, and X5 represents N, K or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in this sequence, X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I') to (III) above; (V) The reverse amino acid sequence of any of the amino acid sequences in (I') to (IV) above.

[0014] [3] The peptide according to [1] or [2] comprises any of the amino acid sequences (i) to (iii) below, wherein the symbol [D] has the same meaning as described above: (i)[D]H[D]P[D]N[D]E[D]V[D]R[D]S; (ii)[D]S[D]T[D]S[D]R[D]N[D]T[D]L; (iii)[D]Q[D]Y[D]A[D]T[D]N[D]L[D]K.

[0015] [4] The peptide according to any one of [1] to [3] comprises the amino acid sequence of (i) below, wherein the symbol [D] has the same meaning as described above: (i)[D]H[D]P[D]N[D]E[D]V[D]R[D]S.

[0016] [5] A conjugate comprising any one of the peptides described in [1] to [4] and one or more components.

[0017] [6] According to the conjugate described in [5], one or more of the aforementioned components contain a boron compound or a gadolinium compound.

[0018] [7] According to the conjugate described in [6], one or more of the aforementioned components include a boron compound.

[0019] [8] According to the conjugate described in [7], wherein the aforementioned conjugate is of the following formula (I): [Chemistry 1] Or the following formula (II): [Chemistry 2] .

[0020] [9] According to the conjugate described in [5], one or more of the aforementioned components contain an anticancer agent.

[0021]

[10] According to the conjugate described in [5], one or more of the aforementioned components contain a detectable substance.

[0022]

[11] According to the conjugate described in

[10] , the aforementioned detectable substance can be detected in vivo by a method selected from X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound examination, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy.

[0023]

[12] According to the conjugate described in

[10] or

[11] , wherein the aforementioned detectable substance is a radioactive isotope, an MRI enhancer, a radioactive impermeable substance, a contrast agent, or a fluorescent substance.

[0024]

[13] A pharmaceutical composition comprising any one of the peptides described in [1] to [4] or any one of the conjugates described in [5] to

[12] .

[0025]

[14] Neutron capture therapy agent comprising any one of the conjugates described in [6] to [8].

[0026]

[15] According to the neutron-capturing therapy agent described in

[14] , wherein the aforementioned conjugate contains a boron compound.

[0027]

[16] Neutron-capturing therapy agents as described in

[14] or

[15] are used for the treatment or prevention of solid cancers.

[0028]

[17] According to the neutron capture therapy agent described in

[16] , the aforementioned solid cancer is selected from skin cancer, brain and nervous system cancer, laryngeal cancer, oral cancer, salivary gland cancer, sinus cancer, thyroid cancer, bladder cancer, prostate cancer, renal pelvis and ureter cancer, and osteosarcoma.

[0029]

[18] A cancer chemotherapy agent comprising the conjugate described in [9].

[0030]

[19] A reagent for cancer examination, comprising any one of the conjugates described in

[10] to

[12] .

[0031]

[20] Neutron capture therapy, comprising administering to a subject an effective amount of the conjugate described in any one of [6] to [8].

[0032] [twenty one] According to the neutron capture therapy described in

[20] , the aforementioned subject has solid cancer.

[0033] [twenty two] According to the neutron capture therapy described in

[21] , the aforementioned solid cancer is selected from skin cancer, brain and nervous system cancer, laryngeal cancer, oral cancer, salivary gland cancer, sinus cancer, thyroid cancer, bladder cancer, prostate cancer, renal pelvis and ureter cancer, and osteosarcoma.

[0034] [twenty three] A method of cancer treatment or prevention, comprising administering to a subject an effective amount of the conjugate described in any one of [6] to [9].

[0035] [twenty four] A method for detecting cancer, comprising administering to a subject an effective amount of the conjugate described in any one of

[10] to

[12] .

[0036]

[25] According to the method described in

[23] or

[24] , wherein the aforementioned cancer is a solid cancer.

[0037]

[26] According to the neutron capture therapy agent described in

[16] or the method described in

[25] , wherein the aforementioned solid cancer is an annexin A1 positive solid cancer.

[0038]

[27] The neutron capture therapy agent according to any one of

[14] to

[17] , or the cancer chemotherapy agent according to

[18] , is used to administer to subjects with annexin A1-positive cancer selected from subjects with cancer.

[0039]

[28] A companion diagnostic for any of the neutron capture therapy agents described in any one of

[14] to

[17] , or the cancer chemotherapy agents described in

[18] , comprising an annexin A1 binding molecule.

[0040]

[29] A method for treating or preventing cancer, comprising: screening subjects with cancer containing annexin A1 positive cells from among subjects with cancer, and administering to the screened subjects a neutron capture therapy agent as described in any one of

[14] to

[17] , or a cancer chemotherapy agent as described in

[18] .

[0041]

[30] The peptide or conjugate of any one of [1] to [4], or the conjugate of any one of [5] to

[12] , used for the treatment or prevention of cancer.

[31] Use of any peptide or conjugate in any one of [1] to [4], or in any one of [5] to

[12] , for the treatment or prevention of cancer.

[0043]

[32] Use of any peptide in any one of [1] to [4], or any conjugate in any one of [5] to

[12] , in the manufacture of a drug for the treatment or prevention of cancer.

[0044] Invention Effects According to the present invention, the obtained D-type peptide can bind to ANXA1 with high affinity, and ANXA1 also has high specificity among tumor angiogenesis-specific marker molecules. Furthermore, this D-type peptide is more soluble than previously known peptides, thus it is extremely useful for PDCs targeting ANXA1. In addition, this D-type peptide can persistently and significantly accumulate at tumor sites, for example, in bladder cancer, prostate cancer, head and neck cancer, etc., and compared with previously known peptides, it is expected to improve the efficiency of drug uptake into cancer cells and the anti-tumor effect, for example, it is extremely useful in medical applications (e.g., cancer treatment, more specifically (boron) neutron capture therapy, cancer chemotherapy, etc.; cancer detection, more specifically monitoring the therapeutic effect of disease using in vivo imaging, etc.). Furthermore, in the case of PDCs, this D-type peptide is also expected to reduce the side effects of the drug. Attached Figure Description

[0045] Figure 1 : Figure 1 This paper presents an outline of a mirror phage display screening method for identifying L-amino acid peptide sequences that bind to the N-terminal 15 residue peptide of Annexin A1, which consists of D-type amino acids.

[0046] Figure 2 : Figure 2 This outlines the identification of L-type peptide sequences binding to D-MC16 by mirror phage display screening of the 7-residue peptide sequences presented by the phage mixtures obtained in rounds 1 through 5.

[0047] Figure 3 : Figure 3 The genome copy number of the lysed phage in each round is shown by the 7-residue peptide sequence presented by the phage mixtures obtained in rounds 1 through 5.

[0048] Figure 4 : Figure 4 The concentration of the L-amino acid sequence presented by the phage mixtures obtained in rounds 1 through 5, relative to the round 1 library, is shown.

[0049] Figure 5 : Figure 5 The alignment results of the top 10 peptide sequences are shown for the 7-residue peptide sequences presented by the phage mixtures obtained in rounds 1 through 5.

[0050] Figure 6 : Figure 6The diagram shows the KD and Rmax values ​​of L-MC16 binding to D-type peptides relative to either L-MC16 or D-MC16. A: Sensing plots and equilibrium dissociation constant KD values ​​of L-MC16 or D-MC16 peptides relative to a dhp7 immobilized sensor. B: Sensing plots and equilibrium dissociation constant KD values ​​of L-MC16 peptides relative to a dst7 immobilized sensor. C: Sensing plots and equilibrium dissociation constant KD values ​​of L-MC16 peptides relative to a dqy7 immobilized sensor.

[0051] Figure 7 : Figure 7 This diagram shows the KD values ​​and isoaffinity plots of the dhp7 peptide relative to the L-MC16 mutant. A: Binding-dissociation related sensing plots of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides relative to the dhp7 immobilized sensor. B: Binding-dissociation related isoaffinity plots and equilibrium dissociation constant KD values ​​of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides relative to the dhp7 immobilized sensor. Two-dimensional affinity kinetics plots of rate constants grouped according to the L-MC16 peptide series. The dashed diagonal line represents the equilibrium binding constant, shown to aid in visualization of affinity distribution.

[0052] Figure 8 : Figure 8 This involves the binding of the dhp7 peptide to HEK293 overexpressing Anxa1-c-His and its uptake into the cell.

[0053] Figure 9 : Figure 9 The results show the binding of the dhp7 peptide to HEK293 overexpressing Anxa1-c-His and its uptake into the cell.

[0054] Figure 10 : Figure 10 The results show the expression analysis of ANXA1 on the surface of cancer cell lines using flow cytometry.

[0055] Figure 11 : Figure 11 The results of a study show the tumor aggregation of Cy7.5-dhp7 in mice carrying bladder cancer.

[0056] Figure 12 : Figure 12 The results of a study show the tumor aggregation of Cy7.5-dhp7 in nude mice carrying prostate cancer.

[0057] Figure 13 : Figure 13The results of a study show the tumor aggregation of Cy7.5-dhp7 in nude mice carrying bladder cancer.

[0058] Figure 14 : Figure 14 The results of a study show the tumor aggregation of Cy7.5-dhp7 in nude mice carrying cancer (head and neck cancer).

[0059] Figure 15 : Figure 15 Showing targets 10 Rabbit antibodies against BSH-BSA 10 Results of immunogenicity and affinity analysis of BSH polyclonal antibodies (#52 and #53).

[0060] Figure 16 : Figure 16 This shows the use of immunofluorescence staining. 10 Visualization results of BSH-dhp7 peptide uptake in ANXA1-positive cancer cells.

[0061] Figure 17 : Figure 17 Showing cancer-carrying mice 10 Results of neutron capture therapy following administration of BSH-dhp7.

[0062] Figure 18 : Figure 18 Showing cancer-carrying mice 10 Results of neutron capture therapy following administration of BSH-dhp7.

[0063] Figure 19 : Figure 19 Show 10 Immunostaining results of tumor removal after neutron capture therapy following administration of BSH-dhp7.

[0064] Figure 20 : Figure 20 The results of a study show the tumor aggregation of Cy7.5-vc-dhp7 in mice carrying prostate cancer with tumors 7 mm in diameter.

[0065] Figure 21 : Figure 21 The results of a study show the tumor aggregation of Cy7.5-vc-dhp7 in mice carrying prostate cancer with tumors 7 mm in diameter.

[0066] Figure 22 : Figure 22 The results of a study show the tumor aggregation of Cy7.5-vc-dhp7 in mice carrying prostate cancer with a tumor diameter of 15 mm.

[0067] Figure 23 : Figure 23 This study presents the results of an in vitro study of the aggregation of Cy7.5-vc-dhp7 in various organs of mice carrying various cancers (prostate cancer, head and neck cancer, and osteosarcoma) with tumors of 15 mm in diameter.

[0068] Figure 24 : Figure 24 This study presents the results of an in vitro study of the aggregation of Cy7.5-vc-dhp7 in various organs of mice with tumors (prostate cancer) with a diameter of 15 mm.

[0069] Figure 25 : Figure 25 The results of a study show the tumor aggregation of Cy7.5-vc-dhp7 in mice carrying prostate cancer with tumors 7 mm in diameter.

[0070] Figure 26 : Figure 26 The results show the tumor aggregation of Cy7.5-vc-dhp7 in mice with tumors of 7 mm diameter (head and neck cancer and osteosarcoma).

[0071] Figure 27 : Figure 27 This shows the use of immunofluorescence staining. 10 Results of a study visualizing BSH uptake in PC3-Anxa1 positive / negative cancer cells.

[0072] Figure 28 : Figure 28 This shows the use of immunofluorescence staining. 10 Results of a study visualizing BSH uptake in PC3-Anxa1 positive / negative cancer cells.

[0073] Figure 29 : Figure 29 This shows the use of immunofluorescence staining. 10 Results of a visualization study on BSH uptake in various cells (PC3, SAS, and MG-63).

[0074] Figure 30 : Figure 31 This shows the use of immunofluorescence staining. 10 Results of a visualization study on BSH uptake in various cells (PC3, SAS, and MG-63).

[0075] Figure 31 : Figure 31 This shows the use of immunofluorescence staining. 10 Results of a visualization study on BSH uptake in various cells (PC3, SAS, and MG-63).

[0076] Figure 32 : Figure 32Showing various [indications] in mice carrying cancer (prostate cancer) 10 Results of fluorescence aggregation at the time of sacrifice in mice treated with drug B.

[0077] Figure 33 : Figure 33 Show various 10 Utilization of anti-inflammatory drugs in various tissues 24 hours after administration of drug B 10 Immunohistochemical staining of tissues with BSH antibodies 10 Visualization results of BSH intake.

[0078] Figure 34 : Figure 34 Show various 10 Utilization of anti-inflammatory drugs in various tissues 24 hours after administration of drug B 10 Immunohistochemical staining of tissues with BSH antibodies 10 Visualization results of BSH intake.

[0079] Figure 35 : Figure 35 Show various 10 Utilization of anti-inflammatory drugs in various tissues 24 hours after administration of drug B 10 Immunohistochemical staining of tissues with BSH antibodies 10 Visualization results of BSH intake.

[0080] Figure 36 : Figure 36 This shows the cancer in mice carrying prostate cancer. 10 Mice were administered BSH-vc-dhp7. 10 The result of time-dependent changes in BSH aggregation.

[0081] Figure 37 : Figure 37 This shows the cancer in mice carrying prostate cancer. 10 24 hours after administration of BSH-VC-DHP7, in various organs 10 The result of BSH aggregation.

[0082] Figure 38 : Figure 38 The EMCS- model is shown in both an ANXA1-negative prostate cancer (PC3-ANXA1-) model and an ANXA1-positive head and neck cancer (SAS) model. 10 BSH-vc-dhp7 administration + BNCT treatment group and 10 Comparative results of the anti-tumor effects of the BSH+BNCT treatment group.

[0083] Figure 39 : Figure 39The EMCS- model is shown in both an ANXA1-negative prostate cancer (PC3-ANXA1-) model and an ANXA1-positive head and neck cancer (SAS) model. 10 BSH-vc-dhp7 administration + BNCT treatment group and 10 Comparative results of the anti-tumor effects of the BSH+BNCT treatment group.

[0084] Figure 40 : Figure 40 The cytotoxicity and IC50 values ​​against Anxa1-positive / negative PC3 cells are shown.

[0085] Figure 41 : Figure 41 The results show the shrinkage effect of luciferase-luminescent tumors and the changes in body weight during treatment with vcMMAE or vcMMAE-dhp7.

[0086] Figure 42 : Figure 42 The appearance of the removed tumor and HE staining results are shown during single-dose or single-dhp7 treatment with vcMMAE, along with post-treatment blood test results.

[0087] Figure 43 : Figure 43 The appearance of the removed tumor and HE staining results are shown during single-dose or single-dhp7 treatment with vcMMAE, along with post-treatment blood test results.

[0088] Figure 44 : Figure 44 Immunostaining results of tumors removed during single-dose vcMMAE or vcMMAE-dhp7 treatment are shown.

[0089] Figure 45 : Figure 45 Immunostaining results of tumors removed during single-dose vcMMAE or vcMMAE-dhp7 treatment are shown.

[0090] Figure 46 : Figure 46 The results show the shrinkage of luciferase-luminescent tumors and the changes in body weight during treatment with VCMMAE single-dose or VCMMAE-DHP7.

[0091] Figure 47 : Figure 47 The appearance of the removed tumor is shown during treatment with a single dose of vcMMAE or vcMMAE-dhp7.

[0092] Figure 48 : Figure 48 The appearance of the removed tumor is shown during treatment with a single dose of vcMMAE or vcMMAE-dhp7. Detailed Implementation

[0093] 1. The peptide of the present invention The present invention provides peptides comprising specific amino acids that bind to Annexin A1. More specifically, the present invention provides peptides comprising any of the amino acid sequences of the following formulas (I) to (V), wherein each amino acid preceding the sequence with the symbol [D] indicates the D-type of that amino acid.

[0094] (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S (In this sequence, X1 represents H or Q, X2 represents P or S, and X3 represents N or K) (Seq ID NO:1); (II) [D]Q [D] (X2) [D]A [D]T [D] (X5) [D]L [D]K (In this sequence, X2 represents Y or L, and X5 represents N, K, or Y) (Seq ID NO:2); (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L (In this sequence, X1 represents S or R, X4 represents R or W, and X5 represents N or I) (Seq ID NO:3). (IV) An amino acid sequence having one or more amino acid insertions, substitutions or deletions or combinations thereof in any of the amino acid sequences in (I) to (III) above; (V) The reverse amino acid sequence of any of the amino acid sequences in (I) to (IV) above.

[0095] In this specification, single-letter and three-letter representations of amino acids are used in accordance with the IUPAC-IUB Joint Committee on Biochemical Nomenclature (JCBN).

[0096] In this specification, the amino acid sequences of (chain-like) peptides are described according to peptide representation conventions, with the left side representing the N-terminus and the right side representing the C-terminus. Furthermore, amino acid symbols preceded by the symbol [D] in the amino acid sequence indicate the D-type of that amino acid, while amino acid symbols not preceded by the symbol [D] in the amino acid sequence indicate the L-type of that amino acid, provided it does not violate the context. In this specification, peptides containing any of the amino acid sequences (I) to (V) above are collectively referred to as the peptides of the present invention. Additionally, the peptides of the present invention may also consist of any of the amino acid sequences (I) to (V) above.

[0097] In one manner, the above (I) is (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S (in this sequence, X1 represents H or Q, and X2 represents P or S) (Seq ID NO:4).

[0098] In one embodiment, (I) above is [D]H[D]P[D]N[D]E[D]V[D]R[D]S (Seq ID NO:5), [D]Q[D]P[D]N[D]E[D]V[D]R[D]S (Seq ID NO:6), [D]H[D]S[D]N[D]E[D]V[D]R[D]S (Seq ID NO:7), [D]Q[D]S[D]N[D]E[D]V[D]R[D]S (Seq ID NO:8), or [D]Q[D]P[D]K[D]E[D]V[D]R[D]S (Seq ID NO:9), preferably [D]H[D]P[D]N[D]E[D]V[D]R[D]S (Seq ID NO:10).

[0099] In one embodiment, (II) above is [D]Q[D]Y[D]A[D]T[D]N[D]L[D]K (Seq ID NO:11), [D]Q[D]Y[D]A[D]T[D]K[D]L[D]K (Seq ID NO:12), or [D]Q[D]L[D]A[D]T[D]Y[D]L[D]K (Seq ID NO:13), preferably [D]Q[D]Y[D]A[D]T[D]N[D]L[D]K (Seq ID NO:14).

[0100] In one embodiment, (III) above is [D]S[D]T[D]S[D]R[D]N[D]T[D]L (Seq ID NO:15) or [D]R[D]T[D]S[D]W[D]I[D]T[D]L (Seq ID NO:16), preferably [D]S[D]T[D]S[D]R[D]N[D]T[D]L (Seq ID NO:17).

[0101] The number of amino acid insertions (or additions), substitutions, deletions, or combinations thereof in (IV) above is 1 to several (2, 3, 4, 5, 6, 7, 8, 9, etc.), more specifically 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1. The inserted, substituted, etc., amino acids can be either D-type or L-type, and from the viewpoint of protease tolerance, D-type amino acids are preferred. In addition, the peptide of the present invention containing the amino acid sequence of (IV) above may contain a partial sequence of 4 consecutive amino acids, a partial sequence of 5 consecutive amino acids, or a partial sequence of 6 consecutive amino acids from the amino acid sequences of (I) to (III) above.

[0102] The amino acids inserted (or added), substituted, or deleted in the amino acid sequence (IV) above can be any amino acid as described below. This amino acid can be an L-type or D-type amino acid; for example, it can be a naturally occurring L-type amino acid as described below, or a D-type amino acid as its optical isomer. Furthermore, this amino acid can accept various chemical modifications as described below.

[0103] The insertion (or addition) of the amino acid in (IV) above can be made at the N-terminal or C-terminal side of the original sequence, or inside the sequence, as long as it can bind to ANXA1.

[0104] The amino acid substitutions in (IV) above are preferably made with amino acids that have similar physicochemical properties (“similar amino acids”), such as aromatic amino acids (Phe (F), Trp (W), Tyr (Y)), aliphatic amino acids (Ala (A), Leu (L), Ile (I), Val (V)), polar amino acids (Gln (Q), Asn (N)), basic amino acids (Lys (K), Arg (R), His (H)), acidic amino acids (Glu (E), Asp (D)), amino acids with hydroxyl groups (Ser (S), Thr (T)), and amino acids with small side chains (Gly (G), Ala (A), Ser (S), Thr (T), Met (M)), etc., which are classified as amino acids in the same group. It is predicted that substitutions based on these similar amino acids will not cause changes in the protein phenotype (i.e., they are conserved amino acid substitutions). Specific examples of conserved amino acid substitutions are well known in the art and described in various publications (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)). In this specification, conserved amino acid substitutions may also include replacing D-type amino acids with their optical isomers, namely L-type amino acids. Conserved amino acid substitutions can be substitutions of L-type amino acids with each other, substitutions of D-type amino acids with each other, or substitutions of L-type amino acids with D-type amino acids.

[0105] The deletion of the amino acid in (IV) above can occur at any position in the original sequence, as long as it can bind to ANXA1. It can be deleted from the N-terminal or C-terminal side of the original sequence, or it can be deleted from inside the sequence.

[0106] The length of the peptides of the present invention is not particularly limited, as long as they can bind to ANXA1. For example, they may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acids. Furthermore, the peptides of the present invention may, for example, consist of up to 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11, at most 10, at most 9, at most 8, or at most 7 amino acids. Alternatively, the peptides of the present invention may be, for example, 3-10, 3-15, 3-20, 3-25, 3-30, 3-40, 3-50, 4-10, 4-15, 4-20, 4-25, 4-30, 4-40, 4-50, 5-10, 5-15, 5-20, 5-25, 5-30, 5-40, 5- The peptides of this invention can be 50, 6-10, 6-15, 6-20, 6-25, 6-30, 6-40, 6-50, 7-10, 7-15, 7-20, 7-25, 7-30, 7-40, 7-50, 8-10, 8-15, 8-20, 8-25, 8-30, 8-40, or 8-50 amino acids in length. For example, the peptides of this invention can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0107] The peptides of the present invention can be composed of a combination of D-type and L-type amino acids. More specifically, the amino acids constituting a peptide comprising any of the amino acid sequences (I) to (V) above can be all D-type amino acids, or may contain L-type amino acids in addition to D-type amino acids. L-type amino acids can be naturally occurring L-type amino acids, and examples include, for example, L-type amino acids such as glycine, alanine, leucine, proline, phenylalanine, tyrosine, methionine, serine, threonine, cysteine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, hydroxylysine, histidine, tryptophan, and valine. Examples of D-type amino acids include, for example, optical isomers of the L-type amino acids described above. It should be noted that, in this specification, the amino acid that does not exhibit optical activity, namely glycine, can be interpreted as an L-type and D-type amino acid, provided it does not deviate from the context.

[0108] The peptides of this invention may contain any of the modified or unusual amino acids mentioned in 37 CFR 1.821-1.822. There are no particular limitations on the modified or unusual amino acids, and examples include, for instance, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmodium, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmodium, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, valine, leucine, ornithine, etc.

[0109] The amino and / or carboxyl termini of the peptides of the present invention can be modified. Modifications to the amino termini can include, for example, methylation (e.g., -NHCH3, -N(CH3)2, etc.), acetylation (e.g., using acetic acid or its halogenated derivatives (e.g., α-chloroacetic acid, α-bromoacetic acid, α-iodoacetic acid, etc.), ureation, carbamate esterification, formylation, Bocation, Fmocation, etc. Alternatively, any protecting group can be introduced, such as a benzyloxycarbonyl group, a carboxylic acid ester functional group (RCOO-), or a sulfonyl functional group (R-SO2-) (where R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc.).

[0110] Examples of modifications to the carboxyl terminus include amidation (-CONH2) and esterification (-COOR). Here, the R in the ester can be, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1～6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. (C60) 3～8 Cycloalkyl; for example, phenyl, α-naphthyl, etc. 6～12 Aryl; for example, benzyl, phenethyl, etc. phenyl-C 1～2 Alkyl; α-Naphthyl-C 1～2 Alkyl and other C 7～14 Aryl groups; neopentyloxymethyl, etc.

[0111] The peptides of the present invention can be modified in various ways, even outside the N-terminus or C-terminus. Chemical modifications can include, for example, methylation, acetylation, phosphorylation, etc. When the peptides of the present invention have a carboxyl group (carboxylic acid ester) outside the C-terminus, the carboxyl group can be amidated or esterified. For example, the C-terminal esters described above can be used as the ester in this case. Alternatively, substituents on the side chains of amino acids within the molecule (e.g., -OH, -SH, amino, imidazole, indole, guanidinyl, etc.) can be protected by suitable protecting groups (e.g., formyl, acetyl, etc.). 1～6 Alkyl (C1～6 (Acyl group, etc.) protection.

[0112] For (V) above, a retro-inverso isomer of a peptide is a peptide in which the chirality of each amino acid residue is reversed (“inverso”) and the direction of the amino acid sequence is reversed (“retro”) relative to the original peptide. In addition, retro-inverso isomers are known to exhibit similar structures and functions to the original peptide (e.g., Acc. Chem. Res., 1993, 26(5), pp266-273, PLoS One. 2013 Dec 2; 8(12): e80390, etc.). Specifically, examples of amino acid sequences in (V) above include, for instance, reverse sequences of (I) above, such as SRVENPH (Seq ID NO:18), SRVENPQ (Seq ID NO:19), SRVENSH (Seq ID NO:20), SRVENSQ (Seq ID NO:21), SRVEKPQ (Seq ID NO:22), etc.; reverse sequences of (II) above, such as KLNTAYQ (Seq ID NO:23), KLKTAYQ (Seq ID NO:24), KLYTALQ (Seq ID NO:25), etc.; and reverse sequences of (III) above, such as LTNRSTS (Seq ID NO:26), LTIWSTR (Seq ID NO:27), etc.

[0113] The peptides of the present invention may comprise two or more sequences selected from (I) to (V) above. In one embodiment, the peptides of the present invention comprise tandem repeat sequences of any of the sequences (I) to (V) above (i.e., structures formed by direct linkage of the same sequence portions). In another embodiment, the peptides of the present invention comprise structures formed by direct linkage of two or more different sequences (I) to (V) above. Alternatively, the peptides of the present invention may be constructed as multivalent peptides using dendritic macromolecules.

[0114] The peptides of the present invention can be free peptides or in the form of salts. Examples of salts of the peptides of the present invention include, for example, pharmaceutically acceptable acid addition salts and base addition salts. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, malic acid, succinic acid, tartaric acid, and citric acid. Examples of base addition salts include salts with alkali metals such as sodium and potassium, salts with alkaline earth metals such as calcium and magnesium, and salts with amines such as ammonium and triethylamine.

[0115] The peptide of the present invention can bind to annexin A1. ANXA1 is a well-known protein belonging to the annexin family, also known as lipocortin 1. The base sequence and amino acid sequence of ANXA1 DNA are known for various biological species. The peptide of the present invention can bind to the N-terminal region of ANXA1. More specifically, the peptide of the present invention can bind to the N-terminal 15 residues (MAMVSEFLKQAWFIE (Seq ID NO:28)) of ANXA1 (e.g., human ANXA1, mouse ANXA1, etc.). In one embodiment, the peptide of the present invention can bind to the N-terminal 15 residues (MAMVSEFLKQAWFIE (Seq ID NO:28)) of ANXA1 (e.g., human ANXA1, mouse ANXA1, etc.) within 5 minutes at 37°C. The peptides of the present invention, for example, when measured using biolayer interferometry (BLI) to determine their intermolecular interactions with ANXA1 in mice or humans, may preferably have 10 -6 M or less, preferably 5.0×10 -7 The equilibrium dissociation constant (KD value) below M.

[0116] The peptides of the present invention can be manufactured according to known peptide synthesis methods. These methods can be, for example, solid-phase synthesis or liquid-phase synthesis. When a portion of the peptide or amino acid that constitutes the peptide of the present invention is condensed with the remaining portion, and the product has a protecting group, the target peptide can be manufactured by removing the protecting group.

[0117] Here, the condensation and removal of the protecting group can be carried out by methods known to them, such as those described in (1) to (8) below: (1) M. Bodanszky&M. A. Ondetti, Peptide Synthesis, IntersciencePublishers, New York (1966) (2) Schroeder&Luebke, The Peptide, Academic Press, New York (1965) (3) Nobuo Izumiya, Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975) (4) Haruaki Yajima and Shunpei Sakakibara, Lectures on Biochemistry Experiments 1: Chemistry of Proteins IV 205 (1977) (5) Haruaki Yajima, Development of Subsequent Pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten. (6) Stewart, JM & Young, JD, “Solid phase peptide synthesis (2nded.)”, Pierce Chemical Company, Rockford (1984) (7) Atherton, E.&Sheppard, RC, "Solid Phase peptide synthesis: a practical approach", IRL Press, Oxford (1989) (8) "Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series)", Oxford University Press (2000).

[0118] The peptides obtained in this way can be purified and separated using known purification methods. Examples of purification methods include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof. When the peptide obtained by the above methods is a free peptide, it can be converted into a suitable salt using known methods or methods based thereon. Conversely, when the peptide is obtained in salt form, the salt can be converted into a free peptide or other salts using known methods or methods based thereon.

[0119] The peptides of the present invention can be safely administered orally or non-orally to the subjects described below. Non-oral administration includes intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, ocular, intracerebral, rectal, vaginal, intraperitoneal, intratumoral, proximal to tumor, and direct administration to the lesion.

[0120] As described above, annexin A1 is known to be highly specific among currently known tumor angiogenesis-specific marker molecules. It is expressed intracellularly in normal cells, but strongly expressed on the luminal surface of blood vessels in tumor neovascular endothelial cells (Oh et al., Nature 429: 629-35, 2004). Therefore, the peptide of the present invention can selectively bind to angiogenic tumors in vivo. Furthermore, ANXA1 is expressed on the blood side of neovascular endothelial cells formed within tumors. When bound to a ligand such as the peptide of the present invention on the blood side, the ligand is transported to the basal side via transcytosis and actively released into the stroma where cancer cells reside. Therefore, the peptide of the present invention can target malignant tumors in vivo. Thus, the peptide of the present invention can be used for, for example, malignant tumor targeting, (molecular) imaging, companion diagnostics, etc.

[0121] For example, for the aforementioned companion diagnostics, annexin A1-positive cancer tissue (cancer tissue containing annexin A1-positive cells or annexin A1-positive (solid) cancer) can be determined, for example, by screening specific cell populations in the cancer tissue and determining the extent to which the cancer tissue contains annexin A1-positive cells. The aforementioned screening and determination can be performed according to known methods. Specifically, examples include immunohistostaining. In this method, annexin A1-positive cells in the cancer tissue are stained with anti-human annexin A1 antibodies (fluorescence or chromogenic methods), and a positive or negative result is determined based on the amount of pigment emitted by the stained cells (e.g., fluorescence or visible light intensity). This staining typically utilizes annexin A1-binding molecules. Therefore, in one aspect, the present invention relates to companion diagnostics (hereinafter also referred to as diagnostics of the present invention) for the neutron capture therapy agents or cancer chemotherapeutic agents of the present invention, which contain annexin A1-binding molecules. The effectiveness of the cancer treatment or preventive agent (cancer treatment agent, preventive agent) of the present invention can be predicted by a method including the step of contacting annexin A1-binding molecules with cells in the cancerous tissue of the subject (recipient).

[0122] There are no particular limitations on annexin A1-binding molecules, as long as they are capable of binding to annexin A1 (preferably specifically). Examples of annexin A1-binding molecules include antibodies, and more specifically, immunoglobulins, Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, single-chain antibody, etc.

[0123] The aforementioned screening and determination process can be performed, for example, using microscopic observation. The determination of positive or negative results is based on known methods and the amount of pigment produced by the stained cells. This determination can be performed using known techniques. For example, the amount of pigment produced by negative control cells (specifically, cells stained with an isotype control antibody) can be set as the background pigment level. Cells with a pigment level below this background level are considered negative, and cells with a fluorescence level above this background level are considered positive. As a simpler method, a method could also be considered that uses mRNA extracted from cancer tissue, performs reverse transcription quantitative PCR of annexin A1, and determines the positive result based on the amount of annexin A1 mRNA.

[0124] In one approach, subjects with cancerous tissue (subjects with cancer) containing annexin A1-positive cells (or subjects with cancer containing annexin A1-positive cells (annexin A1-positive cancer)) are screened from subjects with cancerous tissue (subjects with cancer), and the cancer treatment or preventive agent of the present invention is administered to the screened subjects. The cancer treatment or preventive agent of the present invention can be used by administering to subjects with cancerous tissue containing annexin A1-positive cells (or subjects with cancer containing annexin A1-positive cells (annexin A1-positive cancer)) screened from subjects with cancerous tissue (subjects with cancer).

[0125] 2. The conjugate of the present invention Furthermore, the present invention also provides conjugates (hereinafter also referred to as conjugates of the present invention) comprising one or more components on the peptide of the present invention. The component is not particularly limited as long as it can be linked to the peptide of the present invention, and can be those suitable for administration to animals (e.g., humans) and capable of performing certain functions in the animal. Additionally, the component can be natural or non-natural.

[0126] Examples of components include, but are not limited to, biological materials (e.g., cells, bacteriophages, viruses, etc.), oligonucleotides, nucleic acids (e.g., DNA, RNA, DNA / RNA chimeras, etc.), peptides, polypeptides, proteins, antibodies, lipids, polysaccharides, low molecular weight compounds (organic or inorganic, etc. (e.g., boron compounds, gadolinium compounds, etc.)), particles (e.g., gold particles, various nanoparticles, etc.), and combinations thereof.

[0127] This component can exert a given function at a target site in an animal (e.g., a human). The type of this function is not particularly limited. Through the action of the portion corresponding to the peptide of this invention, the conjugate of this invention can target malignant tumors, etc.; therefore, preferred examples of this function include the imparting of anticancer activity and detectability. Thus, this component can be, for example, a low-molecular-weight compound, an anticancer agent, a detectable substance, etc.

[0128] (Boron compounds) As a boron compound, there are no particular limitations as long as it contains boron atoms; for example, compounds containing boron-10 nuclides (…). 10 Compounds of type B). Furthermore, this boron compound is preferably used in neutron capture therapy. Specifically, examples include RB(OH)2 (boronic acid (where R is a substituent (e.g., selected from alkyl, aryl, heteroaryl, alkylaryl, etc.))), BPA (p-boron phenylalanine), Borax (borax), PCPB (4-carboxyphenylboronic acid), BODIPY (dipyrrolemethylene boron), boron clusters, etc.

[0129] In this specification, the term primarily refers to ionic or nonionic substances having a structure in which multiple boron atoms are aggregated and bonded together, preferably boron clusters having 3 to 20 boron atoms, more preferably boron clusters having 8 to 20 boron atoms, and particularly preferably boron clusters having 10 to 12 boron atoms.

[0130] Examples of boron clusters include, for instance, decaborane (B 10 H 14 ), decahydrodecaborate ([B) 10 H 10 ] 2- (GB-10) (sometimes also called sodium decaborate) , dodecaborate ([B 12 H 12 ] 2- Nested, nested-octadecylborane (22) (B) 18 H 22 Boronhydrides such as CAS RN: 21107-56-2, but not limited to these.

[0131] In addition to boron atoms, the boron cluster may also contain carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, etc., as the framework atoms constituting it. The number of other atoms such as carbon atoms, nitrogen atoms, oxygen atoms, and sulfur atoms in the boron cluster is preferably 0 to 5, more preferably 0 to 2.

[0132] Boron clusters, whose basic framework contains atoms other than boron atoms, include, for example, carboranes (including so-called carborane isomers) containing both boron and carbon atoms. Among carboranes, closed carboranes ([CB) containing one carbon atom are known, for example. 11 H 12 ] - Closed carboranes containing two carbon atoms (C2B) 10 H 12 Nested carborane ([C2B9H) 11 ] - Or, for example, the molecular formula is M(C2B9H) 10 The sandwich-type compounds shown in Figure 2 are examples, but not limited to these. In the above molecular formula, M represents a transition metal element, which can be, for example, Fe, Ni, Co, Mo, etc. That is, the boron cluster can be a metal complex with transition metals such as Fe, Ni, Co, Mo, etc.

[0133] Boron clusters are preferably ionic, and even more preferably water-soluble. Examples of ionic or water-soluble boron clusters include, for instance, dodecaborate ([B...12 H 12 ] 2- Dodecoborate with a thiol group ([B)) 12 H 11 SH] 2- BSH (sometimes also called borate (sodium) or mercaptoundecylhydrododecoborate), and dodecoborate with hydroxyl groups ([B 12 H 11 OH] 2- ), dodecoborate with an amino group ([B 12 H 11 NH3] - )wait.

[0134] (Gadolinium compounds) As for gadolinium compounds, there are no particular limitations as long as they contain gadolinium atoms. For example, compounds containing gadolinium atoms... 157 Gd, 157 Compounds of Gd.

[0135] (Anticancer agent) In this specification, an anticancer agent refers to a drug intended to inhibit the proliferation of malignant tumors (cancer). The mechanism of action of anticancer agents is not particularly limited. Anticancer agents can be metabolic antagonists, alkylating agents, anticancer antibiotics, microtubule inhibitors, platinum preparations, topoisomerase inhibitors, molecularly targeted drugs, etc. The conjugates of this invention may contain two or more identical or different anticancer agents.

[0136] Metabolic antagonists can be, for example, folic acid metabolism antagonists, dihydropteroate synthase inhibitors, dihydrofolate reductase inhibitors (DHFR inhibitors), pyrimidine metabolism inhibitors, thymidylate synthase inhibitors, purine metabolism inhibitors, IMPDH inhibitors, ribonucleotide reductase inhibitors, nucleoside analogs, L-asparaginase, etc. Specific examples of metabolic antagonists include enoxabin (Sunrabin), capecitabine (Xeloda), carmoflu (Pyriflu), cladribine (Leustatin), gemcitabine (Genza), cytarabine (Cylocide), cytarabine alkylphosphamide (Starasid), tegafur (Atilon, Aftofur, Tefsil, Futraful, Lunasin, etc.), tegafur-uracil (UFT), tegafur-gemeracil-oteracil potassium (TS-1: TS-One), deoxyfluorouridine (Furtulon), nerabine (Arranon G), hydroxyurea (Hydrea), fluorouracil (5-FU, Carzonal, Bennan, Lunacor, Lunabon), fludarabine (Fludara), pemetrexed (Alimta), pentostatin (Cohorin), leuchelin, and methotrexate.

[0137] Specific examples of alkylating agents include nitrogen mustard alkylating agents such as cyclophosphamide (Endoxan), ifomide, melphalan (Alkerans), busulfan, and tespamine (Tespamin), and nitrosourea alkylating agents such as nimustine (Nidran), ramustine (Cymerin), dacarbazine, procarbazine (procarbazine hydrochloride), temozolomide (Temodal), carmustine (Gliadel), streptozotocin (Zanosar), and bendamustine (Treakisym).

[0138] Specific examples of anticancer antibiotics include actinomycin D (Cosmegen), aclacinon, calsed, idarubicin, epirubicin (epirarubicin hydrochloride, Farmorubicin), smansine temalame (SMANCS), daunomycin, adriacin, pinorubin (THERARUBICIN), bleomycin (Bleo), pepleomycin (Pepleo), mitomycin C, mitoxantrone (NOVANTRON), and liposomal doxorubicin (DOXIL).

[0139] Examples of microtubule inhibitors include, for instance, vinca alkaloid-based microtubule polymerization inhibitors such as Exal, Oncovin, and Fildesin, and taxane-based microtubule depolymerization inhibitors such as TAXOL, TAXOTERE, and Vittin. Another example is Monomethylauristatin E (MMAE).

[0140] Examples of platinum-based preparations include oxaliplatin (ELPLAT), carboplatin (Carboplatin, Carbomerck, Paraplatin), cisplatin (IA-call, Konaburi, Cisplatin, etc.), and nedaplatin (Akpra).

[0141] Examples of topoisomerase inhibitors include, for instance, type I topoisomerase inhibitors such as camptothecin and its derivatives (e.g., irinotecan, hycamtin, SN-38, etc.); type II topoisomerase inhibitors such as anthracycline drugs such as doxorubicin, epipodophyllotoxin drugs such as etoposide (Lasted, Bepside), and quinolone drugs such as levofloxacin (CRAVIT) and ciprofloxacin (Ciproxan).

[0142] Examples of molecularly targeted drugs include regorafenib (Stivarga), cetuximab (ERBITUX), panitumumab (Vectibix), ramucirumab (Cyramza), gefitinib (IRESSA), erlotinib (TARCEVA), afatinib (Giotrif), crizotinib (XALKORI), alectinib (ALECENSA), ceritinib, lenvatinib (Lenvima), trastuzumab (HERCEPTIN), and lapatinib (Ty Kerb), Pertuzumab (PERJETA), Sunitinib (SUTENT), Sorafenib (Nexavar), Axitinib (Inlyta), Pazopanib (Votrient), Nirumab (OPDIVO), Pembrolizumab, Ipilimumab (YERVOY), Vemurafenib (ZELBORAF), Everolimus (AFINITOR), Tesseirolimus (TORISEL), Rituximab (Rituxan), Bevacizumab (AVASTIN), Geldermycin, etc.

[0143] In addition, anticancer agents can also be anti-angiogenic agents. Anti-angiogenic agents can be vascular endothelial growth factor (VEGF) or other angiogenic factors, or those that inhibit their receptors. Specific examples of anti-angiogenic agents include angiostatin, endostatin, migration inhibitory factor, anti-VEGF antibodies (e.g., AVASTIN), VEGFR-2 inhibitors (e.g., SU5416, SU6668), etc.

[0144] (Detectable substances) In this specification, a detectable substance refers to any substance that enables the conjugates of the present invention, containing therein, to be detected. Preferably, the detectable substance enables the conjugates of the present invention to be detected in vivo, either directly or indirectly using a suitable visualization or imaging (imaging) method. Examples of visualization or imaging methods include, but are not limited to, X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy. Detectable substances may be, for example, radioactive isotopes, MRI enhancers (e.g., paramagnetic ions), radioactive impermeable substances, contrast agents, fluorescent substances, etc.

[0145] Examples of radionuclides useful for PET include, for instance. 18 F, 51 Mn, 52m Mn, 52 Fe、 55 Co、 62 Cu、 64 Cu、 68 Ga、 72 As、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 89 Zr、 94m Tc, 110 In、 120 I, 124 I. Examples of radionuclides that can be used for the detection of gamma rays include, for example, 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 67 Ga、 75 Se、 90 Y、 97 Ru、 99m Tc, 111 In、 114m In、123 I, 125 I, 131 I, 169 Yb、 177 Lu、 192 Ir、 197 Hg, 198 AU 201 Tl、 211 At、 225 Ac、 223 Ra et al.

[0146] Examples of preferred paramagnetic ions include, for example, chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and erbium (III), with gadolinium being particularly preferred. Additionally, metals such as lanthanum (III), gold (III), lead (II), and bismuth (III) can also be used for applications such as X-ray imaging.

[0147] As radioactive impermeable substances and contrast agents, such as iodine compounds (e.g., organic iodic acids such as iodocarboxylic acid, iodoform, triiodophenol, tetraiodoethylene, etc.), barium compounds (e.g., barium sulfate, etc.), gallium compounds (e.g., gallium citrate, etc.), thallium compounds (e.g., thallium chloride, etc.).

[0148] Examples of fluorescent substances include rhodamine, fluorescein, Cy dyes (e.g., Cy3, Cy5, Cy5.5, Cy7, Cy7.5, etc.), Alexa Fluor (registered trademark), phycoerythrin (PE), allophycocyanin (APC), and their derivatives. In addition, near-infrared fluorescent reagents such as indocyanine green are also cited as preferred fluorescent substances.

[0149] (The combination of peptides and components in this invention) The manner in which the peptide of the present invention binds to one or more components in the conjugates of the present invention is not particularly limited. Binding can be direct or indirect, such as through a linker. Binding can be based on covalent bonds, non-covalent bonds, or combinations thereof. One or more components can bind directly or indirectly to the N-terminus, C-terminus, or other positions of the peptide of the present invention. The linking of peptides to other components (or second peptides) is well known in the art, and in the conjugates of the present invention, this binding can also be based on any known method.

[0150] As an example, in cases where the crosslinking occurs via a connector, known crosslinkers such as NHS esters, Sulfo-NHS esters, imide esters, maleimides, carbodiimides, allyl azides, diazacyclopropane, isocyanates, psoralen, and combinations thereof (e.g., homodifunctional crosslinkers, heterodifunctional crosslinkers, etc.) can be used. Examples of homodifunctional crosslinkers include DST, BS2G, DSG, BS3, DSS, DSP, DTSSP, DSSeb, EGS, and Sulfo-EGS. Additionally, examples of heterogeneous bifunctional crosslinks include SBA, SIA, Sulfo-SIA, BMPS, SPDP, GMBS, MBS, Sulfo-MBS, ANB-NOS, SMCC, Sulfo-SMCC, PDPH, EMCS (N-(6-maleimide hexanoyloxy)succinimide), SMPB, SMPH, LC-SPDP, Sulfo-LC-SPDP, and Sulfo-SNAPAH. Alternatively, peptide linkers, such as dipeptide linkers (e.g., Val-Cit linkers), can be used. These linkers can be protected with Fmoc or Alloc groups, or maleimide groups can be introduced. Furthermore, two or more of the linkers described above can be combined. Additionally, the peptides of the present invention can be modified according to the crosslinking agent used. For example, a cysteine ​​residue can be pre-added to the C-terminus of the peptide of the present invention for binding with a maleimide linker.

[0151] In addition, in order to link the radioactive materials (nuclides), paramagnetic ions, and peptides of the present invention as described above, suitable chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 4,7,10-tetraazacyclododecane-N-N',N'',N'''-tetraacetic acid (DOTA) etc.) and / or metallothioneins, etc. (see, for example, Cumali Aktolun et al., “Nuclear Medicine Therapy: Principles and Clinical Applications”, Springer, 2013, etc.).

[0152] The conjugates of the present invention can be safely administered orally or non-orally to the subjects described below. Non-oral administration includes intravenous, intramuscular, subcutaneous, intra-visceral, intranasal, intradermal, ocular, intracerebral, rectal, vaginal, intraperitoneal, intratumoral, proximal to tumor, and direct administration to the lesion.

[0153] 3. The pharmaceutical composition of the present invention In addition, the present invention provides pharmaceutical compositions comprising the peptides or conjugates of the present invention (hereinafter also referred to as pharmaceutical compositions of the present invention). The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutical compositions may be provided in dosage forms suitable for oral or non-oral administration. Non-oral administration includes intravenous, intramuscular, subcutaneous, intra-visceral, intranasal, intradermal, ophthalmic, intracerebral, rectal, vaginal, intraperitoneal, intratumoral, proximal to tumor, and direct administration to the lesion. This administration can be performed using, for example, injection, endoscopy, catheters, etc.

[0154] As a pharmaceutical composition for non-oral administration, injections, suppositories, etc., can be used, and injections can include dosage forms such as intravenous injections, subcutaneous injections, intraperitoneal injections, intradermal injections, intramuscular injections, and intravenous drips. Such injections can be prepared according to known methods. As a method of preparing the injection, for example, it can be prepared by dissolving, suspending, or emulsifying the peptide or conjugate of the present invention in a sterile aqueous liquid or oily liquid commonly used in injections. As an aqueous liquid for injection, physiological saline, isotonic solutions containing glucose or other adjuvants, etc., can be used, and suitable dissolving agents can also be used, such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)), etc. As an oily liquid, castor oil, soybean oil, etc., can be used, and benzyl benzoate, benzyl alcohol, etc., can also be used as dissolving agents. The prepared injection solution is preferably filled into a suitable ampoule. Suppositories used for rectal administration can be prepared by mixing the peptides or conjugates of the present invention into a conventional suppository matrix.

[0155] Examples of pharmaceutical compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such pharmaceutical compositions are manufactured by known methods and may also include carriers, diluents, excipients, etc., commonly used in the pharmaceutical industry. Examples of carriers and excipients used for tablets include, for instance, lactose, starch, sucrose, and magnesium stearate.

[0156] In the pharmaceutical composition of the present invention, the content of the peptide or conjugate of the present invention varies depending on the form of the pharmaceutical composition (formulation). Generally, the amount of the peptide or conjugate of the present invention relative to the whole pharmaceutical composition (whole formulation) is about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0157] It should be noted that the pharmaceutical compositions of the present invention may also contain other active ingredients, provided that no undesirable interactions occur due to their combination with the peptides or conjugates of the present invention.

[0158] The above-mentioned non-oral or oral pharmaceutical compositions are preferably prepared in dosage forms that correspond to the dosage of the active ingredient. Examples of such dosage forms include tablets, pills, capsules, ampoules, and suppositories. The content of the peptide or conjugate is not particularly limited as long as the desired efficacy can be obtained; typically, each dosage form is 0.01 mg to 50 g, injections may be 0.01 mg to 25 g, and other dosage forms may be 0.01 mg to 50 g.

[0159] The pharmaceutical compositions of the present invention can target tumors, particularly angiogenic malignant tumors, and preferably cause the peptide or conjugate to aggregate in the tumor. Therefore, pharmaceutical compositions of the present invention containing anticancer agents, or boron compounds, gadolinium compounds, etc., in their conjugates can be used for the targeted treatment or prevention of malignant tumors. Furthermore, compositions of the present invention containing detectable substances, etc., in their conjugates can be used for the examination and diagnosis of malignant tumors.

[0160] Malignant tumors (cancer) can be any type of cancer, and can be solid or liquid-filled cancer. As solid cancer, ANXA1-positive solid cancer is preferred, and solid cancer expressing ANXA1 on the cell surface is more preferred. Therefore, solid cancers with angiogenesis can be cited as examples. Examples of solid cancers include, for example, cancers of the brain and nervous system (e.g., brain tumors, spinal cord tumors), head and neck cancers (e.g., laryngeal cancer, oral cancer, salivary gland cancer, sinus cancer, thyroid cancer), digestive system cancers (e.g., stomach cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer), urinary or reproductive system cancers (e.g., kidney cancer, renal cell carcinoma, bladder cancer, prostate cancer, renal pelvis and ureter cancer, gallbladder cancer, bile duct cancer, testicular cancer, penile cancer, uterine cancer, endometrial cancer, uterine sarcoma, cervical cancer, vaginal cancer, vulvar cancer, ovarian cancer, fallopian tube cancer), respiratory system cancers (e.g., lung cancer (including small cell lung cancer, non-small cell lung cancer, metastatic lung cancer), bronchial cancer), breast cancer, skin cancer (e.g., malignant melanoma), bone cancer (e.g., osteosarcoma), and muscle cancer (e.g., rhabdomyosarcoma). Preferred solid cancers include skin cancer (e.g., malignant melanoma), brain and nervous system cancers (e.g., refractory brain tumors, spinal cord tumors), laryngeal cancer, oral cancer, salivary gland cancer, sinus cancer, thyroid cancer, bladder cancer, prostate cancer, renal pelvis and ureter cancer, osteosarcoma, etc.

[0161] Examples of liquid-borne cancers include leukemia, malignant lymphoma, multiple myeloma, and myelodysplastic syndrome. Examples of leukemia include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphoblastic leukemia. Malignant lymphomas are classified into Hodgkin's lymphoma and non-Hodgkin's lymphoma. Examples of non-Hodgkin's lymphoma include B-cell lymphoma, adult T-cell lymphoma, lymphoblastic lymphoma, diffuse large cell lymphoma, Burkitt lymphoma, follicular lymphoma, MALT lymphoma, peripheral T-cell lymphoma, and mantle cell lymphoma.

[0162] The peptides or conjugates of the present invention can efficiently cross the blood-brain tumor barrier; therefore, brain tumors are, for example, preferred targets. The brain tumor can be a primary brain tumor or a metastatic brain tumor. Furthermore, the brain tumor can be benign (e.g., meningioma, pituitary adenoma, schwannoma, etc.) or malignant, preferably a malignant brain tumor. Examples of malignant brain tumors include grade 2 brain tumors such as astrocytoma and oligodendroglioma, grade 3 brain tumors such as anaplastic astrocytoma, anaplastic oligodendroglioma, and anaplastic oligodendroastrocytoma, and grade 4 brain tumors such as glioblastoma.

[0163] The pharmaceutical composition of the present invention can be administered to objects expressing annexin A1, such as animals, particularly mammals. Examples of mammals include, but are not limited to, rodents such as mice, rats, hamsters, and guinea pigs, laboratory animals such as rabbits, livestock such as pigs, cattle, goats, horses, sheep, and mink, pets such as dogs and cats, humans, monkeys, macaques, marmosets, orangutans, chimpanzees, and other primates.

[0164] The dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, the recipient, the disease, symptoms, route of administration, etc. For example, when used for the treatment or prevention of cancer as described above, the conjugate of the present invention, containing an anticancer agent, a boron compound, and a gadolinium compound, is typically administered once weekly by intravenous injection or oral administration at a dose of about 0.01 mg to 50 g / kg body weight. Alternatively, when used for the examination or diagnosis of cancer as described above, the conjugate of the present invention, containing a detectable substance, is typically administered intravenously or orally before the examination at a dose of about 0.01 mg to 50 g / kg body weight.

[0165] 4. The agents and reagents of the present invention (Neutron capture therapy agent) Furthermore, the present invention also provides a neutron capture therapy agent comprising the conjugate of the present invention (hereinafter also referred to as the neutron capture therapy agent of the present invention). This conjugate comprises a boron compound or a gadolinium compound as described above. The neutron capture therapy agent of the present invention can aggregate on malignant tumors (cancers) expressing ANXA1 as described above on the cell surface through the peptide of the present invention contained therein. This aggregation means selectively tending towards (binding to) or localizing to cancerous tissue in subjects (e.g., animals, more specifically mammals (e.g., humans, etc.)) compared to non-cancer tissue (e.g., normal tissue, etc.). Therefore, the neutron capture therapy agent of the present invention is preferably used for neutron capture therapy (NCT), and more specifically preferably for boron neutron capture therapy (BNCT) and gadolinium neutron capture therapy (GdNCT). Therefore, the present invention further provides a neutron capture therapy comprising administering to a subject an effective amount of the conjugate of the present invention comprising a boron compound, a gadolinium compound, etc., as described above. The neutron capture therapy of the present invention can be used for the treatment of a disease suffered by the subject, and can also be used for prevention (e.g., prevention of recurrence of the disease).

[0166] In the neutron capture therapy agent of the present invention, the content of the conjugate of the present invention varies depending on the form of the formulation. Generally, the amount of the conjugate of the present invention relative to the whole formulation is about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0167] In neutron capture therapy, after the neutron capture therapy agent of the present invention reaches the tumor, the site is irradiated with an effective amount of low-energy thermal neutron rays, such as thermal neutron rays or hyperthermal neutron rays. The site can be irradiated through the skin, or the site can be fully or partially exposed before irradiation. Furthermore, irradiation can be performed simultaneously from multiple directions. The administration of the neutron capture therapy agent of the present invention and subsequent irradiation with thermal or hyperthermal neutron rays can be repeated as needed. For example, multiple irradiations can be performed at intervals of approximately several months. The total number of irradiations is preferably 1 to 10.

[0168] The neutron capture therapy agent of the present invention can typically be administered to mammals suffering from malignant tumors (cancer) within 72 hours prior to irradiation with thermal neutron rays or hyperthermal neutron rays (preferably 5 minutes to 48 hours prior, more preferably 30 minutes to 30 hours prior). Alternatively, if necessary, it can also be administered during irradiation with thermal neutron rays or hyperthermal neutron rays. The typical dosage of the neutron capture therapy agent of the present invention, for a single irradiation with thermal neutron rays or hyperthermal neutron rays, is in the range of 0.01 mg to 50 g / kg body weight. The irradiation time for a single irradiation with thermal neutron rays or hyperthermal neutron rays is preferably 1 minute to 5 hours, more preferably 10 minutes to 2 hours. The amount of irradiation with thermal neutron rays or hyperthermal neutron rays is not particularly limited, as long as it is the usual amount used in neutron capture therapy.

[0169] The capture therapy using the neutron capture therapy agent of the present invention can be applied as a standalone treatment or in conjunction with conventional surgery or chemotherapy. If necessary, after surgical removal of the tumor as much as possible, the capture therapy using the neutron capture therapy agent of the present invention can also be used to destroy any remaining tumor.

[0170] In neutron capture therapy, the neutron capture therapy agent of the present invention can be used in combination with, for example, anticancer agents, pharmaceutical compositions of the present invention, and chemotherapeutic agents of the present invention described later. When using combination drugs, there is no limitation on the timing of administration of the neutron capture therapy agent of the present invention and the combination drug; the neutron capture therapy agent of the present invention and the combination drug can be administered simultaneously to a subject suffering from a malignant tumor (cancer), or they can be administered at time intervals. When administering at time intervals, there is no particular limitation on the order of administration.

[0171] All other necessary matters related to the neutron capture therapy agent of the present invention, other than those described above, are pursuant to the contents of "1. the peptide of the present invention" and "2. the conjugate of the present invention".

[0172] (Chemotherapy drugs for cancer) Furthermore, the present invention also provides a cancer chemotherapeutic agent comprising the conjugate of the present invention (hereinafter also referred to as the cancer chemotherapeutic agent of the present invention). This conjugate comprises the anticancer agent as described above. The cancer chemotherapeutic agent of the present invention can aggregate on malignant tumors (cancers) expressing ANXA1 as described above on the cell surface through the peptide of the present invention contained therein. This aggregation means selectively tending towards (binding to) or being locally present in cancerous tissue compared to non-cancer tissue (e.g., normal tissue) in the subjects described above (e.g., animals, more specifically mammals (e.g., humans, etc.)). Therefore, the cancer chemotherapeutic agent of the present invention is preferably used for the treatment of cancer. Therefore, the present invention further provides a method for the treatment or prevention of cancer, comprising administering to a subject an effective amount of the conjugate of the present invention comprising an anticancer agent, a boron compound, a gadolinium compound, etc., as described above.

[0173] In the cancer chemotherapy agent of the present invention, the content of the conjugate of the present invention varies depending on the form of the formulation. Generally, the amount of the conjugate of the present invention relative to the whole formulation is about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0174] All other necessary matters related to the cancer chemotherapy agents of the present invention, other than those described above, are pursuant to the contents of "1. the peptide of the present invention" and "2. the conjugate of the present invention".

[0175] (Reagents used for cancer screening) Furthermore, the present invention also provides a cancer examination reagent comprising the conjugate of the present invention (hereinafter also referred to as the cancer examination reagent of the present invention). The conjugate comprises the detectable substance as described above. The cancer examination reagent of the present invention can aggregate on malignant tumors (cancers) expressing ANXA1 as described above on the cell surface by means of the peptide of the present invention contained in the reagent. This aggregation means selectively tending towards (binding to) or being locally present in cancerous tissue compared to non-cancer tissue (e.g., normal tissue) in the subject as described above (e.g., animals, more specifically mammals (e.g., humans, etc.)). Therefore, the cancer examination reagent of the present invention is preferably used for cancer examination. Therefore, the present invention further provides a cancer detection method comprising administering to a subject an effective amount of the conjugate of the present invention comprising the detectable substance, etc., as described above. Examples of cancer examinations include X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound examination, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy as described above. In addition, the testing reagents of the present invention may also include companion diagnostic drugs that pre-determine the likelihood of the efficacy of anticancer agents such as molecularly targeted drugs.

[0176] In the cancer examination reagent of the present invention, the content of the conjugate of the present invention varies depending on the form of the preparation. Generally, the amount of the conjugate of the present invention relative to the whole preparation is about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0177] All other necessary matters related to the cancer examination reagents of the present invention, except as described above, are pursuant to the contents of "1. the peptide of the present invention" and "2. the conjugate of the present invention".

[0178] The following examples illustrate the invention in more detail, but the invention is not limited thereto. Example

[0179] Example 1: Screening for D-type peptide sequences that bind to the N-terminal 15 residues of Annexin A1 within 5 minutes at 37°C using mirror phage display. To screen for 7-residue D-type peptide sequences that selectively bind to the N-terminal 15 residues of Annexin A1 (ANXA1) within 5 minutes at 37°C, mirror phage display screening was performed. Figure 1 and Figure 2 ).

[0180] A peptide (D-MC16) with d-cys residues added to the N-terminal 15-residue amino acid sequence of ANXA1 synthesized using D-type amino acids (mamvseflkqawfie (SeqID NO:29): Hereinafter, D-type amino acids are represented by lowercase letters) was synthesized by adding d-cys residues. This was achieved through the SH group of cys. Figure 1 The concentrations shown (10 nM, 1 nM, 0.1 nM, 0.01 nM) were immobilized on maleimide-coated plates. 200 μl of a T7 phage mixture presenting a 7-residue L-amino acid peptide library was added to the L-cysteine ​​immobilization wells and stirred at room temperature for 1 hour. 200 μl of the T7 phage mixture not bound to the L-cysteine ​​immobilization wells was recovered and added to the 10 nM D-MC16 peptide immobilization wells. The mixture was incubated at 37°C for 5 minutes. After washing, the phage bound to the 10 nM D-MC16 peptide was eluted with 100 μl of 1% SDS-PBS (first round output).

[0181] The first-round output phage mixture was added to 15 ml of logarithmically proliferating *E. coli* BL21 culture medium and incubated at 37°C for 3 hours with stirring. The amplified first-round output phage mixture was obtained as the supernatant after centrifugation. Next, 200 μl of the amplified first-round output phage mixture was added to 1 nM D-MC16 peptide immobilization wells and incubated at 37°C for 5 minutes. After washing, the phage bound to the 1 nM D-MC16 peptide was eluted with 100 μl of 1% SDS-PBS (second-round output). The second-round output phage mixture was added to 15 ml of logarithmically proliferating *E. coli* BL21 culture medium and incubated at 37°C for 3 hours with stirring. The amplified second-round output phage mixture was obtained as the supernatant after centrifugation.

[0182] Next, 200 μl of the second-round output phage mixture was added to the 0.1 nM D-MC16 peptide immobilization wells and incubated at 37°C for 5 minutes. After washing, the phages bound to the 0.1 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (third-round output). The third-round output phage mixture was added to 15 mL of logarithmically growing E. coli BL21 culture medium and incubated at 37°C for 3 hours with stirring. The amplified third-round output phage mixture was obtained as the supernatant after centrifugation. Then, 200 μl of the third-round output phage mixture was added to the 0.01 nM D-MC16 peptide immobilization wells and incubated at 37°C for 5 minutes. After washing, the phages bound to the 0.01 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (fourth-round output). The fourth round of output phage mixture was added to 15 mL of logarithmic growth Escherichia coli BL21 culture medium and cultured at 37°C for 3 hours with stirring. The amplified fourth round of output phage mixture was obtained as the supernatant after centrifugation.

[0183] Next, 200 μl of the fourth round of output phage mixture was added to the 10 nM L-MC16 peptide (L-MC16) immobilization wells and incubated at 37°C for 5 minutes. The remaining 200 μl of the fourth round of output phage mixture that did not bind to L-MC16 was then recovered. 200 μl of the fourth round of output phage mixture that did not bind to L-MC16 peptide was then added to the 10 nM D-MC16 peptide immobilization wells and incubated at 37°C for 5 minutes. After washing, the phage bound to the 10 nM D-MC16 peptide (fifth round output) was eluted with 100 μl of 1% SDS-PBS.

[0184] The copy number of phages eluted in each round was calculated by absolute quantification of the genome copy number of T7 phage using digital PCR. Figure 3 The copy number of the phage mixtures obtained from rounds 1 to 5 was quantified. The results showed that the copy number of phages binding to D-MC16 reached its maximum in round 4. In round 5, the copy number of phages binding to L-MC16 was subtracted from the copy number of phages binding to D-MC16, indicating that the phages specifically binding to D-MC16 were concentrated.

[0185] Amplicon sequencing was performed on the 7-residue peptide sequences presented by the phage mixtures obtained from rounds 1 to 5 using a next-generation sequencer. The frequency of peptide sequence clones presented by each phage mixture was analyzed. Figure 4 ).

[0186] Based on amplicon sequencing analysis using a next-generation sequencer, the most frequent 7-residue peptide sequence HPNEVRS (Seq ID NO:30) in the fifth round output was designated as the dMC16-binding peptide sequence, named HP7 peptide, and synthesized as the D-type amino acid peptide hpnevrs (dhp7) (Seq ID NO:31). Additionally, the second and third most frequent 7-residue peptide sequences STSRNTL (Seq ID NO:32) and QYATNLK (Seq ID NO:33) were named ST7 and QY7 peptides, respectively, and synthesized as the D-type amino acid peptides stsrntl (dst7) (Seq ID NO:34) and qyatnlk (dqy7) (Seq ID NO:35). Among the top 10 sequences, half of them identified common sequences for H / QP / S-NEVRS (i.e., HPNEVRS (Seq ID NO:30), QPNEVRS (Seq ID NO:36), HSNEVRS (Seq ID NO:37), and QSNEVRS (Seq ID NO:38)). Figure 5 ).

[0187] Example 2: Binding affinity analysis of D-type peptides bound to L-MC16 to ANXA1 N-terminal MC16 To determine the binding affinity of the candidate D-peptide selected in Example 1 for L-MC16 to L-MC16 or D-MC16, biolayer interferometry (BLI) was performed using a ForteBio Octet K2 device (Sartorius, Gottingen, Germany). It was assumed that the interaction between the L-MC16 sequence (MAMVSEFLKQAWFIE) (Seq ID NO:28) within the N-terminal domain of ANXA1 and dhp7 occurs when L-MC16 is localized to the cell membrane.

[0188] In mirror phage display screening, the C-terminal side of the presented L-peptide is exposed to the outermost edge of the phage coat protein. Therefore, biotin-modified D-peptides (Biotin-dhp7, Biotin-dst7, Biotin-dqy7) with biotin at the N-terminus were synthesized by Biologica Inc. Each biotinylated D-peptide was diluted at 50 μg / ml with Kinetic buffer and immobilized on an Octet superstreptavidin (SSA) biosensor (Sartorius) in a manner consistent with the peptide presentation pattern of the phage coat, ensuring that the C-terminal side of each D-peptide is exposed to the outermost edge of the SSA biosensor.

[0189] The reference method involved immobilizing 50 μg / ml biotin onto an SSA biosensor. Each immobilized sensor was immersed in kinetic buffer containing 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, or 1.5625 μM L-MC16 for 120 seconds (association phase), followed by dissociation at 37°C for 120 seconds (dissociation phase) to regenerate the biosensor.

[0190] The biosensor was regenerated using a 0.1 mM glycine hydrochloride solution (pH 2.0), applied in three pulses for 5 seconds each time. This association-dissociation-regeneration process was performed on L-MC16 or D-MC16 solutions of various concentrations. Data were analyzed using Octet Dataanalysis HT software version 10 (Sartorius). The obtained data were fitted to a 1:1 binding model to determine the values ​​of Kon and Koff, and the equilibrium dissociation constant KD was calculated. Figure 6 (A~C).

[0191] according to Figure 6 Results A through C show that, among the D-type peptide sequences tested, dhp7 exhibited a KD value of 0.48 μM for the L-MC16 sequence, which is the highest affinity compared to dqy7 (0.99 μM) and dst7 (0.72 μM). Furthermore, no binding of dhp7 to D-MC16 was observed, indicating that dhp7 specifically binds to L-MC16. Based on these results, dhp7 was used in subsequent studies.

[0192] Example 3: Analysis of the amino acid sequence of L-MC16, an important link between dhp7 peptide and the N-terminal L-MC16 of L-type ANXA1. To determine the L-MC16 amino acid crucial for the binding of the dhp7 peptide to L-MC16, biolayer interferometry (BLI) was performed using a ForteBio Octet K2 device (Sartorius, Gottingen, Germany), as in Example 2. 50 μg / ml of Biotin-dhp7 was diluted with Kinetic buffer and immobilized on the Octet Super Streptavidin (SSA) biosensor (Sartorius) in a manner consistent with the peptide presentation pattern of the phage coat, with the C-terminal side of dhp7 exposed to the outermost edge of the SSA biosensor. As a reference, 50 μg / ml of biotin was immobilized on the SSA biosensor. To evaluate the specificity of dhp7 binding to L-MC16 mutants, binding affinity was analyzed for 100 μM of wild-type (L-MC16WT) and mutant L-MC16. Figure 7 (A and B).

[0193] The results showed that the binding affinity of dhp7 to the L-MC16 mutants E6A, F7A, K9A, Q10A, W11A, F13A, and E15A was significantly lower than that to L-MC16WT, indicating that the E6, F7, K9, Q10, W11, F13, and E15 residues of L-MC16 are important amino acids for binding to dhp7. Figure 7 (A and B).

[0194] Example 4: Visualization of dhp7 peptide uptake in ANXA1-positive HEK293 cells using immunofluorescence staining The uptake of the dhp7 sequence, which selectively binds to the MC16 sequence of ANXA1 identified in Examples 1-3, by ANXA1-positive HEK293 cells was investigated. HEK293T cells were transfected with the pCMV3-hANXA1-c-His plasmid, which incorporates a His tag fused to the C-terminus of human ANXA1. After 72 hours, HEK293 cells were supplemented with either a biotinylated dhp7 peptide (50 μg / mL) at the N-terminus or an anti-MC16 antibody (5 μg / mL) reacting with the N-terminus of ANXA1. Cells were incubated at 37°C for 10 minutes and then fixed with 1% PFA. To detect Biotin-dhp7, streptavidin-Alexa Fluor 555 (500-fold dilution) was added, or anti-mouse IgG-Alexa Fluor 555 (500-fold dilution) was added to detect anti-MC16 antibody. After incubation at 37°C for 10 minutes, followed by washing with PBS, the binding of the dph7 peptide to HEK293 overexpressing Anxa1-c-His and its intracellular uptake were investigated using a fluorescence microscope (Keyence BZ9000). Figure 8 and 9 ).

[0195] As a result, compared with HEK293 cells that had been introduced with a negative control plasmid, the Biotin-dhp7 peptide showed higher staining intensity in HEK293 cells overexpressing ANXA1-c-His. Similar to ANXA1 detected using anti-MC16 antibody, a dotted staining pattern was observed within the cells, and subsequently, the cytoplasm was also diffusely stained. This suggests that dhp7 may bind to ANXA1 and be taken up into the cells.

[0196] Example 5: In vivo imaging of tumor aggregation of EMCS-Cy7.5-c-hpnevrs peptide Since Example 4 clearly demonstrated that the dhp7 peptide is taken up by ANXA1-positive cells, an in vivo imaging study was then conducted by administering fluorescently labeled dhp7 to cancer-carrying mice. c(EMCS-Cy7.5)-hpnevrs(Cy7.5-dhp7) (Seq ID NO:31) (Chem. 3) was synthesized with Cy7.5 fluorescent dye bound to the side chain of the N-terminal d-cys residue of the c-hpnevrs sequence (Seq ID NO:31) via an EMCS linker.

[0197] [Chemistry 3] Cell suspensions of human head and neck cancer cell line SAS, osteosarcoma cell line MG-63, prostate-specific membrane antigen (PSMA) negative or PSMA positive prostate cancer cell line PC3-PSMA±, and mouse bladder cancer cell line MBT2 were reacted with anti-MC16 antibody (1 μg / ml) reacting with the N-terminus of ANXA1 for 1 hour at room temperature. After washing the cell clumps, they were reacted with APC-labeled anti-mouse IgG antibody for 1 hour at room temperature. After washing the cell clumps, the expression of ANXA1 on the cell surface was studied by flow cytometry. The results are shown in Figure 1. Figure 10 Antibody reactions are carried out under non-fixed, cell membrane-impermeable conditions.

[0198] Figure 10 The results showed that there were differences in the expression intensity of ANXA1 among different cancer cell lines, but all of them were expressed on the cell surface. This indicates that in PSMA-negative PC3 (PC3-PSMA-) cell lines, the expression of ANXA1 on the cell surface was lower than that in PSMA-positive PC3 (PC3-PSMA+) cell lines. The expression of ANXA1 on the cell surface in MG-63 and SAS cells was at the same level. In MBT2 cells, there were cell populations with strong and weak expression of ANXA1 on the cell surface.

[0199] The tumor aggregation of Cy7.5-c-dhp7 in ANXA1-positive bladder cancer-carrying mice was investigated. 4×10⁻⁶ cells were used. 5 MBT2 mouse bladder cancer cells were inoculated into the right hind thigh of C3H / HeN mice. One week later, mice with tumors reaching a diameter of 5 mm were intraperitoneally administered Cy7.5-dhp7 dissolved in physiological saline at a dose of 10 mg / kg (0.2 mg / 20 g mouse). Post-administration monitoring was performed using in vivo imaging (IVIS) at 0, 30, 90, 120 minutes, 24, 48, 72, and 96 hours. Figure 11 ).

[0200] As a result, the fluorescence intensity of Cy7.5-dhp7 in tumor tissue increased starting 30 minutes after administration, accumulating rapidly until 2 hours later, reaching a plateau after 24 hours, and exhibiting strong tumor-specific aggregation that persisted until 96 hours later. Furthermore, mice were sacrificed at 48 and 96 hours post-administration, and major organs were removed for in vitro imaging. The results showed strong aggregation of Cy7.5-dhp7 in tumor tissue and the kidney, as well as in the intestine, but no aggregation was observed in other organs. This suggests that Cy7.5-dhp7 may be a renal excretory form.

[0201] Furthermore, the uptake of Cy7.5-dhp7 in other cancer cells was investigated. 1×10 6Personal prostate cancer cells PC3-PSMA- and PC3-PSMA+, mouse bladder cancer cells MBT2, and human head and neck cancer cells SAS were inoculated into the backs of nude mice. Two weeks later, in cancer-carrying mice with tumors reaching a diameter of 15 mm, Cy7.5, Cy7.5-dhp7, or the L-type control peptide RRQRRAP (R7)-EMCS-Cy7.5 (Seq ID NO:39 (RRQRRAP)) dissolved in physiological saline was administered intraperitoneally at a dose of 10 mg / kg (0.2 mg / 20 g mouse). Post-administration monitoring was performed using in vivo imaging (IVIS) at 0, 20, 30, 90, 120 minutes, 24, 48, 72, and 96 hours. Figures 12-14 ).

[0202] As a result, in all cancer-carrying nude mice, Cy7.5-dhp7 reached its maximum 24 hours after administration, and its aggregation gradually decreased. Furthermore, mice were sacrificed 96 hours after administration, and major organs were removed for in vitro imaging. The results showed that Cy7.5-dhp7 exhibited strong aggregation in tumor tissue, kidney, and intestine, but no aggregation was observed in other organs, suggesting that Cy7.5-dhp7 may be renally excreted. Neither Cy7.5 alone nor the control L-type R7 peptide-Cy7.5 showed tumor-specific aggregation, thus demonstrating the tumor specificity of dhp7 for prostate cancer, bladder cancer, and head and neck cancer.

[0203] Example 6: EMCS using immunofluorescence staining- 10 Visualization of BSH-dhp7 peptide uptake in ANXA1-positive cancer cells Example 5 clearly demonstrated that Cy7.5-dhp7 was ingested into ANXA1-positive tumor tissues in a mouse model carrying cancer. Therefore, the EMCS- in various ANXA1-positive cancer cells was subsequently investigated. 10 BSH-dhp7 ( 10 The intake of BSH-dhp7 was investigated to confirm whether it was present in the cancer cells studied in Example 5. 10 BSH, created with 10 Rabbit antibodies that specifically respond to BSH 10 BSH polyclonal antibody. 200 μg 10 Two mice were immunized with BSH-KLH as the antigen. Whole blood was collected after the fourth booster immunization to obtain samples containing BSH-KLH. 10 Rabbit antibodies that specifically respond to BSH 10 Serum samples (#52 and #53) containing BSH polyclonal antibodies were purified to IgG fractions using a protein A column. IgG fractions were analyzed by ELISA and BLI assay. 10 Rabbit antibodies against BSH-BSA 10Affinity of BSH polyclonal antibodies (#52 and #53), results shown in Figure 15 .

[0204] Depend on Figure 15 The results confirmed that rabbit anti- 10 BSH polyclonal antibodies (#52 and #53) are both... 10 The BSH-BSA reaction occurs in a concentration-dependent manner. In the following experiments, in order to... 10 The BSH test used antibody #52 or #53.

[0205] Next, a compound was synthesized that binds to the side chain of the N-terminal His residue in the hpnevrs(dhp7) (Seq ID NO:31) sequence via an EMCS linker. 10 BSH's EMCS- 10 BSH-hpnevrs ( 10 BSH-dhp7) (Seq ID NO:31) (Chemistry 4).

[0206] [Chemistry 4] The study investigated cancer cells 10 BSH-dhp7 uptake. 50 μg of BSH-dhp7 was added to MBT2 and PC3-PSMA+ cells seeded in 3.5 cm culture dishes (ib81156) manufactured by Ibidi. 10 BSH-dhp7 or 10 After 24 hours of BSH treatment, cells were washed three times with PBS and then fixed with 4% PFA. After blocking with 3% BSA BD PhosFlow perm / wash buffer I (BD557885), cells were then treated with rabbit anti-BSA containing 1 μg / ml. 10 BSH polyclonal antibody #52 was reacted with 3% BSA BD PhosFlow perm / washbuffer I at room temperature for 1 hour to allow it to react with the ingested intracellular... 10 BSH reaction. After washing three times with PBS, rabbit anti-BSH was detected. 10 BSH polyclonal antibody was reacted with APC-labeled anti-mouse IgG antibody (1000-fold dilution) at room temperature for 1 hour. After washing three times with PBS, the antibody was analyzed relative to various cancer cells using a fluorescence microscope (Keyence BZ9000). 10 BSH-dhp7 or 10 Intracellular uptake of BSH ( Figure 16 ).

[0207] As a result, along with each cancer cell, and 10 Compared to BSH, it adds10 In BSH-dhp7 cells, punctate fluorescence was observed within the cells. 10 The molecular weight of BSH is 219.87. 10 BSH-dhp7 was 1197.5, clarifying that at a concentration of 1 / 5.44, there was even more... 10 BSH is taken up into the cells. 10 BSH has low cell membrane permeability, thus indicating that it can be transported via ANXA1 on the cell surface through binding to the dhp7 peptide. 10 BSH uptake efficiency is improved in cells.

[0208] Example 7: Using EMCS- 10 BSH-hpnevrs ( 10 Boron neutron capture therapy (BSH-dhp7) Example 6 clarifies 10 BSH-dhp7 was ingested by Anxa1-positive cancer cells, therefore its utilization in a mouse bladder cancer model was investigated. 10 Boron neutron capture therapy with BSH-dhp7. 2×10 5 Two weeks after inoculating the right hind leg thigh of C3H / HeN mice with MBT2 mouse bladder cancer cells, mice with tumors reaching 5 mm in diameter were intraperitoneally administered EMCS- dissolved in physiological saline at a dose of 20 mg / kg (0.4 mg / 20 g mouse). 10 BSH-dhp7.

[0209] Following drug administration, starting 48 hours later, the tumor was irradiated with thermal neutrons at 20 MeV and 100 mA for 60 minutes using a Sumitomo Heavy Industries accelerator (dose 1.63 × 10⁻⁶). 12 / cm 2 (Average dose: 0.23 Gy). One week after irradiation, a second BNCT treatment was administered. The medication was administered intraperitoneally 24 hours before thermal neutron irradiation. 10 BSH-dhp7 20 mg / kg (0.4 mg / 20 g mouse) was administered followed by thermal neutron irradiation. One week after the second irradiation, mice in each group were sacrificed, and tumor diameter and weight were measured. Pathological analysis of excised tissues was performed to verify the antitumor effect. Tumor volume was measured from the start of drug administration until sacrifice, according to the following formula (V = major diameter x minor diameter x minor diameter / 2). Figure 17 As a result, in 10 In the BSH-dhp7 administration + non-BNCT treatment group, no tumor shrinkage effect was observed at all. In contrast, in 10In the BSH-dhp7 administration + BNCT treatment group, significant tumor shrinkage was observed immediately after the first BNCT treatment, and the tumors almost completely disappeared one week after the second irradiation. Figure 18 ).

[0210] Pathological analysis of the excised tissue is shown in Figure 19 As a result, in 10 In the BSH-dhp7 administration + BNCT treatment group, no residual tumor was observed in 4 out of 8 cases. Furthermore, in the remaining 4 cases, significant intratumoral infiltration of CD8α-positive immune cells was observed, indicating significantly enhanced ANXA1 expression in residual tumor cells. Currently, in actual clinical practice... 10 To achieve the therapeutic effect of BNCT, BPA needs to be administered in large doses at 500 mg / kg via intravenous route over 2–3 hours, which places a heavy burden on patients and limits the treatment time. 10 BSH-dhp7, with a dosage that is 1 / 25th of what was previously used, allows boron to continue accumulating at the tumor site for 2 to 24 hours after administration. Therefore, compared to previous methods, it is considered a drug that is less burdensome for patients and offers greater flexibility in the timing of treatment.

[0211] Example 8: In vivo imaging of tumor aggregation associated with c(MI-Cy7.5)-vc-hpnevrs peptide In Example 5, the tumor aggregation of EMCS-Cy7.5-c-hpnevrs peptide (Seq ID NO:31) in a mouse model of cancer carriers was studied. To investigate the aggregation when other linkers besides EMCS were changed, the fluorescently labeled peptides shown below were synthesized.

[0212] [Chemistry 5] [Chemistry 6] A C(MI-Cy7.5)-vc-HPNEVRS (Cy7.5-vc-LHP7) (Seq ID NO:30 (HPNEVRS)) containing Cy7.5 fluorescent dye via a Val-Cit linker was synthesized (Chem. 5). A c(MI-Cy7.5)-vc-hpnevrs (Cy7.5-vc-dhp7) (Seq ID NO:31 (hpnevrs)) containing Cy7.5 fluorescent dye via a Val-Cit linker was also synthesized (Chem. 6).

[0213] Next, 1×10 6 Personal prostate cancer cells PC3-PSMA- / ANXA1- and PC3-PSMA+ / ANXA1+ were inoculated into the backs of nude mice. One week later, mice carrying tumors with a diameter of 7 mm were intraperitoneally administered Cy7.5 at 10 mg / kg (0.2 mg / 20 g mouse), Cy7.5-vc-dhp7 at 28.7 mg / kg (0.574 mg / 20 g mouse), or Cy7.5-vc-LHP7 (L-type HP7 peptide) at 28.7 mg / kg (0.574 mg / 20 g mouse), dissolved in physiological saline. The Cy7.5 concentration of each fluorescent compound was prepared as a dosage of 10 mg / kg (0.2 mg / 20 g mouse). Following drug administration, monitoring was performed using an in vivo imaging (IVIS) device at 0, 15, 30, 60, 90, 120 minutes, 24, 48, 72, and 96 hours post-administration. Figure 20 and 21 ).

[0214] As a result, Cy7.5-vc-dhp7 selectively accumulated in tumor tissue 24 hours after administration, reaching its maximum, regardless of ANXA1 expression in the tumor. Its aggregation gradually decreased but continued until 96 hours later. On the other hand, no aggregation to the tumor was observed with Cy7.5-vc-LHP7 and Cy7.5. Therefore, dhp7, as a D-amino acid peptide, may selectively bind to ANXA1 expressed in tumor blood vessels or within the tumor and deliver fluorescent dye. This indicates that even with the same sequence, the D-amino acid composition is important for tumor-targeting activity. Furthermore, strong aggregation was observed in the kidneys for all agents, suggesting renal excretion.

[0215] Furthermore, changes in tumor aggregation based on tumor diameter were investigated. For cancer-carrying mice with tumors reaching 15 mm in diameter, Cy7.5-vc-dhp7 28.7 mg / kg (0.574 mg / 20 g mouse) dissolved in physiological saline or Cy7.5-vc-LHP7 (L-type HP7 peptide) 28.7 mg / kg (0.574 mg / 20 g mouse) were administered intraperitoneally, and tumor aggregation was studied in the same manner as described above. Figure 22 ).

[0216] As a result, Cy7.5-vc-dhp7 selectively accumulated in tumor tissue starting 60 minutes after administration, regardless of ANXA1 expression in the tumor, reaching its maximum between 2 and 24 hours, with aggregation gradually decreasing but continuing until 96 hours. On the other hand, no aggregation to the tumor was observed with Cy7.5-vc-LHP7, suggesting that the larger the tumor diameter, the faster the aggregation of D-amino acid-based dhp7 into the tumor tissue.

[0217] Mice were sacrificed 96 hours after drug administration, and major organs were removed for in vitro imaging. The results are shown below. Figure 23 .according to Figure 23 The results showed that Cy7.5-vc-dhp7 exhibited strong aggregation in tumor tissue, kidney, and intestine, but low aggregation in other organs, suggesting that Cy7.5-vc-dhp7 may be renally excretable. Cy7.5-vc-LHP7 did not show tumor-specific aggregation, thus indicating the tumor specificity of dhp7 for prostate cancer.

[0218] Because Cy7.5-vc-dhp7 exhibits tumor-specific aggregation in a mouse model of prostate cancer, therefore, in conjunction with... Figure 17 Under the same conditions (tumor diameter 7 mm), the tumor aggregation of Cy7.5-vc-dhp7 on other cancer cells besides prostate cancer cells (human head and neck cancer cells SAS, human osteosarcoma cancer cells MG-63) was studied. Figure 24 As a result, Cy7.5-vc-dhp7 selectively accumulated in tumor tissue starting 60 minutes after administration, regardless of ANXA1 expression in the tumor, reaching its maximum between 2 and 24 hours, with aggregation gradually decreasing but continuing until 96 hours. On the other hand, no aggregation to the tumor was observed with Cy7.5-vc-LHP7, thus indicating that dhp7, composed of D-amino acids, aggregates in tumor tissue in the same way as in the prostate cancer model.

[0219] Mice were sacrificed 96 hours after drug administration, and major organs were removed for in vitro imaging. The results are shown below. Figure 25 and 26 .according to Figure 25 and 26 The results showed that Cy7.5-vc-dhp7 exhibited strong aggregation in tumor tissue, kidney, and intestine, but low aggregation in other organs, suggesting that Cy7.5-vc-dhp7 may be renally excreted. Cy7.5-vc-LHP7 did not show tumor-specific aggregation, thus indicating the tumor specificity of dhp7 for head and neck cancer and osteosarcoma.

[0220] Example 9: EMCS using immunofluorescence staining- 10Visualization of BSH-vc-dhp7 peptide uptake in ANXA1-positive cancer cells Example 8 demonstrated that Cy7.5-vc-dhp7 was taken up in an ANXA1-positive cancer cell mouse model. Therefore, a EMCS-linked compound was subsequently synthesized that binds to the N-terminus of the hpnevrs (dhp7) sequence (Seq ID NO:31) via a Val-Cit adapter. 10 BSH's EMCS- 10 BSH-val-cit-hpnevrs ( 10 BSH-vc-dhp7)(Seq ID NO:31(hpnevrs))(Chem.7) was used to study its uptake in ANXA1-positive cancer cells.

[0221] [Chemistry 7] The study investigated cancer cells 10 BSH-VC-DHP7 intake. 330 μg of BSH-VC-DHP7 was added to PC3-PSMA+ cells and PC3-PSMA- cells seeded in 3.5 cm culture dishes (ib81156) manufactured by Ibidi. 10 BSH-vc-dhp7, 403.6 μg of IFLLWQRK (EMCS- 10 BSH)RR (IF7- 10 BSH (the compound described in WO2019244954) (Seq ID NO:40 (IFLLWQRK)) or 50 μg 10 After 24 hours of BSH treatment, cells were washed three times with PBS and then fixed with 4% PFA. The amount of each peptide-boron compound added was calculated as a final addition of 50 μg based on boron concentration. 10 BSH. After blocking with 3% BSA BD PhosFlowperm / wash buffer I (BD557885), prepare a solution containing 1 μg / ml of rabbit anti-BSH. 10 BSH polyclonal antibody #52 was reacted with 3% BSA BD PhosFlow perm / wash buffer I at room temperature for 1 hour to allow it to react with the ingested intracellular... 10 BSH reaction. After washing three times with PBS, rabbit anti-BSH was detected. 10 BSH polyclonal antibody was reacted with APC-labeled anti-mouse IgG antibody (1000-fold dilution) at room temperature for 1 hour, followed by three washes with PBS. The effects relative to individual cancer cells were then investigated using a fluorescence microscope (Keyence BZ9000). 10 BSH-vc-dhp7, IF7- 10 BSH or10 Intracellular uptake of BSH ( Figure 27 and 28 ).

[0222] As a result, along with each cancer cell, and 10 Compared to BSH, it adds 10 In BSH-vc-dhp7 cells, more punctate fluorescence was observed intracellularly. Quantification of fluorescence intensity relative to cells indicated significantly high uptake efficiency. In this study, each drug was prepared in 50 μg doses. 10 BSH levels indicate that BSH has been added. 10 In BSH-vc-dhp7 cells, more 10 BSH is taken up into the cells. 10 BSH has low cell membrane permeability, thus indicating that it can be transported via ANXA1 on the cell surface through binding to the dhp7 peptide. 10 BSH uptake efficiency is enhanced in cells. Additionally, PSMA-negative cells with low ANXA1 expression on their cell surface ( Figure 25 and 26 In ), compared with PSMA-positive cells, 10 Less BSH-vc-dhp7 uptake suggests that ANXA1 expression on the cell surface may affect the efficiency of uptake via the dhp7 peptide. In PSMA-positive cells with high ANXA1 expression, BSH alone or IF7- 10 Compared to BSH (the compound described in WO2019244954), it indicates that in 10 Add BSH-vc-dhp7 to the group. 10 BSH intake efficiency increased significantly, reaching 9.5 to 11.7 times.

[0223] Figure 27 and 28 The results show that 10 BSH-vc-dhp7 processing and IF7- 10 BSH and 10 Compared to BSH treatment, this was relative to ANXA1-positive PC3 cells. 10 BSH showed statistically significantly higher intracellular uptake efficiency. Therefore, in comparison with... Figure 27 and 28 Under the same conditions, other cancer cells besides PC3 cells (human head and neck cancer cells SAS, human osteosarcoma cancer cells MG-63) were studied. 10 Intracellular uptake efficiency of BSH, results are shown in Figures 29-31 .

[0224] according to Figures 29-31 The result, with 10Compared with the group without BSH-vc-dhp7, statistically significant differences were observed in all cells. 10 Intracellular uptake of BSH. Furthermore, among the cell lines tested, PC3-PSMA+ cells, which showed the highest ANXA1 expression on their cell surface, exhibited the highest uptake efficiency. In SAS, MG63, and PC3-PSMA- cells, which showed weakly positive ANXA1 expression on their cell surface, although there were variations depending on the cell type, all showed a similar degree of efficiency. 10 BSH uptake efficiency. Additionally, the uptake by each cell type... 10 The localization of BSH indicates co-localization with lysosomes stained with LysoBright Green. This suggests that in various cells, 10 BSH-vc-dhp7 may be taken up via ANXA1-mediated endocytosis expressed on the cell surface. Within the lysosome, the Val-Cit linker is cleaved by cathepsin B, thereby releasing 10 BSH molecules intracellularly. Figures 29-31 ).

[0225] Example 10: In cancer-carrying mice 10 A study on the association of BSH-vc-dhp7 peptide with boron tumor aggregation Example 9 clarifies EMCS- 10 BSH-vc-dhp7 is taken up by Anxa1-positive cancer cells. Therefore, by using... 10 Immunohistochemical staining with BSH antibody was used to investigate the activity of BSH antibodies in tumor tissue, normal kidney tissue, and thigh muscle tissue in a human prostate cancer model. 10 Does the aggregation concentration of B reach a level sufficient to exert the anti-tumor effect of neutron capture therapy? 10 B concentration. 1×10 6 PC3-PSMA- / ANXA1- cancer cells were inoculated into the left back of nude mice, and PC3-PSMA+ / ANXA1+ cells were inoculated into the right back of nude mice. Two weeks later, in mice carrying tumors with a tumor diameter of 10 mm, the cancer cells dissolved in physiological saline were... 10 BSH 50 mg / kg (1.25 mg / 25 g mouse), EMCS- 10 BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse), IFLLWQRK (EMCS- 10 BSH)RR (IF7- 10 BSH (a compound described in WO2019244954) 403.5 mg / kg (10.08 mg / 25 g mice) (Seq ID NO:40 (IFLLWQRK)) was administered intraperitoneally to each mouse.

[0226] 10 BSH single dose, 10 BSH-VC-DHP7 is dissolved in physiological saline at this concentration, IF7- 10 BSH has low solubility at this concentration, resulting in a turbid state. This indicates that... 10 BSH-VC-DHP7 has a higher solubility than IF7- 10 BSH offers high flexibility in dosage form. To confirm dhp7 aggregation, mice were simultaneously administered 0.574 mg / kg of Cy7.5-vc-dhp7 (…). Figure 32 ).

[0227] Cy7.5-vc-dhp7 began to rise in tumor tissue 120 minutes after administration, and strong aggregation was observed in tumor tissue and kidney 24 hours after administration to euthanize the patient. Figure 32 Tumor tissues showing strong aggregation of Cy7.5-vc-dhp7, kidney tissue, and thigh muscle tissue showing low aggregation were extracted, fixed in formalin, embedded in paraffin, and sectioned. Each tissue section was analyzed using an anti-inflammatory method. 10 Immunostaining with BSH antibodies 10 Immunohistochemical staining was performed using BSH antibody (#53, 1 μg / ml). Results are shown in... Figures 33-35 .

[0228] according to Figures 33-35 As a result, in 10 In the tumor tissue and kidney tissue of the BSH-vc-dhp7 administration group, and with 10 BSH treatment group, IF7- 10 Strong staining was observed compared to the BSH-treated group.

[0229] The above research shows that 10 BSH-vc-dhp7 is taken up by Anxa1-positive cancer cells and excreted by the kidneys. Therefore, BSH-vc-dhp7 in a human prostate cancer model... 10 The aggregation of B is tagged with anti-B via Alexa Fluor 750. 10 BSH antibodies were visualized, and their in vivo distribution was investigated using IVIS. 1×10 6 PC3-PSMA- / ANXA1- cancer cells were inoculated into the left thigh of nude mice, and PC3-PSMA+ / ANXA1+ cells were inoculated into the right thigh of nude mice. Two weeks later, cancer-carrying mice with tumors reaching a diameter of 10 mm were injected with saline-soluble prostate cancer cells. 10 BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse) was administered intraperitoneally to each mouse. 10BSH-VC-DHP7 is dissolved in physiological saline at this concentration. To confirm... 10 BSH aggregation was observed in mice by simultaneous intraperitoneal administration of 0.05 mg / kg of AlexaFluor750-labeled anti-BSH. 10 BSH antibody (#52).

[0230] The results showed that, 10 Like Cy7.5-vc-dhp7, BSH accumulates immediately near the kidneys and testes after intraperitoneal administration. Figure 36 Furthermore, renal aggregation disappeared 24 hours after administration, and strong aggregation was observed at the tumor site regardless of ANXA1 expression. Figure 36 The results of in vitro imaging of each removed organ 24 hours later are shown in... Figure 37 .

[0231] As a result, strong aggregation was observed at the tumor site in all mice 24 hours after administration, indicating that... 10 BSH-vc-dhp7, 10 BSH selectively accumulates at the tumor site.

[0232] Example 11: Utilizing 10 BSH-val-cit-dhp7(EMCS- 10 Boron neutron capture therapy (BSH-vc-dhp7) Example 10 clarifies that through 10 BSH-vc-dhp7 administration resulted in sufficient boron concentration in tumor tissue for the antitumor effect of neutron capture therapy (BNCT). Therefore, EMCS- was tested in ANXA1-negative human prostate cancer models and ANXA1-positive head and neck cancer models. 10 Boron neutron capture therapy with BSH-vc-dhp7. 1×10 6 Personal prostate cancer cells PC3-PSMA- / ANXA1- or human head and neck cancer cells SAS were inoculated into the right hind leg thigh of nude mice. One week later, tumor-carrying mice with tumors reaching 5 mm in diameter were injected with saline-dissolved prostate cancer cells. 10 BSH 50 mg / kg (1.25 mg / 25 g mouse) or 10 BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse) was administered intraperitoneally to each mouse. 24 hours after administration, the tumor was irradiated with thermal neutrons at 20 MeV 100 mA for 60 minutes using a Sumitomo Heavy Industries accelerator (dose 1.63 × 10⁻⁶). 12 / cm 2(Average dose: 0.23 Gy). One week after irradiation, a second BNCT treatment was administered.

[0233] 24 hours before thermal neutron irradiation, the solution dissolved in physiological saline will be... 10 BSH 50 mg / kg (1.25 mg / 25 g mouse) or 10 BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse) was administered intraperitoneally to mice, followed by thermal neutron irradiation 24 hours later. One week after the second irradiation, mice in each group were sacrificed, and tumor diameter and weight were measured. Histopathological analysis of excised tissues was performed to verify the antitumor effect. Tumor volume was measured from the start of administration until sacrifice, according to the following formula (V = major diameter × minor diameter × minor diameter / 2). Figure 38 and 39 ).

[0234] As a result, in the ANXA1-negative prostate cancer model, in EMCS- 10 BSH-vc-dhp7 administration + BNCT treatment group and 10 No tumor shrinkage was observed in either of the two groups treated with BSH plus BNCT. This suggests that in ANXA1-negative cancer tissue, tumor shrinkage is not observed in either group. 10 BSH exhibits low boron aggregation, potentially failing to achieve sufficient tissue boron concentrations for the antitumor effect of BNCT. On the other hand, in an ANXA1-positive head and neck cancer model, in EMCS- 10 In the BSH-vc-dhp7 administration + BNCT treatment group, significant improvements were observed immediately after the first BNCT treatment. 10 The BSH+BNCT treatment group showed a more significant tumor growth inhibition effect; one week after the second irradiation, the tumor volume was smaller than [previous value]. 10 BSH+BNCT treatment group ( Figure 38 and 39 ).

[0235] Based on the above results, EMCS- 10 BSH-vc-dhp7 showed relatively good efficacy against ANXA1-positive cancers. 10 BSH monomers exhibit superior antitumor effects and are therefore considered, compared to previous methods, agents with a higher boron delivery capacity to ANXA1-positive cancer tissues. ANXA1-positive cancers are represented by head and neck cancers, and have been reported in many other cancer types, including breast cancer, lung cancer, melanoma, brain tumors, kidney cancer, and bladder cancer. The peptide-boron compound conjugates of this invention are considered promising candidate agents for BNCT in ANXA1-positive cancers.

[0236] Example 12: Treatment with an anticancer agent using c(vcMMAE)-hpnevrs(vcMMAE-dhp7) Examples 8-11 demonstrate the tumor aggregation and antitumor effects observed in compounds that bind to dhp7 via the Val-Cit linker. Therefore, c(vcMMAE)-hpnevrs(vcMMAE-dhp7) (Seq ID NO:31(hpnevrs)) (Chem. 7) was subsequently synthesized by adding a Cys residue to the N-terminus of the hpnevrs(dhp7) sequence (Seq ID NO:31) and binding the microtubule inhibitor monomethylaurestatin E (MMAE) via the val-cit linker.

[0237] [Chemistry 7] To test the cytotoxicity of the synthesized drug on ANXA1-positive / negative PC3 cells in vitro, the peptide drug conjugate and various concentrations of VCMMAE or VCMMAE-DHP7 were added to PC3 cells and incubated at 37°C for 24 hours. Cell viability was analyzed using a cell counting kit (Tongrentang), and the IC50 value was calculated. Figure 40 The IC50 values ​​of PC3-Luc2-PSMA+ / ANXA1-positive cells treated with vcMMAE or vcMMAE-dhp7 (129.9 or 122.7 ng, respectively) were twice that of PC3-Luc2-PSMA- / ANXA1-negative cells (283.3 or 204.5 ng, respectively). This suggests that in vitro cytotoxicity varies depending on the presence or absence of ANXA1 expression in PC3 cells. Specifically, higher ANXA1 expression levels in PC3 cells correlate with higher in vitro sensitivity to vcMMAE; therefore, higher ANXA1 expression in tumor cells in vcMMAE-dhp7 leads to a greater antitumor effect.

[0238] 1×10 6 One PSMA-negative / ANXA1-negative human prostate cancer cell line PC3 was inoculated into the left back of nude mice, 1×10⁶ 6PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right back. Two weeks later, cancer-carrying mice with tumors reaching 7 mm in diameter were intraperitoneally administered 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) of saline-soluble dhp7 (molecular weight 2257.5) every 4 days. As a control, a single dose of 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse) of luciferase-expressing tumor was administered. Tumor size was monitored by measuring the mean brightness (photons / second / cm² / sr) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse) using an IVIS system. After three cycles of treatment, tumor size, body weight, pathological examination, blood biochemistry, and blood cell count were evaluated. Figure 41 ).

[0239] In vitro, Anxa1-positive cells showed twice the sensitivity to vcMMAE and vcMMAE-dhp7 compared to ANXA1-negative cells, indicating a high cytotoxic effect. On the other hand, in mouse models, the mean brightness of PC3 tumor model mice treated with vcMMAE-dhp7, regardless of ANXA1 expression levels within the tumor, decreased almost completely over all three cycles. Figure 41 In addition, no significant changes such as weight loss were observed in any of the groups. Figure 41 The appearance of the removed tumor and the results of post-treatment blood tests are shown in the figure. Figure 42 and 43 .

[0240] The appearance of the excised tumors and the results of HE staining in the vcMMAE-dhp7 administration group showed significant changes in necrosis and less residual tumor compared to the vcMMAE monotherapy group. Figure 42 The blood biochemical values ​​in the VCMMAE-DHP7 group were not significantly different from those in the VCMMAE-only group, but a decrease in white blood cell count was observed in the VCMMAE-only group. Figure 43 Additionally, the results of immunostaining are shown in... Figure 44 and 45 .

[0241] Results of immunostaining ( Figure 44 and 45The results showed that there were no statistically significant differences in Ki67 index and ANXA1 expression in residual tumors, but the CD31-positive vessel density in residual tumors was significantly reduced in the vcMMAE-dhp7 treatment group compared to the vcMMAE treatment group. These results indicate that, regardless of ANXA1 expression, the number of blood vessels in residual tumors was significantly reduced in the vcMMAE-dhp7 treatment group after treatment. This suggests that the significant change in necrosis in the vcMMAE-dhp7 treatment group is not only due to the direct anti-tumor effect of vcMMAE-dhp7 but also to the reduction of tumor nutrient vessels caused by its cytotoxic effect on tumor nutrient vessels. These results clarify that vcMMAE-dhp7 can reduce the dosage of vcMMAE by approximately 30% while achieving superior anti-tumor effects compared to vcMMAE.

[0242] Based on the above results, and given the excellent anti-tumor effect of vcMMAE-dhp7 observed in a subcutaneous tumor model of the back, a similar treatment experiment was conducted in a tumor model with subcutaneous inoculation in the thighs of both limbs. 1×10 6 PSMA-negative / ANXA1-negative human prostate cancer cells PC3 were inoculated into the left thigh of nude mice, 1×10⁶ cells / year. 6 Two weeks later, mice with tumors reaching a diameter of 7 mm were intraperitoneally injected with 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) of saline-soluble dhp7 (molecular weight 2257.5) every 4 days. As a control, a single dose of 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse) of luciferase was administered. Tumor size was measured using an IVIS system to determine the average brightness (photons / second / cm) of the luciferase-expressing tumor after injection of luciferin (150 mg / mouse). 2 Monitoring was performed using / sr. After three cycles of treatment, the mean brightness and body weight of the tumor were evaluated based on luciferase expression. Figure 46 ).

[0243] In a subcutaneous seeding model of PC3 tumors in the thigh of mice treated with vcMMAE-dhp7, the average brightness decreased uniformly across three treatment cycles, regardless of ANXA1 expression levels or tumor size. Conversely, the vcMMAE treatment group showed a tendency to increase in brightness. Figure 46 In addition, no significant changes such as weight loss were observed in any of the groups. Figure 46 (The exterior is shown.) Figure 47The results above clearly demonstrate that VCMMAE-DHP7 can reduce the dosage of VCMMAE by approximately 30% and achieve a superior anti-tumor effect compared to VCMMAE.

[0244] Based on the above results, excellent antitumor effects of VCMMAE-DHP7 were observed in a subcutaneous tumor model of the thigh. Therefore, the antitumor effects at the same volume were then compared with those of VCMMAE single-dose. (2×10) 6 PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right thigh. One week later, mice with tumors reaching 7 mm in diameter were intraperitoneally administered 2.14 mg / kg (equivalent to 31.25 ng vcMMAE / 25 g mouse) dissolved in physiological saline every 4 days. As a control, a single dose of vcMMAE (molecular weight 1316.6) at 1.25 mg / kg (31.25 ng vcMMAE / 25 g mouse) was administered. Tumor size was measured using an IVIS system to determine the average brightness (photons / second / cm) of the luciferase-expressing tumor after injection of luciferin (150 mg / mouse). 2 The tumor was monitored using / sr. After four cycles of treatment, the tumor's luciferase expression, mean brightness, and body weight were evaluated. Figure 48 ).

[0245] The mean luminance of PC3-PSMA-positive / ANXA1-positive tumors subcutaneously inoculated into the thigh of mice treated with vcMMAE-dhp7 was significantly reduced during four cycles of vcMMAE-dhp7 treatment, while the luminance showed a tendency to increase in the vcMMAE treatment group. Figure 48 In addition, no significant changes such as weight loss were observed in any of the groups. Figure 48 (The exterior is shown.) Figure 48 In the vcMMAE treatment group, the weight of the removed tumor was significantly smaller compared to the vcMMAE treatment group, indicating that vcMMAE-dhp7 can be administered at the same amount as vcMMAE and can achieve a better anti-tumor effect than vcMMAE on ANXA1 positive tumors.

[0246] Industrial practicality According to the present invention, the obtained D-type peptide can bind with high affinity to ANXA1, a tumor angiogenesis-specific marker molecule that also exhibits high specificity, and is therefore useful. Furthermore, this D-type peptide is more soluble than previously known peptides, making it extremely useful for PDCs targeting ANXA1. Moreover, this D-type peptide can persistently and significantly accumulate at tumor sites, for example in bladder cancer, prostate cancer, head and neck cancer, etc., and compared to previously known peptides, it is expected to improve drug uptake efficiency and anti-tumor effects in cancer cells, for example, making it extremely useful in medical applications (e.g., cancer treatment, more specifically (boron) neutron capture therapy, cancer chemotherapy, etc.; cancer detection, more specifically monitoring the therapeutic effects of diseases using in vivo imaging, etc.). In addition, this D-type peptide is expected to reduce drug side effects when used in PDCs, and is therefore useful.

[0247] This application is based on Japanese Special Purpose Application 2023-137833 (filed on August 28, 2023), the contents of which are included in this specification.

Claims

1. A peptide comprising any of the amino acid sequences of formulas (I) to (V) below, wherein in the sequences below, each amino acid preceded by the symbol [D] indicates the D-type of that amino acid: (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S, in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in this sequence, X2 represents Y or L, and X5 represents N, K or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in this sequence, X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I) to (III); (V) The reverse amino acid sequence of any of the amino acid sequences in (I) to (IV).

2. The peptide according to claim 1, comprising any one of the amino acid sequences (I') to (V) below, wherein the symbol [D] has the same meaning as described above: (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S, in which X1 represents H or Q, and X2 represents P or S; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in this sequence, X2 represents Y or L, and X5 represents N, K or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in this sequence, X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I') to (III); (V) The reverse amino acid sequence of any of the amino acid sequences in (I') to (IV).

3. The peptide according to claim 1 or 2, comprising any of the amino acid sequences (i) to (iii) below, wherein the symbol [D] has the same meaning as described above: (i)[D]H[D]P[D]N[D]E[D]V[D]R[D]S; (ii)[D]S[D]T[D]S[D]R[D]N[D]T[D]L; (iii)[D]Q[D]Y[D]A[D]T[D]N[D]L[D]K.

4. The peptide according to any one of claims 1 to 3, comprising the amino acid sequence of (i) below, wherein the symbol [D] has the same meaning as described above: (i)[D]H[D]P[D]N[D]E[D]V[D]R[D]S.

5. A conjugate comprising the peptide of any one of claims 1 to 4 and one or more components.

6. The conjugate according to claim 5, wherein, The one or more components include boron compounds or gadolinium compounds.

7. The conjugate according to claim 6, wherein, The one or more components include boron compounds.

8. The conjugate according to claim 7, wherein, The conjugate is of the following formula (I): [Chemistry 1] 、 Or the following formula (II): [Chemistry 2] 。 9. The conjugate according to claim 5, wherein, The one or more ingredients contain an anticancer agent.

10. The conjugate according to claim 5, wherein, The one or more components contain detectable substances.

11. The conjugate according to claim 10, wherein, The detectable substance can be detected in vivo by a method selected from X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound examination, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy.

12. The conjugate according to claim 10 or 11, wherein, The detectable substance is a radioactive isotope, an MRI enhancer, a radioactive impermeable substance, a contrast agent, or a fluorescent substance.

13. A pharmaceutical composition comprising the peptide of any one of claims 1 to 4 or the conjugate of any one of claims 5 to 12.

14. A neutron-capturing therapeutic agent comprising the conjugate according to any one of claims 6 to 8.

15. The neutron-trapping therapeutic agent according to claim 14, wherein, The conjugate contains a boron compound.

16. The neutron-capturing therapeutic agent according to claim 14 or 15, used for the treatment or prevention of solid cancer.

17. The neutron-trapping therapeutic agent according to claim 16, wherein, The solid tumor mentioned is an annexin A1-positive solid tumor.

18. A cancer chemotherapy agent comprising the conjugate of claim 9.

19. A reagent for cancer examination, comprising the conjugate according to any one of claims 10 to 12.

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