Complexes containing ligands, spacers, peptide linkers and biomolecules

Abstract

The present invention provides a complex comprising a ligand, a spacer, and a peptide linker, which is useful for in vivo diagnostic drugs and internal irradiation therapy, using an anti-human MUC1 antibody Fab fragment whose binding activity is not reduced by labeling with a metal, a fluorescent dye, etc. The complex comprising three-arm DOTA, a specific spacer, a specific peptide linker, and a biomolecule including an anti-human MUC1 antibody Fab fragment has a binding activity that is not reduced by labeling with a metal, a fluorescent dye, etc., and can be used as a diagnostic composition and / or a pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to a complex comprising an anti-human CEACAM5 antibody Fab fragment or a human MUC1 antibody Fab fragment and a ligand. In addition, the present invention relates to a diagnostic composition and / or pharmaceutical composition comprising the above-mentioned complex, and a method for diagnosing and / or treating cancer using the complex, etc. It also relates to a complex comprising a ligand, a spacer, a peptide linker and a biomolecule, a diagnostic composition and / or pharmaceutical composition comprising the complex, and a method for diagnosing and / or treating a disease related to the biomolecule using the complex, etc. It also relates to a complex comprising a complex formed by the ligand and a metal and the anti-human CEACAM5 antibody Fab fragment or the human MUC1 antibody Fab fragment. It also relates to a complex comprising the complex, a spacer, a peptide linker and a biomolecule. Background Art

[0002] CEA (Carcinoembryonic antigen) or CEACAM (Carcinoembryonic antigen-related cell adhesion molecule) is a tumor marker discovered in 1965 (J. Exp. Med.; 1965; 121: 439-462, PNAS; 1969; 64: 161-167), and 23 CEA-related molecules are known so far (BioMed Central Biology; 2010; 8: 12-33). Among them, CEACAM5 is hardly expressed in normal tissues, but is expressed in fetal digestive tract and colorectal cancer (BBA; 1990; 1032: 177-189, J. Clin. Mol. Pathol.; 1999; 52: 174-178). In addition, CEACAM5 is known to be expressed in breast cancer, lung cancer, and thyroid cancer (Diagn. Cytopathol.; 1993; 9: 377-382, Cancer Res.; 1990; 50: 6987-6994, Histopathology; 2000; 37: 530-535).

[0003] Compared with healthy people, patients with colorectal cancer have higher blood CEACAM5 concentrations (J. Exp. Med.; 1965; 121: 439-462), and CEACAM5 has been used as a tumor marker. According to histological studies of colorectal cancer patients, CEACAM5 is highly expressed in more than 90% of tissues (British J. Cancer; 2013; 108: 662-667).

[0004] Early metastasis of colorectal cancer is confined to the liver. Therefore, if liver metastasis can be detected and treated at an early stage, the recurrence rate can be reduced (Cell Mol. Gastroenterol. Hepatol.; 2017; 3: 163-173).

[0005] Mucin 1 (MUC1) is a membrane-bound glycoprotein expressed on the lumen side of epithelial cells constituting epithelial tissues such as the mammary gland, trachea, and digestive tract (Nat. Rev. Cancer, 2004 Jan; 4(1): 45-60). MUC1 is expressed in breast cancer (Mod. Pathol., 2005 Oct; 18(10): 1295-304), lung cancer (Hum. Pathol., 2008 Jan; 39(1): 126-36), colorectal cancer (Int. J. Oncol., 2000 Jan; 16(1): 55-64), bladder cancer (PloS One, 2014 Mar; 9 (3): e92742), skin cancer (Histopathology, 2000 Sep; 37 (3): 218-23), thyroid cancer (J. Pathol., 2003 Jul; 200 (3): 357-69), gastric cancer (J. Pathol., 2000 Mar; 190 (4): 437-43), pancreatic cancer (Int. J. Oncol., 2004 Jan; 24 (1): 107-13), renal cancer (Mod. Pathol., 2004 Feb; MUC1 is overexpressed in cancer cells such as ovarian cancer (Gynecol. Oncol., 2007 Jun; 105(3): 695-702) and cervical cancer (Am. J. Clin. Pathol., 2004 Jul; 122(1): 61-9), and is useful as a target molecule for detecting cancer lesions (Nat. Rev. Cancer, 2004 Jan; 4(1): 45-60, Pathol. Res. Pract., 2010 Aug 15; 206(8): 585-9).

[0006] In MUC1, the threonine at position 9 of the 20-amino acid tandem repeat sequence HGVTSAPDTRPAPGSTAPPA (SEQ ID NO. 19 in the sequence list of the present application) present in the extracellular domain is O-glycosylated. It is known that this O-glycosylation is incomplete in cancer cells, and O-glycosylation of T (Galβ1-3GalNAcα1-O-Ser / Thr), Tn (GalNAcα1-O-Ser / Thr) and 2,3ST (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser / Thr) etc. occurs in a cancer-specific manner (Patent Document 1, Non-Patent Document 1). MUC1 of normal tissues is not subjected to these cancer-specific O-glycosylation, so human cancer-specific MUC1 is particularly useful as a target molecule for treating various human cancers. As such anti-human cancer-specific MUC1 antibodies, for example, 1B2 antibody (Patent Document 1), PankoMab antibody (Non-Patent Document 2), 5E5 antibody (Patent Document 2) and the like are known. Among these antibodies, the 1B2 antibody is reported to have higher specificity for human cancer-specific MUC1 than the PankoMab antibody (Patent Document 1).

[0007] In the diagnosis of liver metastasis, CT (computed tomography), MRI (magnetic resonance imaging), and FDG-PET (Fluorodeoxyglucose-positron emission tomography) are used. The detection sensitivity of CT, MRI, and FDG-PET are 74.4, 80.3, and 81.4%, respectively. The detection sensitivity of tumors smaller than 1 cm for CT drops to 47.3%, and the detection sensitivity of tumors smaller than 1 cm for MRI drops to 60.2% (Radiology; 2010; 257: 674-684). Although liver-specific contrast agents are also used in MRI, the detection sensitivity of tumors smaller than 1 cm is 29-38% (Radiology; 2005; 237: 89-98).

[0008] Anticancer agents and antibodies conjugated with metal radioisotopes are used for the diagnosis and treatment of cancer. Targeting with antibodies is highly specific to tumor cells and has few side effects. So far, several monoclonal antibodies labeled with metal radioisotopes have been clinically used for diagnosis and treatment (Cancer Control; 2012; 19: 196-203).

[0009] On the other hand, antibodies generally have a long half-life in the blood, and after administration into the body, it takes a long time of 4 to 5 days to reach a tumor / blood ratio that provides a signal sufficient to visualize cancer (Clin. Pharmacol. Ther.; 2010; 87: 586-592). In addition, the Fc region of an antibody causes the pharmacological effects of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Glycoconj. J.; 2013; 30: 227-236, Curr. Opin. Biotechnol.; 2002; 13: 609-614). In addition, since antibodies are metabolized in the liver, they are highly accumulated in the liver regardless of the target. Since early metastasis of colorectal cancer is confined to the liver, it is difficult to detect liver metastasis lesions using antibodies (Clin. Pharmacol. Ther.; 2010; 87: 586-592).

[0010] Low-molecular-weight recombinant antibody fragments such as Fab, scFV, and diabodies have high tissue permeability and can therefore easily reach lesions. They can be expected to be produced at low cost using expression systems in Escherichia coli and yeast, and can therefore be used as therapeutic antibodies. On the other hand, they have a short half-life in the blood and are excreted through the kidneys, so their use as diagnostic drugs has also been reported (Nat. Biotechnol.; 2005; 23: 1126-1136).

[0011] As an anti-human CEACAM5 antibody used as a diagnostic drug, a humanized antibody M5A of the mouse monoclonal antibody T84.66 is known (Patent Document 3). 64 Cu-labeled M5A requires more than 22 hours after administration to obtain good PET images (Non-patent Document 3). In addition, in an experiment using a liver metastasis model mouse, the uptake into normal liver tissue and liver lesion sites was equivalent at 3 hours after administration, and there was a significant difference after 24 hours (Non-patent Document 4).

[0012] Regarding the fragments of anti-human CEACAM5 antibodies, it is reported that the Fab′ of mouse monoclonal antibody NP-4 was used 99mTc-labeled CEA-Scan can be used for the diagnosis of colorectal cancer (Non-patent Document 5). However, the uptake of CEA-Scan into the lesion site does not exceed that into the normal liver, and the detection sensitivity of liver metastasis is lower than that of FDG-PET (Non-patent Document 6). Although CEA-Scan was approved by the FDA as a diagnostic drug for colorectal cancer in 1999, it has long been discontinued (Non-patent Document 7).

[0013] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) has a clinically effective and widely used chelator for radioactive metals. In recent years, studies have been reported on using DOTA to label metals and bind to peptides and antibodies for targeting (Non-Patent Document 8).

[0014] In cancer treatment, as a drug having DOTA, Satoreotidetetraxetan (Non-patent Document 9) is reported to be in clinical phase I development. 90 Y-Tetanus Epratuzumab ( 90 Y-epratuzumab tetraxetan) (Non-Patent Document 10).

[0015] Generally, complexes formed by binding of chelating agents such as DOTA to low-molecular-weight antibodies or peptides are taken up into the kidneys in large quantities and are retained or accumulated therein (Non-Patent Documents 11 and 12).

[0016] Thus, the accumulation of the complex in the kidneys causes inconvenience in correct diagnosis and treatment (Non-Patent Document 13).

[0017] For example, it has been reported that in the use of [ 111 In a mouse experiment on a complex of In]DOTA-rituximab Fab, it was found to be highly accumulated in the kidney (Non-Patent Document 11).

[0018] In order to avoid such high accumulation in the kidney, first, research on complexes using fragments of antibodies such as Fab, scFV, Fab', dsFV, etc. can be listed. Secondly, research on complexes using a linker (also called a peptide linker) between the chelate and the antibody that is specifically cut in the kidney can be listed (Non-Patent Document 12).

[0019] Initially, it was reported that iodine hippuric acid-Gly-Lys-Fab with a peptide linker of Gly (glycine)-Lys (lysine) can be cut by renal brush border membrane enzymes, and iodine hippuric acid is contained in urine as a metabolite and excreted (Non-Patent Document 14). In addition, iodine hippuric acid-Gly-Tys-Fab with a peptide linker of Gly-Tyr (tyrosine) was reported (Non-Patent Document 13).

[0020] On the other hand, the organic rhenium complex CpTR-COOH, which replaces hippuric acid, was reported to be linked to a peptide linker Gly-Lys. 188 Re]CpTR-Gly-Lys-Fab (Non-patent Document 15).

[0021] In addition, a peptide linker of Gly-Phe (phenylalanine)-Lys was reported. 99m Tc-PGGFML-IT-Fab (Patent Document 4).

[0022] In addition, attention has been paid to NOTA as a chelate, and a complex comprising NOTA and a peptide linker has been reported (Patent Document 5).

[0023] Although the complex with the above-mentioned peptide linker has been created, the problem that cannot be cut by the enzyme is generated according to the type of chelating agent, and a complex (patent document 6) that solves this problem by introducing a connecting portion (-CH2-Ph-CO-NH-) of a specific structure between the chelating agent and the peptide linker is reported. The goal is to obtain a complex with a ligand of an atom with a larger atomic radius such as indium that can coordinate widely used as a radioisotope. The following situation is mentioned: a substance with a thiourea structure recorded in patent document 5 is introduced as a spacer between the chelate and the peptide linker, but it is not decomposed by the renal brush border membrane enzyme.

[0024] Based on the following findings, a complex with a peptide linker of Met (methionine)-Ile (isoleucine) that is targeted for lysosomal cleavage is reported. 67 Ga-NOTA-Met-Ile-Fab (Non-Patent Document 16).

[0025] Report on the metabolites generated by lysosomal cleavage of the complex NOTA-(p-SCN-Bz)-dsFv without a peptide linker 67 Ga-NOTA-Bn-Met is excreted in urine (Non-patent Document 17). The second position from the N-terminus of the light chain of the dsFv in the above-mentioned complex NOTA-(p-SCN-Bz)-dsFv is Ile, so the complex was designed. 67 Ga-NOTA-Met-Ile-Her2 (Herceptin) Fab (Non-Patent Document 19).

[0026] Prior art literature

[0027] Non-patent literature

[0028] Non-patent literature 1: Glycoconj. J., 2013Apr; 30(3): 227-36.

[0029] Non-patent document 2: Cancer Immunol Immunother, 2006 Nov; 55(11): 1337-47

[0030] Non-patent document 3: Bioconjug. Chem.; 2008; 19: 89-96

[0031] Non-patent literature 4: PLOS ONE; 2014; 9(9): e106921

[0032] Non-patent literature 5: Ann. Surg.; 1997; 226: 621-631

[0033] Non-patent document 6: J. Nucl. Med.; 2000; 41: 1657-1663

[0034] Non-patent document 7: Kenneth T. Cheng, “99mTc-Arcitumomab”, [online], Update: March 17, 2008., Molecular Imaging and Contrast Agent Database, [retrieved on May 17, 2017], URL: https: / / www.ncbi.nlm.nih.gov / books / NBK23676 /

[0035] Non-patent literature 8: Bioorg. Med. Chem.; 2019; 27: 3248-3253

[0036] Non-patent literature 9: Clinical Trials.gov Identifier: NCT02592707

[0037] Non-patent literature 10: Eur J Haematol. 2013 Dec; 91(6): 552-6

[0038] Non-patent document 11: Bioconjugate Chem. 2001, 12, 264-270

[0039] Non-patent document 12: Bioconjugate Chem. 2002, 13, 985-995

[0040] Non-patent document 13: Bioconjugate Chem. 2013, 24, 291-299

[0041] Non-patent document 14: Cancer Res. 1999, 59, 128-134

[0042] Non-patent document 15: Bioconjugate Chem. 2007, 18, 190-198

[0043] Non-patent literature 16: Bioconjugate Chem. 2014, 25, 2038-2045

[0044] Non-patent document 17: Bioconjugate Chem. 1997, 8, 365-369

[0045] Patent Literature

[0046] Patent Document 1: International Publication No. WO2010 / 050528 Pamphlet

[0047] Patent Document 2: International Publication No. WO2008 / 040362

[0048] Patent Document 3: International Publication No. WO2005 / 086875 Pamphlet

[0049] Patent Document 4: International Publication No. WO2013 / 081091 Pamphlet

[0050] Patent Document 5: International Publication No. WO2017 / 150549 Pamphlet

[0051] Patent Document 6: International Publication No. WO2019 / 065774 Pamphlet

[0052] Patent Document 7: International Publication No. WO2018 / 092885 Pamphlet Summary of the invention

[0053] Problems to be solved by the invention

[0054] The molecular weight of the monovalent Fab fragment is about 50 kDa, which is smaller than the antibody of about 150 kDa, and it is excreted through the kidneys, and the half-life in the blood is also short. Therefore, within 2 to 32 hours after administration, the tumor / blood ratio that provides a signal sufficient to visualize the cancer is reached. Since it does not have an Fc region, it will not cause ADCC or CDC. The Fab fragment is mainly excreted through the kidneys, so it will not hinder the detection of liver metastasis. Due to such features, it can be expected that the Fab fragment is more effective as an in vivo diagnostic drug compared to antibodies.

[0055] However, since the Fab fragment is a monovalent antibody, its binding activity is often weakened. In addition, in order to use antibodies as in vivo diagnostic drugs or drugs used in photoimmunotherapy, they must be labeled with metals, fluorescent dyes, etc., but there is a problem that the binding activity of the antibody is weakened by labeling with such substances.

[0056] An object of the present invention is to provide a labeled complex useful as an in vivo diagnostic drug and internal radiation therapy using an anti-human CEACAM5 antibody Fab fragment whose binding activity is not reduced by labeling with a metal, a fluorescent dye, or the like.

[0057] Provided are complexes comprising an anti-human MUC1 antibody Fab fragment (Patent Document 7) and a peptide linker and a ligand, and complexes comprising an anti-human MUC1 antibody Fab fragment and a ligand. In addition, another object of the present invention is to provide a diagnostic composition comprising the complex and a diagnostic method using the diagnostic composition, as well as a pharmaceutical composition comprising the complex and a therapeutic method using the pharmaceutical composition.

[0058] Another object of the present invention is to provide a complex that allows a complex having a chelating agent and a biomolecule accumulated in the kidney to be excreted more rapidly.

[0059] Methods used to solve problems

[0060] The present inventors first prepared an anti-human CEACAM5 antibody Fab fragment having good affinity for human CEACAM5 (International Application PCT / JP2018 / 025618). Further in-depth research was conducted, and as a result, a complex formed by binding a ligand for labeling to the anti-human CEACAM5 antibody Fab fragment with the aid of (or without the aid of) a peptide linker was prepared. It was found that the complex had the same affinity for human CEACAM5 as the anti-human CEACAM5 antibody Fab fragment itself, that is, the binding activity was not weakened by the binding of the label portion to the Fab fragment, thereby completing the present invention. That is, the present invention provides a complex comprising an anti-human CEACAM5 antibody Fab fragment, a peptide linker and a ligand, and a complex comprising an anti-human CEACAM5 antibody Fab fragment and a specific ligand. It was confirmed that the binding activity of the complex to human CEACAM5 was not weakened by the binding of the label portion to the Fab fragment, and a good binding activity to human CEACAM5 was maintained. Based on these results, a diagnostic method and a therapeutic method using the complex of the present invention are provided.

[0061] In addition, the present inventors prepared an anti-human MUC1 antibody Fab fragment having good affinity for human cancer-specific MUC1, and further conducted in-depth research, resulting in the preparation of a complex formed by binding a ligand to the anti-human MUC1 antibody Fab fragment with the aid of (or without the aid of) a peptide linker. The complex has the same affinity for human cancer-specific MUC1 as the anti-human MUC1 antibody Fab fragment itself. That is, the present invention provides a complex comprising an anti-human MUC1 antibody Fab fragment, a peptide linker, and a ligand, and a complex comprising an anti-human MUC1 antibody Fab fragment and a specific ligand. Moreover, it was confirmed that the binding activity of the complex to human cancer-specific MUC1 is not weakened by the binding of the label portion, and the good binding activity to human cancer-specific MUC1 is maintained. Based on these results, a diagnostic method and a therapeutic method using the complex of the present invention are provided.

[0062] In addition, the present inventors were concerned that complexes composed of complexes formed by ligands and metals (also called metal complexes), spacers, peptide linkers, and biomolecules, such as antibodies useful as contrast agents and anticancer drugs, would accumulate in the kidneys, so they studied complexes that could be excreted more quickly. As a result, they focused on DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which has a clinically effective and widely used chelator for radioactive metals, and found that complexes composed of three-arm DOTA (3arm DOTA), a specific spacer, a specific peptide linker, and biomolecules could be decomposed and excreted in the kidneys.

[0063] Based on the above, the present invention relates to the following complex containing a Fab antibody to CEACAM5, a diagnostic composition and / or a pharmaceutical composition containing the complex, and a method for diagnosing and / or treating cancer using the complex, etc. In addition, the present invention relates to the following complex containing a Fab antibody to MUC1, a diagnostic composition and / or a pharmaceutical composition containing the complex, and a method for diagnosing and / or treating cancer using the complex, etc. In addition, the present invention relates to a complex containing DOTA, a spacer, a peptide linker and a biomolecule that is rapidly excreted from the kidney, an intermediate of the complex, and a method for diagnosing and / or treating a biomolecule-related disease using the complex, etc.

[0064] [1] A complex represented by the following formula (I).

[0065] (Y-S1-X) p -Fab 1 (I)

[0066] [Wherein,

[0067] Fab 1is an anti-human CEACAM5 antibody Fab fragment selected from the group consisting of the following (a) and (b), wherein the Fab 1 With the help of this Fab 1 The p amino or thiol groups present in the bond with X:

[0068] (a) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2 and a light chain fragment comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4; and

[0069] (b) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2, in which glutamic acid at amino acid 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4;

[0070] X is a peptide linker or bond;

[0071] S1 is a spacer or bond;

[0072] Y is a ligand; and

[0073] p is a natural number from 1 to 25, indicating 1 The number of bound groups (Y-S1-X);

[0074] Wherein, when X is a bond, S1 is -CH2-(1,4-phenylene)-NH-C(=S)- or a bond, and Y is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (hereinafter sometimes abbreviated as DOTA)]

[0075] [2] The complex according to [1] above, wherein Fab 1 Selected from the group consisting of (a) and (b) below:

[0076] (a) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 4; and

[0077] (b) An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2, in which glutamic acid at amino acid No. 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0078] [3] The complex according to [2] above, wherein Fab 1A Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 4 (hereinafter referred to as Fab 2 ).

[0079] [4] The complex according to any one of [1] to [3] above, wherein X is a peptide linker comprising a peptide consisting of 2 to 4 amino acids having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme.

[0080] [5] The composite according to any one of [1] to [4] above, wherein S1 is:

[0081] -C(=O)-CH2O-(1,3-phenylene)-C(=O)-,

[0082] -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-,

[0083] -C(=O)-(1,3-phenylene)-C(=O)-,

[0084] -NH-CH2-(1,3-phenylene)-C(=O)-,

[0085] -NH-(CH2)2-C(=O)-,

[0086] -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-,

[0087] -NH-CH2-(1,4-phenylene)-NH-C(=O)-,

[0088] -NH-(CH2)3-C(=O)-,

[0089] -NH-(CH2CH2O)3-CH2-C(=O)-、

[0090] -NH-CH2-(1,4-phenylene)-C(=O)-,

[0091] -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-,

[0092] -CH2-(1,4-phenylene)-NH-C(=S)-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-,

[0093] A spacer or bond represented by the following formulae (a) to (q),

[0094]

[0095] In the above formula, R 1 Represents a hydrogen atom, a halogen, or C 1-6 Alkyl. The same applies below. X is selected from

[0096] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0097] (2)-Gly-Lys*-Z2-,

[0098] (3)-Gly-Phe-Lys*-Z2-,

[0099] (4)-Met-Val-Lys*-Z2-,

[0100] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0101] (6)-Gly-Lys-Lys*-Z2-,

[0102] (7)-Gly-Arg-Lys*-Z2-,

[0103] (8) -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0104] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0105] (10)-Asp-Gly-Lys*-Z2-,

[0106] (11)-Met-Gly-Lys*-Z2-,

[0107] (12)-Met-Ile-Lys*-Z2-,

[0108] (13)-Gly-Tyr*-CH2-C(=O)-Lys*-Z2-、

[0109] (14)-Val-NH-(CH2)2-Z1-,

[0110] (15)-Ile-NH-(CH2)2-Z1-,

[0111] (16)-Gly-Val-NH-(CH2)2-Z1-,

[0112] (17)-Gly-Ile-NH-(CH2)2-Z1-,

[0113] (18)-Met-Phe-Lys*-Z2-,

[0114] (19)-Gly-Tyr-Lys*-Z2-,

[0115] (20)-Gly-Tyr*-CH2-C(=O)-NH-(CH2O)3-CH2-C(=O)-NH-(CH2)2-Z1-,

[0116] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0117] (22)-Gly-diphenylalanine-Lys*-Z2-,

[0118] (23)-Gly-Tyr-NH-(CH2)5-Z1-,

[0119] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0120] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0121] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0122] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0123] (28)-Met-Gly-Lys*-Z3-, or

[0124] (29) key,

[0125] Here, Met represents methionine, Ile represents isoleucine, Gly represents glycine, Lys represents lysine, Phe represents phenylalanine, Val represents valine, Tyr represents tyrosine, Arg represents arginine, Asp represents aspartic acid, Z1 represents a group represented by the following formula (II-I) or (II-II), -Lys*-Z2- represents a group represented by the following formula (III-I) or (III-II), -Tyr*-CH2- represents a group represented by the following formula (IV), -Lys*-C(=S)- represents a group represented by the following formula (V), -Lys*-Z3- represents a group represented by the following formula (III-III), and the group (A-3), (A-4) or (A-5) is represented by the following formula.

[0126]

[0127] [6] The complex according to any one of [1] to [5] above, wherein S1 is -C(=O)-CH2O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-, or -C(=O)-(1,3-phenylene)-C(=O)-,

[0128] X is the choice of freedom

[0129] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0130] (2)-Gly-Lys*-Z2-,

[0131] (3)-Gly-Phe-Lys*-Z2-,

[0132] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0133] (6)-Gly-Lys-Lys*-Z2-, and

[0134] (8) A peptide linker in the group consisting of -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-.

[0135] [7-1] The complex according to any one of [1] to [5] above, wherein S1 is -C(=O)-CH2O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -NH-CH2-(1,4-phenylene)-C(=O)-, -C(=O)-(1,3-phenylene)-C(=O)-, or is the following formula:

[0136]

[0137] X is the choice of freedom

[0138] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0139] (2)-Gly-Lys*-Z2-,

[0140] (3)-Gly-Phe-Lys*-Z2-,

[0141] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0142] (6)-Gly-Lys-Lys*-Z2-,

[0143] (8) -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0144] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0145] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0146] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0147] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0148] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0149] [7-2] The complex according to [7-1] above, wherein S1 is -NH-CH2-(1,4-phenylene)-NH-C(=O)-, or is the following formula:

[0150]

[0151] X is the choice of freedom

[0152] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0153] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0154] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0155] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0156] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0157] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0158] [8] The complex according to any one of [1] to [5] above, wherein S1 is -NH-CH2-(1,3-phenylene)-C(=O)-, -NH-(CH2)2-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -NH-(CH2)3-C(=O )-, -NH-(CH2CH2O)3-CH2-C(=O)-, -NH-CH2-(1,4-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, a spacer or bond represented by the following formulae (a) to (q),

[0159]

[0160] X is the choice of freedom

[0161] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0162] (2)-Gly-Lys*-Z2-,

[0163] (3)-Gly-Phe-Lys*-Z2-,

[0164] (4)-Met-Val-Lys*-Z2-,

[0165] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0166] (7)-Gly-Arg-Lys*-Z2-,

[0167] (8) -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0168] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-, (10) -Asp-Gly-Lys*-Z2-,

[0169] (11)-Met-Gly-Lys*-Z2-,

[0170] (12)-Met-Ile-Lys*-Z2-,

[0171] (13)-Gly-Tyr*-CH2-C(=O)-Lys*-Z2-、

[0172] (14)-Val-NH-(CH2)2-Z1-,

[0173] (15)-Ile-NH-(CH2)2-Z1-,

[0174] (16)-Gly-Val-NH-(CH2)2-Z1-,

[0175] (17)-Gly-Ile-NH-(CH2)2-Z1-,

[0176] (18)-Met-Phe-Lys*-Z2-,

[0177] (19)-Gly-Tyr-Lys*-Z2-,

[0178] (20)-Gly-Tyr*-CH2-C(=O)-NH-(CH2O)3-CH2-C(=O)-NH-(CH2)2-Z1-,

[0179] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0180] (22)-Gly-diphenylalanine-Lys*-Z2-,

[0181] (23)-Gly-Tyr-NH-(CH2)5-Z1-

[0182] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0183] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0184] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0185] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0186] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0187] [9] The complex according to any one of [1] to [5] above, wherein S1 is -NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, a spacer represented by the following formula (e) to (i) or (k), or a bond,

[0188]

[0189] X is the choice of freedom

[0190] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0191] (2)-Gly-Lys*-Z2-,

[0192] (3)-Gly-Phe-Lys*-Z2-,

[0193] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0194] (10)-Asp-Gly-Lys*-Z2-,

[0195] (11)-Met-Gly-Lys*-Z2-,

[0196] (12)-Met-Ile-Lys*-Z2-,

[0197] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0198] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0199] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0200] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0201] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0202] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0203]

[10] The complex according to any one of [1] to [5] above, wherein S1 is a group selected from the group consisting of -NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, a spacer represented by the following formula (f) or (g), or a bond;

[0204]

[0205] X is the choice of freedom

[0206] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0207] (4)-Met-Val-Lys*-Z2-,

[0208] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0209] (11)-Met-Gly-Lys*-Z2-,

[0210] (12)-Met-Ile-Lys*-Z2-,

[0211] (18)-Met-Phe-Lys*-Z2-,

[0212] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0213] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0214] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0215] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0216] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0217]

[11] The complex according to any one of [1] to [5] above, wherein X is selected from (1) -Met-Ile-NH-(CH2)2-Z1-,

[0218] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0219] (12)-Met-Ile-Lys*-Z2-,

[0220] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0221] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0222] (26)-Met-Ile-NH-(CH2)2-(II-II)-, and

[0223] (27)-Met-Ile-NH-(CH2)2-(A-3)-.

[0224]

[12] The complex according to any one of [1] to [5] above, wherein X is selected from (4)-Met-Val-Lys*-Z2-,

[0225] (11)-Met-Gly-Lys*-Z2-,

[0226] (18)-Met-Phe-Lys*-Z2-, and

[0227] (28) Peptide linker in the group consisting of Met-Gly-Lys*-Z3-.

[0228]

[13] The complex according to any one of [1] to [5] above, wherein X is selected from (11)-Met-Gly-Lys*-Z2-, and

[0229] (28)-Met-Gly-Lys*-Z3- in the peptide linker group.

[0230]

[14] The complex according to any one of [1] to [5] above, which is a complex selected from the group consisting of compounds represented by the following formula: Fab 1 With the help of this Fab 1 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0231]

[0232]

[0233]

[15] The complex according to any one of [1] to

[14] above, wherein Y is deferoxamine (hereinafter sometimes abbreviated as DFO) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (hereinafter sometimes abbreviated as DOTA).

[0234]

[16] The complex according to

[15] above, wherein Y is DFO.

[0235]

[17] The complex according to the above

[15] , wherein Y is DOTA.

[0236]

[18] The complex according to

[17] above, wherein Y is three-arm DOTA or four-arm DOTA.

[0237]

[19] The complex according to the above

[18] , wherein Y is three-arm DOTA.

[0238]

[20] The complex according to the above-mentioned

[18] , wherein Y is four-arm DOTA.

[0239]

[21] The complex according to any one of

[15] or

[17] to

[20] , which is a complex selected from the group consisting of compounds represented by the following formula: Fab 1 With the help of this Fab 1 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0240]

[0241]

[0242]

[0243]

[0244]

[0245] In addition, the composite of the present invention may be a mixture of the following two composites.

[0246]

[0247]

[22] The complex according to any one of

[15] or

[17] to

[20] , which is a complex selected from the group consisting of compounds represented by the following formula: Fab 1 With the help of this Fab 1 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0248]

[0249]

[23] The complex according to any one of [1] to

[22] above, wherein p is a natural number of 1 to 5.

[0250]

[24] The complex according to any one of [1] to

[20] above, wherein Y is coordinated with a metal.

[0251]

[25] The complex according to any one of

[21] to

[22] above, wherein a metal is coordinated thereto.

[0252]

[26] The complex according to

[24] or

[25] above, wherein the metal is a metal radioisotope.

[0253]

[27] The composite according to

[26] above, wherein the metal is 89 Zr.

[0254]

[28] The complex according to

[24] or

[25] above, wherein the metal is a paramagnetic metal ion.

[0255]

[29] The composite according to

[28] above, wherein the metal is Gd 3+ .

[0256]

[30] The complex according to any one of

[24] to

[29] above, which is a PET tracer.

[0257]

[31] A diagnostic composition comprising one or more of the complexes described in any one of

[24] to

[30] above and a pharmaceutically acceptable carrier.

[0258]

[32] The diagnostic composition according to

[31] above, which is used as an early diagnosis drug or a staging diagnosis drug.

[0259]

[33] The diagnostic composition according to any one of

[31] or

[32] above, which is used for diagnosing cancer expressing human CEACAM5.

[0260]

[34] The diagnostic composition according to

[33] above, wherein the cancer is colorectal cancer, breast cancer, lung cancer, thyroid cancer, or a cancer metastasized from these cancers.

[0261]

[35] A pharmaceutical composition comprising one or more of the complexes described in any one of

[24] to

[29] above and a pharmaceutically acceptable carrier.

[0262]

[36] The pharmaceutical composition according to

[35] above, which is a pharmaceutical composition for treating cancer expressing human CEACAM5.

[0263]

[37] The pharmaceutical composition according to

[36] above, wherein the cancer is colorectal cancer, breast cancer, lung cancer, thyroid cancer or a cancer metastasized from these cancers.

[0264]

[38] Use of one or more of any one of

[24] to

[29] above in the manufacture of a composition for diagnosing cancer and / or a pharmaceutical composition for treating cancer.

[0265]

[39] The complex according to any one of

[24] to

[30] above, which is used for diagnosis and / or treatment of cancer.

[0266]

[40] A method for diagnosing cancer, comprising the step of administering one or more of the complexes described in any one of

[24] to

[30] above to a subject.

[0267]

[41] A method for treating cancer, comprising the step of administering to a subject a therapeutically effective amount of the complex described in any one of

[24] to

[30] above.

[0268]

[42] A complex represented by the following formula (Ia).

[0269]

[0270] [Wherein,

[0271] DOTA 1 : Three-arm DOTA,

[0272] U: bond or -NH(CH2)2O(CH2)2O(CH2)2OCH2C(=O)-,

[0273] Q: -C(=O)-, -NH-C(=O)- or -NH-C(=S)-,

[0274] X: C or N,

[0275] R 1a , R 1b : Same or different, H or C 1-6 alkyl,

[0276] It should be noted that R 1a and R 1b Can form C 1-6 Alkylene.

[0277] p is a natural number from 1 to 25, using Biomolecule 1 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0278] R 1 :H, halogen, C1-6 Alkyl or halogenated C 1-6 alkyl,

[0279] R 2 :C 1-6 Alkyl or halogenated C 1-6 alkyl,

[0280] L 2 : Ile, Gly, Ala, Val, Phe, -NHCH(CHCH3NR 3 R 4 )C(=O)-, -NHCH(CHCH3N3)C(=O)-, or -NHCH(CHCH3CH2CH2CH3)C(=O)-,

[0281] R 3 :H、C 1-6 alkyl,

[0282] R 4 :H、C 1-6 alkyl,

[0283] L 3 : bond, Arg or His,

[0284] L 4 :-NH-(CH2)2-, -NHCH(C(=O)OH)(CH2)4-, or a bond,

[0285] V 1 : A group represented by the following formulae (A-1) to (A-5),

[0286]

[0287] Alternatively, the group -L 3 -L 4 -V 1 -together,

[0288]

[0289] Biomolecule 1 :Biomolecules

[0290] dotted line Q is bonded to any one of the two carbon atoms on the ring.

[0291] dotted line single or double bond]

[0292]

[43] The complex according to

[42] , wherein L 3 , L 4 and V 1 is the following group.3 : bond, Arg or His,

[0293] L 4 :-NH-(CH2)2-, -NHCH(C(=O)OH)(CH2)4-, or a bond,

[0294] V 1 : A group represented by any one of the following formulae (A-1) to (A-5).

[0295]

[0296]

[44] The complex according to

[43] , wherein the group -L 3 -L 4 -V 1 -together they are as follows.

[0297]

[0298]

[45] A complex represented by the following formula (Ib).

[0299]

[0300] [Wherein,

[0301] DOTA 1 : Three-arm DOTA,

[0302] U: bond or -NH(CH2)2O(CH2)2O(CH2)2OCH2C(=O)-,

[0303] Q: -C(=O)-, -NH-C(=O)-, or -NH-C(=S)-,

[0304] X: C or N,

[0305] R 1a , R 1b : Same or different, H or C 1-6 alkyl,

[0306] It should be noted that R 1a and R 1b Can form C 1-6 Alkylene.

[0307] p is a natural number from 1 to 25, using Biomolecule 2 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0308] R 1 :H, halogen, C 1-6 Alkyl or halogenated C 1-6 alkyl,

[0309] R 2 :C 1-6 Alkyl or halogenated C 1-6 alkyl,

[0310] L 2 : Ile, Gly, Ala, Val, Phe, -NHCH(CHCH3NR 3 R 4 )C(=O)-, -NHCH(CHCH3N3)C(=O)-, or -NHCH(CHCH3CH2CH2CH3)C(=O)-,

[0311] R 3 :H、C 1-6 alkyl,

[0312] R 4 :H、C 1-6 alkyl,

[0313] L 3 : bond, Arg or His,

[0314] L 4 :-NH-(CH2)2-, -NHCH(C(=O)OH)(CH2)4-, or a bond,

[0315] V 1 : a group described in any one of the following formulae (A-1) to (A-5),

[0316]

[0317] Alternatively, the group -L 3 -L 4 -V 1 -together they are as follows.

[0318]

[0319] Biomolecule 2 : Antibody Fab fragment,

[0320] dotted line Q is bonded to any one of the two carbon atoms on the ring.

[0321] dotted line single or double bond]

[0322] [46-1] The complex according to any one of

[42] to

[45] above, which is represented by the following formula (Ic).

[0323]

[0324] [Wherein,

[0325] L 3 :key,

[0326] L 4 :-NH-(CH2)2- or -NHCH(C(=O)OH)(CH2)4-,

[0327] Biomolecule: Biomolecule 1 or Biomolecule 2 ]

[0328] [46-2] The complex according to the above-mentioned [46-1], wherein in formula (Ic),

[0329] L 3 is the key,

[0330] L 4 It is -NH-(CH2)2-.

[0331] [47-1] The complex according to any one of

[42] to

[45] above, wherein the complex described in

[42] to [46-2] above is represented by the following formula (Id).

[0332]

[0333] [Wherein,

[0334] L 3 :key,

[0335] L 4 :-NH-(CH2)2- or -NHCH((C=O)OH)(CH2)4-,

[0336] Biomolecule: Biomolecule 1 or Biomolecule 2 ]

[0337] [47-2] The complex according to the above-mentioned [47-1], wherein in formula (Id),

[0338] L 3 is the key,

[0339] L 4 It is -NH-(CH2)2-.

[0340]

[48] ​​The complex according to any one of

[42] to [47-2] above, wherein V 1 It is any one of the following formulae (A-3) to (A-5).

[0341]

[0342]

[49] The complex according to any one of

[42] to

[48] , wherein the group (B) in formula (Ia) or (Ib) is

[0343]

[0344] And it is a group selected from the group consisting of the following formulae (B-1) to (B-7).

[0345]

[0346]

[0347]

[50] The complex according to any one of

[42] to

[49] , which is selected from the group consisting of compounds represented by the following formula.

[0348]

[0349]

[0350]

[0351] [51-1] The complex according to any one of

[42] to

[50] , wherein the Biomolecule 1 and Biomolecule 2 A biological molecule or an antibody Fab fragment other than the following antibody Fab fragments:

[0352] An anti-human CEACAM5 antibody Fab fragment selected from the group consisting of the following (a) and (b),

[0353] (a) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2 and a light chain fragment comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4; and

[0354] (b) An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2, in which glutamic acid at amino acid 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4.

[0355] [51-2] The complex according to [51-1], wherein the Biomolecule 1and Biomolecule 2 A biological molecule or an antibody Fab fragment other than the following antibody Fab fragments:

[0356] (a) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2 and a light chain fragment comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4;

[0357] (b) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2, in which glutamic acid at amino acid 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of an amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4;

[0358] (c) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 4; or

[0359] (b) An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2, in which glutamic acid at amino acid No. 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain comprising the light chain shown in SEQ ID NO: 4.

[0360]

[52] The complex according to any one of

[42] to [51-2], wherein the Biomolecule 1 or Biomolecule 2 An anti-human MUC1 antibody Fab fragment selected from the following groups (a) and (b):

[0361] An anti-human MUC1 antibody Fab fragment selected from the group consisting of (a) and (b) below,

[0362] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain fragment comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; and

[0363] (b) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14, in which the glutamine at amino acid number 1 of SEQ ID NO: 12 or SEQ ID NO: 14 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0364]

[53] The complex according to

[52] , wherein the Biomolecule 1 or Biomolecule 2 An anti-human MUC1 antibody Fab fragment selected from the following groups (a) and (b):

[0365] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10; and

[0366] (b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0367]

[54] The complex according to

[53] , wherein the Biomolecule 1 or Biomolecule 2 The following anti-human MUC1 antibody Fab fragment:

[0368] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0369]

[55] The complex according to any one of

[42] to

[54] above, wherein p is a natural number of 1 to 4.

[0370]

[56] The complex according to any one of

[42] to

[55] , wherein DOTA 1 There is metal on the top.

[0371]

[57] The complex according to

[56] above, wherein the metal is a metal radioisotope.

[0372]

[58] The composite according to

[57] above, wherein the metal is 89 Zr.

[0373]

[59] The complex according to

[56] above, wherein the metal is a paramagnetic metal ion.

[0374]

[60] The composite according to

[59] above, wherein the metal is Gd 3+ .

[0375]

[61] The complex according to any one of

[56] to

[60] above, which is a PET tracer.

[0376]

[62] A diagnostic composition comprising one or more of the complexes described in any one of

[56] to

[61] above and a pharmaceutically acceptable carrier.

[0377]

[63] The diagnostic composition according to

[62] above, which is used as an early diagnosis drug or a staging diagnosis drug.

[0378]

[64] The diagnostic composition according to any one of

[62] or

[63] , which is used in Biomolecule 1 or Biomolecule 2 Diagnosis of related diseases.

[0379]

[65] The diagnostic composition according to

[64] above, wherein the Biomolecule 1 or Biomolecule 2 Related diseases are MUC1-related diseases.

[0380]

[66] The diagnostic composition according to

[65] above, which is used for diagnosing cancer expressing MUC1.

[0381]

[67] The diagnostic composition according to

[66] above, wherein the cancer is breast cancer, lung cancer, colon cancer, bladder cancer, skin cancer, thyroid cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer or cervical cancer.

[0382]

[68] A pharmaceutical composition comprising one or more of the complexes described in any one of

[56] to

[60] above and a pharmaceutically acceptable carrier.

[0383]

[69] The pharmaceutical composition according to

[68] , which is used for Biomolecule 1 or Biomolecule 2 Diagnosis of related diseases.

[0384]

[70] The pharmaceutical composition according to

[69] above, wherein the Biomolecule 1 or Biomolecule 2 Related diseases are MUC1-related diseases.

[0385]

[71] The pharmaceutical composition according to the above-mentioned

[70] , which is a pharmaceutical composition for treating cancer expressing MUC1.

[0386]

[72] The pharmaceutical composition according to

[71] above, wherein the cancer is breast cancer, lung cancer, colorectal cancer, bladder cancer, skin cancer, thyroid cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer or cervical cancer.

[0387]

[73] Use of one or more of any one of

[56] to

[60] above in the manufacture of a composition for diagnosing cancer and / or a pharmaceutical composition for treating cancer.

[0388]

[74] The complex according to any one of

[56] to

[60] above, which is used for diagnosis and / or treatment of cancer.

[0389]

[75] A method for diagnosing cancer, comprising the step of administering to a subject one or more of the complexes described in any one of

[56] to

[60] above.

[0390]

[76] A method for treating cancer, comprising the step of administering to a subject a therapeutically effective amount of the complex described in any one of

[56] to

[60] above.

[0391] Effects of the Invention

[0392] The complex comprising an anti-human CEACAM5 antibody Fab fragment, a peptide linker and a ligand, and the complex comprising an anti-human CEACAM5 antibody Fab fragment and a specific ligand described in the present invention have excellent binding activity to human CEACAM5. Therefore, the complex of the present invention further comprising a metal can be expected to be useful in the diagnosis and / or treatment of cancer. In addition, the complex comprising a human MUC1 antibody Fab fragment, a peptide linker and a ligand described in the present invention has excellent binding activity to human MUC1. The complex comprising a three-arm DOTA, a spacer, a peptide linker and a biomolecule described in the present invention is excreted more rapidly through the kidneys. Therefore, the complex of the present invention further comprising a metal can be expected to be useful in the diagnosis and / or treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0393] Figure 1 Show that the drug administration includes 64 PET / CT images of Cu-protein complex solution (A) obtained after about 3 hours in PBS solution.

[0394] Figure 2 Show that the drug administration includes 64 PET / CT image of Cu-protein complex solution (B) obtained after about 3 hours in PBS solution.

[0395] Figure 3 An SUV is shown. DETAILED DESCRIPTION

[0396] The present invention will be described in detail below, but the present invention is not limited thereto. In this specification, unless otherwise specified, scientific terms and technical terms used in connection with the present invention have the meanings generally understood by those skilled in the art.

[0397] "Alkyl" means a linear or branched saturated hydrocarbon chain and a monovalent group.

[0398] “C 1-6 The term "alkyl" refers to an alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc. In one embodiment, C 1-4 In one embodiment, the alkyl group is a methyl group or an ethyl group. In one embodiment, the alkyl group is a methyl group.

[0399] “C 1-6 "Alkylene" is the above C 1-6 A divalent group formed by removing hydrogen from an alkyl group. In one embodiment, it is a methylene group, an ethylene group, a propylene group, or a methylmethylene group.

[0400] "Halogen" means F, Cl, Br, I.

[0401] "Halogenated C 1-6 "Alkyl" is a C 1-6 In one embodiment, it is C substituted with 1 to 5 halogens 1-6 In one embodiment, the alkyl group is CF3.

[0402] 1-1. Composite of the present invention

[0403] The complex of the present invention is a complex represented by the following formula (I).

[0404] (Y-S1-X) p -Fab 1 (I)

[0405] [Wherein,

[0406] Fab 1 is an anti-human CEACAM5 antibody Fab fragment. 1 With the help of this Fab 1 The p amino groups or thiol groups present in the

[0407] X is a peptide linker or bond,

[0408] S1 is a spacer or bond,

[0409] Y is a ligand, and

[0410] p is a natural number from 1 to 25, indicating 1 The number of combined (Y-S1-X),

[0411] Wherein, when X is a bond, S1 is -CH2-(1,4-phenylene)-NH-C(=S)- or a bond, and Y is a group represented by the following formula]

[0412]

[0413] Anti-human CEACAM5 antibody Fab fragment (Fab 1 )

[0414] In the above formula (I), "Fab 1 ” is used to illustrate the anti-human CEACAM5 antibody Fab fragment shown in FIG.

[0415] The basic structure of antibody molecules is common to all types, and is composed of a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with a molecular weight of 20,000 to 30,000. The heavy chain is composed of a polypeptide chain usually containing about 440 amino acids, and each type has a characteristic structure, and is called γ, μ, α, δ, and ε chains corresponding to IgG, IgM, IgA, IgD, and IgE. In addition, there are IgG1, IgG2, IgG3, and IgG4 in IgG, which are called γ1, γ2, γ3, and γ4, respectively. The light chain is composed of a polypeptide chain usually containing about 220 amino acids, and two types of L and K are known, which are called λ and κ chains, respectively. The peptide structure of the basic structure of the antibody molecule is that two identical heavy chains and two light chains are bound by disulfide bonds (SS bonds) and non-covalent bonds, and the molecular weight is 150,000 to 190,000. Two light chains can be paired with any heavy chain. Each antibody molecule is always formed by the same two light chains and the same two heavy chains.

[0416] There are four intrachain SS bonds in the heavy chain (five in μ and ε chains) and two in the light chain. Every 100 to 110 amino acid residues form a ring, and its three-dimensional structure is similar between each ring, which is called a structural unit or domain. For the domain located at the N-terminus of both the heavy chain and the light chain, even if it is a standard product of the same class (subclass) from the same animal, its amino acid sequence is not constant, and it is called a variable region. Each domain is called a heavy chain variable region (V H ) and light chain variable region (V L The amino acid sequence closer to the C-terminal side of this domain is basically constant in each class or subclass, and is called the constant region. Each domain is represented by C H 1. C H 2. C H 3 or C L .

[0417] The specificity of antibody-antigen binding depends on the H and V LThe amino acid sequence of the part that constitutes it. On the other hand, the biological activities such as binding to complement and various cells reflect the structural differences of the constant regions of various Igs. It is known that the variability of the variable regions of heavy and light chains is basically limited to three small hypervariable regions that exist in any chain. These regions are called complementary determining regions (CDR; CDR1, CDR2, and CDR3 from the N-terminal side). The rest of the variable region is called the framework region (FR) and is relatively constant.

[0418] C present in the heavy chain constant region of an antibody H 1 domain and C H The region between the two domains is called the hinge region, which contains a large number of proline residues and includes multiple interchain SS bonds connecting the two heavy chains. For example, the hinge regions of human IgG1, IgG2, IgG3, and IgG4 contain 2, 4, 11, and 2 cysteine ​​residues, respectively, that constitute the SS bonds between the heavy chains. The hinge region is a region that is highly sensitive to proteolytic enzymes such as papain and pepsin. When the antibody is digested with papain, the heavy chain is cut at a position in the hinge region that is closer to the N-terminal side than the SS bonds between the heavy chains, and is decomposed into two Fab fragments and an Fc fragment. The Fab fragment consists of a light chain and a heavy chain variable region (V H ), C H The Fab fragment contains the variable region and has antigen-binding activity.

[0419] In one embodiment, the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is a Fab fragment having the following characteristics:

[0420] An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence of amino acids 1 to 121 of SEQ ID NO: 2 and a light chain fragment comprising a light chain variable region consisting of the amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4.

[0421] As the heavy chain constant region of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention, any constant region of Igγ1, Igγ2, Igγ3 or Igγ4 can be selected. In one embodiment, the heavy chain constant region of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is the human Igγ1 constant region.

[0422] As the light chain constant region of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention, either Igλ or Igκ can be selected. In one embodiment, the light chain constant region of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is a human Igκ constant region.

[0423] In one embodiment, the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is the following Fab 2 Snippet:

[0424] An anti-human CEACAM5 antibody Fab fragment (referred to as Fab) comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 4 2 ).

[0425] When antibodies including Fab fragments are expressed in cells, it is known that antibodies are modified after translation. Examples of post-translational modifications include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid at the N-terminus of the heavy chain and light chain to pyroglutamic acid by pyroglutamylation, glycosylation, oxidation, deamidation, glycation, etc. Such post-translational modifications are known to occur in various antibodies (J. Pharm. Sci.; 2008; 97: 2426-2447).

[0426] The anti-CEACAM5 antibody Fab fragment contained in the complex of the present invention may also include a Fab fragment generated by post-translational modification. Examples of the anti-human CEACAM5 antibody Fab fragment of the present invention that can be generated by post-translational modification include an anti-human CEACAM5 antibody Fab fragment in which the N-terminus of the heavy chain is pyroglutamylated. It is well known in the art that such post-translational modification by pyroglutamylation of the N-terminus does not affect the activity of the antibody (Anal. Biochem.; 2006; 348: 24-39).

[0427] In one embodiment, the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is an anti-human CEACAM5 antibody Fab fragment having the following characteristics:

[0428] An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence of amino acid numbers 1 to 121 of SEQ ID NO: 2, in which glutamic acid at amino acid number 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of an amino acid sequence of amino acid numbers 1 to 112 of SEQ ID NO: 4.

[0429] In one embodiment, the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is an anti-human CEACAM5 antibody Fab fragment having the following characteristics:

[0430] An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2, wherein the glutamic acid at amino acid No. 1 of SEQ ID NO: 2 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0431] In another embodiment, the anti-CEACAM5 antibody Fab fragment contained in the complex of the present invention is an anti-human CEACAM5 antibody Fab fragment having the following characteristics:

[0432] An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment and a light chain, wherein the heavy chain fragment comprises a CDR1 consisting of an amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2, a CDR2 consisting of an amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 2, and a heavy chain variable region consisting of a CDR3 consisting of an amino acid sequence of amino acid numbers 99 to 110 of SEQ ID NO: 2, and the light chain comprises a CDR1 consisting of an amino acid sequence of amino acid numbers 24 to 38 of SEQ ID NO: 4, a CDR2 consisting of an amino acid sequence of amino acid numbers 54 to 60 of SEQ ID NO: 4, and a light chain variable region consisting of a CDR3 consisting of an amino acid sequence of amino acid numbers 93 to 101 of SEQ ID NO: 4.

[0433] The anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention binds to human CEACAM5. As a method for measuring the binding activity of the obtained anti-human CEACAM5 antibody Fab fragment to human CEACAM5, there are methods such as analysis based on the surface plasmon resonance (SPR) method and ELISA. For example, in the case of using an analysis based on the SPR method, a biotin capture kit (GE Healthcare Japan) and biotinylated human CEACAM5 are solid-phased on a sensor chip using Biacore T200 (GE Healthcare Japan), and serially diluted Fab fragments are added, thereby measuring the association rate constant (ka), dissociation rate constant (kd) and dissociation constant (K D ).

[0434] A person skilled in the art can easily prepare the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention based on the sequence information of the heavy chain fragment and the light chain of the anti-human CEACAM5 antibody Fab fragment of the present invention disclosed in the present specification by using methods known in the art. The anti-human CEACAM5 antibody Fab fragment of the present invention can be produced, for example, by the method described in <Method for producing anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention> described later, but is not particularly limited.

[0435] 1-2. About ligand

[0436] "Ligand" is a part of the complex that can form a chelate complex with a metal, and refers to a group composed of a chelating agent. The composed group refers to a group having a binding bond by removing a proton from the chelating agent. The group composed of the chelating agent is directly bound to the anti-human CEACAM5 antibody Fab fragment or through a spacer and / or a peptide linker.

[0437] "Chelating agent" is a compound that can coordinate with metal. As the "chelating agent" in this specification, iron carriers and non-iron carriers can be listed. As iron carriers, hydroxamic acid type, catechol type or mixed ligand type can be listed. As hydroxamic acid type iron carriers, for example, iron pigments, the following formula:

[0438]

[0439] As shown in the deferoxamine (DFO), sickle C, ornibactin, rhodotorulic acid. Examples of catechol-type iron carriers include enterobactin, bacillus, and vibrioin. Examples of mixed ligand-type iron carriers include azotocin, pyocyanin, and yersinibactin. It should be noted that in the case of the above-mentioned iron carriers, DFO can react with a spacer or a peptide linker by means of its reactive functional group -NH2. In the case of iron carriers other than DFO, it can also react with a spacer or a peptide linker by means of reactive functional groups such as carboxyl, hydroxyl, and amino groups using methods commonly used by those skilled in the art.

[0440] As the non-iron carrier, for example, the following formula can be mentioned:

[0441]

[0442] The DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, CAS No.: 60239-18-1), DTPA (diethylenetriaminepentaacetic acid, CAS No.: 67-43-6), DTPA-BMA (1,7-bis(methylcarbamoylmethyl)-1,4,7-triazaheptane-1,4,7-triacetic acid, CAS No.: 119895-95-3), EOB-DTPA (N-[(2S)-2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[ 2-[Bis(carboxymethyl)amino]ethyl]glycine, CAS No.: 158599-72-5), TTHA (triethylenetetraaminehexaacetic acid, CAS No.: 869-52-3), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, CAS No.: 217973-03-0), HP-DO3A (10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, CAS No.: 120041-08-9), and their known reactive derivatives.

[0443] DOTA can react with a spacer or a peptide linker via a carboxylic acid as its reactive functional group (hereinafter, DOTA having three carboxylic acids bound in this way is sometimes referred to as three-arm DOTA (PloS One. 2019 Mar 22; 14(3): e0213397).).

[0444] Among DOTA, examples of three-arm DOTA include the following compounds.

[0445]

[0446] Alternatively, DOTA having four carboxylic acids (hereinafter sometimes referred to as four-arm-DOTA) may be reacted with a spacer or a peptide linker using a reagent of the following formula p-SCN-Bn-DOTA.

[0447]

[0448] As one embodiment of the "chelating agent" forming the ligand contained in the complex of the present invention, DFO, DOTA, DTPA, DTPA-BMA, EOB-DTPA, DO3A, and HP-DO3A can be mentioned. As one embodiment, DFO and DOTA can be mentioned.

[0449] It should be noted that, unless otherwise specified, the compounds and complexes in this specification also include free bodies and their salts. Here, "its salt" means that depending on the type of the substituent of the compound and the complex, an acid addition salt or a salt with a base is sometimes formed, which is a salt that the compound and the complex can form. Specifically, it can be listed with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, glutamic acid and other organic acid acid addition salts; with inorganic bases such as sodium, potassium, magnesium, calcium, aluminum, methylamine, ethylamine, ethanolamine, lysine, ornithine and other organic bases; with various amino acids and amino acid derivatives such as acetyleucine; ammonium salts, etc. For example, DFO can exist in the form of deferoxamine mesylate or in the form of other salts. DTPA can exist in the form of DTPA sodium salt or in the form of free form.

[0450] The metal-containing complex of the present invention can be used in various contrast agents and / or therapeutic agents for cancer, for example, agents used in MRI contrast agents, PET tracers, and the like.

[0451] One embodiment of the "chelating agent" used as an MRI contrast agent is the above-mentioned siderophore and non-siderophore chelating agent.

[0452] One embodiment of the "chelating agent" used as a PET tracer is the above-mentioned siderophore and non-siderophore chelating agent, and one embodiment is DFO or DOTA.

[0453] In the complex of the present invention, the chelating agent may contain a metal. In this specification, "metal" means a paramagnetic metal ion or a metal radioisotope. As the metal, there is no particular limitation as long as it is a metal that is coordinated and bound to each chelating agent. The combination of the appropriate chelating agent and the metal is selected according to the purpose of use of the complex.

[0454] Paramagnetic metal ions are suitable for use as MRI contrast agents. Examples of paramagnetic metal ions include Fe 2+ , Fe 3+ , Cu 2+ 、Ni 2+ , Rh 2+ 、Co 2+ , Gd 3+ 、Eu 3+ 、Dy 3+ , Tb 3+ 、Pm 3+ 、Nd 3+ 、Tm 3+ 、Ce 3+ , Y3+ 、Ho 3+ , Er 3+ ,La 3+ , Yb 3+ , Mn 3+ or Mn 2+ , but not limited to these. As one embodiment, Gd 3+ , Mn 3+ , Mn 2+ , Fe 2+ or Fe 3+ As one embodiment, Gd 3+ As one embodiment, Mn 3+ or Mn 2+ In this case, a halogen or the like can be used as a counter anion in the complex. Alternatively, the counter anion may be C(=O)O of the ligand. - In addition, the complex can also have Na + Equivalent counter cations.

[0455] Metal radioisotopes are used as PET tracers, etc. As one embodiment of the metal radioisotopes, there can be mentioned 89 Zr, 52 Mn, 52 Fe, 64 Cu, 67 Ga, 68 Ga, 72 As, 90 Y. 99m Tc, 111 In or 177 Lu, but not limited to these. As a certain form of metal radioisotopes used for PET tracers, SPECT tracers, etc., there can be cited 89 Zr, 64 Cu, 67 Ga, 68 Ga, 99m Tc or 111 In. As one embodiment, a radioactive isotope of zirconium (Zr) can be listed. As one embodiment, a radioactive isotope of zirconium (Zr) can be listed. 89 Zr. As one embodiment of the metal radioisotope used for cancer treatment, there are 90 Y or 177 Lu.

[0456] In one embodiment of the complex of the present invention, Y is a coordinating 89 As another example, Y is coordinated with a Zr-DFO complex. 90 Y. 67 Ga, 68 Ga and 177In another embodiment, Y is a complex of DOTA composed of a metal radioisotope of Lu. 3+ and Y 3+ As another embodiment, Y is a complex of DOTA with a paramagnetic metal ion. 3+ The DOTA complex.

[0457] 1-3. Peptide linkers or bonds

[0458] In the complex of the present invention, the ligand (Y) or the spacer (S1) and the Fab 1 The binding may be direct (ie, X is a bond) or may be via a peptide linker (ie, X is a peptide linker).

[0459] In the present specification, a "peptide linker" is a linker comprising a peptide consisting of 2 to 4 amino acids, and may have linkers Z1 to Z3 and the like suitable for binding to an anti-human CEACAM5 antibody Fab fragment as desired. Here, the peptide included in the peptide linker is not particularly limited, but is preferably a peptide consisting of 2 to 4 amino acids selected from the group consisting of glycine (Gly), lysine (Lys), methionine (Met), isoleucine (Ile), phenylalanine (Phe), valine (Val), tyrosine (Tyr), arginine (Arg), alanine (Ala), glutamine (Gln), glutamic acid (Glu), asparagine (Asn), aspartic acid (Asp), histidine (His), leucine (Leu), 3-(2-naphthyl)alanine and diphenylalanine, and more preferably a peptide consisting of 2 to 4 amino acids selected from the group consisting of glycine, lysine, methionine, isoleucine, phenylalanine, valine, tyrosine, aspartic acid, arginine, 3-(2-naphthyl)alanine and diphenylalanine. It should be noted that, unless otherwise specified, the configuration of the amino acid residues other than glycine is L-form.

[0460] One embodiment of the peptide linker is a peptide consisting of 2 to 4 amino acids having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme, and may further have a linker between the peptide linker and a biomolecule or an antibody Fab fragment. It is reported that a peptide linker having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme can be specifically cleaved by these enzymes present in the kidney, thereby reducing the accumulation of the labeled part in the kidney, and it is expected to reduce the risk of radiation exposure to the kidney and kidney damage. For example, Adv Drug Deliv Rev. 2008 Sep; 60 (12): 1319-28., Bioconjug Chem. 2005 Nov-Dec; 16 (6): 1610-6., and Cancer Res. 1999 Jan 1; 59 (1): 128-34. describe that a glycine-lysine linker can be specifically cleaved by a renal brush border membrane enzyme present in the kidney. Japanese Patent No. 6164556 states that a glycine-phenylalanine-lysine linker can be specifically cleaved by a renal brush border membrane enzyme in the kidney, and Bioconjug Chem. 2002 Sep-Oct; 13(5): 985-95. states that a linker comprising a glycine-leucine-glycine-lysine sequence can be specifically cleaved by a renal brush border membrane enzyme, and Bioconjug Chem. 2013 Feb 20; 24(2): 291-9. states that a glycine-tyrosine linker can be specifically cleaved by a renal brush border membrane enzyme. In addition, Bioconjug Chem. 2014 Nov 19; 25(11): 2038-45. states that a linker comprising a methionine-isoleucine sequence can be specifically cleaved by a lysosomal enzyme present in the kidney. As one embodiment of the peptide linker, it is a peptide linker comprising an amino acid sequence selected from the group consisting of methionine-isoleucine, glycine-lysine, glycine-phenylalanine-lysine, methionine-valine-lysine, glycine-tyrosine, glycine-lysine-lysine, and glycine-arginine-lysine, aspartic acid-glycine-lysine, methionine-glycine-lysine, methionine-isoleucine-lysine, glycine-tyrosine-lysine, glycine-valine, glycine-isoleucine, methionine-phenylalanine-lysine, glycine-(3-(2-naphthyl)alanine)-lysine, and glycine-diphenylalanine-lysine. In one embodiment, the peptide linker comprises an amino acid sequence selected from the group consisting of methionine-isoleucine aspartic acid-glycine-lysine, glycine-phenylalanine-lysine, methionine-glycine-lysine, glycine-lysine, and glycine-(3-(2-naphthyl)alanine)-lysine.

[0461] The "peptide linker" may optionally have a linker suitable for binding to the anti-human CEACAM5 antibody Fab fragment. Here, the linker suitable for binding to the anti-human CEACAM5 antibody Fab fragment is a group that allows the peptide linker to bind to the amino group or thiol group of the anti-human CEACAM5 antibody Fab fragment by organic chemical means. In one embodiment, it is a group containing a maleimide-derived group (for example, a group represented by the following formula (II-I) or (II-II)) or an isothiocyanate-derived group (-NH-C(=S)-) at the terminal. In one embodiment, it is -NH-(CH2)2-Z1-, -CH2-C(=O)-NH-(CH2)2-Z1-, -C(=S)-NH-(1,4-phenylene)-NH-C(=S)-, or -NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-, where Z1 is represented by the following formula (II-I) or (II-II). In addition, the following formula (II-I) is sometimes represented by -Z1(#N)-, and the following formula (II-II) is sometimes represented by -Z1(#S)-.

[0462] The connector is combined with the amino group or carboxyl group of the amino acid at the end of the peptide, or with the amino group (e.g., lysine) or the hydroxyl group (e.g., tyrosine) in the side chain of the amino acid to form a peptide linker. As an example of a connector that is combined with a functional group in the side chain of the amino acid at the end of the peptide to form a peptide linker, for example, as a connector integrated with Lys, the group shown in the following formula (III-I) or (III-II) can be cited, and both are collectively recorded as -Lys*-Z2- in this specification. The following formula (III-I) is sometimes recorded as -Lys*-Z2(#N)-, and the following formula (III-II) is sometimes recorded as -Lys*-Z2(#S)-. The following formula (III-III) is sometimes recorded as -Lys*-Z3-.

[0463]

[0464] In the present specification, the group represented by the following formula (IV) having a structure in which a functional group in the side chain of the terminal amino acid is bound to a linker is represented by -Tyr*-CH2-, and the group represented by the following formula (V) is represented by -Lys*-C(=S)-.

[0465]

[0466] In one embodiment, the peptide linker of "X" is a peptide linker comprising a connector. In one embodiment, the peptide linker is a peptide linker selected from the group consisting of the following peptide linkers:

[0467] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0468] (2)-Gly-Lys*-Z2-、

[0469] (3)-Gly-Phe-Lys*-Z2-、

[0470] (4)-Met-Val-Lys*-Z2-、

[0471] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0472] (6)-Gly-Lys-Lys*-Z2-、

[0473] (7)-Gly-Arg-Lys*-Z2-、

[0474] (8)-Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-、

[0475] (9)-Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-、

[0476] (10)-Asp-Gly-Lys*-Z2-、

[0477] (11)-Met-Gly-Lys*-Z2-、

[0478] (12)-Met-Ile-Lys*-Z2-、

[0479] (13)-Gly-Tyr*-CH2-C(=O)-Lys*-Z2-、

[0480] (14)-Val-NH-(CH2)2-Z1-、

[0481] (15)-Ile-NH-(CH2)2-Z1-、

[0482] (16)-Gly-Val-NH-(CH2)2-Z1-、

[0483] (17)-Gly-Ile-NH-(CH2)2-Z1-、

[0484] (18)-Met-Phe-Lys*-Z2-、

[0485] (19)-Gly-Tyr-Lys*-Z2-、

[0486] (20)-Gly-Tyr*-CH2-C(=O)-NH-(CH2O)3-CH2-C(=O)-NH-(CH2)2-Z1-,

[0487] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0488] (22)-Gly-diphenylalanine-Lys*-Z2-,

[0489] (23)-Gly-Tyr-NH-(CH2)5-Z1-,

[0490] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0491] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0492] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0493] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0494] (28)-Met-Gly-Lys*-Z3-.

[0495] Furthermore, one embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0496] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0497] (2)-Gly-Lys*-Z2-,

[0498] (3)-Gly-Phe-Lys*-Z2-,

[0499] (4)-Met-Val-Lys*-Z2-,

[0500] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0501] (6)-Gly-Lys-Lys*-Z2-,

[0502] (7)-Gly-Arg-Lys*-Z2-,

[0503] (8)-Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-, and

[0504] (9)-Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-.

[0505] In one embodiment, the peptide linker is selected from the group consisting of the following peptide linkers:

[0506] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0507] (2)-Gly-Lys*-Z2-,

[0508] (3)-Gly-Phe-Lys*-Z2-,

[0509] (10)-Asp-Gly-Lys*-Z2-,

[0510] (11)-Met-Gly-Lys*-Z2-,

[0511] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0512] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0513] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0514] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0515] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0516] (28)-Met-Gly-Lys*-Z3-.

[0517] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0518] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0519] (2)-Gly-Lys*-Z2-,

[0520] (3)-Gly-Phe-Lys*-Z2-,

[0521] (4)-Met-Val-Lys*-Z2-,

[0522] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0523] (7)-Gly-Arg-Lys*-Z2-,

[0524] (8) -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0525] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0526] (10)-Asp-Gly-Lys*-Z2-,

[0527] (11)-Met-Gly-Lys*-Z2-,

[0528] (12)-Met-Ile-Lys*-Z2-,

[0529] (13)-Gly-Tyr*-CH2-C(=O)-Lys*-Z2-、

[0530] (14)-Val-NH-(CH2)2-Z1-,

[0531] (15)-Ile-NH-(CH2)2-Z1-,

[0532] (16)-Gly-Val-NH-(CH2)2-Z1-,

[0533] (17)-Gly-Ile-NH-(CH2)2-Z1-,

[0534] (18)-Met-Phe-Lys*-Z2-,

[0535] (19)-Gly-Tyr-Lys*-Z2-,

[0536] (20)-Gly-Tyr*-CH2-C(=O)-NH-(CH2O)3-CH2-C(=O)-NH-(CH2)2-Z1-,

[0537] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-,

[0538] (22)-Gly-diphenylalanine-Lys*-Z2-,

[0539] (23)-Gly-Tyr-NH-(CH2)5-Z1-,

[0540] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0541] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0542] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0543] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0544] (28)-Met-Gly-Lys*-Z3-.

[0545] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0546] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0547] (2)-Gly-Lys*-Z2-,

[0548] (3)-Gly-Phe-Lys*-Z2-,

[0549] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0550] (6)-Gly-Lys-Lys*-Z2-,

[0551] (7)-Gly-Arg-Lys*-Z2-, and

[0552] (8)-Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-.

[0553] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0554] (2)-Gly-Lys*-Z2-,

[0555] (3)-Gly-Phe-Lys*-Z2-,

[0556] (10)-Asp-Gly-Lys*-Z2-,

[0557] (11)-Met-Gly-Lys*-Z2-, and

[0558] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-.

[0559] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0560] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0561] (2)-Gly-Lys*-Z2-,

[0562] (3)-Gly-Phe-Lys*-Z2-,

[0563] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0564] (6)-Gly-Lys-Lys*-Z2-, and

[0565] (8)-Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-.

[0566] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0567] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0568] (2)-Gly-Lys*-Z2-,

[0569] (3)-Gly-Phe-Lys*-Z2-,

[0570] (5)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1-、

[0571] (6)-Gly-Lys-Lys*-Z2-,

[0572] (8) -Gly-Lys*-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0573] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0574] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0575] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0576] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0577] (28)-Met-Gly-Lys*-Z3-.

[0578] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0579] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0580] (2)-Gly-Lys*-Z2-,

[0581] (3)-Gly-Phe-Lys*-Z2-,

[0582] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0583] (10)-Asp-Gly-Lys*-Z2-,

[0584] (11)-Met-Gly-Lys*-Z2-,

[0585] (12)-Met-Ile-Lys*-Z2-,

[0586] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2- and

[0587] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0588] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0589] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0590] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0591] (28)-Met-Gly-Lys*-Z3-.

[0592] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0593] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0594] (4)-Met-Val-Lys*-Z2-,

[0595] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0596] (11)-Met-Gly-Lys*-Z2-,

[0597] (12)-Met-Ile-Lys*-Z2-,

[0598] (18)-Met-Phe-Lys*-Z2-,

[0599] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0600] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0601] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0602] (27)-Met-Ile-NH-(CH2)2-(A-3)- and

[0603] (28)-Met-Gly-Lys*-Z3-.

[0604] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0605] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0606] (9) -Met-Ile-NH-(CH2)2-NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

[0607] (12)-Met-Ile-Lys*-Z2-,

[0608] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0609] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0610] (26)-Met-Ile-NH-(CH2)2-(II-II)-, and

[0611] (27)-Met-Ile-NH-(CH2)2-(A-3)-.

[0612] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0613] (4)-Met-Val-Lys*-Z2-,

[0614] (11)-Met-Gly-Lys*-Z2-,

[0615] (18)-Met-Phe-Lys*-Z2-, and

[0616] (28)-Met-Gly-Lys*-Z3-.

[0617] One embodiment of the peptide linker of "X" is a peptide linker selected from the group consisting of the following peptide linkers:

[0618] (11)-Met-Gly-Lys*-Z2-, and

[0619] (28)-Met-Gly-Lys*-Z3-.

[0620] 1-4. Spacer or bond (S1)

[0621] In the complex of the present invention, the ligand (Y) and the peptide linker (X) may be directly bound (ie, S1 is a bond) or bound via a spacer (ie, S1 is a spacer).

[0622] In this specification, the "spacer" of S1 is used to allow the ligand to be bound to the peptide linker or Fab. 1 The group introduced to space a certain distance between the ligands or to bind the ligand to the peptide linker can be listed as a certain embodiment, including -C(=O)-CH2O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-, -C(=O)-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,3-phenylene)-C(=O)-, -NH-(CH2)2-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)- C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -NH-(CH2)3-C(=O)-, -NH-(CH2CH2O)3-CH2-C(=O)-, -NH-CH2-(1,4-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, and spacers represented by the following formulae (a) to (q), etc.

[0623]

[0624] In one embodiment, S1 is -C(=O)-CH2O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-, or -C(=O)-(1,3-phenylene)-C(=O)-. In one embodiment, S1 is -NH-CH2-(1,3-phenylene)-C(=O)-, -NH-(CH2)2-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-, -NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -NH-(CH2)3-C(=O)-, -NH-(CH In one embodiment, S1 is -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-CH2-(1,3-phenylene)-C(=O)-, -CH2-(1,4-phenylene)-NH-C(=S)-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-phenylene)-C(=O)-, or a spacer represented by the above formulae (a) to (q). In one embodiment, S1 is -NH-CH2-(1,4-phenylene)-NH-C(=O)-, or a spacer represented by the above formulae (g), (i), or (k).

[0625] As a certain embodiment, S1 is -C(=O)-CH2O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH2CH2O)4-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,3-phenylene)-C(=O)-, -NH-CH2-(1,4-phenylene)-NH-C(=O)-, -NH-CH2-(1,4-phenylene)-C(=O)-, -C(=O)-(1,3-phenylene)-C(=O)-, or the following formula.

[0626]

[0627] As a certain aspect, S1 is a key.

[0628] In addition, in the complex wherein Y is DOTA of the present invention, DOTA and the anti-human CEACAM5 antibody Fab fragment (Fab 1 ) can be directly bound or bound with the aid of a spacer (-CH2-(1,4-phenylene)-NH-C(=S)-). Among them, DOTA and anti-human CEACAM5 antibody Fab fragment (Fab 1) is a complex formed by bonding with -CH2-(1,4-phenylene)-NH-C(=S)- as a spacer, which is a complex represented by the following formula (VI).

[0629]

[0630] In addition, the S1 spacer described in the specification of the present application includes a novel spacer, a spacer represented by the above formula (g) and (l), and these are particularly useful as a spacer when a peptide linker containing a peptide consisting of 2 to 4 amino acids having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme is bound to DOTA. As shown in the kidney aggregation evaluation test of Example 4 described later, it has been confirmed that a complex composed of a peptide linker that can be cleaved by an enzyme, DOTA, and a spacer represented by the above formula (g) can reduce the aggregation of the labeled portion to the kidney. As a certain embodiment, S1 is a spacer represented by the above formula (g) and (l).

[0631] As one embodiment of the complex of the present invention, Y is DOTA, S1 is a spacer represented by formula (g) and (l), and X is a peptide linker comprising a peptide composed of 2 to 4 amino acids having an amino acid sequence that is cleaved by renal brush border membrane enzymes or lysosomal enzymes.

[0632] In one embodiment, Y is DOTA, S1 is a spacer represented by formula (g) and (l), and X is a peptide linker selected from the group consisting of the following peptide linkers:

[0633] (2)-Gly-Lys*-Z2-,

[0634] (3)-Gly-Phe-Lys*-Z2-,

[0635] (10)-Asp-Gly-Lys*-Z2-,

[0636] (11)-Met-Gly-Lys*-Z2-, and

[0637] (21)-Gly-(3-(2-naphthyl)alanine)-Lys*-Z2-.

[0638] In one embodiment, Y is DOTA, S1 is -NH-CH2-(1,4-phenylene)-NH-C(=O)-, a spacer represented by the above formula (g), (i), or (k), and X is a peptide linker selected from the group consisting of the following peptide linkers:

[0639] (1)-Met-Ile-NH-(CH2)2-Z1-,

[0640] (11)-Met-Gly-Lys*-Z2-,

[0641] (12)-Met-Ile-Lys*-Z2-,

[0642] (24)-Met-Ile-NH-(CH2)2-(A-4)-、

[0643] (25)-Met-Ile-NH-(CH2)2-(A-5)-、

[0644] (26)-Met-Ile-NH-(CH2)2-(II-II)-,

[0645] (27)-Met-Ile-NH-(CH2)2-(A-3)-, and

[0646] (28)-Met-Gly-Lys*-Z3-.

[0647] In one embodiment, Y is DOTA, S1 is a spacer represented by formula (g), and X is a peptide linker selected from the group consisting of the following peptide linkers:

[0648] (11)-Met-Gly-Lys*-Z2-, and

[0649] (28)-Met-Gly-Lys*-Z3-.

[0650] The composite of the present invention includes a combination of the above aspects.

[0651] Specific embodiments of the complex of the present invention are as follows.

[0652] In the formula, Fab 2 It is a Fab fragment containing a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0653] p is a natural number from 1 to 25. 2 With the help of this Fab 2 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

[0654]

[0655]

[0656] It should be noted that the tag portion of the combination in which Y is DOTA, S1 is a spacer represented by formula (g) and (l), and X is a peptide linker comprising a peptide consisting of 2 to 4 amino acids having an amino acid sequence cleaved by a renal brush border membrane enzyme or a lysosomal enzyme is not limited to the Fab of the present invention. 1The combination is useful as a labeling part that is expected to reduce nephrotoxicity, etc. in the same complex using various Fab antibodies, and the present invention also includes the invention of the labeling part itself.

[0657] In the production of the complex of the present invention, the binding of the anti-CEACAM5 antibody Fab fragment to the ligand, spacer and / or peptide linker, and the binding of the ligand to the spacer and / or peptide linker can be appropriately performed by those skilled in the art by known methods.

[0658] In this specification, the "labeling part" refers to, for example, Fab in formula (I). 1 In formula (Ia) or (Ib), "labeled part" means a Biomolecule 1 or Biomolecule 2 Parts other than the above. It is (i) a ligand and a peptide linker (Y-S1-X: wherein S1 is a bond and X is a peptide linker.), (ii) a ligand (Y-S1-X: wherein S1 and X are bonds respectively.), or (iii) a ligand, a spacer and a peptide linker (Y-S1-X: wherein S1 is a spacer and X is a peptide linker.). As a certain embodiment, (ii) a ligand can be listed. As a certain embodiment, (i) a ligand and a peptide linker or (iii) a ligand, a spacer and a peptide linker can be listed. Here, the ligand of the "labeling part" may further contain a metal. As a certain embodiment, it is (i) a ligand and a peptide linker, (ii) a ligand or (iii) a ligand, a spacer and a peptide linker containing a metal. In other words, it is (i) a ligand and a peptide linker that form a chelate complex with a metal, (ii) a ligand that forms a chelate complex with a metal, or (iii) a ligand, a spacer and a peptide linker that form a chelate complex with a metal. There is also a case where (iv) the peptide linker further includes a spacer in the above-mentioned peptide linker. 1 or Biomolecule 2 Other parts.

[0659] 1-5. Labeling part (Y-S1-X) relative to anti-human CEACAM5 antibody Fab fragment (Fab 1 ) and the number of binding sites (p) and the labeling site relative to the biomolecule of formula (Ia) or (Ib) 1 and Biomolecule 2 The number of combinations (p)

[0660] The complex of the present invention is formed by the anti-human CEACAM5 antibody Fab fragment (Fab 1) is a complex in which one or more amino groups or thiol groups in the compound are bonded to one or more labeling parts (Y-S1-X). In addition, the complex of the present invention is a Biomolecule of formula (Ia) or (Ib) 1 and biomolecule 2 A complex in which one or more amino groups or thiol groups in the Fab are bonded with one or more labeling moieties. The complex of the present invention may be a mixture of complexes in which the number of the bonded labeling moieties is different from each other. Formula (I) represents Fab 1 Any one of the complexes having 1 to 25 labeling parts (Y-S1-X) bound thereto or a mixture thereof. In one embodiment, the complex of the present invention is 1 It includes 1 to 25 marking parts (Y-S1-X), as a certain method, it includes 1 to 23 marking parts (Y-S1-X), as a certain method, it includes 1 to 15 marking parts (Y-S1-X), as a certain method, it includes 1 to 11 marking parts (Y-S1-X), as a certain method, it includes 1 to 9 marking parts (Y-S1-X), as a certain method, it includes 1 to 7 marking parts (Y-S1-X), as a certain method, it includes 1 to 5 marking parts (Y-S1-X), and further, as a certain method, it includes 1 to 4 marking parts (Y-S1-X).

[0661] In formula (Ia) or (Ib), Biomolecule 1 and Biomolecule 2 Indicates Fabs with 1 to 25 of the labeled moieties 1 As one embodiment, the complex of the present invention is relative to one Biomolecule 1 and Biomolecule 2 It contains 1 to 25 of the marking parts, as a certain method, it contains 1 to 23 of the marking parts, as a certain method, it contains 1 to 15 of the marking parts, as a certain method, it contains 1 to 11 of the marking parts, as a certain method, it contains 1 to 9 of the marking parts, as a certain method, it contains 1 to 7 of the marking parts, as a certain method, it contains 1 to 5 of the marking parts, and further, as a certain method, it contains 1 to 4 of the marking parts.

[0662] That is, it indicates that the label part (Y-S1-X) is relative to one Fab 1 The number of binding "p" indicates the number of binding sites per biomolecule. 1 and Biomolecule 2The "p" of the combination number is the same or different, and as a certain method, it is a natural number from 1 to 25, as a certain method, it is a natural number from 1 to 23, as a certain method, it is a natural number from 1 to 15, as a certain method, it is a natural number from 1 to 11, as a certain method, it is a natural number from 1 to 9, as a certain method, it is a natural number from 1 to 7, as a certain method, it is a natural number from 1 to 6, as a certain method, it is a natural number from 1 to 5, as a certain method, it is a natural number from 1 to 4, and further, as a certain method, it is a natural number from 1 to 3.

[0663] 2. Polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention

[0664] In a certain embodiment, the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is encoded by a polynucleotide, wherein the polynucleotide is: a polynucleotide comprising a base sequence encoding a heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment, and a polynucleotide comprising a base sequence encoding a light chain of the anti-human CEACAM5 antibody Fab fragment.

[0665] In a certain embodiment, the polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is: a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region composed of the amino acid sequence shown by amino acid numbers 1 to 121 of SEQ ID NO: 2, or a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region composed of the amino acid sequence shown by amino acid numbers 1 to 112 of SEQ ID NO: 4.

[0666] Examples of polynucleotides containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acid numbers 1 to 121 of SEQ ID NO: 2 include, for example, a polynucleotide containing a base sequence of base numbers 1 to 363 of SEQ ID NO: 1. Examples of polynucleotides containing a base sequence encoding a light chain comprising a light chain variable region consisting of an amino acid sequence represented by amino acid numbers 1 to 112 of SEQ ID NO: 4 include, for example, a polynucleotide containing a base sequence of base numbers 1 to 336 of SEQ ID NO: 3.

[0667] In one embodiment, the polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is: a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2, or a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0668] Examples of polynucleotides comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 include, for example, a polynucleotide comprising the base sequence shown in SEQ ID NO: 1. Examples of polynucleotides comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 include, for example, a polynucleotide comprising the base sequence shown in SEQ ID NO: 3.

[0669] The polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention can be synthesized based on the base sequence designed based on the amino acid sequence of the heavy chain fragment and the light chain of the anti-human CEACAM5 antibody Fab fragment and using a gene synthesis method known in the art. As such a gene synthesis method, various methods known to those skilled in the art such as the method for synthesizing antibody genes described in International Publication No. 90 / 07861 can be used.

[0670] 3. Expression vector of the polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention

[0671] The expression vector of the polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention includes: an expression vector containing a polynucleotide comprising a base sequence encoding the heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment, an expression vector containing a polynucleotide comprising a base sequence encoding the light chain of the anti-human CEACAM5 antibody Fab fragment, and an expression vector containing a polynucleotide comprising a base sequence encoding the heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment and a polynucleotide comprising a base sequence encoding the light chain of the anti-human CEACAM5 antibody Fab fragment.

[0672] Preferred expression vectors include: an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 121 of SEQ ID NO: 2, an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 112 of SEQ ID NO: 4, and an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 121 of SEQ ID NO: 2, and an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 112 of SEQ ID NO: 4.

[0673] Preferred expression vectors include: an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in sequence number 2, an expression vector containing a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in sequence number 4, and an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in sequence number 2 and a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in sequence number 4.

[0674] These expression vectors are not particularly limited as long as they can produce the polypeptide encoded by the polynucleotide of the present invention in various host cells of prokaryotic cells and / or eukaryotic cells. Such expression vectors include, for example, plasmid vectors, viral vectors (e.g., adenovirus, retrovirus), etc., and pEE6.4 and pEE12.4 (Lonza) can be preferably used.

[0675] In addition, these expression vectors may contain a promoter linked to the gene encoding the heavy chain fragment and / or light chain of the polynucleotide encoding the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention in a functional manner. As a promoter for expressing the Fab fragment in a host cell, when the host cell is an Escherichia bacterium, for example, Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter, etc. can be cited. As a promoter for expression in yeast, for example, PH05 promoter, PGK promoter, GAP promoter, ADH promoter can be cited, and as a promoter for expression in Bacillus bacteria, SL01 promoter, SP02 promoter, penP promoter, etc. can be cited. In addition, when the host is a eukaryotic cell such as a mammalian cell, promoters derived from viruses such as CMV, RSV, SV40, promoters of retroviruses, actin promoters, EF (elongation factor, elongation factor) 1α promoters, heat shock promoters, etc. can be cited.

[0676] When using bacteria, particularly Escherichia coli as host cells, these expression vectors may further include start codons, stop codons, terminator regions and reproducible units. On the other hand, when using yeast, animal cells or insect cells as hosts, expression vectors may include start codons and stop codons. In addition, in this case, enhancer sequences, non-translated regions on the 5' side and 3' side of the gene encoding the heavy chain fragment of the present invention and / or light chain, secretion signal sequences, splicing junctions, polyadenylation sites or reproducible units, etc. may be included. In addition, commonly used selection markers (e.g., tetracycline resistance genes, ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, dihydrofolate reductase genes) may be included according to the purpose.

[0677] 4. Host cells transformed with expression vectors

[0678] Host cells transformed with the expression vector include host cells in the group consisting of the following (a) to (d):

[0679] (a) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment;

[0680] (b) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a light chain of an anti-human CEACAM5 antibody Fab fragment;

[0681] (c) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment and a polynucleotide comprising a base sequence encoding a light chain of an anti-human CEACAM5 antibody Fab fragment; and

[0682] (d) A host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain of an anti-human CEACAM5 antibody Fab fragment.

[0683] In one embodiment, the host cell transformed with the expression vector is a host cell transformed with an expression vector selected from the group consisting of the following (a) to (d):

[0684] (a) a host cell transformed with an expression vector containing a polynucleotide having a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence represented by amino acid numbers 1 to 121 of SEQ ID NO: 2;

[0685] (b) a host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence represented by amino acid numbers 1 to 112 of SEQ ID NO: 4;

[0686] (c) a host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising the heavy chain variable region consisting of the amino acid sequence shown in amino acids 1 to 121 of SEQ ID NO: 2 and a polynucleotide containing a base sequence encoding a light chain comprising the light chain variable region consisting of the amino acid sequence shown in amino acids 1 to 112 of SEQ ID NO: 4; and

[0687] (d) A host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 121 of SEQ ID NO: 2, and an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 112 of SEQ ID NO: 4.

[0688] In one embodiment, the host cell transformed with the expression vector is a host cell transformed with an expression vector selected from the group consisting of the following (a) to (d):

[0689] (a) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2;

[0690] (b) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4;

[0691] (c) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and

[0692] (d) A host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0693] Preferred host cells transformed with expression vectors include: host cells transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and a polynucleotide comprising a base sequence encoding a light chain of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention; and host cells transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention.

[0694] The host cell transformed with the expression vector is not particularly limited as long as it is suitable for the expression vector used and can be transformed with the expression vector to express the Fab fragment, and various cells such as natural cells or artificially established cells commonly used in the technical field of the present invention (for example, bacteria (Escherichia bacteria, Bacillus bacteria), yeast (Saccharomyces, Pichia, etc.), animal cells or insect cells (for example, Sf9), etc.), and mammalian cell lines (for example, cultured cells such as CHO-K1SV cells, CHO-DG44 cells, and 293 cells) can be exemplified. The transformation itself can be performed by a known method such as the calcium phosphate method and the electroporation method.

[0695] 5. Method for producing the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention

[0696] The production of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention comprises the following steps: culturing the above-mentioned transformed host cells to express the anti-human CEACAM5 antibody Fab fragment.

[0697] In one embodiment, the transformed host cell cultured for producing the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is selected from the group consisting of the following (a) to (c):

[0698] (a) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and a polynucleotide comprising a base sequence encoding a light chain of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention;

[0699] (b) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention; and

[0700] (c) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention, and a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a light chain fragment of an anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention.

[0701] In one embodiment, the transformed host cell cultured for producing the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is selected from the group consisting of the following (a) to (c):

[0702] (a) a host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2 and a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4;

[0703] (b) a host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in amino acids 1 to 121 of SEQ ID NO: 2 and an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence shown in amino acids 1 to 112 of SEQ ID NO: 4; and

[0704] (c) A host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 121 of SEQ ID NO: 2, and a host cell transformed with an expression vector having a polynucleotide containing a base sequence encoding a light chain comprising a light chain variable region consisting of the amino acid sequence shown in amino acid numbers 1 to 112 of SEQ ID NO: 4.

[0705] In one embodiment, the transformed host cell cultured for producing the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention is selected from the group consisting of the following (a) to (c):

[0706] (a) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4;

[0707] (b) a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2 and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; and

[0708] (c) A host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 2, and a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0709] Preferably, the transformed host cell used is: a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and a polynucleotide comprising a base sequence encoding a light chain of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention; or, a host cell transformed with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain fragment of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention and an expression vector containing a polynucleotide comprising a base sequence encoding a light chain of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention.

[0710] In the production of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention, the transformed host cells can be cultured in a nutrient medium. The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source required for the growth of the transformed host cells. As a carbon source, examples include glucose, dextran, soluble starch, sucrose, etc., and as an inorganic nitrogen source or an organic nitrogen source, examples include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, etc. In addition, other nutrients (such as inorganic salts (such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (such as tetracycline, neomycin, ampicillin, kanamycin, etc.) can be contained as desired.

[0711] The culture of the transformed host cells is carried out by a known method. The culture conditions, such as temperature, pH of the culture medium and culture time, can be appropriately selected. For example, when the host is an animal cell, as a culture medium, MEM culture medium (Science; 1955; 122: 501.), DMEM culture medium (Virology; 1959; 8: 396-97.), RPMI1640 culture medium (J. Am. Med. Assoc.; 1967; 199: 519-24.), 199 culture medium (Proc. Soc. Exp. Biol. Med.; 1950; 73: 1-8.) and the like containing about 5% to about 20% fetal bovine serum can be used. The pH of the culture medium is preferably about 6 to about 8, and the culture is usually carried out at about 30° C. to about 40° C. for about 15 hours to about 336 hours, and ventilation and stirring can also be performed as needed. When the host is an insect cell, for example, Grace's medium (PNAS; 1985; 82: 8404-8.) containing fetal bovine serum can be cited, and its pH is preferably about 5 to about 8. Cultivation is usually carried out at about 20°C to about 40°C for 15 to 100 hours, and ventilation and stirring can be performed as needed. When the host is bacteria, actinomycetes, yeast, or filamentous fungi, for example, a liquid culture medium containing the above-mentioned nutrient source is appropriate. A culture medium with a pH of 5 to 8 is preferred. When the host is Escherichia coli (E. coli), LB culture medium, M9 culture medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory; 1972: 431.) and the like can be exemplified as preferred culture media. In this case, the culture can be carried out at 14 to 43°C for about 3 to about 24 hours while aerating and stirring as needed. When the host is a bacillus, the culture can be carried out at 30 to 40°C for about 16 to 96 hours with ventilation and stirring as needed. When the host is yeast, the culture medium includes, for example, Burkholder minimal medium (PNAS; 1980; 77: 4505-4508.), and the pH is preferably 5 to 8. The culture is usually carried out at about 20°C to about 35°C for about 14 to about 144 hours, and ventilation and stirring can be carried out as needed.

[0712] In the production of the anti-human CEACAM5 antibody Fab fragment contained in the complex of the present invention, in addition to the step of culturing the transformed host cells to express the anti-human CEACAM5 antibody Fab fragment, the steps of recovering, preferably isolating and purifying the expressed anti-human CEACAM5 antibody Fab fragment may be included. Examples of the isolation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, methods utilizing molecular weight differences such as dialysis, ultrafiltration, gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, methods utilizing charge such as ion exchange chromatography and hydroxyapatite chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing hydrophobicity differences such as reversed-phase high performance liquid chromatography, and methods utilizing isoelectric point differences such as isoelectric point electrophoresis.

[0713] 6. Method for producing the complex of the present invention

[0714] The method for producing the complex of the present invention may include the step of covalently binding the anti-human CEACAM5 antibody Fab fragment to the labeling part (Y-S1-X). A person skilled in the art can appropriately bind the components in the above-mentioned labeling part (Y-S1-X) using a known method. As a reaction example, after the ligand (Y) is bound to the peptide linker (X) directly or via a spacer (S1), the above-mentioned peptide linker is bound to the anti-human CEACAM5 antibody Fab fragment. In addition, after the anti-human CEACAM5 antibody Fab fragment is bound to the peptide linker (X), the above-mentioned peptide linker is bound to the ligand (Y) directly or via a spacer (S1). It should be noted that as a raw material, a compound formed by pre-binding a ligand and a spacer (S1) can also be used.

[0715] In addition, the method for producing the complex of the present invention may include: the step of culturing the above-mentioned transformed host cells to express the anti-human CEACAM5 antibody Fab fragment; and the step of covalently binding the Fab fragment to the labeling part (Y-S1-X). In addition, the method for producing the complex of the present invention may include: the step of culturing the above-mentioned transformed host cells to express the anti-human CEACAM5 antibody Fab fragment; the step of recovering the expressed Fab fragment; and the step of covalently binding the Fab fragment to the labeling part (Y-S1-X). In addition, the method for producing the complex of the present invention may include the step of adding a metal. The chelating agent, peptide linker, spacer, number of labeling parts, metal, etc. used can use the substances described in this specification.

[0716] The method for producing the complex of the present invention can be implemented as a method including two or more steps defined above in the form of a series of steps, or as a method including at least one step defined above. In addition, for example, a method including the step of binding an anti-human CEACAM5 antibody Fab fragment to a labeling portion (Y-S1-X), and a method including the step of coordinating a metal to an anti-human CEACAM5 antibody Fab fragment bound to a labeling portion (Y-S1-X) are also included in the method for producing the complex of the present invention. In addition, the method for producing the complex of the present invention also includes a method in which the order of the steps is different. For example, a method in which a labeling portion (Y-S1-X) after the metal is coordinated to a ligand is covalently bound to an anti-human CEACAM5 antibody Fab fragment is also included in the method for producing the complex of the present invention.

[0717] Alternatively, the anti-human CEACAM5 antibody Fab fragment can be produced in the same manner by using the biomolecule described in the present invention instead of the anti-human CEACAM5 antibody Fab fragment.

[0718] 7. Complexes containing ligands, spacers, peptide linkers and biomolecules

[0719] The complex of the present invention represented by the following formula and comprising a ligand, a spacer, a peptide linker and a biomolecule will be described.

[0720]

[0721] The following formula (S2) corresponds to the spacer:

[0722] The following methods can be listed:

[0723]

[0724] As another embodiment, formula S2 can be listed as The group.

[0725] As another embodiment, formula S2 can be listed as

[0726]

[0727] The group.

[0728] As another embodiment, formula S2 can be listed as

[0729]

[0730] The group.

[0731] Examples of the form of the group Q in the formula (S2) include -C(=O)-, -NH-C(=O)-, and -NH-C(=S)-.

[0732] As the form of the group Q in the formula (S2), -C(=O)- or -NH-C(=O)-.

[0733] As an embodiment of the group Q in the formula (S2), -NH-C(=O)- can be mentioned.

[0734] As the group L in formula (Ia) or (Ib) 2 In the case of, Ile, Phe, and Gly can be mentioned.

[0735] As the group L in formula (Ia) or (Ib) 2 The way can be listed as Ile.

[0736] As the group L in formula (Ia) or (Ib) 2 As an example, Phe can be cited.

[0737] As the group L in formula (Ia) or (Ib) 2 As an example, Gly can be mentioned.

[0738] As the group L in formula (Ia) or (Ib) 3 In the case of, bond, Arg, His can be listed.

[0739] As the group L in formula (Ia) or (Ib) 3 The keys can be listed in a .

[0740] As the group L in formula (Ia) or (Ib) 3 In the method, Arg can be listed.

[0741] As the group L in formula (Ia) or (Ib) 3 , His can be listed.

[0742] As the group L in formula (Ia) or (Ib) 4 The form of can be -NH-(CH2)2-, -NHCH(C(=O)OH)(CH2)4-, or a bond.

[0743] As the group L in formula (Ia) or (Ib) 4 The form of can be -NH-(CH2)2-.

[0744] As the group L in formula (Ia) or (Ib) 4 The form of -NHCH(C(=O)OH)(CH2)4- can be mentioned.

[0745] As the group L in formula (Ia) or (Ib) 4 The keys can be listed in a .

[0746] As group V in formula (Ia) or (Ib) 1 In the embodiment, groups represented by the following formulae (A-1) to (A-5) can be mentioned.

[0747]

[0748] As group V in formula (Ia) or (Ib) 1 In the embodiment, a group represented by the following formula (A-1) can be mentioned.

[0749]

[0750] As group V in formula (Ia) or (Ib) 1 In the embodiment, a group represented by the following formula (A-2) can be mentioned.

[0751]

[0752] As group V in formula (Ia) or (Ib) 1 In the embodiment, a group represented by the following formula (A-3) can be mentioned.

[0753]

[0754] As group V in formula (Ia) or (Ib) 1 In the embodiment, a group represented by the following formula (A-4) can be mentioned.

[0755]

[0756] As group V in formula (Ia) or (Ib) 1 In the embodiment, a group represented by the following formula (A-5) can be mentioned.

[0757]

[0758] The following formula (B) in formula (Ia) or (Ib) is a group consisting of a spacer and a peptide linker.

[0759]

[0760] Examples include groups selected from the group consisting of the following formulae (B-1) to (B-7).

[0761]

[0762]

[0763] In one embodiment, the compound may be selected from the group consisting of the following formula: wherein p is a natural number of 1 to 25; 1The complex described by the above formula (Ia) in which p amino groups or thiol groups present in the compound are bonded to adjacent carbon atoms.

[0764]

[0765]

[0766] An embodiment of the present invention is a complex represented by the following formula.

[0767]

[0768] [Wherein, Fab 5 The following antibody Fab fragments:

[0769] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0770] An embodiment of the present invention is a complex represented by the following formula.

[0771]

[0772] [Wherein, Fab 5 The following antibody Fab fragments:

[0773] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0774] An embodiment of the present invention is a complex represented by the following formula.

[0775]

[0776] [Wherein, Fab 5 The following antibody Fab fragments:

[0777] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0778] An embodiment of the present invention is a complex represented by the following formula.

[0779]

[0780] [Wherein, Fab 5 The following antibody Fab fragments:

[0781] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0782] An embodiment of the present invention is a complex represented by the following formula.

[0783]

[0784] [Wherein, Fab 5 The following antibody Fab fragments:

[0785] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0786] An embodiment of the present invention is a complex represented by the following formula.

[0787]

[0788] [Wherein, Fab 5 The following antibody Fab fragments:

[0789] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0790] An embodiment of the present invention is a complex represented by the following formula.

[0791]

[0792] [Wherein, Fab 5 The following antibody Fab fragments:

[0793] An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8, in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.]

[0794] In addition, as embodiments of the present invention, the following formulas (Ie) and (If) can be mentioned.

[0795]

[0796] The compound of the present invention sometimes exists in geometric isomers and tautomers. In addition, the compound of the present invention sometimes has chiral carbon. The present invention includes products obtained by separating these isomers, or mixtures thereof. In addition, labeled compounds, i.e. compounds obtained by replacing one or more atoms in the compound of the present invention with radioactive isotopes or non-radioactive isotopes, are also included in the present invention.

[0797] As a form of biomolecule, any biomolecule may be used as long as it has physiological activity, and examples thereof include peptides, proteins, hormones, drugs, nucleotides, oligonucleotides, nucleic acids, polysaccharides, etc. In the case of proteins, antibodies, enzymes, receptors, and fragments thereof are included.

[0798] Other forms of biomolecules include peptides and proteins.

[0799] Another example of biomolecules is protein.

[0800] Other forms of biomolecules include Fab fragments of proteins, enzymes, receptors, and fragments thereof. For example, somatostatin, PSMA ligand, RGD (Arg-Gly-Asp) peptide, ATSM (Diacetyl-bis (N4-methylthiosemicarbazone), diacetyl-bis (N4-methylthiosemicarbazone)), 211 At-AITM(4- 211 At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide, 4- 211 At-astato-N-[4-(6-(isopropylamino)pyridin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide) is exemplified.

[0801] Other forms of biomolecules include commercially available antibodies or Fab fragments thereof.

[0802] Examples thereof include nivolumab, pembrolizumab, bevacizumab, rituximab, pertuzumab, daratumumab, denosumab, cetuximab, ipilimumab, panitumumab, brentuximab, ramucirumab, atezolizumab, obinutuzumab, elotuzumab, avelumab, ibritumomab tiuxetan, alemtuzumab, gemtuzumab ozogamicin, and necituzumab.

[0803] In addition, as another aspect of the biomolecule, there are biomolecules that can be expected to be used in α-ray therapy and β-ray therapy.

[0804] For example, octreotide (Octreoscan), 131 I-MIBG (I-131metaiodobenzylguanidine, I-131 metaiodobenzylguanidine), 211 At-MABG( 211 At-astato-benzylguanidine), trastuzumab, humanized antibody A33 (Cancer Biotherapy & Radiopharmaceuticals 2005Oct; 20(5): 514-23.), Omburtamab, tetumomab, CD45 antibody (ClinicalTrials.gov Identifier: NCT03128034; 9595; NCI-2017-00452), ibritumomab tiuxetan, Actimab.

[0805] In addition, other forms of biomolecules include HuM195 described in Cancer studies and molecular medicine, MX35-F(ab')2 monoclonal antibody, mouse 9.2.27, proteoglycan (MCSP) antigen, CD20 antigen, CD30 antigen, etc. (Cancer studies and molecular medicine (2004), 1, 1, 1-7).

[0806] In addition, other aspects of the biomolecule include CD19, GD2, VE-cadherin, and the like.

[0807] As a form of biomolecule, a MUC1 antibody Fab fragment (called Fab 3 Fab 4 or Fab 5 ), examples described in International Publication WO2018 / 092885 can be cited.

[0808] [1M] Specifically, the Fab 3 or Fab 4 The anti-human MUC1 antibody Fab fragment selected from the group consisting of the following (a) and (b), wherein the Fab fragment having a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12 is referred to as Fab 3 The Fab fragment having a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 14 is referred to as Fab 4 :

[0809] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain fragment comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; and

[0810] (b) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14, in which the glutamine at amino acid number 1 of SEQ ID NO: 12 or SEQ ID NO: 14 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0811] [2M] In one embodiment, the anti-human MUC1 antibody Fab fragment described in [1M] above is selected from the group consisting of the following (a) and (b):

[0812] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 8 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10; and

[0813] (b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8, in which the glutamine at amino acid number 1 of SEQ ID NO: 6 or SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[0814] [3M] In one embodiment, the anti-human MUC1 antibody Fab fragment described in [1M] above is selected from the group consisting of the following (a) and (b):

[0815] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14 and a light chain fragment comprising a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16; and

[0816] (b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 14 and in which glutamine at amino acid No. 1 of SEQ ID NO: 10 is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0817] [4M] In one embodiment, the anti-human MUC1 antibody Fab fragment described in [3M] is selected from the group consisting of the following (a) and (b):

[0818] (a) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10; and

[0819] (b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0820] [5M] In one embodiment, the anti-human MUC1 antibody Fab fragment described in [4M] above is an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0821] [6M] In one embodiment, the anti-human MUC1 antibody Fab fragment is the anti-human MUC1 antibody Fab fragment described in [4M] above, which is a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid number 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 10.

[0822] [7M] P10-1 or P10-2 (referred to as Fab) as an anti-MUC1 antibody Fab fragment described in International Publication WO2018 / 092885 5 ).

[0823] Furthermore, the composite of the present invention may be composed of a combination of the above-mentioned various aspects.

[0824] 7. Diagnostic composition and diagnostic method

[0825] The present invention relates to a diagnostic composition containing a metal-containing complex of the present invention (hereinafter referred to as a detectable complex of the present invention). The diagnostic composition of the present invention may contain more than one complex of the present invention. That is, the diagnostic composition of the present invention may contain one complex of the present invention, or may contain a combination of two or more complexes of the present invention. The detectable complex of the present invention can be formulated according to conventional methods and used as an early diagnosis drug or a staging diagnosis drug (particularly a cancer diagnosis drug).

[0826] Early diagnostic drugs are diagnostic drugs that are used to diagnose at an early stage when the disease is not observed or the course of the disease is not observed. For example, in cancer, it refers to diagnostic drugs used at a stage when the disease is not observed or at stage 0 or stage 1.

[0827] Staging diagnostic drugs are diagnostic drugs that can check how far a disease has progressed. For example, in the case of cancer, they are diagnostic drugs that can check its stage.

[0828] The cancer that can be diagnosed using the diagnostic composition of the present invention is expected to be a cancer that expresses human CEACAM5. As a certain embodiment, colorectal cancer, breast cancer, lung cancer, thyroid cancer and cancers metastasized from them can be listed. As a certain embodiment, the cancer is colorectal cancer or a cancer metastasized from colorectal cancer. More preferably, the cancer is a cancer metastasized from colorectal cancer, and such cancers include metastatic liver cancer. In addition, it is a cancer that expresses human MUC1. As a certain embodiment, breast cancer, lung cancer, colorectal cancer, bladder cancer, skin cancer, thyroid cancer, gastric cancer, pancreatic cancer, kidney cancer, ovarian cancer or cervical cancer can be listed. In a certain embodiment, the cancer is breast cancer or bladder cancer.

[0829] The amount of the complex of the present invention added when the diagnostic composition of the present invention is formulated varies depending on the severity of the patient's symptoms and age, the dosage form of the preparation used, or the binding titer of the Fab fragment or biomolecule, for example, about 0.001 mg / kg to about 100 mg / kg can be used based on the mass of the Fab fragment or biomolecule per unit body weight of the patient.

[0830] As examples of the dosage form of the diagnostic composition of the present invention, parenteral agents such as injections and drips can be listed, which can be administered by intravenous injection, intramuscular injection to the target tissue local, subcutaneous injection, intravesical administration, etc. In addition, when formulated, carriers and additives corresponding to these dosage forms can be used within a pharmaceutically acceptable range. The types of pharmaceutically acceptable carriers and additives are not particularly limited, and carriers and additives known to those skilled in the art can be used.

[0831] In addition, the present invention relates to the use of the detectable complex of the present invention in the manufacture of a composition for early diagnosis or staging diagnosis of cancer. The present invention also relates to the detectable complex of the present invention for use in early diagnosis or staging diagnosis of cancer.

[0832] In addition, the present invention also relates to a method for diagnosing cancer, which comprises administering a detectable complex of the present invention to a subject. Here, "subject" refers to a person or other mammal who needs to receive the diagnosis. As a certain embodiment, it is a person who needs to receive the diagnosis. The effective amount of the detectable complex of the present invention in the diagnostic method of the present invention can be the same amount as the effective amount of the complex of the present invention when formulated as described above. In the diagnostic method of the present invention, the detectable complex of the present invention can be administered by local intramuscular injection, subcutaneous injection, etc. to the target tissue.

[0833] In another embodiment, the present invention also relates to the use of an anti-human CEACAM5 antibody Fab fragment in the manufacture of a complex comprising the anti-human CEACAM5 antibody Fab fragment of the present invention, a peptide linker and / or a ligand. As a certain embodiment, the present invention also relates to the use of an anti-human CEACAM5 antibody Fab fragment in the manufacture of a diagnostic composition comprising the complex of the present invention.

[0834] In addition, as a mode for providing the diagnostic composition of the present invention containing a metal radioisotope, the composition may be labeled with a metal radioisotope immediately before use, or may be provided as a diagnostic composition containing a metal radioisotope.

[0835] 8. Pharmaceutical compositions and methods of treatment

[0836] In the present invention, the 90 Y or 177 One or more metal radioisotopes such as Lu can be used based on the mass of the complex Fab fragment of the present invention at about 0.001 mg / kg to about 100 mg / kg.

[0837] The pharmaceutical composition comprising the complex of the present invention can be used for the treatment of cancer. Cancers that can be expected to be treated using the pharmaceutical composition comprising the complex of the present invention are cancers that express human CEACAM5, and examples thereof include colorectal cancer, breast cancer, lung cancer, thyroid cancer, and cancers metastasized therefrom. Alternatively, cancers that can be expected to be treated using the pharmaceutical composition comprising the complex of the present invention are cancers that express human MUC1, and examples thereof include breast cancer, lung cancer, colorectal cancer, bladder cancer, skin cancer, thyroid cancer, gastric cancer, pancreatic cancer, kidney cancer, ovarian cancer, or cervical cancer. In a certain embodiment, the cancer is breast cancer or bladder cancer.

[0838] The present invention includes a pharmaceutical composition for treating colorectal cancer or cancer metastasized from colorectal cancer, comprising the complex of the present invention. In addition, the present invention includes a method for treating colorectal cancer or cancer metastasized from colorectal cancer, the method comprising the step of administering a therapeutically effective amount of the complex of the present invention. In addition, the present invention includes a method for inducing cell death of cancer cells of colorectal cancer or cancer metastasized from colorectal cancer, comprising the step of administering a therapeutically effective amount of the complex of the present invention.

[0839] The pharmaceutical composition for treating cancer can also be used for the diagnosis of cancer. For example, the pharmaceutical composition for treating colorectal cancer or cancer metastasized from colorectal cancer can also be used for the diagnosis of the cancer.

[0840] In addition, the present invention includes the complex of the present invention for treating colorectal cancer or cancer metastasized from colorectal cancer. Further, the present invention includes the use of the complex of the present invention in the manufacture of a pharmaceutical composition for treating colorectal cancer or cancer metastasized from colorectal cancer.

[0841] In another embodiment, the present invention also relates to the use of an anti-human CEACAM5 antibody Fab fragment in the manufacture of a pharmaceutical composition comprising the complex of the present invention.

[0842] The above-mentioned embodiments or modes may also be performed by using a biomolecule instead of the anti-human CEACAM5 antibody Fab fragment. The complex of the present invention may be provided in the form of a diagnostic composition or a pharmaceutical composition for a disease associated with the biomolecule.

[0843] The present invention has been described as a whole, but for further understanding, reference is made to specific examples. However, these examples are intended to illustrate and not to limit the present invention.

[0844] Example

[0845] In the following examples and tables described below, the following abbreviations may be used.

[0846] Gd / DOTA: three-arm DOTA labeled with Gd, Gd / four-arm DOTA: four-arm DOTA labeled with Gd, MS: mass spectrometry, ESI+: m / z value (ionization method ESI, [M+H]+ unless otherwise specified), ESI-: m / z value (ionization method ESI, [MH]- unless otherwise specified), APCI / ESI+: m / z value (ionization method APCI / ESI, APCI / ESI means simultaneous measurement of APCI and ESI. [M+H]+ unless otherwise specified), APCI / ESI-: m / z value (ionization method APCI / ESI, [M+H]- unless otherwise specified), Ex-No: complex number, SNo: manufacturing example number, Str: chemical structure, Me: methyl, Et: ethyl, tBu: tert-butyl, 1,3-Ph: 1,3-phenylene, 1,4-Ph: 1,4-phenylene, diph-Ala: diphenylalanine, and naph-Ala: 3-(2-naphthyl)alanine.

[0847] (Example 1: Preparation of anti-human CEACAM5 antibody Fab fragment)

[0848] After humanizing T84.66, a mouse-derived anti-human CEACAM5 antibody, using the method described in the reference (Protein Eng. Des. Sel.; 2004; 17: 481-489), a molecular model of a humanized antibody constructed according to the reference (Proteins: Structure, Function, and Bioinformatics; 2014; 82: 1624-1635) was used to design an antibody having a variable region that is expected not to reduce affinity even when bound to a tag portion.

[0849] A gene encoding a signal sequence (Protein Engineering; 1987; 1: 499-505) was linked to the 5' side of the heavy chain variable region gene of the above-mentioned antibody, and a Fab region gene of human Igγ1 (composed of a base sequence of base numbers 364 to 678 of SEQ ID NO: 1) was linked to the 3' side, and the heavy chain fragment gene was inserted into the GS vector pEE6.4 (Lonza). In addition, a gene encoding a signal sequence was linked to the 5' side of the light chain variable region gene of the antibody, and a constant region gene of human Igκ (composed of a base sequence of base numbers 337 to 657 of SEQ ID NO: 3) was linked to the 3' side, and the light chain gene was inserted into the GS vector pEE12.4 (Lonza). The pEE vector inserted with the genes of the heavy chain fragment and light chain of the antibody was restriction enzyme cut with NotI and PvuI, and ligated using the ligation kit TAKARA ligation kit Ver2.1 (Takara) to construct a GS vector inserted with both the heavy chain fragment and light chain genes.

[0850] Using the above-mentioned GS vectors with heavy chain fragments and light chain genes inserted, antibodies were expressed using transient expression and stable expression. For transient expression, Expi293F cells (Thermo Fisher Scientific) cultured to about 3 million / mL in Expi293 expression medium (Thermo Fisher Scintific) were transfected with the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific) and cultured for 5 to 7 days. The culture supernatant was purified using KappaSelect (General Electric Healthcare Japan) to obtain Fab fragments. For stable expression, the GS vectors with heavy chain fragments and light chain genes inserted, which were formed into straight chains using PvuI, were transfected into CHOK1SV cells (Lonza) by electroporation using Gene Pulser (Bio-Rad). Methionine sulfoximine was added the day after transfection and cultured for 5 to 7 days. The cells were inoculated into a semisolid medium containing methylcellulose, and after colonies were formed, ClonePix FL (Molecular Devices) was used to obtain cells with high expression of Fab fragments. The culture supernatant of the cells was purified using Capto L (GE Healthcare Japan), Q Sepharose Fast Flow (GE Healthcare Japan), and BioPro S75 (YMC) to obtain Fab fragments.

[0851] The Fab fragments of the anti-human CEACAM5 antibody (called PB009-01 or Fab 2 ) is shown in sequence number 1, the amino acid sequence encoded thereby is shown in sequence number 2, the base sequence encoding the light chain of PB009-01 is shown in sequence number 3, and the amino acid sequence encoded thereby is shown in sequence number 4. The heavy chain variable region of PB009-01 is composed of the amino acid sequence of amino acid numbers 1 to 121 of sequence number 2, and the CDR1, CDR2, and CDR3 of the heavy chain are composed of the amino acid sequences of amino acid numbers 31 to 35, 50 to 66, and 99 to 110 of sequence number 2, respectively. The light chain variable region of PB009-01 is composed of the amino acid sequence of amino acid numbers 1 to 112 of sequence number 4, and the CDR1, CDR2, and CDR3 of the light chain are composed of the amino acid sequences of amino acid numbers 24 to 38, 54 to 60, and 93 to 101 of sequence number 4, respectively.

[0852] It should be noted that the variable region and CDR sequences are determined according to the Kabat numbering (Kabat et al., 1991, Sequences of Proteins of Protein Sequences of Immunological Interest, 5th edition, U.S. Public Health Service, National Institutes of Health, Bethesda).

[0853] (Example 2: Preparation of anti-human CEACAM5 antibody Fab fragment complex)

[0854] This example discloses a manufacturing example of a complex. In each manufacturing example in this example, "Example 2" is used to record the "complex number" with a hyphen. For example, "Example 2-11" indicates the manufacturing example of complex 11 in this example. In addition, Fab in this example 2 The anti-human CEACAM5 antibody Fab fragment (PB009-01 / Fab 2 ), "p-Fab" means Fab 2 The p amino groups thereof are bound to the p labeling parts described in [] or () to form a complex. It should be noted that the Fab of each complex confirmed by MS analysis is described in the following examples. 2 The number of the labeled parts (Y-S1-X) bound, but this result does not mean that the complexes with other binding numbers are not included. It is easy to understand that there may be complexes with binding numbers whose existence cannot be confirmed due to the accuracy of the MS analysis equipment. In addition, in the structural formula of the present embodiment, the structural formula of DOTA bound to Gd schematically represents DOTA labeled with Gd.

[0855] (Example 2-11. ([Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Asp-Gly-Lys*-Z2(#N)]p-Fab 2 )

[0856]

[0857] (i)O 4 -tert-butyl-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-α-aspartylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0858] Under ice cooling, N-(tert-butyloxycarbonyl)glycyl-N 6-[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester (1.00 g) in dichloromethane (6 mL) was added trifluoroacetic acid (hereinafter referred to as TFA) (3 mL) and stirred at the same temperature for 2 hours. After concentration under reduced pressure, saturated sodium bicarbonate aqueous solution was added and extracted twice with dichloromethane. The combined organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and then concentrated. N-[(9H-fluorene-9-ylmethoxy)carbonyl]-L-aspartic acid hydrogen O was added to the residue. 4 -tert-butyl ester (920 mg), dichloromethane (10 mL), diisopropylethylamine (hereinafter referred to as DIPEA) (2 mL), hexafluorophosphate (1-) 1-(dimethylamino)-N,N-dimethyl-1-[(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)oxy]methanimine (hereinafter referred to as HATU) (1.2 g), stirred at room temperature for 16 hours. Saturated sodium bicarbonate aqueous solution was added and extracted twice with dichloromethane. The combined organic layer was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and then concentrated to obtain the title compound (2.86 g). MS (ESI+): 809.5 [M+Na] +

[0859] (ii)O 4 -tert-butyl-L-α-aspartylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0860] To O 4 -tert-butyl-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-α-aspartylglycyl-N 6 Morpholine (6 mL) was added to a solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (2.86 g) in tetrahydrofuran (hereinafter referred to as THF) (10 mL), and the mixture was stirred at room temperature for 1 hour. After cooling in an ice bath, the generated solid was filtered out and the residue was washed with methanol. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→4% methanol / chloroform) to obtain the title compound (1.04 g). MS (ESI+): 565.5

[0861] (iii) N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-O 4 -tert-butyl-L-α-aspartylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0862] O 4 -tert-butyl-L-α-aspartylglycyl-N 6A mixture of tert-butyl-[(benzyloxy)carbonyl]-L-lysine (570 mg), dichloromethane (6 mL), 3-{[(tert-butyloxycarbonyl)amino]methyl}benzoic acid (305 mg), DIPEA (520 μL), and HATU (575 mg) was stirred at room temperature for 43 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→80% ethyl acetate / hexane) to obtain the title compound (712 mg). MS (ESI+): 798.6

[0863] (iv) N-[3-(aminomethyl)benzoyl]-O 4 -tert-butyl-L-α-aspartylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0864] Under ice cooling, N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-O 4 -tert-butyl-L-α-aspartylglycyl-N 6 TFA (2 mL) was added to a solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (712 mg) in dichloromethane (2 mL), and the mixture was stirred at the same temperature for 7 hours. Triethylamine and amino silica gel were added, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (amino silica gel, solvent gradient; 0→2% methanol / chloroform) to obtain the title compound (495 mg). MS (ESI+): 698.4

[0865] (v)O 4 -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0866] N-[3-(aminomethyl)benzoyl]-O 4 -tert-butyl-L-α-aspartylglycyl-N 6A mixture of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (495 mg), N,N-dimethylformamide (hereinafter referred to as DMF) (5 mL), [4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetic acid (hereinafter referred to as DOTA-tris(t-Bu)ester) (446 mg), DIPEA (400 μL), and HATU (404 mg) was stirred at room temperature for 66 hours. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (solvent gradient; 0→20% methanol / chloroform) to obtain the title compound (387 mg). MS (ESI+): 1275.5 [M+Na]+

[0867] (vi)O 4 Synthesis of tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-L-lysine tert-butyl ester

[0868] O 4 -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-N 6 A mixture of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (387 mg), 10% palladium-supported carbon (50% water content, 65 mg), and ethanol (4 mL) was stirred at room temperature for 5 hours under a hydrogen atmosphere (1 atm). The mixture was filtered and concentrated using diatomaceous earth. 10% palladium-supported carbon (50% water content, 650 mg) and ethanol (8 mL) were added to the residue, and the mixture was stirred at room temperature for 20 hours under a hydrogen atmosphere (1 atm). The mixture was filtered using diatomaceous earth and concentrated to obtain the title compound (336 mg). MS (ESI+): 1140.5 [M+Na]+

[0869] (vii)O 4 Synthesis of tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0870] Under ice cooling, 4-tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-L-lysine tert-butyl ester (336 mg) and a saturated sodium bicarbonate aqueous solution (2.5 mL) were added with a solution of 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester (56 mg) and THF (5 mL), and the mixture was stirred at the same temperature for 2 hours. After adding 1M sodium hydroxide aqueous solution (60 μL) under ice cooling, the mixture was stirred at room temperature for 1 hour. After adding ethyl acetate and 10% citric acid aqueous solution, the organic layer was separated. The aqueous layer was extracted with 10% methanol / chloroform, and the collected organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→20% methanol / chloroform) to obtain the title compound (341 mg). MS (ESI+): 1198.5

[0871] (viii) Synthesis of N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0872] To O 4 -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (341 mg) in dichloromethane (2 mL) was added with TFA (2 mL), and the mixture was stirred at room temperature for 5 hours. After concentration under reduced pressure, the residue was purified by reverse phase column chromatography (solvent gradient; 0→20% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (99.6 mg). MS (ESI+): 918.3

[0873] (ix) [N-{3-[(2-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 Synthesis of 3-(3-)-Gadolinium-1,1-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-Norleucine

[0874] To a mixture of N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide}methyl)benzoyl]-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (20.0 mg), water (2 mL), and gadolinium chloride (6 mg) was added an aqueous sodium bicarbonate solution (0.1 M, 800 μL), and the mixture was stirred at room temperature for 10 minutes. An aqueous sodium bicarbonate solution (0.1 M, 60 μL) was added, and the mixture was stirred at room temperature for 1 hour. An aqueous ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) solution (0.5 M, 300 μL) was added, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was purified by reverse phase column chromatography (solvent gradient; 0→25% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (12.0 mg). MS (ESI-): 1071.4

[0875] (x) Synthesis of Composite No. 11

[0876] [N-{3-[(2-{4,7,10-tri[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κO)methyl]benzoyl}-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (3-)]gadolinium (1 mg) was dissolved in DMSO (40 μL). 40 μL of the solution was dispensed, 0.1 M boric acid buffer (40 μL) was added, and 0.1 M sodium carbonate aqueous solution was added to adjust the pH to 6.6.

[0877] To 4.45mg / mL Fab 2 The previously prepared solution was added to the boric acid buffer (160 μL) and kept warm at 30°C for 2 hours. After adding 0.05M EDTA aqueous solution (40 μL), it was kept warm at 30°C for 10 minutes. Purification was performed using a PD-10 column and recovery was performed using an Amicon Ultra-0.5mL centrifugal filter (Merck Millipore). The recovered solution was repeatedly washed 7 times with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.9 kDa. 2A mixture of a complex having one [Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Asp-Gly-Lys*-Z2(#N)] with a molecular weight of 1073, a complex having two, and a complex having three.

[0878] (Example 2-12. ([Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)-Gly-Phe-Lys*-Z2(#N)]p-Fab 2 )

[0879]

[0880] (i) N-(tert-butyloxycarbonyl)-L-phenylalanyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0881] To a mixture of N-(tert-butyloxycarbonyl)-L-phenylalanine (1.5 g) and dichloromethane (30 ml) were added N 6 -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester monohydrochloride (2.1 g), Et3N (2.4 mL) and HATU (2.5 g) were reacted at room temperature overnight.

[0882] The reaction mixture was concentrated and purified using a silica gel column (solvent gradient; 10→50% ethyl acetate / hexane) to give the title compound (3.16 g). MS (ESI+): 606.4 [M+Na]+

[0883] (ii) Synthesis of N-(tert-butyloxycarbonyl)-L-phenylalanyl-L-lysine tert-butyl ester

[0884] N-(tert-butyloxycarbonyl)-L-phenylalanyl-N 6 A mixture of tert-butyl -[(benzyloxy)carbonyl]-L-lysine (3150 mg) in ethanol (60 ml) and 10% palladium-supported carbon (50% water content, 1000 mg) was stirred at room temperature for 4.5 hours under a hydrogen atmosphere (1 atm). Since there was residual raw material, the system was purged with argon, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated. The residue was dissolved in methanol (60 ml), 10% palladium-supported carbon (50% water content, 1000 mg) was added, and the mixture was stirred at room temperature for 5 hours under a hydrogen atmosphere (1 atm). After confirming that the raw material disappeared, the system was purged with argon, the mixture was filtered through diatomaceous earth, and the filtrate was concentrated to give the title compound (2520 mg). MS (ESI+): 450.4

[0885] (iii) Synthesis of N-(tert-butyloxycarbonyl)-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0886] Under ice cooling, to a suspension of N-(tert-butoxycarbonyl)-L-phenylalanyl-L-lysine tert-butyl ester (825 mg) in saturated sodium bicarbonate aqueous solution (6 ml) was added a solution of 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylic acid methyl ester (340 mg) in THF (12 ml). After stirring at the same temperature for 2 hours, 1M sodium hydroxide aqueous solution (0.36 ml) was added and stirred at room temperature for 1 hour. The mixture was diluted with ethyl acetate and water and then made acidic with 10% citric acid aqueous solution. The organic layer was separated, washed with saturated sodium bicarbonate aqueous solution and saturated brine, dried over magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column (solvent gradient; 0→8% methanol / chloroform) to obtain the title compound (743 mg). MS (ESI+): 552.4 [M+Na] +

[0887] (iv) Synthesis of L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetate) salt

[0888] Under ice cooling, TFA (2 ml) was added dropwise to a solution of N-(tert-butyloxycarbonyl)-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (325 mg) in dichloromethane (4 ml). The mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain a crude product of the title compound (418 mg). MS (ESI+): 430.4

[0889] (v) Synthesis of N-(tert-butyloxycarbonyl)glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0890] To a mixture of L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt (415 mg) in dichloromethane (10 ml) were added N-(tert-butyloxycarbonyl)glycine (118 mg), triethylamine (hereinafter referred to as TEA) (0.26 ml), and HATU (256 mg) in sequence, and the mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and purified using a silica gel column (solvent gradient; 0→30% acetone / chloroform) to obtain the title compound (206 mg). MS (ESI+): 609.4 [M+Na) +

[0891] (vi) Synthesis of glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt

[0892] Using N-(tert-butyloxycarbonyl)glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (205 mg), the same procedure as in Example 2-12 (iv) was performed to obtain a crude product of the title compound (223 mg). MS (ESI+): 487.4

[0893] (vii) Synthesis of Glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine Mono(trifluoroacetic acid) Salt

[0894] To a mixture of glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt (9 mg) in dichloromethane (1 ml) was added TFA (1 ml) and stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain a crude product of the title compound (8 mg). MS (ESI+): 431.3

[0895] (viii) Synthesis of N-[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane-2-yl]methyl}phenyl)thiocarbamoyl]glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0896] To a mixture of glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetic acid) salt (8 mg) and 2,2',2",2"'-{2-[(4-isothiocyanatophenyl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl}tetraacetic acid (abbreviated as p-SCN-Bn-DOTA) (8 mg) in DMF (1 ml) was added DIPEA (35 μL) and stirred at room temperature for 1.5 hours. The mixture was diluted with 1% TFA aqueous solution (about 5 ml) and purified by reverse phase column chromatography (solvent gradient 5-50% acetonitrile / 0.1% TFA aqueous solution) to give the title compound (13 mg). MS (ESI+): 982.3

[0897] (ix) hydrogen = [N-{[4-({1,4,7,10-tetra[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-2-yl-κ 4 N1 ,N 4 ,N 7 ,N 10 Synthesis of 1-(4-)-[(4-methyl)phenyl]thiocarbamoyl]glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine]gadotinate

[0898] N-[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane-2-yl]methyl}phenyl)thiocarbamoyl]glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (12 mg) was diluted with water (5 ml) and gadolinium chloride (4 mg) was added. The pH of the mixture was adjusted to 5-6 with 0.1 M sodium bicarbonate and stirred at room temperature for 1 hour. 0.5 M EDTA aqueous solution (80 μL) was added to the mixture and stirred at room temperature for 5 minutes. After adding TFA (10 μL), the title compound (5 mg) was purified by reverse phase column chromatography (solvent gradient 5-50% acetonitrile / 0.1% TFA aqueous solution). MS (ESI+): 1137.0

[0899] (x) Synthesis of Composite No. 12

[0900] Using hydrogen = [N-{[4-({1,4,7,10-tetrakis[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-2-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 The same method as step (x) of Example 2-11 was used to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex with one [Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)-Gly-Phe-Lys*-Z2(#N)] of molecular weight 1136, a complex with two, and a complex with three.

[0901] (Example 2-13. ([Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#N)]p-Fab 2 )

[0902]

[0903] (i) N-(tert-butyloxycarbonyl)-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0904] Under ice cooling, N-(tert-butyloxycarbonyl)glycyl-N 6 -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester (1.00g) was dissolved in dichloromethane (6mL) and TFA (3mL) was added, followed by stirring at the same temperature for 2 hours. After concentration under reduced pressure, a saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted twice with dichloromethane. The combined organic layers were washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated. N-(tert-butoxycarbonyl)-L-methionine (556mg), dichloromethane (10mL), DIPEA (2mL), and HATU (1.16g) were added to the residue, and the mixture was stirred at room temperature for 1 hour. After the reaction solution was concentrated, a saturated aqueous sodium bicarbonate solution was added to the residue, and the mixture was extracted twice with dichloromethane. The combined organic layers were washed with a 1M aqueous sodium hydroxide solution and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated to obtain the title compound (1.70g). MS (ESI+): 625.5

[0905] (ii) L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0906] Under ice cooling, N-(tert-butyloxycarbonyl)-L-methionylglycyl-N 6 TFA (4 mL) was added to a solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (1.70 g) in dichloromethane (10 mL), and the mixture was stirred at the same temperature for 7 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (amino silica gel, solvent gradient; 0→4% methanol / chloroform) to obtain the title compound (663 mg). MS (ESI+): 525.4

[0907] (iii) N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0908] L-methionylglycyl-N 6A mixture of tert-butyl-[(benzyloxy)carbonyl]-L-lysine (663 mg), dichloromethane (6 mL), 3-{[(tert-butyloxycarbonyl)amino]methyl}benzoic acid (480 mg), DIPEA (700 μL), and HATU (960 mg) was stirred at room temperature for 39 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→100% ethyl acetate / hexane) to obtain the title compound (935 mg). MS (ESI+): 758.5

[0909] (iv) N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0910] Under ice cooling, N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-N 6 TFA (2 mL) was added to a solution of -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester (935 mg) in dichloromethane (2 mL), and the mixture was stirred at the same temperature for 1 hour. Et3N and amino silica gel were added, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (amino silica gel, solvent gradient; 0→10% methanol / chloroform) to obtain the title compound (260 mg). MS (ESI+): 658.5

[0911] (v) N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[0912] Use N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-N 6 -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester (260 mg) was subjected to the same procedure as in step (v) of Example 2-11 to give the title compound (353 mg). MS (ESI-): 1210.4

[0913] (vi) Synthesis of N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-L-lysine tert-butyl ester

[0914] N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-N 6 -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester (353 mg) was subjected to the same procedure as step (vi) of Example 2-11 to obtain the title compound (245 mg). MS (ESI+): 1078.8

[0915] (vii) Synthesis of N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0916] Using N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-L-lysine tert-butyl ester (245 mg), the same procedure as step (vii) of Example 2-11 was performed to obtain the title compound (139 mg). MS (ESI+): 1158.8

[0917] (viii) Synthesis of N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0918] Using N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (341 mg), the same method as step (viii) of Example 2-11 was used to obtain the title compound (38.2 mg). MS (ESI+): 934.4

[0919] (ix) [N-{3-[(2-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10Synthesis of 3-(3-)-Gadolinium-1,1-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-Norleucine}-acetamido-κO)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0920] Using N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (28 mg), the same method as step (ix) of Example 2-11 was used to obtain the title compound (13.8 mg). MS (ESI+): 1089.2

[0921] (x) Synthesis of Composite No. 13

[0922] [N-{3-[(2-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κO)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (3-)]gadolinium, and the same method as step (x) of Example 2-11 was used to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex having one [Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#N)] with a molecular weight of 1089, a complex having two, and a complex having three.

[0923] (Example 2-15. ([Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Gly-Lys*-Z2(#N)]p-Fab 2 )

[0924]

[0925] (i) Synthesis of glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt

[0926] Under ice cooling, TFA (4 mL) was added to a solution of N-(tert-butyloxycarbonyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (400 mg) in dichloromethane (4 mL), and the mixture was stirred for 1 hour under ice cooling. The reaction mixture was concentrated under reduced pressure to obtain the title compound (642 mg). MS (ESI+): 340.4

[0927] (ii) Synthesis of N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0928] Under ice cooling, 3-{[(tert-butoxycarbonyl)amino]methyl}benzoic acid (228 mg), DIPEA (1.5 mL) and HATU (345 mg) were added to a mixture of glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt (642 mg) and dichloromethane (6 mL), and the mixture was stirred at room temperature for 2 hours. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (solvent gradient; 0→4% methanol / chloroform) to obtain the title compound (489 mg). MS (ESI+): 573.4

[0929] (iii) Synthesis of N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0930] Under ice cooling, TFA (2 mL) was added to a mixture of N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (489 mg), anisole (280 μL), and dichloromethane (5 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and dichloromethane (7 mL) was added. 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-oic acid (289 mg), DIPEA (1.5 mL), and HATU (487 mg) were added under ice cooling, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→1% methanol / chloroform) to obtain the title compound (427 mg). MS (ESI+): 762.5

[0931] (iv) Synthesis of N-(3-{3,15-dioxo-16-[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]-5,8,11-trioxa-2,14-diazahexadec-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0932] Under ice cooling, TFA (1.3 mL) was added to a mixture of N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (427 mg), anisole (200 μL), and dichloromethane (4 mL), and the mixture was stirred at the same temperature for 1 hour. After the reaction mixture was concentrated under reduced pressure, DMF (6 mL) was added, and DOTA-tris(t-Bu)ester (353 mg), DIPEA (0.96 mL), and HATU (320 mg) were added under ice cooling, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→10% methanol / chloroform) to obtain the title compound (863 mg). MS (ESI+): 1238.9 [M+Na]+

[0933] (v) Synthesis of N-(3-{3,15-dioxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]-5,8,11-trioxa-2,14-diazahexadecane-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0934] The title compound (40.0 mg) was obtained in the same manner as in step (viii) of Example 2-11 using N-(3-{3,15-dioxo-16-[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]-5,8,11-trioxa-2,14-diazahexadec-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (863 mg). MS (ESI+): 992.5

[0935] (vi) [N-{3-[3-oxo-15-(oxo-κO)-16-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 Synthesis of 5-(3-)-[(-5,8,11-trioxa-2,14-diazahexadecane-1-yl)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine]gadolinium

[0936] Using N-(3-{3,15-dioxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]-5,8,11-trioxa-2,14-diazahexadecane-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (40 mg), the same method as step (ix) of Example 2-11 was used to obtain the title compound (15.9 mg). MS (ESI-): 1145.0

[0937] (vii) Synthesis of Compound No. 15

[0938] [N-{3-[3-oxo-15-(oxo-κO)-16-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}-5,8,11-trioxa-2,14-diazahexadecane-1-yl]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (3-)]gadolinium, and the same method as step (x) of Example 2-11 was used to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex containing one [Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Gly-Lys*-Z2(#N)] with a molecular weight of 1147, a complex containing two, and a complex containing three.

[0939] (Example 2-24. ([Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)]p-Fab 2 )

[0940] Synthesis of Compound No.24 (coupling via iminothiolane)

[0941] To 4.45mg / mL Fab 2 2 mg / mL 2-iminothiolane (hereinafter referred to as 2-IT) in 0.1 M boric acid buffer (5 μL) was added to 160 μL boric acid buffer and incubated at 37°C for 40 minutes. Excess 2-IT was washed three times with phosphate-buffered saline using an Amicon Ultra-0.5 mL centrifugal filter and finally concentrated.

[0942] [N-{3-[(2-{4,7,10-tri[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κO)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (3-)]gadolinium (1 mg) was dissolved in DMSO (40 μL). 0.1 M boric acid buffer (40 μL) was added to the solution, and 0.1 M sodium carbonate aqueous solution was added to adjust the pH to 6.0.

[0943] The prepared linker solution was added to the obtained filtrate containing the antibody and incubated at 30°C for 2 hours. After adding phosphate-buffered saline (pH 6.0) containing EDTA, it was incubated at 30°C for 10 minutes. Purification was performed using a PD-10 column and recovery was performed using an Amicon Ultra-0.5mL centrifugal filter. The recovered solution was washed 7 times with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex containing one [Gd / DOTA-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)] with a molecular weight of 1190 and a complex containing two.

[0944] (Example 2-29. ([Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#N)]p-Fab 2 )

[0945]

[0946] (i) Synthesis of methyl 3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoate

[0947] To a solution of 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-oic acid (396 mg) in dichloromethane (10 mL) was added methyl 3-(aminomethyl)benzoate monohydrochloride (286 mg), HATU (590 mg), and Et3N (900 μL), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (solvent gradient; 2→6% methanol / chloroform) to obtain the title compound (598 mg). MS (ESI+): 455.2

[0948] (ii) Synthesis of 3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoic acid

[0949] To a solution of methyl 3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoate (597 mg) in THF (15 mL) was added 1 M aqueous sodium hydroxide solution (4 mL) and water (5 mL), and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with water, 10% citric acid was added, and then extracted with ethyl acetate, and the collected organic layer was washed with saturated brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound (662 mg). MS (ESI+): 441.1

[0950] (iii) Synthesis of N-(tert-butyloxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0951] Under ice cooling, TFA (3 mL) was added to a mixture of N-(tert-butoxycarbonyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (1.63 g) and dichloromethane (5 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. DIPEA (5 mL) was added to a mixture of N-(tert-butoxycarbonyl)-L-methionine (1.11 g), dichloromethane (8 mL), and HATU (2.12 g), and the mixture was stirred at room temperature for 5 minutes. A dichloromethane (4 mL) solution of the residue obtained in the previous reaction was added to the reaction mixture, and the mixture was stirred at room temperature for 20 hours. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (solvent gradient; 0→5% methanol / chloroform) to obtain the title compound (1.63 g). MS (APCI / ESI+): 571.3

[0952] (iv) Synthesis of N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0953] Under ice cooling, TFA (2 mL) was added to a mixture of N-(tert-butyloxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (150 mg) and dichloromethane (2 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. DIPEA (360 μL) was added to a mixture of 3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoic acid (120 mg), dichloromethane (3 mL), and HATU (150 mg), and the mixture was stirred at room temperature for 5 minutes. A dichloromethane (2 mL) solution of the residue obtained in the previous reaction was added to the reaction mixture, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→5% methanol / chloroform) to obtain the title compound (113 mg). MS (ESI+): 893.4

[0954] (v) Synthesis of N-(3-{3,15-dioxo-16-[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]-5,8,11-trioxa-2,14-diazahexadec-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester tetra(trifluoroacetate)

[0955] Under ice cooling, TFA (2 mL) was added to a mixture of N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecane-1-yl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (113 mg) and dichloromethane (2 mL), and the mixture was stirred at the same temperature for 1 hour, and then concentrated under reduced pressure to obtain a crude product. DIPEA (0.22 mL) was added to a mixture of DOTA-tris(t-Bu)ester (80 mg), HATU (80 mg) and dimethylacetamide (hereinafter referred to as DMAc) (1 mL) at room temperature, and after stirring for 10 minutes, a mixture of the crude product obtained above and DMAc (1 mL) was added, and stirred at the same temperature for 2 hours. The reaction mixture was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (178 mg). MS (ESI-): 1345.8

[0956] (vi) Synthesis of N-(3-{3,15-dioxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]-5,8,11-trioxa-2,14-diazahexadecane-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0957] The title compound (60.0 mg) was obtained in the same manner as in step (viii) of Example 2-11 using N-(3-{3,15-dioxo-16-[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]-5,8,11-trioxa-2,14-diazahexadec-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester tetra(trifluoroacetate) salt (178 mg). MS (APCI / ESI+): 1123.4

[0958] (vii) [N-{3-[3-oxo-15-(oxo-κO)-16-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 Synthesis of 3-(3-)-Gadolinium-5,8,11-Trioxa-2,14-diazahexadecane-1-yl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0959] The title compound (10.3 mg) was obtained in the same manner as in step (ix) of Example 2-11 using N-(3-{3,15-dioxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]-5,8,11-trioxa-2,14-diazahexadecane-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (40 mg). MS (ESI+): 1278.3

[0960] (viii) Synthesis of Compound No. 29

[0961] [N-{3-[3-oxo-15-(oxo-κO)-16-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 The same method as step (x) of Example 2-11 was used to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A complex containing one [Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#N)] with a molecular weight of 1278 and a mixture of two complexes.

[0962] (Example 2-33. ([Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys*-Z2(#N)]p-Fab 2 )

[0963]

[0964] (i) Synthesis of N-[2-(tert-butyloxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0965] Under ice cooling, TFA (2 mL) was added to a mixture of N-(tert-butoxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (150 mg) and dichloromethane (2 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. A dichloromethane (2 mL) solution of the residue obtained in the previous reaction was added to a mixture of 2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carboxylic acid (88 mg), dichloromethane (2 mL), HATU (150 mg), and DIPEA (360 μL), and the mixture was stirred at room temperature for 1 hour. After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (solvent gradient; 0→6% methanol / chloroform) to obtain the title compound (231 mg). MS (APCI / ESI+): 752.2 [M+Na]+

[0966] (ii) Synthesis of N-(2-{[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester tetra(trifluoroacetate)

[0967] Using N-[2-(tert-butyloxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (231 mg), the same procedure as step (v) of Example 2-29 was performed to obtain the title compound (222 mg). MS (ESI-): 1182.7

[0968] (iii) Synthesis of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0969] The title compound (53 mg) was obtained in the same manner as in step (viii) of Example 2-11 using N-(2-{[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester tetra(trifluoroacetate) salt (222 mg). MS (APCI / ESI+): 960.2

[0970] (iv) {N-[2-({4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 Synthesis of [(3-)-Gadolinium]-[(((3-)}-acetyl-κO)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine]

[0971] Using N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (22 mg), the same procedure as step (ix) of Example 2-11 was performed to obtain the title compound (7.0 mg). MS (ESI+): 1115.3

[0972] (v) Synthesis of Compound No. 33

[0973] The compound (iv) was used in the same manner as step (x) of Example 2-11 to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2A mixture of a complex in which one [Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(═O)-Met-Gly-Lys*-Z2(#N)] having a molecular weight of 1115 is bound and a complex in which two are bound.

[0974] (Example 2-35. ([Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)]p-Fab 2 )

[0975] Synthesis of Compound No.35

[0976] [N-{3-[3-oxo-15-(oxo-κO)-16-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 The steps of Example 2-24 were carried out in the same manner to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 It is bound to a complex of [Gd / DOTA-NH-(CH2CH2O)3-CH2-C(=O)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)] with a molecular weight of 1380.

[0977] (Example 2-40. ([Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)]p-Fab 2 )

[0978]

[0979] (i) Synthesis of N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[0980] Using N-(tert-butyloxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (690 mg), the same method as step (i) of Example 2-33 was used to obtain the title compound (710 mg). MS (ESI+): 726.5 [M+Na]+

[0981] (ii) Synthesis of N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetate) salt

[0982] Using N-(3-{[(tert-butyloxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (41 mg), the same method as in Example 2-12 (iv) was used to obtain a crude product of the title compound (42 mg). MS (ESI+): 604.3

[0983] (iii) Synthesis of N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetic acid) salt

[0984] To a mixture of N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester mono(trifluoroacetic acid) salt (41 mg) in dichloromethane (4 ml) was added TFA (2 ml) and stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and subjected to toluene azeotropy to obtain a crude product (43 mg). 16 mg of the product was purified by reverse phase column chromatography (solvent gradient; 5→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (11 mg). MS (ESI+): 548.2

[0985] (iv) Synthesis of N-[3-({[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane-2-yl]methyl}phenyl)thiocarbamoyl]amino}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0986] The title compound (17 mg) was obtained by the same method as step (viii) of Example 2-12 using N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetic acid) salt (11 mg). MS (ESI+): 1099.5

[0987] (v) hydrogen = [N-{3-[({[4-({1,4,7,10-tetra[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-2-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 Synthesis of 1-(4-)-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine)-carbamoyl-(4-)-(1-)-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[0988] The title compound (6 mg) was obtained in the same manner as in step (ix) of Example 2-12 using N-[3-({[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane-2-yl]methyl}phenyl)carbamothioyl]amino}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (16 mg). MS (ESI+): 1254.6

[0989] (vi) Synthesis of Composite No. 40

[0990] Using hydrogen = [N-{3-[({[4-({1,4,7,10-tetra[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-2-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10 The same steps as in Example 2-24 were performed to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2A complex containing one [Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)-NH-CH2-(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z2(#S)] with a molecular weight of 1355 and a mixture of complexes containing two.

[0991] (Example 2-47: ([Gd / DOTA]p-Fab 2 )

[0992]

[0993] Under ice cooling, 1M sodium hydroxide aqueous solution (80 μL) was added to a mixed solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (16 mg) and water (810 μL) to adjust the pH to 6. Under ice cooling, 1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate sodium (2.3 mg) dissolved in water (117 μL) was added to the obtained solution (239 μL). Thereafter, 3-{[(ethylimino)methylene]amino}-N,N-dimethylpropane-1-amine monohydrochloride (hereinafter referred to as EDCHCl) aqueous solution (8.3 μL, 25 mg / mL) was added, and the mixture was stirred for 30 minutes under ice cooling to prepare an N-hydroxysulfonylsuccinimide DOTA solution. Fab 2 Prior to the addition, a 0.2 M sodium dihydrogen phosphate aqueous solution (pH 9) (40 μL) was added to adjust the pH to 7.

[0994] (i) To 17.8 mg / mL Fab 2 The prepared N-hydroxysulfonylsuccinimidyl DOTA solution (200 μL) was added to a 0.1 M sodium dihydrogen phosphate aqueous solution (390 μL) (60 μL), and the mixture was incubated at 4°C for 23 hours. The excess linker was washed three times with 10 mM phosphate buffer using an Amicon Ultra-0.5 mL centrifugal filter, washed with 0.3 M ammonium acetate buffer, and finally concentrated, and then filtered with a membrane filter to obtain a complex.

[0995]

[0996] (ii) The 0.3 M ammonium acetate buffer containing the complex prepared in (i) was diluted with the same buffer and adjusted to pH 6.62 using 0.25 M acetate buffer. 2A 0.057M GdCl3 aqueous solution (35μL) was added to the solution and kept warm at 37°C for 0.5 hours. After the reaction, 0.05M EDTA (525μL) was added, and then purified using a PD-10 column and recovered using an Amicon Ultra-0.5mL centrifugal filter. The recovered solution was washed 5 times with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.9kDa. 2 A mixture of complexes with one or two [Gd / DOTA] molecules of molecular weight 542.

[0997] (Example 2-48: ([Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)]p-Fab 2 )

[0998]

[0999] (i) When DOTA as a chelating agent is combined with Fab 2 p-SCN-Bn-DOTA (Macrocyclics) was used for the binding. Fab prepared at 2.54 mg / mL in phosphate buffered saline (pH 7.4) and glycerol was added. 2 0.1M sodium carbonate solution (pH 9.0) was added to the solution to adjust the pH to 8.8-9.5. p-SCN-Bn-DOTA was added thereto and incubated at 37°C for 2 hours. After the reaction, the complex was recovered using an Amicon Ultra-0.5mL centrifugal filter and purified. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A complex with one or two [quadruple-DOTA-CH2-(1,4-Ph)-NH-C(=S)] molecules of molecular weight 553.

[1000]

[1001] (ii) Using the complex prepared in (i), the same process as step (ii) of Example 2-47 was performed to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A complex of [Gd / four-arm-DOTA-CH2-(1,4-Ph)-NH-C(=S)] with a molecular weight of 706 is bound.

[1002] (Example 2-60: ([DFO-C(=O)-CH2O-(1,3-Ph)-C(=O)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1(#S)] p -Fab 2 )

[1003]

[1004] (i) Synthesis of 3-[2-(Benzyloxy)-2-oxoethoxy]benzoic acid

[1005] To a mixture of (3-formylphenoxy)benzyl acetate (2.90 g), 2-methylpropane-2-ol (60 mL) and water (30 mL) was added 2-methylbutan-2-one (6 mL), sodium dihydrogen phosphate dihydrate (3.35 g) and sodium chlorite (3.64 g) at room temperature, and the mixture was stirred for 2 hours. Ethyl acetate and 1M hydrochloric acid (60 mL) were added to the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated to obtain the title compound (2.93 g). MS (ESI-): 285.1

[1006] (ii) Synthesis of N-[(Benzyloxy)carbonyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester

[1007] Dissolve N-[(Benzyloxy)carbonyl]glycyl-L-tyrosine tert-butyl ester (2.49 g) in acetone (50 mL), add ethyl bromoacetate (2.05 g) and potassium carbonate (2.41 g), and stir at room temperature for 4 hours. Filter out insoluble matter, concentrate the filtrate, and purify the obtained residue by silica gel column chromatography (ethyl acetate: hexane = 30: 70-60: 40) to obtain the title compound (2.99 g). MS (ESI+): 537.4 [M+Na]+

[1008] (iii) Synthesis of N-{3-[2-(benzyloxy)-2-oxoethoxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester

[1009] N-[(Benzyloxy)carbonyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester (2.75 g) was dissolved in ethanol (55 mL), and 10% palladium-supported carbon (50% water content, 550 mg) was added under argon atmosphere, and stirred overnight at room temperature under hydrogen atmosphere (1 atm). After replacing the system with argon, the insoluble matter was filtered off, and the filtrate was concentrated. The residue was dissolved in dichloromethane (55 mL), and 3-[2-(benzyloxy)-2-oxoethoxy]benzoic acid (1.99 g), HATU (2.84 g), and Et3N (1.5 mL) were added, and stirred at room temperature for 1 hour. The reaction solution was concentrated and purified by silica gel column chromatography (ethyl acetate:hexane=30:70-70:30) to obtain the title compound (3.07 g). MS (ESI-): 647.4

[1010] (iv) Synthesis of N-[3-(carboxymethoxy)benzoyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester

[1011] Dissolve N-{3-[2-(benzyloxy)-2-oxoethoxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester (3282 mg) in THF (66 mL), add 10% palladium-supported carbon (50% water content, 328 mg) under argon atmosphere, and stir overnight at room temperature under hydrogen atmosphere. Filter the reaction solution with celite, and concentrate the filtrate to obtain the title compound (2.54 g). MS (ESI-): 557.4

[1012] (v) Synthesis of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester

[1013] To N 4 -{5-[Acetyl(hydroxy)amino]pentyl}-N 1 -(5-{4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutyramide}pentyl)-N 1-Hydroxysuccinamide monomethanesulfonate (DFO.MeSO3H) (500 mg), N-[3-(carboxymethoxy)benzoyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester (446 mg), EDC HCl (175 mg), 1H-benzotriazol-1-ol (hereinafter referred to as HOBt) (123 mg) were added with DMF (5 mL) and Et3N (0.16 mL), and the mixture was stirred at room temperature overnight. 0.1% TFA aqueous solution (1 ml) and TFA (0.03 mL) were added to the reaction solution, and the title compound (548 mg) was obtained by purification by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-0:100). MS (ESI-): 1099.7

[1014] (vi) Synthesis of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(carboxymethyl)-L-tyrosine tert-butyl ester

[1015] Methanol (5 mL) and DMF (5 mL) were added to N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosine tert-butyl ester (500 mg), and the mixture was dissolved by slight heating. A 1M aqueous sodium hydroxide solution (600 μL) was added, and the mixture was stirred at room temperature overnight. A 1M aqueous sodium hydroxide solution (600 μL) was further added, and the mixture was stirred at room temperature overnight. TFA (90 μL) was added under ice cooling, and the methanol was distilled off under reduced pressure. The obtained solution was purified by reverse phase column chromatography (0.1% aqueous TFA solution: acetonitrile = 95:5-0:100) to obtain the title compound (315 mg). MS (ESI-): 1071.6

[1016] (vii) Synthesis of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)-L-tyrosine tert-butyl ester

[1017] To a mixture of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(carboxymethyl)-L-tyrosine tert-butyl ester (269 mg), EDC HCl (58 mg), and HOBt (41 mg) was added DMF (4 mL), and then 1-(2-aminoethyl)-1H-pyrrole-2,5-dione monohydrochloride (44 mg) and Et3N (35 μL), and the mixture was stirred at room temperature for 4 hours. 0.1% TFA aqueous solution (1 mL) was added to the reaction solution, and the mixture was purified by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-0:100) to obtain a mixture of the starting carboxylic acid and the title compound (155 mg, about 6:4). Dissolve it in DMSO (1 mL) and DMF (2 mL), add EDC HCl (22 mg) and HOBt (15 mg), stir at room temperature for 5 minutes, then add 1-(2-aminoethyl)-1H-pyrrole-2,5-dione monohydrochloride (15.5 mg) and Et3N (12 μL), and stir at room temperature for 1 hour. Add 0.1% TFA water (1 mL) to the reaction solution, and purify it by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-10:90) to obtain the title compound (118 mg). MS (ESI-): 1193.6

[1018] (viii) Synthesis of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)-L-tyrosine

[1019] N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)-L-tyrosine tert-butyl ester (118 mg) was dissolved in TFA (1.2 mL) and stirred at room temperature for 1.5 hours. The reaction solution was concentrated, and DMF (1.5 ml) and water (0.5 ml) were added, and purified by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-10:90) to obtain the title compound (82 mg). MS (ESI-): 1137.5

[1020] (ix) Synthesis of Composite No. 60

[1021] Fab prepared at 4.45 mg / mL in 0.1 M borate buffer 2 A 2-IT solution prepared with 0.1 M boric acid buffer was added to the solution and incubated at 37°C for 30 minutes. Excess 2-IT was washed with phosphate-buffered saline (pH 6.0) containing EDTA using an Amicon Ultra-0.5 mL centrifugal filter, and finally concentrated and filtered with a membrane filter. N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)-L-tyrosine dissolved in DMF was added to the obtained filtrate, diluted with 0.1 M boric acid buffer (pH 8.5), and incubated at room temperature for 2 hours. Excess reagents were washed with phosphate-buffered saline (pH 6.0) containing EDTA using an Amicon Ultra-0.5 mL centrifugal filter, and this was repeated three times. Finally, the mixture was concentrated and filtered using a membrane filter.

[1022] Next, a 10 mg / mL 2-iodoacetamide solution prepared with phosphate buffered saline (pH 6.0) was added to the obtained supernatant, and then the mixture was incubated at 37°C for 30 minutes. The excess iodoacetamide was washed three times with phosphate buffered saline using an Amicon Ultra-0.5 mL centrifugal filter, and finally concentrated and filtered with a membrane filter to separate the Fab-bound 2 The complex was purified. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex with a molecular weight of 1241 [DFO-C(=O)-CH2O-(1,3-Ph)-C(=O)-Gly-Tyr*-CH2-C(=O)-NH-(CH2)2-Z1(#S)].

[1023] (Example 2-61: ([DFO-C(=O)-(CH2CH2O)4-(1,3-Ph)-C(=O)-Gly-Lys*-Z2(#S)] p -Fab 2 )

[1024]

[1025] (i) Synthesis of N-(3-hydroxybenzoyl)glycine tert-butyl ester

[1026] Under ice cooling, HATU (3.3 g) and DIPEA (3 mL) were added to a mixture of 3-hydroxybenzoic acid (1.0 g) and glycine tert-butyl ester monohydrochloride (1.2 g) in DMF (10 mL), and the mixture was stirred at room temperature for 1 hour. Water and ethyl acetate were added to the mixture, and liquid extraction was performed twice. The organic layer was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous magnesium sulfate, filtered, and the filtrate was then concentrated under reduced pressure. Ethyl acetate was added to the obtained solid, and the insoluble matter was filtered out after stirring at room temperature, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (hexane / ethyl acetate = 95 / 5-50 / 50), and the target fraction was collected, concentrated, and dried under reduced pressure to obtain the title compound (1.23 g). MS (ESI+): 274.2 [M+Na] +

[1027] (ii) Synthesis of benzyl 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propionate

[1028] 4M hydrogen chloride / dioxane (10 mL) was added to tert-butyl 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate (3.0 g) under ice cooling, and the mixture was stirred at room temperature for 1 hour. After concentration, azeotropic drying was performed twice with toluene, and then methanol (20 mL) and 1M aqueous sodium hydroxide solution (13 mL) were added at room temperature, and stirred at the same temperature for 1.5 hours. After the mixture was concentrated, THF (10 mL), methanol (10 mL) and benzyl bromide (1.8 mL) were added, and stirred at room temperature for 3 days. The mixture was concentrated, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 95 / 5-0 / 100 → chloroform / methanol = 90 / 10) to obtain the title compound (2.19 g). MS (ESI+): 313.2

[1029] (iii) Synthesis of N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycine tert-butyl ester

[1030] N-(3-hydroxybenzoyl)glycine tert-butyl ester (915 mg), 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propionic acid benzyl ester (1.38 g), diethyl azodicarboxylate (40% toluene solution, about 2.2 M, 3.4 mL), THF (20 mL) were added, and triphenylphosphine (2.0 g) was added little by little at room temperature, and the mixture was stirred overnight at a bath temperature of 60°C. Magnesium chloride (1.5 g) and toluene (20 mL) were added, and the mixture was heated and stirred at a bath temperature of 60°C for 2 hours. After cooling naturally to room temperature, the precipitated solid was removed by filtration, and the solvent was distilled off. The residue was purified by silica gel column chromatography (silica gel; hexane / ethyl acetate = 95 / 5-20 / 80), and then purified again by silica gel column chromatography (amino silica gel; hexane / ethyl acetate = 95 / 5-50 / 50) to obtain the title compound (1.12 g). MS (ESI+): 546.4

[1031] (iv) Synthesis of N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycine

[1032] N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycine tert-butyl ester (1.11 g) was dissolved in 4M hydrogen chloride / dioxane (5 mL) at room temperature and stirred at the same temperature for 2 hours. The mixture was concentrated and dried under reduced pressure to give the title compound (1.12 g). MS (ESI+): 490.3

[1033] (v) N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[1034] Under ice cooling, N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycine (1300 mg), N 6 HATU (1.2 g) and DIPEA (1.4 mL) were added to a solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester monohydrochloride (1.0 g) in DMF (20 mL), and the mixture was stirred at room temperature for 2 hours. Water and ethyl acetate were added to the mixture for separation and extraction. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (amino silica gel; hexane / ethyl acetate = 90 / 10-0 / 100) to obtain the title compound (1.14 g). MS (ESI+): 808.5

[1035] (vi) Synthesis of N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-L-lysine tert-butyl ester

[1036] At room temperature, N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradec-14-yl)oxy]benzoyl}glycyl-N 6 10% palladium on carbon (50% water, 200 mg) was added to a mixture of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (1.12 g), Et3N (20 μL) and ethanol (20 mL), and stirred overnight at the same temperature under a hydrogen atmosphere. Set as an open system, stir at room temperature for more than 30 minutes, then filter the reaction solution with diatomaceous earth, and concentrate the filtrate to obtain the title compound (825 mg). MS (ESI+): 584.5

[1037] (vii) Synthesis of N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[1038] At room temperature, methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (150 mg) and Et3N (500 μL) were added to a mixture of tert-butyl N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-L-lysine (350 mg), THF (3 mL) and DMF (1 mL), and the mixture was stirred at a bath temperature of 60°C for 4 days. After the mixture was cooled to room temperature, TFA (300 μL) and water (500 μl) were added to neutralize it. Appropriate amounts of water and DMF were added to the mixture, and it was purified by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-0:100). The fractions of the target product were collected, concentrated, and dried under reduced pressure to obtain the title compound (250 mg). MS (ESI+): 664.5

[1039] (viii) Synthesis of N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19,24,30,35,41-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester

[1040] Under ice cooling, 4 -{5-[Acetyl(hydroxy)amino]pentyl}-N 1-(5-{4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutyramide}pentyl)-N 1 To a mixture of DMF (3 mL) and DMSO (1 mL) of 1-hydroxysuccinamide monomethanesulfonate (DFO.MeSO3H) (240 mg), N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (243 mg) was added EDC HCl (140 mg), HOBt (100 mg) and DIPEA (200 μL), and the mixture was stirred at room temperature for 3 hours. TFA (100 μL) and water (500 μL) were added, and the solution was purified by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-10:90) and freeze-dried to obtain the title compound (207 mg). MS (ESI+): 1228.5 [M+Na]+

[1041] (ix) Synthesis of N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19,24,30,35,41-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

[1042] At room temperature, TFA (1 mL) was added to N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19,24,30,35,41-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine tert-butyl ester (202 mg), and the mixture was stirred at the same temperature for 1 hour. The mixture was concentrated, DMF (4 ml) and water (500 μL) were added, and the mixture was purified by reverse phase column chromatography (0.1% TFA aqueous solution: acetonitrile = 95:5-10:90) and freeze-dried to obtain the title compound (137 mg). MS (ESI-): 1148.7

[1043] (x) Synthesis of Composite No. 61

[1044] The complex was obtained by using N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19,24,30,35,41-hexaazatricarboxan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine in the same manner as step (ix) of Example 2-60. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A complex containing one [DFO-C(=O)-(CH2CH2O)4-(1,3-Ph)-C(=O)-Gly-Lys*-Z2(#S)] with a molecular weight of 1252 and a mixture of complexes containing two.

[1045] (Example 2-18. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#N)]p-Fab 2 )

[1046]

[1047] (i) Synthesis of 4-nitrophenyl (4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamate

[1048] Under a nitrogen atmosphere, a solution of 4-nitrophenyl chloroformate (952 mg) in dichloromethane (10 ml) was ice-cooled, and a mixed solution of [(4-aminophenyl)methyl]carbamic acid tert-butyl ester (1000 mg) and pyridine (430 μL) in dichloromethane (10 mL) was added dropwise, and stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 10→100% ethyl acetate / hexane) to obtain a crude product. Ethyl acetate was added to the crude product, stirred, and the solid was crushed. The solid was filtered, washed with ethyl acetate, and dried under reduced pressure to obtain the title compound (861 mg). MS (ESI+): 410.3 [M+Na] +

[1049] (ii) L-methionyl-N 1 Synthesis of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide mono(trifluoroacetic acid) salt

[1050] Under ice cooling, N-(tert-butyloxycarbonyl)-L-methionyl-N 1TFA (2 ml) was added dropwise to a solution of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide (257 mg) in dichloromethane (4 ml). The mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure, isopropyl ether was added, and decantation was performed twice. The precipitated solid was washed to obtain a crude product of the title compound (248 mg). MS (ESI+): 385.3

[1051] (iii) N-[(4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N 1 Synthesis of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide

[1052] To 4-nitrophenyl (4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamate (54 mg) and L-methionyl-N- 1 TEA (59 μL) was added to a mixed solution of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide mono(trifluoroacetic acid) salt (70 mg) in dichloromethane (5 ml), and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 2→6% methanol / chloroform) to obtain the title compound (55 mg). MS (ESI+): 633.5

[1053] (iv) N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N 1 Synthesis of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide mono(trifluoroacetic acid) salt

[1054] Under ice cooling, N-[(4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N 1 TFA (2 ml) was added dropwise to a solution of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide (54 mg) in dichloromethane (4 ml), and the mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure, isopropyl ether was added, and decantation was performed twice. The precipitated solid was washed to obtain a crude product of the title compound (65 mg). MS (ESI+): 555.4 [M+Na]+

[1055] (v) N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 Synthesis of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide tetra(trifluoroacetic acid) salt

[1056] To N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N 1 HATU (66 mg) and DIPEA (45 μL) were added to a mixed solution of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide mono(trifluoroacetic acid) salt (64 mg) and DOTA-tris(t-Bu)ester (50 mg) in DMAc (2 mL), and the mixture was stirred at room temperature for 2 hours. After adding 1% TFA aqueous solution (about 4 ml) and acetonitrile (about 1 ml) to the reaction mixture, the diluted solution was purified by reverse phase column chromatography (solvent gradient; 20→100% 0.1% TFA acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (92 mg). MS (ESI+): 1109.4 [M+Na]+

[1057] (vi) N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 Synthesis of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide tetra(trifluoroacetic acid) salt

[1058] To N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 TFA (4 mL) was added to a solution of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide tetra(trifluoroacetic acid) salt (90 mg) in dichloromethane (4 mL), and the mixture was stirred overnight at room temperature. After concentration under reduced pressure, the residue was purified by reverse phase column chromatography (solvent gradient; 5→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (57 mg). MS (ESI+): 919.4

[1059] (vii) [N-({4-[(2-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 Synthesis of 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-L-isoleucineamide (3-)-gadolinium

[1060] To N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 Gadolinium chloride (8 mg) was added to a mixture of -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucine amide tetra(trifluoroacetic acid) salt (22 mg) and water (3 mL), and a 0.1 M aqueous sodium bicarbonate solution was added to adjust the pH to 5-6, and the mixture was stirred at room temperature for 1 hour. After TFA (10 μL) was added to the reaction mixture, the solution was purified by reverse phase column chromatography (solvent gradient; 5→50% acetonitrile / 0.1% aqueous TFA solution) to obtain the title compound (16 mg). MS (ESI-): 1072.1

[1061] (viii) Synthesis of Compound No. 18

[1062] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)] gadolinium (1 mg) was dissolved in DMSO (40 μL). 0.1 M boric acid buffer (40 μL) was added to the solution, and the pH was adjusted to 7.3 using 0.1 M sodium carbonate aqueous solution and 0.25 M acetate buffer.

[1063] To 4.45mg / mL Fab 240 μL of the previously prepared solution was added to the boric acid buffer (160 μL) and kept at 30°C for 2 hours. Purification was performed using a PD-10 column and recovery was performed using an Amicon Ultra-0.5mL centrifugal filter. The recovered solution was washed 7 times with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex containing one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#N)] with a molecular weight of 1074, a complex containing two, and a complex containing three.

[1064] (Example 2-66. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-4 and / or A-5)]p-Fab 2 )

[1065] (i) Synthesis of Compound No. 66

[1066] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)] gadolinium (1 mg) was dissolved in DMSO (40 μL). 0.1 M boric acid buffer (40 μL) was added to the solution, and the pH was adjusted to 6.5 using 0.1 M sodium carbonate aqueous solution.

[1067] The previously prepared solution was added to 4.45 mg / mL Fab2 in borate buffer (320 μL), and the mixture was incubated at 30°C for 2 hours. 10 μL of 0.05 M EDTA was added, and the mixture was incubated at 30°C for 10 minutes. The mixture was washed twice with phosphate-buffered saline using an Amicon Ultra-15 mL centrifugal filter, and the solution was recovered.

[1068] Add 20mM Bis-Tris propane buffer (pH 9.5) to the recovered solution, dilute it to 5 times the amount, load it onto a 5mL HiTrap Q column (GE Healthcare), and let it stand at room temperature for 6 hours. Wash the column with 20mM Bis-Tris propane buffer (pH 9.5) and 1M sodium chloride aqueous solution to recover the loaded solution. After buffer exchange with phosphate-buffered saline using an Amicon Ultra-15mL centrifugal filter, the solution is purified with a PD-10 column and recovered with an Amicon Ultra-15mL centrifugal filter. The recovered solution is washed twice with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex is relative to a Fab with a molecular weight of 47.9kDa. 2 A mixture of a complex having one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-4 and / or A-5)] with a molecular weight of 1092, a complex having two, a complex having three and a complex having four.

[1069] (Example 2-67. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#S)]p-Fab 2 )

[1070] (i) Synthesis of Compound No. 67

[1071] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)]gadolinium (2 mg) was dissolved in DMSO (80 μL), 0.1 M boric acid buffer (80 μL) was added, and the pH was adjusted to 7.3 with 0.1 M sodium carbonate aqueous solution.

[1072] To 4.45mg / mL Fab 2A 2 mg / mL 2-IT solution (10 μL) prepared with 0.1 M boric acid buffer was added to a boric acid buffer (320 μL) and incubated at 37°C for 40 minutes. For excess 2-IT, an Amicon Ultra-15 mL centrifugal filter was used, washed with phosphate-buffered saline, concentrated to 200 μL, and then the previously prepared solution was added and incubated at 30°C for 2 hours. 0.05 M EDTA (10 μL) was added, incubated at 30°C for 10 minutes, purified with a PD-10 column, and recovered with an Amicon Ultra-15 mL centrifugal filter. The recovered solution was washed twice with phosphate-buffered saline, and finally concentrated, and then filtered with a membrane filter to obtain a complex.

[1073] MS analysis confirmed that the complex was a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex containing one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#S)] with a molecular weight of 1175, a complex containing two, and a complex containing three.

[1074] (Example 2-68. [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-3)]p-Fab 2 )

[1075]

[1076] (i)N 1 -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-N 2 Synthesis of -(tert-Butyloxycarbonyl)-L-isoleucine amide

[1077] To a mixed solution of N-(tert-butyloxycarbonyl)-L-isoleucine (3.00 g) and HATU (5.92 g) in dichloromethane (30 ml) were added DIPEA (6.6 mL) and (2-aminoethyl) benzyl carbamate monohydrochloride (3.29 g) in sequence, and the mixture was stirred at room temperature for 1 hour. A saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the mixture was extracted twice with dichloromethane. The combined organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated. 200 mL of ethyl acetate / dichloromethane (1:2) solution was added to the residue, and the mixture was concentrated to obtain 100 mL of solution, which was then ice-cooled. The precipitated solid was filtered out and washed with ethyl acetate / dichloromethane (1:1) solution to obtain a solid. The filtrate was concentrated again, and then ice-cooled, and the precipitated solid was filtered out and washed with ethyl acetate / dichloromethane (1:1) solution to obtain a solid. The obtained solids were combined and dried under reduced pressure to obtain the title compound (4.10 g). MS (ESI+): 408.3

[1078] (ii) N-(tert-butyloxycarbonyl)-L-methionyl-N 1 Synthesis of -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide

[1079] Under ice cooling, 1 -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-N 2 TFA (7.51 mL) was added to a solution of -(tert-butoxycarbonyl)-L-isoleucine amide (2.00 g) in dichloromethane (10 mL), and the mixture was stirred at the same temperature for 1 hour. After concentration under reduced pressure, N-(tert-butoxycarbonyl)-L-methionine (1.35 g), DIPEA (4.2 mL), dichloromethane (20 mL) and HATU (2.24 g) were added to the residue, and the mixture was stirred at room temperature for 1 hour. The precipitated solid was filtered, washed with dichloromethane and methanol, and dried under reduced pressure to obtain the title compound (1.58 g). MS (ESI+): 539.4

[1080] (iii) L-methionyl-N 1 Synthesis of -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide

[1081] Under ice cooling, N-(tert-butyloxycarbonyl)-L-methionyl-N 1TFA (3 ml) was added dropwise to a solution of -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide (1.05 g) in dichloromethane (6 ml). The mixture was stirred at the same temperature for 2 hours. The mixture was concentrated under reduced pressure, dichloromethane and saturated aqueous sodium bicarbonate solution were added, and extracted twice with dichloromethane. The combined organic layer was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated to obtain a crude product of the title compound (690 mg). MS (ESI+): 439.4

[1082] (iv) N-[(4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N 1 Synthesis of -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide

[1083] Under argon atmosphere, diphenylphosphoryl azide (680 μL) was added to a mixed solution of 4-{[(tert-butyloxycarbonyl)amino]methyl}benzoic acid (395 mg), TEA (660 μL) and toluene (20 mL), and the mixture was stirred at room temperature for 1 hour and then stirred at a bath temperature of 100°C for 2 hours. The reaction solution was cooled to room temperature and L-methionyl-N-(2-methoxy-4-yl)-1-nitropropene was added. 1 -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide (690 mg) in THF (7 mL) was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and sodium bicarbonate aqueous solution and ethyl acetate were added. The generated solid was filtered out and washed with ethyl acetate, and then dried under reduced pressure to obtain the title compound (790 mg). MS (ESI+): 687.5

[1084] (v) N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N 1 Synthesis of -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide

[1085] Under ice cooling, N-[(4-{[(tert-butyloxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N 1 TFA (2 ml) was added dropwise to a solution of -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide (790 mg) in dichloromethane (3 ml). The mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure and a saturated aqueous sodium bicarbonate solution was added. The precipitated solid was filtered and washed with water, and then dried under reduced pressure to obtain a crude product of the title compound (720 mg). MS (ESI+): 587.6

[1086] (vi) N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 Synthesis of -(2-{[(Benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide

[1087] To N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N 1 HATU (280 mg) was added to a mixed solution of -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide (360 mg), DOTA-tris(t-Bu)ester (386 mg), and DIPEA (320 μL) in DMAc (2 mL), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by silica gel column chromatography (solvent gradient; 0→20% methanol / chloroform) to obtain the title compound (683 mg). MS (ESI+): 1141.9

[1088] (vii) N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 Synthesis of -(2-aminoethyl)-L-isoleucine amide

[1089] N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 A mixture of -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucine amide (683 mg), 10% palladium on carbon (50% water content, 382 mg) and methanol (10 mL) was stirred at room temperature for 18 hours under a hydrogen atmosphere (1 atm). The mixture was filtered to remove insoluble matter, and then concentrated. 10% palladium on carbon (50% water content, 382 mg) and methanol (10 mL) were added to the residue, and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere (1 atm). The mixture was filtered to remove insoluble matter, and then concentrated to obtain the title compound (417 mg). MS (ESI+): 1007.7

[1090] (viii) N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1Synthesis of -(2-aminoethyl)-L-isoleucine amide penta(trifluoroacetic acid) salt

[1091] N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 A mixed solution of -(2-aminoethyl)-L-isoleucine amide (417 mg), water (210 μL), tri(propane-2-yl)silane (210 μL) and TFA (8 mL) was stirred at room temperature for 3 hours. After concentration under reduced pressure, the residue was purified by reverse phase column chromatography (solvent gradient; 0→33% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (255 mg). MS (ESI+): 839.7

[1092] (ix) N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 Synthesis of -(2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucine amide tetra(trifluoroacetic acid) salt

[1093] Under ice cooling, N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 -(2-aminoethyl)-L-isoleucine amide penta(trifluoroacetic acid) salt (255 mg) in DMAc (2 mL) was added with TEA (200 μL), and the mixture was stirred at the same temperature for 10 minutes. To the solution was added a solution of 1,4-diisothiocyanatobenzene (174 mg) in DMAc (2 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction solution was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (136 mg). MS (ESI+): 1031.4

[1094] (x) [N-({4-[(2-{4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1Synthesis of (2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucineamide (3-)]gadolinium

[1095] To N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N 1 To a mixture of -(2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucineamide tetra(trifluoroacetic acid) salt (36 mg) and water (1.8 mL) was added gadolinium chloride (10 mg) and 0.1 M sodium bicarbonate aqueous solution (1.7 mL) and the pH was adjusted to 5.4, followed by stirring at room temperature for 30 minutes. The reaction mixture was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (33.0 mg). MS (ESI-): 1184.3

[1096] (xi) Synthesis of Compound No. 68

[1097] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -(2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucineamido(3-)]gadolinium (1 mg) was dissolved in DMSO (310 μL).

[1098] To 4.45mg / mL Fab 2 30 μL of the previously prepared solution was added to a phosphate buffered saline solution (320 μL), adjusted to about pH 9.0 with a 0.1 M sodium carbonate aqueous solution, and kept at 37°C for 24 hours. Purified with a PD-10 column and recovered with an Amicon Ultra-15 mL centrifugal filter. The recovered solution was washed twice with phosphate buffered saline, finally concentrated, and then filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.9 kDa. 2A mixture of a complex having one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-3)] with a molecular weight of 1187, a complex having two, and a complex having three.

[1099] (Example 2-69. ([Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys*-C(=S)-NH-(1,4-Ph)-NH-C(=S)]p-Fab 2 )

[1100]

[1101] (i) N-[2-(tert-butyloxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(Benzyloxy)carbonyl]-L-lysine

[1102] To L-methionylglycyl-N 6 TEA (0.24 mL) and HATU (241 mg) were added to a mixed solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (303 mg) and 2-(tert-butyloxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carboxylic acid (160 mg) in dichloromethane (10 mL), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (solvent gradient; 0→4% methanol / chloroform) to obtain the title compound (191 mg). MS (ESI+): 784.4

[1103] (ii) N-(1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(benzyloxy)carbonyl]-L-lysine mono(trifluoroacetate) salt

[1104] Under ice cooling, N-[2-(tert-butyloxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N 6 TFA (1 ml) was added dropwise to a solution of -[(benzyloxy)carbonyl]-L-lysine tert-butyl ester (190 mg) in dichloromethane (3 ml). The mixture was stirred at the same temperature for 1 hour and then concentrated under reduced pressure to obtain a crude product of the title compound (262 mg). MS (ESI+): 684.5

[1105] (iii) N-(2-{[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N 6 Synthesis of tert-butyl-[(benzyloxy)carbonyl]-L-lysine tetra(trifluoroacetate) salt

[1106] HATU (184 mg) and DIPEA (124 μL) were added to a solution of DOTA-tris(t-Bu)ester (138 mg) in DMAc (1 mL), and the mixture was stirred at room temperature for 5 minutes. N-(1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N ... 6 A solution of -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester mono(trifluoroacetic acid) salt (261 mg) in DMAc (1 mL) was stirred at room temperature for 2 hours. The reaction mixture was diluted with 1% TFA aqueous solution (about 1 ml), and the solution was purified by reverse phase column chromatography (solvent gradient; 20→100% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (231 mg). MS (ESI+): 1260.7 [M+Na]+

[1107] (iv) Synthesis of N-(2-{[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysine tert-butyl ester tetra(trifluoroacetate)

[1108] N-(2-{[4,7,10-tri(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N 6 A mixture of -[(Benzyloxy)carbonyl]-L-lysine tert-butyl ester tetra(trifluoroacetic acid) salt (230 mg), 10% palladium on carbon (50% water content, 300 mg), and methanol (10 mL) was stirred at room temperature for 10 minutes. The mixture was filtered to remove insoluble matter, and the filtrate was concentrated. 10% palladium on carbon (50% water content, 300 mg) and methanol (10 mL) were added to the residue, and stirred at room temperature for 18 hours under a hydrogen atmosphere (3 atm). The mixture was filtered to remove insoluble matter and then concentrated to obtain the title compound (152 mg). MS (ESI+): 1104.5

[1109] (v) Synthesis of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysine penta(trifluoroacetate) salt

[1110] To a solution of N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysine tert-butyl ester tetra(trifluoroacetate) salt (152 mg) in dichloromethane (1 mL) was added TFA (1.5 mL), and the mixture was stirred at room temperature for 4 hours. After concentration under reduced pressure, the residue was purified by reverse phase column chromatography (solvent gradient; 0→33% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (76 mg). MS (ESI+): 880.4

[1111] (vi) N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N 6 Synthesis of -[(4-isothiocyanatophenyl)thiocarbamoyl]-L-lysine tetra(trifluoroacetic acid) salt

[1112] Under ice cooling, TEA (60 μL) was added to a solution of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysine penta(trifluoroacetic acid) salt (76 mg) and 1,4-diisothiocyanatobenzene (50 mg) in DMAc (1 mL), and the mixture was stirred at the same temperature for 1 hour. The reaction solution was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (46 mg). MS (ESI-): 1070.6

[1113] (vii) {N-[2-({4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetyl-κO)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N 6 Synthesis of 3-[(4-isothiocyanatophenyl)thiocarbamoyl]-L-lysine Gadolinium

[1114] N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N 6 Gadolinium chloride (8 mg) and 0.1 M sodium bicarbonate aqueous solution (700 μL) were added to a mixture of -[(4-isothiocyanatophenyl)thiocarbamoyl]-L-lysine tetra(trifluoroacetic acid) salt (16 mg) and water (800 μL), and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified by reverse phase column chromatography (solvent gradient; 0→40% acetonitrile / 0.1% TFA aqueous solution) to obtain the title compound (7.4 mg). MS (ESI-): 1225.3

[1115] (viii) Synthesis of Compound No.69

[1116] The {N-[2-({4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetyl-κO)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N 6 -[(4-Isothiocyanatophenyl)thiocarbamoyl]-L-lysine (3-)}gadolinium (1 mg) was dissolved in DMSO (310 μL).

[1117] To 4.45mg / mL Fab 2 30 μL of the previously prepared solution was added to a phosphate buffered saline solution (320 μL), adjusted to about pH 9.0 with a 0.1 M sodium carbonate aqueous solution, and kept at 37°C for 24 hours. Purified with a PD-10 column and recovered with an Amicon Ultra-15 mL centrifugal filter. The recovered solution was washed twice with phosphate buffered saline, finally concentrated, and then filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.9 kDa. 2 A mixture of a complex having 1 [Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys*-C(=S)-NH-(1,4-Ph)-NH-C(=S)] with a molecular weight of 1228, a complex having 2, a complex having 3, a complex having 4 and a complex having 5.

[1118] The composites obtained in the above-mentioned production examples are shown in Tables 1 and 2.

[1119] [Table 1]

[1120] (Y-S1-X) p -Fab 2

[1121]

[1122] [Table 2]

[1123] (Y-S1-X) p -Fab 2

[1124]

[1125] In the above table and Tables 15 to 18 described later, - represents a bond, and the symbols (a) to (q) in the S1 column represent spacers represented by the following formulas (a) to (q), respectively.

[1126]

[1127] The compounds of Production Example Nos. A1 to A43 and B1 to B4 in Tables 3 to 14 were synthesized by the same method as in the above-mentioned Production Examples or a method known to those skilled in the art, and the composites shown in Tables 15 to 18 described below were obtained using these.

[1128] [Table 3]

[1129]

[1130] [Table 4]

[1131]

[1132] [Table 5]

[1133]

[1134] [Table 6]

[1135]

[1136] [Table 7]

[1137]

[1138] [Table 8]

[1139]

[1140] [Table 9]

[1141]

[1142] [Table 10]

[1143]

[1144] [Table 11]

[1145]

[1146] [Table 12]

[1147]

[1148] [Table 13]

[1149]

[1150] [Table 14]

[1151]

[1152] [Table 15]

[1153] (Y-S1-X)pp-Fab2

[1154]

[1155] [Table 16]

[1156] (YSX)Fab 2

[1157]

[1158] [Table 17]

[1159] (YSX)p-Fab

[1160]

[1161] [Table 18]

[1162] (Y-S1-X) p -Fab 2

[1163]

[1164] MS analyses of the example compounds described in Tables 15 to 18 are shown in Tables 19 to 23 below.

[1165] [Table 19]

[1166]

[1167] [Table 20]

[1168]

[1169] [Table 21]

[1170]

[1171] [Table 22]

[1172]

[1173] [Table 23]

[1174]

[1175] Example 3-1: Evaluation of Binding Activity of CEACAM5 Antibody Fab Fragment Complex

[1176] ELISA was performed on each anti-human CEACAM5 antibody Fab fragment complex prepared in Example 2 to evaluate the binding activity to CEACAM5. In this test, as the solvent of the antigen solid phase solution, a phosphate buffer (NAKALI TESQUE, 0.1 mol / L-phosphate buffer (pH 7.2)) diluted 10 times with distilled water to prepare 10 mM was used, and as the washing liquid, a 20X PBS / Tween 20 buffer (Thermo, 28352) diluted 20 times with distilled water was used. In addition, bovine serum albumin (BSA) used in this test was used by adding a 30% bovine serum albumin solution (SIGMA, A9576-50ML) at an appropriate ratio. CEACAM5 (R&D Systems, 4128-CM-050) was diluted to 0.1 μg / mL with 10 mM phosphate buffer (pH 7.2), and 30 μL was added to each well of Nunc Maxisorp White 384 Plate (Nunc, 460372), and the plate was incubated at 4°C overnight for solid phase. After the CEACAM5 solid phase solution was removed by reverse centrifugation, PBS / Tween 20 buffer containing 5.0% BSA was added for blocking. After that, the blocking solution was removed by reverse centrifugation, and PBS / Tween 20 buffer containing 1.0% BSA was used to block the above CEACAM5 antibody Fab. 2 The fragment complex or the Fab without labeling part as a control 2Fragment (PB009-01), a solution of about 10000 ng / mL was diluted 3-fold for 14 times, 30 μL was added to each well, and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS / Tween 20 buffer, and 30 μL of horseradish peroxidase (HRP) labeled goat anti-human IgG (H+L chain) antibody (MBL Life Science, 206) diluted to 1000 times with PBS / Tween 20 buffer containing 5.0% BSA was added to each well, and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS / Tween 20 buffer, and 30 μL of ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232) as a substrate was added to each well. After incubation at room temperature for 15 minutes, the signal value was measured using an Envision counter (Parkin Elmer).

[1177] The experiments for each antibody were performed in duplicate, and the EC was calculated using a four-parameter logistic curve model. 50 The EC values ​​of the 9 tests for PB009-01 50 The geometric mean, standard deviation, and lower and upper limits of the 95% confidence interval (95% CI of geo.mean) are shown in Table 24. 50 The values ​​are shown in Table 25. It should be noted that Ex-No in the table represents the composite number in Example 2.

[1178] [Table 24]

[1179] PB009-01 Geometric mean (nM) 0.20 Standard Deviation 1.45 Lower limit of 95% confidence interval (nM) 0.15 Upper limit of 95% confidence interval (nM) 0.27

[1180] [Table 25]

[1181]

[1182] Example 3-2:

[1183] ELISA was performed on each anti-human CEACAM5 antibody Fab fragment complex prepared in Example 2 to evaluate the binding activity to CEACAM5. In this test, as the solvent of the antigen solid phase solution, a phosphate buffer (NAKALI TESQUE, 0.1 mol / L-phosphate buffer (pH 7.2)) diluted 10 times with distilled water to prepare 10 mM was used, and as the washing liquid, a 20X PBS / Tween 20 buffer (Thermo, 28352) diluted 20 times with distilled water was used. In addition, bovine serum albumin (BSA) used in this test was used by adding a 30% bovine serum albumin solution (SIGMA, A9576-50ML) at an appropriate ratio. CEACAM5 (R&D Systems, 4128-CM-050) was diluted to 0.1 μg / mL with 10 mM phosphate buffer (pH 7.2), and 30 μL was added to each well of Nunc Maxisorp White 384 Plate (Nunc, 460372), and the plate was incubated at 4°C overnight for solid phase. After the CEACAM5 solid phase solution was removed by reverse centrifugation, PBS / Tween 20 buffer containing 5.0% BSA was added for blocking. After that, the blocking solution was removed by reverse centrifugation, and PBS / Tween 20 buffer containing 1.0% BSA was used to block the above CEACAM5 antibody Fab. 2 The fragment complex or the Fab without labeling part as a control 2 Fragment (PB009-01), about 10000 ng / mL solution was diluted 3-fold for 14 times, 30 μL was added to each well, and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS / Tween 20 buffer, and 30 μL of horseradish peroxidase (HRP) labeled goat anti-human IgG (H+L chain) antibody (MBL Life Science, 206) diluted to 1000 times with PBS / Tween 20 buffer containing 5.0% BSA was added to each well, and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS / Tween 20 buffer, and 30 μL of ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232) as a substrate was added to each well. After incubation at room temperature for 15 minutes, the signal value was measured using an Envision counter (Parkin Elmer).

[1184] The experiments for each antibody were repeated and the EC values ​​were calculated using a four-parameter logistic curve model. 50 The EC values ​​of the two tests for PB009-01 were50 The values ​​(nM) are shown in Table 26. In addition, the EC values ​​of each complex obtained by repeated experiments were 50 The values ​​are shown in Table 27. It should be noted that Ex-No in the table represents the composite number of Example 2.

[1185] [Table 26]

[1186]

[1187] [Table 27]

[1188]

[1189] Example 4-1: Kidney aggregation evaluation test of Gd-labeled complex (normal mice)

[1190] The complex includes a complex containing a peptide linker having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme as "X". The complex of the present invention having such a linker can be specifically cleaved at the linker portion by these enzymes present in the kidney, and therefore is expected to reduce the aggregation of the labeled portion in the kidney. The evaluation results of the renal aggregation of such a complex of the present invention are shown below.

[1191] (Test method)

[1192] The Gd-labeled complex containing the peptide linker prepared in the above example was administered to mice (BALB / c or BALB / c nu / nu) via the tail vein at a protein amount of 0.02 mg (100 μL) per mouse. The mice were killed and the kidneys were removed after about 24 hours, and the amount of Gd in the kidneys was measured by ICP-MS. The experiment was carried out with 3 cases per group, and the average value of the 3 cases was calculated, and the amount of Gd contained in each kidney after administration was shown as a percentage (% of dose / tissue) relative to the total amount of Gd administered. The results are shown in Table 28 described below. Ex-No in the table represents the complex number of Example 2. In addition, the same experiment was carried out using Ex-No.47 ([Gd / DOTA]p-Fab) produced in the above-mentioned Production Example 2-47 without a peptide linker as a control complex, and the geometric mean (Geometric mean), standard deviation (Geometric SD factor), and lower limit (Lower) and upper limit (Upper) of the 95% confidence interval (95% CI of geo.mean) of 5 experiments were calculated.

[1193] (result)

[1194] The results are shown in Table 29 described below. The reduction ratio of the amount of Gd contained in the kidney of each of the above-mentioned complexes with a peptide linker was calculated based on the percentage of the control complex (percentage of the dose / tissue), and is shown in Table 28. As shown in Table 28, the aggregation of the labeled portion in the kidney of each of the above-mentioned complexes with a peptide linker was reduced by 39.6 to 82.9 percentage points relative to the control complex.

[1195] [Table 28]

[1196] Ex-No Percentage of drug dose / tissue Reduction ratio (%) 11 14.8 52.2 12 9.9 68.0 13 15.4 50.3 15 17.1 44.8 24 12.9 58.3 25 14.2 54.1 29 6.8 78.0 31 18.7 39.6 33 5.3 82.9 34 11.9 61.6 35 7.0 77.4 40 8.4 72.9

[1197] [Table 29]

[1198] Ex-No 47 Geometric mean (percentage of dose / tissue) 31.0 Standard Deviation 1.2 Lower limit of 95% confidence interval (percentage of dose / tissue) 25.3 Upper limit of 95% confidence interval (percentage of dose / tissue) 37.9

[1199] Example 4-2: Kidney aggregation evaluation test of Gd-labeled complex (normal mice)

[1200] (Test method)

[1201] The Gd-labeled complex containing the peptide linker prepared in the above example was administered to mice (BALB / c or BALB / c nu / nu) via the tail vein at a protein amount of 0.02 mg (100 μL) per mouse. The mice were killed and the kidneys were removed after about 24 hours, and the amount of Gd in the kidneys was measured by ICP-MS. The experiment was carried out with 3 cases per group, and the average value of the 3 cases was calculated, and the amount of Gd contained in each kidney after administration was shown as a percentage relative to the total amount of Gd administered (percentage of the administered amount / tissue). The results are shown in Table 30 described below. In the table, Ex-No represents the complex number of Example 2. In addition, the same experiment was carried out using Ex-No 47 ([Gd / DOTA]p-Fab) prepared in the above-mentioned manufacturing example 2 without a peptide linker as a control complex.

[1202] (result)

[1203] The results are shown in Table 30 described below. The reduction ratio of the amount of Gd contained in the kidney of each of the above-mentioned complexes with a peptide linker was calculated based on the percentage of the control complex (percentage of the dose / tissue), and is shown in Table 30. As shown in Table 30, the aggregation of the labeled portion in the kidney of each of the above-mentioned complexes with a peptide linker was reduced by 92.15 to 95.38 percentage points relative to the control complex 47.

[1204] [Table 30]

[1205] Kidney residual volume (normal mice)

[1206]

[1207] Example 4-3: Kidney aggregation evaluation test of Gd-labeled complex (normal mice)

[1208] (Test method)

[1209] The Gd-labeled complex containing the peptide linker prepared in the above example was administered to mice (BALB / c or BALB / c nu / nu) via the tail vein at a protein amount of 0.02 mg (100 μL) per mouse. The mice were killed and the kidneys were removed after about 24 hours, and the amount of Gd in the kidneys was measured by ICP-MS. The experiment was carried out with 3 cases per group, and the average value of the 3 cases was calculated, and the amount of Gd contained in each kidney after administration was shown as a percentage relative to the total amount of Gd administered (percentage of the administered amount / tissue). The test was similarly carried out using Ex-No.47 prepared in the above-mentioned manufacturing example 2 without a peptide linker as a control complex, and the average of the residual amount in the kidney of 5 tests for Ex-No.47 is shown in Table 31.

[1210] (result)

[1211] The reduction ratio (%) of the amount of Gd contained in the kidney of each of the above-mentioned complexes with a peptide linker was calculated based on the percentage of the control complex (percentage of the dose / tissue), and is shown in Table 32. As shown in Table 32, the aggregation of the labeled portion in the kidney of each of the above-mentioned complexes with a peptide linker was reduced by 21.00 to 94.66 percentage points relative to the control complex.

[1212] [Table 31]

[1213] Kidney residual volume (normal mice)

[1214]

[1215] [Table 32]

[1216] Kidney residual volume (normal mice)

[1217]

[1218] Example 4-4: Kidney aggregation evaluation test of Gd-labeled complexes (tumor-bearing mice)

[1219] (Test method)

[1220] The Gd-labeled complex containing the peptide linker prepared in the above example was administered to the tumor-bearing mice via the tail vein in a dose of 0.02 mg (100 μL) per mouse. 6Mice (BALB / cnu / nu) were tumor-bearing mice (BALB / cnu / nu) transplanted subcutaneously with human colon cancer cell line LS174T cells (ATCC (registered trademark); CL-188) in the back. The mice were killed 24 hours after the sample administration, and the kidneys and tumors were removed. The amount of Gd in the kidneys and tumors was measured using ICP-MS. This experiment was carried out with 3 cases in each group, and the average value of the 3 cases was calculated. The amount of Gd contained in each kidney after administration is shown as a percentage relative to the amount of Gd administered (percentage of dose / tissue), and the amount of Gd contained in each 1g of tumor tissue is shown as a percentage relative to the total amount of Gd administered (% of dose / g (percentage of dose / g)). The results are shown in Tables 35 and 36. In the table, Ex-No represents the complex number of Example 2. In addition, Ex-No.47 produced in Production Example 2-47 without a peptide linker as a control complex was tested in the same manner. The average values ​​of the two tests on Ex-No.47 are shown in Tables 33 and 34.

[1221] (result)

[1222] As shown in Tables 35 and 36, the aggregation of the labeled portion in the kidney of each complex having a peptide linker was reduced by 52.26 to 77.68 percentage points compared to the control complex.

[1223] [Table 33]

[1224] Kidney residual volume (tumor-bearing mice)

[1225]

[1226] [Table 34]

[1227] Tumor aggregation

[1228]

[1229] [Table 35]

[1230] Kidney residual volume (tumor-bearing mice)

[1231]

[1232] [Table 36]

[1233] Tumor accumulation (tumor-bearing mice)

[1234] Ex-No. Percentage of dosage / g 18 10.63 24 10.50 29 4.37 33 4.53 35 6.90

[1235] (Example 5-1: Anti-human MUC1 antibody Fab 5 Preparation of fragment complexes

[1236] This embodiment discloses a manufacturing embodiment of a complex. In each manufacturing embodiment of this embodiment, "Example 5" is used to record the "complex number" with a hyphen. For example, "Example 5-101" indicates the manufacturing embodiment of complex 101 in this embodiment. In addition, Fab in this embodiment 5 The (P10-2) described in International Publication WO2018 / 092885 was used. 5 "Fab 5 The p amino groups thereof are bound to the p labeling parts described in [] or () to form a complex. It should be noted that the Fab of each complex confirmed by MS analysis is described in the following examples. 5 The number of the labeled part bound) is not shown in Table 1, but this result does not mean that the complex having a binding number other than this is not included. It is easy to understand that there may be a complex with a binding number whose existence cannot be confirmed due to the accuracy of the MS analysis equipment. In addition, in the structural formula of the present embodiment, the structural formula of DOTA bound to Gd schematically represents DOTA labeled with Gd.

[1237] (Example 5-101. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#N)]p-Fab 5 )

[1238] (i) Synthesis of Composite No. 101

[1239] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)] gadolinium (1 mg) was dissolved in DMSO (40 μL). 0.1 M boric acid buffer (40 μL) was added to the solution, and the pH was adjusted to 6.6 with 0.25 M acetate buffer.

[1240] To 5.2mg / mL Fab 5Add 80 μL of the previously prepared solution to the boric acid buffer (240 μL) and incubate at 30°C for 2 hours. Add 15 μL of 0.05M EDTA and incubate at 30°C for 10 minutes. Purify using a PD-10 column and recover using an Amicon Ultra-0.5mL centrifugal filter. Wash the recovered solution 7 times with phosphate-buffered saline, and finally concentrate it and filter it with a membrane filter to obtain a complex. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.5 kDa. 5 A complex containing one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#N)] with a molecular weight of 1074 and a mixture of complexes containing two.

[1241] (Example 5-104. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-4 and / or A-5)]p-Fab 5 )

[1242] (i) Synthesis of Composite No. 104

[1243] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)] gadolinium (1 mg) was dissolved in DMSO (40 μL). 0.1 M boric acid buffer (40 μL) was added to the solution, and the pH was adjusted to 10.2 using 0.1 M sodium carbonate aqueous solution.

[1244] Add all of the previously prepared solution to 5.2 mg / mL Fab 5 The mixture was added to a borate buffer (240 μL) and incubated at 30°C for 2 hours. The mixture was recovered using an Amicon Ultra-15 mL centrifugal filter and washed with phosphate-buffered saline.

[1245] The buffer was exchanged twice with 3 mL of HEPES-NaOH buffer (pH=4.5), concentrated to obtain 100 μL of a solution with a pH of 4.52, and then the solution was incubated at 45°C for 30 minutes.

[1246] The solution was purified by PD-10 column and recovered by Amicon Ultra-15mL centrifugal filter. The recovered solution was washed three times with phosphate buffered saline, and finally concentrated and filtered with a membrane filter to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.5 kDa. 5 A mixture of a complex having one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-4 and / or A-5)] with a molecular weight of 1092, a complex having two such complexes, and a complex having three such complexes.

[1247] (Example 5-107. ([Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#S)]p-Fab 5 )

[1248] (i) Synthesis of Composite No. 107

[1249] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucineamide (3-)]gadolinium (0.75 mg) was dissolved in DMSO (40 μL), 0.1 M boric acid buffer (40 μL) was added, and then the pH was adjusted to 8.2 with 0.1 M sodium carbonate aqueous solution and 0.25 M acetate buffer.

[1250] To 5.2mg / mL Fab 5Add 2mg / mL 2-IT solution (7.5μL) prepared with 0.1M boric acid buffer to boric acid buffer (240μL) and incubate at 37°C for 40 minutes. For excess 2-IT, use Amicon Ultra-0.5mL centrifugal filter, wash with phosphate buffered saline, concentrate, add the previously prepared solution, and incubate at 30°C for 2 hours. Purify with PD-10 column and recover with Amicon Ultra-15mL centrifugal filter. Wash the recovered solution twice with phosphate buffered saline, finally concentrate, and then filter with a membrane filter to obtain a complex.

[1251] MS analysis confirmed that the complex was a Fab with a molecular weight of 47.5 kDa. 5 A mixture of a complex containing one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-Z1(#S)] with a molecular weight of 1175, a complex containing two, and a complex containing three.

[1252] (Example 5-112. [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-3)]p-Fab 5 )

[1253] (i) Synthesis of Compound No. 112

[1254] [N-({4-[(2-{4,7,10-tris[(carboxyl-κO)methyl]-1,4,7,10-tetraazacyclododecane-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetamide-κ(O)methyl]phenyl}carbamoyl)-L-methionyl-N 1 -(2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucineamido(3-)]gadolinium (0.46 mg) was dissolved in DMSO (155 μL).

[1255] Prepare Fab at 5.2 mg / mL using borate buffer and glycerol solution. 5A 0.1M sodium carbonate aqueous solution and 30μL of the previously prepared solution were added to the solution (240μL), the pH was adjusted to 8.8, and the mixture was kept at 37°C for 2 hours. The mixture was purified using a PD-10 column and recovered using an Amicon Ultra-15mL centrifugal filter. The recovered solution was washed twice with phosphate-buffered saline, and finally concentrated and filtered using a membrane filter to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.5kDa. 5 A mixture of a complex in which one [Gd / DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-3)] with a molecular weight of 1187 is bound and a complex in which two are bound.

[1256] (Example 5-113. ([Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys*-C(=S)-NH-(1,4-Ph)-NH-C(=S)]p-Fab 5 )

[1257] (i) Synthesis of Composite No. 113

[1258] The {N-[2-({4,7,10-tris[(carboxy-κO)methyl]-1,4,7,10-tetraazacyclododec-1-yl-κ 4 N 1 ,N 4 ,N 7 ,N 10}acetyl-κO)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N 6 -[(4-Isothiocyanatophenyl)carbamothioyl]-L-lysine (3-)}gadolinium (0.22 mg) was dissolved in DMSO (72 μL).

[1259] Prepare Fab at 5.2 mg / mL using borate buffer and glycerol solution. 5 A 0.1M sodium carbonate aqueous solution and 30μL of the previously prepared solution were added to the solution (240μL), the pH was adjusted to 9.3, and the mixture was kept at 37°C for 2 hours. The mixture was purified using a PD-10 column and recovered using an Amicon Ultra-15mL centrifugal filter. The recovered solution was washed twice with phosphate-buffered saline, and finally concentrated and filtered using a membrane filter to obtain a complex. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.5kDa. 5A mixture of a complex containing one [Gd / DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys*-C(=S)-NH-(1,4-Ph)-NH-C(=S)] having a molecular weight of 1228 and a complex containing two such complexes.

[1260] Tables 37 and 38 show the chemical structural formula (Str) of the complex obtained in the above-mentioned production example.

[1261] [Table 37]

[1262]

[1263] [Table 38]

[1264]

[1265] The compounds of Preparation Example No. C1 and C2 in Table 39 were synthesized by the same method as in the above-mentioned Preparation Example or a method known to those skilled in the art, and the compounds synthesized in these or Example 2 were used to obtain the Fab-like compounds shown in Tables 40 and 41 described below. 5 of the complex.

[1266] [Table 39]

[1267]

[1268] [Table 40]

[1269]

[1270] [Table 41]

[1271]

[1272] The MS analysis of the above-mentioned example compounds described in Tables 40 to 41 is shown in the following table.

[1273] [Table 42]

[1274]

[1275] The example compounds in Table 43 were obtained using the same method as in the above-mentioned preparation examples or a method known to those skilled in the art.

[1276] [Table 43]

[1277]

[1278] The MS analysis of the above-mentioned example compounds described in Table 43 is shown in Table 44 below.

[1279] [Table 44]

[1280]

[1281] Furthermore, the composites of Tables 45 to 50 can be obtained by the same method as in the above-mentioned Production Example or a method known to those skilled in the art.

[1282] [Table 45]

[1283]

[1284] [Table 46]

[1285]

[1286] [Table 47]

[1287]

[1288] [Table 48]

[1289]

[1290] [Table 49]

[1291]

[1292] [Table 50]

[1293]

[1294] Example 6-1: Anti-human MUC1 antibody Fab 5 Evaluation of binding activity of fragment complexes

[1295] Each anti-human MUC1 antibody Fab prepared by the method of Example 5 5The fragment complex was subjected to ELISA to evaluate the binding activity to human cancer-specific MUC1. In this test, 20X PBS / Tween 20 buffer (Thermo Company, 28352) diluted to 20 times with distilled water was used as a washing solution. In addition, the bovine serum albumin (BSA) used in this test was used by adding a 30% bovine serum albumin solution (SIGMA Company, A9576-50ML) at an appropriate ratio. The human cancer-specific MUC1 peptide (Patent Document 1) was added to the Nunc Maxisorp white 384 plate (Nunc Company, 460372) at 0.5μmol / L and 30μL per well, and the plate was incubated at 4°C overnight for solid phase. After removing the MUC1 peptide by reverse centrifugation, PBS / Tween 20 buffer containing 5.0% BSA was added for blocking. Afterwards, the blocking solution was removed by reverse centrifugation, and the anti-human MUC1 antibody Fab was lysed using PBS / Tween 20 buffer containing 1.0% BSA. 5 The fragment complex (Ex-No. 104, 108, 112) was diluted 14 times in a 3-fold manner at a concentration of about 10,000 ng / mL, and 30 μL was added to each well, and the mixture was incubated at room temperature for 60 minutes. The plate was washed three times with PBS / Tween 20 buffer, and 30 μL of horseradish peroxidase-labeled goat anti-human Igκ antibody (Southern Biotechnology Associates) diluted 10,000-fold with PBS / Tween 20 buffer containing 5.0% BSA was added to each well, and the mixture was incubated at room temperature for 30 minutes. The plate was washed three times with PBS / Tween 20 buffer, and 30 μL of ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232) as a substrate was added to each well. After incubation at room temperature for 15 minutes, the signal value was measured using an Envision counter (Parkin Elmer).

[1296] The experiments for each antibody were performed in duplicate, and the EC was calculated using a four-parameter logistic curve model. 50 The EC values ​​of the three tests for P10-2 were 50 The values ​​(nM) are shown in Table 51. In addition, the EC values ​​of each complex obtained by repeated experiments were 50 The values ​​are shown in Table 52.

[1297] [Table 51]

[1298] P10-2(nM) 0.06 0.06 0.14

[1299] [Table 52]

[1300]

[1301] Example 6-2: Anti-human MUC1 antibody Fab 5 Evaluation of binding activity of fragment complexes

[1302] Each anti-human MUC1 antibody Fab prepared by the method of Example 5 5 The fragment complex was subjected to ELISA to evaluate the binding activity to human cancer-specific MUC1. In this test, 20X PBS / Tween 20 buffer (Thermo Company, 28352) diluted to 20 times with distilled water was used as a washing solution. In addition, the bovine serum albumin (BSA) used in this test was used by adding a 30% bovine serum albumin solution (SIGMA Company, A9576-50ML) at an appropriate ratio. The human cancer-specific MUC1 peptide (Patent Document 1) was added to the Nunc Maxisorp white 384 plate (Nunc Company, 460372) at 0.5μmol / L and 30μL per well, and the plate was incubated at 4°C overnight for solid phase. After removing the MUC1 peptide by reverse centrifugation, PBS / Tween 20 buffer containing 5.0% BSA was added for blocking. Afterwards, the blocking solution was removed by reverse centrifugation, and the anti-human MUC1 antibody Fab was lysed using PBS / Tween 20 buffer containing 1.0% BSA. 5 The fragment complex (Ex-No.101-113) was diluted 14 times in a 3-fold manner at a concentration of about 10,000 ng / mL, and 30 μL was added to each well, and the mixture was incubated at room temperature for 60 minutes. The plate was washed three times with PBS / Tween 20 buffer, and 30 μL of horseradish peroxidase-labeled goat anti-human Igκ antibody (Southern Biotechnology Associates) diluted 10,000 times with PBS / Tween 20 buffer containing 5.0% BSA was added to each well, and the mixture was incubated at room temperature for 30 minutes. The plate was washed three times with PBS / Tween 20 buffer, and 30 μL of ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232) as a substrate was added to each well. After incubation at room temperature for 15 minutes, the signal value was measured using an Envision counter (Parkin Elmer).

[1303] The experiments for each antibody were performed in duplicate, and the EC was calculated using a four-parameter logistic curve model. 50 The EC values ​​of the three tests for P10-2 were 50The values ​​(nM) are shown in Table 53. In addition, the EC values ​​of each complex obtained by repeated experiments were 50 The values ​​are shown in Table 54.

[1304] [Table 53]

[1305] P10-2(nM) 0.06 0.06 0.14

[1306] [Table 54]

[1307]

[1308] Example 7: Kidney aggregation evaluation test of Gd-labeled complex (normal mice)

[1309] (Test method)

[1310] The Gd-labeled complex containing the complex of the peptide linker manufactured in the above-mentioned embodiment was administered to mice (BALB / c or BALB / c nu / nu) through the tail vein in a manner of 0.02 mg (100 μL) per mouse in terms of protein amount. The mice were killed and the kidneys were removed after about 24 hours, and the amount of Gd in the kidneys was measured by ICP-MS. The experiment was carried out with 3 cases per group, and the average value of the 3 cases was calculated, and the amount of Gd contained in each kidney after administration was shown as a percentage relative to the total amount of Gd administered (percentage of the administered amount / tissue). The experiment was similarly carried out using Ex-No 108 manufactured in the above-mentioned manufacturing example 5 without a peptide linker as a control complex. The average values ​​of the 2 experiments on Ex-No 108 are shown in Table 55.

[1311] (result)

[1312] The results are shown in Table 56. The reduction ratio of the amount of Gd contained in the kidney of each of the above-mentioned complexes with a peptide linker was calculated based on the amount of Gd contained in the kidney of the control complex. As shown in Table 56, the aggregation of the labeled portion in the kidney of each of the above-mentioned complexes with a peptide linker was reduced by 91.52 to 93.03 percentage points relative to the control complex.

[1313] [Table 55]

[1314] Kidney residual volume (normal mice)

[1315] Ex-No.108 Average value (percentage of dose / tissue) 33.00

[1316] [Table 56]

[1317] Kidney residual volume (normal mice

[1318]

[1319] Example 8: Kidney aggregation evaluation test of Gd-labeled complexes (tumor-bearing mice)

[1320] (Test method)

[1321] The Gd-labeled complex containing the peptide linker prepared in the above example was administered to the tumor-bearing mice via the tail vein in a dose of 0.02 mg (100 μL) per mouse. 6 Mice (BALB / cnu / nu) in a tumor-bearing state were transplanted subcutaneously with human breast cancer cell line MDA-MB-468 (ATCC (registered trademark); HTB-132) on the back. The mice were killed 24 hours after the sample administration, and the kidneys and tumors were removed. The amount of Gd in the kidneys and tumors was measured using ICP-MS. This experiment was carried out with 3 cases in each group, and the average value of the 3 cases was calculated. The amount of Gd contained in each kidney after administration is shown as a percentage relative to the amount of Gd administered (percentage of the administered amount / tissue), and the amount of Gd contained in each 1g of tumor tissue is shown as a percentage relative to the total amount of Gd administered (percentage of the administered amount / g). The results are shown in Tables 59 and 60. In the table, Ex-No represents the complex number of Example 5. In addition, the same experiment was performed using Ex-No.108 produced in the above-mentioned manufacturing example 5 without a peptide linker as a control complex. The average values ​​of the two tests on Ex-No.108 are shown in Tables 57 and 58.

[1322] (result)

[1323] The results are shown in Tables 57 to 60. In each of the complexes having a peptide linker, the aggregation of the labeled portion in the kidney was reduced by 88.08 to 90.87 percentage points compared to the control complex.

[1324] [Table 57]

[1325] Renal residual volume

[1326]

[1327] [Table 58]

[1328] Tumor aggregation

[1329]

[1330] [Table 59]

[1331] Kidney residual volume (tumor-bearing mice)

[1332]

[1333] [Table 60]

[1334] Tumor accumulation (tumor-bearing mice)

[1335] Ex-No. Percentage of dosage / g 104 1.04 112 1.27

[1336] Example 9: Anti-human MUC1 antibody Fab 5 Preparation of fragment complexes

[1337] (Example 9-115. [DOTA] p-Fab 5 Synthesis of

[1338]

[1339] Under ice cooling, a 1M sodium hydroxide aqueous solution (80 μL) was added to a mixed solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (16 mg) and water (810 μL) to adjust the pH to 5.5-6. Under ice cooling, sodium 1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate (2.3 mg) dissolved in water (117 μL) was added to the obtained solution (239 μL). Thereafter, an EDC HCl aqueous solution (8.3 μL, 25 mg / mL) was added, and the mixture was stirred for 30 minutes under ice cooling to prepare an N-hydroxysulfonylsuccinimide DOTA solution. After adding Fab 5 The pH was adjusted to 7 by adding 0.2 M sodium dihydrogen phosphate aqueous solution (pH 9) (40 μL).

[1340] To 20.8mg / mL Fab 5 The prepared N-hydroxysulfonylsuccinimide DOTA solution (300 μL) was added to a 0.1M sodium dihydrogen phosphate aqueous solution (585 μL) (90 μL) and incubated at 4°C for 23 hours. For the excess linker, an Amicon Ultra-15 mL centrifugal filter was used, and it was repeatedly washed twice with 10 mM phosphate buffer, washed with 0.3 M ammonium acetate buffer, and finally concentrated and filtered with a membrane filter to obtain a complex Ex-No.115. MS analysis confirmed that the complex was a Fab with a molecular weight of 47.5 kDa. 5 A mixture of a complex to which one [DOTA] with a molecular weight of 387 is bound, a complex to which two are bound, a complex to which three are bound, and a complex to which four are bound.

[1341] (Example 9-116. [DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH2)2-(A-3)]p-Fab 5 Synthesis

[1342]

[1343] The N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]acetamide}methyl)phenyl]carbamoyl}-L-methionyl-N- 1 -(2-{[(4-isothiocyanatophenyl)thiocarbamoyl]amino}ethyl)-L-isoleucine amide tetra(trifluoroacetic acid) salt (1 mg) was dissolved in DMSO (336 μL).

[1344] To 5.2mg / mL Fab 5 Add 30 μL of the previously prepared solution to a phosphate-buffered saline solution (240 μL), adjust the pH to about 8.0 with a 0.1 M sodium carbonate solution, and keep warm at 30°C for 2 hours. Purify with a PD-10 column and recover with an Amicon Ultra-15 mL centrifugal filter. The recovered solution was washed twice with phosphate-buffered saline, and finally concentrated and filtered with a membrane filter to obtain complex Ex-No.116. Perform the same operation again and mix the obtained complex Ex-No.116. MS analysis confirmed that the complex was relative to a Fab with a molecular weight of 47.5 kDa. 5 A mixture of a complex having one [DOTA-NH-CH2-(1,4-Ph)-NH-C(=O)-Met-lle-NH-(CH2)2-(A-3)] with a molecular weight of 1032, a complex having two, a complex having three, and a complex having four.

[1345] Example 10: Study on the dynamics of metal complex compounds in marmosets

[1346] 64 Preparation Example 1 of Cu-labeled protein

[1347] Towards[ 64 10 μL of 0.05 mol / L hydrochloric acid solution of Cu]CuCl2 (19.8 MBq, Fujifilm Toyama Chemical) was added with 139 μL of 0.1 mol / L sodium acetate buffer (pH 6.5) and mixed. 51 μL of the protein complex prepared in Example 9-115 was further added and incubated at room temperature for 60 minutes to react. The reaction solution was added to an ultrafiltration membrane (Amicon Ultra10K, Millipore), and 50 mmol / L sodium acetate buffer was further added for ultrafiltration purification, thereby obtaining the target 64Cu-protein complex solution (A) 140 μL of the protein complex and 145 μL of the PBS solution were mixed with the obtained solution, and filtered using a syringe filter (Millex-GV 0.22 μm, Millipore) to prepare a solution containing 64 Cu-protein complex (A) in PBS (11.5 MBq, 19.17 MBq / mg).

[1348] 64 Preparation Example 2 of Cu-labeled protein

[1349] Towards[ 64 20 μL of 0.05 mol / L hydrochloric acid solution of Cu]CuCl2 (37.1 MBq, Fujifilm Toyama Chemical) was added with 285 μL of 0.1 mol / L sodium acetate buffer (pH 6.5) and mixed. 94.7 μL of the protein complex prepared in Example 9-116 was further added and incubated at room temperature for 60 minutes to react. The reaction solution was added to an ultrafiltration membrane (Amicon Ultra10K, Millipore), and 50 mmol / L sodium acetate buffer was further added for ultrafiltration purification to obtain the target 64 Cu-protein complex solution (B) 260 μL of the protein complex and 377 μL of the PBS solution were mixed with the obtained solution, and filtered using a syringe filter (Millex-GV 0.22 μm, Millipore) to prepare a solution containing 64 Cu-protein complex (B) in PBS (31.5 MBq, 26.27 MBq / mg).

[1350] PET / CT

[1351] Marmosets (2 years old) were anesthetized with isoflurane and administered via the tail vein with 64 175-185 μL of the Cu-protein complex solution (A) or (B) in PBS was used. After administration, images were acquired under anesthesia using a PET / CT imaging device (PET: Clairvivo PET, manufactured by Shimadzu Corporation, CT: Aquilion, manufactured by TOSHIBA).

[1352] Dosage includes 64 The PET / CT images obtained after about 3 hours of the Cu-protein complex solution (A) in PBS solution are as follows: Figure 1 In addition, the administration comprises 64 The PET / CT images obtained after about 3 hours of the Cu-protein complex solution (B) in PBS solution are as follows: Figure 2 . Figure 3SUV is shown. SUV is the value obtained by dividing the radioactivity per 1g of tissue by the radioactivity per 1g of body weight, that is, the value represented by SUV = radioactivity per 1g of tissue / radioactivity per 1g of body weight. It should be noted that the radioactivity uses the value after attenuation correction. Figure 1 compared to, Figure 2 Less accumulation was observed in the kidney.

[1353] Industrial Applicability

[1354] The present invention includes a complex having excellent binding activity to human CEACAM5, and is expected to be useful in the diagnosis and / or treatment of human CEACAM5-related cancers.

[1355] In addition, the present invention includes a complex having excellent binding activity to MUC1, and is expected to be useful in the diagnosis and / or treatment of MUC1-related cancers.

[1356] In addition, the complex of the present invention, that is, the complex comprising three-arm DOTA, a specific spacer, a specific peptide linker, and a biomolecule, can be degraded and excreted in the kidneys, and therefore is expected to be useful in the diagnosis and / or treatment of biomolecule-related diseases.

[1357] Sequence Listing Free Text

[1358] The description of the representative "artificial sequence" is recorded in the digital title <223> of the following sequence listing. Specifically, the base sequences shown in sequence numbers 1 and 3 of the sequence listing are the base sequences of the heavy chain fragment and light chain of PB009-01, respectively, and the amino acid sequences shown in sequence numbers 2 and 4 are the amino acid sequences of the heavy chain fragment and light chain encoded by sequence numbers 1 and 3, respectively. In addition, the base sequences shown in sequence numbers 5 and 9 of the sequence listing are the base sequences of the heavy chain fragment and light chain of P10-1, respectively, and the amino acid sequences shown in sequence numbers 6 and 10 are the amino acid sequences of the heavy chain fragment and light chain encoded by sequence numbers 5 and 9, respectively. The base sequences shown in sequence numbers 7 and 9 are the base sequences of the heavy chain fragment and light chain of P10-2, respectively, and the amino acid sequences shown in sequence numbers 8 and 10 are the amino acid sequences of the heavy chain fragment and light chain encoded by sequence numbers 7 and 9, respectively. Sequence numbers 11 and 15 are the heavy chain variable region and light chain variable region of P10-1, respectively, and the amino acid sequences shown in sequence numbers 12 and 16 are the amino acid sequences of the heavy chain variable region and light chain variable region encoded by sequence numbers 11 and 15, respectively. Sequence numbers 13 and 15 are the heavy chain variable region and light chain variable region of P10-2, respectively, and the amino acid sequences shown in sequence numbers 14 and 16 are the amino acid sequences of the heavy chain variable region and light chain variable region encoded by sequence numbers 13 and 15, respectively. Sequence Listing <110> Astellas Pharma Inc. <120> Complexes containing ligands, spacers, peptide linkers and biomolecules <130> A20002A00 <150> JP 2019-000530 <151> 2019-01-07 <150> JP 2019-206560 <151> 2019-11-14 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 678 <212> DNA <213> Artificial sequence <220> <223> DNA encoding PB009-01 Fab heavy chain <220> <221> CDS <222> (1)..(678) <400> 1 gaa gtg cag ctg gtg gaa tct ggc ggc gga ctg gtg cag cct ggc gga 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 tct ctg aga ctg agc tgt gcc gcc agc ggc ttc aac atc cgg gac acc 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 tac atg cac tgg gtg cgc cag gcc cct ggc aag gga ctg gaa tgg gtg 144 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gcc aga atc gac ccc gcc aac ggc aac agc aga tac gtg ccc aag ttc 192 Ala Arg Ile Asp Pro Ala Asn Gly Asn Ser Arg Tyr Val Pro Lys Phe 50 55 60 cag ggc cgg ttc acc atc agc gcc gac acc agc aga aac acc gcc tac 240 Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Arg Asn Thr Ala Tyr 65 70 75 80 ctg cag atg aac agc ctg cgg gcc gag gac acc gcc gtg tac tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcc ccc ttc ggc tac tac gtg tcc gac tac gcc atg gcc tat tgg ggc 336 Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110 cag ggc acc ctc gtg aca gtg tcc tca gcc tcc acc aag ggc cca tcg 384 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg 432 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg 480 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct 528 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 gtc cta cag tcc tca gga ctc tac tcc ctt agt agc gtg gtg acc gtg 576 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac 624 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 aag ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct tgt 672 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 gac tga 678 Asp 225 <210> 2 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 2 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Arg Asp Thr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Asp Pro Ala Asn Gly Asn Ser Arg Tyr Val Pro Lys Phe 50 55 60 Gln Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Arg Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp 225 <210> 3 <211> 657 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding the light chain of PB009-01 <220> <221> CDS <222> (1)..(657) <400> 3 gac atc cag ctg acc cag agc cct agc agc ctg tct gcc agc gtg ggc 48 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtg acc atc acc tgt aga gcc ggc gag agc gtg gac atc ttc 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30 ggc gtg gga ttt ctg cac tgg tat cag cag aag ccc ggc aag gcc ccc 144 Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 aag ctg ctg atc tac aga gcc agc aac ctg gaa agc ggc atc ccc agc 192 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser 50 55 60 aga ttc agc ggc agc ggc tcc aga acc gac ttc acc ctg acc atc agc 240 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 agc ctg cag ccc gag gac ttc gcc acc tac tac tgc cag cag acc aac 288 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 gag gac ccc tac acc ttt ggc cag ggc acc aag gtg gaa atc aag cgt 336 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 acg gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag 384 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat 432 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg 480 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc acc 528 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 tac agc ctg agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa 576 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 cac aaa gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc 624 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 gtc aca aag agc ttc aac agg gga gag tgt tag 657 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 4 <211> 218 <212> PRT <213> artificial sequence <220> <223> synthetic construction <400> 4 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30 Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding the P10-1 Fab heavy chain fragment <400> 5 caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 60 tcctgcaagg ccagcggcta caccttcacc aactacggcc tgagctgggt gcgccaggct 120 cctggacagg gactggaatg gatgggcgag aaccaccctg gcagcggcat catctaccac 180 aacgagaagt tccggggcag agtgaccctg accgccgacc ggagcaccag caccgcctac 240 atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgtgc cagaagcagc 300 ggcaccagag gctttgccta ttggggacag ggcaccctcg tgaccgtgtc ctcagcctcc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctta gtagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgactga 669 <210> 6 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> P10-1 Fab Heavy Chain Fragment <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Asn His Pro Gly Ser Gly Ile Ile Tyr His Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Gly Thr Arg Gly Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 <210> 7 <211> 669 <212> DNA <213> Artificial sequence <220> <223> DNA encoding P10-2 Fab heavy chain fragment <400> 7 caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 60 tcctgcaagg ccagcggcta caccttcacc aactacggcc tgagctgggt gcgccaggct 120 cctggacagg gactggaatg gatgggcgag aaccaccctg gcagcggcat catctaccac 180 aacgagaagt tccggggcag agtgaccctg accgccgacc ggagcaccag caccgcctac 240 atggaactga gcagcctgcg gagcgaggac accgccgtgt actactgtgc cagaagcagc 300 ggcaccagag gctttgacta ttggggacag ggcaccctcg tgaccgtgtc ctcagcctcc 360 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 420 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540 tactccctta gtagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 600 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660 tgtgactga 669 <210> 8 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> P10-2 Fab Heavy Chain Fragment <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Asn His Pro Gly Ser Gly Ile Ile Tyr His Asn Glu Lys Phe 50 55 60 Arg Gly Arg Val Thr Leu Thr Ala Asp Arg Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Gly Thr Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 <210> 9 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding antibody light chain <400> 9 gacgtcgtga tgacccagac ccctctgagc ctgagcgtga cacctggaca gcctgccagc 60 atcagctgca gatccagcca gagcatcgtg cacagcaacg gcaacaccta cctggaatgg 120 tatctgcaga agcccggcca gagcccccag ctgctgatct acagggtgtc caaccggttc 180 agcggcgtgc ccgacagatt ttctggcagc ggctccggca ccgacttcac cctgaagatc 240 tcccgggtgg aagccgagga cgtgggcgtg tactactgtt ttcaaggcag ccacggcccc 300 tggacctttg gccagggaac aaagctggaa atcaagcgta cggtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctg 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgttag 660 <210> 10 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> Antibody Light Chain <400> 10 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro...

Claims

1. A complex of the following formula, In the formula, Fab 5 The following antibody Fab fragments: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, p is a natural number from 1 to 25, Fab 5 With the help of this Fab 5 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

2. A complex of the following formula, In the formula, Fab 5 The following antibody Fab fragments: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, p is a natural number from 1 to 25, Fab 5 With the help of this Fab 5 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

3. A complex of the following formula, In the formula, Fab 5 The following antibody Fab fragments: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, p is a natural number from 1 to 25, Fab 5 With the help of this Fab 5 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

4. A complex of the following formula, In the formula, Fab 5 The following antibody Fab fragments: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, p is a natural number from 1 to 25, Fab 5 With the help of this Fab 5 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

5. A complex of the following formula: In the formula, Fab 5 The following antibody Fab fragments: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence shown in SEQ ID NO: 8 in which the glutamine at amino acid No. 1 of SEQ ID NO: 8 is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10, p is a natural number from 1 to 25, Fab 5 With the help of this Fab 5 The p amino groups or thiol groups present in the moiety are bonded to adjacent carbon atoms.

6. The composite according to any one of claims 1 to 5, wherein p is a natural number from 1 to 9.

7. The composite according to claim 6, wherein p is a natural number ranging from 1 to 4.

8. The composite according to any one of claims 1 to 5, wherein Metal is coordinated on the three-arm DOTA.

9. The composite according to claim 8, wherein Metals are radioactive isotopes of metals.

10. The composite according to claim 9, wherein p is a natural number from 1 to 9.

11. The composite according to claim 10, wherein p is a natural number ranging from 1 to 4.

12. The composite according to claim 9, wherein Metal 89 Zr.

13. The composite according to claim 8, wherein The metal is a paramagnetic metal ion.

14. The composite according to claim 13, wherein Metal is Gd 3+ . The complex according to claim 8 , which is a PET tracer. 16 . The complex according to claim 8 , which is used for diagnosis of cancer expressing human MUCl and / or treatment of cancer expressing human MUCl , wherein the cancer is breast cancer.

17. A diagnostic composition comprising one or more complexes according to any one of claims 8 to 15 and a pharmaceutically acceptable carrier, wherein the diagnostic composition is used for diagnosing cancer expressing human MUC1, wherein the cancer is breast cancer.

18. The diagnostic composition according to claim 17, which is used as an early diagnosis drug or a staging diagnosis drug.

19. A pharmaceutical composition comprising one or more complexes according to any one of claims 8 to 14 and a pharmaceutically acceptable carrier. 20 . The pharmaceutical composition according to claim 19 , which is a pharmaceutical composition for treating cancer expressing human MUC1, wherein the cancer is breast cancer.

21. Use of the complex according to any one of claims 8 to 14 for producing a composition for diagnosing cancer expressing human MUC1 and / or a pharmaceutical composition for treating cancer expressing human MUC1, wherein the cancer is breast cancer.

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