D-peptide and use thereof

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480055570.2 (22) Application Date 2024.08.27 (30) Priority Data 2023-137833 2023.08.28 JP (85) PCT International Application Entering National Phase Date 2026.02.27 (86) PCT International Application Application Data PCT / JP2024 / 030462 2024.08.27 (87) PCT International Application Publication Data WO2025 / 047727 JA 2025.03.06 (71) Applicant: Hirosaki University, National University Corporation, Address: Aomori Prefecture, Japan (72) Inventors: Tetsuya Yoneyama, Riki Yamazaki, Shingo Hatakeyama, Motohiro Nonaka (74) Patent Agency: China Patent Agency (Hong Kong) Limited, 72001 Patent Attorneys: Wei Lu, Chang Peng (51) Int.Cl. C07K 7 / 06 (2006.01) A61K 38 / 08 (2019.01) A61K 41 / 00 (2020.01) A61K 47 / 64 (2017.01) A61K 49 / 00 (2006.01) A61K 49 / 04 (2006.01) A61K 49 / 14 (2006.01) A61K 51 / 08 (2006.01) A61P 35 / 00(2006.01) (54) Invention Title: D-type Peptide and Its Uses (57) Abstract: This invention provides a peptide comprising any of the amino acid sequences of the following formulas (I) to (V), wherein each amino acid symbol preceded by the symbol [D] in the following sequences represents the D-type of that amino acid. (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S, in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D] Q [D] (X2) [D] A [D] T [D] (X5) [D] L [D] K, in which X2 represents Y or L, and X5 represents N, K, or Y; (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in which X1 X4 represents S or R, X5 represents R or W, and X5 represents N or I; (IV) an amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I) to (III) above; (V) the reverse amino acid sequence of any of the amino acid sequences in (I) to (IV) above. Claims: 2 pages; Description: 32 pages; Sequence Listing (electronic publication); Figures: 29 pages. CN 121752583 A 2026.03.27 CN 1 21 75 25 83 A1. A peptide comprising any of the amino acid sequences in formulas (I) to (V) below, wherein each amino acid preceding the symbol [D] in the following sequences represents the D-type of that amino acid: (I) [D](X1)[D](X2)[D](X3)[D]E[D]V[D]R[D]S, in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in which X2 represents Y or L, and X5 represents N, K, or Y; (III) [D](X1)[D]T[D]S[D](X4)[D](X5)[D]T[D]L, in which X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions or deletions or combinations thereof in any of the amino acid sequences in (I) to (III); (V) A reverse amino acid sequence of any of the amino acid sequences in (I) to (IV). 2. The peptide according to claim 1, comprising any one of the following amino acid sequences (I') to (V), wherein the symbol [D] has the same meaning as described: (I') [D](X1) [D](X2) [D]N [D]E [D]V [D]R [D]S, wherein 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, 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 more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I') to (III); (V) A reversed amino acid sequence of any of the amino acid sequences in (I') to (IV). 3. The peptide according to claim 1 or 2, comprising any of the following amino acid sequences (i) to (iii), wherein the symbol [D] has the same meaning as described: (i) [D]H[D]P[D]N[D]E[D]V[D]R[D]S; (ii) [D]S[D]T[D]S[D]R[D]N[D]T[D]L; (iii) [D]Q[D]Y[D]A[D]T[D]N[D]L[D]K. 4. The peptide according to any one of claims 1 to 3, comprising the amino acid sequence of (i) below, wherein the symbol [D] has the same meaning as described: (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 claims 1 to 4 and one or more components.6. The conjugate according to claim 5, wherein one or more components comprise a boron compound or a gadolinium compound. 7. The conjugate according to claim 6, wherein one or more components comprise a boron compound. 8. The conjugate according to claim 7, wherein the conjugate is of the following formula (I): [Chemical 1] Claims 1 / 2 page 2 CN 121752583 A, or the following formula (II): [Chemical 2]. 9. The conjugate according to claim 5, wherein one or more components comprise an anticancer agent. 10. The conjugate according to claim 5, wherein one or more components comprise a detectable substance. 11. The conjugate according to claim 10, wherein the detectable substance can be detected in vivo by a method selected from X-ray photography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound examination, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy. 12. The conjugate according to claim 10 or 11, wherein the detectable substance is a radioactive isotope, an MRI enhancer, a radioactive impermeable substance, a contrast agent, or a fluorescent substance. 13. A pharmaceutical composition comprising the peptide of any one of claims 1-4 or the conjugate of any one of claims 5-12. 14. A neutron capture therapy agent comprising the conjugate of any one of claims 6-8. 15. The neutron capture therapy agent according to claim 14, wherein the conjugate comprises a boron compound. 16. The neutron capture therapy agent according to claim 14 or 15, used 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 cancer examination comprising the conjugate of any one of claims 10-12. Claims 2 / 2 Page 3 CN 121752583 AD-type peptides and their uses Technical Field

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

[0002] In anticancer therapy, in recent years, antibody-drug conjugates (ADCs), which combine anticancer agents with high cell-killing effects but strong side effects with antibodies targeting molecules on the surface of cancer cells, have attracted attention. ADCs are low-molecular-weight compounds with high cell-killing effects combined with antibodies, enabling them to be released at the target site.ADCs are anticipated as next-generation antibody drugs. Because antibodies bind with strong affinity to antigens present at the target site, ADCs can selectively deliver the bound low-molecular-weight compound to the target site. Therefore, the characteristics of both antibodies and low-molecular-weight anticancer agents can be simultaneously utilized. That is, antibodies have very high affinity for antigens and a long half-life, but can only target antigens present on the cell surface. On the other hand, low-molecular-weight anticancer agents are selective, have a short residence time in vivo, but have high permeability and can also target intracellular proteins. ADCs, which target intracellular proteins by binding low-molecular-weight compounds to antibodies with long half-lives and releasing them at the target site, are also a very suitable form of drug delivery system (DDS) formulation.

[0003] However, the research and development costs of ADC-based antibody drugs are soaring, increasing the economic burden on patients. In the future, with advancements in medical technology such as gene-based diagnostic systems, there are concerns that this trend will further intensify. Furthermore, the impact of these high drug prices on national healthcare costs is immeasurable. Furthermore, the high cost of antibody drugs will make them unaffordable for underdeveloped countries, leading to further global healthcare disparities. To address these issues, a longer-term strategy is needed, in addition to pursuing superior drug seeds, to explore the possibility of inexpensive biopharmaceuticals such as short-chain peptides.

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

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

[0006] As peptides targeting ANXA1, IF7 and dTIT7 are known, but even when attempting to treat cancer using PDCs containing these peptides, sufficient efficacy cannot be expected due to the poor solubility and low stability of these peptides in vivo.The risk. Therefore, it has been hoped to develop peptides suitable for targeting ANXA1 PDC.

[0007] Prior art literature Patent literature Specification 1 / 32 pages 4 CN 121752583 A Patent literature 1: International Publication No. 2018 / 034356 Non-patent literature Non-patent literature 1: Fukuda et al., Cancer Res., 60:450-6, 2000 Non-patent literature 2: Hatakeyama et al., Proc. Natl. Acad. Sci. USA,108: 19587-92, 2011 Non-patent literature 3: Oh et al., Nature, 429: 629-35, 2004 Non-patent literature 4: Lauren K et al., Immunology, 166(1):2-16, 2022.

[0008] Summary of the Invention Therefore, the object of the present invention is to provide a peptide comprising a specific amino acid that binds to Annexin A1, and a conjugate comprising the peptide and one or more components. Furthermore, the object of the present invention is also to provide compositions comprising the peptide or conjugate and their uses.

[0009] Means for Solving the Problem The inventors conceived a method different from dTIT7, namely, screening only for D-type peptide sequences that bind to the L-type peptide sequence of the N-terminus 15 residues of Annexin A1 within 5 minutes under biological conditions at 37°C. Therefore, a mirror-image T7 phage library screening was performed to attempt the identification of D-type peptides that selectively bind to the N-terminus of ANXA1. As a result, a novel peptide with a specific 7-residue D-type amino acid having high affinity for ANXA1 was successfully obtained. In addition, the obtained peptide exhibits excellent solubility. Furthermore, surprisingly, the obtained peptide showed tumor-specific aggregation in bladder cancer, prostate cancer, and head and neck cancer models 24 hours after administration to mice. In view of these results, the inventors believe that the obtained peptide is suitable for DDS of boron compounds and anticancer agents.

[0010] The inventors have previously confirmed that since the IF7-10B compound formed by combining boron compounds with IF7 accumulates in tumor tissue, this compound can exert the therapeutic effect of boron neutron capture therapy (BNCT), a heavy particle beam therapy that shows specific cell damage to tumors and has been approved in recent years (International Publication No. 2019 / 244954, Yoneyama et al., BMC cancer, 21:72, 2021). However, IF7 has the problem of poor solubility and low stability in blood. As a fundamental solution, novel peptides for PDC have been explored. Therefore, it is necessary to investigate whether the obtained easily soluble peptide can be used for BNCT.Research was conducted. Further research revealed that in BNCT using the obtained peptide, the conjugate (peptide-drug conjugate) formed by combining a boron compound with the peptide exhibited excellent tumor aggregation and antitumor effects. Based on these insights, further repeated research resulted in the development of the peptide comprising a specific amino acid bound to Annexin A1, and conjugates comprising the peptide with one or more components, as described in this invention. Furthermore, compositions comprising the peptide or conjugates of this invention and their uses (e.g., for neutron capture therapy, cancer chemotherapy, cancer examination, etc.) have also been developed.

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

[0012] [1] A peptide comprising any of the amino acid sequences of the following formulas (I) to (V), wherein in the following sequences, each amino acid symbol preceded by the symbol [D] indicates the D type of that amino acid: (I) [D](X1)[D](X2)[D](X3)[D]E[D]V[D]R[D]S, in which X1 represents H or Q, X2 represents P or S, and X3 represents N or K; (II) [D]Q[D](X2)[D]A[D]T[D](X5)[D]L[D]K, in which X2 represents Y or L, and X5 represents N, K, or Y; Specification 2 / 32 pages 5 CN 121752583 A (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L, in which X1 represents S or R, X4 represents R or W, and X5 represents N or I; (IV) An amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences in (I) to (III) above; (V) The reverse amino acid sequence of any of the amino acid sequences in (I) to (IV) above.

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

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

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

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

[0017] [6] The conjugate according to [5], wherein the aforementioned one or more components comprise a boron compound or a gadolinium compound.

[0018] [7] The conjugate according to [6], wherein the aforementioned one or more components comprise a boron compound.

[0019] [8] The conjugate according to [7], wherein the aforementioned conjugate is of the following formula (I): [Chemical 1] Specification 3 / 32 page 6 CN 121752583 A, or the following formula (II): [Chemical 2].

[0020] [9] The conjugate according to [5], wherein the aforementioned one or more components comprise an anticancer agent.

[0021]

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

[0022]

[11] The conjugate according to

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

[0023]

[12] The conjugate according to

[10] or

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

[0024]

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

[12] .

[0025]

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

[0026]

[15] The neutron capture therapy agent according to

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

[0027]

[16] The neutron-capturing therapy agent according to

[14] or

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

[0028]

[17] Specification 4 / 32Page 7 CN 121752583 A Neutron capture therapy agent according to

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

[0029]

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

[0030]

[19] Cancer examination reagent comprising the conjugate described in any one of

[10] to

[12] .

[0031]

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

[0032]

[21] Neutron capture therapy according to

[20] , wherein the aforementioned subject has solid cancer.

[0033]

[22] The neutron capture therapy according to

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

[0034]

[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].

[0035]

[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] .

[0036]

[25] The method according to

[23] or

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

[0037]

[26] The neutron capture therapy agent according to

[16] or the method according to

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

[0038]

[27] A neutron-capturing therapy agent according to any one of

[14] to

[17] , or a cancer chemotherapeutic agent according to

[18] , for administration to a subject selected from subjects with cancer who has annexin A1-positive cancer.

[0039]

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

[14] to

[17] , or a cancer chemotherapeutic agent according to

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

[0040]

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

[14] to

[17] , or a cancer chemotherapeutic agent according to

[18] .

[0041]

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

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

[0042]

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

[12] , for use in the specification, page 5 / 32, CN 121752583 ACancer treatment or prevention.

[0043]

[32] Use of the peptide or conjugate of any one of [1] to [4] in the manufacture of a cancer treatment or prevention drug.

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

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

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

[0047] Figure 3: Figure 3 shows the genome copy number of the lysed phages in each round of the 7-residue peptide sequences presented by the phage mixtures obtained in rounds 1 to 5.

[0048] Figure 4: Figure 4 shows the concentration of the L-amino acid sequences presented by the phage mixtures obtained in rounds 1 to 5 relative to the round 1 library, based on the 7-residue peptide sequences presented.

[0049] Figure 5: Figure 5 shows the alignment results of the top 10 peptide sequences presented by the 7-residue peptide sequences obtained in rounds 1 to 5.

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

[0051] Figure 7: Figure 7 shows the KD value and is affinity plot of dhp7 peptide relative to L-MC16 mutant. A: Relative to dhp7B: Sensing plots related to the binding and dissociation of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides of the immobilized sensor. B: Isoaffinity plots and equilibrium dissociation constants (KD) related to the binding and dissociation of L-MC16WT, E6A, F7A, K9A, Q10A, W11A, F13A, and E15A peptides of the dhp7 immobilized sensor. Two-dimensional affinity kinetic plots of rate constants grouped according to the L-MC16 peptide series. The dashed diagonal line represents the equilibrium binding constant, shown to aid in visualization of affinity distribution.

[0052] Figure 8: Figure 8 relates to the binding and intracellular uptake of dhp7 peptides with HEK293 overexpressing Anxa1-c-His.

[0053] Figure 9: Figure 9 shows the results of the binding and intracellular uptake of dhp7 peptide with HEK293 overexpressing Anxa1-c-His.

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

[0055] Figure 11: Figure 11 shows the results of a study on the tumor aggregation of Cy7.5-dhp7 in mice carrying cancer (bladder cancer).

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

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

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

[0059] Figure 15: Figure 15 shows the results of immunoassay and affinity analysis of rabbit anti-10BSH polyclonal antibodies (#52 and #53) against 10BSH-BSA.

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

[0061] Figure 17: Figure 17 shows the results of neutron capture therapy following administration of 10BSH-dhp7 in cancer-carrying mice.

[0062] Figure 18: Figure 18 shows the results of neutron capture therapy following administration of 10BSH-dhp7 in cancer-carrying mice.

[0063] Figure 19: Figure 19 shows the results of immunostaining of excised tumors after neutron capture therapy following administration of 10BSH-dhp7.

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

[0065] Figure 21: Figure 21 shows the results of a study on the tumor aggregation of Cy7.5-vc-dhp7 in mice with tumors 7 mm in diameter.

[0066] Figure 22: Figure 22 shows the results of the study on the tumor aggregation of Cy7.5-vc-dhp7 in mice with tumors of 15 mm diameter (prostate cancer).

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

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

[0069] Figure 25: Figure 25 shows the results of the study on the tumor aggregation of Cy7.5-vc-dhp7 in mice with tumors of 7 mm diameter (prostate cancer).

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

[0071] Figure 27: Figure 27 shows the results of a visualization study on the uptake of 10BSH in PC3-Anxa1 positive / negative cancer cells using immunofluorescence staining.

[0072] Figure 28: Figure 28 shows the results of a visualization study on the uptake of 10BSH in PC3-Anxa1 positive / negative cancer cells using immunofluorescence staining.

[0073] Figure 29: Figure 29 shows the results of a visualization study on the uptake of 10BSH in each cell type (PC3, SAS, and MG-63) using immunofluorescence staining.

[0074] Figure 30: Figure 31 shows the results of a visualization study on the uptake of 10BSH in each cell type (PC3, SAS, and MG-63) using immunofluorescence staining.

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

[0076] Figure 32: Figure 32 shows the results of fluorescence aggregation at sacrifice in mice treated with various 10B agents in cancer-carrying (prostate cancer) mice. Specification 7 / 32 pages 10 CN 121752583 A

[0077] Figure 33: Figure 33 shows the results of visualization of intra-tissue 10BSH uptake in various tissues 24 hours after administration of various 10B agents using immunohistochemical staining with anti-10BSH antibodies.

[0078] Figure 34: Figure 34 shows the results of visualization of intra-tissue 10BSH uptake in various tissues 24 hours after administration of various 10B agents using immunohistochemical staining with anti-10BSH antibodies.

[0079] Figure 35: Figure 35 shows the results of visualization of various 10B agent uptake in various tissues 24 hours after administration of various 10B agents.Visualization of 10BSH uptake in tissues 24 hours after administration of 10B agent using immunohistochemical staining with anti-10BSH antibody.

[0080] Figure 36: Figure 36 shows the time-dependent changes in 10BSH accumulation in mice treated with 10BSH-vc-dhp7 in prostate cancer-carrying mice.

[0081] Figure 37: Figure 37 shows the results of 10BSH accumulation in various organs 24 hours after administration of 10BSH-vc-dhp7 in prostate cancer-carrying mice.

[0082] Figure 38: Figure 38 shows the comparison of antitumor effects between the EMCS-10BSH-vc-dhp7 treatment group and the 10BSH+BNCT treatment group in ANXA1-negative prostate cancer (PC3-ANXA1-) and ANXA1-positive head and neck cancer (SAS) models.

[0083] Figure 39: Figure 39 shows the comparison results of the antitumor effects of the EMCS-10BSH-vc-dhp7 treatment group and the 10BSH+BNCT treatment group in the ANXA1-negative prostate cancer (PC3-ANXA1-) model and the ANXA1-positive head and neck cancer (SAS) model.

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

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

[0086] Figure 42: Figure 42 shows the appearance of the excised tumors and HE staining and post-treatment blood examination results during vcMMAE single-dose or vcMMAE-dhp7 treatment.

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

[0088] Figure 44: Figure 44 shows the immunostaining results of the removed tumor during treatment with vcMMAE single-dose or vcMMAE-dhp7.

[0089] Figure 45: Figure 45 shows the immunostaining results of the removed tumor during treatment with vcMMAE single-dose or vcMMAE-dhp7.

[0090] Figure 46: Figure 46 shows the shrinkage effect of luciferase-luminescent tumor and the results of weight change during treatment with vcMMAE single-dose or vcMMAE-dhp7.

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

[0092] Figure 48: Figure 48 shows the appearance of the removed tumor during treatment with vcMMAE single-dose or vcMMAE-dhp7. Detailed Embodiments

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

[0094] (I) [D] (X1) [D] (X2) [D] (X3) [D] E [D] V [D] R [D] S (in this sequence, X1 represents H or Q, X2 represents P or S, X3 represents N or K) (Seq ID NO:1); (II) [D] Q [D] (X2) [D] A [D] T [D] (X5) [D] L [D] K (in this sequence, X2 represents Y or L, X5 represents N, K or Y) (Seq ID NO:2); Specification 8 / 32 page 11 CN 121752583 A (III) [D] (X1) [D] T [D] S [D] (X4) [D] (X5) [D] T [D] L (in this sequence, X1 represents S or R, X4 represents R or W, X5 represents N or I) (Seq ID NO:3); (IV) An amino acid sequence having one or more amino acid insertions, substitutions, or deletions, or combinations thereof, in any of the amino acid sequences described in (I) to (III) above; (V) A reverse amino acid sequence of any of the amino acid sequences described in (I) to (IV) above.

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

[0096] In this specification, the amino acid sequences of (chain-like) peptides are described according to the convention of peptide representation, with the left side being the N-terminal side and the right side being the C-terminal side. In addition, each amino acid symbol in the amino acid sequence preceded by the symbol [D] indicates the D-type of the amino acid, and each amino acid symbol in the amino acid sequence without the symbol [D] before it indicates the L-type of the amino acid, provided that it does not violate the context. In this specification, peptides containing any of the amino acid sequences described in (I) to (V) above are collectively referred to as peptides of the present invention. In addition, the peptides of the present invention may also be composed of any of the amino acid sequences described in (I) to (V) above.

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

[0098] In one embodiment, the above (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), preferably [D]H[D]P[D]N[D]E[D]V[D]R[D]S (Seq ID NO:10).

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

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

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

[0102] The amino acid inserted (or added), replaced, or deleted in the amino acid sequence of (IV) above can be any amino acid as described below. The amino acid can be an L-type or D-type amino acid, for example, it can be a naturally occurring L-type amino acid as described below, or a D-type amino acid as its optical isomer. In addition, the amino acid can be subject to various chemical modifications as described below.

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

[0104] The substitution of the amino acid of (IV) above is preferably replaced with an amino acid with similar physicochemical properties ("similar amino acid").Amino acids classified as "acids" include, for example, aromatic amino acids (Phe (F), Trp (W), Tyr (Y)), aliphatic amino acids (Ala (A), Leu (L), Ile (I), Val (V)), polar amino acids (Gln (Q), Asn (N)), basic amino acids (Lys (K), Arg (R), His (H)), acidic amino acids (Glu (E), Asp (D)), amino acids with hydroxyl groups (Ser (S), Thr (T)), and amino acids with small side chains (Gly (G), Ala (A), Ser (S), Thr (T), Met (M)). It is predicted that substitutions based on these similar amino acids will not alter the protein's phenotype (i.e., conserved amino acid substitutions). Specific examples of conserved amino acid substitutions are well known in the art and documented in various literatures (see, for example, Bowie et al., Science, ...). 247:1306-1310 (1990). In this specification, the conserved amino acid substitution may also include replacing the D-type amino acid with its optical isomer, namely the L-type amino acid. The conserved amino acid substitution may be the substitution of L-type amino acids with each other, the substitution of D-type amino acids with each other, or the substitution of L-type amino acids with D-type amino acids.

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

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

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

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

[0109] The amino terminus and / or carboxyl terminus of the peptide of the present invention may be modified. Modifications to the amino terminus can include, for example, methylation (e.g., -NHCH3, -N(CH3)2, etc.), acetylation (e.g., using acetic acid or its halogenated derivatives (e.g., α-chloroacetic acid, α-bromoacetic acid, α-iodoacetic acid, etc.), ureation, carbamate esterification, formylation, Bocation, Fmocation, etc. Alternatively, see specification 10 / 32 page 13 CN 121752583 AIt is also possible to introduce any protecting group such as benzyloxycarbonyl, carboxylic acid ester functional group (RCOO-), sulfonyl functional group (R-SO2-) (here, R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc.).

[0110] As a modification of the carboxyl terminus, examples include amidation (-CONH2), esterification (-COOR), etc. Here, as R in the ester, for example, C1-6 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.; for example, C3-8 cycloalkyl such as cyclopentyl, cyclohexyl, etc.; for example, C6-12 aryl such as phenyl, α-naphthyl, etc.; for example, phenyl-C1-2 alkyl such as benzyl, phenethyl, etc.; C7-14 aryl such as α-naphthyl-C1-2 alkyl; neopentyloxymethyl, etc.

[0111] The peptide of the present invention can accept various modifications even outside the N-terminus or C-terminus. Chemical modifications can be, for example, methylation, acetylation, phosphorylation, etc. When the peptide of the present invention has a carboxyl group (carboxylic acid ester) outside the C-terminus, the carboxyl group can be amidated or esterified. As an ester at this time, for example, the C-terminal ester described above can be used. Alternatively, substituents on the side chains of amino acids within the molecule (e.g., -OH, -SH, amino, imidazole, indole, guanidinyl, etc.) can be protected by appropriate protecting groups (e.g., C1-6 alkyl acyl groups (C1-6 acyl groups) such as formyl or acetyl).

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

[0113] The peptide of the present invention may contain two or more sequences selected from (I) to (V) above. In one embodiment, the present invention...The peptide comprises a tandem repeat sequence of any of the sequences (I) to (V) above (i.e., a structure formed by directly linking the same sequence portions together). In another embodiment, the peptide of the present invention comprises a structure formed by directly linking two or more different sequences (I) to (V) above. Alternatively, the peptide of the present invention can be a multivalent peptide formed by dendritic macromolecules.

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

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

[0116] The peptides of the present invention can be manufactured according to known peptide synthesis methods. Peptide synthesis methods may be, for example, solid-phase synthesis or liquid-phase synthesis. When a portion of the peptide or amino acid that constitutes the peptide of the present invention is condensed with the residual portion, and the product has a protecting group, the target peptide can be manufactured by removing the protecting group. Instruction manual 11 / 32 pages 14 CN 121752583 A

[0117] Here, the condensation and removal of the protecting group can be carried out by methods known to them, for example, the methods 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) Nobuo Izumiya, Fundamentals and Experiments of Peptide Synthesis, Maruzen (Co., Ltd.) (1975) (4) Haruaki Yajima and Shunpei Sakakibara, Lectures on Biochemistry Experiments 1, Chemistry of Proteins IV 205 (1977) (5) Haruaki Yajima (supervisor), Development of Subsequent Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten (6) Stewart, J. M. & Young, J. D., “Solid phase peptide synthesis (2nd ed.)”, Pierce Chemical Company, Rockford (1984) (7) Atherton, E. & Sheppard, R. C., “Solid Phase peptide synthesis: a practical approach”, IRL Press, Oxford (1989) (8) “Fmoc Solid Phase Peptide Synthesis: A "Practical Approach (Practical Approach Series)", Oxford University Press (2000).

[0118] The peptides thus obtained can be purified and separated 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 methods is a free peptide, the free peptide can be converted into a suitable salt by a known method or a method based thereon. Conversely, when the peptide is obtained in the form of a salt, the salt can be converted into a free peptide or other salt by a known method or a method based thereon.

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

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

[0121] For example, for 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, for example, by screening specific cell populations in the cancer tissue and determining whether the cancer tissue contains annexin A1 positive cells to what extent. The aforementioned screening and determination can be performed according to known methods. Specifically, for example, immunohistostaining can be used. In this method, annexin A1 positive cells in the cancer tissue are stained with anti-human annexin A1 antibody (fluorescence or chromogenic method), and the positive or negative result is determined based on the amount of pigment emitted by the stained cells (e.g., fluorescence or visible light). This staining can typically be performed using annexin A1 binding molecules. Therefore, in one embodiment, the present invention relates to a companion diagnostic agent (hereinafter also referred to as the diagnostic agent of the present invention) for the neutron capture therapy agent or cancer chemotherapeutic agent of the present invention, which comprises an annexin A1 binding molecule. By means of a method including the step of contacting the annexin A1 binding molecule with cells in the cancer tissue of a subject (recipient), the effectiveness relative to the cancer therapeutic agent or preventive agent (cancer therapeutic agent, preventive agent) of the present invention can be predicted. Specification 12 / 32 pages 15 CN 121752583 A

[0122] The annexin A1 binding molecule is not particularly limited as long as it is a molecule that can bind to annexin A1 (preferably specifically). Examples of annexin A1 binding molecules include, for example, antibodies, and more specifically, for example, immunoglobulins, Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, single-chain antibody, etc.

[0123] The series of screening and determination operations described above can be performed, for example, by microscopic observation. The determination of positive or negative is based on known methods and on the amount of pigment produced by the stained cells. This determination can be performed using known methods. For example, the amount of pigment produced by negative control cells (specifically, cells stained with an allotype control antibody, for example) is set as the background pigment amount. Cells with a pigment amount lower than this background are determined to be negative cells, and cells with a fluorescence amount higher than this background are determined to be positive cells. As a simpler method, reverse transcription quantitative PCR of annexin A1 can also be performed using mRNA extracted from cancer tissue, and the results can be obtained by...The method for determining the amount of mRNA is as follows.

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

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

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

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

[0128] (Boron compounds) As boron compounds, there is no particular limitation as long as the compound contains boron atoms, and examples include, for example, compounds containing boron 10 nuclide (10B). In addition, the boron compound is preferably used in neutron capture therapy. Specifically, examples include R-B(OH)2 (boric acid (where R is a substituent (e.g., selected from alkyl, aryl, heteroaryl, alkylaryl, etc.))), BPA (p-boron phenylalanine), Borax, PCPB (4-carboxyphenylboronic acid), BODIPY (dipyrrolemethylene boron), boron clusters, etc.

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

[0130] Examples of boron clusters include, for example, decaborane (B10H14), decahydrodecaborate ([B10H10]2-) (GB-10) (sometimes also called sodium decahydrodecaborate)), dodecaborate ([B12H12]2-) and nested, nested-octadecoborane (22) (B18H22, CAS RN: 21107-56-2), but are not limited thereto.

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

[0132] Examples of boron clusters containing atoms other than boron atoms in their basic framework include, for example, carboranes (including so-called carborane isomers) containing boron atoms and carbon atoms. As carboranes, examples include, for example, closed-cell carboranes ([CB11H12]-) containing 1 carbon atom, closed-cell carboranes (C2B10H12) containing 2 carbon atoms, nested carboranes ([C2B9H11]-), or, for example, sandwich-type ones with the molecular formula M(C2B9H10)2, but are not limited thereto. In the above molecular formula, M represents a transition metal element, which can be, for example, Fe, Ni, Co, Mo, etc. That is, the boron cluster can be a metal complex with transition metals such as Fe, Ni, Co, Mo, etc.

[0133] The boron cluster is preferably ionic, and even more preferably water-soluble. Examples of ionic or water-soluble boron clusters include, for example, dodecaborate ([B12H12]2-), dodecaborate with a thiol group ([B12H11SH]2-, BSH (sometimes also called boroca (sodium) or mercaptoundecylhydrododecanoate)), dodecaborate with a hydroxyl group ([B12H11OH]2-), and dodecaborate with an amino group ([B12H11NH3]-).

[0134] (Gadolinium compounds) As gadolinium compounds, there is no particular limitation as long as the compound contains gadolinium atoms, and examples include, for example, compounds containing 157Gd, 157Gd.

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

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

[0137] Specific examples of alkylating agents include nitrogen mustard alkylating agents such as cyclophosphamide (Endoxan), ifomide, melphalan (Alkerans), busulfan, and thiotepamin, as well as nitrosourea alkylating agents such as nimustine (Nidran), ramustine (Cymerin), dacarbazine, procarbazine (procarbazine hydrochloride), temozolomide (Temodal), carmustine (Gliadel), streptozotocin (Zanosar), and bendamustine (Treakisym). Instructions for Use, Page 14 / 32, 17 CN 121752583 A

[0138] Specific examples of anticancer antibiotics include actinomycin D (Cos meggen), aclacinon, calsed, idarubicin, epirubicin (epirarubicin hydrochloride, Farmorubicin), smancozeb (SMANCS), daunomycin, and doxorubicin.Adriacin, Pinorubin (THERARUBICIN), Bleomycin, Pepleo, Mitomycin C, Novantron, and liposomal doxorubicin (DOXIL) are examples of microtubule inhibitors.

[0139] Examples of microtubule inhibitors include, for instance, Vincristine (Exal), Oncovin, Fildesin, and other Vinca minor alkaloid-based microtubule polymerization inhibitors, as well as Taxane-based microtubule depolymerization inhibitors such as Taxol, Docetaxel, and Vittin. Additionally, Monomethyl auristatin E (MMAE) is another example.

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

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

[0142] Examples of molecularly targeted drugs include, for instance, 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), pertuzumab (PERJETA), and sunitinib (SUTENT). Sorafenib (Nexavar), axitinib (Inlyta), pazopanib (Votrient), nilumumab (OPDIVO), pembrolizumab, ipilimumab (YERVOY), vemurafenib (ZELBORAF), everolimus (AFINITOR), tesimolimus (TORISEL), rituximab (Rituxan), bevacizumab (AVASTIN), geldmycin, etc.

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

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

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

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

[0147] Examples of radioactively impermeable substances and contrast agents include, for example, iodine compounds (e.g., organic iodic acids such as iodocarboxylic acid, iodine...).Examples of fluorescent substances include: barium compounds (e.g., barium sulfate), gallium compounds (e.g., gallium citrate), and thallium compounds (e.g., thallium chloride).

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

[0149] (Binding of peptides and components of the present invention) There is no particular limitation on the manner in which the peptides of the present invention in the conjugates of the present invention bind to one or more components. The binding can be direct or indirect, such as through a linker. The binding can be based on a common bond, a non-covalent bond, or a combination thereof. One or more components can bind directly or indirectly to the N-terminus, C-terminus, or other positions of the peptides of the present invention. The linking of peptides to other components (or second peptides) is well known in the art, and in the conjugates of the present invention, this linking can also be based on any known method.

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

[0151] In addition, in order to link the radioactive material (nuclide) and paramagnetic ion as described above with the peptide of the present invention, it is possible toSuitable chelating agents are used (e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 4,7,10-tetraazacyclododecane-N-N',N'',N'''-tetraacetic acid (DOTA), etc.) and / or metallothioneins, etc. (see, for example, Cumali Aktolun et al., “Nuclear Medicine Therapy: Principles and Clinical Applications”, Springer, 2013, etc.).

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

[0153] 3. Pharmaceutical Compositions of the Invention In addition, 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 comprise pharmaceutically acceptable carriers. The pharmaceutical composition can be provided in dosage forms suitable for oral or non-oral administration. Non-oral administration includes intravenous, intramuscular, subcutaneous, intra-visceral, intranasal, intradermal, ocular, intracerebral, rectal, vaginal, intraperitoneal, intratumoral, proximal to tumor, and direct lesion administration. This administration can be performed, for example, by injection, endoscopy, catheter, etc.

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

[0155] As a pharmaceutical composition for oral administration, solid or liquid dosage forms can be cited, specifically tablets (containing sugar)Coated tablets, film-coated tablets, pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such pharmaceutical compositions are manufactured by known methods and may also contain carriers, diluents, excipients, etc., commonly used in the field of pharmaceutical preparations. For example, lactose, starch, sucrose, magnesium stearate can be used as carriers and excipients for tablets.

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

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

[0158] The above-described non-oral or oral pharmaceutical compositions are preferably prepared in dosage forms with dosage units adapted to the dosage of the active ingredient. Examples of dosage forms for such administration units 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 can be obtained; typically, each dosage unit is 0.01 mg to 50 g, injections are 0.01 mg to 25 g, and other dosage forms are 0.01 mg to 50 g.

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

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

[0161] Examples of solid cancers include leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, etc. Examples of leukemia include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, etc. Malignant lymphomas are classified into Hodgkin lymphomas and non-Hodgkin lymphomas. Examples of non-Hodgkin lymphomas include B-cell lymphomas, adult T-cell lymphomas, lymphoblastic lymphomas, diffuse large cell lymphomas, Burkitt lymphomas, follicular lymphomas, MALT lymphomas, peripheral T-cell lymphomas, and mantle cell lymphomas.

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

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

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

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

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

[0167] In the neutron capture therapy, after the neutron capture therapy agent of the present invention reaches the tumor, the site is irradiated with an effective amount of low-energy thermal neutron rays such as thermal neutron rays or hyperthermal neutron rays. The site can be irradiated through the skin, or the site can be irradiated by fully or partially exposing it before irradiation. In addition, irradiation can be performed simultaneously from multiple directions (pages 18 / 32 of the specification, CN 121752583 A). The administration of the neutron capture therapy agent of the present invention and subsequent irradiation with thermal neutron rays or hyperthermal neutron rays can be repeated as needed. For example, multiple irradiations can be performed at intervals of about several months. The total number of irradiations is preferably 1 to 10.

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

[0169] The capture therapy using the neutron capture therapy agent of the present invention can be used as a standalone treatment, or it can be used in conjunction with conventional surgery or chemotherapy. If necessary, after surgically removing the tumor as much as possible, the remaining tumor can also be destroyed by using the capture therapy using the neutron capture therapy agent of the present invention.

[0170] In neutron capture therapy, the neutron capture therapy agent of the present invention can be used in combination with drugs such as anticancer agents, pharmaceutical compositions of the present invention, and cancer chemotherapy agents of the present invention described later. When using combination drugs, the timing of administration of the neutron capture therapy agent of the present invention and the combination drug is not limited; the neutron capture therapy agent of the present invention and the combination drug can be administered simultaneously to a subject suffering from a malignant tumor (cancer), or they can be administered at time intervals. When administering at time intervals, the order of administration is not particularly limited.

[0171] All necessary matters related to the neutron capture therapy agent of the present invention other than those described above are referred to in the contents of "1. Peptide of the present invention" and "2. Conjugate of the present invention".

[0172] (Cancer Chemotherapy Agent) In addition, the present invention also provides a cancer chemotherapy agent comprising the conjugate of the present invention (hereinafter also referred to as the cancer chemotherapy agent of the present invention). The conjugate comprises the anticancer agent as described above. The cancer chemotherapy agent of the present invention can aggregate on malignant tumors (cancers) expressing ANXA1 as described above on the cell surface by means of the peptide of the present invention contained in the agent. This aggregation means selectively tending to (bind to) or being locally present in cancerous tissues compared with non-cancer tissues (e.g., normal tissues) in the subjects described above (e.g., animals, more specifically mammals (e.g., humans, etc.)). Therefore, the cancer chemotherapy agent of the present invention is preferably used for the treatment of cancer. Therefore, the present invention also provides a method for the treatment or prevention of cancer, which includes administering to a subject an effective amount of the conjugate of the present invention comprising the anticancer agent, boron compound, gadolinium compound, etc. as described above.

[0173] 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. Generally, the amount of the conjugate of the present invention relative to the whole formulation is about 0.01 to 100% by weight, preferably about 0.1 to 50% by weight.

[0174] All necessary matters related to the cancer chemotherapeutic agent of the present invention other than those described above are hereinafter referred to in "1. Peptide of the present invention" and "2. Conjugate of the present invention".

[0175] (Cancer examination reagent) In addition, the present invention also provides a cancer examination reagent (hereinafter also referred to as the cancer examination reagent of the present invention) comprising the conjugate of the present invention. The conjugate comprises the detectable substance as described above. The cancer examination reagent of the present invention canThe peptides of the present invention contained in this agent aggregate on malignant tumors (cancers) expressing ANXA1 as described above on the cell surface. This aggregation refers to the selective tendency (binding) or local presence in cancerous tissue compared to non-cancer tissue (e.g., normal tissue) in the subjects described above (e.g., animals, more specifically mammals, such as humans). Therefore, the cancer detection reagent of the present invention is preferably used for cancer detection. Therefore, the present invention further provides a cancer detection method, as described on pages 19 / 32 of CN 121752583 A, which includes administering an effective amount of the conjugate of the present invention containing detectable substances as described above to the subject. Examples of cancer detection methods include X-ray imaging, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, scintillation scanning, positron emission tomography (PET), intravenous RI therapy, endoscopy, and laparoscopy as described above. Furthermore, the detection reagent of the present invention may also contain companion diagnostic drugs that pre-judge the likelihood of the efficacy of anticancer agents such as molecularly targeted drugs.

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

[0177] All other necessary matters related to the cancer examination reagent of the present invention, other than those mentioned above, are denoted by the contents of "1. Peptide of the present invention" and "2. Conjugate of the present invention".

[0178] The present invention is illustrated in more detail by the following examples, but the present invention is not limited thereto. Examples

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

[0180] A peptide (D-MC16) with d-cys residues was added to the N-terminal 15 residues of the amino acid sequence of ANXA1 synthesized from D-type amino acids (mamvseflkqawfie (Seq ID NO:29): Hereinafter, D-type amino acids are represented by lowercase letters). The peptide was immobilized on a maleimide-coated plate at concentrations shown in Figure 1 (10 nM, 1 nM, 0.1 nM, 0.01 nM) via the SH group of cys residues. 200 μl of a T7 phage mixture presenting a 7-residue L-type amino acid peptide library was added to the L-cysteine ​​immobilization wells, and the reaction was carried out with stirring at room temperature for 1 hour. 200 μl of the T7 phage mixture that did not bind to the L-cysteine ​​immobilization wells was then recovered and added to a 10 nM plate.D-MC16 peptide immobilization wells. Incubate at 37°C for 5 minutes, wash, and elute with 100 μl of 1% SDS-PBS to remove phage bound to 10 nM D-MC16 peptide (first round output).

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

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

[0183] Next, 200 μl of the fourth-round output phage mixture was added to the 10 nM L-amino acid MC16 peptide (L-MC16) immobilization wells, incubated at 37°C for 5 minutes, and 200 μl of the fourth-round output phage mixture that was not bound to L-MC16 was recovered. Add 200 μl of the fourth-round output phage mixture (page 23, CN 121752583 A, unspecified) bound to the L-MC16 peptide to a 10 nM D-MC16 peptide immobilization well. Incubate at 37°C for 5 minutes. After washing, elute the phage bound to the 10 nM D-MC16 peptide with 100 μl of 1% SDS-PBS (fifth-round output).

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

[0185] The 7-residue peptide sequences presented by the phage mixtures obtained from rounds 1 to 5 were amplicon sequenced using a next-generation sequencer, and the frequency of peptide sequence clones presented by each phage mixture was analyzed (Figure 4).

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

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

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

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

[0190] The biosensor was regenerated using a 0.1 mM glycine hydrochloride (pH 2.0) solution, applied in 3 pulses for 5 seconds each time. This association-dissociation-regeneration process was performed for each concentration of L-MC16 or D-MC16 solution. Data were analyzed using Octet Data analysis HT software version 10 (Sartorius). The obtained data were fitted to a 1:1 binding model to determine the values ​​of Kon and Koff, and the equilibrium dissociation constant KD was calculated (Figures 6A-C).

[0191] According to the results in Figures 6A-C, among the tested D-type peptide sequences, dhp7 had a KD value of 0.48 μM for the L-MC16 sequence, which was the highest affinity compared to dqy7 (0.99 μM) and dst7 (0.72 μM). Furthermore, no binding of dhp7 to D-MC16 was observed, indicating that dhp7 specifically binds to L-MC16. Based on these results, dhp7 was used in subsequent studies. Instruction manual, pages 21 / 32, 24, CN 121752583 A

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

[0193] As a result, the binding affinity of dhp7 to 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 (Figures 7A and B).

[0194] Example 4: Visualization of the uptake of dhp7 peptide by immunofluorescence staining in ANXA1-positive HEK293 cells investigated whether the dhp7 sequence selectively binding to the MC16 sequence of ANXA1 identified in Examples 1-3 was taken up by ANXA1-positive HEK293 cells. After transfecting HEK293T cells with the pCMV3-hANXA1-c-His plasmid containing the ANXA1-c-His fusion gene fused with a His tag at the C-terminus of human ANXA1, HEK293 cells were 72 hours later. Then, the cells were added with either biotin-labeled dhp7 peptide (50 μg / mL) at the N-terminus or 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 anti-mouse IgG-Alexa Fluor 555 (500-fold dilution) was added to detect anti-MC16 antibody. After incubation at 37°C for 10 minutes, followed by washing with PBS, the binding of the dph7 peptide to HEK293 cells overexpressing Anxa1-c-His and its intracellular uptake were studied using a fluorescence microscope (Keyence BZ9000) (Figures 8 and 9).

[0195] As a result, compared with HEK293 cells introduced with a negative control plasmid, the Biotin-dhp7 peptide showed higher staining intensity in HEK293 cells overexpressing ANXA1-c-His, and was observed intracellularly, similar to ANXA1 detected using anti-MC16 antibody.Dotted staining patterns were observed, and the cytoplasm was diffusely stained. This revealed that dhp7 may bind to ANXA1 and be taken up into the cell.

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

[0197] [Chemical 3] Specification 22 / 32 pages 25 CN 121752583 A Cell suspensions of human head and neck cancer cell line SAS, osteosarcoma cell line MG-63, prostate-specific membrane antigen (PSMA) negative or PSMA positive prostate cancer cell line PC3-PSMA±, and mouse bladder cancer cell line MBT2 were reacted with anti-MC16 antibody (1 μg / ml) reacting with the N-terminus of ANXA1 for 1 hour at room temperature. After washing the cell clumps, they were reacted with APC-labeled anti-mouse IgG antibody for 1 hour at room temperature. After washing the cell clumps, the expression of ANXA1 on the cell surface was studied by flow cytometry, and the results are shown in Figure 10. The antibody reaction was performed under non-fixed, cell membrane impermeable conditions.

[0198] The results in Figure 10 show that there were differences in the expression intensity of ANXA1 among the cancer cell lines, but all were expressed on the cell surface. This indicates that in PSMA-negative PC3 (PC3-PSMA-) cell lines, the expression of ANXA1 on the cell surface is low compared to PSMA-positive PC3 (PC3-PSMA+) cell lines. The expression of ANXA1 on the cell surface of MG-63 and SAS cells is at the same level. There are cell populations with strong and weak expression of ANXA1 on the cell surface in MBT2 cells.

[0199] The tumor aggregation of Cy7.5-c-dhp7 in ANXA1-positive bladder cancer carrier mice was studied. 4 × 10⁵ mouse bladder cancer cells MBT2 were inoculated into the right hind leg thigh of C3H / HeN mice. One week later, cancer carrier mice with tumors reaching 5 mm in diameter were intraperitoneally administered Cy7.5-dhp7 dissolved in physiological saline at a dose of 10 mg / kg (0.2 mg / 20 g mouse). Following drug administration, monitoring was performed using an in vivo imaging system (IVIS) at 0, 30, 90, 120 minutes, 24, 48, 72, and 96 hours (Figure 11).

[0200] As a result, the fluorescence intensity of Cy7.5-dhp7 in tumor tissue increased starting 30 minutes after administration, and then rapidly accumulated 2 hours after administration, reaching a plateau phase after 24 hours. The strong tumor-specific aggregation continued until 96 hours later. Furthermore, mice were sacrificed at 48 and 96 hours after administration, and major organs were removed for in vitro imaging. Strong aggregation of Cy7.5-dhp7 was observed in tumor tissue and kidneys, and also in the intestines, but no aggregation was observed in other organs. This suggests that Cy7.5-dhp7 may be renally excreted.

[0201] Further, the uptake of Cy7.5-dhp7 in other cancer cells was investigated. One × 10⁶ 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 backs of nude mice. Two weeks later, in mice carrying tumors with a tumor diameter of 15 mm, Cy7.5 or Cy7.5-dhp7 or L-type peptide RRQRRAP (R7)-EMCS-Cy7.5 (Seq ID NO:39 (RRQRRAP)) dissolved in physiological saline was administered intraperitoneally at 10 mg / kg (0.2 mg / 20 g mouse). Post-administration monitoring was performed using in vivo imaging (IVIS) at 0, 20, 30, 90, 120 minutes, 24, 48, 72, and 96 hours (Figures 12–14).

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

[0203] Example 6: Visualization of EMCS-10BSH-dhp7 peptide uptake in ANXA1 positive cancer cells using immunofluorescence staining. Example 5 clarified that Cy7.5-dhp7 was taken up in ANXA1 positive tumor tissue of a mouse model carrying cancer. Therefore, the uptake of EMCS-10BSH-dhp7 (10BSH-dhp7) in various ANXA1 positive cancer cells was subsequently investigated. To confirm whether 10BSH was taken up in the cancer cells studied in Example 5, rabbit anti-10BSH polyclonal antibodies specifically reacting with 10BSH were prepared.Antibodies. Two mice were immunized with 200 μg of 10BSH-KLH as the antigen. Whole blood was collected after the fourth booster immunization to obtain serum containing rabbit anti-10BSH polyclonal antibodies that specifically react with 10BSH (#52 and #53). The IgG fraction of each serum was purified by passing it through a protein A column. The affinity of the rabbit anti-10BSH polyclonal antibodies (#52 and #53) against 10BSH-BSA was analyzed by ELISA and BLI methods. The results are shown in Figure 15.

[0204] The results in Figure 15 confirm that the rabbit anti-10BSH polyclonal antibodies (#52 and #53) both react in a 10BSH-BSA concentration-dependent manner. In the following experiments, either antibody #52 or #53 was used for 10BSH detection.

[0205] Next, EMCS-10BSH-hpnevrs (10BSH-dhp7) (Seq ID NO:31) (Chemical 4) was synthesized, in which 10BSH was bound to the side chain of the N-terminal His residue of the hpnevrs (dhp7) (Seq ID NO:31) sequence via the EMCS linker.

[0206] [Chemical 4] The uptake of 10BSH-dhp7 in cancer cells was studied. MBT2 cells and PC3-PSMA+ cells seeded in 3.5 cm culture dishes (ib81156) manufactured by Ibidi were supplemented with 50 μg of 10BSH-dhp7 or 10BSH. After 24 hours, the cells were washed three times with PBS and then fixed with 4% PFA. After blocking with 3% BSA BD PhosFlow perm / wash buffer I (BD557885), the mixture containing 1 μg / ml rabbit anti-10BSH polyclonal antibody #52 was reacted with 10BSH taken up into the cells for 1 hour at room temperature. After washing three times with PBS, to detect the rabbit anti-10BSH polyclonal antibody, it was reacted with APC-labeled anti-mouse IgG antibody (1000-fold dilution) for 1 hour at room temperature. After washing three times with PBS, the intracellular uptake of 10BSH-dhp7 or 10BSH relative to each cancer cell was studied using a fluorescence microscope (Keyence BZ9000) (Figure 16).

[0207] As a result, in addition to each cancer cell, compared with 10BSH, punctate fluorescence was observed in cells with added 10BSH-dhp7 compared to 10BSH. The molecular weight of 10BSH is 219.87, and that of 10BSH-dhp7 is 1197.5. It was clarified that at a concentration of 1 / 5.44, there was even more...10BSH was taken up into the cells. 10BSH has low cell membrane permeability, thus indicating that the uptake efficiency of 10BSH in cells is enhanced via ANXA1 on the cell surface through binding to the dhp7 peptide.

[0208] Example 7: Boron neutron capture therapy using EMCS-10BSH-hpnevrs (10BSH-dhp7) As demonstrated in Example 6, 10BSH-dhp7 was taken up in Anxa1-positive cancer cells; therefore, boron neutron capture therapy using 10BSH-dhp7 was attempted in a mouse bladder cancer model. Two × 10⁵ mouse bladder cancer cells MBT2 were inoculated into the right hind leg thigh of C3H / HeN mice. Two weeks later, cancer-carrying mice with tumors reaching a diameter of 5 mm were intraperitoneally administered EMCS-10BSH-dhp7 dissolved in physiological saline at a dose of 20 mg / kg (0.4 mg / 20 g mouse).

[0209] 48 hours after administration, the tumor was irradiated with thermal neutrons at 20 MeV 100 mA for 60 minutes using a Sumitomo Heavy Industries accelerator (dose 1.63 × 10¹² / cm², average 0.23 Gy). One week after irradiation, a second BNCT treatment was administered. 24 hours before thermal neutron irradiation, 10BSH-dhp7 20 mg / kg (0.4 mg / 20 g mouse) was administered intraperitoneally, followed by thermal neutron irradiation. One week after the second irradiation, mice in each group were sacrificed, and tumor diameter and weight were measured. Pathological analysis of the excised tissue was performed to verify the antitumor effect. Tumor volume was measured from the start of administration until sacrifice, according to the following formula (V = major diameter x minor diameter x minor diameter / 2) (Figure 17). As a result, no tumor shrinkage effect was observed in the 10BSH-dhp7 administration + non-BNCT treatment group. In contrast, in the 10BSH-dhp7 administration + BNCT treatment group, a significant tumor shrinkage effect was observed immediately after the first BNCT treatment, and the tumor almost completely disappeared one week after the second irradiation (Figure 18).

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

[0211] Example 8: In vivo imaging of tumor aggregation associated with c(MI-Cy7.5)-vc-hpnevrs peptide. In Example 5, the tumor aggregation of EMCS-Cy7.5-c-hpnevrs peptide (Seq ID NO:31) in a mouse model carrying cancer was studied. In order to study the aggregation when the linker was changed to other than EMCS linker, the fluorescently labeled peptide shown below was synthesized.

[0212] [Chemical 5] Specification 25 / 32 pages 28 CN 121752583 A [Chemical 6] C(MI-Cy7.5)-vc-HPNEVRS (Cy7.5-vc-LHP7) (Seq ID NO:30 (HPNEVRS)) (Chemical 5) was synthesized with Cy7.5 fluorescent dye bound to the side chain of the N-terminal Cys residue of the C-HPNEVRS sequence (Seq ID NO:30) with L-Cys residue added via the Val-Cit linker. c(MI-Cy7.5)-vc-hpnevrs (Cy7.5-vc-dhp7) (Seq ID NO:31(hpnevrs)) (Chemical 6) were synthesized with Cy7.5 fluorescent dye bound to the side chain of the N-terminal cys residue of the c-hpnevrs sequence (Seq ID NO:31) with added d-cys residues.

[0213] Next, 1×10⁶ human prostate cancer cells PC3-PSMA- / ANXA1- and PC3-PSMA+ / ANXA1+ were inoculated into the backs of nude mice. One week later, mice carrying tumors with a diameter of 7 mm were intraperitoneally administered Cy7.5 at 10 mg / kg (0.2 mg / 20 g mouse), Cy7.5-vc-dhp7 at 28.7 mg / kg (0.574 mg / 20 g mouse), or Cy7.5-vc-LHP7 (L-type HP7 peptide) at 28.7 mg / kg (0.574 mg / 20 g mouse), dissolved in physiological saline. The Cy7.5 concentration of each fluorescent compound was prepared as a dosage of 10 mg / kg (0.2 mg / 20 g mouse). Following administration, in vivo imaging (IVIS) was used to monitor the drug at 0, 15, 30, 60, 90, 120 minutes, 24, 48, 72, and 96 hours (Figures 20 and 21).

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

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

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

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

[0218] Since Cy7.5-vc-dhp7 showed tumor-specific aggregation in a mouse model of prostate cancer, the tumor aggregation of Cy7.5-vc-dhp7 in other cancer cells (human head and neck cancer cells SAS, human osteosarcoma cancer cells MG-63) was studied under the same conditions as in Figure 17 (tumor diameter 7 mm) (Figure 24). As a result, Cy7.5-vc-dhp7 selectively aggregated in tumor tissue from 60 minutes after administration, regardless of the expression of ANXA1 in the tumor, reaching its maximum at 2 to 24 hours, and its aggregation gradually decreased but continued until 96 hours. On the other hand, no aggregation to the tumor was observed in Cy7.5-vc-LHP7, thus indicating that dhp7 composed of D-amino acids aggregated in tumor tissue in the same way as in the prostate cancer model.

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

[0220] Example 9: Visualization of the uptake of EMCS-10BSH-vc-dhp7 peptide in ANXA1 positive cancer cells using immunofluorescence staining. In Example 8, it was confirmed that Cy7.5-vc-dhp7 was taken up in a mouse model of ANXA1 positive cancer cells. Therefore, EMCS-10BSH-val-cit-hpnevrs (10BSH-vc-dhp7) (Seq ID NO:31 (hpnevrs)) (Chem. 7) was synthesized with EMCS-10BSH bound to the N-terminus of the hpnevrs (dhp7) sequence (Seq ID NO:31) via the Val-Cit linker, and its uptake in ANXA1 positive cancer cells was studied.

[0221] [Chem. 7] Specification 27 / 32 pages 30 CN 121752583 A The uptake of 10BSH-vc-dhp7 in cancer cells was studied. PC3-PSMA+ and PC3-PSMA- cells seeded in 3.5 cm culture dishes (ib81156) manufactured by Ibidi were supplemented with either 330 μg of 10BSH-vc-dhp7, 403.6 μg of IFLLWQRK (EMCS-10BSH)RR (IF7-10BSH (a compound described in WO2019244954)) (Seq ID NO:40 (IFLLWQRK)) or 50 μg of 10BSH. 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 calculated based on boron concentration to prepare a final addition of 50 μg of 10BSH. After blocking with 3% BSA BD PhosFlow perm / wash buffer I (BD557885), the rabbit anti-10BSH polyclonal antibody #52 containing 1 μg / ml of 3% BSA BD PhosFlow perm / wash buffer I was reacted with intracellular 10BSH for 1 hour at room temperature. After washing three times with PBS, to detect the rabbit anti-10BSH polyclonal antibody, it was reacted with APC-labeled anti-mouse IgG antibody (1000-fold dilution) for 1 hour at room temperature, followed by three washes with PBS. The results were then analyzed using a fluorescence microscope (Keyence BZ9000).The intracellular uptake of 10BSH-vc-dhp7, IF7-10BSH, or 10BSH relative to individual cancer cells was investigated (Figures 27 and 28).

[0222] As a result, more punctate fluorescence was observed intracellularly in cells supplemented with 10BSH-vc-dhp7 compared to 10BSH, along with the cancer cells. Quantification of fluorescence intensity relative to cells showed that the uptake efficiency was significantly higher. In this study, since each agent was prepared in 50 μg of 10BSH, it indicates that more 10BSH was taken up intracellularly in cells supplemented with 10BSH-vc-dhp7. The low cell membrane permeability of 10BSH indicates that the uptake efficiency of 10BSH in cells is enhanced via ANXA1 on the cell surface through binding to the dhp7 peptide. In addition, in PSMA-negative cells with low ANXA1 expression on the cell surface (Figs. 25 and 26), 10BSH-vc-dhp7 uptake was less compared to PSMA-positive cells, suggesting that ANXA1 expression on the cell surface may affect the uptake efficiency via the dhp7 peptide. In PSMA-positive cells with high ANXA1 expression, the uptake efficiency of 10BSH was significantly higher in the 10BSH-vc-dhp7 addition group, reaching 9.5 to 11.7 times, compared to BSH alone or IF7-10BSH (the compound described in WO2019244954).

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

[0224] According to the results in Figures 29-31, statistically significant intracellular uptake of 10BSH was observed in all cells compared to the group without 10BSH-vc-dhp7. In addition, among the cell lines tested, the uptake efficiency was highest in PC3-PSMA+ cells, which had the highest ANXA1 expression level on the cell surface. In SAS, MG63, and PC3-PSMA-, which had weakly positive ANXA1 expression on the cell surface, although there were biases due to cell type, they all showed the same level of 10BSH uptake efficiency. In addition, the localization of 10BSH uptake in each cell showed co-localization with lysosomes stained with LysoBright Green. This suggests that 10BSH-vc-dhp7 may be taken up in individual cells via ANXA1-mediated endocytosis expressed on the cell surface, and within lysosomes,The Val-Cit linker is cleaved by cathepsin B, thereby releasing 10BSH into the cell (Figures 29-31).

[0225] Example 10: Study on the Boron-related Tumor Aggregation of 10BSH-vc-dhp7 Peptide in Cancer-Carrying Mice 28 / 32 pages 31 CN 121752583 A In Example 9, it was established that EMCS-10BSH-vc-dhp7 is taken up by Anxa1-positive cancer cells. Therefore, by using immunohistochemical staining with 10BSH antibody, the aggregation concentration of 10B in tumor tissue, normal kidney tissue, and thigh muscle tissue in a human prostate cancer model was investigated to determine whether it reached a concentration sufficient to exert the antitumor effect of neutron capture therapy. 1×10⁶ human prostate cancer cells PC3-PSMA- / ANXA1- were inoculated into the left back of nude mice, and PC3-PSMA+ / ANXA1+ were inoculated into the right back of nude mice. Two weeks later, in mice carrying tumors with a diameter of 10 mm, 10BSH 50 mg / kg (1.25 mg / 25 g mouse), EMCS-10BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse), and IFLLWQRK(EMCS-10BSH)RR (IF7-10BSH (a compound described in WO2019244954)) 403.5 mg / kg (10.08 mg / 25 g mouse) (Seq ID NO:40 (IFLLWQRK)) were administered intraperitoneally.

[0226] 10BSH and 10BSH-vc-dhp7 were dissolved in saline at this concentration, while IF7-10BSH had low solubility at this concentration and was turbid. This indicates that the solubility of 10BSH-vc-dhp7 is higher than that of IF7-10BSH, and the dosage form has a high degree of freedom. To confirm the aggregation of dhp7, each mouse was simultaneously administered 0.574 mg / kg of Cy7.5-vc-dhp7 (Figure 32).

[0227] Cy7.5-vc-dhp7 began to rise in the tumor tissue 120 minutes after administration, and strong aggregation was observed in the tumor and kidney 24 hours after sacrifice (Figure 32). The tumor tissue, kidney, and thigh muscle tissue with low aggregation of Cy7.5-vc-dhp7 were removed and prepared into tissue sections by formalin fixation and paraffin embedding. Immunohistochemical staining was performed on each tissue section using anti-10BSH antibody (#53, 1 μg / ml) based on anti-10BSH antibody. The results are shown in Figures 33-35.

[0228] According to the results in Figures 33-35, in the tumor tissue and kidney tissue of the 10BSH-vc-dhp7 administration group, compared with the 10BSH-administered group...Strong staining was observed in the drug group compared to the IF7-10BSH administration group.

[0229] The above studies show that 10BSH-vc-dhp7 is taken up by Anxa1-positive cancer cells and excreted by the kidneys. Therefore, the aggregation of 10B in a human prostate cancer model was visualized by labeling anti-10BSH antibody with Alexa Fluor 750, and the in vivo distribution was investigated by IVIS. 1×106 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. Two weeks later, 330 mg / kg (8.25 mg / 25 g mouse) of 10BSH-vc-dhp7 dissolved in physiological saline was administered intraperitoneally to cancer-carrying mice with tumor diameters reaching 10 mm. 10BSH-vc-dhp7 was dissolved in saline at this concentration. To confirm the aggregation of 10BSH, mice were simultaneously administered 0.05 mg / kg of Alexa Fluor 750-labeled anti-10BSH antibody (#52) intraperitoneally.

[0230] The results showed that 10BSH, like Cy7.5-vc-dhp7, aggregated immediately near the kidneys and testes after intraperitoneal administration (Fig. 36). In addition, the aggregation in the kidneys disappeared 24 hours after administration, and strong aggregation was observed at the tumor site regardless of ANXA1 expression (Fig. 36). The results of ex vivo imaging of each excised organ 24 hours later are shown in Fig. 37.

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

[0232] Example 11: Boron neutron capture therapy using 10BSH-val-cit-dhp7 (EMCS-10BSH-vc-dhp7) Example 10 demonstrated that the boron concentration in tumor tissue after administration of 10BSH-vc-dhp7 was sufficient for the antitumor effect of neutron capture therapy (BNCT). Therefore, boron neutron capture therapy using EMCS-10BSH-vc-dhp7 was attempted in ANXA1-negative human prostate cancer models and ANXA1-positive head and neck cancer models. 1 × 10⁶ human prostate cancer cells PC3-PSMA- / ANXA1- or human head and neck cancer cells SAS were inoculated into the right hind leg thigh of nude mice. One week later, mice with tumors reaching 5 mm in diameter were administered either 50 mg / kg (1.25 mg / 25 g mouse) of 10BSH dissolved in physiological saline or 330 mg / kg (8.25 mg / 25 g mouse) of 10BSH-VC-DHP7 intraperitoneally. Following administration, 24 hours later, the mice were given a home remedy.Yuhe Heavy Industries accelerator was used to irradiate the tumor with thermal neutrons at 20 MeV 100 mA for 60 minutes (dose 1.63×10¹² / cm², average 0.23 Gy). One week after irradiation, a second BNCT treatment was administered.

[0233] 24 hours before thermal neutron irradiation, mice were intraperitoneally administered 10BSH 50 mg / kg (1.25 mg / 25 g mouse) dissolved in physiological saline or 10BSH-vc-dhp7 330 mg / kg (8.25 mg / 25 g mouse), and thermal neutron irradiation was performed 24 hours later. One week after the second irradiation, mice in each group were sacrificed, tumor diameter and weight were measured, and pathological analysis of the extracted tissue was performed to verify the anti-tumor effect. Tumor volume was measured from the start of administration to irradiation using the following formula (V = major diameter × minor diameter × minor diameter / 2) (Figures 38 and 39).

[0234] As a result, in the ANXA1-negative prostate cancer model, no tumor shrinkage effect was observed in either the EMCS-10BSH-vc-dhp7 administration + BNCT treatment group or the 10BSH administration + BNCT treatment group. This suggests that the accumulation effect of boron from 10BSH in ANXA1-negative cancer tissue is low, and the tissue boron concentration may not reach a sufficient level for the antitumor effect of BNCT. On the other hand, in the ANXA1-positive head and neck cancer model, a more significant tumor growth inhibition effect was observed immediately after the first BNCT in the EMCS-10BSH-vc-dhp7 administration + BNCT treatment group than in the 10BSH + BNCT treatment group, and the tumor volume was smaller than that in the 10BSH + BNCT treatment group one week after the second irradiation (Figures 38 and 39).

[0235] Based on the above results, EMCS-10BSH-vc-dhp7 showed superior antitumor effects against ANXA1-positive cancers compared to 10BSH monomers, and is therefore considered to be an agent with a higher boron delivery capacity to ANXA1-positive cancer tissues compared to the past. ANXA1-positive cancers are represented by head and neck cancers, and have been reported in many types of cancers such as breast cancer, lung cancer, melanoma, brain tumors, kidney cancer, and bladder cancer. The peptide-boron compound conjugate of the present invention is considered a candidate agent useful for BNCT of ANXA1-positive cancers.

[0236] Example 12: Treatment of cancer with anticancer agent using c(vcMMAE)-hpnevrs(vcMMAE-dhp7) In Examples 8-11, tumor aggregation and antitumor effects were observed in the compound bound to dhp7 via the Val-Cit linker. Therefore, a compound with the addition of boron to the N-terminus of the hpnevrs(dhp7) sequence (Seq ID NO:31) was then synthesized.Cys residues, and via the val-cit linker, bind the microtubule inhibitor monomethylaurestatin E (MMAE) to form c(vcMMAE)-hpnevrs (vcMMAE-dhp7) (Seq ID NO:31(hpnevrs)) (Chem. 7).

[0237] [Chem. 7] To test the cytotoxicity of the synthesized agent on ANXA1-positive / negative PC3 cells in vitro, the peptide drug conjugate and various concentrations of vcMMAE or vcMMAE-dhp7 were added to PC3 cells and incubated at 37°C for 24 hours. Cell viability was analyzed using a cell counting kit8 (Tongrentang), and the IC50 value was calculated (Figure 40). The IC50 values ​​of PC3-Luc2-PSMA+ / ANXA1-positive cells treated with vcMMAE or vcMMAE-dhp7 (129.9 or 122.7 ng, respectively) were confirmed to be twice that of PC3-Luc2-PSMA- / ANXA1-negative cells (283.3 or 204.5 ng, respectively). This suggests that the presence or absence of ANXA1 expression, as described in the PC3 cell datasheet (pages 30 / 32, CN 121752583 A), affects in vitro cytotoxicity. Specifically, higher ANXA1 expression levels in PC3 cells correlate with higher in vitro sensitivity to vcMMAE; therefore, higher ANXA1 expression in tumor cells, as described in vcMMAE-dhp7, indicates a stronger antitumor effect.

[0238] One × 10⁶ PSMA-negative / ANXA1-negative human prostate cancer cells (PC3) were seeded into the left back of nude mice, and one × 10⁶ PSMA-positive / ANXA1-positive PC3 cells were seeded into the right back. Two weeks later, mice with tumors reaching a diameter of 7 mm were intraperitoneally administered 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) dissolved in physiological saline every 4 days. As a control, a single dose of vcMMAE (molecular weight 1316,6) at 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse) was administered. Tumor size was monitored by measuring the average brightness (photons / second / cm² / sr) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse) using an IVIS system. After three courses of treatment, the tumor and body weight, pathological examination, blood biochemistry examination, and blood cell count were evaluated (Figure 41).

[0239] In vitro, Anxa1 positive cells were twice as sensitive to vcMMAE and vcMMAE-dhp7 as ANXA1 negative cells, indicating a high cell-killing effect. On the other hand, in a mouse model, the PC3 tumor model treated with vcMMAE-dhp7 showed significantly higher sensitivity.The average brightness of the mice, regardless of the ANXA1 expression level within the tumor, decreased almost completely over the three cycles (Fig. 41). Furthermore, no significant changes such as weight loss were observed in any group (Fig. 41). The appearance of the removed tumor and the results of post-treatment blood tests are shown in Figs. 42 and 43.

[0240] The appearance of the removed tumor and HE staining results in the vcMMAE-dhp7 treatment group showed significant necrosis and less residual tumor compared to the vcMMAE monotherapy group (Fig. 42). There were no significant differences in blood biochemical values ​​between the vcMMAE-dhp7 treatment group and the vcMMAE monotherapy group, but a decrease in leukocytes was observed in the vcMMAE monotherapy group (Fig. 43). Additionally, the results of immunostaining are shown in Figs. 44 and 45.

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

[0242] Based on the above results, since the excellent anti-tumor effect of vcMMAE-dhp7 was observed in the subcutaneous tumor model of the back, the same treatment experiment was carried out in the tumor model of subcutaneous inoculation in the thighs of both limbs. 1 × 10⁶ PSMA-negative / ANXA1-negative human prostate cancer cells PC3 were inoculated into the left thigh of nude mice, and 1 × 10⁶ PSMA-positive / ANXA1-positive PC3 cells were inoculated into the right thigh. Two weeks later, in cancer-carrying mice with tumors reaching a diameter of 7 mm, vcMMAE-dhp7 (molecular weight 2257.5) dissolved in physiological saline was administered intraperitoneally at a dose of 1.25 mg / kg (equivalent to 16.75 ng vcMMAE / 25 g mouse) every 4 days. As a control, a single dose of vcMMAE (molecular weight 1316.6) of 0.919 mg / kg (22.975 ng vcMMAE / 25 g mouse) was administered. Tumor size was determined by injecting fluorescein (150 mg / min) using an IVIS system.The average brightness (photons / second / cm2 / sr) of luciferase-expressing tumors was monitored after three treatment cycles. The average brightness of luciferase-expressing tumors and body weight were evaluated after three treatment cycles (Fig. 46).

[0243] The average brightness of PC3 tumors in the thigh subcutaneous seeding model mice treated with vcMMAE-dhp7 decreased in the same way during the three treatment cycles, regardless of the ANXA1 expression level or tumor size. In contrast, the vcMMAE treatment group showed a tendency to increase in size (Fig. 46). In addition, no significant changes such as weight loss were observed in any group (Fig. 46). The appearance of the extracted tumor is shown in Fig. 47. The above results show that vcMMAE-dhp7 can reduce the dosage of vcMMAE by about 30% and obtain a better anti-tumor effect than vcMMAE.

[0244] Based on the above results, excellent antitumor effects of vcMMAE-dhp7 were observed in a subcutaneous tumor model of the thigh. Therefore, the antitumor effects at the same volume were compared with those of vcMMAE single dose. Two × 10⁶ PSMA-positive / ANXA1-positive PC3 cells were seeded into the right thigh. One week later, in cancer-carrying mice with tumors reaching a diameter of 7 mm, vcMMAE-dhp7 (molecular weight 2257.5) dissolved in physiological saline was administered intraperitoneally at a dose of 2.14 mg / kg (equivalent to 31.25 ng vcMMAE / 25 g mouse) every 4 days. As a control, a single dose of vcMMAE (molecular weight 1316.6) was administered at 1.25 mg / kg (31.25 ng vcMMAE / 25 g mouse). Tumor size was monitored by measuring the average brightness (photons / second / cm² / sr) of luciferase-expressing tumors after injection of luciferin (150 mg / mouse) using an IVIS system. After four treatment cycles, the mean brightness and weight of the luciferase-expressing tumors were evaluated (Fig. 48).

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

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

[0247] This application is based on Japanese Patent Application No. 2023-137833 (filed on August 28, 2023), the entire contents of which are included in this specification. Instruction Manual Page 32 / 32 35 CN 121752583 A Figure 1 Figure 2 Instruction Manual Appendix 1 / 29 Page 36 CN 121752583 A Figure 3 Figure 4 Instruction Manual Appendix 2 / 29 Page 37 CN 121752583 A Figure 5 Figure 6 Figure 7 Instruction Manual Appendix 3 / 29 Page 38 CN 121752583 A Figure 8 Figure 9 Instruction Manual Appendix 4 / 29 Page 39 CN 121752583 A Figure 10 Figure 11 Instruction Manual Appendix 5 / 29 Page 40 CN 121752583 A Figure 12 Figure 13 Instruction Manual Appendix 6 / 29 Page 41 CN 121752583 A Figure 14 Figure 15 Instruction Manual Appendix 7 / 29 Page 42 CN 121752583 A Figure 16 Instruction Manual Appendix 8 / 29 Page 43 CN 121752583 A Figure 17 Figure 18 Instruction manual figures 9 / 29, page 44, CN 121752583 A, Figure 19, Figure 20, Figure 21; Instruction manual figures 10 / 29, page 45, CN 121752583 A, Figure 22, Figure 23; Instruction manual figures 11 / 29, page 46, CN 121752583 A, Figure 24; Instruction manual figures 12 / 29, page 47, CN 121752583 A, Figure 25; Instruction manual figures 13 / 29, page 48, CN 121752583 A, Figure 26; Instruction manual figures 14 / 29, page 49, CNFigure 27, Figure 28, Appendix Figure 15 / 29, Page 50, CN 121752583 A; Figure 29, Appendix Figure 16 / 29, Page 51, CN 121752583 A; Figure 30, Appendix Figure 17 / 29, Page 52, CN 121752583 A; Figure 31, Appendix Figure 18 / 29, Page 53, CN 121752583 A; Figure 32, Figure 33, Appendix Figure 19 / 29, Page 54, CN 121752583 A; Figure 34, Figure 35, Appendix Figure 20 / 29, Page 55, CN 121752583 A; Figure 36, Figure 37, Appendix Figure 21 / 29, Page 56, CN 121752583 A; Figure 38, Figure 39, Appendix Figure 22 / 29, Page 57, CN 121752583 A; Figure 40, Appendix Figure 23 / 29, Page 58 CN 121752583 A Figure 41 Appendix to the Instruction Manual 24 / 29 Page 59 CN 121752583 A Figure 42 Figure 43 Appendix to the Instruction Manual 25 / 29 Page 60 CN 121752583 A Figure 44 Figure 45 Appendix to the Instruction Manual 26 / 29 Page 61 CN 121752583 A Figure 46 Appendix to the Instruction Manual 27 / 29 Page 62 CN 121752583 A Figure 47 Appendix to the Instruction Manual 28 / 29 Page 63 CN 121752583 A Figure 48 Appendix to the Instruction Manual 29 / 29 Page 64 CN 121752583 A

Claims

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Images