D-peptide and use thereof

Abstract

The present invention provides a peptide comprising any one of the amino acid sequences represented by formulae (I) to (V) (in the following sequences, each amino acid symbol to which a sign [D] is attached immediately before the amino acid symbol refers to a D-form of the amino acid). (I) [D](X1)[D](X2)[D](X3)[D]E[D]V[D]R[D]S (wherein 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 (wherein 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 (wherein X1 represents S or R, X4 represents R or W, and X5 represents N or I); (IV) an amino acid sequence that has a structure having insertion, substitution or deletion of one or several amino acid residues or a combination of the insertion, substitution and deletion in any one of the amino acid sequences (I) to (III); and (V) an amino acid sequence that is a retro-inverso sequence of any one of the amino acid sequences (I) to (IV).

Description

D-type peptides and their uses

[0001] The present invention relates to peptides containing specific amino acids that bind to Annexin A1, and conjugates containing the peptides and one or more components. The present invention also relates to compositions containing the peptides or conjugates, and uses thereof.

[0002] In recent years, antibody-drug conjugates (ADCs), which combine an anticancer drug with a high cytocidal effect but strong side effects with an antibody that targets cancer cell surface molecules, have attracted attention in anticancer drug therapy. ADCs are compounds in which a highly cytocidal small molecule compound is conjugated to an antibody so that it can be released at the target site, and are expected to be next-generation antibody drugs. ADCs allow selective delivery of the conjugated small molecule compound to the target site because the antibody binds with high affinity to the antigen present at the target site. This allows ADCs to simultaneously utilize the characteristics of both antibodies and small molecule anticancer drugs. Specifically, antibodies have high affinity for antigens and a long half-life, but can only target antigens present on the cell surface. In contrast, small molecule anticancer drugs have low selectivity and a short residence time in the body, but are highly permeable and can also target intracellular proteins. ADCs, which bind small molecular weight compounds to antibodies with long half-lives to release small molecular weight anticancer drugs in a sustained manner at the target site and target intracellular proteins, are also very suitable as drug delivery system (DDS) formulations.

[0003] However, the rising costs of research and development of antibody drugs based on ADCs are placing a growing financial burden on patients. It is feared that this trend will accelerate further in the future as medical advances, such as diagnostic systems using genetic information, advances in medicine. Furthermore, the impact of these rising drug prices on national medical expenses is immeasurable. Furthermore, expensive antibody drugs will become unaffordable in developing countries, further widening healthcare disparities on a global scale. To address these issues from a longer-term perspective, in addition to pursuing the search for superior drug seeds, we must also explore the potential of inexpensive biopharmaceuticals, such as short-chain peptides.

[0004] Peptide-drug conjugates (PDCs) are a strategy similar to the ADCs described above. Recently, the world's first successful inhibition of glycan-dependent cancer metastasis using PDCs was reported (Non-Patent Document 1). Furthermore, in the course of investigating the vascular endothelial receptors with which these glycomimetic peptides interact, a peptide named IF7 was reported to bind to Annexin A1 (ANXA1) (Non-Patent Document 2). Similarly, a peptide named dTIT7 is known to be capable of binding to ANXA1, and it has also been reported that it can be used as a PDC for the delivery system of anticancer drugs (Patent Document 1).

[0005] ANXA1 has been shown to be highly specific among currently known tumor vascular marker molecules, and is expressed intracellularly in normal cells, but is strongly expressed on the luminal surface of tumor-forming endothelial cells that are in contact with the bloodstream (Non-Patent Document 3). It has also been reported that ANXA1 expression is elevated in several types of cancer and is associated with prognosis (Non-Patent Document 4).

[0006] Although the above-mentioned IF7 and dTIT7 are known as peptides that target ANXA1, even if cancer treatment or the like is attempted using PDC using these peptides, there is a risk that sufficient effects cannot be expected due to the poor solubility of the peptides and their low stability in vivo, etc. Therefore, there has been a demand for the development of peptides suitable for PDC that target ANXA1.

[0007] International Publication No. 2018 / 034356

[0008] Fukuda et al., Cancer Res., 60:450-6, 2000Hatakeyama et al., Proc. Natl. Acad. Sci. USA,108: 19587-92, 2011Oh et al., Nature, 429: 629-35, 2004Lauren K et al., Immunology, 166(1):2-16, 2022

[0009] Therefore, an object of the present invention is to provide a peptide containing a specific amino acid that binds to Annexin A1, and a conjugate containing the peptide and one or more components. Another object of the present invention is to provide a composition containing the peptide or the conjugate, and uses thereof.

[0010] The present inventors conceived a method to screen for D-peptide sequences that bind to the N-terminal 15-residue L-peptide sequence of Annexin A1, which is distinct from dTIT7, within 5 minutes at 37°C in vivo. Therefore, we performed mirror-image T7 phage library screening to identify D-peptides that selectively bind to the N-terminus of ANXA1. As a result, we successfully isolated a novel peptide containing seven specific D-amino acid residues that exhibited high affinity for ANXA1. Furthermore, the isolated peptide exhibited excellent solubility. Furthermore, surprisingly, the isolated peptide was found to accumulate in tumor-specific manner in bladder cancer, prostate cancer, and head and neck cancer models within 24 hours of administration to mice. Based on these results, the inventors believed that the isolated peptide would be suitable for use in DDS for boron compounds and anticancer drugs.

[0011] The present inventors have previously synthesized IF7- 10 Because B compounds accumulate in tumor tissue, they have been shown to be effective in boron neutron capture therapy (BNCT), a recently approved heavy ion radiotherapy method that exhibits tumor-specific cytotoxicity (WO 2019 / 244954, Yoneyama et al., BMC Cancer, 21:72, 2021). However, IF7 suffers from poor solubility and low stability in the blood. To address this issue, we sought novel peptides for PDC. Therefore, we investigated whether the readily soluble peptides we obtained could be used in BNCT. After extensive investigation, we found that conjugates (peptide-drug complexes) in which boron compounds were bound to the peptides exhibited excellent tumor accumulation and antitumor effects in BNCT using the peptides. Based on these findings, we further developed peptides containing specific amino acids that bind to Annexin A1, as well as conjugates containing the peptides and one or more components. Furthermore, compositions containing the peptides or conjugates of the present invention, and uses thereof (for example, for neutron capture therapy, cancer chemotherapy, cancer testing, etc.) have also been completed.

[0012] That is, the present invention is as follows: [1] A peptide comprising an amino acid sequence of any one of the following formulas (I) to (V) (in the following sequences, each amino acid symbol immediately preceded by the symbol [D] indicates the D-form of the amino acid). (I) [D](X1) [D](X2) [D](X3) [D]E[D]V[D]R[D]S (in the sequence, 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 the 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 the 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 several amino acid insertions, substitutions, or deletions, or a combination thereof, in any of the amino acid sequences of (I) to (III). (V) An amino acid sequence which is a retro-inverso of any of the amino acid sequences (I) to (IV) above. [2] The peptide according to [1], which contains any of the amino acid sequences (I') to (V) below (in the sequences below, the symbol [D] has the same meaning as above): (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S (in the sequence, 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 the 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 the 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 several amino acid insertions, substitutions, or deletions, or a combination thereof, in any of the amino acid sequences of (I') to (III). (V) An amino acid sequence which is a retro-inverso of any of the amino acid sequences (I') to (IV) above. [3] The peptide according to [1] or [2], which contains any of the amino acid sequences (i) to (iii) below (in the sequences below, the symbol [D] has the same meaning as 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] A peptide according to any one of [1] to [3], comprising the amino acid sequence of (i) below (in the sequences below, the symbol [D] has the same meaning as above): (i) [D]H[D]P[D]N[D]E[D]V[D]R[D]S [5] A conjugate comprising the peptide according to any one of [1] to [4] and one or more components. [6] The conjugate according to [5], wherein the one or more components comprise a boron compound or a gadolinium compound. [7] The conjugate according to [6], wherein the one or more components comprise a boron compound. [8] The conjugate is represented by the following formula (I):

[0013]

[0014] or the following formula (II):

[0015]

[0016] The conjugate according to [7], wherein the one or more components comprise an anticancer drug. [9] The conjugate according to [5], wherein the one or more components comprise a detectable substance.

[10] The conjugate according to [5], wherein the one or more components comprise a detectable substance.

[11] The conjugate according to

[10] , wherein the detectable substance enables in vivo detection of the conjugate by a means selected from the group consisting of X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintigraphy, positron emission tomography (PET), radiotherapy, endoscopy, and laparoscopy.

[12] The conjugate according to

[10] or

[11] , wherein the detectable substance is a radioisotope, an MRI enhancing agent, a radiopaque substance, a contrast agent, or a fluorescent substance.

[13] A pharmaceutical composition comprising the peptide according to any one of [1] to [4] or the conjugate according to any one of [5] to

[12] .

[14] A neutron capture therapy agent comprising the conjugate according to any one of [6] to [8].

[15] The neutron capture therapy agent according to

[14] , wherein the conjugate comprises a boron compound.

[16] The neutron capture therapy agent according to

[14] or

[15] , for the treatment or prevention of solid cancer.

[17] The neutron capture therapy agent according to

[16] , wherein the solid cancer is selected from the group consisting of skin cancer, cancer of the brain and nervous system, laryngeal cancer, oral cancer, salivary gland cancer, paranasal cancer, thyroid cancer, bladder cancer, prostate cancer, renal pelvis and ureter cancer, and osteosarcoma.

[18] A cancer chemotherapy agent comprising the conjugate according to [9].

[19] A reagent for cancer testing, comprising the conjugate according to any one of

[10] to

[12] .

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

[21] The neutron capture therapy according to

[20] , wherein the subject is suffering from a solid cancer.

[22] The neutron capture therapy according to

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

[23] A method for treating or preventing cancer, comprising administering to a subject an effective amount of the conjugate according to any one of [6] to [9].

[24] A method for detecting cancer, comprising administering to a subject an effective amount of the conjugate according to any one of

[10] to

[12] .

[25] The method according to

[23] or

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

[26] The neutron capture therapy agent according to

[16] or the method according to

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

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

[14] to

[17] or the cancer chemotherapy agent according to

[18] , for administration to a subject suffering from an annexin A1-positive cancer selected from subjects suffering from cancer.

[28] A companion diagnostic for the neutron capture therapy agent according to any one of

[14] to

[17] or the cancer chemotherapy agent according to

[18] , comprising an annexin A1-binding molecule.

[29] A method for treating or preventing cancer, comprising selecting from among subjects suffering from cancer a subject suffering from a cancer containing annexin A1-positive cells, and administering to the selected subject the neutron capture therapy agent according to any one of

[14] to

[17] or the cancer chemotherapeutic agent according to

[18] .

[30] A peptide according to any one of [1] to [4] or a conjugate according to any one of [5] to

[12] for use in treating or preventing cancer.

[31] Use of the peptide according to any one of [1] to [4] or the conjugate according to any one of [5] to

[12] for treating or preventing cancer.

[32] Use of the peptide according to any one of [1] to [4] or the conjugate according to any one of [5] to

[12] in the manufacture of a drug for treating or preventing cancer.

[0017] According to the present invention, the obtained D-peptide can bind with high affinity to ANXA1, a highly specific tumor vascular marker molecule. Furthermore, the D-peptide is more soluble than previously known peptides, making it extremely useful for PDC targeting ANXA1. Furthermore, the D-peptide continuously and significantly accumulates at tumor sites, and is expected to improve drug uptake into cancer cells and antitumor effects compared to previously known peptides, for example, in bladder cancer, prostate cancer, head and neck cancer, etc., making it extremely useful for medical applications (e.g., cancer treatment, more specifically, (boron) neutron capture therapy, cancer chemotherapy, etc., cancer testing, more specifically, monitoring the effectiveness of disease treatment by in vivo imaging, etc.). Furthermore, when used for PDC, the D-peptide may also be expected to reduce drug side effects.

[0018] Figure 1 shows an overview of mirror-image phage display screening to identify L-amino acid peptide sequences that bind to the N-terminal 15-residue peptide of Annexin A1, which consists of D-amino acids. Figure 2 shows an overview of mirror-image phage display screening to identify L-amino acid peptide sequences that bind to D-MC16, for the 7-residue peptide sequences displayed by the phage mixtures obtained in the first to fifth outputs. Figure 3 shows the genome copy number of phage eluted in each round for the 7-residue peptide sequences displayed by the phage mixtures obtained in the first to fifth outputs. Figure 4 shows the enrichment rate of L-amino acid sequences displayed by phages eluted in each round relative to the first-round pool for the 7-residue peptide sequences displayed by the phage mixtures obtained in the first to fifth outputs. Figure 5 shows the alignment results of the top 10 ranking peptide sequences for the 7-residue peptide sequences displayed by the phage mixtures obtained in the first to fifth outputs. Figure 6 shows the KD values ​​and Rmax values ​​of L-MC16-bound D-type peptides for L-MC16 or D-MC16. A: Sensorgrams showing the binding and dissociation of L-MC16 or D-MC16 peptides to a dhp7-immobilized sensor and the equilibrium dissociation constant KD values. B: Sensorgrams showing the binding and dissociation of L-MC16 peptides to a dst7-immobilized sensor and the equilibrium dissociation constant KD values. C: Sensorgrams showing the binding and dissociation of L-MC16 peptides to a dqy7-immobilized sensor and the equilibrium dissociation constant KD values. Figure 7 shows the KD values ​​and iso-affinity graphs of dhp7 peptides for L-MC16 mutants. A: Sensorgrams showing the binding and dissociation of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides to a dhp7-immobilized sensor. B: Iso-Affinity Graph and equilibrium dissociation constant KD values ​​for binding and dissociation of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides to the dhp7-immobilized sensor. Two-dimensional affinity kinetic plot of rate constants grouped by the L-MC16 peptide series. The dashed diagonal line represents the equilibrium binding constant and is shown to aid in visualization of the affinity distribution.Figure 8 shows the binding and cellular uptake of the dhp7 peptide to HEK293 cells overexpressing Anxa1-c-His. Figure 9 shows the binding and cellular uptake of the dhp7 peptide to HEK293 cells overexpressing Anxa1-c-His. Figure 10 shows the results of flow cytometry analysis of ANXA1 expression on the cell surface of cancer cell lines. Figure 11 shows the results of an investigation into the tumor accumulation of Cy7.5-dhp7 in tumor-bearing (bladder cancer) mice. Figure 12 shows the results of an investigation into the tumor accumulation of Cy7.5-dhp7 in tumor-bearing (prostate cancer) nude mice. Figure 13 shows the results of an investigation into the tumor accumulation of Cy7.5-dhp7 in tumor-bearing (bladder cancer) nude mice. Figure 14 shows the results of an investigation into the tumor accumulation of Cy7.5-dhp7 in tumor-bearing (head and neck cancer) nude mice. Figure 15 shows the results of an investigation into the tumor accumulation of Cy7.5-dhp7 in tumor-bearing (head and neck cancer) nude mice. 10 Rabbit antibody against BSH-BSA 10 Figure 16 shows the results of immunofluorescence staining of BSH polyclonal antibodies (#52 and #53) and their affinity analysis. 10 Figure 17 shows the results of visualization of the uptake of BSH-dhp7 peptide into ANXA1-positive cancer cells. 10 Figure 18 shows the results of neutron capture therapy after administration of BSH-dhp7 in tumor-bearing mice. 10 Figure 19 shows the results of neutron capture therapy after administration of BSH-dhp7. 10Figure 20 shows the results of immunostaining of excised tumors after neutron capture therapy following administration of BSH-dhp7. Figure 21 shows the results of examining the tumor accumulation of Cy7.5-vc-dhp7 in tumor-bearing (prostate cancer) mice with tumors 7 mm in diameter. Figure 22 shows the results of examining the tumor accumulation of Cy7.5-vc-dhp7 in tumor-bearing (prostate cancer) mice with tumors 15 mm in diameter. Figure 23 shows the results of ex vivo examination of the accumulation of Cy7.5-vc-dhp7 in each organ in tumor-bearing (prostate cancer, head and neck cancer, and osteosarcoma) mice with tumors 15 mm in diameter. Figure 24 shows the results of ex vivo examination of the accumulation of Cy7.5-vc-dhp7 in each organ in tumor-bearing (prostate cancer) mice with tumors 15 mm in diameter. Figure 25 shows the results of investigating the tumor accumulation of Cy7.5-vc-dhp7 in tumor-bearing mice (prostate cancer) with a tumor diameter of 7 mm. Figure 26 shows the results of investigating the tumor accumulation of Cy7.5-vc-dhp7 in mice bearing each tumor (head and neck cancer and osteosarcoma) with a tumor diameter of 7 mm. Figure 27 shows the results of investigating the tumor accumulation of Cy7.5-vc-dhp7 in mice bearing each tumor (head and neck cancer and osteosarcoma) with a tumor diameter of 7 mm. 10 Figure 28 shows the results of visualization of BSH uptake into PC3-Anxa1 positive / negative cancer cells by immunofluorescence staining. 10 Figure 29 shows the results of visualization of BSH uptake into PC3-Anxa1 positive / negative cancer cells by immunofluorescence staining. 10 Figure 31 shows the results of visualization of the uptake of BSH into various cells (PC3, SAS, and MG-63) by immunofluorescence staining. 10 Figure 31 shows the results of visualization of the uptake of BSH into various cells (PC3, SAS, and MG-63) by immunofluorescence staining. 10 Figure 32 shows the results of visualization of the uptake of BSH into various cells (PC3, SAS, and MG-63). 10 B shows the results of fluorescence accumulation at the time of sacrifice of drug-administered mice. 10 B Antibody levels in each tissue 24 hours after drug administration 10 Immunohistochemical staining of tissues with BSH antibody 10The results of visualization of BSH uptake are shown in Figure 34. 10 B Antibody levels in each tissue 24 hours after drug administration 10 Immunohistochemical staining of tissues with BSH antibody 10 The results of visualization of BSH uptake are shown in Figure 35. 10 B Antibody levels in each tissue 24 hours after drug administration 10 Immunohistochemical staining of tissues with BSH antibody 10 Figure 36 shows the results of visualization of BSH uptake in tumor-bearing (prostate cancer) mice. 10 BSH-vc-dhp7 drug-administered mice 10 Figure 37 shows the time course of BSH accumulation in tumor-bearing (prostate cancer) mice. 10 BSH-vc-dhp7 in each organ 24 hours after drug administration 10 Figure 38 shows the results of BSH accumulation. 10 BSH-vc-dhp7 + BNCT treatment group 10 Figure 39 shows the results of comparing the antitumor effects of the BSH + BNCT treatment groups. 10 BSH-vc-dhp7 + BNCT treatment group 10The figures show a comparison of the antitumor effects of the BSH + BNCT treatment groups. Figure 40 shows the cytotoxicity and IC50 values ​​against Anxa1-positive / -negative PC3 cells. Figure 41 shows the results of luciferase-luminescent tumor shrinkage and body weight changes following vcMMAE or vcMMAE-dhp7 treatment. Figure 42 shows the appearance of excised tumors and HE staining results after treatment with vcMMAE alone or vcMMAE-dhp7, as well as the results of blood tests following treatment with vcMMAE alone or vcMMAE-dhp7. Figure 43 shows the appearance of excised tumors and HE staining results after treatment with vcMMAE alone or vcMMAE-dhp7, as well as the results of blood tests following treatment with vcMMAE alone or vcMMAE-dhp7. Figure 44 shows the results of immunostaining of excised tumors following treatment with vcMMAE alone or vcMMAE-dhp7. Figure 45 shows the results of immunostaining of excised tumors following treatment with vcMMAE alone or vcMMAE-dhp7. Figure 46 shows the results of luciferase-luminescent tumor shrinkage and body weight changes following treatment with vcMMAE alone or vcMMAE-dhp7. Figure 47 shows the appearance of excised tumors after treatment with vcMMAE alone or vcMMAE-dhp7. Figure 48 shows the appearance of excised tumors after treatment with vcMMAE alone or vcMMAE-dhp7.

[0019] 1. Peptides of the Present Invention The present invention provides peptides containing specific amino acids that bind to Annexin A1. More specifically, the present invention provides peptides containing any of the amino acid sequences of the following formulas (I) to (V) (in the sequences below, each amino acid symbol immediately preceded by the symbol [D] indicates the D-form of the amino acid). (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S (in the 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 the 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 the 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 a combination thereof, in any of the amino acid sequences (I) to (III), (V) an amino acid sequence that is a retro-inverso of any of the amino acid sequences (I) to (IV). In this specification, the one-letter and three-letter codes for amino acids as defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used.

[0020] In this specification, the amino acid sequence of a (linear) peptide is written with the N-terminus on the left and the C-terminus on the right, in accordance with the convention for notating peptides. Furthermore, an amino acid symbol immediately preceded by the symbol [D] in an amino acid sequence indicates the D-form of that amino acid, and an amino acid symbol not immediately preceded by the symbol [D] in an amino acid sequence indicates the L-form of that amino acid, unless the context indicates otherwise. 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. Furthermore, the peptides of the present invention may consist of any of the amino acid sequences (I) to (V) above.

[0021] In one aspect, (I) is (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S (in the sequence, X1 represents H or Q, and X2 represents P or S) (SEQ ID NO: 4).

[0022] In one embodiment, (I) 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), and preferably [D]H[D]P[D]N[D]E[D]V[D]R[D]S (SEQ ID NO: 10).

[0023] In one aspect, (II) 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).

[0024] In one aspect, (III) 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).

[0025] The number of amino acid insertions (or additions), substitutions, or deletions, or combinations thereof, in (IV) above is 1 to several (e.g., 2, 3, 4, 5, 6, 7, 8, 9), more specifically 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1. The amino acids to be inserted, substituted, etc. may be either D- or L-amino acids, but D-amino acids are preferred from the viewpoint of protease resistance, etc. Furthermore, the peptide of the present invention containing the amino acid sequence of (IV) above may contain a partial sequence consisting of 4 consecutive amino acids, a partial sequence consisting of 5 consecutive amino acids, or a partial sequence consisting of 6 consecutive amino acids from the amino acid sequences of (I) to (III) above.

[0026] The amino acids inserted (or added), substituted, or deleted in the amino acid sequence (IV) above may be any amino acids as described below. The amino acids may be L- or D-amino acids, for example, naturally occurring L-amino acids as described below, or D-amino acids that are optical isomers thereof. Furthermore, the amino acids may be chemically modified in various ways as described below.

[0027] The insertion (or addition) of the amino acid (IV) above may be inserted (or added) at the N-terminus or C-terminus of the original sequence, or may be inserted (or added) within the sequence, as long as it is capable of binding to ANXA1.

[0028] The substitution of the amino acid (IV) above is preferably with an amino acid from the same group, such as an aromatic amino acid (Phe (F), Trp (W), Tyr (Y)), an aliphatic amino acid (Ala (A), Leu (L), Ile (I), Val (V)), a polar amino acid (Gln (Q), Asn (N)), a basic amino acid (Lys (K), Arg (R), His (H)), an acidic amino acid (Glu (E), Asp (D)), an amino acid with a hydroxyl group (Ser (S), Thr (T)), or an amino acid with a small side chain (Gly (G), Ala (A), Ser (S), Thr (T), Met (M)). It is predicted that substitution with such an analogous amino acid will not alter the phenotype of the protein (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various documents (see, for example, Bowie et al., Science, 247:1306-1310 (1990)). As used herein, conservative amino acid substitutions may also include substitutions of D-amino acids with their optical isomers, L-amino acids. Conservative amino acid substitutions may be substitutions of L-amino acids with L-amino acids, substitutions of D-amino acids with D-amino acids, or substitutions of L-amino acids with D-amino acids.

[0029] The deletion of the amino acid (IV) above may occur at any position in the original sequence, as long as it is capable of binding to ANXA1, and may be deleted from the N-terminus or C-terminus of the original sequence, or from within the sequence.

[0030] The length of the peptides of the present invention is not particularly limited as long as they can bind to ANXA1, but may include, for example, 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. In addition, the peptides of the present invention may be, for example, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, or up to 7 amino acids. Alternatively, the peptides of the present invention may be, for example, 3 to 10, 3 to 15, 3 to 20, 3 to 25, 3 to 30, 3 to 40, 3 to 50, 4 to 10, 4 to 15, 4 to 20, 4 to 25, 4 to 30, 4 to 40, 4 to 50, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 40, 5 to 5 The peptides of the invention may be 0, 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, peptides of the invention may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0031] The peptides of the present invention may be composed of a combination of D-amino acids and L-amino acids. More specifically, the amino acids constituting a peptide containing any of the amino acid sequences (I) to (V) above may all be D-amino acids, or may contain D-amino acids and L-amino acids. L-amino acids may be naturally occurring L-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, all of which are L-forms. D-amino acids include, for example, optical isomers of the L-amino acids described above. In this specification, glycine, an amino acid that does not exhibit optical activity, can be interpreted as being both an L- and D-amino acid, unless the context indicates otherwise.

[0032] The peptides of the present invention may include modified or unusual amino acids, such as those referred to in 37 CFR 1.821-1.822. Modified or abnormal amino acids include, but are not limited to, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine, and ornithine.

[0033] The amino terminus and / or carboxy terminus of the peptide of the present invention may be modified. Modification of the amino terminus may be, for example, methylation (e.g., -NHCH3, -N(CH3)2, etc.), acetylation (e.g., with acetic acid or a halogenated derivative thereof (e.g., α-chloroacetic acid, α-bromoacetic acid, α-iodoacetic acid, etc.)), ureation, carbamate conversion, formylation, Boc conversion, Fmoc conversion, etc. Alternatively, any protecting group such as a benzyloxycarbonyl group, a carboxylate functional group (RCOO-), or a sulfonyl functional group (R-SO2-) (where R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc.) may be introduced.

[0034] Modification of the carboxy terminus includes amidation (-CONH2) and esterification (-COOR). Here, R in the ester is, for example, C, such as methyl, ethyl, n-propyl, isopropyl, and n-butyl. 1～6 Alkyl group: for example, C cyclopentyl, cyclohexyl, etc. 3～8 Cycloalkyl groups such as phenyl and α-naphthyl 6～12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1～2 Alkyl group: α-naphthyl-C 1～2 C such as alkyl group 7～14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0035] The peptides of the present invention may also be modified in various ways at positions other than the N-terminus or C-terminus. Chemical modifications may include, for example, methylation, acetylation, phosphorylation, etc. When the peptides of the present invention have a carboxyl group (carboxylate) at a position other than the C-terminus, the carboxyl group may be amidated or esterified. In this case, for example, the above-mentioned C-terminal esters are used as the esters. Alternatively, substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) may be protected by suitable protecting groups (e.g., C-terminal protecting groups such as formyl group, acetyl group, etc.). 1～6 Alkanoyl group (C 1～6 It may be protected by an alkyl group such as an acyl group.

[0036] Regarding (V) above, a retro-inverso isomer of a peptide refers to a peptide in which the chirality of each amino acid residue is opposite to that of the original peptide ("inverso") and the direction of the amino acid sequence is reversed ("retro"). Retro-inverso isomers are known to exhibit similar structures and functions to the original peptide (e.g., Acc. Chem. Res., 1993, 26(5), pp. 266-273; PLoS One. 2013 Dec. 2; 8(12): e80390, etc.). Specifically, examples of the amino acid sequence (V) above include retro-inversos of the sequence (I) above, such as SRVENPH (SEQ ID NO: 18), SRVENPQ (SEQ ID NO: 19), SRVENSH (SEQ ID NO: 20), SRVENSQ (SEQ ID NO: 21), and SRVEKPQ (SEQ ID NO: 22); retro-inversos of the sequence (II) above, such as KLNTAYQ (SEQ ID NO: 23), KLKTAYQ (SEQ ID NO: 24), and KLYTALQ (SEQ ID NO: 25); and retro-inversos of the sequence (III) above, such as LTNRSTS (SEQ ID NO: 26) and LTIWSTR (SEQ ID NO: 27).

[0037] The peptide of the present invention may comprise two or more sequences selected from the above (I) to (V). In one embodiment, the peptide of the present invention comprises tandem repeats of any of the above sequences (I) to (V) (i.e., a structure in which identical sequence portions are directly linked to each other). In another embodiment, the peptide of the present invention comprises a structure in which two or more different sequences from the above (I) to (V) are directly linked. Alternatively, the peptide of the present invention may comprise a multivalent peptide formed by a dendrimer.

[0038] The peptides of the present invention may be in the form of free peptides or salts. Examples of salts of the peptides of the present invention include 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.

[0039] The peptides of the present invention can bind to annexin A1. ANXA1, also known as lipocortin 1, is a known protein belonging to the annexin family. The DNA nucleotide and amino acid sequences of ANXA1 are known for various species. The peptides of the present invention can bind to the N-terminal region of ANXA1. More specifically, the peptides 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 peptides 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.) at 37°C within 5 minutes. When the intermolecular interaction of the peptides of the present invention with mouse or human ANXA1 is measured using biolayer interferometry (BLI), for example, the peptides of the present invention preferably bind to the N-terminal 15 residues (MAMVSEFLKQAWFIE (SEQ ID NO: 28)) of ANXA1 (e.g., human ANXA1, mouse ANXA1, etc.). -6 M or less, more preferably 5.0 × 10 -7 The antibody may have an equilibrium dissociation constant (KD value) of M or less.

[0040] The peptides of the present invention can be produced according to known peptide synthesis methods. The peptide synthesis method may be, for example, either solid-phase synthesis or liquid-phase synthesis. The desired peptide may be produced by condensing a partial peptide or amino acid that can constitute the peptide of the present invention with the remaining portion, and, if the product contains a protecting group, removing the protecting group. Here, the condensation and removal of the protecting group can be carried out according to known methods, such as those described in (1) to (8) below. (1) M. Bodanszky & M.A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966) (2) Schroeder & Luebke, The Peptide, Academic Press, New York (1965) (3) N. Izumiya et al., Fundamentals and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975) (4) H. Yajima & Shunpei Sakakibara, Biochemistry Experiment Course 1, Chemistry of Proteins IV 205 (1977) (5) H. Yajima (ed.), Continued Drug Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten (6) J. Stewart & J. Young, J.D., “Solid Phase Peptide Synthesis (2nd ed.)”, Pierce Chemical Company, Rockford (1984) (7) E. Atherton & R.C. Sheppard, “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)

[0041] The peptide thus obtained can be purified and isolated by 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 method is a free peptide, the free peptide can be converted into an appropriate salt by known methods or methods equivalent thereto. Conversely, when the peptide is obtained as a salt, the salt can be converted into the free peptide or another salt by known methods or methods equivalent thereto.

[0042] The peptide of the present invention can be safely administered orally or parenterally to subjects as described below. Parenteral administration includes intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, ocular, intracerebral, intrarectal, intravaginal, intraperitoneal, intratumor, administration in the vicinity of a tumor, and administration directly to a lesion.

[0043] As mentioned above, annexin A1 is known to be one of the most highly specific tumor vascular marker molecules currently known. While it is expressed intracellularly in normal cells, it is highly expressed on the luminal surface of tumor neovascular endothelial cells, which are in contact with the bloodstream (Oh et al., Nature 429: 629-35, 2004). Therefore, the peptides of the present invention can selectively bind to neovascular tumors in vivo. Furthermore, ANXA1 is expressed on the blood-brain side of neovascular endothelial cells formed within tumors. When ANXA1 binds to a ligand, such as the peptides of the present invention, on the blood-brain side, it transports the ligand to the basolateral side by transcytosis and actively releases it into the interstitium where cancer cells reside. Therefore, the peptides of the present invention can target malignant tumors in vivo. Thus, the peptides of the present invention are useful for applications such as targeting malignant tumors, (molecular) imaging, and companion diagnostics.

[0044] For example, in the above-mentioned companion diagnostics, annexin A1-positive cancer tissue (cancer tissue containing annexin A1-positive cells or annexin A1-positive (solid) cancer) can be determined by, for example, selecting a specific cell population in the cancer tissue and determining the level of annexin A1-positive cells in the cancer tissue. The selection and determination can be performed according to known methods. Specific examples include immunohistochemical staining. In this method, annexin A1-positive cells in the cancer tissue are stained with an anti-human annexin A1 antibody (fluorescence or chromogenic), and the positive or negative result is determined based on the amount of dye emitted by the stained cells (e.g., the amount of fluorescence or visible light). The staining can typically be performed using an annexin A1-binding molecule. Therefore, in one aspect, the present invention relates to a companion diagnostic agent (hereinafter also referred to as the diagnostic agent of the present invention) for a neutron capture therapy agent or a cancer chemotherapy agent of the present invention, which comprises an annexin A1-binding molecule. The effectiveness of the cancer treatment and prevention drugs (cancer therapeutic agents and preventive agents) of the present invention can be predicted by a method including a step of contacting an annexin A1-binding molecule with cells in the cancer tissue of a subject (test subject).

[0045] The annexin A1-binding molecule is not particularly limited as long as it is a molecule capable of binding (preferably specifically binding) to annexin A1. Examples of the annexin A1-binding molecule include antibodies, more specifically, immunoglobulins, Fab, F(ab')2, minibodies, scFv-Fc, Fv, scFv, diabodies, triabodies, tetrabodies, and monobodies.

[0046] The above series of selection and determination steps can be performed, for example, using microscopic observation. Positive or negative determination is performed based on the amount of dye emitted by the stained cells according to known methods. This determination can be performed according to known techniques. For example, the amount of dye emitted by negative control cells (specifically, cells stained with an isotype control antibody, for example) is used as the background amount of dye, and a dye amount sufficiently high relative to the background is used as a standard. Cells with a lower amount of dye than this standard are determined to be negative cells, and cells with a higher amount of fluorescence than this standard are determined to be positive cells. A simpler method is to perform reverse transcription quantitative PCR of annexin A1 on mRNA extracted from cancer tissue and determine the amount of annexin A1 mRNA.

[0047] In one embodiment, subjects having cancer tissue containing annexin A1-positive cells (or subjects suffering from cancer containing annexin A1-positive cells (annexin A1-positive cancer)) are selected from subjects having cancer tissue (subjects suffering from cancer), and the cancer therapeutic agent or preventive agent of the present invention is administered to the selected subjects. The cancer therapeutic agent or preventive agent of the present invention can be administered to subjects having cancer tissue containing annexin A1-positive cells (or subjects suffering from cancer containing annexin A1-positive cells (annexin A1-positive cancer)) selected from subjects having cancer tissue (subjects suffering from cancer).

[0048] 2. Conjugates of the Present Invention The present invention also provides conjugates (hereinafter also referred to as conjugates of the present invention) comprising one or more components in the peptides of the present invention. The components are not particularly limited as long as they can be linked to the peptides of the present invention, and may be suitable for administration to animals (e.g., humans) and perform some function in the animal's body. Furthermore, the components may be natural or non-natural.

[0049] Examples of components include, but are not limited to, biological materials (e.g., cells, phages, viruses, etc.), oligonucleotides and nucleic acids (e.g., DNA, RNA, DNA / RNA chimeras, etc.), peptides, polypeptides, proteins, antibodies, lipids, polysaccharides, low molecular weight compounds (organic or inorganic, e.g., boron compounds, gadolinium compounds, etc.), particles (e.g., gold particles, various nanoparticles, etc.), and combinations thereof.

[0050] The component can perform a given functionality at a target site in the animal (e.g., human) body. The type of functionality is not particularly limited. Since the conjugate of the present invention can be targeted to a malignant tumor by the action of the portion corresponding to the peptide of the present invention, preferred examples of the functionality include anticancer activity and conferring detectability. Therefore, the component may be, for example, a low-molecular-weight compound, an anticancer drug, a detectable substance, etc.

[0051] (Boron Compound) The boron compound is not particularly limited as long as it is a compound containing a boron atom, and for example, boron-10 nuclide ( 10 Examples of suitable boron compounds include compounds containing RB(OH)2 (boronic acid (wherein R is a substituent group (e.g., alkyl, aryl, heteroaryl, alkylaryl, etc.))), BPA (p-boronophenylalanine), Borax, PCPB (4-carboxybenzeneboronic acid), BODIPY (boron dipyrromethene), and boron clusters.

[0052] In this specification, the term mainly refers to an ionic or nonionic substance having a structure obtained by bonding a plurality of boron atoms together, and is preferably a boron cluster having 3 to 20 boron atoms, more preferably a boron cluster having 8 to 20 boron atoms, and particularly preferably a boron cluster having 10 to 12 boron atoms.

[0053] Examples of boron clusters include decaborane (B 10 H 14)), decahydrodecaborate ([B 10 H 10 ] 2- ) (GB-10) (sometimes called sodium decahydrodecaborate)), dodecaborate ([B 12 H 12 ] 2- )), nido, nido-Octadecaborane (22) (B 18 H 22 , CAS RN: 21107-56-2).

[0054] The boron cluster may contain, as skeletal atoms constituting the cluster, carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, etc., in addition to boron atoms. 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, and more preferably 0 to 2.

[0055] Examples of boron clusters containing atoms other than boron atoms in the basic skeleton include carboranes (including so-called carborane isomers) containing boron atoms and carbon atoms. Examples of carboranes include closo-carboranes ([CB 11 H 12 ] - ), closo-carborane containing two carbon atoms (C2B 10 H 12 ) and nido-carborane ([C2B9H 11 ] - ), or, for example, the molecular formula M(C2B9H 10 Sandwich-type boron clusters represented by the formula (I) are known, but are not limited to these. In the above molecular formula, M represents a transition metal element, such as Fe, Ni, Co, or Mo. That is, the boron cluster may be a metal complex with a transition metal such as Fe, Ni, Co, or Mo.

[0056] The boron cluster is preferably ionic and water-soluble. Examples of ionic or water-soluble boron clusters include dodecaborate ([B 12 H 12 ] 2- )), dodecaborate with thiol groups ([B 12 H 11 SH] 2- , BSH (sometimes called borocaptate (sodium) or mercaptoundecahydrododecaborate)), dodecaborate with hydroxyl groups ([B 12 H 11 OH] 2- ), dodecaborate with amino groups ([B 12 H 11 NH3] - ) etc.

[0057] (Gadolinium Compound) The gadolinium compound is not particularly limited as long as it is a compound containing a gadolinium atom, and examples thereof include: 157 Gd, 157 Compounds containing Gd are also included.

[0058] (Anticancer Agent) As used herein, an anticancer agent refers to a drug intended to suppress the growth of malignant tumors (cancer). The mechanism of action of the anticancer agent is not particularly limited. The anticancer agent may be an antimetabolite, an alkylating agent, an anticancer antibiotic, a microtubule inhibitor, a platinum compound, a topoisomerase inhibitor, a molecular targeted drug, or the like. The conjugate of the present invention may contain two or more of the same or different anticancer agents.

[0059] Examples of the antimetabolite may include antifolates, dihydropteroate synthase inhibitors, dihydrofolate reductase inhibitors (DHFR inhibitors), pyrimidine metabolism inhibitors, thymidylate synthase inhibitors, purine metabolism inhibitors, IMPDH inhibitors, ribonucleotide reductase inhibitors, ribonucleotide reductase inhibitors, nucleotide analogs, and L-asparaginase. Specific examples of the antimetabolite include enocitabine (Sanrabine), capecitabine (Xeloda), carmofur (Miflor), cladribine (Leustatin), gemcitabine (Gemzar), cytarabine (Kilocide), cytarabine ocfosfate (Staracid), tegafur (Atilon, Aftofur, Tefsil, Futraful, Lunasin, etc.), tegafur / uracil (UFT), tegafur / gimeracil / ote These include tracil potassium (TS-1), doxifluridine (Furtulon), nelarabine (Alanonzi), hydroxycarbamide (Hydrea), fluorouracil (5-FU, Calzoner, Bennan, Lunacol, Lunabon), fludarabine (Fludara), pemetrexed (Alimta), pentostatin (Coforin), mercaptopurine (Leukerin), and methotrexate (Methotrexate).

[0060] Specific examples of alkylating agents include nitrogen mustard alkylating agents such as cyclophosphamide (Endoxan), ifosfamide (Ifomide), melphalan (Alkeran), busulfan, and thiotepa (Tespamine), and nitrosourea alkylating agents such as nimustine (Nidran), ranimustine (Cymelin), dacarbacin (Dacarbacin), procarbacin (Procarbacin Hydrochloride), temozolomide (Temodar), carmustine (Gliadel), streptozotocin (Zanosar), and bendamustine (Treaximin).

[0061] Specific examples of anticancer antibiotics include actinomycin D (Cosmegen), aclarubicin (Acrasinon), amrubicin (Calced), idarubicin (Idamycin), epirubicin (epirubicin hydrochloride, famorubicin), zinostatin stimalamer (Smanx), daunorubicin (Daunomycin), doxorubicin (Adriacin), pirarubicin (pinorubin, terarubicin), bleomycin (Bleo), peplomycin (Pepleo), mitomycin C (Mitomycin), mitoxantrone (Novantrone), and liposomal doxorubicin (Doxil).

[0062] Examples of microtubule inhibitors include vinca alkaloid microtubule polymerization inhibitors such as vinblastine (Exal), vincristine (Oncovin), and vindesine (Fordesine), and taxane microtubule depolymerization inhibitors such as paclitaxel (Taxol), docetaxel (Taxotere), and vedotin. Other examples include monomethyl auristatin E (MMAE).

[0063] Examples of platinum preparations include oxaliplatin (Elplat), carboplatin (Carboplatin, Carbomerc, Paraplatin), cisplatin (IA-Call, Conaburi, Cisplatin, etc.), and nedaplatin (Acupra).

[0064] Examples of topoisomerase inhibitors include type I topoisomerase inhibitors such as camptothecin and its derivatives (e.g., irinotecan (Campto), nogitecan (Hycamtin), SN-38, etc.); type II topoisomerase inhibitors such as anthracycline drugs such as doxorubicin (Adriamycin), epipodophyllotoxin drugs such as etoposide (Lasted, Bepcid), and quinolone drugs such as levofloxacin (Cravit) and ciprofloxacin (Ciproxan).

[0065] Examples of molecular 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), lapatinib (Tykerb), and Pertz These include izumab (Perjeta), sunitinib (Sutent), sorafenib (Nexavar), axitinib (Inlyta), pazopanib (Votrient), nivolumab (Opdivo), pembrolizumab, ipilimumab (Yervoy), vemurafenib (Zelboraf), everolimus (Afinitor), temsirolimus (Torisel), rituximab (Rituxan), bevacizumab (Avastin), and geldanamycin.

[0066] The anticancer drug may also be an antiangiogenic agent. The antiangiogenic agent may inhibit vascular endothelial growth factor (VEGF) or other angiogenic factors or their receptors. Specific examples of antiangiogenic agents include angiostatin, endostatin, metastatin, anti-VEGF antibodies (e.g., Avastin), and VEGFR-2 inhibitors (e.g., SU5416 and SU6668).

[0067] (Detectable Substance) As used herein, the term "detectable substance" refers to any substance that enables the detection of the conjugate of the present invention containing it. Preferably, the detectable substance enables the detection of the conjugate of the present invention in vivo, either directly or indirectly using an appropriate visualization or imaging means. Examples of visualization or imaging means include, but are not limited to, X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintigraphy, positron emission tomography (PET), RI internal therapy, endoscopy, laparoscopy, etc. The detectable substance may be, for example, a radioisotope, an MRI enhancing agent (e.g., a paramagnetic ion), a radiopaque substance, a contrast agent, a fluorescent substance, etc.

[0068] Radionuclides useful for PET include, for example: 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 Radionuclides useful for detecting 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 Examples include Ra.

[0069] Suitable paramagnetic ions include, for example, chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), erbium(III), and the like, with gadolinium being particularly preferred. Additionally, metals such as lanthanum(III), gold(III), lead(II), and bismuth(III) are also useful in applications such as x-ray imaging.

[0070] Examples of radiopaque substances and contrast agents include 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.), and thallium compounds (e.g., thallium chloride, etc.).

[0071] Examples of fluorescent substances include rhodamine, fluorescein, Cy dyes (e.g., Cy3, Cy5, Cy5.5, Cy7, Cy7.5, etc.), Alexa (registered trademark) Fluor, phycoerythrin (PE), allophycocyanin (APC), and derivatives thereof. Near-infrared fluorescent reagents such as indocyanine are also preferred examples of fluorescent substances.

[0072] (Binding between the peptide of the present invention and a component) The mode of binding between the peptide of the present invention and one or more components in the conjugate of the present invention is not particularly limited. The binding may be direct or indirect via a linker or the like. The binding may be by a covalent bond, a non-covalent bond, or a combination thereof. One or more components may be bound directly or indirectly to the N-terminus, C-terminus, or other position of the peptide of the present invention. Linking of a peptide to another component (or a second peptide) is well known in the art, and in the conjugate of the present invention, the binding may be by any known means.

[0073] For example, when the bond is mediated by a linker, known crosslinkers (crosslinkers) such as NHS ester, sulfo-NHS ester, imidoester, maleimide, carbodiimide, allyl azide, diazirine, isocyanate, psoralen, and combinations thereof (e.g., homobifunctional crosslinkers, heterobifunctional crosslinkers, etc.) can be used. Examples of homobifunctional crosslinkers include DST, BS2G, DSG, BS3, DSS, DSP, DTSSP, DSSeb, EGS, and sulfo-EGS. Examples of heterobifunctional crosslinkers include SBA, SIA, sulfo-SIA, BMPS, SPDP, GMBS, MBS, sulfo-MBS, ANB-NOS, SMCC, sulfo-SMCC, PDPH, EMCS (N-(6-maleimidocaproyloxy)succinimide), SMPB, SMPH, LC-SPDP, sulfo-LC-SPDP, and sulfo-SNAPAH. Alternatively, a peptide linker, such as a dipeptide linker (e.g., a Val-Cit linker), may be used. The N-terminus of the linker may be protected with an Fmoc group, an Alloc group, or the like, or a maleimide group or the like may be introduced. Furthermore, two or more of the above-mentioned linkers may be used in combination. Furthermore, the peptide of the present invention may be appropriately modified depending on the crosslinker used. For example, a cysteine ​​may be added in advance to the C-terminus of the peptide of the present invention for binding to a maleimide linker.

[0074] Furthermore, for linking the above-mentioned radioactive substances (nuclides) or paramagnetic ions to the peptides of the present invention, 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 metallothionein, etc., can be used (see, for example, Cumali Aktolun et al., "Nuclear Medicine Therapy: Principles and Clinical Applications", Springer, 2013, etc.).

[0075] The conjugate of the present invention can be safely administered orally or parenterally to subjects as described below. Parenteral administration includes intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, ocular, intracerebral, intrarectal, intravaginal, intraperitoneal, intratumor, administration in the vicinity of a tumor, and administration directly to a lesion.

[0076] 3. Pharmaceutical Compositions of the Present Invention The present invention also 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 contain a pharmaceutically acceptable carrier. The pharmaceutical compositions may be provided in dosage forms suitable for oral or parenteral administration. Parenteral administration includes intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, ocular, intracerebral, rectal, intravaginal, intraperitoneal, intratumor, administration proximal to a tumor, and administration directly to a lesion. Such administration may be performed, for example, using an injection, an endoscope, a catheter, or the like.

[0077] Pharmaceutical compositions for parenteral administration include, for example, injections and suppositories. Injections may be in the form of intravenous injections, subcutaneous injections, intraperitoneal injections, intradermal injections, intramuscular injections, or drip infusions. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying the peptide or conjugate of the present invention in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline and isotonic solutions containing glucose and other adjuvants, which may be used in combination with appropriate solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injection solution is preferably filled into a suitable ampule. Suppositories for rectal administration can be prepared by mixing the peptide or conjugate of the present invention with a conventional suppository base.

[0078] 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 produced by known methods and may contain carriers, diluents, excipients, etc. commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

[0079] 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 (preparation), but is usually about 0.01 to 100 wt %, preferably about 0.1 to 50 wt %, in terms of the amount of the peptide or conjugate of the present invention relative to the total pharmaceutical composition (total preparation).

[0080] The pharmaceutical composition of the present invention may contain other active ingredients as long as they do not cause undesirable interactions when combined with the peptide or conjugate of the present invention.

[0081] The parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit forms appropriate for the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, injections (ampoules), and suppositories. The content of the peptide or conjugate is not particularly limited as long as the desired efficacy is achieved, but is typically 0.01 mg to 50 g per dosage unit, 0.01 mg to 25 g for injections, and 0.01 mg to 50 g for other dosage forms.

[0082] The pharmaceutical compositions of the present invention can target tumors, particularly angiogenic malignant tumors, and preferably accumulate the peptide or conjugate in the tumor. Therefore, pharmaceutical compositions of the present invention containing an anticancer drug, a boron compound, a gadolinium compound, or the like in the conjugate are useful for the targeted treatment or prevention of malignant tumors. Furthermore, compositions of the present invention containing a detectable substance or the like in the conjugate are useful for the examination and diagnosis of malignant tumors.

[0083] The malignant tumor (cancer) may be any type of cancer, and may be a solid cancer or a liquid cancer. The solid cancer is preferably an ANXA1-positive solid cancer, more preferably an ANXA1-expressing solid cancer on the cell surface, and therefore an angiogenic solid cancer. Examples of solid cancers include brain and nervous system cancers (e.g., brain tumors, spinal cord tumors, etc.), head and neck cancers (e.g., laryngeal cancer, oral cancer, salivary gland cancer, paranasal sinus cancer, thyroid cancer, etc.), digestive cancers (e.g., stomach cancer, esophageal cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, etc.), 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, etc.), respiratory system cancers (e.g., lung cancer (including small cell lung cancer, non-small cell lung cancer, and metastatic lung cancer), bronchial cancer, etc.), breast cancer, skin cancer (e.g., malignant melanoma, etc.), bone cancer (e.g., osteosarcoma, etc.), and muscle cancer (e.g., rhabdomyosarcoma, etc.). Preferred solid cancers include skin cancer (e.g., malignant melanoma, etc.), brain and nervous system cancer (e.g., treatment-resistant brain tumor, spinal cord tumor, etc.), laryngeal cancer, oral cancer, salivary gland cancer, paranasal sinus cancer, thyroid cancer, bladder cancer, prostate cancer, renal pelvis and ureter cancer, and osteosarcoma.

[0084] Liquid cancers include leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, etc. Leukemias include acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia. Malignant lymphomas are classified into Hodgkin's lymphoma and non-Hodgkin's lymphoma, and non-Hodgkin's lymphomas include B-cell lymphoma, adult T-cell lymphoma, lymphoblastic lymphoma, diffuse large cell lymphoma, Burkitt's lymphoma, follicular lymphoma, MALT lymphoma, peripheral T-cell lymphoma, and mantle cell lymphoma.

[0085] Since the peptides or conjugates of the present invention can efficiently cross the blood-brain tumor barrier, brain tumors are a preferred target. Brain tumors may be primary or metastatic. Furthermore, brain tumors may be benign (e.g., meningiomas, pituitary adenomas, schwannomas, etc.) or malignant, with malignant brain tumors being preferred. Malignant brain tumors include grade 2 brain tumors such as astrocytomas and oligodendrogliomas, grade 3 brain tumors such as anaplastic astrocytomas, anaplastic oligodendrogliomas, and anaplastic oligoastrocytomas, and grade 4 brain tumors such as glioblastomas.

[0086] The pharmaceutical composition of the present invention can be administered to subjects 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, cows, goats, horses, sheep, and mink, pets such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees.

[0087] The dosage of the pharmaceutical composition of the present invention varies depending on the purpose of administration, the subject of administration, the target disease, symptoms, administration route, etc. For example, when used for the treatment or prevention of cancer as described above, a single dose of the conjugate of the present invention containing an anticancer agent, a boron compound, or a gadolinium compound is typically administered intravenously or orally at about 0.01 mg to 50 g / kg body weight once a week. Alternatively, when used for the examination or diagnosis of cancer as described above, a single dose of the conjugate of the present invention containing a detectable substance is typically administered intravenously or orally at about 0.01 mg to 50 g / kg body weight prior to the examination.

[0088] 4. Agents and Reagents of the Present Invention (Neutron Capture Therapy Agent) 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). The conjugate comprises a boron compound or gadolinium compound as described above. The neutron capture therapy agent of the present invention can accumulate in malignant tumors (cancers) expressing ANXA1 on the cell surface as described above by virtue of the peptide of the present invention contained therein. This accumulation means that the agent selectively targets (binds to) or localizes in cancerous tissues over non-cancer tissues (e.g., normal tissues) in subjects such as those described above (e.g., animals, more specifically, mammals (e.g., humans)). Therefore, the neutron capture therapy agent of the present invention is suitable for use in neutron capture therapy (NCT), more specifically, boron neutron capture therapy (BNCT) and gadolinium neutron capture therapy (GdNCT). Therefore, the present invention also provides neutron capture therapy, which comprises administering to a subject an effective amount of the conjugate of the present invention, which comprises the above-mentioned boron compound, gadolinium compound, etc. The neutron capture therapy of the present invention may be performed for the treatment of a disease from which the subject is suffering, or may be performed for prevention (for example, prevention of recurrence of the disease).

[0089] 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 preparation, but the amount of the conjugate of the present invention relative to the total preparation is usually about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0090] In neutron capture therapy, after the neutron capture therapy agent of the present invention has reached the tumor, the tumor is irradiated with an effective amount of low-energy neutrons, such as thermal or epithermal neutrons. The tumor may be irradiated through the skin, or the tumor may be completely or partially exposed prior to irradiation. Irradiation may also be performed simultaneously from multiple directions. Administration of the neutron capture therapy agent of the present invention and subsequent irradiation with thermal or epithermal neutrons may be repeated as necessary. For example, multiple irradiations may be performed at intervals of several months. The total number of irradiations is preferably 1 to 10.

[0091] The neutron capture therapy agent of the present invention can typically be administered to a mammal suffering from a malignant tumor (cancer) within 72 hours (preferably 5 minutes to 48 hours, more preferably 30 minutes to 30 hours) before thermal or epithermal neutron irradiation. It may also be administered during thermal or epithermal neutron irradiation, if necessary. A typical dose of the neutron capture therapy agent of the present invention is in the range of 0.01 mg to 50 g / kg body weight per thermal or epithermal neutron irradiation. The duration of a single thermal or epithermal neutron irradiation is preferably 1 minute to 5 hours, more preferably 10 minutes to 2 hours. The dose of thermal or epithermal neutron irradiation is not particularly limited, and may be within the range of a typical dose used in neutron capture therapy.

[0092] Capture therapy using the neutron capture therapy agent of the present invention may be administered as a sole treatment or in conjunction with conventional surgery or chemotherapy. If desired, after surgically removing as much of the tumor as possible, the remaining tumor can be destroyed by capture therapy using the neutron capture therapy agent of the present invention.

[0093] In neutron capture therapy, the neutron capture therapy agent of the present invention may be used together with a concomitant drug such as an anticancer agent, the pharmaceutical composition of the present invention, or a cancer chemotherapy agent of the present invention described below. When a concomitant drug is used, the timing of administration of the neutron capture therapy agent of the present invention and the concomitant drug is not limited, and the neutron capture therapy agent of the present invention and the concomitant drug may be administered simultaneously or at staggered times to a subject suffering from a malignant tumor (cancer). When administered at staggered times, the order of administration is not particularly limited.

[0094] For all other necessary matters concerning the neutron capture therapy agent of the present invention other than those mentioned above, the contents of "1. Peptides of the present invention" and "2. Conjugates of the present invention" are incorporated herein by reference.

[0095] (Cancer Chemotherapeutic Agent) 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). The conjugate comprises an anticancer agent as described above. The cancer chemotherapeutic agent of the present invention can accumulate in malignant tumors (cancers) expressing ANXA1 on the cell surface as described above due to the peptide of the present invention contained in the agent. This accumulation means that the agent selectively targets (binds to) or localizes in cancerous tissues over non-cancer tissues (e.g., normal tissues) in a subject as described above (e.g., an animal, more specifically, a mammal (e.g., a human)). Therefore, the cancer chemotherapeutic agent of the present invention is suitable for use in cancer treatment. Therefore, the present invention also provides a method for treating or preventing cancer, comprising administering to a subject an effective amount of the conjugate of the present invention comprising the anticancer agent, a boron compound, a gadolinium compound, or the like as described above.

[0096] In the cancer chemotherapeutic agent of the present invention, the content of the conjugate of the present invention varies depending on the form of the formulation, but is usually about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight, in terms of the amount of the conjugate of the present invention relative to the total weight of the formulation.

[0097] For all other necessary matters concerning the cancer chemotherapeutic agent of the present invention other than those mentioned above, the contents of "1. Peptide of the present invention" and "2. Conjugate of the present invention" are incorporated herein by reference.

[0098] (Cancer Testing Reagent) The present invention also provides a cancer testing reagent comprising the conjugate of the present invention (hereinafter also referred to as the cancer testing reagent of the present invention). The conjugate comprises a detectable substance as described above. The cancer testing reagent of the present invention can accumulate in malignant tumors (cancers) expressing ANXA1 on the cell surface as described above due to the peptide of the present invention contained in the agent. This accumulation means that in a subject (e.g., an animal, more specifically a mammal (e.g., a human)), the conjugate preferentially targets (binds to) or localizes in cancer-affected tissue over non-cancer tissue (e.g., normal tissue). Therefore, the cancer testing reagent of the present invention is suitable for use in cancer testing. Therefore, the present invention also provides a method for detecting cancer, comprising administering to a subject an effective amount of the conjugate of the present invention comprising the detectable substance as described above. Cancer tests include the above-mentioned X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintigraphy, positron emission tomography (PET), RI internal therapy, endoscopy, and laparoscopy. The test reagent of the present invention may also include a companion diagnostic agent for determining in advance the likelihood that an anticancer drug such as a molecular targeted drug will be effective.

[0099] In the cancer detection reagent of the present invention, the content of the conjugate of the present invention varies depending on the form of the preparation, but is usually about 0.01 to 100 wt %, preferably about 0.1 to 50 wt %, in terms of the amount of the conjugate of the present invention relative to the total preparation.

[0100] For all other necessary matters concerning the cancer testing reagent of the present invention other than those mentioned above, the contents of "1. Peptide of the present invention" and "2. Conjugate of the present invention" are incorporated by reference.

[0101] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0102] 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-image 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-image phage display screening was performed (Figures 1 and 2).

[0103] A peptide (D-MC16) consisting of the N-terminal 15 amino acid sequence of ANXA1 synthesized using D-amino acids (mamvseflkqawfie (SEQ ID NO: 29); D-amino acids are represented in lowercase letters in the following Examples) with a d-Cys residue added was immobilized on maleimide-coated plates via the SH group of the Cys residue at the concentrations shown in Figure 1 (10 nM, 1 nM, 0.1 nM, 0.01 nM). Two hundred microliters of a T7 phage mixture displaying a 7-residue L-amino acid peptide library was added to L-cysteine-immobilized wells and stirred at room temperature for 1 hour. The remaining 200 μl of the T7 phage mixture that did not bind to the L-cysteine-immobilized wells was collected and added to 10 nM D-MC16 peptide-immobilized wells. After incubation at 37°C for 5 minutes and washing, phages binding to 10 nM D-MC16 peptide were eluted with 100 µl of 1% SDS-PBS (1st output).

[0104] The first-output phage mixture was added to 15 ml of logarithmic-phase E. coli BL21 culture medium and cultured at 37°C for 3 hours with agitation. The amplified first-output phage mixture was obtained as the supernatant after centrifugation. Next, 200 μl of the amplified first-output phage mixture was added to 1 nM D-MC16 peptide-immobilized wells and incubated at 37°C for 5 minutes. After washing, phages that bound to 1 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (second output). The second-output phage mixture was added to 15 ml of logarithmic-phase E. coli BL21 culture medium and cultured at 37°C for 3 hours with agitation. The amplified second-output phage mixture was obtained as the supernatant after centrifugation.

[0105] Next, 200 μl of the 2nd output phage mixture was added to 0.1 nM D-MC16 peptide-immobilized wells, incubated at 37°C for 5 minutes, and washed. Phages binding to 0.1 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (3rd output). The 3rd output phage mixture was added to 15 mL of logarithmic growth phase E. coli BL21 culture medium, and cultured with agitation at 37°C for 3 hours. The amplified 3rd output phage mixture was obtained as the supernatant after centrifugation. Next, 200 μl of the 3rd output phage mixture was added to 0.01 nM D-MC16 peptide-immobilized wells, incubated at 37°C for 5 minutes, and washed. Phages binding to 0.01 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (4th output). The 4th output phage mixture was added to 15 mL of logarithmic growth phase E. coli BL21 culture medium, and the mixture was cultivated at 37°C with stirring for 3 hours. The amplified 4th output phage mixture was obtained as a supernatant after centrifugation.

[0106] Next, 200 μl of the 4th output phage mixture was added to wells immobilized with 10 nM L-amino acid MC16 peptide (L-MC16) and incubated at 37°C for 5 minutes. 200 μl of the 4th output phage mixture that did not bind to L-MC16 was collected. 200 μl of the 4th output phage mixture that did not bind to L-MC16 peptide was added to wells immobilized with 10 nM D-MC16 peptide and incubated at 37°C for 5 minutes. After washing, phages that bound to 10 nM D-MC16 peptide were eluted with 100 μl of 1% SDS-PBS (5th output).

[0107] The copy number of phage eluted in each round was calculated by absolute quantification of the genome copy number of T7 phage using digital PCR (Figure 3). Quantification of the copy number of the phage mixture obtained from the first to fifth outputs revealed that the copy number of phage binding to D-MC16 reached a maximum in the fourth round, and in the fifth round, the copy number of phage binding to L-MC16 was subtracted from the copy number of phage binding to D-MC16, indicating that phages specifically binding to D-MC16 were enriched.

[0108] The 7-residue peptide sequences displayed by the phage mixtures obtained in the 1st to 5th outputs were subjected to amplicon sequencing using a next-generation sequencer, and the frequency of peptide sequence clones displayed by each phage mixture was analyzed (Figure 4).

[0109] Based on amplicon sequence analysis using a next-generation sequencer, the seven-residue peptide sequence HPNEVRS (SEQ ID NO: 30) that appeared most frequently in the 5th output was designated the HP7 peptide as the dMC16-binding peptide sequence, and was synthesized as the D-amino acid peptide hpnevrs (dhp7) (SEQ ID NO: 31). The second and third most frequently occurring seven-residue peptide sequences STSRNTL (SEQ ID NO: 32) and QYATNLK (SEQ ID NO: 33) were also designated ST7 and QY7 peptides, respectively, and were synthesized as the D-amino acid peptides stsrntl (dst7) (SEQ ID NO: 34) and qyatnlk (dqy7) (SEQ ID NO: 35). Half of the top 10 sequences contained the H / QP / S-NEVRS consensus sequence (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).

[0110] Example 2: Analysis of binding affinity of D-type peptides that bind to L-MC16 to ANXA1 N-terminal MC16 To measure the binding affinity of the D-type peptide candidates that bind to L-MC16 selected in Example 1 to L-MC16 or D-MC16, biolayer interferometry (BLI) was performed using a ForteBio Octet K2 instrument (Sartorius, Göttingen, Germany). The interaction of the L-MC16 sequence (MAMVSEFLKQAWFIE) (SEQ ID NO: 28) in the ANXA1 N-terminal domain with dhp7 was thought to occur when L-MC16 is localized to the cell membrane.

[0111] In mirror-image phage display screening, the C-terminus of the displayed L-peptide is exposed on the outermost surface of the phage shell protein. Therefore, candidate D-peptides were modified with biotin at the N-terminus (Biotin-dhp7, Biotin-dst7, Biotin-dqy7) by Biologica Co., Ltd. 50 μg / ml of each biotinylated D-peptide was diluted with kinetic buffer and immobilized on an Octet Super Streptavidin (SSA) biosensor (Sartorius). The C-terminus of each D-peptide was exposed on the outermost surface of the SSA biosensor, similar to the peptide display mode on the phage shell.

[0112] For the reference, 50 μg / ml biocytin was immobilized on 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 or D-MC16 for 120 seconds (association phase), followed by dissociation at 37°C for 120 seconds (dissociation phase) to regenerate the biosensor.

[0113] The biosensor was regenerated by applying three 5-second pulses of 0.1 mM glycine-HCl (pH 2.0). This association-dissociation-regeneration process was performed for various concentrations of L-MC16 or D-MC16. Data were analyzed using Octet Data analysis HT software version 10 (Sartorius). The data were fitted to a 1:1 binding model to determine the K and K values, and the equilibrium dissociation constant, K, was calculated (Figure 6A–C).

[0114] The results in Figures 6A-C show that of the D-peptide sequences tested, dhp7 had the highest affinity for the L-MC16 sequence, with a KD of 0.48 μM, compared to dqy7 (0.99 μM) and dst7 (0.72 μM). Furthermore, no binding of dhp7 to D-MC16 was observed, demonstrating that dhp7 specifically binds to L-MC16. Based on these results, dhp7 was used in subsequent studies.

[0115] Example 3: Analysis of the amino acid sequence of L-MC16 important for the binding of dhp7 peptide to L-type ANXA1 N-terminal L-MC16. To determine the L-MC16 amino acids important for the binding of dhp7 peptide to L-MC16, biolayer interferometry (BLI) was performed using a ForteBio Octet K2 instrument (Sartorius, Göttingen, Germany) as in Example 2. 50 μg / ml of biotin-dhp7 was diluted with kinetic buffer and immobilized on an Octet super streptavidin (SSA) biosensor (Sartorius). The C-terminus of dhp7 was exposed on the outermost surface of the SSA biosensor, similar to the peptide display format on the phage shell. For reference, 50 μg / ml of biocytin was immobilized on the SSA biosensor. To evaluate the specificity of dhp7 binding to L-MC16 mutants, the binding affinity was analyzed against 100 μM L-MC16 wild type (L-MC16WT) and mutants (FIGS. 7A and 7B).

[0116] As a result, 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 (Fig. 7A and B).

[0117] Example 4: Visualization of dhp7 peptide uptake into ANXA1-positive HEK293 cells by immunofluorescence staining We investigated whether the dhp7 sequence, which selectively binds to the MC16 sequence of ANXA1 as determined in Examples 1 to 3, is uptake by ANXA1-positive HEK293 cells. HEK293T cells were transfected with the pCMV3-hANXA1-c-His plasmid, which contained an ANXA1-c-His fusion gene in which a His tag was fused to the C-terminus of human ANXA1. After 72 hours, the HEK293 cells were treated with N-terminally biotin-labeled dhp7 peptide (50 μg / mL) or an anti-MC16 antibody (5 μg / mL) that reacts with the N-terminus of ANXA1. After incubation at 37°C for 10 minutes, the cells were fixed with 1% PFA. To detect biotin-dhp7, streptavidin-Alexa Fluor 555 (500-fold dilution) was added, or to detect anti-MC16 antibodies, anti-mouse IgG-Alexa Fluor 555 (500-fold dilution) was added. After incubation at 37°C for 10 minutes, the cells were washed with PBS and examined for binding and intracellular uptake of the dph7 peptide to HEK293 cells overexpressing Anxa1-c-His under a fluorescence microscope (Keyence BZ9000) (Figures 8 and 9).

[0118] The biotin-dhp7 peptide stained more intensely in HEK293 cells overexpressing ANXA1-c-His than in HEK293 cells transfected with a negative control plasmid. Similar to ANXA1 detected with the anti-MC16 antibody, a dotted pattern was observed within the cells, and the cytoplasm was also diffusely stained. These results suggest that dhp7 may bind to ANXA1 and be internalized into the cells.

[0119] Example 5: In vivo imaging of tumor accumulation of EMCS-Cy7.5-c-hpnevrs peptide. Since Example 4 demonstrated that the dhp7 peptide is taken up by ANXA1-positive cells, we next attempted in vivo imaging by administering fluorescently labeled dhp7 to tumor-bearing mice. The c-hpnevrs sequence (SEQ ID NO: 31) was conjugated to the side chain of the N-terminal d-cys residue via an EMCS linker to synthesize c(EMCS-Cy7.5)-hpnevrs (Cy7.5-dhp7) (SEQ ID NO: 31) (Chemical Formula 3).

[0120]

[0121] Cell suspensions of the 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 incubated with anti-MC16 antibody (1 μg / ml) that reacts with the N-terminus of ANXA1 at room temperature for 1 hour. After washing, the cell pellet was incubated with APC-labeled anti-mouse IgG antibody at room temperature for 1 hour. After washing, the cell pellet was examined for ANXA1 expression on the cell surface by flow cytometry. The results are shown in Figure 10. The antibody reaction was performed under non-fixed, non-permeable cell membrane conditions.

[0122] The results in Figure 10 reveal that ANXA1 is expressed on the cell surface of each cancer cell line, although there are differences in the intensity of expression. PSMA-negative PC3 (PC3-PSMA-) cell lines have lower ANXA1 cell surface expression than PSMA-positive PC3 (PC3-PSMA+) cell lines. MG-63 and SAS cells have similar levels of ANXA1 cell surface expression, while MBT2 cells have both strong and weak ANXA1 cell surface expression.

[0123] We investigated the tumor accumulation of Cy7.5-c-dhp7 in mice bearing ANXA1-positive bladder cancer. 5The mouse bladder cancer cell line MBT2 was inoculated into the right hind thigh of C3H / HeN mice. One week later, tumors reached 5 mm in diameter. Cy7.5-dhp7 dissolved in saline was intraperitoneally administered to the tumor-bearing mice at 10 mg / kg (0.2 mg / 20 g mouse). The tumors were monitored using an in vivo imaging (IVIS) system for 0, 30, 90, 120 minutes, 24, 48, 72, and 96 hours after administration (Figure 11).

[0124] Cy7.5-dhp7 showed an increase in fluorescence intensity in tumor tissue 30 minutes after administration, rapidly accumulated within 2 hours, reached a plateau at 24 hours, and maintained strong tumor-specific accumulation for up to 96 hours. Mice were sacrificed 48 and 96 hours after administration, and major organs were removed for ex vivo imaging. Cy7.5-dhp7 showed strong accumulation in tumor tissue and kidneys, as well as in the intestine, but not in other organs. This suggests that Cy7.5-dhp7 may be excreted through the kidney.

[0125] Furthermore, we examined the uptake of Cy7.5-dhp7 in other cancer cells. 6 Human 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 dorsal region of nude mice. Two weeks later, tumors reached 15 mm in diameter. Cy7.5 or Cy7.5-dhp7, or the L-type control peptide RQRRAP(R7)-EMCS-Cy7.5 (SEQ ID NO: 39 (RRQRRAP)), dissolved in saline, was intraperitoneally administered to each mouse at 10 mg / kg (0.2 mg / 20 g mouse). Cells were monitored using an in vivo imaging (IVIS) system at 0, 20, 30, 90, 120 minutes, 24, 48, 72, and 96 hours after administration (Figures 12–14).

[0126] In tumor-bearing nude mice, Cy7.5-dhp7 accumulation reached a maximum 24 hours after administration and then gradually decreased. Mice were sacrificed 96 hours after administration, and major organs were removed for ex vivo imaging. Cy7.5-dhp7 showed strong accumulation in tumor tissue, kidney, and intestine, but not in other organs, suggesting that Cy7.5-dhp7 is likely excreted by the kidney. Cy7.5 alone and the control L-type R7 peptide-Cy7.5 did not show tumor-specific accumulation, demonstrating the tumor specificity of dhp7 for prostate cancer, bladder cancer, and head and neck cancer.

[0127] Example 6: EMCS- by immunofluorescence staining 10 Visualization of BSH-dhp7 peptide uptake into ANXA1-positive cancer cells. In Example 5, it was revealed that Cy7.5-dhp7 was taken up into ANXA1-positive tumor tissues in a cancer-bearing mouse model. Next, we investigated the uptake of EMCS- 10 BSH-dhp7 ( 10 The uptake of BSH-dhp7 into the cancer cells examined in Example 5 was investigated. 10 To check if BSH is installed, 10 Rabbit antibody specifically reacting with BSH 10 BSH polyclonal antibody was produced. 200 μg 10 Two mice were immunized with BSH-KLH as an antigen, and whole blood was collected after the fourth booster vaccination. 10 Rabbit antibody specifically reacting with BSH 10 Sera containing BSH polyclonal antibodies (#52 and #53) were obtained. The IgG fraction of each serum was purified using a Protein A column. 10 Rabbit antibody against BSH-BSA 10 The affinity of the BSH polyclonal antibodies (#52 and #53) was analyzed by ELISA and BLI, and the results are shown in FIG.

[0128] From the results in Figure 15, the rabbit anti- 10 Both BSH polyclonal antibodies (#52 and #53) 10It was confirmed that the reaction was dependent on the concentration of BSH-BSA. 10 Antibodies #52 and #53 were used to detect BSH.

[0129] Next, the side chain of the N-terminal His residue of the hpnevrs(dhp7) (SEQ ID NO: 31) sequence was 10 BSH was linked via an EMCS linker to EMCS- 10 BSH-hpnevrs ( 10 BSH-dhp7) (SEQ ID NO: 31) was synthesized (Chemical Formula 4).

[0130]

[0131] to cancer cells 10 The uptake of BSH-dhp7 was examined. 50 μg of BSH-dhp7 was administered to MBT2 cells and PC3-PSMA+ cells seeded on a 3.5 cm culture dish (Ibidi ib81156). 10 BSH-dhp7 or 10 BSH was added, and after 24 hours, the cells were washed three times with PBS and fixed with 4% PFA. After blocking with 3% BSA BD PhosFlow perm / wash buffer I (BD557885), 1 μg / ml of rabbit anti-PFA was added. 10 The antibody was incubated with 3% BSA BD PhosFlow perm / wash buffer I containing BSH polyclonal antibody #52 at room temperature for 1 hour to detect the intracellular uptake. 10 After washing three times with PBS, the antibody was reacted with rabbit anti- 10 To detect BSH polyclonal antibodies, the cells were reacted with APC-labeled anti-mouse IgG antibody (1000-fold dilution) at room temperature for 1 hour, washed three times with PBS, and then examined under a fluorescence microscope (Keyence BZ9000) for the antibodies against each cancer cell. 10 BSH-dhp7 or 10 The uptake of BSH into cells was examined (Fig. 16).

[0132] As a result, each cancer cell 10 Compared to BSH, 10 In cells to which BSH-dhp7 had been added, dot-like fluorescence was observed within the cells. 10 The molecular weight of BSH is 219.87.10 BSH-dhp7 was 1197.5, and at 1 / 5.44 the concentration, 10 It was revealed that BSH was taken up into cells. 10 Because BSH has low cell membrane permeability, it binds to the dhp7 peptide and is transported via ANXA1 on the cell surface. 10 It was revealed that the efficiency of BSH uptake into cells was enhanced.

[0133] Example 7: EMCS- 10 BSH-hpnevrs ( 10 Boron neutron capture therapy using BSH-dhp7) in Example 6 10 Since it was revealed that BSH-dhp7 is taken up by Anxa1-positive cancer cells, it was confirmed that it is effective in mouse bladder cancer models. 10 Boron neutron capture therapy was attempted using BSH-dhp7. 5 The mouse bladder cancer cell line MBT2 was inoculated into the right hind thigh of C3H / HeN mice, and after two weeks, tumor-bearing mice with tumors reaching 5 mm in diameter were treated with EMCS- 10 BSH-dhp7 was administered intraperitoneally at 20 mg / kg (0.4 mg / 20 g mouse).

[0134] 48 hours after administration, 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 12 / cm 2 One week after irradiation, the second BNCT treatment was performed. 24 hours before thermal neutron irradiation. 10 After intraperitoneal administration of 20 mg / kg (0.4 mg / 20 g mouse) of BSH-dhp7, thermal neutron irradiation was performed. One week after the second irradiation, mice from each group were sacrificed, and tumor diameter and tumor weight were measured. Pathological analysis of the excised tissue was performed to verify the antitumor effect. Tumor volume was calculated from the start of administration until sacrifice using the following formula (V = major diameter x minor diameter x minor diameter / 2) (Figure 17). The results were as follows: 10 In the BSH-dhp7 + non-BNCT treatment group, no tumor shrinkage effect was observed, 10In the BSH-dhp7 + BNCT treatment group, a significant tumor shrinkage effect was observed immediately after the first BNCT, and the tumors had almost completely disappeared one week after the second irradiation (Figure 18).

[0135] The pathological analysis of the excised tissue is shown in Figure 19. 10 In the BSH-dhp7 + BNCT treatment group, no residual tumors were observed in 4 out of 8 cases. In the remaining 4 cases, significant infiltration of CD8α-positive immune cells into the tumor was observed, and it was revealed that the expression of ANXA1 in the residual tumor cells was significantly increased. 10 To achieve the therapeutic effect of BNCT, BPA must be administered intravenously at a dose of 500 mg / kg over a period of 2 to 3 hours, which places a heavy burden on patients and limits the treatment time. 10 BSH-dhp7 requires a low dose, 1 / 25 of the conventional dose, and boron continues to accumulate at the tumor site for 2 to 24 hours after administration, making it a drug that places less of a burden on patients and allows greater flexibility in the timing of treatment.

[0136] Example 8: In vivo imaging of tumor accumulation of c(MI-Cy7.5)-vc-hpnevrs peptide In Example 5, the tumor accumulation of the EMCS-Cy7.5-c-hpnevrs peptide (SEQ ID NO: 31) in a cancer-bearing mouse model was investigated. To investigate accumulation when a linker other than the EMCS linker was used, the fluorescently labeled peptide shown below was synthesized.

[0137]

[0138]

[0139] C(MI-Cy7.5)-vc-HPNEVRS (Cy7.5-vc-LHP7) (SEQ ID NO: 30(HPNEVRS)) was synthesized by attaching the Cy7.5 fluorescent dye to the side chain of the N-terminal Cys residue of the C-HPNEVRS sequence (SEQ ID NO: 30) via a Val-Cit linker (Chemical Formula 5). c(MI-Cy7.5)-vc-hpnevrs (Cy7.5-vc-dhp7) (SEQ ID NO: 31(hpnevrs)) was synthesized by attaching the Cy7.5 fluorescent dye to the side chain of the N-terminal Cys residue of the c-hpnevrs sequence (SEQ ID NO: 31) via a Val-Cit linker (Chemical Formula 6).

[0140] Next, 1×10 6 Human prostate cancer cells PC3-PSMA- / ANXA1- and PC3-PSMA+ / ANXA1+ were inoculated into the dorsal region of nude mice. One week later, tumors reached 7 mm in diameter. The mice were intraperitoneally injected with Cy7.5 (10 mg / kg, 0.2 mg / 20 g mouse), Cy7.5-vc-dhp7 (28.7 mg / kg, 0.574 mg / 20 g mouse), or Cy7.5-vc-LHP7 (L-type HP7 peptide) (28.7 mg / kg, 0.574 mg / 20 g mouse), dissolved in saline. The Cy7.5 concentration of each fluorescent compound was adjusted to a dose of 10 mg / kg (0.2 mg / 20 g mouse). After administration, monitoring was performed using an in vivo imaging (IVIS) device at 0, 15, 30, 60, 90, 120 minutes, 24 hours, 48 ​​hours, 72 hours, and 96 hours (FIGS. 20 and 21).

[0141] Cy7.5-vc-dhp7 selectively accumulated in tumor tissues 24 hours after administration, reaching a maximum, regardless of ANXA1 expression in the tumor. The accumulation gradually decreased, but persisted for up to 96 hours. In contrast, Cy7.5-vc-LHP7 and Cy7.5 administration showed no tumor accumulation. This suggests that the D-amino acid peptide dhp7 selectively binds to ANXA1 expressed in tumor vasculature or tumors, delivering the fluorescent dye. It was revealed that the D-amino acid structure of the peptide is important for tumor targeting activity, even though the sequence is the same. Furthermore, both drugs showed strong accumulation in the kidney, suggesting that they are excreted in the kidney.

[0142] We further investigated the effect of tumor size on accumulation. Mice bearing tumors reaching 15 mm in diameter were intraperitoneally administered 28.7 mg / kg (0.574 mg / 20 g mouse) of Cy7.5-vc-dhp7 or 28.7 mg / kg (0.574 mg / 20 g mouse) of Cy7.5-vc-LHP7 (L-type HP7 peptide) dissolved in saline, and tumor accumulation was examined in the same manner as above (Figure 22).

[0143] As a result, regardless of the expression of ANXA1 in the tumor, Cy7.5-vc-dhp7 began to selectively accumulate in tumor tissue 60 minutes after administration, reaching a maximum between 2 and 24 hours, and then gradually decreasing in accumulation, which persisted for up to 96 hours. On the other hand, Cy7.5-vc-LHP7 showed no tumor accumulation at all, suggesting that dhp7, which is composed of D-amino acids, may accumulate in tumor tissue more quickly the larger the tumor diameter.

[0144] Mice were sacrificed 96 hours after administration, and major organs were removed and then subjected to ex vivo imaging. The results are shown in Figure 23. The results in Figure 23 indicate that Cy7.5-vc-dhp7 accumulated strongly in tumor tissue, kidney, and intestine, but not in other organs, suggesting that Cy7.5-vc-dhp7 may be excreted through the kidney. Cy7.5-vc-LHP7 did not show tumor-specific accumulation, demonstrating the tumor specificity of dhp7 for prostate cancer.

[0145] Since tumor-specific accumulation of Cy7.5-vc-dhp7 in a mouse model of prostate cancer was demonstrated, we investigated the tumor accumulation of Cy7.5-vc-dhp7 in other cancer cells (human head and neck cancer cells SAS and human osteosarcoma cancer cells MG-63) under the same conditions as in Figure 17 (tumor diameter 7 mm) (Figure 24). Cy7.5-vc-dhp7 selectively accumulated in tumor tissue starting at 60 min after administration, reaching a peak between 2 and 24 h, and then gradually decreased, but persisted for up to 96 h. In contrast, Cy7.5-vc-LHP7 showed no tumor accumulation at all, indicating that dhp7, composed of D-amino acids, accumulates in tumor tissue, similar to the prostate cancer model.

[0146] Mice were sacrificed 96 hours after administration, and major organs were removed and then subjected to ex vivo imaging. The results are shown in Figures 25 and 26. The results in Figures 25 and 26 indicate that Cy7.5-vc-dhp7 accumulated strongly in tumor tissue, the kidney, and the intestine, but accumulated poorly in other organs, suggesting that Cy7.5-vc-dhp7 may be excreted through the kidney. Cy7.5-vc-LHP7 did not show tumor-specific accumulation, demonstrating the tumor specificity of dhp7 for head and neck cancer and osteosarcoma.

[0147] Example 9: EMCS- by immunofluorescence staining 10Visualization of uptake of BSH-vc-dhp7 peptide into ANXA1-positive cancer cells Since it was revealed in Example 8 that Cy7.5-vc-dhp7 was taken up into an ANXA1-positive cancer cell mouse model, we next synthesized EMCS- 10 BSH-conjugated EMCS- 10 BSH-val-cit-hpnevrs ( 10 BSH-vc-dhp7) (SEQ ID NO: 31 (hpnevrs)) (Chemical Formula 7) was synthesized and its uptake into ANXA1-positive cancer cells was examined.

[0148]

[0149] to cancer cells 10 The uptake of BSH-vc-dhp7 was examined. 330 μg of BSH-vc-dhp7 was administered to PC3-PSMA+ cells and PC3-PSMA- cells seeded on a 3.5 cm culture dish (Ibidi ib81156). 10 BSH-vc-dhp7, 403.6μg IFLLWQRK(EMCS- 10 BSH)RR (IF7- 10 BSH (a compound described in WO2019244954) (SEQ ID NO: 40 (IFLLWQRK)) or 50 μg 10 BSH was added, and after 24 hours, the cells were washed three times with PBS and then fixed with 4% PFA. The amount of each peptide-boron compound added was 50 μg in terms of boron concentration. 10 After blocking with 3% BSA BD PhosFlow perm / wash buffer I (BD557885), 1 μg / ml of rabbit anti- antibody was added. 10 The antibody was incubated with 3% BSA BD PhosFlow perm / wash buffer I containing BSH polyclonal antibody #52 at room temperature for 1 hour to detect the intracellular uptake. 10 After washing three times with PBS, the antibody was reacted with rabbit anti- 10To detect BSH polyclonal antibodies, the cells were incubated with APC-labeled anti-mouse IgG antibody (1000-fold dilution) at room temperature for 1 hour, and then washed three times with PBS. After that, the cells were examined for BSH polyclonal antibodies using a fluorescence microscope (Keyence BZ9000). 10 BSH-vc-dhp7, IF7- 10 BSH or 10 The uptake of BSH into cells was examined (Figs. 27 and 28).

[0150] As a result, each cancer cell 10 Compared to BSH, 10 In cells to which BSH-vc-dhp7 had been added, more dot-shaped fluorescence was observed within the cells. Quantitation of the fluorescence intensity per cell showed that the uptake efficiency was significantly higher. In this study, 50 μg of each drug was administered. 10 Since it is prepared so that the amount of BSH is 10 More cells treated with BSH-vc-dhp7 10 It was revealed that BSH was taken up into cells. 10 Because BSH has low cell membrane permeability, it binds to the dhp7 peptide and is transported via ANXA1 on the cell surface. 10 It was revealed that the efficiency of BSH uptake into cells was enhanced. In addition, in PSMA-negative cells (Figures 25 and 26), which have low cell surface expression of ANXA1, compared with PSMA-positive cells, 10 The low uptake of BSH-vc-dhp7 suggested that the expression of ANXA1 on the cell surface may affect the efficiency of dhp7 peptide-mediated uptake. In PSMA-positive cells with high ANXA1 expression, BSH alone or IF7- 10 Compared to BSH (a compound described in WO2019244954), 10 The BSH-vc-dhp7 group showed a significant increase of 9.5 to 11.7 times 10 It was found that the uptake efficiency of BSH increased.

[0151] From the results in Figures 27 and 28, 10 BSH-vc-dhp7 processing fails with IF7- 10 BSH and 10Compared with BSH treatment, ANXA1-positive PC3 cells 10 The intracellular uptake efficiency of BSH was statistically significantly higher. Therefore, the efficacy of BSH in other cancer cells (human head and neck cancer cell line SAS, human osteosarcoma cancer cell line MG-63) was also demonstrated. 10 The efficiency of BSH uptake into cells was investigated under the same conditions as in Figures 27 and 28, and the results are shown in Figures 29 to 31.

[0152] From the results in Figures 29 to 31, 10 Statistically significant differences were observed in all cells compared with the BSH-vc-dhp7-untreated group. 10 BSH was observed to be taken up into cells. Furthermore, the efficiency of uptake was highest in PC3-PSMA+ cells, which had the highest expression of ANXA1 on the cell surface among the cell lines tested. In SAS, MG63, and PC3-PSMA-, which had weakly positive ANXA1 expression on the cell surface, the uptake was similar, although there was some variation between cells. 10 The BSH uptake efficiency was also shown to be 10 BSH was shown to be co-localized with lysosomes stained with LysoBright Green. This suggests that BSH is endocytosed via ANXA1 expressed on the cell surface in each cell type. 10 It was suggested that BSH-vc-dhp7 was taken up, and the Val-Cit linker was cleaved by cathepsin B in the lysosome, resulting in the release of 10BSH into the cell (Figures 29-31).

[0153] Example 10: In tumor-bearing mice 10 Study on the boron tumor accumulation of BSH-vc-dhp7 peptide In Example 9, EMCS- 10 BSH-vc-dhp7 was found to be taken up by Anxa1-positive cancer cells. 10 The accumulated concentration of B is sufficient to exert the antitumor effect of neutron capture therapy. 10 Regarding whether concentration B has been reached 10 The results were analyzed by immunohistochemical staining using BSH antibody. 6Human prostate cancer cells PC3-PSMA- / ANXA1- were inoculated into the left dorsal region of nude mice, and PC3-PSMA+ / ANXA1+ were inoculated into the right dorsal region of nude mice. After two weeks, tumors in the tumor-bearing mice, which had reached a diameter of 10 mm, were dissolved in saline. 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 mouse) (SEQ ID NO: 40 (IFLLWQRK)) was intraperitoneally administered to each mouse.

[0154] 10 BSH alone, 10 BSH-vc-dhp7 was dissolved in physiological saline at the concentration indicated, and IF7- 10 BSH had low solubility at this concentration and was in a cloudy state. 10 BSH-vc-dhp7 is IF7- 10 It was shown to have higher solubility than BSH and to have greater flexibility in formulation. Each mouse was simultaneously administered 0.574 mg / kg of Cy7.5-vc-dhp7 to confirm the accumulation of dhp7 (Figure 32).

[0155] Cy7.5-vc-dhp7 increased in the tumor tissue 120 minutes after administration, and strong accumulation was observed in the tumor and kidney 24 hours after sacrifice (Figure 32). The tumor tissue and kidney, which showed strong accumulation of Cy7.5-vc-dhp7, and the thigh muscle tissue, which showed low accumulation, were each excised, fixed in formalin, embedded in paraffin, and tissue sections were prepared. 10 Immunostaining with BSH antibody 10 Immunohistochemical staining was performed using BSH antibody (#53, 1 μg / ml), and the results are shown in Figures 33 to 35.

[0156] From the results in Figures 33 to 35, 10 ​In tumor and kidney tissues of the BSH-vc-dhp7-treated group 10 BSH-treated group and IF7- 10 Compared to the BSH-administered group, stronger staining was observed.

[0157] From the above consideration, 10 It was revealed that BSH-vc-dhp7 is taken up by Anxa1-positive cancer cells and excreted by the kidney. 10 Accumulation of B was measured by AlexaFluor 750-labeled antibody 10 The biodistribution was investigated by IVIS using BSH antibody visualization. 6 Human prostate cancer cells PC3-PSMA- / ANXA1- were inoculated into the left thigh of nude mice, and PC3-PSMA+ / ANXA1+ were inoculated into the right thigh of nude mice. After two weeks, tumors in the tumor-bearing mice, which had reached a diameter of 10 mm, were dissolved in saline. 10 BSH-vc-dhp7 was intraperitoneally administered to each mouse at 330 mg / kg (8.25 mg / 25 g mouse). 10 BSH-vc-dhp7 was dissolved in physiological saline at the relevant concentration, and each mouse was 10 To confirm the accumulation of BSH, 0.05 mg / kg of AlexaFluor 750-labeled antibody was added. 10 BSH antibody (#52) was simultaneously administered intraperitoneally.

[0158] the result, 10 Similar to Cy7.5-vc-dhp7, BSH immediately accumulated near the kidney and testis after intraperitoneal administration (Figure 36). Furthermore, 24 hours after administration, the accumulation in the kidney disappeared, and strong accumulation was observed at the tumor site, regardless of ANXA1 expression (Figure 36). Ex vivo imaging of each excised organ 24 hours later is shown in Figure 37.

[0159] As a result, strong accumulation was observed at the tumor site in each mouse 24 hours after administration. 10 BSH-vc-dhp7 allows 10 It was shown that BSH selectively accumulates at tumor sites.

[0160] Example 11: 10BSH-val-cit-dhp7(EMCS- 10 Boron neutron capture therapy using BSH-vc-dhp7) in Example 10 10 Administration of BSH-vc-dhp7 revealed that the boron concentration in tumor tissue was sufficient for the antitumor effect of BNCT. Therefore, we investigated the efficacy of EMCS- 10 Boron neutron capture therapy was attempted using BSH-vc-dhp7. 6 Human prostate cancer cells PC3-PSMA- / ANXA1- or human head and neck cancer cells SAS were inoculated into the right hind thigh of nude mice. After one week, tumors of 5 mm in diameter were inoculated into each tumor-bearing mouse. 10 BSH 50 mg / kg (1.25 mg / 25 g mouse) or 10 BSH-vc-dhp7 was intraperitoneally administered to each mouse at 330 mg / kg (8.25 mg / 25 g mouse). 24 hours after administration, 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 12 / cm 2 (Average dose: 0.23 Gy) A second BNCT treatment was performed one week after irradiation.

[0161] Dissolved in saline 24 hours before thermal neutron irradiation 10 BSH 50 mg / kg (1.25 mg / 25 g mouse) or 10 BSH-vc-dhp7 was administered intraperitoneally to each mouse at 330 mg / kg (8.25 mg / 25 g mouse) and then irradiated with thermal neutrons 24 hours later. One week after the second irradiation, mice from each group were sacrificed. Tumor diameter and weight were measured, and pathological analysis of excised tissue was performed to verify the antitumor effect. Tumor volume was calculated from the start of administration until sacrifice using the following formula: V = long diameter × short diameter × short diameter / 2 (Figures 38 and 39).

[0162] As a result, in the ANXA1-negative prostate cancer model, EMCS- 10 BSH-vc-dhp7 + BNCT treatment group10 In both the BSH administration + BNCT treatment groups, no tumor shrinkage effect was observed. Therefore, in ANXA1-negative cancer tissue, 10 The accumulation of boron derived from BSH was low, suggesting that the tissue boron concentration may not have been sufficient for the antitumor effect of BNCT. 10 In the BSH-vc-dhp7 + BNCT treatment group, immediately after the first BNCT, 10 A more significant tumor growth suppression effect was observed than in the BSH + BNCT treatment group, and the tumor growth suppression effect was observed one week after the second irradiation. 10 The tumor volume was smaller than that of the BSH+BNCT treatment group (Figures 38 and 39).

[0163] From the above results, EMCS- 10 BSH-vc-dhp7 inhibits ANXA1-positive cancer 10 Since it showed a superior antitumor effect to BSH alone, it is believed to be a drug with a higher boron delivery capacity to ANXA1-positive cancer tissue than conventional drugs. ANXA1-positive cancers have been reported in many types, including head and neck cancer, as well as breast cancer, lung cancer, melanoma, brain tumors, kidney cancer, and bladder cancer. The conjugate of this invention's peptide and boron compound is thought to be a useful drug candidate for BNCT of ANXA1-positive cancers.

[0164] Example 12: Anticancer drug treatment with c(vcMMAE)-hpnevrs (vcMMAE-dhp7) Examples 8 to 11 demonstrated that compounds linked via a Val-Cit linker in dhp7 exhibit tumor accumulation and antitumor effects. Therefore, a Cys residue was added to the N-terminus of the hpnevrs (dhp7) sequence (SEQ ID NO: 31), and the tubulin inhibitor monomethyl auristatin E (MMAE) was linked via a Val-Cit linker to synthesize c(vcMMAE)-hpnevrs (vcMMAE-dhp7) (SEQ ID NO: 31 (hpnevrs)) (Chemical Formula 7).

[0165]

[0166] To test the cytotoxicity of the synthesized drugs against ANXA1-positive / -negative PC3 cells in vitro, the peptide-drug conjugates 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 Cell Counting Kit 8 (Dojindo), and IC50 values ​​were 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 confirmed to be twice that of PC3-Luc2-PSMA- / ANXA1-negative cells (283.3 or 204.5 ng, respectively). These results suggest that the presence or absence of ANXA1 expression in PC3 cells may affect their in vitro cytotoxicity. Specifically, the higher the expression level of ANXA1 in PC3 cells, the higher their sensitivity to vcMMAE in vitro. This suggests that the higher the expression level of ANXA1 in tumor cells, the greater the antitumor effect of vcMMAE-dhp7.

[0167] 1×10 6 1×10 PSMA-negative / ANXA1-negative human prostate cancer cells PC3 were injected into the left dorsal region of nude mice. 6 PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right dorsal region. Two weeks later, tumors reached 7 mm in diameter and were intraperitoneally administered vcMMAE-dhp7 (molecular weight 2257.5) dissolved in saline at 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) every four days. As a control, vcMMAE alone (molecular weight 1316.6) was administered at 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse). Tumor size was monitored by measuring the mean luminance (photons / second / cm² / sr) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse) using an IVIS system. After three courses of treatment, tumor size, body weight, pathology, blood chemistry, and hematology were evaluated (Figure 41).

[0168] In vitro, Anxa1-positive cells were twice as sensitive to vcMMAE and vcMMAE-dhp7 as ANXA1-negative cells, demonstrating a high cytocidal effect. In mouse models, the mean brightness of PC3 tumor model mice treated with vcMMAE-dhp7 was almost completely reduced after three courses, regardless of the amount of ANXA1 expression in the tumor (Figure 41). Furthermore, no significant changes such as weight loss were observed in either group (Figure 41). The appearance of the excised tumors and the results of blood tests after treatment are shown in Figures 42 and 43.

[0169] The appearance and HE staining of the excised tumors in the vcMMAE-dhp7 group revealed significant necrotic changes and little residual tumor, unlike the vcMMAE-only group (Figure 42). Blood biochemistry values ​​in the vcMMAE-dhp7 group were not significantly different from those in the vcMMAE-only group, but the vcMMAE-only group showed a decrease in leukocyte counts (Figure 43). The results of immunostaining are shown in Figures 44 and 45.

[0170] Immunostaining results (Figures 44 and 45) showed no statistically significant differences in the Ki67 index or ANXA1 expression in the residual tumors. However, the CD31-positive vascular density in the residual tumors was statistically significantly reduced in the vcMMAE-dhp7-treated group compared to the vcMMAE-treated group. These results indicate that the residual tumors after treatment had fewer blood vessels than the vcMMAE-dhp7-treated group, regardless of ANXA1 expression. The significant necrotic changes in the vcMMAE-dhp7-treated group are likely due not only to the direct antitumor effect of vcMMAE-dhp7 on the tumor but also to the reduction of tumor-feeding vessels due to the cytotoxic effect of vcMMAE-dhp7 on the tumor. These results demonstrate that vcMMAE-dhp7 can reduce the dose of vcMMAE by approximately 30% and achieve superior antitumor effects compared to vcMMAE.

[0171] Based on the above results, the excellent antitumor effect of vcMMAE-dhp7 was confirmed in the dorsal subcutaneous tumor model. Therefore, a similar treatment experiment was also carried out in a tumor model in which tumors were inoculated subcutaneously in the thighs of both legs. 61×10 PSMA-negative / ANXA1-negative human prostate cancer cells PC3 were injected into the left thigh of nude mice. 6 PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right thigh. Two weeks later, tumors reached 7 mm in diameter. Tumor-bearing mice received intraperitoneal injections of vcMMAE-dhp7 (molecular weight 2257.5) dissolved in saline at 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) every four days. As a control, vcMMAE alone (molecular weight 1316.6) was administered at 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse). Tumor size was measured by the average brightness (photons / second / cm) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse). 2 The mean tumor brightness and body weight of luciferase-expressing tumors were assessed after three courses of administration (Figure 46).

[0172] The average luminescence intensity of PC3 tumor-inoculated subcutaneously in the thigh model mice treated with vcMMAE-dhp7 decreased over three courses of treatment, regardless of the amount of ANXA1 expression in the tumor or the size of the tumor. However, the vcMMAE-treated group showed a tendency to increase (Figure 46). Furthermore, no significant changes such as weight loss were observed in either group (Figure 46). The appearance of the excised tumors is shown in Figure 47. These results demonstrate that vcMMAE-dhp7 can reduce the dose of vcMMAE by approximately 30% and achieves superior antitumor effects compared to vcMMAE.

[0173] The above results demonstrated the excellent antitumor effect of vcMMAE-dhp7 in the thigh subcutaneous tumor model. Next, we compared the antitumor effect of vcMMAE-dhp7 with that of vcMMAE alone at the same dose. 6PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right thigh. One week later, tumors reached 7 mm in diameter. Tumor-bearing mice were intraperitoneally administered vcMMAE-dhp7 (molecular weight 2257.5) dissolved in saline at 2.14 mg / kg (equivalent to 31.25 ng vcMMAE / 25 g mouse) every 4 days. As a control, vcMMAE alone (molecular weight 1316.6) was administered at 1.25 mg / kg (31.25 ng vcMMAE / 25 g mouse). Tumor size was measured by the average brightness (photons / second / cm) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse). 2 The mean tumor brightness and body weight of luciferase-expressing tumors were assessed after four courses of treatment (Figure 48).

[0174] The average luminescence intensity of the vcMMAE-dhp7-administered PC3-PSMA-positive / ANXA1-positive tumor subcutaneously disseminated in the thigh model mice was significantly reduced after four courses of vcMMAE-dhp7 treatment, whereas it tended to increase in the vcMMAE-treated group (Figure 48). Furthermore, no significant changes such as weight loss were observed in either group (Figure 48). Figure 48 shows the appearance of the excised tumor. The excised tumor weight was significantly smaller in the vcMMAE-treated group compared to the vcMMAE-treated group, demonstrating that vcMMAE-dhp7 can be administered at the same dose as vcMMAE and provides superior antitumor effects against ANXA1-positive tumors compared to vcMMAE.

[0175] According to the present invention, the obtained D-peptides are useful because they can bind with high affinity to ANXA1, a highly specific tumor vascular marker molecule. Furthermore, because the D-peptides are more soluble than previously known peptides, they are extremely useful for PDC, which targets ANXA1. Furthermore, the D-peptides continuously and significantly accumulate at tumor sites, and are expected to improve drug uptake into cancer cells and antitumor effects compared to previously known peptides, for example, in bladder cancer, prostate cancer, head and neck cancer, etc., making them extremely useful for medical applications (e.g., cancer treatment, more specifically, (boron) neutron capture therapy, cancer chemotherapy, etc., cancer testing, more specifically, monitoring the effectiveness of disease treatment by in vivo imaging, etc.). Furthermore, when used for PDC, the D-peptides are expected to reduce drug side effects, making them useful.

[0176] This application is based on patent application No. 2023-137833 filed in Japan (filing date: August 28, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A peptide comprising any of the amino acid sequences of the following formulas (I) to (V) (in the following sequences, each amino acid symbol immediately preceded by the symbol [D] indicates the D-form of that amino acid). (I) [D](X1) [D](X2) [D](X3) [D]E[D]V[D]R[D]S (wherein 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 (wherein 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 (wherein X1 represents S or R, X4 represents R or W, and X5 represents N or I); (IV) an amino acid sequence having one or several amino acid insertions, substitutions, or deletions, or a combination thereof, in any of the amino acid sequences (I) to (III). (V) An amino acid sequence which is a retro-inverse of any one of the amino acid sequences (I) to (IV).

2. The peptide according to claim 1, comprising any one of the amino acid sequences (I') to (V) below (in the sequences below, the symbol [D] has the same meaning as above). (I') [D] (X1) [D] (X2) [D] N [D] E [D] V [D] R [D] S (in the sequence, 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 the 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 the 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 several amino acid insertions, substitutions or deletions, or a combination thereof, in any of the amino acid sequences of (I') to (III); (V) An amino acid sequence which is a retro-inverse of any one of the amino acid sequences (I') to (IV).

3. The peptide according to claim 1 or 2, comprising any one of the following amino acid sequences (i) to (iii) (in the sequences below, the symbol [D] has the same meaning as 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. A peptide according to any one of claims 1 to 3, comprising the amino acid sequence (i) below (in the sequences below, the symbol [D] has the same meaning as above): (i) [D]H[D]P[D]N[D]E[D]V[D]R[D]S 5. A conjugate comprising a peptide according to any one of claims 1 to 4 and one or more moieties.

6. The conjugate of claim 5, wherein the one or more components comprises a boron compound or a gadolinium compound.

7. The conjugate of claim 6, wherein the one or more components comprises a boron compound.

8. The conjugate has the following formula (I): Or the following formula (II): The conjugate of claim 7, 9. The conjugate of claim 5, wherein the one or more components comprises an anti-cancer drug.

10. The conjugate of claim 5, wherein the one or more moieties comprises a detectable substance.

11. The conjugate of claim 10, wherein the detectable substance allows detection of the conjugate in vivo by a means selected from the group consisting of X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound examination, scintigraphy, positron emission tomography (PET), radioisotope therapy, endoscopy, and laparoscopy.

12. The conjugate of claim 10 or 11, wherein the detectable substance is a radioisotope, an MRI enhancing agent, a radiopaque substance, a contrast agent, or a fluorescent substance.

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

14. A neutron capture therapy agent comprising the conjugate according to any one of claims 6 to 8.

15. The neutron capture therapy agent of claim 14, wherein the conjugate comprises a boron compound.

16. A neutron capture therapy agent according to claim 14 or 15 for the treatment or prevention of solid cancer.

17. The neutron capture therapy agent according to claim 16, wherein the solid cancer is an annexin A1-positive solid cancer.

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

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

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