RU2807081C2 - Conjugate including ligand, spacer, peptide linker and biomolecule - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: conjugate of Formula (Ic), including a ligand, a spacer and a peptide linker, useful for an in-vivo diagnostic agent and internal radiation therapy, using a Fab fragment of an antibody to human MUC1, the binding activity of which is not weakened even when labeled with a fluorescent metal dye or so.

biomolecule

EFFECT: diagnostic and pharmaceutical compositions intended for diagnosing or treating cancer expressing human MUC1 are provided.

31 cl, 3 dwg, 60 tbl, 10 ex

Description

Field of technology to which the invention relates

[0001] The present invention relates to a conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody or a Fab fragment of an anti-human MUC1 antibody and a ligand. The present invention also relates to a diagnostic composition and/or pharmaceutical composition including the conjugate, a method for diagnosing and/or treating cancer using the conjugate, and the like. In addition, a conjugate comprising a ligand, a spacer, a peptide linker and a biomolecule, a diagnostic composition and/or a pharmaceutical composition including the conjugate, a method for diagnosing and/or treating a disease associated with the biomolecule using the conjugate, etc. Furthermore, the present invention provides a conjugate comprising a complex formed from a ligand and a metal and a Fab fragment of an anti-human CEACAM5 antibody or a Fab fragment of an anti-human MUC1 antibody. In addition, the present invention relates to a conjugate comprising a complex, a spacer, a peptide linker and a biomolecule.

Prerequisites for creating the invention

[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 to date 23 CEA-related molecules have been identified (BioMed Central Biology; 2010;8:12-33). Of these, CEACAM5 is rarely expressed in normal tissues, but is expressed in the fetal digestive tract and colorectal cancer (BBA; 1990;1032:177-189, J. Clin. Mol. Pathol.; 1999; 52:174-178). In addition, CEACAM5 is also 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] The concentration of CEACAM5 in the blood is higher in patients with colorectal cancer than in healthy subjects (J. Exp. Med.; 1965; 121:439-462), and CEACAM5 is used as a tumor marker. In a histological study of patients with colorectal cancer, high expression of CEACAM5 was found in 90% or more tissues (British J. Cancer; 2013; 108:662-667).

Early metastases of colorectal cancer are localized to the liver, and thus the recurrence rate can be reduced if liver metastases can be detected and treated at an early stage (Cell Mol. Gastroenterol. Hepatol.; 2017; 3:163-173).

Mucin 1 (Mucin 1: MUC1) is a membrane-bound glycoprotein expressed on the luminal side of epithelial cells forming the epithelial tissues of the mammary gland, trachea of the digestive tract, etc. (Nat. Rev. Cancer, 2004 Jan; 4(1):45-60). MUC1 is overexpressed in cancer cells of 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), kidney cancer (Mod. Pathol., 2004 Feb; 17(2):180-8), ovarian cancer (Gynecol. Oncol., 2007 Jun; 105(3 ):695-702), cervical cancer (Am. J. Clin. Pathol., 2004 Jul; 122(1):61-9), etc., and MUC1 is useful as a target molecule for the detection of cancerous lesions (Nat. Rev. Cancer 2004 Jan; 4(1):45-60, Pathol. Res. Pract. 2010 Aug15; 206(8):585-9).

MUC1 is O-glycosylated at threonine 9 HGVTSAPDTRPAPGSTAPPA (SEQ ID NO: 19 in the sequence listing herein), which is a 20 amino acid tandem repeat sequence present in the extracellular domain. It is known that this O-glycosylation is incomplete in cancer cells and that O-glycosylations such as T (Galβ1-3GalNAcα1-O-Ser/Thr), Tn (GalNAcα1-O-Ser/Thr) and 2,3ST (Neu5Acα2- 3Galβ1-3GalNAcα-O-Ser/Thr) occur in a cancer-specific manner (PTL 1 and NPL 1). MUC1 in normal tissues does not undergo these cancer-specific O-glycosylations, and thus human cancer-specific MUC1 is particularly useful as a target molecule for the treatment of various types of human cancer. Such antibodies to human cancer-specific MUC1 are, for example, 1B2 antibody (PTL 1), PankoMab antibody (NPL 2) and 5E5 antibody (PTL 2). Of these antibodies, the 1B2 antibody has been reported to have higher specificity for human cancer-specific MUC1 than the PankoMab antibody (PTL 1).

CT (computed tomography), MRI (nuclear magnetic resonance imaging) and FDG-PET (fluorodeoxyglucose positron emission tomography) are used to diagnose liver metastases. The detection sensitivities of CT, MRI, and FDG-PET are 74.4, 80.3, and 81.4%, respectively, and for tumors 1 cm or smaller, the detection sensitivity drops to 47.3% for CT and 60.2% for MRI. . (Radiology; 2010;257:674-684). Contrast-enhanced MRI of the liver is also used, and the detection sensitivity of this method is 29-38% for tumors 1 cm or smaller (Radiology; 2005; 237:89-98).

[0004] Anticancer agents and antibodies associated with metal radioisotopes are used for the diagnosis and treatment of cancer. Antibody targeting is highly specific for tumor cells and has few side effects. Today, some metal radiolabeled monoclonal antibodies are used clinically in diagnosis and treatment (Cancer Control; 2012; 19:196-203).

On the other hand, antibodies generally have a long half-life in the blood, and after their introduction into the body, a period of 4 days to 5 days is required to achieve a ratio of their content in the tumor to the content in the blood that gives a sufficient signal for cancer imaging (Clin Pharmacol Ther 2010;87:586-592). In addition, the Fc region of an antibody causes pharmacological effects of antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) (Glycoconj. J.; 2013; 30:227-236, Curr. Opin. Biotechnol.; 2002; 13: 609-614). In addition, antibodies are metabolized in the liver, and thus they accumulate to a high degree in the liver regardless of the target, but early metastases of colorectal cancer are localized in the liver, and thus it is difficult to detect lesions such as liver metastases using antibodies (Clin. Pharmacol. Ther.; 2010; 87:586-592).

Low molecular weight recombinant antibody fragments such as Fab, scFv and diabody have high tissue penetration and easy access to lesions and are expected to be produced at low cost using an Escherichia coli or yeast based expression system and thus Thus, they are used as antibodies for treatment, where they are characterized as having a short half-life in the blood and being excreted by the kidneys and thus they have been described for use as diagnostic drugs (Nat. Biotechnol.; 2005; 23: 1126-1136).

[0005] As an antibody to human CEACAM5 used as a diagnostic drug, M5A (PTL 3), which is a humanized antibody from the murine monoclonal antibody T84.66, is known. For ⁶⁴ Cu-labeled M5A, a trial using a mouse subcutaneously transplanted with cancer cells reported that 22 hours or more must pass after administration to obtain a good PET image (NPL 3), and further, a trial using a mouse model liver metastases, it was reported that uptake in normal liver tissue and uptake in diseased liver tissue was almost the same 3 hours after administration, and that there was a significant difference after 24 hours (NPL 4).

Regarding the anti-human CEACAM5 antibody fragment, it has been reported that CEA-Scan, which is a ^99m Tc-labeled mouse monoclonal antibody NP-4 Fab', can be used for the diagnosis of colorectal cancer (NPL 5). However, CEA-Scan uptake in diseased areas does not exceed that in normal liver tissue, and the detection sensitivity of liver metastases is lower than FDG-PET (NPL 6). CEA-Scan was approved by the FDA as a diagnostic drug for colorectal cancer in 1999, but it is no longer marketed (NPL 7).

DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) has clinical results and is widely used as a radioactive metal chelator. In recent years, a study has been reported in which metal labeling is carried out using DOTA, followed by peptide and antibody binding and targeting (NPL 8).

In terms of cancer treatment, Satoreotide tetraxetane (NPL 9) has been reported to be in Phase I development as a DOTA drug. It was reported that ⁹⁰ Y-epratuzumab tetraxetane was administered to a patient having a hematological tumor (NPL 10).

Typically, the conjugate to which a chelating agent, such as DOTA, and a small molecule antibody or peptide is bound is highly absorbed, retained, or accumulated in the kidney (NPLs 11 and 12).

As described above, accumulation of the conjugate in the kidneys is inconvenient for accurate diagnosis and treatment (NPL 13).

[0006] For example, in a test in mice treated with the [ ¹¹¹ In]DOTA-Rituxan Fab conjugate, it was reported to accumulate highly in the kidneys (NPL 11).

To avoid such high accumulation in the kidney, reference may be made to a first study of a conjugate modified to an antibody fragment such as Fab, scFV, Fab' or dsFV, and a second study of a conjugate containing a linker (also referred to as a peptide linker) specifically cleaved in the kidney , between the chelate and the antibody (NPL 12).

It was reported that initially, iodohippuric acid-Gly-Lys-Fab, in which the peptide linker is Gly (glycine)-Lys (lysine), is cleaved by the renal brush border membrane enzyme, and iodohypuric acid is contained in the urine as a metabolite and excreted (NPL 14). In addition, iodohippuric acid-Gly-Tys-Fab, in which the peptide linker is Gly-Tyr (tyrosine) (NPL 13), has been described.

On the other hand, [ ¹⁸⁸ Re]CpTR-Gly-Lys-Fab has been reported in which the Gly-Lys peptide linker is linked to the organorhenium complex CpTR-COOH rather than to hippuric acid (NPL 15).

Additionally, ^{a 99m} Tc-PGGFML-IT-Fab in which the peptide linker is Gly-Phe (phenylalanine)-Lys (PTL 4) has been reported.

Additionally, focusing on NOTA as a chelate, a conjugate consisting of NOTA and a peptide linker (PTL 5) has been described.

A conjugate containing a peptide linker has been created, but depending on the type of chelating agent, there is a problem of lack of enzyme cleavage, and the conjugate is designed to solve the problem by introducing a linking moiety (-CH ₂ -Ph-CO-NH-), having a specific structure, between chelating agent and peptide linker (PTL 6). The goal is to obtain a conjugate containing a ligand that can coordinate an atom having a relatively large atomic radius, such as indium, which is commonly used as a radioisotope. A spacer having the thiourea structure described in PTL 5 was introduced as a spacer via a chelate and a peptide linker, but it was stated that the enzyme did not degrade the bud brush border membrane.

The ⁶⁷ Ga-NOTA-Met-Ile-Fab conjugate, in which the peptide linker targeted for lysosome degradation is Met (methionine)-Ile (isoleucine), was reported based on the following data (NPL 16).

Metabolite ⁶⁷ Ga-NOTA-Bn-Met, produced by lysosomal cleavage of the NOTA-(p-SCN-Bz)-dsFv conjugate lacking a peptide linker, was reported to be excreted in urine (NPL 17), and the second from the N-terminus of the light chain The dsFv of the above NOTA-(p-SCN-Bz)-dsFv conjugate is Ile, and therefore ^{the 67} Ga-NOTA-Met-Ile-Her2 (Herceptin) Fab (NPL 19) conjugate was designed.

List of cited documents

Non-patent literature

[0007] NPL 1: Glycoconj. J., 2013 Apr;30(3):227-36.

NPL 2: Cancer Immunol Immunother, 2006 Nov; 55(11): 1337-47

NPL 3: Bioconjug. Chem.; 2008; 19:89-96

NPL 4: PLOS ONE; 2014; 9(9): e106921

NPL 5: Ann. Surg.; 1997; 226:621-631

NPL 6: J. Nucl. Med.; 2000; 41: 1657-1663

NPL 7: Kenneth T. Cheng, "99mTc-Arcitumomab", [online], Update: March 17, 2008., Molecular Imaging and Contrast Agent Database, [searched on May 17, 2017], Internet <URL: https:// www.ncbi.nlm.nih.gov/books/NBK23676/>

NPL 8: Bioorg. Med. Chem.; 2019; 27: 3248-3253

NPL 9: Clinical Trials. gov Identifier: NCT02592707

NPL 10: Eur J Haematol. Dec 2013; 91(6): 552-6

NPL 11: Bioconjugate Chem. 2001, 12, 264-270

NPL 12: Bioconjugate Chem. 2002, 13, 985-995

NPL 13: Bioconjugate Chem. 2013, 24, 291-299

NPL 14: Cancer Res. 1999, 59, 128-134

NPL 15: Bioconjugate Chem. 2007, 18, 190-198

NPL 16: Bioconjugate Chem. 2014, 25, 2038-2045

NPL 17: Bioconjugate Chem. 1997, 8, 365-369 CITATION LIST

Patent literature

[0008] PTL 1: International publication WO2010/050528

PTL 2: International publication WO2008/040362

PTL 3: International publication WO2005/086875

PTL 4: International publication WO2013/081091

PTL 5: International publication WO2017/150549

PTL 6: International publication WO2019/065774

PTL 7: International publication WO2018/092885

The essence of the invention

Technical problem

[0009] The monovalent Fab fragment has a molecular weight of about 50 kDa, less than an antibody with a molecular weight of about 150 kDa, is excreted by the kidneys, and has a short half-life in the blood. Because of this, within 2-32 hours after injection, a tumor/blood ratio is reached that provides sufficient signal for cancer imaging. The Fab fragment does not have an Fc region and therefore does not cause ADCC or CDC. The Fab fragment is mainly excreted by the kidneys and thus does not interfere with the detection of liver metastases. Based on these characteristics, the Fab fragment would be expected to be more effective as an in vivo diagnostic drug than an antibody.

However, in the Fab fragment, the binding activity of the Fab fragment is often weakened due to the fact that it is monovalent rather than divalent like an antibody. In addition, in order to use an antibody as an in vivo diagnostic drug or an agent used in photoimmunotherapy, the antibody must be labeled with a metal, a fluorescent dye, etc., but the problem is that when labeled with such a substance, the binding activity of the antibody is weakened.

An object of the present invention is to provide a labeled conjugate useful for in vivo diagnostic drug and internal radiotherapy using the Fab fragment of anti-human CEACAM5 antibody, the binding activity of which is not attenuated even by labeling with a metal, fluorescent dye or the like. An object of the present invention is to provide a conjugate comprising an anti-human MUC1 antibody Fab fragment (PTL 7), a peptide linker and a ligand, and a conjugate comprising an anti-human MUC1 antibody Fab fragment and a ligand. Moreover, another object of the present invention is to provide a diagnostic composition comprising the above conjugate and a diagnostic method using it, as well as a pharmaceutical composition including the above conjugate and a treatment method using it.

It is also an object of the present invention to provide a conjugate containing a chelator and a biomolecule that accumulates in the kidneys and is excreted more quickly.

The solution of the problem

[0010] The present inventors have previously obtained a Fab fragment of an anti-human CEACAM5 antibody having good affinity for human CEACAM5 (International Application PCT/JP2018/025618). As a result of additional careful research, the present inventors obtained a conjugate in which the ligand used for labeling is associated with the Fab fragment of the anti-human CEACAM5 antibody through a peptide linker (or without it), and found that the conjugate has the same affinity for human CEACAM5, as the Fab fragment of the anti-human CEACAM5 antibody itself, that is, the binding activity is not weakened even by binding between the labeling part and the Fab fragment, which led to the present invention. Thus, the present invention provides a conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody, a peptide linker and a ligand, and a conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody and a specific ligand. It was confirmed that the conjugate does not weaken the binding activity to human CEACAM5 even when bound between the labeling part and the Fab fragment, and retains good binding activity to human CEACAM5, and based on these results, a diagnostic agent and a therapeutic agent using the conjugate are provided according to the present invention.

[0011] In addition, the inventors of the present invention have obtained a Fab fragment of an anti-human MUC1 antibody having good affinity for human cancer-specific MUC1, and through further careful studies have obtained a conjugate in which a ligand is linked to a Fab fragment of an anti-human MUC1 antibody through peptide linker (or without it). The conjugate has the same affinity for human cancer-specific MUC1 as the Fab fragment of the anti-human MUC1 antibody itself. Thus, the present invention provides a conjugate comprising a Fab fragment of an anti-human MUC1 antibody, a peptide linker and a ligand, and a conjugate comprising a Fab fragment of an anti-human MUC1 antibody and a specific ligand. In addition, it was confirmed that the conjugate does not weaken the binding activity to human cancer-specific MUC1 even when the labeling part is bound and retains good binding activity to human cancer-specific MUC1, and based on these results, a diagnostic agent and a treatment agent using conjugate of the present invention.

In addition, the inventors of the present invention have studied a conjugate that is excreted faster because a conjugate consisting of a complex formed of a ligand and a metal (also referred to as a metal complex), a spacer, a peptide linker, and a biomolecule such as an antibody useful as a drug agents, such as a contrast agent or an anticancer agent, may accumulate in the kidneys. As a result, the inventors of the present invention focused on DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which has clinical results and is widely used as a radioactive metal chelator, and found that a conjugate consisting of 3arm DOTA, a specific spacer, a specific peptide linker and a biomolecule is degraded in the kidneys and excreted.

Based on the above, the present invention relates to the following conjugate comprising Fab antibodies to CEACAM5, a diagnostic composition and/or pharmaceutical composition including the conjugate, a method for diagnosing and/or treating cancer using the conjugate, and the like. In addition, the present invention relates to the following conjugate comprising anti-MUC1 Fab antibodies, a diagnostic composition and/or a pharmaceutical composition including the conjugate, a method for diagnosing and/or treating cancer using the conjugate, and the like. Furthermore, the present invention provides a conjugate comprising DOTA, a spacer, a peptide linker and a biomolecule that is rapidly excreted by the kidneys, a conjugate intermediate, and a method for diagnosing and/or treating a disease associated with the biomolecule using the conjugate and the like.

[0012] [1] The conjugate represented by the following formula (I):

(YS ₁ -X) _p -Fab ¹ (I)

Where

Fab¹represents A Fab fragment of an anti-human CEACAM5 antibody selected from the group consisting of

(a) a Fab fragment of an anti-human CEACAM5 antibody, 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 comprising a light chain variable region consisting of the amino acid sequence amino acids 1-112 SEQ ID NO: 4, and

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

Fab ¹ is linked to X through p number of amino groups or thiol groups in Fab ¹ ;

X represents a peptide linker or bond;

S₁represents spacer or connection;

Y is a ligand; And

p is a natural number from 1 to 25 and represents the number (Y-S1-X) associated with Fab ¹ ;

with the proviso that when X is a bond, S ₁ is -CH ₂ -(1,4-phenylene)-NH-C(=S)- or bond, and Y is 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (hereinafter sometimes abbreviated as DOTA).

[0013] [2] Conjugate in accordance with [1], where

Fab ¹ is selected from the group consisting of

(a) an anti-human CEACAM5 Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, and

(b) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 2 and in which glutamic acid, amino acid 1 in SEQ ID NO: 2, is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

[0014] [3] A conjugate according to [2], where Fab ¹ includes a Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence presented in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by in SEQ ID NO: 4 (hereinafter referred to as Fab ² ).

[4] A conjugate according to any of [1]-[3], wherein X is a peptide linker comprising a peptide of 2-4 amino acids having an amino acid sequence cleavable by a renal brush border membrane enzyme or a lysosomal enzyme.

[0015] [5] A conjugate according to any of [1]-[4], where

S ₁ represents

-C(=O)-CH ₂ O-(1,3-phenylene)-C(=O)-,

-C(=O)-(CH ₂ CH ₂ O) ₄ -(1,3-phenylene)-C(=O)-,

-C(=O)-(1,3-phenylene)-C(=O)-,

-NH-CH ₂ -(1,3-phenylene)-C(=O)-,

-NH-(CH ₂ ) ₂ -C(=O)-,

-CH ₂ -(1,4-phenylene)-NH-C(=S)-, -NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1, 3-phenylene)-C(=O)-,

-NH-CH ₂ -(1,4-phenylene)-NH-C(=O)-,

-NH-(CH ₂ ) ₃ -C(=O)-,

-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-,

-NH-CH ₂ -(1,4-phenylene)-C(=O)-,

-CH ₂ -(1,4-phenylene)-NH-C(=S)-NH-CH ₂ -(1,3-phenylene)-C(=O)-,

-CH ₂ -(1,4-phenylene)-NH-C(=S)-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3- phenylene)-C(=O)-,

a spacer represented by any of the following formulas (a)-(q), or a bond,

[0016] [Chemical formula 1]

[0017] where R ¹ means a hydrogen atom, halogen or C _1-6 alkyl, and the same applies to the following; And

X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(4) -Met-Val-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ -,

(7) -Gly-Arg-Lys*-Z ₂ -,

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

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(13) -Gly-Tyr*-CH ₂ -C(=O)-Lys*-Z ₂ -,

(14) -Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(15) -Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(16) -Gly-Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(17) -Gly-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(19) -Gly-Tyr-Lys*-Z ₂ -,

(20) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ O) ₃ -CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(22) -Gly-diphenylalanine-Lys*-Z ₂ -,

(23) -Gly-Tyr-NH-(CH ₂ ) ₅ -Z ₁ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ - or

(29) communication,

where Met is methionine, Ile is isoleucine, Gly is glycine, Lys is lysine, Phe is phenylalanine, Val is valine, Tyr is tyrosine, Arg is arginine, Asp is aspartic acid, Z ₁ is is a group represented by the following formula (II-I) or (II-II), and -Lys*-Z ₂ is a group represented by the following formula (III-I) or (III-II), Tyr*-CH ₂ - is a group represented by the following formula (IV), -Lys*-C(=S)- is a group represented by the following formula (V), -Lys*-Z ₃ - is a group represented by the following formula (III- III), and

group (A-3), (A-4) or (A-5) is as represented by the following formulas.

[0018] [Chemical formula 2]

[0019] [6] A conjugate according to any of [1]-[5], wherein S ₁ is -C(=O)-CH ₂ O-(1,3-phenylene)-C(=O)- , -C(=O)-(CH ₂ CH ₂ O) ₄ -(1,3-phenylene)-C(=O)- or -C(=O)-(1,3-phenylene)-C(= O)-, and

X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ - and

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

[7-1] A conjugate according to any of [1]-[5], wherein S ₁ is -C(=O)-CH ₂ O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH ₂ CH ₂ O) ₄ -(1,3-phenylene)-C(=O)-, -NH-CH ₂ -(1,3-phenylene)-C(=O) -, -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)-, -NH-CH ₂ -(1,4-phenylene)-C(=O)-, -C(= O)-(1,3-phenylene)-C(=O)- or the following formula

[0020] [Chemical formula 3]

,

And

X is a peptide linker selected from the group consisting of

[0021] (1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6)-Gly-Lys-Lys*-Z ₂ -,

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

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[7-2] The conjugate according to [7-1], where S ₁ is -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)- or the following formula

[0022] [Chemical formula 4]

,

And

[0023] X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[8] A conjugate according to any of [1]-[5], wherein S ₁ is -NH-CH ₂ -(1,3-phenylene)-C(=O)-, -NH-(CH ₂ ) ₂ -C(=O)-, -CH ₂ -(1,4-phenylene)-NH-C(=S)-, -NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O) -NH-CH ₂ -(1,3-phenylene)-C(=O)-, -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)-, -NH-(CH ₂ ) ₃ -C(=O)-, -NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-, -NH-CH ₂ -(1,4-phenylene)-C(=O )-, -CH ₂ -(1,4-phenylene)-NH-C(=S)-NH-CH ₂ -(1,3-phenylene)-C(=O)-, -CH ₂ -(1, 4-phenylene)-NH-C(=S)-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-phenylene)-C(=O )-, spacer represented by any of the following formulas (a)-(q), or bond

[0024] [Chemical formula 5]

,

And

[0025] X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(4) -Met-Val-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(7) -Gly-Arg-Lys*-Z ₂ -,

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

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(13) -Gly-Tyr*-CH ₂ -C(=O)-Lys*-Z ₂ -,

(14) -Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(15) -Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(16) -Gly-Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(17) -Gly-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(19) -Gly-Tyr-Lys*-Z ₂ -,

(20) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ O) ₃ -CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(22) -Gly-diphenylalanine-Lys*-Z ₂ -,

(23) -Gly-Tyr-NH-(CH ₂ ) ₅ -Z ₁ -

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[0026] [9] A conjugate according to any of [1]-[5], wherein S ₁ is -NH-CH ₂ -(1,3-phenylene)-C(=O)-, -CH ₂ - (1,4-phenylene)-NH-C(=S)-, -NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-phenylene) -C(=O)-, -CH ₂ -(1,4-phenylene)-NH-C(=S)-NH-CH ₂ -(1,3-phenylene)-C(=O)-, -NH -CH ₂ -(1,4-phenylene)-NH-C(=O)-, a spacer represented by any of the following formulas (e)-(i) or (k), or a bond

[0027] [Chemical formula 6]

,

And

[0028] X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[10] A conjugate according to any of [1]-[5], where S₁represents the 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

[0029] [Chemical formula 7]

,

And

[0030] X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(4) -Met-Val-Lys*-Z ₂ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[11] A conjugate according to any one of [1]-[5], wherein X is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(12) -Met-Ile-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)- and

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)-.

[12] A conjugate according to any one of [1]-[5], wherein X is a peptide linker selected from the group consisting of

(4) -Met-Val-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(18) -Met-Phe-Lys*-Z ₂ - and

(28) -Met-Gly-Lys*-Z ₃ -.

[13] A conjugate according to any one of [1]-[5], wherein X is a peptide linker selected from the group consisting of

(11) -Met-Gly-Lys*-Z ₂ - and

(28) -Met-Gly-Lys*-Z ₃ -.

[14] A conjugate according to any one of [1] to [5], wherein the conjugate is a conjugate selected from the group consisting of compounds represented by the following formulas, and Fab ¹ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ¹ .

[0031] [Chemical formula 8]

[0032] [Chemical formula 9]

[0033] [Chemical formula 10]

[0034] [Chemical formula 11]

[0035] [Chemical formula 12]

[0036] [Chemical formula 13]

[0037] [15] A conjugate according to any of [1]-[14], wherein Y is deferoxamine (hereinafter sometimes abbreviated as DFO) or 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (sometimes abbreviated as DOTA).

[16] Conjugate according to [15], where Y is DFO.

[17] Conjugate according to [15], where Y is DOTA.

[18] Conjugate according to [17], where Y is 3arm DOTA or 4arm DOTA.

[19] Conjugate according to [18], where Y is 3arm DOTA.

[20] Conjugate according to [18], where Y is 4arm DOTA.

[21] A conjugate according to any one of [15] or [17]-[20], where the conjugate is a conjugate selected from the group consisting of compounds represented by the following formulas, and Fab ¹ is bonded to an adjacent carbon atom through a p number amino groups or thiol groups in Fab ¹ .

[0038] [Chemical formula 14]

,

[0039] [Chemical formula 15]

,

[0040] [Chemical formula 16]

,

[0041] [Chemical formula 17]

,

[0042] [Chemical formula 18]

,

[0043] [Chemical formula 19]

,

[0044] [Chemical formula 20]

,

[0045] [Chemical formula 21]

,

[0046] [Chemical formula 22]

,

[0047] [Chemical formula 23]

,

[0048] [Chemical formula 24]

[0049] [Chemical formula 25]

[0050] [Chemical formula 26]

[0051] [Chemical formula 27]

[0052] [Chemical formula 28]

[0053] [Chemical formula 29]

[0054] [Chemical formula 30]

[0055] [Chemical formula 31]

[0056]

Moreover, the conjugate of the present invention may be a mixture of the following two conjugates.

[0057] [Chemical formula 27]

[0058] [Chemical formula 28]

[22] A conjugate according to any one of [15] or [17]-[20], where the conjugate is a conjugate selected from the group consisting of compounds represented by the following formulas, and Fab ¹ is bonded to an adjacent carbon atom through a p number amino groups or thiol groups in Fab ¹ .

[0059] [Chemical formula 32]

[0060] [23] A conjugate according to any of [1]-[22], where p is a natural number from 1 to 5.

[24] A conjugate according to any of [1]-[20], where the metal is coordinated to Y.

[25] A conjugate according to any of [21]-[22], where the metal is coordinated.

[26] A conjugate according to [24] or [25], where the metal is a radioisotope of the metal.

[27] Conjugate according to [26], where the metal is ⁸⁹ Zr.

[28] A conjugate according to [24] or [25], where the metal is a paramagnetic metal ion.

[29] Conjugate according to [28], where the metal is Gd ³⁺ .

[30] A conjugate according to any one of [24]-[29], wherein the conjugate is a PET radiopharmaceutical.

[0061] [31] A diagnostic composition comprising one or more conjugates according to any of [24]-[30] and a pharmaceutically acceptable carrier.

[32] The diagnostic composition according to [31], wherein the diagnostic composition is used as a drug for early diagnosis or a drug for determining the stage of a disease.

[33] A diagnostic composition according to any one of [31] or [32], wherein the diagnostic composition is used for diagnosing cancer expressing human CEACAM5.

[34] The diagnostic composition according to [33], wherein the cancer is colorectal cancer, breast cancer, lung cancer, thyroid cancer or cancer resulting from metastasis thereof.

[0062] [35] A pharmaceutical composition comprising one or more conjugates according to any of [24]-[29] and a pharmaceutically acceptable carrier.

[36] The pharmaceutical composition according to [35], wherein the pharmaceutical composition is a pharmaceutical composition for treating cancer expressing human CEACAM5.

[37] The pharmaceutical composition according to [36], wherein the cancer is colorectal cancer, breast cancer, lung cancer, thyroid cancer or cancer resulting from metastasis thereof.

[38] Use of one or more in accordance with any of [24]-[29] for the preparation of a composition for diagnosing cancer and/or a pharmaceutical composition for treating cancer.

[0063] [39] A conjugate according to any of [24]-[30], wherein the conjugate is used for diagnosing cancer and/or treating cancer.

[40] A method for diagnosing cancer, comprising administering one or more conjugates according to any one of [24]-[30] to a subject.

[41] A method for treating cancer, comprising administering to a subject a therapeutically effective amount of a conjugate in accordance with any one of [24]-[30].

[42] The conjugate represented by the following formula (Ia)

[0064] [Chemical formula 33]

[0065] where

DOTA ¹ : 3arm DOTA,

U: bond or -NH(CH ₂ ) ₂ O(CH ₂ ) ₂ O(CH ₂ ) ₂ OCH ₂ C(=O)-

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

X: C or N

R ^1a , R ^1b : the same or different from each other, H or C _1-6 alkyl,

with the proviso that R ^1a and R ^1b together can form C _1-6 alkylene;

p represents a natural number from 1 to 25 and is bonded to an adjacent carbon atom via p amino groups or thiol groups in Biomolecule ¹ ;

R ¹ : H, halogen, C _1-6 alkyl or halogen C _1-6 alkyl,

R ² : C _1-6 alkyl or halogen C _1-6 alkyl,

L ² : Ile, Gly, Ala, Val, Phe, -NHCH(CHCH ₃ NR ³ R ⁴ )C(=O)-, -NHCH(CHCH ₃ N ₃ )C(=O)- or -NHCH(CHCH ₃ CH ₂ CH ₂ CH ₃ )C(=O)-,

R ³ : H, C _1-6 alkyl,

R ⁴ : H, C _1-6 alkyl,

L ³ : bond, Arg or His,

L ⁴ : -NH-(CH ₂ ) ₂ -, -NHCH(C(=O)OH)(CH ₂ ) ₄ - or bond,

V ¹ : group represented by any of the following formulas (A-1)-(A-5),

[0066] [Chemical formula 34]

[0067] or groups -L ³ -L ⁴ -V ¹ - together form the following formula

[0068] [Chemical formula 35]

[0069] Biomolecule ¹ : biomolecule

[0070] [Chemical formula 36]

dotted line Q is bonded to either of the two carbon atoms on the ring; dotted line single bond or double bond.

[0071] [43] The conjugate according to [42], where L ³ , L ⁴ and V ¹ represent the following groups.

L ³ : bond, Arg or His,

L ⁴ : -NH-(CH ₂ ) ₂ -, -NHCH(C(=O)OH)(CH ₂ ) ₄ - or bond,

V ¹ : group represented by any of the following formulas (A-1)-(A-5),

[0072] [Chemical formula 37]

[0073] [44]

Conjugate in accordance with [43], where

groups -L ³ -L ⁴ -V ¹ - together form the following formula.

[0074] [Chemical formula 38]

[0075] [45]

The conjugate represented by the following formula (Ib)

[0076] [Chemical formula 39]

[0077] where

DOTA ¹ : 3arm DOTA,

U: bond or -NH(CH ₂ ) ₂ O(CH ₂ ) ₂ O(CH ₂ ) ₂ OCH ₂ C(=O)-

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

X: C or N

R ^1a , R ^1b : the same or different from each other, H or C _1-6 alkyl,

with the proviso that R ^1a and R ^1b together can form C _1-6 alkylene;

p represents a natural number from 1 to 25 and is bonded to an adjacent carbon atom via p amino groups or thiol groups in Biomolecule ² ;

R ¹ : H, halogen, C _1-6 alkyl or halogen C _1-6 alkyl,

R ² : C _1-6 alkyl or halogen C _1-6 alkyl,

L ² : Ile, Gly, Ala, Val, Phe, -NHCH(CHCH ₃ NR ³ R ⁴ )C(=O)-, -NHCH(CHCH ₃ N ₃ )C(=O)- or -NHCH(CHCH ₃ CH ₂ CH ₂ CH ₃ )C(=O)-,

R ³ : H, C _1-6 alkyl,

R ⁴ : H, C _1-6 alkyl,

L ³ : bond, Arg or His,

L ⁴ : -NH-(CH ₂ ) ₂ -, -NHCH(C(=O)OH)(CH ₂ ) ₄ - or bond,

V ¹ : group represented by any of the following formulas (A-1)-(A-5),

[0078] [Chemical formula 40]

[0079] or groups -L ³ -L ⁴ -V ¹ - together form the following formula

[0080] [Chemical formula 41]

[0081]

Biomolecule ² : Antibody Fab fragment

[0082] [Chemical formula 42]

dotted line Q is bonded to either of the two carbon atoms on the ring; dotted line single bond or double bond.

[0083] [46-1] A conjugate according to any of [42]-[45], wherein the conjugate has the following formula (Ic):

[0084] [Chemical formula 43]

[0085] where

L ³ : communication,

L ⁴ : -NH-(CH ₂ ) ₂ - or -NHCH(C(=O)OH)(CH ₂ ) ₄ -,

Biomolecule: Biomolecule ¹ or Biomolecule ² .

[46-2] Conjugate in accordance with [46-1], where in formula (Ic),

L ³ represents a bond, and

L ⁴ represents -NH-(CH ₂ ) ₂ -.

[47-1] A conjugate according to any one of [42]-[46-2], wherein the conjugate is represented by the following formula (Id):

[0086] [Chemical formula 44]

[0087] where

L ³ : communication,

L ⁴ : -NH-(CH ₂ ) ₂ - or -NHCH(C(=O)OH)(CH ₂ ) ₄ -,

Biomolecule: Biomolecule¹or Biomolecule²

a conjugate according to any one of [42]-[45].

[47-2] Conjugate in accordance with [47-1], where in formula (Id),

L ³ represents a bond, and

L ⁴ represents -NH-(CH ₂ ) ₂ -.

[48]

[0089] A conjugate according to any of [42]-[47-2], wherein V ¹ is any of the following formulas (A-3)-(A-5).

[0088] [Chemical formula 45]

[49]

A conjugate according to any one of [42]-[48], where group (B) in formula (Ia) or (Ib)

[0090] [Chemical formula 46]

[0091] is a group selected from the group consisting of the following formulas (B-1) to (B-7).

[0092] [Chemical formula 47]

[0093] [Chemical formula 48]

[0094] [50] A conjugate according to any one of [42]-[49], wherein the conjugate is selected from the group consisting of compounds represented by the following formulas.

[0095] [Chemical formula 49]

[0096] [Chemical formula 50]

[0097] [Chemical formula 51]

[0098] [Chemical formula 52]

[0099] [Chemical formula 53]

[0100] [Chemical formula 54]

[0101] [Chemical formula 55]

[51-1]

A conjugate according to any one of [42]-[50], where Biomolecule¹And Biomolecule²each is a biomolecule or Fab fragment of an antibody, different from the following Fab fragments of antibodies:

(a) a Fab fragment of an anti-human CEACAM5 antibody, 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 comprising a light chain variable region consisting of the amino acid sequence amino acids 1-112 SEQ ID NO: 4, and

(b) a Fab fragment of an anti-human CEACAM5 antibody selected from the group consisting of a Fab fragment of an anti-human CEACAM5 antibody comprising a heavy chain fragment comprising a heavy chain variable region that consists of the amino acid sequence of amino acids 1-121 SEQ ID NO: 2 and wherein glutamic acid, amino acid 1 in SEQ ID NO: 2, is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 1-112 of SEQ ID NO: 4.

[51-2]

A conjugate according to [51-1], wherein Biomolecule ¹ and Biomolecule ² are each a biomolecule or Fab fragment of an antibody other than the following Fab antibody fragments:

(a) a Fab fragment of an anti-human CEACAM5 antibody, 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 comprising a light chain variable region consisting of the amino acid sequence amino acids 1-112 SEQ ID NO: 4, and

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

(c) an anti-human CEACAM5 Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4, or

(b) an anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 2 and in which glutamic acid, amino acid 1 in SEQ ID NO: 2, is modified to pyroglutamic acid, and a light chain including the light chain shown in SEQ ID NO: 4.

[0102] [52] A conjugate according to any one of [42]-[51-2], wherein Biomolecule¹or Biomolecule²represents An anti-human MUC1 antibody Fab fragment selected from the group consisting of the following (a) and (b):

(a) a Fab fragment of an anti-human MUC1 antibody 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 and a light chain including a light chain variable region consisting of the amino acid sequence presented in SEQ ID NO: 16,

And

(b) an anti-human MUC1 antibody Fab fragment selected from the group consisting of an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region that consists of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14, and wherein glutamine, amino acid 1 in 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.

[53] Conjugate in accordance with [52], where Biomolecule¹or Biomolecule²represents An anti-human MUC1 antibody Fab fragment selected from the group consisting of the following (a) and (b):

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

(b) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[0103] [54] Conjugate in accordance with [53], where Biomolecule¹or Biomolecule²is the following Fab fragment of an antibody to human MUC1:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

[55] A conjugate according to any of [42]-[54], where p is a natural number from 1 to 4.

[56] A conjugate according to any of [42]-[55], wherein the metal is coordinated to DOTA ¹ .

[57] A conjugate according to [56], where the metal is a radioisotope of the metal.

[58] A conjugate according to [57], where the metal is ⁸⁹ Zr.

[59] A conjugate according to [56], where the metal is a paramagnetic metal ion.

[60] Conjugate according to [59], where the metal is Gd ³⁺ .

[61] A conjugate according to any one of [56]-[60], wherein the conjugate is a PET radiopharmaceutical.

[62] A diagnostic composition comprising one or more conjugates according to any of [56]-[61] and a pharmaceutically acceptable carrier.

[63] The diagnostic composition according to [62], wherein the diagnostic composition is used as a drug for early diagnosis or a drug for determining the stage of a disease.

[64] A diagnostic composition according to any one of [62] or [63], wherein the diagnostic composition is used to diagnose a disease associated with the Biomolecule¹or Biomolecule².

[65] The diagnostic composition according to [64], wherein the disease associated with Biomolecule ¹ or Biomolecule ² is a disease associated with MUC1.

[66] A diagnostic composition according to [65], wherein the diagnostic composition is used for diagnosing cancer expressing MUC1.

[67] The diagnostic composition according to [66], 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.

[0104] [68] A pharmaceutical composition comprising one or more conjugates according to any of [56]-[60] and a pharmaceutically acceptable carrier.

[69] The pharmaceutical composition according to [68], wherein the pharmaceutical composition is used for diagnosing a disease associated with Biomolecule ¹ or Biomolecule ² .

[70] Pharmaceutical composition according to [69], wherein the disease associated with the Biomolecule¹or Biomolecule²is a MUC1-related disease.

[71] The pharmaceutical composition according to [70], wherein the pharmaceutical composition is a pharmaceutical composition for treating cancer expressing MUC1.

[72] The pharmaceutical composition according to [71], 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.

[73] Use of one or more in accordance with any of [56]-[60] for the preparation of a composition for diagnosing cancer and/or a pharmaceutical composition for treating cancer.

[74] A conjugate according to any one of [56]-[60], wherein the conjugate is used for diagnosing cancer and/or treating cancer.

[75] A method for diagnosing cancer, comprising administering to a subject one or more conjugates according to any one of [56]-[60].

[76] A method for treating cancer, comprising administering to a subject a therapeutically effective amount of a conjugate in accordance with any one of [56]-[60].

Beneficial effects of the invention

[0105] A conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody, a peptide linker and a ligand, and a conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody and a specific ligand described in the present invention have excellent binding activity to human CEACAM5. Based on this, it is expected that the conjugate of the present invention further comprising a metal will be useful for the diagnosis and/or treatment of cancer. In addition, the conjugate comprising the Fab fragment of an anti-human MUC1 antibody, a peptide linker and a ligand described in the present invention has excellent binding activity to human MUC1. The conjugate consisting of 3arm DOTA, spacer, peptide linker and biomolecule described in the present invention is excreted by the kidneys more quickly. Based on this, it is expected that the conjugate of the present invention further comprising a metal will be useful for the diagnosis and/or treatment of cancer.

Brief description of drawings

[0106] [Fig. 1] Fig. 1 is a PET/CT image obtained approximately 3 hours after administration of a PBS solution containing conjugate⁶⁴Cu protein (A).

[Fig. 2] Fig. 2 is a PET/CT image obtained approximately 3 hours after administration of a PBS solution containing conjugate⁶⁴Cu protein (B).

[Fig. 3] Fig. 3 represents SUV (standardized accumulation rate).

Description of Embodiments

[0107] The present invention will be described in detail below, but the present invention is not limited thereto. Unless otherwise specified, scientific and technical terms used in the context of the present invention should have the meanings commonly known to those skilled in the art.

"Alkyl" means a straight or branched saturated hydrocarbon chain and means a monovalent group.

"C _1-6 alkyl" refers to an alkyl containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n -hexyl. In one embodiment, C _1-6 alkyl is C _1-4 alkyl, in one embodiment, C _1-6 alkyl is methyl or ethyl, and in one embodiment, C _1-6 alkyl is methyl.

"C _1-6 alkylene" is a divalent group obtained by removing hydrogen from the above C _1-6 alkyl. In one embodiment, C _1-6 alkylene is methylene, ethylene, propylene, methylmethylene, or the like.

"Halogen" means F, Cl, Br or I.

"C _1-6 alkyl halogen" is a C _1-6 alkyl substituted with one or more halogens. In one embodiment, a C _1-6 alkyl halogen is a C _1-6 alkyl substituted with 1-5 halogens, and in one embodiment, a C _1-6 alkyl halogen is CF ₃ .

[0108] 1-1. Conjugate of the present invention

The conjugate of the present invention is the conjugate represented by the following formula (I):

(YS ₁ -X) _p -Fab ¹ (I)

Where

Fab ¹ is a Fab fragment of the anti-human CEACAM5 antibody, and Fab ¹ is linked to X through p the number of amino groups or thiol groups in Fab ¹ .

X represents a peptide linker or bond,

S₁represents spacer or connection,

Y is a ligand, and

p is a natural number from 1 to 25 and represents the number of (YS ₁ -X) associated with Fab ¹ ,

with the proviso that when X is a bond, S ₁ is -CH ₂ -(1,4-phenylene)-NH-C(=S)- or bond, and Y is

[0110] group represented by the following formula

[0109] [Chemical formula 56]

.

Fab fragment of antibody to human CEACAM5 (Fab ¹ )

Next, the Fab fragment of the anti-human CEACAM5 antibody, represented as "Fab ¹ " in formula (I), will be described.

The basic structure of the antibody molecule is common to each class and consists 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 usually consists of a polypeptide chain of about 440 amino acids, having a characteristic structure for each class and is called γ, μ, α, δ or chain, corresponding to IgG, IgM, IgA, IgD or IgE, respectively. In addition, IgG includes IgG1, IgG2, IgG3 and IgG4, which are called γ1, γ2, γ3 and γ4, respectively. The light chain generally consists of a polypeptide chain of about 220 amino acids, and two types are known, L-type and K-type, which are called λ and κ chains, respectively. The peptide configuration of the basic structure of an antibody molecule is such that two homologous heavy chains and two homologous light chains are linked by disulfide bonds (S-S bonds) and non-covalent bonds, and the molecular weight ranges from 150,000 to 190,000. The two light chains can be paired with any heavy chain. Each antibody molecule always consists of two identical light chains and two identical heavy chains.

There are four intrachain SS bonds in the heavy chain (five for the μ and ε chains) and two intrachain SS bonds in the light chain; one loop is formed for every 100-110 amino acid residues, and this steric structure is the same among the loops and is called a structural unit or domain. The domains located at the N-termini of both the heavy chain and the light chain are called variable regions because their amino acid sequences are variable even in an authentic sample from the same class (subclass) of the same animal species, and their corresponding domains are called the heavy chain variable region ( _VH ) and the light chain variable region ( _VL ). The amino acid sequence located closer to the C-terminal side than the N-terminal side is almost constant for each class or subclass and is called the constant region, and each of the domains is represented as CH ₁ , _CH 2, _CH 3 or C _L.

[0111] The specificity of antibody-antigen binding depends on the amino acid sequence of the portion consisting of V _H and V _L . On the other hand, biological activities such as binding to complements and various cells reflect differences in constant region structure between Ig classes. It has been found that the variability of the heavy chain and light chain variable regions is mainly limited to three small hypervariable regions present in both chains, and these regions are called complementarity determining regions (CDRs; CDR1, CDR2 and CDR3, starting from the N-terminus). The remainder of the variable region is called the framework region (FR) and is relatively constant.

[0112] The region between the C _H 1 domain and the C _H 2 domain of the heavy chain constant region of an antibody is called the hinge region, and this region includes many proline residues and includes many interchain SS bonds connecting the two heavy chains. For example, the hinge regions of human IgG1, IgG2, IgG3 and IgG4 include 2, 4, 11 and 2 cysteine residues, respectively, which constitute the SS linkages between the heavy chains. The hinge region is an area very sensitive to proteolytic enzymes such as papain or pepsin. When an antibody is cleaved by papain, its heavy chain is cleaved at a position closer to the N-terminal side than the SS linkage of the hinge region between the heavy chains, and the antibody is cleaved into two Fab fragments and one Fc fragment. The Fab fragment consists of a light chain and a heavy chain fragment including a heavy chain variable region ( _VH ), a _CH1 domain, and part of the hinge region. The Fab fragment includes a variable region and has antigen-binding activity.

In one embodiment, the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is a Fab fragment having the following characteristics:

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

[0113] As the heavy chain constant region of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention, any constant region such as Igγ1, Igγ2, Igγ3 or Igγ4 can be selected. In one embodiment, the heavy chain constant region of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is a human Igγ1 constant region.

As the light chain constant region of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention, any Igλ or Igκ constant region can be selected. In one embodiment, the light chain constant region of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is a human Igκ1 constant region.

In one embodiment, the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is the following Fab ² fragment:

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

It is known that when an antibody comprising a Fab fragment is expressed in a cell, the antibody undergoes post-translational modification. Examples of post-translational modification include cleavage of lysine at the C-terminus of the heavy chain by carboxypeptidase, modification of glutamine or glutamic acid at the N-termini of the heavy chain and light chain to pyroglutamic acid by pyroglutamylation, glycosylation, oxidation, deamidation and glycosylation, and such post-translational modification is known to occur in various antibodies (J. Pharm. Sci.; 2008; 97: 2426-2447).

The anti-CEACAM5 Fab fragment included in the conjugate of the present invention may also include a Fab fragment resulting from post-translational modification. Examples of the Fab fragment of the anti-human CEACAM5 antibody of the present invention that can be obtained by post-translational modification include a Fab fragment of a pyroglutamylated anti-human CEACAM5 antibody at the N-terminus of the heavy chain. It is known in the art that such post-translational modification by N-terminal polyglutamylation does not affect the activity of the antibody (Anal. Biochem.; 2006; 348:24-39).

[0114] in one embodiment, the anti-human CEACAM5 Fab fragment included in the conjugate of the present invention is an anti-human CEACAM5 Fab fragment having the following characteristics:

An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region that consists of the amino acid sequence of amino acids 1-121 of SEQ ID NO: 2 and in which glutamic acid, amino acid 1 in SEQ ID NO: 2, is modified to pyroglutamic acid, and a light chain comprising a light chain variable region consisting of the amino acid sequence of amino acids 1-112 SEQ ID NO: 4.

In one embodiment, the anti-human CEACAM5 Fab fragment included in the conjugate of the present invention is an anti-human CEACAM5 Fab fragment having the following characteristics:

An anti-human CEACAM5 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 2 and in which glutamic acid, amino acid 1 in 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.

In another embodiment, the anti-CEACAM5 Fab fragment included in the conjugate of the present invention is an anti-human CEACAM5 Fab fragment having the following characteristics:

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

The anti-human CEACAM5 Fab fragment included in the conjugate of the present invention binds to human CEACAM5. Examples of a method for measuring the binding activity of a produced anti-human CEACAM5 antibody Fab fragment to human CEACAM5 include methods such as surface plasmon resonance (SPR) analysis and ELISA. For example, when SPR analysis is used, the binding rate constant (ka), dissociation rate constant (kd) and dissociation constant ( _KD ) can be measured by immobilizing a Biotin CAPture Kit (GE Healthcare Japan Corporation) and biotinylated human CEACAM5 on a sensor chip using Biacore T200 (GE Healthcare Japan Corporation) and addition of serially diluted Fab fragment.

The Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention can be easily prepared by one skilled in the art using a method known in the art based on the sequence information of the heavy chain fragment and the light chain of the Fab fragment of the anti-human CEACAM5 antibody according to the present invention disclosed in this application. The Fab fragment of the anti-human CEACAM5 antibody of the present invention is not particularly limited, and can be obtained, for example, in accordance with the method described in the section <Method for producing the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention> described below.

[0115] 1-2. Ligand

"Ligand" is a conjugate moiety that can chelate with a metal and means the group formed by the chelating agent. A formed group refers to a group that is bonded as a result of the removal of a proton from the chelating agent. The group formed by the chelating agent binds to the Fab fragment of the anti-human CEACAM5 antibody directly or through a spacer and/or peptide linker.

"Chelating agent" refers to a compound that can coordinate with a metal. Examples of a "chelating agent" as used herein include a siderophore and a non-siderophore. Examples of the siderophore include hydroxamic acid type, catechol type and mixed-ligand type siderophore. Examples of hydroxamic acid type siderophore include ferrichrome,

[0117] deferoxamine (DFO), represented by the following formula:

[0116] [Chemical formula 57]

fusarinin C, ornibactin and rhodotorulic acid. Examples of catechol-type siderophore include enterobactin, bacillibactin, and vibriobactin. Examples of mixed-ligand type siderophore include azotobactin, pyoverdine, and yersiniabactin. In the case of the siderophores described above, DFO may interact with the spacer or peptide linker via -NH ₂ , which is its reactive functional group, and in the case of siderophores other than DFO, they may also interact with the spacer or peptide linker via the reactive functional group. a functional group, such as a carboxyl group, hydroxyl group or amino group, in a manner commonly used by those skilled in the art.

Examples of a non-siderophore include

[0119] DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, CAS number: 60239-18-1) represented by the following formula:

[0118] [Chemical formula 58]

DTPA (diethylenetriaminepentaacetic acid, CAS number: 67-43-6), DTPA-BMA (1,7-bis(methylcarbamoylmethyl)-1,4,7-triazaheptane-1,4,7-triacetic acid, CAS number: 119895- 95-3), EOB-DTPA (N-[(2S)-2-[bis(carboxymethyl)amino]-3-(4-ethoxyphenyl)propyl]-N-[2-[bis(carboxymethyl)amino]ethyl] glycine, CAS number: 158599-72-5), TTHA (triethylenetetramine hexacetic acid, CAS number: 869-52-3), DO3A (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, CAS number : 217973-03-0), HP-DO3A (10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, CAS number: 120041-08-9) and their known reactive derivatives.

DOTA can interact with a spacer or peptide linker through one of the carboxylic acids, which are its reactive functional groups (hereinafter DOTA containing three carboxylic acids linked in this way is sometimes represented as 3arm DOTA (PLoS One. 2019 Mar 22;14(3 ):e0213397)).

From DOTA, examples of 3arm DOTA include the following.

[0120] [Chemical formula 59]

[0121] Alternatively, DOTA in which four carboxylic acids are supported (hereinafter sometimes represented as 4arm-DOTA) using the p-SCN-Bn-DOTA reagent of the following formula can also be reacted with a spacer or peptide linker.

[0122] [Chemical formula 60]

[0123] Examples of an embodiment of a “chelating agent” that forms a ligand included in the conjugate of the present invention include DFO, DOTA, DTPA, DTPA-BMA, EOB-DTPA, DO3A, and HP-DO3A. In one embodiment, the chelating agent is DFO or DOTA.

The compounds and conjugates provided herein also include their free forms and salts unless otherwise indicated. “Their salts” are salts that, when an acid addition salt or a salt with a base, can be formed depending on the type of substituent of the compound and its conjugate, can be formed by the compound and the conjugate. Specific examples thereof include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid, as well as with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid 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 acid and glutamic acid, salts with inorganic bases such as sodium, potassium, magnesium, calcium and aluminum, and with organic bases such as methylamine, ethylamine, ethanolamine, lysine and ornithine, salts with various amino acids and amino acid derivatives such as acetylleucine , and ammonium salts. For example, DFO also exists as deferoxamine methanesulfonate and another salt. DTPA exists as the sodium salt of DTPA and also in free form.

[0124] The metal conjugate of the present invention can be used for various contrast agents and/or therapeutic agents for the treatment of cancer and is used, for example, for an MRI contrast agent and an agent used for PET imaging.

Examples of the "chelating agent" embodiment when used for an MRI contrast agent include the siderophore and non-siderophore chelating agents described above.

Examples of the "chelating agent" embodiment when used for PET imaging include the siderophore and non-siderophore chelating agents described above, and in one embodiment, the chelating agent is DFO or DOTA.

In the conjugate of the present invention, the chelating agent may include a metal. As used herein, the term “metal” means a paramagnetic metal ion or radioisotope of a metal. The metal is not particularly limited as long as it is a metal that coordinates with each chelating agent. The appropriate combination of chelating agent and metal is selected according to the intended use of the conjugate.

[0125] A paramagnetic metal ion is preferably used as a contrast agent in MRI. Examples of the paramagnetic metal ion embodiment include, but are not limited to, 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⁺ , Y ³⁺ , Ho ³⁺ , Er ³⁺ , La ³⁺ , Yb ³⁺ , Mn ³⁺ or Mn ²⁺ . In one embodiment, the paramagnetic metal ion is Gd ³⁺ , Mn ³⁺ , Mn ²⁺ , Fe ²⁺ or Fe ³⁺ . In one embodiment, the paramagnetic metal ion is Gd ³⁺ . In one embodiment, the paramagnetic metal ion is Mn ³⁺ or Mn ²⁺ . In this case, halogen or the like. can be used as a counteranion in the conjugate. In addition, the counteranion may be a C(=O)O ⁻ ligand, and further, the conjugate may contain a countercation such as Na ⁺ .

The radioisotope of the metal is used for radiopharmaceuticals for PET, etc. Examples of a metal radioisotope embodiment include, but are not limited to, ⁸⁹ Zr, ⁵² Mn, ⁵² Fe, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁹⁰ Y, ^99m Tc, ¹¹¹ In, or ¹⁷⁷ Lu. Examples of an embodiment of a metal radioisotope used for a PET radiopharmaceutical, a SPECT radiopharmaceutical and the like include ⁸⁹ Zr, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^99m Tc, or ¹¹¹ In. In one embodiment, the metal radioisotope is a zirconium (Zr) radioisotope. In one embodiment, the radioisotope of the metal is ⁸⁹ Zr. Examples of an embodiment of a metal radioisotope used for the treatment of cancer include ⁹⁰ Y or ¹⁷⁷ Lu.

An embodiment of the conjugate of the present invention is a conjugate in which Y is a DFO with which the⁸⁹Zr. Another embodiment is a conjugate in which Y is DOTA to which it is coordinated radioisotope of a metal consisting of⁹⁰Y,⁶⁷Ga,⁶⁸Ga and¹⁷⁷Lu. Another embodiment is a conjugate in which Y is DOTA to which a paramagnetic metal ion consisting of Gd is coordinated³⁺ and Y³⁺. Another embodiment is a conjugate in which Y DOTA to which Gd is coordinated³⁺.

[0126] 1-3. Peptide linker or bond

In the conjugate of the present invention, the ligand (Y) or spacer (S ₁ ) and Fab ¹ can be directly linked (i.e. X is a bond) or can be linked through a peptide linker (i.e. X is a peptide linker) .

As used herein, a “peptide linker” is a linker comprising a peptide of 2-4 amino acids and, if desired, has Z ₁ - Z ₃ or the like attachments suitable for binding to the Fab fragment of an anti-human CEACAM5 antibody . Here, the peptide included in the peptide linker is not particularly limited, but is preferably a peptide consisting of 2-4 amino acids, each of which is 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) and leucine (Leu), 3-(2-naphthyl)alanine and diphenylalanine, and more preferably a peptide consisting of 2-4 amino acids, each selected from the group consisting of glycine, lysine, methionine, isoleucine, phenylalanine, valine, tyrosine, aspartic acid, arginine, 3-(2-naphthyl)alanine and diphenylalanine. Unless otherwise stated, the configuration of amino acid residues other than glycine is the L form.

[0127] An embodiment of a peptide linker is a peptide linker that includes a peptide consisting of 2-4 amino acids having an amino acid sequence cleavable by a renal brush border membrane enzyme or a lysosomal enzyme, and may also have an attachment located between the peptide linker and the biomolecule or Fab fragment of an antibody. It has been reported that a peptide linker having an amino acid sequence that is cleaved by a renal brush border membrane enzyme or a lysosomal enzyme is specifically cleaved by these enzymes present in the kidney, thereby reducing the accumulation of the labeling moiety in the kidney, and that the risk of renal and renal effects can be expected to be reduced. disorders. 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. disclose that the glycine-lysine linker is specifically cleaved by a renal brush border membrane enzyme present in the kidney. Japanese Patent No. 6164556 discloses that the glycine-phenylalanine-lysine linker is specifically cleaved in the kidney by a renal brush border membrane enzyme; in addition, Bioconjug Chem. 2002 Sep-Oct; 13(5):985-95. discloses that a linker comprising the sequence glycine-leucine-glycine-lysine is specifically cleaved in the kidney by a renal brush border membrane enzyme; and in addition, Bioconjug Chem. 2013 Feb 20;24(2):291-9. reveals that the glycine-tyrosine linker is specifically cleaved by a renal brush border membrane enzyme. In addition, Bioconjug Chem. 2014 Nov 19;25(11):2038-45 discloses that the linker comprising the methionine-isoleucine sequence is specifically cleaved by a lysosomal enzyme present in the kidney. An embodiment of the peptide linker 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. An embodiment is a peptide linker comprising 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.

[0128] The "peptide linker" may optionally have an attachment suitable for binding to a Fab fragment of an anti-human CEACAM5 antibody, wherein the attachment suitable for binding to a Fab fragment of an anti-human CEACAM5 antibody is a group that, according to the laws of organic chemistry, forms a bond between the peptide linker part and the amino group or thiol group of the Fab fragment of the anti-human CEACAM5 antibody, and an embodiment thereof is a group including a group formed from a maleimide (for example, a group represented by the following formula (II-I) or (II- II)) or an isothiocyanate-derived group (-NH-C(=S)-) at the end. The embodiment is -NH-(CH ₂ ) ₂ -Z ₁ -, -CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -, -C(=S)-NH-(1 ,4-phenylene)-NH-C(=S)- or -NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)- , where Z ₁ is represented by the following formula (II-I) or (II-II). In addition, the following formula (II-I) can be written as -Z ₁ (#N)-, and the following formula (II-II) can be written as -Z ₁ (#S)-.

The addition forms a peptide linker by binding to the amino group or carboxyl group of an amino acid at the end of the peptide, or to an amino group (eg, lysine) or hydroxyl group (eg, tyrosine) on the side chain of an amino acid. Examples of an attachment that forms a peptide linker by linking to a functional group on an amino acid side chain at the end of the peptide include the group represented by the following formula (III-I) or (III-II) as a Lys-integrated attachment, and these two are summarized herein are indicated as -Lys*-Z ₂ -. The following formula (III-I) can be represented as -Lys*-Z ₂ (#N)-, and the following formula (III-II) can be represented as -Lys*-Z ₂ (#S)-. The following formula (III-III) can be represented as -Lys*-Z ₃ -.

[0129] [Chemical formula 61]

[0130] Moreover, similarly, in the present application, the group represented by the following formula (IV) and the group represented by the following formula (V), which have a structure in which the functional group in the side chain of the terminal amino acid and the attachment are linked, are represented as - Tyr*-CH ₂ - and -Lys*-C(=S)-, respectively.

[0131] [Chemical formula 62]

[0132] Peptide linker embodiment "X" is a peptide linker that includes attachment. An embodiment is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(4) -Met-Val-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ -,

(7) -Gly-Arg-Lys*-Z ₂ -,

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

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(13) -Gly-Tyr*-CH ₂ -C(=O)-Lys*-Z ₂ -,

(14) -Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(15) -Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(16) -Gly-Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(17) -Gly-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(19) -Gly-Tyr-Lys*-Z ₂ -,

(20) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ O) ₃ -CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(22) -Gly-diphenylalanine-Lys*-Z ₂ -,

(23) -Gly-Tyr-NH-(CH ₂ ) ₅ -Z ₁ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[0133] Additionally, peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(4) -Met-Val-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ -,

(7) -Gly-Arg-Lys*-Z ₂ -,

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

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-.

An embodiment is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(4) -Met-Val-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(7) -Gly-Arg-Lys*-Z ₂ -,

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

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(13) -Gly-Tyr*-CH ₂ -C(=O)-Lys*-Z ₂ -,

(14) -Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(15) -Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(16) -Gly-Val-NH-(CH ₂ ) ₂ -Z ₁ -,

(17) -Gly-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(19) -Gly-Tyr-Lys*-Z ₂ -,

(20) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ O) ₃ -CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(22) -Gly-diphenylalanine-Lys*-Z ₂ -,

(23) -Gly-Tyr-NH-(CH ₂ ) ₅ -Z ₁ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[0134] Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ -,

(7) -Gly-Arg-Lys*-Z ₂ - and

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

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ - and

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -.

[0135] Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ - and

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

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(5) -Gly-Tyr*-CH ₂ -C(=O)-NH-(CH ₂ ) ₂ -Z ₁ -,

(6) -Gly-Lys-Lys*-Z ₂ -,

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

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[0136] Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(4) -Met-Val-Lys*-Z ₂ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(18) -Met-Phe-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28) -Met-Gly-Lys*-Z ₃ -.

[0137] Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(1) -Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(9) -Met-Ile-NH-(CH ₂ ) ₂ -NH-C(=S)-NH-(1,4-phenylene)-NH-C(=S)-,

(12) -Met-Ile-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26) -Met-Ile-NH-(CH ₂ ) ₂ -(II-II)- and

(27) -Met-Ile-NH-(CH ₂ ) ₂ -(A-3)-.

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(4) -Met-Val-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(18) -Met-Phe-Lys*-Z ₂ - and

(28) -Met-Gly-Lys*-Z ₃ -.

Peptide linker embodiment "X" is a peptide linker selected from the group consisting of

(11) -Met-Gly-Lys*-Z ₂ - and

(28) -Met-Gly-Lys*-Z ₃ -.

[0138] 1-4. Spacer or bond (S ₁ )

In the conjugate of the present invention, the ligand (Y) and the peptide linker (X) are directly linked (ie S ₁ is a bond) or linked through a spacer (ie S ₁ is a spacer).

In the context of this application S₁"spacer" is a group introduced to create a specific distance between a ligand and a peptide linker or Fab¹, or for coupling between a ligand and a peptide linker, and examples of an embodiment thereof include -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 formulas (a)-(q).

[0139] [Chemical formula 63]

[0140] In one embodiment, S ₁ is -C(=O)-CH ₂ O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH ₂ CH ₂ O ) ₄ -(1,3-phenylene)-C(=O)- or -C(=O)-(1,3-phenylene)-C(=O)-. In one embodiment, S ₁ is -NH-CH ₂ -(1,3-phenylene)-C(=O)-, -NH-(CH ₂ ) ₂ -C(=O)-, -CH ₂ -( 1,4-phenylene)-NH-C(=S)-, -NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-phenylene)- C(=O)-, -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)-, -NH-(CH ₂ ) ₃ -C(=O)-, -NH-( CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-, -NH-CH ₂ -(1,4-phenylene)-C(=O)-, -CH ₂ -(1,4-phenylene) -NH-C(=S)-NH-CH ₂ -(1,3-phenylene)-C(=O)-, -CH ₂ -(1,4-phenylene)-NH-C(=S)-NH -(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-phenylene)-C(=O)- or a spacer represented by any of the above formula(a) -(q). In one embodiment, S ₁ is -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)- or a spacer represented by formula (g), (i) or (k) above.

In one embodiment, S ₁ is -C(=O)-CH ₂ O-(1,3-phenylene)-C(=O)-, -C(=O)-(CH ₂ CH ₂ O) ₄ - (1,3-phenylene)-C(=O)-, -NH-CH ₂ -(1,3-phenylene)-C(=O)-, -NH-CH ₂ -(1,4-phenylene)- NH-C(=O)-, -NH-CH ₂ -(1,4-phenylene)-C(=O)- or -C(=O)-(1,3-phenylene)-C(=O) -, or

[0142] represents

[0141] [Chemical formula 64]

.

In one embodiment, S ₁ is a bond.

Moreover, in a conjugate in which Y is DOTA according to the present invention, DOTA and the Fab fragment of the anti-human CEACAM5 antibody (Fab ¹ ) can be directly linked or linked through a spacer (-CH ₂ -(1,4-phenylene )-NH-C(=S)-). However, a conjugate in which DOTA and the Fab fragment of anti-human CEACAM5 antibody (Fab ¹ ) are linked through -CH ₂ -(1,4-phenylene)-NH-C(=S)-, which is a spacer, is the conjugate represented by the following formula (VI).

[0143] [Chemical formula 65]

[0144] In addition, S₁The spacer described herein includes the novel spacer and the spacers represented by formulas (g) and (l), which are particularly useful as a spacer when DOTA is linked to a peptide linker comprising a 2-4 amino acid peptide having the amino acid sequence , cleaved by a renal brush border membrane enzyme or a lysosomal enzyme. As shown in the renal accumulation assay in Example 4 described below, in a conjugate in which an enzyme-cleavable peptide linker is combined with DOTA and a spacer represented by formula (g), it was confirmed that the accumulation of the labeling moiety in the kidney is reduced. In one embodiment, S₁represents a spacer represented by formula (g) or (l).

An embodiment of the conjugate of the present invention is a conjugate in which Y is DOTA, S₁represents a spacer represented by formula (g) or (l), and X is a peptide linker comprising a peptide of 2-4 amino acids having an amino acid sequence cleavable by a renal brush border enzyme or a lysosomal enzyme.

An embodiment thereof is a conjugate in which Y is DOTA, S₁represents a spacer represented by formula (g) or (l), and X is a peptide linker selected from the group consisting of

(2) -Gly-Lys*-Z ₂ -,

(3) -Gly-Phe-Lys*-Z ₂ -,

(10) -Asp-Gly-Lys*-Z ₂ -,

(11) -Met-Gly-Lys*-Z ₂ - and

(21) -Gly-(3-(2-naphthyl)alanine)-Lys*-Z ₂ -.

An embodiment is a conjugate in which Y is DOTA, S ₁ is -NH-CH ₂ -(1,4-phenylene)-NH-C(=O)-, or a spacer represented by any of formula (g), (i) or (k), and X is a peptide linker selected from the group consisting of

(1)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ -,

(11) -Met-Gly-Lys*-Z ₂ -,

(12) -Met-Ile-Lys*-Z ₂ -,

(24) -Met-Ile-NH-(CH ₂ ) ₂ -(A-4)-,

(25) -Met-Ile-NH-(CH ₂ ) ₂ -(A-5)-,

(26)-Met-Ile-NH-(CH ₂ ) ₂ -(II-II)-,

(27)-Met-Ile-NH-(CH ₂ ) ₂ -(A-3)- and

(28)-Met-Gly-Lys*-Z ₃ -.

An embodiment is a conjugate in which Y is DOTA, S₁represents a spacer represented by formula (g), and X is a peptide linker selected from the group consisting of

(11) -Met-Gly-Lys*-Z ₂ - and

(28) -Met-Gly-Lys*-Z ₃ -.

[0145] The conjugate of the present invention consists of a combination of the above embodiments.

Specific embodiments of the conjugate of the present invention are as follows.

In Fab formulas²represents A Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

p represents a natural number from 1 to 25. Fab ² is bonded to an adjacent carbon atom through p is the number of amino groups or thiol groups in Fab ² .

[0146] [Chemical formula 66]

[0147] [Chemical formula 67]

[0148] [Chemical formula 68]

[0149] [Chemical formula 69]

[0150] [Chemical formula 70]

[0151] [Chemical formula 71]

[0152] [Chemical formula 72]

[0153] A labeling portion of a combination in which Y is DOTA, S₁represents a spacer represented by formula (g) or (l), and X is a peptide linker including a peptide consisting of 2-4 amino acids having an amino acid sequence cleavable by a renal brush border membrane enzyme or a lysosomal enzyme is useful as a labeling part, which is expected to reduce nephrotoxicity, etc. in similar conjugates using different Fab antibodies, not limited to combination with Fab¹according to the present invention, and the present invention also includes the invention of a marking part as such.

When preparing the conjugate of the present invention, the binding of the Fab fragment of the anti-CEACAM5 antibody to the ligand, spacer and/or peptide linker and the binding of the ligand to the spacer and/or peptide linker can be suitably carried out by one skilled in the art in a known manner.

[0154] In the context of the present application, "tag part" means, for example, a part other than Fab¹in formula (I), and the part other than the Biomolecule¹or Biomolecules²in formula (Ia) or (Ib). The labeling portion is (i) a ligand and a peptide linker (Y-S₁-X: where S₁represents a bond and X represents a peptide linker), (ii) a ligand (Y-S₁-X: where S₁And X each represents a bond) or (iii) a ligand, spacer and peptide linker (Y-S₁-X: where S₁represents spacer and X represents a peptide linker). In one embodiment, the labeling moiety is (ii) a ligand. In one embodiment, the labeling portion is (i) a ligand and a peptide linker or (iii) a ligand, a spacer, and a peptide linker. The "labeling moiety" ligand may further include a metal, and some embodiments are (i) a ligand including a metal and a peptide linker, (ii) a ligand, or (iii) a ligand, a spacer, and a peptide linker, and are (i) a metal chelating ligand and a peptide linker, (ii) a metal chelating ligand, or (iii) a metal chelating ligand, a spacer and a peptide linker. In addition, there is the case of (iv) peptide linker, in which a spacer is also included in the peptide linker. Moreover, in formula (Ia) or (Ib), the labeling part means a part other than the Biomolecule¹or Biomolecules².

[0155] 1-5. The number (p) of a labeling moiety (YS ₁ -X) associated with the Fab fragment of the anti-human CEACAM5 antibody (Fab ¹ ), and the number (p) of a labeling moiety associated with Biomolecule ¹ and Biomolecule ² formula (Ia) or (Ib)

The conjugate of the present invention is a conjugate in which one or more labeling portions (Y-S₁-X) are linked through one or more amino groups or thiol groups in the Fab fragment of the anti-human CEACAM5 antibody (Fab¹). Moreover, the conjugate of the present invention is a conjugate in which one or more labeling moieties are linked through one or more amino groups or thiol groups in the Biomolecule¹And Biomolecule²formulas (Ia) or (Ib). The conjugate of the present invention may be a mixture of conjugates in which the number of labeling parts that are connected is different from each other, and this indicates that the conjugate is any of the conjugates in which 1-25 labeling parts (Y-S₁-X) are associated with Fab¹in formula (I), or a mixture thereof. For one Fab¹ the conjugate of the present invention includes 1-25 labeling moieties (Y-S₁-X) in one embodiment, includes 1-23 marking parts (Y-S₁-X) in one embodiment, includes 1-15 marking parts (Y-S₁-X) in one embodiment, includes 1-11 marking parts (Y-S₁-X) in one embodiment, includes 1-9 marking parts (Y-S₁-X) in one embodiment, includes 1-7 marking parts (Y-S₁-X) in one embodiment, includes 1-5 marking parts (Y-S₁-X) in one embodiment and further includes 1-4 marking parts (Y-S₁-X) in one embodiment.

It is shown that in formula (Ia) or (Ib) Biomolecule¹And Biomolecule²are any of the Fab¹, in which 1-25 tagging parts are connected, or a mixture thereof. For one Biomolecule¹And Biomolecules², the conjugate of the present invention includes 1-25 labeling moieties in one embodiment, includes 1-23 labeling moieties in one embodiment, includes 1-15 labeling moieties in one embodiment, includes 1-11 labeling moieties in one embodiment, includes 1-9 marking parts in one embodiment, includes 1-7 marking parts in one embodiment, includes 1-5 marking parts in one embodiment, and further includes 1-4 marking parts in one embodiment.

Those. "p" which represents the amount of marking part (Y-S₁-X) associated with one Fab¹, And "p" which represents the amount of labeling moiety associated with one Biomolecule¹And Biomolecule², are the same or different from each other, and each is a natural number from 1 to 25 in one embodiment, is a natural number from 1 to 23 in one embodiment, is a natural number from 1 to 15 in one embodiment, is is a natural number from 1 to 11 in one embodiment, is a natural number from 1 to 9 in one embodiment, is a natural number from 1 to 7 in one embodiment, is a natural number from 1 to 6 in one embodiment , is a natural number from 1 to 5 in one embodiment, is a natural number from 1 to 4 in one embodiment, and is furthermore a natural number from 1 to 3 in one embodiment.

[0156] 2. A polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention

In one embodiment, the Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention is encoded by a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody and a polynucleotide comprising a nucleotide sequence encoding a Fab light chain anti-human CEACAM5 antibody fragment.

In one embodiment, a polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention is a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequence represented by amino acids 1- 121 SEQ ID NO: 2, or a polynucleotide comprising a nucleotide sequence encoding a light chain including a light chain variable region consisting of the amino acid sequence represented by amino acids 1-112 SEQ ID NO: 4.

Examples of a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment including a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2 include a polynucleotide comprising the nucleotide sequence of nucleotides 1 to 363 of SEQ ID NO: 1. Examples of a polynucleotide comprising a nucleotide sequence encoding a light chain including a light chain variable region consisting of the amino acid sequence of amino acids 1 to 112 of SEQ ID NO: 4 include a polynucleotide comprising the nucleotide sequence of nucleotides 1 to 336 of SEQ ID NO: 3.

In one embodiment, a polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention is a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4.

Examples of a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of an amino acid sequence set forth in SEQ ID NO: 2 include a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1. Examples of a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4 include a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3.

The polynucleotide encoding the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention can be synthesized using a gene synthesis method known in the art based on a nucleotide sequence designed from the amino acid sequences of the Fab heavy chain fragment and the light chain. anti-human CEACAM5 antibody fragment. As such a gene synthesis method, various methods known to those skilled in the art can be used, such as the antibody gene synthesis method disclosed in International Publication No. 90/07861.

[0157] 3. Expression vector of a polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention

An expression vector of a polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention includes an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody, an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain of a Fab fragment of an anti-human CEACAM5 antibody, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody, and a polynucleotide comprising a nucleotide sequence encoding a light chain of a Fab fragment antibodies to human CEACAM5.

Examples of preferred expression vectors include an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, an expression vector comprising a polynucleotide comprising a nucleotide a light chain coding sequence comprising a light chain variable region consisting of an amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment including a heavy chain variable region, consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain including a light chain variable region consisting of an amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4.

Preferred expression vectors include an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain fragment consisting of the amino acid sequence presented in SEQ ID NO: 4, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence presented in SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain consisting from the amino acid sequence shown in SEQ ID NO: 4.

[0158] These expression vectors are not particularly limited, provided that they can produce the polypeptide encoded by the polynucleotide of the present invention in various host cells, such as prokaryotic cells and/or eukaryotic cells. Examples of such expression vectors include a plasmid vector and a viral vector (eg adenovirus or retrovirus), and preferably pEE6.4 or pEE12.4 (Lonza) can be used.

In addition, these expression vectors may include a promoter operably linked to a gene encoding a heavy chain fragment and/or light chain of a polynucleotide encoding a Fab fragment of an anti-human CEACAM5 antibody included in the conjugate of the present invention. Examples of a promoter for expressing a Fab fragment in a host cell include, when the host cell is a bacterium of the genus Escherichia, Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and tac promoter. Examples of the promoter for expression in yeast include the PH05 promoter, PGK promoter, GAP promoter and ADH promoter, and examples of the promoter for expression in the Bacillus genus include SL01 promoter, SP02 promoter and penP promoter. Further, examples thereof include when the host is a eukaryotic cell such as a mammalian cell, a promoter derived from a virus such as CMV, RSV or SV40, a retroviral promoter, an actin promoter, an EF (elongation factor) 1α promoter and a heat shock promoter.

[0159] These expression vectors may further include, when a bacterium, particularly E. coli, is used as a host cell, a start codon, a stop codon, a stop region, and a replication unit. On the other hand, when a yeast, animal cell or insect cell is used as a host, the expression vectors may include a start codon and a stop codon. In addition, in this case, the expression vectors may include an enhancer sequence, 5' and 3' untranslated regions of a gene encoding a heavy chain fragment and/or light chain of the invention, a secretory signal sequence, a splice junction, a polyadenylation site, a replication unit, or the like. .P. In addition, the expression vectors may include a selectable marker typically used depending on the intended target (eg, a tetracycline resistance gene, an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, or a dihydrofolate reductase gene).

[0160] 4. Host cell transformed with expression vector

The host cells transformed with the expression vector include a host cell selected from the group consisting of the following (a)-(d):

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

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

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody, and a polynucleotide comprising a nucleotide sequence encoding a light chain of a Fab fragment of an anti-human CEACAM5 antibody; And

(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain fragment of an anti-human Fab fragment CEACAM5.

[0161] In one embodiment, the host cell transformed with the expression vector is a host cell transformed with the expression vector selected from the group consisting of the following (a)-(d):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide 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;

(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain including a light chain variable region consisting of the amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4;

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, and a polynucleotide comprising a nucleotide a light chain coding sequence comprising a light chain variable region consisting of the amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4; And

(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain, including a light chain variable region consisting of the amino acid sequence represented by amino acids 1-112 of SEQ ID NO: 4.

[0162] In one embodiment, the host cell transformed with the expression vector is a host cell transformed with the expression vector selected from the group consisting of the following (a)-(d):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2;

(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 4;

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid the sequence shown in SEQ ID NO: 4; And

(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0163] Examples of a host cell transformed with a preferred expression vector include a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, and a polynucleotide comprising a nucleotide sequence encoding the light chain of a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, and a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in the conjugate of the present invention, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention.

[0164] A host cell transformed with an expression vector is not particularly limited as long as it is compatible with the expression vector used and can be transformed with an expression vector for expressing a Fab fragment, and examples thereof include various cells such as a natural cell or an artificially generated one. cell (for example, a bacterium (a bacterium from the genus Escherichia or a bacterium from the genus Bacillus), a yeast (from the genus Saccharomyces, the genus Pichia, etc.) and an animal or insect cell (for example, Sf9)), a mammalian cell line (for example, a cultured cell, such as a CHO-K1SV cell, a CHO-DG44 cell, or a 293 cell) commonly used in the art to which the present invention relates. The transformation itself can be carried out by a known method, such as the calcium phosphate method or the electroporation method.

5. Method for producing a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention

Production of the anti-human CEACAM5 Fab fragment included in the conjugate of the present invention involves the step of culturing the transformed host cell described above to express the anti-human CEACAM5 Fab fragment.

[0165] In one embodiment, the transformed host cell cultured to obtain the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is selected from the group consisting of the following (a)-(c):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, and a polynucleotide comprising a nucleotide sequence encoding a light chain of a Fab fragment antibodies to human CEACAM5 included in the conjugate of the present invention;

(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light a chain of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention; And

(c) a host cell transformed with an expression vector comprising a polynucleotide containing a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in the conjugate of the present invention, and a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain of the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention.

[0166] In one embodiment, the transformed host cell cultured to obtain the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is selected from the group consisting of the following (a)-(c):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of the amino acid sequences shown by amino acids 1 to 121 of SEQ ID NO: 2, and a polynucleotide comprising the nucleotide a light chain coding sequence comprising a light chain variable region consisting of the amino acid sequence represented by amino acids 1 to 112 of SEQ ID NO: 4;

(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment comprising a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain, including a light chain variable region consisting of the amino acid sequence represented by amino acids 1-112 SEQ ID NO: 4; And

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment including a heavy chain variable region consisting of an amino acid sequence represented by amino acids 1 to 121 of SEQ ID NO: 2, and a host cell, transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain including a light chain variable region consisting of the amino acid sequence represented by amino acids 1-112 of SEQ ID NO: 4.

[0167] In one embodiment, the transformed host cell cultured to obtain the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention is selected from the group consisting of the following (a)-(c):

(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid the sequence shown in SEQ ID NO: 4;

(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2 and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of the amino acid sequence presented in SEQ ID NO: 4; And

(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a host cell transformed with an expression vector comprising a polynucleotide comprising the nucleotide sequence a sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.

[0168] The transformed host cell used is preferably a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in the conjugate of the present invention, and a polynucleotide comprising a nucleotide sequence a sequence encoding a light chain Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, or a host cell transformed with an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain fragment of a Fab fragment of an anti-human CEACAM5 antibody included in a conjugate of the present invention, and an expression vector comprising a polynucleotide comprising a nucleotide sequence encoding the light chain of a Fab fragment of an anti-human CEACAM5 antibody included in the conjugate of the present invention.

When obtaining the Fab fragment of the anti-human CEACAM5 antibody included in the conjugate of the present invention, the transformed host cell can be cultured in a nutrient medium. The nutrient medium preferably includes a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch and sucrose, and examples of the inorganic nitrogen source or organic nitrogen source include ammonium salt, nitrate, amino acid, corn extract, peptone, casein, meat extract, soybean starch and potato extract. In addition, the culture medium may optionally include another nutrient (eg, an inorganic salt (eg, calcium chloride, sodium dihydrogen phosphate, or magnesium chloride), a vitamin, and an antibiotic (eg, tetracycline, neomycin, ampicillin, or kanamycin).

The cultivation of the transformed host cell itself is carried out using a known method. Culture conditions such as temperature, pH of the medium and culture time are selected accordingly. For example, when the host is an animal cell, the media may be MEM (Science; 1955; 122:501) containing about 5-20% fetal bovine serum, DMEM (Virology; 1959; 8: 396-97), RPMI 1640 (J. Am. Med. Assoc.; 1967; 199:519-24.), medium 199 (Proc. Soc. Exp. Biol. Med.; 1950; 73:1-8.) or the like. The pH of the medium is preferably about 6-8, and cultivation is usually carried out at a temperature of about 30-40°C for from about 15 to 336 hours, and aeration and stirring can also be carried out if necessary. When the host is an insect cell, examples of the medium include Grace's medium (PNAS; 1985; 82:8404-8) containing fetal bovine serum, and its pH is preferably about 5-8. Cultivation is usually carried out at about 20-40°C for 15-100 hours, if necessary, aeration and stirring can also be carried out. When the host is a bacterium, actinomycete, yeast or filamentous fungus, for example, a liquid medium containing the above nutrient source is suitable. An environment with a pH of 5-8 is preferred. When the host is E. coli, examples of preferred media include LB media and M9 media (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory; 1972:431). In this case, cultivation can usually be carried out at a temperature of 14-43°C for about 3-24 hours, if necessary with aeration and stirring. If the host is a bacterium of the genus Bacillus, cultivation can be carried out at a temperature of 30-40°C for approximately 16-96 hours, with aeration and stirring if necessary. When the host is yeast, examples of the medium include Burkholder's minimal medium (PNAS; 1980; 77:4505-4508), and its pH is desirably 5-8. Cultivation is usually carried out at a temperature of about 20-35°C for 14-144 hours, if necessary, aeration and stirring can also be carried out.

Preparation of the anti-human CEACAM5 antibody Fab fragment included in the conjugate of the present invention may include, in addition to the step of culturing the transformed host cell described above for expressing the anti-human CEACAM5 antibody Fab fragment, the step of isolating, preferably isolating and purifying, the expressed Fab anti-human CEACAM5 antibody fragment. Examples of isolation and purification methods include a solubility-based method such as salting out or a solvent precipitation method, a molecular weight difference method such as dialysis, ultrafiltration, gel filtration, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a using a charge such as ion exchange chromatography or hydroxylapatite chromatography, a method using specific affinity such as affinity chromatography, a method using difference in hydrophobicity such as reverse phase high performance liquid chromatography, and a method using difference in isoelectric point such as isoelectric focusing.

[0170] 6. Method for producing the conjugate of the present invention

The method for preparing the conjugate of the present invention may include the step of covalently linking the Fab fragment of the anti-human CEACAM5 antibody to a labeling moiety (YS ₁ -X). Those skilled in the art will be able to appropriately carry out the coupling between the components in the labeling portion (YS ₁ -X) in a known manner. As an example reaction, after binding a ligand (Y) to a peptide linker (X) directly or through a spacer (S ₁ ), the peptide linker can be linked to a Fab fragment of the anti-human CEACAM5 antibody. In addition, after binding the anti-human CEACAM5 Fab fragment to the peptide linker (X), the peptide linker can also be linked to a ligand (Y) directly or through a spacer (S ₁ ). It is also possible to use a compound in which a ligand and a spacer (S ₁ ) are pre-bound as a starting material.

[0171] The method for producing the conjugate of the present invention may also include the step of culturing the transformed host cell described above to express the Fab fragment of the anti-human CEACAM5 antibody and the step of covalently linking the Fab fragment and the labeling portion (YS ₁ -X). The method for producing the conjugate of the present invention may also include the step of culturing the transformed host cell described above to express the Fab fragment of the anti-human CEACAM5 antibody, the step of isolating the expressed Fab fragment, and the step of covalently linking the Fab fragment and the labeling part (YS ₁ - X). The method for producing the conjugate of the present invention may further include the step of adding a metal. As a chelating agent, a peptide linker, a spacer, a specified number of labeling parts, a metal, etc., described in this application can be used.

[0172] The method for producing a conjugate of the present invention may be carried out as a method including two or more steps as defined above as a series of steps, or may be carried out as a method including at least one step as defined above. For example, a method comprising the step of binding a Fab fragment of an anti-human CEACAM5 antibody to a labeling moiety (YS ₁ -X), and a method comprising the step of coordinating a metal with a Fab fragment of an anti-human CEACAM5 antibody to which a labeling moiety (YS ₁ -X) is bound ), are also included in the method for producing the conjugate of the present invention. In addition, the method for producing the conjugate of the present invention also includes a method in which a different order of steps is used. For example, a method for covalently linking a Fab fragment of an anti-human CEACAM5 antibody to a labeling moiety (YS ₁ -X) in which a metal is coordinated to a ligand is also included in the method for preparing the conjugate of the present invention.

In addition, the conjugate of the present invention can be prepared in the same manner, also with the biomolecule described in the present invention, instead of the Fab fragment of the anti-human CEACAM5 antibody described above.

7. Conjugate consisting of a ligand, spacer, peptide linker and biomolecule

Next, a conjugate consisting of a ligand, a spacer, a peptide linker and a biomolecule according to the present invention is described, represented by the following formula.

[0173] [Chemical formula 73]

[0176] Embodiment

[0174] the following formula (S2) corresponding to the spacer

[0175] [Chemical formula 74]

[0178] represents

[0177] [Chemical formula 75]

.

Another embodiment

[0180] represents a group where

formula (S2) is

[0179] [Chemical formula 76]

.

Another embodiment

[0182] represents a group where

formula (S2) is

[0181] [Chemical formula 77]

.

Another embodiment

[0184] represents a group where

formula (S2) is

[0183] [Chemical formula 78]

.

An embodiment of group Q in formula (S2) is -C(=O)-, -NH-C(=O)-, or -NH-C(=S)-.

An embodiment of group Q in formula (S2) is -C(=O)- or -NH-C(=O)-.

An embodiment of group Q in formula (S2) is -NH-C(=O)-.

An embodiment of the group L ² in formula (Ia) or (Ib) is Ile, Phe or Gly.

An embodiment of the group L ² in formula (Ia) or (Ib) is Ile.

An embodiment of group L ² in formula (Ia) or (Ib) is s Phe.

An embodiment of the group L ² in formula (Ia) or (Ib) is Gly.

An embodiment of the group L ³ in formula (Ia) or (Ib) is a bond, Arg or His.

An embodiment of group L ³ in formula (Ia) or (Ib) is a bond.

An embodiment of group L ³ in formula (Ia) or (Ib) is Arg.

An embodiment of the group L ³ in formula (Ia) or (Ib) is His.

An embodiment of the group L ⁴ in formula (Ia) or (Ib) is -NH-(CH ₂ ) ₂ -, -NHCH(C(=O)OH)(CH ₂ ) ₄ - or a bond.

An embodiment of the group L ⁴ in formula (Ia) or (Ib) is -NH-(CH ₂ ) ₂ -.

An embodiment of group L ⁴ in formula (Ia) or (Ib) is -NHCH(C(=O)OH)(CH ₂ ) ₄ -.

An embodiment of group L ⁴ in formula (Ia) or (Ib) is a bond.

Embodiment of group V ¹ in formula (Ia) or (Ib)

represents

a group represented by any of the following formulas (A-1)-(A-5)

[0185] [Chemical formula 79]

.

[0186] An embodiment of group V ¹ in formula (Ia) or (Ib)

represents

group represented by the following formula (A-1)

[0187] [Chemical formula 80]

.

[0188] An embodiment of group V ¹ in formula (Ia) or (Ib)

represents

group represented by the following formula (A-2)

[0189] [Chemical formula 81]

.

[0190] An embodiment of group V ¹ in formula (Ia) or (Ib)

represents

group represented by the following formula (A-3)

[0191] [Chemical formula 82]

.

[0192] An embodiment of group V ¹ in formula (Ia) or (Ib)

represents

group represented by the following formula (A-4)

[0193] [Chemical formula 83]

.

[0194] An embodiment of group V ¹ in formula (Ia) or (Ib)

represents

group represented by the following formula (A-5)

[0195] [Chemical formula 84]

.

[0196] The following formula (B) in formula (Ia) or (Ib) is a group consisting of a spacer and a peptide linker.

[0197] [Chemical formula 85]

[0198] Examples include a group selected from the group consisting of the following formulas (B-1) to (B-7).

[0199] [Chemical formula 86]

[0200] [Chemical formula 87]

[0201] [Chemical formula 88]

[0202] [Chemical formula 89]

[0203] [Chemical formula 90]

[0204] [Chemical formula 91]

[0205] [Chemical formula 92]

[0206] An embodiment is a conjugate represented by formula (Ia), wherein the conjugate is a conjugate selected from the group consisting of compounds of the following formulas, p is a natural number from 1 to 25, and Biomolecule ¹ is bonded to an adjacent carbon atom through p is the number of amino groups or thiol groups in Biomolecule ¹ .

[0207] [Chemical formula 93]

[0208] [Chemical formula 94]

[0209] [Chemical formula 95]

[0210] [Chemical formula 96]

[0211] [Chemical formula 97]

[0212] An embodiment of the present invention is a conjugate represented by the following formula.

[0213] [Chemical formula 98]

[0214] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0215] [Chemical formula 99]

[0216] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0217] [Chemical formula 100]

[0218] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0219] [Chemical formula 101]

[0220] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0221] [Chemical formula 102]

[0222] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0223] [Chemical formula 103]

[0224] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

An embodiment of the present invention is a conjugate represented by the following formula.

[0225] [Chemical formula 104]

[0226] where Fab ⁵ is the following Fab antibody fragment:

An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in 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.

In addition, examples of the embodiment of the present invention include the following formulas (Ie) and (If).

[0227] [Chemical formula 105]

[0228] [Chemical formula 106]

[0229] The conjugate of the present invention may have a geometric isomer and a tautomer. In addition, the compound of the present invention may have an asymmetric carbon. Individual variants of these isomers or mixtures thereof are included in the present invention. Moreover, a labeled form, which is a compound in which one or more atoms of the compound of the present invention is replaced by a radioactive isotope or a non-radioactive isotope, is also included in the present invention.

The biomolecule embodiment may be any biomolecule as long as it has physiological activity, and examples include peptide, protein, hormone, drug, nucleotide, oligonucleotide, nucleic acid and polysaccharide. In the case of a protein, antibody, enzyme, receptor, and fragments thereof are included.

Another embodiment of the biomolecule is a peptide and a protein.

Another embodiment of the biomolecule is a protein.

Another embodiment of the biomolecule is a Fab fragment, enzyme, receptor or fragment of such a protein. For example, somatostatin, PSMA ligand, RGD (Arg-Gly-Asp) Peptide, ATSM (Diacetyl-bis(N4-methylthiosemicarbazone)) or ²¹¹ At-AITM (4- ²¹¹ At-astato-N-[4-(6-( isopropylamino)pyridin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide) corresponds to the above.

[0230] Another embodiment of the biomolecule is a commercially available antibody or Fab fragment thereof.

Examples include nivolumab, pembrolizumab, bevacizumab, rituximab, pertuzumab, daratumumab, denosumab, cetuximab, ipilimumab, panitumumab, brentuximab, ramucirumab, atezolizumab, obinutuzumab, elotumituzumab, avelumab, ibritumomab, alemtuzumab, gemtuzum ab and necitumomab.

In addition, another embodiment of the biomolecule is one that would be expected for alpha particle therapy or beta particle therapy.

Examples include Octreoscan, ¹³¹ I-MIBG (I-131 metaiodobenzylguanidine), ²¹¹ At-MABG ( ²¹¹ At-astatobenzylguanidine), trastuzumab, humanized antibody A33 (Cancer Biotherapeutics & Radiopharmaceuticals 2005 Oct; 20(5):514-23.) , omburtamab, tenatumomab, anti-CD45 antibody (ClinicalTrials. gov Identifier: NCT03128034; 9595; NCI-2017-00452), ibritumomab and actimab.

In addition, another embodiment of the biomolecule is HuM195, MX35-F(ab') ₂ monoclonal antibodies, mouse 9.2.27, proteoglycan (MCSP) antigen, CD20 antigen, CD30 antigen, or the like, described in cancer research and molecular medicine (Cancer studies and molecular medicine (2004), 1, 1, 1-7).

Additionally, another embodiment of the biomolecule is CD19, GD2, VE cadherin, or the like.

[0231] An embodiment of the biomolecule is a Fab fragment of an anti-MUC1 antibody (identified as Fab ³ , Fab ⁴ or Fab ⁵ ), and examples include those disclosed in international publication WO2018/092885.

[1M] Specifically Fab³or Fab⁴represents An anti-human MUC1 antibody Fab fragment selected from the group consisting of the following (a) and (b), and a 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, listed as Fab³and a Fab fragment containing a heavy chain fragment including a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 14 is specified as Fab⁴:

(a) A Fab fragment of an anti-human MUC1 antibody comprising a heavy chain fragment including a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain including a light chain variable region consisting of the amino acid sequence presented in SEQ ID NO: 16; And

(b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region that consists of the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14, and in which glutamine, amino acid 1 in SEQ ID NO: 12 or SEQ ID NO: 14, 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.

[2M] An embodiment is a Fab fragment of an anti-human MUC1 antibody according to [1M], which is selected from the group consisting of the following (a) and (b):

(a) An anti-human MUC1 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 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10; And

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

[3M] An embodiment is a Fab fragment of an anti-human MUC1 antibody according to [1M], which is selected from the group consisting of the following (a) and (b):

(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 set forth in SEQ ID NO: 14 and a light chain comprising a light chain variable region consisting of the amino acid sequence, presented in SEQ ID NO: 16; And

(b) An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment comprising a heavy chain variable region which consists of the amino acid sequence set forth in SEQ ID NO: 14 and in which glutamine, amino acid 1 in SEQ ID NO: 10, 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.

[0232] [4M] An embodiment is a Fab fragment of an anti-human MUC1 antibody according to [3M], which is selected from the group consisting of the following (a) and (b):

(a) an anti-human MUC1 Fab fragment comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 8 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10; And

(b) an anti-human MUC1 Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[5M] An embodiment is a Fab fragment of an anti-human MUC1 antibody according to [4M], which is a Fab fragment of an anti-human MUC1 antibody comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10.

[6M] An embodiment is a Fab fragment of an anti-human MUC1 antibody according to [4M], which is a Fab fragment of an anti-human MUC1 antibody comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and wherein glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence presented in SEQ ID NO: 10.

[7M] P10-1 or P10-2 (referred to as Fab ⁵ ), which is a Fab fragment of an anti-MUC1 antibody, is disclosed in international publication WO2018/092885.

In addition, the conjugate of the present invention may consist of a combination of the individual embodiments described above.

[0233] 7. Diagnostic composition and method for diagnosis

The present invention relates to a diagnostic composition comprising a metal-containing conjugate of the present invention (hereinafter referred to as the detectable conjugate of the present invention). The diagnostic composition of the present invention may include one or more conjugates of the present invention. That is, the diagnostic composition of the present invention may include a single conjugate of the present invention or may include a combination of two or more conjugates of the present invention. The detectable conjugate of the present invention can be formulated according to a conventional method and used as an early diagnosis drug or a disease staging drug (especially as a cancer diagnostic agent).

Early diagnosis drug means a diagnostic agent intended to make a diagnosis at a stage when no disease state is observed or at an early stage of a disease. For example, in the case of cancer, an early diagnosis agent means a diagnostic agent used at a stage where no disease state is observed, or at stage 0 or stage 1.

A disease staging agent means a diagnostic agent that can examine how far a disease state has progressed. For example, in the case of cancer, a disease staging agent means a diagnostic agent that can determine its stage.

The cancer that is expected to be diagnosed using the diagnostic composition of the present invention is a cancer expressing human CEACAM5. In one embodiment, examples thereof include colorectal cancer, breast cancer, lung cancer, thyroid cancer, and cancer resulting from metastasis thereof. In one embodiment, the cancer is colorectal cancer or cancer resulting from metastasis of colorectal cancer. More preferably, the cancer is a cancer resulting from metastasis of colorectal cancer, and such cancer includes metastatic liver cancer. In addition, the cancer is a cancer expressing human MUC1. In one embodiment, examples of cancer include breast cancer, lung cancer, colorectal cancer, bladder cancer, skin cancer, thyroid cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer, or cervical cancer. In one embodiment, the cancer is breast cancer or bladder cancer.

[0234] The amount of the conjugate of the present invention added when formulating the diagnostic composition of the present invention varies depending on the severity of the symptom and the age of the patient, the dosage form of the composition used, the binding activity of the Fab fragment or biomolecule, etc., and, for example, about 0.001 mg/kg to 100 mg/kg based on the weight of the Fab fragment or biomolecule per unit body weight of the patient may be used.

Examples of the dosage form of the diagnostic composition of the present invention include a parenteral preparation such as injection or infusion, and administration can be accomplished by intravenous injection, intramuscular site injection, subcutaneous injection, intravesical administration or the like. In addition, the composition can use a carrier and an additive in accordance with these dosage forms within pharmaceutically acceptable limits. The types of pharmaceutically acceptable carrier and additive are not particularly limited, and the carrier and additive well known to those skilled in the art may be used.

The present invention also relates to the use of a detectable conjugate of the present invention for the preparation of a composition for early diagnosis or a composition for staging cancer. The present invention also relates to a detectable conjugate of the present invention for use in the early diagnosis and staging of cancer.

In addition, the present invention also relates to a method for diagnosing cancer, comprising administering to a subject a detectable conjugate of the present invention. In this case, "subject" refers to the human or other mammal for which a diagnosis is to be made. In one embodiment, the subject is a person who needs to be diagnosed. The effective amount of the detectable conjugate of the present invention in the diagnostic method of the present invention may be the same amount as the effective amount of the conjugate of the present invention in the composition described above. In the diagnostic method of the present invention, the detectable conjugate of the present invention can be administered by intramuscular injection into a specific tissue site, subcutaneous injection, or the like.

In another embodiment, the present invention also relates to the use of a Fab fragment of an anti-human CEACAM5 antibody to produce a conjugate comprising a Fab fragment of an anti-human CEACAM5 antibody of the present invention, a peptide linker and/or a ligand. In one embodiment, the present invention also relates to the use of a Fab fragment of an anti-human CEACAM5 antibody for the preparation of a diagnostic composition comprising a conjugate of the present invention.

Moreover, in one embodiment, when a diagnostic composition of the present invention is provided including a metal radioisotope, it can be labeled with a metal radioisotope immediately prior to use of the composition, and it can be presented as a diagnostic composition including a metal radioisotope.

[0235] 8. Pharmaceutical composition and method for treatment

In the present invention, from about 0.001 mg/kg to 100 mg/kg, based on the weight of one or more Fab fragments of the conjugate of the present invention, including a radioisotope of a metal such as ⁹⁰ Y or ¹⁷⁷ Lu, can be used.

A pharmaceutical composition comprising the conjugate of the present invention can be used for the treatment of cancer. The cancer that is expected to be treated by the pharmaceutical composition comprising the conjugate of the present invention is a cancer expressing human CEACAM5, and examples thereof include colorectal cancer, breast cancer, lung cancer, thyroid cancer and cancer resulting from its metastasis. Alternatively, the cancer that is expected to be treated by the pharmaceutical composition comprising the conjugate of the present invention is a cancer expressing human MUC1, and examples thereof include breast cancer, lung cancer, colorectal cancer, bladder cancer, skin cancer, thyroid cancer, stomach cancer, pancreatic cancer, kidney cancer, ovarian cancer or cervical cancer. In one embodiment, the cancer is breast cancer or bladder cancer.

The present invention includes a pharmaceutical composition comprising a conjugate of the present invention for the treatment of colorectal cancer or cancer resulting from metastatic colorectal cancer. In addition, the present invention includes a method for treating colorectal cancer or cancer resulting from metastatic colorectal cancer, comprising the step of administering a therapeutically effective amount of a conjugate of the present invention. In addition, the present invention includes a method for inducing cell death of colorectal cancer cells or cancer resulting from metastatic colorectal cancer, comprising the step of administering a therapeutically effective amount of a conjugate of the present invention.

The pharmaceutical composition for treating cancer can also be used for diagnosing cancer. For example, a pharmaceutical composition for treating colorectal cancer or cancer resulting from metastasis of colorectal cancer can also be used for diagnosing cancer.

In addition, the present invention includes a conjugate of the present invention for use in the treatment of colorectal cancer or cancer resulting from metastatic colorectal cancer. In addition, the present invention includes the use of the conjugate of the present invention for the preparation of a pharmaceutical composition for the treatment of colorectal cancer or cancer resulting from metastatic colorectal cancer.

In another embodiment, the present invention also relates to the use of a Fab fragment of an anti-human CEACAM5 antibody to prepare a pharmaceutical composition comprising a conjugate of the present invention.

In the embodiments described above, a biomolecule may be used in place of the Fab fragment of the anti-human CEACAM5 antibody. The conjugate of the present invention can be provided as a diagnostic composition or pharmaceutical composition for a disease associated with a biomolecule.

The present invention has been described in general terms, and specific Examples will now be presented to provide a better understanding of the invention. However, they are presented for illustrative purposes and are not intended to limit the present invention.

EXAMPLES

[0236] The following abbreviations may be used in the following Examples and tables provided below.

Gd/DOTA: 3arm DOTA labeled with Gd, Gd/4arm DOTA: 4arm DOTA labeled with Gd, MS: mass spectrometry, ESI+: m/z value (ESI ionization method unless otherwise stated [M+H]+), ESI-: m/z value (ESI ionization method, unless otherwise specified [M-H]-), APCI/ESI+: m/z value (APCI/ESI ionization method, APCI/ESI means simultaneous measurement of APCI and ESI. Unless specified other [M+H]+), APCI/ESI-: m/z value (APCI/ESI ionization method, unless otherwise specified [M+H]-), Pr.-No.: conjugate number, SNo: preparation example number , Str: chemical structural formula, 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.

[0237] (Example 1: Preparation of Fab fragment of anti-human CEACAM5 antibody)

T84.66, which is a mouse-derived anti-human CEACAM5 antibody, was humanized with reference to a method disclosed in the literature (Protein Eng. Des. Sel.;2004;17:481-489) and then a molecular model of the humanized antibody , created in accordance with the literature (Proteins: Structure, Function and Bioinformatics; 2014; 82:1624-1635), was used to construct an antibody having a variable region, and it was expected that the affinity would not decrease even when the labeling part was bound.

The heavy chain fragment gene was formed by joining the gene encoding the signal sequence (Protein Engineering; 1987; 1:499-505) to the 5' end of the antibody heavy chain variable region gene and the Fab region gene of human Igγ1 (consisting of the nucleotide sequence 364 nucleotides -678 SEQ ID NO: 1) at the 3' end, and this heavy chain fragment gene was inserted into the GS vector pEE6.4 (Lonza). In addition, the light chain gene was formed by connecting the gene encoding the signal sequence to the 5' end of the antibody light chain variable region gene and the human Igκ constant region gene (consisting of the nucleotide sequence of nucleotides 337-657 SEQ ID NO: 3) to 3 ' end, and this light chain gene was inserted into the GS vector pEE12.4 (Lonza). The pEE vectors described above, into which the heavy chain fragment gene and the antibody light chain gene were respectively inserted, were digested with restriction enzymes NotI and PvuI, and ligated using the TAKARA Ligation Kit Ver2.1 (Takara Bio Inc.) for constructing a GS vector into which both the heavy chain fragment gene and the light chain gene are inserted.

[0238] Using the GS vector described above into which both the heavy chain fragment gene and the light chain gene were inserted, antibody expression was carried out in two ways by transient expression and constitutive expression. For transient expression, Expi293F cells (Thermo Fisher Scientific Inc.), cultured to about 3 million cells/ml in Expi293 expression medium (Thermo Fisher Scientific Inc.), were transfected with the GS vector described above into which both the heavy chain fragment gene and the light chain using the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific Inc.) and cultured for 5–7 days. The culture supernatant was purified using KappaSelect (GE Healthcare Japan Corporation) to obtain the Fab fragment. For constitutive expression, CHOK1SV cells (Lonza) were transfected with the GS vector described above into which both the heavy chain fragment gene and the light chain gene linearized with PvuI were inserted by electroporation using a Gene Pulser (Bio-Rad Laboratories, Inc.). The day after transfection, methionine sulfoximine was added and the cells were cultured for 5-7 days. Cells were seeded in semisolid medium containing methylcellulose, and after colonization, cells with high levels of Fab fragment expression were obtained using ClonePix FL (Molecular Devices, LLC). Cell culture supernatant was purified using Capto L (GE Healthcare Japan Corporation), Q Sepharose Fast Flow (GE Healthcare Japan Corporation), and BioPro S75 (YMC Co., Ltd.) to obtain the Fab fragment.

[0239] The nucleotide sequence encoding the heavy chain fragment of the resulting Fab fragment of the anti-human CEACAM5 antibody (referred to as PB009-01 or Fab ² ) is shown in SEQ ID NO: 1, and the amino acid sequence encoding it is shown in SEQ ID NO: 2. the nucleotide sequence encoding the light chain of PB009-01 is shown in SEQ ID NO: 3, and the amino acid sequence encoding it is shown in SEQ ID NO: 4. The heavy chain variable region of PB009-01 consists of the amino acid sequence of amino acids 1-121 of SEQ ID NO: : 2, and the heavy chain CDR1, CDR2 and CDR3 consist of the amino acid sequences of amino acids 31-35, 50-66 and 99-110, respectively, SEQ ID NO: 2. The light chain variable region PB009-01 consists of the amino acid sequence 1-112 SEQ ID NO: 4, and the light chain CDR1, CDR2 and CDR3 consist of the amino acid sequences of amino acids 24-38, 54-60 and 93-101, respectively, SEQ ID NO: 4.

The variable region and CDR sequences were determined according to Kabat numbering (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda).

[0240] (Example 2: Preparation of a conjugate comprising the Fab fragment of anti-human CEACAM5 antibody)

This Example discloses Examples of the preparation of the conjugate. When presenting each Preparation Example in this Example, "Example 2" is followed by a single hyphen followed by a "conjugate number." For example, "Example 2-11" indicates that this is an Example for the preparation of conjugate 11 in this Example. In addition, Fab²V in this Example means the Fab fragment of the antibody to human CEACAM5 (PB009-01/Fab²) obtained in Example 1, and "p-Fab" means that Fab²bonded to p labeling moieties shown in brackets [ ] or ( ), via p of its amino groups to form a conjugate. Some of the following Examples describe the number of marking parts (Y-S₁-X) associated with Fab²each conjugate that has been confirmed by MS analysis, but the result does not indicate that the conjugate having an associated amount of labeling moieties other than the amount specified above is not included. It should be understood that there may still be a conjugate having a certain associated number of labeling moieties, the presence of which could not be confirmed due to the precision of the MS analysis equipment. Moreover, in the structural formulas in this Example, the structural formula of DOTA to which Gd is bound schematically represents DOTA labeled with Gd.

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

[0241] [Chemical formula 107]

[0242] (i) Synthesis of tert-butyl O ⁴ -tert-butyl-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L- lysinate

Trifluoroacetic acid (hereinafter abbreviated as TFA) (3 ml) was added to a solution of tert-butyl N-(tert-butoxycarbonyl)glycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (1.00 g) in dichloromethane (6 ml ) while cooling with ice and the resulting mixture was stirred at the same temperature for 2 hours. The mixture was concentrated under reduced pressure, then a saturated aqueous sodium hydrogen carbonate solution was added, and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated. To this residue was added O ⁴ -tert-butyl hydrogen N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartate (920 mg), dichloromethane (10 ml), diisopropylethylamine (hereinafter abbreviated as DIPEA) (2 ml) , 1-(Dimethylamino)-N, N-dimethyl-1-[(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)oxy]methaniminium hexafluoridophosphate(1-) (hereinafter abbreviated as HATU) (1.2 g) and the resulting mixture was stirred at room temperature for 16 hours. Saturated aqueous sodium hydrogen carbonate was added and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated to give the title compound (2.86 g). MS (ESI+); 809.5 [M+Na]+

[0243] (ii) Synthesis of tert-butyl O ⁴ -tert-butyl-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

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

(iii) Synthesis of tert-butyl N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)-O ⁴ -tert-butyl-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L -lysinate

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

[0244] (iv) Synthesis of tert-butyl N-[3-(aminomethyl)benzoyl]-O ⁴ -tert-butyl-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

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

(v) Synthesis of tert-butyl O ⁴ -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10 -tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

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

[0245] (vi) Synthesis of tert-butyl O ⁴ -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4, 7,10-tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L-α-aspartylglycyl-L-lysinate

A mixture of tert-butyl O ⁴ -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane- 1-yl]acetamido}methyl)benzoyl]-L-α-aspartylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (387 mg), 10% palladium on carbon (water content 50%, 65 mg) and ethanol (4 ml) was stirred under a hydrogen atmosphere (1 atm) at room temperature for 5 hours. The mixture was filtered through Celite and concentrated. 10% palladium on carbon (50% humidity, 650 mg) and ethanol (8 ml) were added to the residue, and the resulting mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature for 20 hours. The mixture was filtered through Celite and then concentrated to give the title compound (336 mg). MS (ESI+); 1140.5 [M+Na]+

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

A solution of methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (56 mg) and THF (5 ml) was added to the mixture of tert-butyl O ⁴ -tert-butyl-N-[3-( {2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L-α-aspartylglycyl- L-lysinate (336 mg) and saturated aqueous sodium hydrogen carbonate (2.5 ml) under ice-cooling, and the resulting mixture was stirred at the same temperature for 2 hours. 1 M sodium hydroxide aqueous solution (60 μL) was added under ice-cooling, and then the resulting mixture was stirred at room temperature for 1 hour. Ethyl acetate and 10% aqueous citric acid were added and then 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 then the residue was purified by silica gel column chromatography (solvent gradient; 0→20% methanol/chloroform) to give the title compound (341 mg). MS (ESI+); 1198.5

[0246] (viii) Synthesis of N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L -α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

TFA (2 ml) was added to the solution of tert-butyl O ⁴ -tert-butyl-N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4 ,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L- norleucinate (341 mg) in dichloromethane (2 ml) and the resulting mixture was stirred at room temperature for 5 hours. The mixture was concentrated under reduced pressure and then the residue was purified by reverse phase column chromatography (solvent gradient; 0→20% acetonitrile/0.1% aqueous TFA) to give the title compound (99.6 mg). MS (ESI+); 918.3

(ix) Synthesis of [N-{3-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ , N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]benzoyl}-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L- norleucinato(3-)]gadolinium

An aqueous solution of sodium hydrogen carbonate (0.1 M, 800 μl) was added to the mixture of N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl ]acetamido}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) and the resulting mixture was stirred at room temperature for 10 minutes. Aqueous sodium hydrogen carbonate (0.1 M, 60 μl) was added and the resulting mixture was stirred at room temperature for 1 hour. Ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA) aqueous solution (0.5 M, 300 μL) was added and the resulting 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% aqueous TFA) to give the title compound (12.0 mg). MS (ESI-); 1071.4

[0247] (x) Synthesis of conjugate no. eleven

[N-{3-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]benzoyl}-L-α-aspartylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3- )]gadolinium (1 mg) was dissolved in DMSO (40 μl). 40 μL of the resulting solution was measured, 0.1 M borate buffer (40 μL) was added, and 0.1 M sodium carbonate aqueous solution was added to obtain a pH of 6.6.

The pre-prepared solution was added to 4.45 mg/ml borate buffer containing Fab ² (160 μl) and the resulting mixture was incubated at 30°C for 2 hours. A 0.05 M aqueous solution of EDTA (40 μl) was added and the resulting mixture was then incubated at 30°C for 10 minutes. The mixture was purified through a PD-10 column and the resulting solution was isolated using an Amicon Ultra-0.5 mL centrifugal filter (Merck Millipore). The isolated solution was washed with phosphate-buffered saline 7 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,3-Ph)-C(=O)-Asp-Gly-Lys*-Z ₂ (# N)], having a molecular weight of 1073, was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

(Example 2-12. Synthesis of ([Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)-Gly-Phe-Lys*-Z ₂ (#N)]p- Fab ² ))

[0248] [Chemical formula 108]

[0249] (i) Synthesis of tert-butyl N-(tert-butoxycarbonyl)-L-phenylalanyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

tert-Butyl N ⁶ -[(benzyloxy)carbonyl]-L-lysinate monohydrochloride (2.1 g), Et ₃ N (2.4 ml) and HATU (2.5 g) were successively added to the liquid mixture of N-(tert -butoxycarbonyl)-L-phenylalanine (1.5 g) in dichloromethane (30 ml) and the resulting mixture was left to react overnight at room temperature.

The reaction mixture was concentrated and then purified through 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]+

(ii) Synthesis of tert-butyl N-(tert-butoxycarbonyl)-L-phenylalanyl-L-lysinate

A mixture of an ethanol solution (60 ml) of tert-butyl N-(tert-butoxycarbonyl)-L-phenylalanyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (3150 mg) and 10% palladium on carbon (water content 50% , 1000 mg) was stirred under a hydrogen atmosphere (1 atm) at room temperature for 4 and a half hours. The starting materials remained and thus the system was purged with argon, then the mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in methanol (60 ml), 10% palladium on carbon (50% humidity, 1000 mg) was added and the resulting mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature for 5 hours. The disappearance of the starting material was confirmed, the system was then purged with argon, the mixture was filtered through Celite and the filtrate was concentrated to give the title compound (2520 mg). MS (ESI+): 450.4

[0250] (iii) Synthesis of tert-butyl N-(tert-butoxycarbonyl)-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

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

(iv) Synthesis of tert-butyl L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono(trifluoroacetate)

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

[0251] (v) Synthesis of tert-butyl N-(tert-butoxycarbonyl)glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

N-(tert-butoxycarbonyl)glycine (118 mg), triethylamine (hereinafter abbreviated as TEA) (0.26 ml) and HATU (256 mg) were successively added to the mixture of tert-butyl L-phenylalanyl-6-(2,5-dioxo -2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono(trifluoroacetate) (415 mg) in dichloromethane (10 ml) and the resulting mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure and then purified through a silica gel column (solvent gradient; 0→30% acetone/chloroform) to give the title compound (206 mg). MS (ESI+): 609.4 [M+Na)+

[0252] (vi) Synthesis of tert-butyl glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono(trifluoroacetate)

Using tert-butyl N-(tert-butoxycarbonyl)glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (205 mg), the crude product of the title compound (223 mg) was prepared in the same manner as Example 2-12(iv) above. MS (ESI+): 487.4

(vii) Synthesis of glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetate)

TFA (1 ml) was added to a mixture of tert-butyl glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono(trifluoroacetate) (9 mg) in dichloromethane (1 ml) and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to give the crude product of the title compound (8 mg). MS (ESI+): 431.3

[0253] (viii) Synthesis of N-[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl]methyl}phenyl)carbamothioyl]glycyl- L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

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

(ix) Synthesis of hydrogen [N-{[4-({1,4,7,10-tetrakis[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-2-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }methyl)phenyl]carbamothioyl}glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato( 4-)]gadolinate(1-)

N-[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl]methyl}phenyl)carbamothioyl]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 a solution of 0.1 M sodium hydrogen carbonate and the mixture was stirred at room temperature for 1 hour. A 0.5 M EDTA aqueous solution (80 μL) was added to the mixture, and the resulting mixture was stirred at room temperature for 5 minutes. TFA (10 μl) was added and the resulting mixture was then purified by reverse phase column chromatography (solvent gradient 5→50% acetonitrile/0.1% aqueous TFA) to give the title compound (5 mg). MS (ESI+): 1137.0

(x) Synthesis of conjugate no. 12

Using hydrogen [N-{[4-({1,4,7,10-tetrakis[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-2-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }methyl)phenyl]carbamothioyl}glycyl-L-phenylalanyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(4- )]gadolinate(1-) conjugate was prepared in the same manner as in step (x) of Example 2-11 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)-Gly-Phe-Lys*-Z ₂ (#N)], having a molecular weight of 1136, was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it .

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

[0254] [Chemical formula 109]

[0255] (i) Synthesis of tert-butyl N-(tert-butoxycarbonyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

TFA (3 ml) was added to a solution of tert-butyl N-(tert-butoxycarbonyl)glycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (1.00 g) in dichloromethane (6 ml) under ice cooling and the resulting the mixture was stirred at the same temperature for 2 hours. The mixture was concentrated under reduced pressure, then a saturated aqueous sodium hydrogen carbonate solution was added, and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated. To this residue was added N-(tert-butoxycarbonyl)-L-methionine (556 mg), dichloromethane (10 ml), DIPEA (2 ml) and HATU (1.16 g) and the resulting mixture was stirred at room temperature for 1 hour . The reaction solution was concentrated, then a saturated aqueous sodium hydrogen carbonate solution was added to the residue, and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with 1 M aqueous sodium hydroxide and saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated to give the title compound (1.70 g). MS (ESI+); 625.5

(ii) Synthesis of tert-butyl L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

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

[0256] (iii) Synthesis of tert-butyl N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

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

(iv) Synthesis of tert-butyl N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

TFA (2 ml) was added to a solution of tert-butyl N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (935 mg ) in dichloromethane (2 ml) under ice cooling and the resulting mixture was stirred at the same temperature for 1 hour. Et ₃ N and amino silica gel were added, the resulting mixture was concentrated under reduced pressure and then the residue was purified by silica gel column chromatography (amino silica gel, solvent gradient; 0→10% methanol/chloroform) to give the title compound (260 mg ). MS (ESI+); 658.5

[0257] (v) Synthesis of tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1 -yl]acetamido}methyl)benzoyl]-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate

Using tert-butyl N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (260 mg), the title compound (353 mg) was prepared in the same manner. as in step (v) of Example 2-11 above. MS (ESI-); 1210.4

(vi) Synthesis of tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl] acetamido}methyl)benzoyl]-L-methionylglycyl-L-lysinate

Using tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido} methyl)benzoyl]-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (353 mg), the title compound (245 mg) was prepared in the same manner as in step (vi) of Example 2-11 above . MS (ESI+): 1078.8

[0258] (vii) Synthesis of tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1 -yl]acetamido}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

Using tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido} methyl)benzoyl]-L-methionylglycyl-L-lysinate (245 mg), the title compound (139 mg) was prepared in the same manner as in step (vii) of Example 2-11 above. MS (ESI+): 1158.8

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

Using tert-butyl N-[3-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido} methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (341 mg) title compound (38.2 mg) was prepared in the same manner as in step (viii) of Example 2-11 above. MS (ESI+): 934.4

(ix) Synthesis of [N-{3-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ , N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato( 3-)]gadolinium

Using N-[3-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)benzoyl]-L-methionylglycyl-6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (28 mg), the title compound (13.8 mg) was prepared in the same manner as in step (ix) Example 2-11 above. MS (ESI+): 1089.2

(x) Synthesis of conjugate no. 13

Using [N-{3-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3- )]gadolinium conjugate was prepared in the same manner as in step (x) of Example 2-11 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z ₂ (# N)], having a molecular weight of 1089, was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

(Example 2-15. Synthesis of ([Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-Ph)-C(=O )-Gly-Lys*-Z ₂ (#N)]p-Fab ² ))

[0259] [Chemical formula 110]

[0260] (i) Synthesis of tert-butyl glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono(trifluoroacetate)

TFA (4 ml) was added to a solution of tert-butyl N-(tert-butoxycarbonyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (400 mg ) in dichloromethane (4 ml) under ice-cooling, and the resulting mixture was stirred under ice-cooling for 1 hour. The reaction mixture was concentrated under reduced pressure to obtain the title compound (642 mg). MS (ESI+); 340.4

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

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

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

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

[0261] (iv) Synthesis of tert-butyl N-(3-{3,15-dioxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7, 10-tetraazacyclododecan-1-yl]-5,8,11-trioxa-2,14-diazahexadecan-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol- 1-yl)-L-norleucinate

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

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

Using tert-butyl N-(3-{3,15-dioxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1 -yl]-5,8,11-trioxa-2,14-diazahexadecan-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- L-norleucinate (863 mg), the title compound (40.0 mg) was prepared in the same manner as in step (viii) of Example 2-11 above. MS (ESI+): 992.5

(vi) Synthesis of [N-{3-[3-oxo-15-(oxo-co)-16-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10- tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }-5,8,11-trioxa-2,14-diazahexadecan-1-yl]benzoyl}glycyl-6-(2,5- dioxo-2,5)-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)]gadolinium

Using N-(3-{3,15-dioxo-16-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]-5,8,11-trioxa -2,14-diazahexadecan-1-yl}benzoyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine (40 mg) title compound (15.9 mg) was prepared in the same manner as in step (ix) of Example 2-11 above. MS (ESI-); 1145.0

(vii) Synthesis of conjugate no. 15

[0262] Using [N-{3-[3-oxo-15-(oxo-co)-16-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10 -tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }-5,8,11-trioxa-2,14-diazahexadecan-1-yl]benzoyl}glycyl-6-(2,5 -dioxo-2,5)-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)]gadolinium conjugate was prepared in the same manner as in step (x) of Example 2-11 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3- Ph)-C(=O)-Gly-Lys*-Z ₂ (#N)], having a molecular weight of 1147, was associated with one Fab ² having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were bound to it, and a conjugate in which three such molecules were bound to it.

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

Synthesis of conjugate no. 24 (conjugation via iminothiolane)

A solution in 0.1 M borate buffer (5 μl) of 2 mg/ml 2-iminothiolane (hereinafter abbreviated as 2-IT) was added to 4.45 mg/ml Fab ² borate buffer (160 μl) and the resulting mixture was incubated at 37°C within 40 minutes. Excess 2-IT was washed with phosphate buffered saline 3 times using an Amicon Ultra-0.5 mL centrifuge filter and finally concentrated.

[N-{3-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)] gadolinium (1 mg) synthesized in Example 2-13 (ix) was dissolved in DMSO (40 μl). 0.1 M borate buffer (40 μL) was added to the resulting solution, and 0.1 M sodium carbonate aqueous solution was added to obtain a pH of 6.0.

The resulting linker solution was added to the resulting filtrate containing the antibody, and the resulting mixture was incubated at 30°C for 2 hours. EDTA-containing phosphate-buffered saline (pH 6.0) was added and the resulting mixture was then incubated at 30°C for 10 minutes. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-0.5 mL centrifuge filter. The isolated solution was washed with phosphate-buffered saline 7 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,3-Ph)-C(=O)-Met-Gly-Lys*-Z ₂ (# S)], having a molecular weight of 1190, was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it.

(Example 2-29. Synthesis of ([Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-Ph)-C(=O )-Met-Gly-Lys*-Z ₂ (#N)]p-Fab ² ))

[0263] [Chemical formula 111]

[0264] (i) Synthesis of methyl 3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecan-1-yl)benzoate

Methyl 3-(Aminomethyl)benzoate monohydrochloride (286 mg), HATU (590 mg) and Et ₃ N (900 μl) were added to the 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5 solution -azahexadecane-16-oic acid (396 mg) in dichloromethane (10 ml) and the resulting 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; 2→6% methanol/chloroform) to give the title compound (598 mg). MS (ESI+); 455.2

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

1 M aqueous sodium hydroxide solution (4 ml) and water (5 ml) were added to the 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) and the resulting mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with water, 10% citric acid was added, then the resulting mixture was extracted with ethyl acetate, and the collected organic layer was washed with brine. The organic layer was dried over anhydrous magnesium sulfate and filtered, and then the filtrate was concentrated under reduced pressure to obtain the title compound (662 mg). MS (ESI+); 441.1

(iii) Synthesis of tert-butyl N-(tert-butoxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

TFA (3 ml) was added to a mixture of tert-butyl N-(tert-butoxycarbonyl)glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (1, 63 g) and dichloromethane (5 ml) under ice-cooling, and the resulting 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 resulting mixture was stirred at room temperature for 5 minutes . A solution of the residue obtained in the previous reaction in dichloromethane (4 ml) was added to this reaction mixture, and the resulting mixture was stirred at room temperature for 20 hours. The reaction mixture was concentrated under reduced pressure and then the residue was purified by silica gel column chromatography (solvent gradient; 0→5% methanol/chloroform) to give the title compound (1.63 g). MS (APCI/ESI+); 571.3

[0265] (iv) Synthesis of tert-butyl N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecan-1-yl)benzoyl] -L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

TFA (2 ml) was added to a mixture of tert-butyl N-(tert-butoxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (150 mg) and dichloromethane (2 ml) under ice-cooling, and the resulting 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-diazaoctadecan-1-yl)benzoic acid (120 mg), dichloromethane (3 ml) and HATU (150 mg) and the resulting mixture was stirred at room temperature for 5 minutes. A solution of the residue obtained in the previous reaction in dichloromethane (2 ml) was added to this reaction mixture, and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then the residue was purified by silica gel column chromatography (solvent gradient; 0→5% methanol/chloroform) to give the title compound (113 mg). MS (ESI+); 893.4

(v) Synthesis of tert-butyl N-(3-{3,15-dioxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane -1-yl)]-5,8,11-trioxa-2,14-diazahexadecan-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole -1-yl)-L-norleucinate tetrakis(trifluoroacetate)

TFA (2 ml) was added to the mixture of tert-butyl N-[3-(17,17-dimethyl-3,15-dioxo-5,8,11,16-tetraoxa-2,14-diazaoctadecan-1-yl)benzoyl ]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (113 mg) and dichloromethane (2 ml) under ice cooling and the resulting mixture was stirred at the same temperature for 1 hour and then concentrated under reduced pressure to obtain the crude product. DIPEA (0.22 ml) was added to a mixture of DOTA-tris(t-Bu)ether (80 mg), HATU (80 mg) and dimethylacetamide (hereinafter abbreviated as DMAc) (1 ml) at room temperature, the resulting mixture was stirred for 10 minutes, then a mixture of the previously obtained crude product in DMAc (1 ml) was added and the resulting mixture was 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% aqueous TFA) to give the title compound (178 mg). MS (ESI-); 1345.8

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

Using tert-butyl N-(3-{3,15-dioxo-16-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1 -yl]-5,8,11-trioxa-2,14-diazahexadecan-1-yl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1- tetrakis(trifluoroacetate) yl)-L-norleucinate (178 mg), the title compound (60.0 mg) was prepared in the same manner as in step (viii) of Example 2-11 above. MS (APCI/ESI+); 1123.4

[0266] (vii) Synthesis of [N-{3-[3-oxo-15-(oxo-co)-16-{4,7,10-tris[(carboxy-co)methyl]-1,4,7 ,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }-5,8,11-trioxa-2,14-diazahexadecan-1-yl]benzoyl}-L-methionylglycyl-6 -(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)]gadolinium

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

(viii) Synthesis of conjugate no. 29

Using [N-{3-[3-oxo-15-(oxo-co)-16-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecane- 1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }-5,8,11-trioxa-2,14-diazahexadecan-1-yl]benzoyl}-L-methionylglycyl-6-(2,5 -Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)]gadolinium conjugate was prepared in the same manner as in step (x) of Example 2-11 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3- Ph)-C(=O)-Met-Gly-Lys*-Z ₂ (#N)], having a molecular weight of 1278, was associated with one Fab ² having a molecular weight of 47.9 kDa, and a conjugate in which two such molecules were associated with it.

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

[0267] [Chemical formula 112]

[0268] (i) Synthesis of tert-butyl N-[2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

TFA (2 ml) was added to a mixture of tert-butyl N-(tert-butoxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (150 mg) and dichloromethane (2 ml) under ice-cooling, and the resulting mixture was stirred at the same temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. A solution of the residue obtained in the previous reaction in dichloromethane (2 ml) 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 resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then the residue was purified by silica gel column chromatography (solvent gradient; 0→6% methanol/chloroform) to give the title compound (231 mg). MS (APCI/ESI+); 752.2 [M+Na]+

(ii) Synthesis of tert-butyl N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}- 1,2,3,4-tetrahydroisoquinolin-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate tetrakis(trifluoroacetate)

Using tert-butyl N-[2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-L-norleucinate (231 mg), the title compound (222 mg) was prepared in the same manner as in step (v) of Example 2-29 above. MS (ESI-); 1182.7

(iii) Synthesis of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-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

Using tert-butyl N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1, 2,3,4-tetrahydroisoquinolin-5-carbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate tetrakis(trifluoroacetate) (222 mg) the title compound (53 mg) was prepared in the same manner as in step (viii) of Example 2-11 above. MS (APCI/ESI+); 960.2

[0269] (iv) Synthesis of {N-[2-({4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ , N ⁴ ,N ⁷ ,N ¹⁰ }acetyl-co)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-L-norleucinato(3-)}gadolinium

Using N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-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 title compound (7.0 mg) was prepared by the same in a manner similar to step (ix) of Example 2-11 above. MS (ESI+): 1115.3

(v) Synthesis of conjugate no. 33

Using compound (iv), a conjugate was prepared in the same manner as in step (x) of Example 2-11 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-[2,5-(1,2,3,4-tetrahydroisoquinoline)]-C(=O)-Met-Gly-Lys* -Z ₂ (#N)], having a molecular weight of 1115, was associated with one Fab ^2, having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it.

(Example 2-35. Synthesis of ([Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-Ph)-C(=O )-Met-Gly-Lys*-Z ₂ (#S)]p-Fab ² ))

Synthesis of conjugate no. 35

Using [N-{3-[3-oxo-15-(oxo-co)-16-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecane- 1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }-5,8,11-trioxa-2,14-diazahexadecan-1-yl]benzoyl}-L-methionylglycyl-6-(2,5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinato(3-)]gadolinium synthesized in Example 2-29, (vii), the conjugate was prepared in the same way as in the step of Example 2 -24 above. MS analysis confirmed that the conjugate was a conjugate in which one [Gd/DOTA-NH-(CH ₂ CH ₂ O) ₃ -CH ₂ -C(=O)-NH-CH ₂ -(1,3-Ph )-C(=O)-Met-Gly-Lys*-Z ₂ (#S)], having a molecular weight of 1380, was associated with one Fab ² having a molecular weight of 47.9 kDa.

(Example 2-40. Synthesis of [Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)-NH-CH ₂ -(1,3-Ph)-C(=O )-Met-Gly-Lys*-Z ₂ (#S)]p-Fab ² ))

[0270] [Chemical formula 113]

[0271] (i) Synthesis of tert-butyl N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-L-norleucinate

Using tert-butyl N-(tert-butoxycarbonyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (690 mg) indicated in The title compound (710 mg) was prepared in the same manner as in step (i) of Example 2-33 above. MS (ESI+): 726.5 [M+Na]+

(ii) Synthesis of tert-butyl N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate mono (trifluoroacetate)

Using tert-butyl N-(3-{[(tert-butoxycarbonyl)amino]methyl}benzoyl)-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )-L-norleucinate (41 mg), the crude product of the title compound (42 mg) was prepared in the same manner as Example 2-12 (iv) above. MS (ESI+): 604.3

(iii) Synthesis of N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetate)

TFA (2 ml) was added to the mixture of tert-butyl N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- L-norleucinate mono(trifluoroacetate) (41 mg) in dichloromethane (4 ml) and the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure and then azeotroped with toluene to give the crude product (43 mg). 16 mg of the crude product was purified by reverse phase column chromatography (solvent gradient; 5→50% acetonitrile/0.1% aqueous TFA) to give the title compound (11 mg). MS (ESI+): 548.2

(iv) Synthesis of N-[3-({[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl]methyl}phenyl)carbamothioyl] amino}methyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

Using N-[3-(aminomethyl)benzoyl]-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine mono(trifluoroacetate) (11 mg) the title compound (17 mg) was prepared in the same manner as in step (viii) of Example 2-12 above. MS (ESI+): 1099.5

[0272] (v) Synthesis of hydrogen [N-{3-[({[4-({1,4,7,10-tetrakis[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecane- 2-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }methyl)phenyl]carbamothioyl}amino)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-L-norleucinato(4-)]gadolinate(1-)

Using N-[3-({[(4-{[1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-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) the title compound (6 mg) was prepared as follows in the same manner as in step (ix) of Example 2-12 above. MS (ESI+): 1254.6

(vi) Synthesis of conjugate no. 40

Using hydrogen [N-{3-[({[4-({1,4,7,10-tetrakis[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-2-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }methyl)phenyl]carbamothioyl}amino)methyl]benzoyl}-L-methionylglycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1 -yl)-L-norleucinato(4-)]gadolinate(1-) conjugate was prepared in the same manner as in Example 2-24 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)-NH-CH ₂ -(1,3 -Ph)-C(=O)-Met-Gly-Lys*-Z ₂ (#S)], having a molecular weight of 1355, was associated with one Fab ² having a molecular weight of 47.9 kDa, and a conjugate in which two such molecules were associated with it.

(Example 2-47: Synthesis of ([Gd/DOTA]p-Fab ² ))

[0273] [Chemical formula 114]

[0274] 1 M 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) while cooling with ice to adjust the pH to 6. Sodium 1-Hydroxy-2,5-dioxopyrrolidine-3-sulfonate (2.3 mg) dissolved in water (117 μL) was added to the resulting solution (239 μL) while cooling with ice. An aqueous solution of 3-{[(ethylimino)methylidene]amino}-N,N-dimethylpropan-1-amine monohydrochloride (hereinafter abbreviated as EDC HCl) (8.3 μL, 25 mg/mL) was then added and the resulting mixture was stirred for 30 minutes while cooling with ice to obtain a solution of N-hydroxysulfosuccinimidyl DOTA. Before adding Fab ^2, 0.2 M aqueous disodium hydrogen phosphate (pH 9) (40 μl) was added to adjust the pH to 7.

(i) The resulting N-hydroxysulfosuccinimidyl DOTA solution (200 µl) was added to a 0.1 M aqueous solution of disodium hydrogen phosphate (390 µl) with 17.8 mg/ml Fab ² (60 µl) and the resulting mixture was incubated at 4°C for 23 hours. Excess linker was washed with 10 mM phosphate buffer using an Amicon Ultra-0.5 ml centrifuge filter 3 times, washed with 0.3 M ammonium acetate buffer, finally concentrated and then filtered through a membrane filter to obtain the conjugate.

[0275] [Chemical formula 115]

[0276] (ii) 0.3 M ammonium acetate buffer containing the conjugate obtained in (i) was diluted with the same buffer and the pH was adjusted to 6.62 using 0.25 M acetate buffer. Aqueous solution of GdCl₃(35 µl) obtained to 0.057 M was added to the Fab solution², obtained to 2.8 mg/ml, and the resulting mixture was incubated at 37°C for 0.5 hours. After the reaction, 0.05 M EDTA (525 μl) was added and then the resulting mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-0.5 ml centrifuge filter. The isolated solution was washed with phosphate-buffered saline 5 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of conjugates in which one or two [Gd/DOTA] having a molecular weight of 542 were bound to one Fab², having a molecular weight of 47.9 kDa.

(Example 2-48. Synthesis of ([Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)]p-Fab ² ))

[0277] [Chemical formula 116]

[0278] (i) to bind DOTA, which is a chelating agent, to Fab² p-SCN-Bn-DOTA (Macrocyclics, Inc.) was used. 0.1 M sodium carbonate solution (pH 9.0) was added to the Fab solution², obtained to 2.54 mg/ml using phosphate-buffered saline (pH 7.4) and glycerol to adjust the pH to 8.8-9.5. P-SCN-Bn-DOTA was added to the mixture and the resulting mixture was incubated at 37°C for 2 hours. After the reaction, the reaction product was isolated using an Amicon Ultra-0.5 ml centrifuge filter to purify the conjugate. MS analysis confirmed that the conjugate was a conjugate in which one or two [4arm-DOTA-CH₂-(1,4-Ph)-NH-C(=S)], having a molecular weight of 553, were associated with one Fab², having a molecular weight of 47.9 kDa.

[0279] [Chemical formula 117]

[0280] (ii) Using the conjugate obtained in (i), the conjugate was prepared in the same manner as in step (ii) of Example 2-47 above. MS analysis confirmed that the conjugate was a conjugate in which one [Gd/4arm-DOTA-CH ₂ -(1,4-Ph)-NH-C(=S)], having a molecular weight of 706, was bound to one of Fab ² , having a molecular weight of 47.9 kDa.

(Example 2-60. Synthesis of ([DFO-C(=O)-CH ₂ O-(1,3-Ph)-C(=O)-Gly-Tyr*-CH ₂ -C(=O)-NH -(CH ₂ ) ₂ -Z ₁ (#S)] _p -Fab ² ))

[0281] [Chemical formula 118]

[0282] (i) Synthesis of 3-[2-(benzyloxy)-2-oxoethoxy]benzoic acid

2-Methylbut-2-ene (6 ml), sodium dihydrogen phosphate dihydrate (3.35 g) and sodium chlorite (3.64 g) were added to a mixture of benzyl (3-formylphenoxy) acetate (2.90 g), 2-methylpropane -2-ol (60 ml) and water (30 ml) at room temperature and the resulting mixture was stirred for 2 hours. Ethyl acetate and a 1 M hydrochloric acid solution (60 ml) were added to the reaction liquid, and the resulting mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous sodium chloride, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated to give the title compound (2.93 g). MS (ESI-); 285.1

(ii) Synthesis of tert-butyl N-[(benzyloxy)carbonyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate

tert-Butyl N-[(benzyloxy)carbonyl]glycyl-L-tyrosinate (2.49 g) was dissolved in acetone (50 ml) and ethyl bromoacetate (2.05 g) and potassium carbonate (2.41 g) were added and the resulting mixture stirred at room temperature for 4 hours. The insoluble material was filtered off, the filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography (ethyl acetate:hexane=30:70→60:40) to give the title compound (2.99 g). MS (ESI+): 537.4 [M+Na]+

(iii) Synthesis of tert-butyl N-{3-[2-(benzyloxy)-2-oxoethoxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate

tert-Butyl N-[(benzyloxy)carbonyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate (2.75 g) was dissolved in ethanol (55 ml), 10% palladium on carbon was added (content water 50%, 550 mg) under argon atmosphere and the resulting mixture was stirred under hydrogen atmosphere (1 atm) overnight at room temperature. The system was purged with argon, then the insoluble matter was filtered off, the filtrate was concentrated, the residue was dissolved in dichloromethane (55 ml), 3-[2-(benzyloxy)-2-oxoethoxy]benzoic acid (1.99 g), HATU (2.84 g) was added ) and Et ³ N (1.5 ml) and the resulting mixture was stirred at room temperature for 1 hour. The reaction liquid 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

[0283] (iv) Synthesis of tert-butyl N-[3-(carboxymethoxy)benzoyl]glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate

tert-Butyl N-{3-[2-(benzyloxy)-2-oxoethoxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate (3282 mg) was dissolved in THF (66 ml), 10% palladium on carbon (50% humidity, 328 mg) was added under argon and the resulting mixture was stirred under hydrogen overnight at room temperature. The reaction liquid was filtered through Celite and the filtrate was concentrated to give the title compound (2.54 g). MS (ESI-); 557.4

(v) Synthesis of tert-butyl N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatritriacontane- 1-yl)oxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate

DMF (5 ml) and Et ₃ N (0.16 ml) were added to N ⁴ -{5-[acetyl(hydroxy)amino]pentyl}-N ¹ -(5-{4-[(5-aminopentyl)(hydroxy )amino]-4-oxobutanamido}pentyl)-N ¹ -hydroxybutanediamide monomethane sulfonate (DFO.MeSO ₃ H) (500 mg), tert-butyl N-[3-(carboxymethoxy)benzoyl]glycyl-O-(2-ethoxy- 2-oxoethyl)-L-tyrosinate (446 mg), EDC HCl (175 mg) and 1H-benzotriazol-1-ol (hereinafter abbreviated as HOBt) (123 mg) and the resulting mixture was stirred overnight at room temperature. A 0.1% aqueous solution of TFA (1 ml) and TFA (0.03 ml) was added to the reaction liquid, and the resulting mixture was purified by reverse phase column chromatography (0.1% aqueous solution TFA:acetonitrile=95:5 → 0:100 ) to obtain the title compound (548 mg). MS (ESI-); 1099.7

(vi) Synthesis of tert-butyl N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatritriacontane- 1-yl)oxy]benzoyl}glycyl-O-(carboxymethyl)-L-tyrosinate

Methanol (5 ml) and DMF (5 ml) were added to tert-butyl N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14 ,20,25,31-hexaazatritriacontan-1-yl)oxy]benzoyl}glycyl-O-(2-ethoxy-2-oxoethyl)-L-tyrosinate (500 mg) and slightly heated to dissolve, add 1 M aqueous hydroxide solution sodium (600 μl) and the resulting mixture was stirred overnight at room temperature. 1 M aqueous sodium hydroxide solution (600 μl) was added and the resulting mixture was stirred overnight at room temperature. TFA (90 μL) was added under ice-cooling, methanol was distilled off under reduced pressure, and the resulting solution was purified by reverse phase column chromatography (0.1% aqueous TFA:acetonitrile=95:5 → 0:100) to obtain the title compound ( 315 mg). MS (ESI-); 1071.6

[0284] (vii) Synthesis of tert-butyl N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31 -hexaazatritriacontan-1-yl)oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2- oxoethyl)-L-tyrosinate

DMF (4 ml) was added to the mixture of tert-butyl N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25, 31-Hexaazatritriacontan-1-yl)oxy]benzoyl}glycyl-O-(carboxymethyl)-L-tyrosinate (269 mg), EDC HCl (58 mg) and HOBt (41 mg), then added 1-(2-aminoethyl) -1H-pyrrole-2,5-dione monohydrochloride (44 mg) and Et ₃ N (35 μl) and the resulting mixture was stirred at room temperature for 4 hours. 0.1% TFA aqueous solution (1 ml) was added to the reaction liquid, and the resulting mixture was purified by reverse phase column chromatography (0.1% TFA aqueous solution:acetonitrile=95:5 → 0:100) to obtain a carboxylic acid starting material mixture and the title compound (155 mg, about 6:4). This mixture was dissolved in DMSO (1 ml) and DMF (2 ml), EDC HCl (22 mg) and HOBt (15 mg) were added, the resulting mixture was stirred at room temperature for 5 minutes, then 1-(2-aminoethyl) was added. -1H-pyrrole-2,5-dione monohydrochloride (15.5 mg) and Et ₃ N (12 μl) and the resulting mixture was stirred at room temperature for 1 hour. 0.1% TFA aqueous solution (1 ml) was added to the reaction liquid, and the resulting mixture was purified 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

(viii) Synthesis of N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatritriacontan-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 N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatritriacontan-1-yl) oxy]benzoyl}glycyl-O-(2-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]amino}-2-oxoethyl)-L-tyrosinate ( 118 mg) was dissolved in TFA (1.2 ml) and the resulting solution was stirred at room temperature for 1.5 hours. The reaction liquid was concentrated, DMF (1.5 ml) and water (0.5 ml) were added, and the resulting mixture was purified by reverse phase column chromatography (0.1% aqueous TFA:acetonitrile=95:5 → 10:90) to obtain of the title compound (82 mg). MS (ESI-); 1137.5

[0285] (ix) Synthesis of conjugate no. 60

The 2-IT solution prepared with 0.1 M borate buffer was added to the Fab solution², obtained to 4.45 mg/ml using 0.1 M borate buffer, and the resulting mixture was incubated at 37°C for 30 minutes. Excess 2-IT was washed with EDTA-containing phosphate-buffered saline (pH 6.0) using an Amicon Ultra-0.5 ml centrifuge filter, finally concentrated and then filtered through a membrane filter. N-{3-[(9,20,31-trihydroxy-2,10,13,21,24,32-hexaoxo-3,9,14,20,25,31-hexaazatriacontan-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 resulting filtrate and the resulting mixture was diluted with 0.1 M borate buffer (pH 8.5) and incubated at room temperature for 2 hours. Excess reagent was washed with EDTA-containing phosphate-buffered saline (pH 6.0) using an Amicon Ultra-0.5 ml centrifuge filter, this was repeated 3 times, finally concentrated and then filtered through a membrane filter.

Then, a solution of 2-iodoacetamide, prepared to 10 mg/ml using phosphate-buffered saline (pH 6.0), was added to the resulting supernatant and then the resulting mixture was incubated at 37°C for 30 minutes. Excess iodoacetamide was washed with phosphate buffered saline using an Amicon Ultra-0.5 ml centrifuge filter 3 times, finally concentrated and then filtered through a membrane filter to purify the Fab ² -bound conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [DFO-C(=O)-CH ₂ O-(1,3-Ph)-C(=O)-Gly-Tyr*-CH ₂ - C(=O)-NH-(CH ₂ ) ₂ -Z ₁ (#S)], having a molecular weight of 1241, was associated with one Fab ² having a molecular weight of 47.9 kDa.

(Example 2-61. Synthesis of ([DFO-C(=O)-(CH ₂ CH ₂ O) ₄ -(1,3-Ph)-C(=O)-Gly-Lys*-Z ₂ (#S )] _p -Fab ² ))

[0286] [Chemical formula 119]

[0287] (i) Synthesis of tert-butyl N-(3-hydroxybenzoyl)glycinate

HATU (3.3 g) and DIPEA (3 ml) were added to a mixture of 3-hydroxybenzoic acid (1.0 g) and tert-butyl glycinate monohydrochloride (1.2 g) in DMF (10 ml) under ice cooling and the resulting mixture was stirred at room temperature for 1 hour. Water and ethyl acetate were added to the mixture, the resulting mixture was subjected to layer separation and extraction twice, the organic layer was washed with water and a saturated aqueous sodium chloride solution, then dried over anhydrous magnesium sulfate and filtered, then the filtrate was concentrated under reduced pressure, and ethyl acetate was added to the resulting solid. The resulting mixture was stirred at room temperature, then the insoluble matter was collected by filtration, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=95/5 → 50/50), and fractions containing the desired product were collected, concentrated and dried under reduced pressure to give the title compound (1.23 g). MS (ESI+): 274.2 [M+Na]+

(ii) Synthesis of benzyl 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate

A 4 M hydrogen chloride/dioxane solution (10 ml) was added to tert-butyl 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate (3.0 g) under ice-cooling, and then the resulting mixture was stirred at room temperature for 1 hour. The mixture was concentrated and then azeotroped twice with toluene, then methanol (20 ml) and 1 M aqueous sodium hydroxide (13 ml) were added at room temperature and the resulting mixture was stirred at the same temperature for 1.5 hours. The mixture was concentrated, then THF (10 ml), methanol (10 ml) and benzyl bromide (1.8 ml) were added and the resulting mixture was 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 give the title compound (2.19 g). MS (ESI+): 313.2

(iii) Synthesis of tert-butyl N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradecane-14-yl)oxy]benzoyl}glycinate

Add tert-butyl N-(3-Hydroxybenzoyl)glycinate (915 mg), benzyl 3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate (1.38 g), diethyl azodicarboxylate (40% solution in toluene, about 2.2 M, 3.4 ml) and THF (20 ml), then triphenylphosphine (2.0 g) was added portionwise at room temperature and the resulting mixture was then stirred overnight at a bath temperature of 60°C. Magnesium chloride (1.5 g) and toluene (20 ml) were added and the resulting mixture was heated and stirred at a bath temperature of 60°C for 2 hours. The mixture was allowed to cool to room temperature, then 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 above in the title compound (1.12 g). MS (ESI+): 546.4

(iv) Synthesis of N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradecan-14-yl)oxy]benzoyl}glycine

tert-Butyl N-{3-[(3-Oxo-1-phenyl-2,6,9,12-tetraoxatetradecane-14-yl)oxy]benzoyl}glycinate (1.11 g) was dissolved in a mixture of 4 M hydrogen chloride /dioxane (5 ml) at room temperature and the resulting solution was 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

[0288] (v) Synthesis of tert-butyl N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradecane-14-yl)oxy]benzoyl}glycyl-N ⁶ -[( benzyloxy)carbonyl]-L-lysinate

HATU (1.2 g) and DIPEA (1.4 ml) were added to the N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradecane-14-yl)oxy]benzoyl solution }glycine (1300 mg), tert-butyl N ⁶ -[(benzyloxy)carbonyl]-L-lysinate monohydrochloride (1.0 g) in DMF (20 ml) under ice cooling and the resulting mixture was stirred at room temperature for 2 hours . Water and ethyl acetate were added to the mixture to separate the layers and extract, the organic layer was washed with water and saturated brine, then dried over anhydrous magnesium sulfate and filtered, and then 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 give the title compound (1.14 g). MS (ESI+): 808.5

(vi) Synthesis of tert-butyl N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-L-lysinate

10% Palladium on carbon (50% moisture, 200 mg) was added to the mixture of tert-butyl N-{3-[(3-oxo-1-phenyl-2,6,9,12-tetraoxatetradecane-14-yl)oxy] benzoyl}glycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (1.12 g), Et ₃ N (20 μl) and ethanol (20 ml) at room temperature and the resulting mixture was stirred under hydrogen atmosphere overnight at the same temperature. The mixture was stirred at room temperature for 30 minutes or more in an open system, then the reaction liquid was filtered through Celite and the filtrate was concentrated to obtain the title compound (825 mg). MS (ESI+): 584.5

(vii) Synthesis of tert-butyl N-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro -1H-pyrrol-1-yl)-L-norleucinate

Methyl 2,5-dioxo-2,5-dihydro-1H-pyrrole-1-carboxylate (150 mg) and Et ₃ N (500 μl) were added to the mixture of tert-butyl N-[3-(2-{2-[ 2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-L-lysinate (350 mg), THF (3 ml) and DMF (1 ml) at room temperature and the resulting mixture was stirred at a bath temperature of 60°C in within 4 days. The mixture was cooled to room temperature and then TFA (300 μL) and water (500 μL) were added to neutralize the mixture. Suitable amounts of water and DMF were added to the mixture, and the resulting mixture was purified by reverse phase column chromatography (0.1% aqueous TFA:acetonitrile=95:5 → 0:100). Fractions containing the desired product were collected, concentrated and dried under reduced pressure to obtain the title compound (250 mg). MS (ESI+): 664.5

[0289] (viii) Synthesis of tert-butyl N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19 ,24,30,35,41-hexaazatritetracontan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate

EDC HCl (140 mg), HOBt (100 mg) and DIPEA (200 μl) were added to the mixture of N ⁴ -{5-[acetyl(hydroxy)amino]pentyl}-N ¹ -(5-{4-[(5- aminopentyl)(hydroxy)amino]-4-oxobutanamido}pentyl)-N ¹ -hydroxybutanediamide monomethanesulfonate (DFO.MeSO ₃ H) (240 mg), tert-butyl N-[3-(2-{2-[2-( 2-carboxyethoxy)ethoxy]ethoxy}ethoxy)benzoyl]glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (243 mg) in DMF (3 ml ) and DMSO (1 ml) under ice cooling and the resulting mixture was stirred at room temperature for 3 hours. TFA (100 μL) and water (500 μL) were added and the resulting solution was purified by reverse phase column chromatography (0.1% aqueous TFA:acetonitrile=95:5 → 10:90) and freeze-dried to give the title compound ( 207 mg). MS (ESI+): 1228.5 [M+Na]+

(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-hexaazatritetracontan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine

TFA (1 ml) was added to tert-butyl N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19 ,24,30,35,41-hexaazatritetracontan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucinate (202 mg) at room temperature and the resulting 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 resulting mixture was purified by reverse phase column chromatography (0.1% aqueous TFA:acetonitrile=95:5 → 10:90) and freeze-dried to give the following: title compound (137 mg). MS (ESI-); 1148.7

(x) Synthesis of conjugate no. 61

Using N-{3-[(19,30,41-trihydroxy-12,20,23,31,34,42-hexaoxo-3,6,9-trioxa-13,19,24,30,35,41 -hexaazatritetetracontan-1-yl)oxy]benzoyl}glycyl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-L-norleucine conjugate was prepared in the same way as in step ( ix) Example 2-60 above. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [DFO-C(=O)-(CH ₂ CH ₂ O) ₄ -(1,3-Ph)-C(=O)-Gly-Lys *-Z ₂ (#S)], having a molecular weight of 1252, was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it.

(Example 2-18. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (# N)]p-Fab ² ))

[0290] [Chemical formula 120]

[0291] (i) Synthesis of 4-nitrophenyl (4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamate

A solution of 4-nitrophenylcarbonochloridate (952 mg) in dichloromethane (10 ml) was cooled with ice under a nitrogen atmosphere, and a mixed solution of tert-butyl [(4-aminophenyl)methyl]carbamate (1000 mg) and pyridine (430 μl) in dichloromethane was added dropwise ( 10 ml) and then the resulting mixture was 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 give the crude product. Ethyl acetate was added to the crude product and the resulting mixture was stirred to tritute the solid into powder. The solid was collected by filtration, washed with ethyl acetate and then dried under reduced pressure to obtain the title compound (861 mg). MS (ESI+): 410.3 [M+Na]+

(ii) Synthesis of L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide mono(trifluoroacetate)

TFA (2 ml) was added dropwise to a solution of N-(tert-butoxycarbonyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl ]-L-isoleucinamide (257 mg) in dichloromethane (4 ml) with ice cooling. The resulting mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure, diisopropyl ether was added, decanting was performed twice, and the precipitated solid was washed to give the crude product of the title compound (248 mg). MS (ESI+): 385.3

(iii) Synthesis of N-[(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)ethyl]-L-isoleucinamide

TEA (59 μl) was added to a mixed solution of 4-nitrophenyl (4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamate (54 mg) and L-methionyl-N ¹ -[2-(2,5-dioxo -2,5-dihydro)-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide mono(trifluoroacetate) (70 mg) in dichloromethane (5 ml) and the resulting 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 give the title compound (55 mg). MS (ESI+): 633.5

[0292] (iv) Synthesis of N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1- yl)ethyl]-L-isoleucinamide mono(trifluoroacetate)

TFA (2 ml) was added dropwise to a solution of N-[(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N ¹ -[2-(2,5-dioxo-2 ,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide (54 mg) in dichloromethane (4 ml) under ice-cooling and the resulting mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure, diisopropyl ether was added, decanting was performed twice, and the precipitated solid was washed to give the crude product of the title compound (65 mg). MS (ESI+): 555.4 [M+Na]+

(v) Synthesis of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido} methyl)phenyl]carbamoyl}-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide tetrakis(trifluoroacetate)

HATU (66 mg) and DIPEA (45 μl) were added to a mixed solution of N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5- Dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide mono(trifluoroacetate) (64 mg) and DOTA-tris(t-Bu)ester (50 mg) in DMAc (2 ml) and the resulting mixture was stirred at room temperature for 2 hours. 1% TFA aqueous solution (about 4 ml) and acetonitrile (about 1 ml) were added to the reaction mixture, then the diluted solution was purified by reverse phase column chromatography (solvent gradient; 20 → 100% 0.1% TFA acetonitrile/0.1% aqueous TFA solution) to obtain the title compound (92 mg). MS (ESI+): 1109.4 [M+Na]+

(vi) Synthesis of N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]carbamoyl}-L -methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide tetrakis(trifluoroacetate)

TFA (4 ml) was added to a solution of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1- yl]acetamido}methyl)phenyl]carbamoyl}-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide tetrakis( trifluoroacetate) (90 mg) in dichloromethane (4 ml) and the resulting mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure and then the residue was purified by reverse phase column chromatography (solvent gradient; 5→50% acetonitrile/0.1% aqueous TFA) to give the title compound (57 mg). MS (ESI+): 919.4

(vii) Synthesis of N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ , N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl )ethyl]-L-isoleucine amidato(3-)]gadolinium

Gadolinium chloride (8 mg) was added to the N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl mixture ]carbamoyl}-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl]-L-isoleucinamide tetrakis(trifluoroacetate) (22 mg) and water (3 ml), 0.1 M aqueous sodium hydrogen carbonate was added to adjust the pH to 5-6 and the resulting mixture was stirred at room temperature for 1 hour. TFA (10 μL) was added to the reaction mixture and the solution was then purified by reverse phase column chromatography (solvent gradient 5→50% acetonitrile/0.1% TFA aqueous solution) to obtain the title compound (16 mg). MS (ESI-); 1072.1

[0293] (viii) Synthesis of conjugate no. 18

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (1 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (40 μl). 0.1 M borate buffer (40 μl) was added to the resulting solution and the pH was adjusted to 7.3 using 0.1 M sodium carbonate aqueous solution and 0.25 M acetate buffer.

40 μl of the pre-prepared solution was added to 4.45 mg/ml Fab²V borate buffer (160 μl) and the resulting mixture was incubated at 30°C for 2 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-0.5 mL centrifuge filter. The isolated solution was washed with phosphate-buffered saline 7 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH₂-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH₂)₂-Z₁(#N)], having a molecular weight of 1074, was associated with one Fab², having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

[0294] (Example 2-66. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -( A-4 and/or A-5)]p-Fab ² ))

(i) Synthesis of conjugate no. 66

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (1 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (40 μl). 0.1 M borate buffer (40 μL) was added to the resulting solution and the pH was adjusted to 6.5 using 0.1 M aqueous sodium carbonate solution.

The pre-prepared solution was added to 4.45 mg/ml Fab²V borate buffer (320 μl) and the resulting mixture was incubated at 30°C for 2 hours. 10 μl of 0.05 M EDTA was added and the resulting mixture was incubated at 30°C for 10 minutes. Using an Amicon Ultra-15 mL centrifuge filter, the mixture was washed twice with phosphate-buffered saline and the resulting solution was isolated.

20 mM bis-tris propane buffer solution (pH 9.5) was added to the isolated solution, diluted to 5-fold, supported on a 5 ml HiTrap Q column (GE Healthcare) and allowed to stand at room temperature for 6 hours. The column was washed using 20 mM bis-tris propane buffer solution (pH 9.5) and 1 M sodium chloride aqueous solution and the supporting solution was isolated. The buffer solution was exchanged with phosphate buffered saline using an Amicon Ultra-15 ml centrifuge filter, then the solution was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-4 and/or A-5)], having a molecular weight of 1092, was associated with one Fab ² having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were associated with it, a conjugate, in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it.

[0295] (Example 2-67. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#S)]p-Fab ² ))

(i) Synthesis of conjugate no. 67

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (2 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (80 μl), 0.1 M borate buffer (80 μl) was added and then the pH was adjusted to 7 ,3 using 0.1 M aqueous sodium carbonate solution.

A solution of 2 mg/ml 2-IT (10 μl) prepared using 0.1 M borate buffer was added to 4.45 mg/ml Fab²V borate buffer (320 μl) and the resulting mixture was incubated at 37°C for 40 minutes. Excess 2-IT was washed with phosphate buffered saline using an Amicon Ultra-15 ml centrifuge filter and concentrated to 200 µl, then the pre-prepared solution was added and the resulting mixture was incubated at 30°C for 2 hours. 0.05 M EDTA (10 μl) was added, the resulting mixture was incubated at 30°C for 10 minutes, then the mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate.

MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#S)], having a molecular weight of 1175, was associated with one Fab ² having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were bound to it.

(Example 2-68. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-3 )]p-Fab ² ))

[0296] [Chemical formula 121]

[0297] (I) Synthesis of N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-N ² -(tert-butoxycarbonyl)-L-isoleucinamide

DIPEA (6.6 ml) and benzyl (2-aminoethyl) carbamate monohydrochloride (3.29 g) were sequentially added to a mixed solution of N-(tert-butoxycarbonyl)-L-isoleucine (3.00 g) and HATU (5.92 d) in dichloromethane (30 ml) and the resulting mixture was stirred at room temperature for 1 hour. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated. 200 ml of ethyl acetate/dichloromethane (1:2) solution was added to the residue, and then the resulting mixture was concentrated to obtain 100 ml of a solution, which was then cooled with ice. The precipitated solid was collected by filtration and washed with a solution of ethyl acetate/dichloromethane (1:1) to obtain a solid. The filtrate was concentrated again and then cooled with ice, and the precipitated solid was collected by filtration and washed with a solution of ethyl acetate/dichloromethane (1:1) to obtain a solid. The resulting solids were combined and dried under reduced pressure to give the title compound (4.10 g). MS (ESI+): 408.3

(ii) Synthesis of N-(tert-butoxycarbonyl)-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide

TFA (7.51 ml) was added to a solution of N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-N ² -(tert-butoxycarbonyl)-L-isoleucinamide (2.00 g) in dichloromethane (10 ml) under ice cooling and the resulting mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure, then N-(tert-butoxycarbonyl)-L-methionine (1.35 g), DIPEA (4.2 ml), dichloromethane (20 ml) and HATU (2.24 g) were added to this residue. and the resulting mixture was stirred at room temperature for 1 hour. The precipitated solid was collected by filtration, washed with dichloromethane and methanol, and then dried under reduced pressure to obtain the title compound (1.58 g). MS (ESI+): 539.4

(iii) Synthesis of L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide

TFA (3 ml) was added dropwise to a solution of N-(tert-butoxycarbonyl)-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide (1.05 g) in dichloromethane (6 ml) with ice cooling. The resulting mixture was stirred at the same temperature for 2 hours. The mixture was concentrated under reduced pressure, dichloromethane and saturated aqueous sodium hydrogen carbonate were added, and the resulting mixture was extracted twice with dichloromethane. The combined organic layers were washed with saturated aqueous sodium chloride and then dried over anhydrous sodium sulfate, filtered and then concentrated to give the crude product of the title compound (690 mg). MS (ESI+): 439.4

[0298] (iv) Synthesis of N-[(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl) -L-isoleucinamide

Diphenylphosphorazitate (680 µl) was added to a mixed solution of 4-{[(tert-butoxycarbonyl)amino]methyl}benzoic acid (395 mg), TEA (660 µl) and toluene (20 ml) under argon and the resulting 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 allowed to cool to room temperature, a solution of L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide (690 mg) in THF (7 ml) was added and the resulting mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, aqueous sodium hydrogen carbonate and ethyl acetate were added, and the resulting solid was filtered, washed with ethyl acetate and then dried under reduced pressure to obtain the title compound (790 mg). MS (ESI+): 687.5

(v) Synthesis of N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide

TFA (2 ml) was added dropwise to a solution of N-[(4-{[(tert-butoxycarbonyl)amino]methyl}phenyl)carbamoyl]-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino }ethyl)-L-isoleucinamide (790 mg) in dichloromethane (3 ml) with ice cooling. The resulting mixture was stirred at the same temperature for 1 hour. The mixture was concentrated under reduced pressure and a saturated aqueous sodium hydrogen carbonate solution was added. The precipitated solid was collected by filtration, washed with water and then dried under reduced pressure to obtain the crude product of the title compound (720 mg). MS (ESI+): 587.6

(vi) Synthesis of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido} methyl)phenyl]carbamoyl}-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide

HATU (280 mg) was added to a mixed solution of N-{[4-(aminomethyl)phenyl]carbamoyl}-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide (360 mg), DOTA-tris(t-Bu)ester (386 mg) and DIPEA (320 μl) in DMAc (2 ml) and the resulting 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 give the title compound (683 mg). MS (ESI+); 1141.9

[0299] (vii) Synthesis of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl ]acetamido}methyl)phenyl]carbamoyl}-L-methionyl-N ¹ -(2-aminoethyl)-L-isoleucinamide

Mixture of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl ]carbamoyl}-L-methionyl-N ¹ -(2-{[(benzyloxy)carbonyl]amino}ethyl)-L-isoleucinamide (683 mg), 10% palladium on carbon (50% moisture, 382 mg) and methanol ( 10 ml) was stirred under a hydrogen atmosphere (1 atm) at room temperature for 18 hours. The mixture was filtered to remove insoluble matter and then concentrated. 10% palladium-carbon (50% water content, 382 mg) and methanol (10 ml) were added to the residue, and the resulting mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature for 2 hours. The mixture was filtered to remove insoluble matter and then concentrated to give the title compound (417 mg). MS (ESI+); 1007.7

(viii) Synthesis of N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]carbamoyl}-L -methionyl-N ¹ -(2-aminoethyl)-L-isoleucinamide pentakis(trifluoroacetate)

Mixed solution of N-{[4-({2-[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl) phenyl]carbamoyl}-L-methionyl-N ¹ -(2-aminoethyl)-L-isoleucinamide (417 mg), water (210 µl), tri(propan-2-yl)silane (210 µl) and TFA (8 ml ) was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure and then the residue was purified by reverse phase column chromatography (solvent gradient; 0→33% acetonitrile/0.1% aqueous TFA) to give the title compound (255 mg). MS (ESI+); 839.7

(ix) Synthesis of N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]carbamoyl}-L -methionyl-N ¹ -(2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl)-L-isoleucinamide tetrakis(trifluoroacetate)

TEA (200 μL) was added to the N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl] solution carbamoyl}-L-methionyl-N ¹ -(2-aminoethyl)-L-isoleucinamide pentakis(trifluoroacetate) (255 mg) in DMAc (2 ml) under ice cooling and the resulting mixture was stirred at the same temperature for 10 minutes. To this solution was added a solution of 1,4-diisothiocyanatobenzene (174 mg) in DMAc (2 ml) and the resulting 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% aqueous TFA) to give the title compound (136 mg). MS (ESI+); 1031.4

[0300] (x) Synthesis of N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl]-L-isoleucine amidato (3-)]gadolinium

Gadolinium chloride (10 mg) and 0.1 M aqueous sodium hydrogen carbonate (1.7 ml) were added to the mixture of N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4, 7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]carbamoyl}-L-methionyl-N ¹ -[2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl]-L-isoleucinamide tetrakis(trifluoroacetate) (36 mg) and water (1.8 ml) to adjust the pH to 5.4 and the resulting mixture was stirred at room temperature for 30 minutes. The reaction mixture was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile/0.1% aqueous TFA) to give the title compound (33.0 mg). MS (ESI-); 1184.3

(xi) Synthesis of conjugate no. 68

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl]-L-isoleucine amidato(3-)]gadolinium (1 mg) synthesized in Example 2-68 (x) was dissolved in DMSO (310 μl).

30 µl of the pre-prepared solution was added to the 4.45 mg/ml Fab solution²V phosphate-buffered saline (320 μl), the pH was adjusted to about 9.0 using 0.1 M aqueous sodium carbonate solution and the resulting mixture was incubated at 37°C for 24 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH₂-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH₂)₂-(A-3)], having a molecular weight of 1187, was associated with one Fab², having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

(Example 2-69. Synthesis of ([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 ))

[0301] [Chemical formula 122]

[0302] (i) Synthesis of tert-butyl N-[2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]- L-lysinate

TEA (0.24 ml) and HATU (241 mg) were added to a mixed solution of tert-butyl L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate (303 mg) and 2-(tert-butoxycarbonyl)- 1,2,3,4-tetrahydroisoquinoline-5-carboxylic acid (160 mg) in dichloromethane (10 ml) and the resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and then the residue was purified by silica gel column chromatography (solvent gradient; 0→4% methanol/chloroform) to give the title compound (191 mg). MS (ESI+); 784.4

(ii) Synthesis of tert-butyl N-(1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate mono(trifluoroacetate)

TFA (1 ml) was added dropwise to a solution of tert-butyl N-[2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N ⁶ -[(benzyloxy) carbonyl]-L-lysinate (190 mg) in dichloromethane (3 ml) with ice cooling. The resulting mixture was stirred at the same temperature for 1 hour and then concentrated under reduced pressure to obtain the crude product of the title compound (262 mg). MS (ESI+): 684.5

(iii) Synthesis of tert-butyl N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}- 1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate tetrakis(trifluoroacetate)

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 resulting mixture was stirred at room temperature for 5 minutes. A solution of tert-butyl N-(1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate mono(trifluoroacetate) (261 mg) was added to the reaction solution ) in DMAc (1 ml) and the resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with 1% aqueous TFA (about 1 ml) and then the resulting solution was purified by reverse phase column chromatography (solvent gradient; 20→100% acetonitrile/0.1% aqueous TFA) to give the title compound (231 mg ). MS (ESI+); 1260.7 [M+Na]+

[0303] (iv) Synthesis of tert-butyl N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl] acetyl}-1,2,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysinate tetrakis(trifluoroacetate)

A mixture of tert-butyl N-(2-{[4,7,10-tris(2-tert-butoxy-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,2 ,3,4-tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-N ⁶ -[(benzyloxy)carbonyl]-L-lysinate tetrakis(trifluoroacetate) (230 mg), 10% palladium on carbon (50% moisture, 300 mg ) and methanol (10 ml) were stirred at room temperature for 10 minutes. The mixture was filtered to remove insoluble matter and then the filtrate was concentrated. To the residue were added 10% palladium on carbon (50% humidity, 300 mg) and methanol (10 ml) and then the resulting mixture was stirred under a hydrogen atmosphere (3 atm) at room temperature for 18 hours. The mixture was filtered to remove insoluble matter and then concentrated to give the title compound (152 mg). MS (ESI+); 1104.5

(v) Synthesis of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5 -carbonyl)-L-methionylglycyl-L-lysine pentakis(trifluoroacetate)

TFA (1.5 ml) was added to the solution of tert-butyl 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-lysinate tetrakis(trifluoroacetate) (152 mg) in dichloromethane (1 ml) and the resulting mixture was stirred at room temperature in within 4 hours. The mixture was concentrated under reduced pressure and then the residue was purified by reverse phase column chromatography (solvent gradient; 0→33% acetonitrile/0.1% aqueous TFA) to give the title compound (76 mg). MS (ESI+); 880.4

(vi) Synthesis of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,2,3,4-tetrahydroisoquinoline-5 -carbonyl)-L-methionylglycyl-N ⁶ -[(4-isothiocyanatophenyl)carbamothioyl]-L-lysine tetrakis(trifluoroacetate)

TFA (60 μl) was added to a solution of N-(2-{[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetyl}-1,2,3,4 -tetrahydroisoquinoline-5-carbonyl)-L-methionylglycyl-L-lysine pentakis(trifluoroacetate) (76 mg) and 1,4-diisothiocyanatobenzene (50 mg) in DMAc (1 ml) under ice cooling and the resulting mixture was stirred at room temperature in within 1 hour. The reaction solution was purified by reverse phase column chromatography (solvent gradient; 0→50% acetonitrile/0.1% aqueous TFA) to give the title compound (46 mg). MS (ESI-); 1070.6

(vii) Synthesis of {N-[2-({4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetyl-co)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N ⁶ -[(4-isothiocyanatophenyl)carbamothioyl]-L-lysinato(3-)} gadolinium

Gadolinium chloride (8 mg) and 0.1 M aqueous sodium hydrogen carbonate solution (700 μl) were added to the mixture 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-N ⁶ -[(4-isothiocyanatophenyl)carbamothioyl]-L-lysine tetrakis(trifluoroacetate) (16 mg) and water (800 μl) and the resulting 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% aqueous TFA) to give the title compound (7.4 mg). MS (ESI-); 1225.3

[0304] (viii) Synthesis of conjugate no. 69

{N-[2-({4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetyl-co)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N ⁶ -[(4-isothiocyanatophenyl)carbamothioyl]-L-lysinato(3-)}gadolinium (1 mg ) synthesized in Example 2-69 (vii) was dissolved in DMSO (310 μl).

30 µl of the pre-prepared solution was added to a solution of 4.45 mg/ml Fab ² in phosphate buffered saline (320 µl), the pH was adjusted to about 9.0 using 0.1 M aqueous sodium carbonate and the resulting mixture was incubated at 37 °C for 24 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 mL centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which 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, was associated with one Fab ² having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, a conjugate in which four such molecules were associated with it, and a conjugate in which five such molecules were associated with it.

The conjugates obtained in the Preparation Examples described above are shown in Tables 1 and 2.

[0305]

Table 1

[0306]

table 2

[0307] In the tables presented above and Tables 15-18 presented below, - represents the bond and the symbols (a)-(q) in the S ₁ column indicate the spacers represented by the following formulas (a)-(q), respectively.

[0308] [Chemical formula 123]

[0309] The compounds of Preparation Examples Nos. A1-A43 and B1-B4 in Tables 3-14 were synthesized using the same methods as those in the above Preparation Examples or methods known to those skilled in the art, and these compounds were used to obtaining the conjugates shown in Tables 15-18 below.

[0310]

Table 3

[0311]

Table 4

[0312]

Table 5

[0313]

Table 6

[0314]

Table 7

[0315]

Table 8

[0316]

Table 9

[0317]

Table 10

[0318]

Table 11

[0319]

Table 12

[0320]

Table 13

[0321]

Table 14

[0322]

Table 15

[0323]

Table 16

[0324]

Table 17

[0325]

Table 18

[0326] MS analyzes of the compounds of the Examples shown in Tables 15-18 are shown in Tables 19-23 below.

[0327]

Table 19 Pr.-No. MS 1 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 937 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 2 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 838 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it 3 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 852 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 4 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1046 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 5 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 926 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 6 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1059 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 7 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1061 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 8 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1027 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 9 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 958 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 10 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1012 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 14 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1093 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 17 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1149 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it .

[0328]

Table 20 Pr.-No. MS 18 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1074 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 19 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 988 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it. 20 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 825 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 21 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 960 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it. 22 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 896 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 23 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 936 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 25 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1248 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 26 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 954 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 27 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 950 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 28 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 908 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. thirty MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1149 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

[0329]

Table 21 Pr.-No. MS 31 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1123 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 32 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1095 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 34 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1101 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 36 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1045 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 38 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1190 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 39 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1384 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 41 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1145 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it. 42 MS analysis confirmed that the conjugate was a conjugate in which one small molecule having a molecular weight of 1107 was associated with one Fab ² having a molecular weight of 47.9 kDa. 43 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1090 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 44 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1259 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it.

It was confirmed by MS analysis that the conjugate was a mixture of a conjugate in which one low molecular weight molecule having a molecular weight of 1259 was bound to one Fab ² having a molecular weight of 47.9 kDa, a conjugate in which two such molecules were bound thereto, and a conjugate in which three such molecules were bound thereto.
[0330]

Table 22 Pr.-No. MS 45 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1296 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 46 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1296 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 49 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1203 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it 50 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1280 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 51 MS analysis confirmed that the conjugate was a mixture of conjugate in which one small molecule having a molecular weight of 1443 was associated with one Fab ² having a molecular weight of 47.9 kDa. 52 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 984 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 53 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1148 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 54 MS analysis confirmed that the conjugate was a mixture of conjugate in which one small molecule having a molecular weight of 1121 was associated with one Fab ² having a molecular weight of 47.9 kDa. 55 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1187 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 56 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1153 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 57 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1079 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 58 MS analysis confirmed that the conjugate was a mixture of conjugate in which one small molecule having a molecular weight of 1244 was associated with one Fab ² having a molecular weight of 47.9 kDa.

[0331]

Table 23 Pr.-No. MS 59 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1304 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 62 MS analysis confirmed that the conjugate was a conjugate in which one small molecule having a molecular weight of 1106 was associated with one Fab ² having a molecular weight of 47.9 kDa. 63 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1253 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it . 64 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1076 was associated with one Fab ² having a molecular mass of 47.9 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 65 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1234 was associated with one Fab ² having a molecular mass of 47.9 kDa, and a conjugate in which two such molecules were associated with it .

[0332] Example 3-1: Evaluation of Anti-CEACAM5 Antibody Fab Conjugate Binding Activity

Each anti-human CEACAM5 Fab conjugate obtained in Example 2 was subjected to ELISA to evaluate its binding activity to CEACAM5. In this test, phosphate buffer (Nacalai Tesque Inc., 0.1 mol/L Phosphate Buffer Solution (pH 7.2)), prepared to 10 mM by diluting 10 times with distilled water, was used as a solvent for the antigen immobilization liquid , and 20X PBS/Tween 20 Buffer (Thermo Fisher Scientific Inc., 28352) diluted 20-fold with distilled water was used as a wash liquid. In addition, regarding the bovine serum albumin (BSA) used in this test, 30% bovine serum albumin solution (Sigma-Aldrich, Inc., A9576-50ML) was added in a suitable proportion for use. CEACAM5 (R&D Systems, 4128-CM-050) was diluted with 10 mM phosphate buffer (pH 7.2) to 0.1 μg/ml and added to a white 384 Nunc MaxiSorp plate (Nunc, 4603272) at 30 μl per well and the plate was incubated overnight at 4°C for immobilization. The CEACAM5 immobilizing fluid was removed by reverse centrifugation and then blocked by adding PBS/Tween 20 buffer containing 5.0% BSA. The blocking solution was then removed by reverse centrifugation, and a solution of each anti-CEACAM5 Fab ² fragment conjugate described above or a Fab ² fragment lacking the labeling moiety (PB009-01) as a control at about 10,000 ng/ml was diluted in 14 steps by 3 -fold dilution using PBS/Tween 20 buffer containing 1.0% BSA, and 30 μl per well was added and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and horseradish peroxidase (HRP)-labeled goat anti-human IgG (H+L chain) antibody (MBL Life Sciences, 206) diluted 1000-fold using PBS/Tween 20 buffer containing 5.0 % BSA was added at 30 μl per well and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and ECL Prime Western Blotting Detection reagent (GE Healthcare, RPN2232) was added as substrate at 30 μl per well. The substrate was incubated at room temperature for 15 minutes and then its signal value was measured using an Envision counter (PerkinElmer, Inc.).

The test for each antibody was performed in duplicate and the EC ₅₀ value was calculated using a 4-parameter logistic curve model. Geometric mean (geometric mean), standard deviation (geometric SD factor) and lower limit (lower value) and upper limit (upper value) 95% confidence interval (95% CI of geometric mean) EC ₅₀ values (nM) for 9 experiments for PB009-01 are shown in Table 24. In addition, the EC ₅₀ value of each conjugate tested in duplicate is shown in Table 25. Example No. in the table represents the conjugate number in Example 2.

[0333]

Table 24 RV009-01 Geometric mean (nM) 0.20 Geometric SD factor 1.45 Lower value 95% CI geom. average (nM) 0.15 Upper value 95% CI geom. average (nM) 0.27

[0334]

Table 25

[0335] Example 3-2:

Each anti-human CEACAM5 Fab conjugate obtained in Example 2 was subjected to ELISA to evaluate its binding activity to CEACAM5. In this test, phosphate buffer (Nacalai Tesque Inc., 0.1 mol/L Phosphate Buffer Solution (pH 7.2)), prepared to 10 mM by diluting 10 times with distilled water, was used as a solvent for the antigen immobilization liquid , and 20X PBS/Tween 20 Buffer (Thermo Fisher Scientific Inc., 28352) diluted 20-fold with distilled water was used as a wash liquid. In addition, regarding the bovine serum albumin (BSA) used in this test, 30% bovine serum albumin solution (Sigma-Aldrich, Inc., A9576-50ML) was added in a suitable proportion for use. CEACAM5 (R&D Systems, 4128-CM-050) was diluted with 10 mM phosphate buffer (pH 7.2) to 0.1 μg/ml and added to a white 384 Nunc MaxiSorp plate (Nunc, 4603272) at 30 μl per well and the plate was incubated overnight at 4°C for immobilization. The CEACAM5 immobilizing fluid was removed by reverse centrifugation and then blocked by adding PBS/Tween 20 buffer containing 5.0% BSA. The blocking solution was then removed by reverse centrifugation, and a solution of each anti-CEACAM5 antibody fragment Fab2 conjugate described above or Fab ² fragment lacking the labeling moiety (PB009-01) as a control at about 10,000 ng/ml was diluted in 14 steps by 3- A fold dilution using PBS/Tween 20 buffer containing 1.0% BSA and 30 μl was added per well and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and horseradish peroxidase (HRP)-labeled goat anti-human IgG (H+L chain) antibody (MBL Life Sciences, 206) diluted 1000-fold using PBS/Tween 20 buffer containing 5.0 % BSA was added at 30 μl per well and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and ECL Prime Western Blotting Detection reagent (GE Healthcare, RPN2232) was added as substrate at 30 μl per well. The substrate was incubated at room temperature for 15 minutes and then its signal value was measured using an Envision counter (PerkinElmer, Inc.).

[0336] The test for each antibody was performed in duplicate and the EC ₅₀ value was calculated using a 4-parameter logistic curve model. The EC ₅₀ value (nM) for each of the 2 experiments for PB009-01 is shown in Table 26. In addition, the EC ₅₀ value of each conjugate tested in duplicate is shown in Table 27. Example No. in the table represents the conjugate number in the Example 2.

[0337]

Table 26 RV009-01 (nM) 0.14 0.21

[0338]

Table 27 Pr.-No. EC ₅₀ (nM) 66 0.16 68 0.31

[0339] Example 4-1: Test to Assess Renal Accumulation of Gd-Labeled Conjugates (Normal Mice)

The conjugate includes a conjugate containing a peptide linker having an amino acid sequence cleavable by a renal brush border membrane enzyme or a lysosomal enzyme as the peptide linker "X." The conjugate of the present invention containing such a linker is specifically cleaved at the linker group by any of these enzymes present in the kidney, and thus the accumulation of the labeling moiety in the kidney is expected to be reduced. The results of assessing the accumulation in the kidneys of such a conjugate of the present invention are shown below.

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to mice (BALB/c or BALB/c nu/nu) such that the protein weight was 0.02 mg ( 100 µl) per animal. After approximately 24 hours, the mice were sacrificed and the kidneys were removed and the amount of Gd in the kidneys was measured using ICP-MS. This test was performed on 3 cases in each group, the average of the 3 cases was calculated, and the amount of Gd contained per kidney after administration was shown as a percentage (% dose/tissue) of the total amount of Gd administered. The results are shown in Table 28 below. Pr.-No. in the table represents the number of the conjugate in Example 2. In addition, the same test was carried out using Pr.-No. 47 ([Gd/DOTA]p-Fab) obtained in Preparation Example 2-47, which did not contain a peptide linker, as a control conjugate, and the geometric mean (geometric mean), standard deviation (geometric SD factor) and value were calculated lower limit (lower value) and upper limit value (upper value) of the 95% confidence interval (95% CI of the geom. mean) for 5 experiments.

[0340] (Results)

The results are shown in Table 29 below. Based on the percentage of control conjugate (% dose/tissue), the percentage reduction in the amount of Gd contained in the kidney was determined for each of the conjugates containing the peptide linker and is shown in Table 28. As shown in Table 28, the accumulation of the labeling moiety in the kidney was 39.6-82.9% points lower in conjugates containing the peptide linker compared to the control conjugate.

[0341]

Table 28

[0342]

Table 29 Ave.-No. 47 Geometric mean (nM) 31.0 Geometric SD factor 1.2 Lower value 95% CI geom. average (nM) (% dose/tissue) 25.3 Upper value 95% CI geom. average (nM) (% dose/tissue) 37.9

[0343] Example 4-2: Test to Assess Renal Accumulation of Gd-Labeled Conjugates (Normal Mice)

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to mice (BALB/c or BALB/c nu/nu) such that the protein weight was 0.02 mg ( 100 µl) per animal. After approximately 24 hours, the mice were sacrificed and the kidneys were removed and the amount of Gd in the kidneys was measured using ICP-MS. This test was performed on 3 cases in each group, the average of the 3 cases was calculated, and the amount of Gd contained per kidney after administration was shown as a percentage (% dose/tissue) of the total amount of Gd administered. The results are shown in Table 30 below. Example No. in the table represents the number of the conjugate in Example 2. In addition, the same test was carried out using Example No. 47 ([Gd/DOTA]p-Fab) obtained in Preparation Example 2, which did not contain the peptide linker, as a control conjugate.

(Results)

The results are shown in Table 30 below. Based on the percentage of control conjugate (% dose/tissue), the percentage reduction in the amount of Gd contained in the kidney was determined for each of the conjugates containing the peptide linker and is shown in Table 30. As shown in Table 30, the accumulation of the labeling moiety in the kidney was 92.15-95.38% points lower in conjugates containing the peptide linker than in the control conjugate 47.

[0344]

Table 30
Residuals in kidneys (normal mice) Pr.-No. % dose/tissue Reduction percentage (%) 66 2.43 92.15 68 1.43 95.38 47 31.33 -

[0345] Example 4-3: Test to Assess Renal Accumulation of Gd-Labeled Conjugates (Normal Mice)

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to mice (BALB/c or BALB/c nu/nu) such that the protein weight was 0.02 mg ( 100 µl) per animal. After approximately 24 hours, the mice were sacrificed and the kidneys were removed and the amount of Gd in the kidneys was measured using ICP-MS. This test was performed on 3 cases in each group, the average of the 3 cases was calculated, and the amount of Gd contained per kidney after administration was shown as a percentage (% dose/tissue) of the total amount of Gd administered. The same test was carried out using Pr.-No. 47 obtained in Preparation Example 2 above, which did not contain the peptide linker, as a control conjugate and the average of the kidney residues for 5 experiments for Pr.-No. 47 is shown in Table 31.

(Results)

Based on the percentage of control conjugate (% dose/tissue), the percentage (%) reduction in the amount of Gd contained in the kidneys for each of the peptide linker-containing conjugates was determined and shown in Table 32. As shown in Table 32, labeling accumulation kidney fraction was 21.00-94.66% points lower in conjugates containing the peptide linker compared to the control conjugate.

[0346] [Table 31]

Table 31
Residuals in kidneys (normal mice) Residual amount in kidneys
(% dose/tissue) Average value 29.33

[0347]

Table 32
Residuals in kidneys (normal mice)

[0348] Example 4-4: Test to evaluate renal accumulation of Gd-labeled conjugates (tumor-bearing mice)

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to tumor-bearing mice such that the protein mass was 0.02 mg (100 μl) per animal. The tumor-bearing mice used in this Example were mice (BALB/c nu/nu) that were in a tumor-bearing state as a result of transplantation of LS174T cells from the human colorectal cancer cell line (ATCC (registered trademark); CL- 188), at 2.0-5.0 × 10 ⁶ cells/mouse, subcutaneously in the dorsal region before introducing the sample. 24 hours after sample administration, mice were sacrificed, kidneys and tumor were removed, and the amount of Gd in the kidney and tumor was measured using ICP-MS. This test was performed on 3 cases in each group and the average of the 3 cases was calculated. The amount of Gd contained per kidney after administration was shown as a percentage of the amount of Gd administered (% dose/tissue), and the amount of Gd contained per g of tumor tissue was shown as a percentage of the total amount of Gd administered (% dose/g). The results are shown in Tables 35 and 36. Pr.-No. in the tables represents the number of the conjugate in Example 2. Moreover, the same test was carried out using Pr.-No. 47 obtained in Preparation Example 2-47, which did not contain the peptide linker, as a control conjugate. Average values for 2 experiments for Pr.-No. 47 are shown in Tables 33 and 34.

(Results)

As shown in Tables 35 and 36, the accumulation of the labeling moiety in the kidneys was 52.26-77.68% points lower in the conjugates containing the peptide linker compared to the control conjugate.

[0349]

Table 33
Residual amounts in kidneys (tumor-bearing mice) Residual amount in kidneys
(% dose/tissue) Average value 25.83

[0350]

Table 34
Accumulation in tumor Amount accumulated in tumor
(% dose/g) Average value 4.92

[0351]

Table 35
Residual amounts in kidneys (tumor-bearing mice)

[0352]

Table 36
Amount accumulated in tumor (tumor-bearing mice) Pr.-No. % dose/g 18 10.63 24 10.50 29 4.37 33 4.53 35 6.90

[0353] (Example 5-1: Preparation of anti-human MUC1 antibody fragment Fab ⁵ conjugate)

This Example discloses Examples of the preparation of the conjugate. When presenting each Preparation Example in this Example, the words “Example 5” are followed by a single hyphen followed by “conjugate number.” For example, "Example 5-101" means that this is an Example for the preparation of conjugate 101 in this Example. Also, as Fab⁵V This Example used (P10-2) disclosed in international publication WO2018/092885. "p-Fab⁵" shows that Fab⁵linked to p labeling moieties enclosed in brackets [ ] or ( ), via p of its amino groups to form a conjugate. Some of the following Examples describe the number of labeling moieties associated with Fab⁵each conjugate that has been confirmed by MS analysis, but the result does not indicate that the conjugate having an associated amount of labeling moieties other than the amount specified above is not included. It should be understood that there may still be a conjugate having a certain associated number of labeling moieties, the presence of which could not be confirmed due to the precision of the MS analysis equipment. Moreover, in the structural formulas in this Example, the structural formula of DOTA to which Gd is bound schematically represents DOTA labeled with Gd.

[0354] (Example 5-101. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#N)]p-Fab ⁵ ))

(i) Synthesis of conjugate no. 101

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (1 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (40 μl). 0.1 M borate buffer (40 μl) was added to the resulting solution and the pH was adjusted to 6.6 using 0.25 M acetate buffer.

80 μl of the pre-prepared solution was added to 5.2 mg/ml Fab ⁵ borate buffer (240 μl) and the resulting mixture was incubated at 30°C for 2 hours. 15 μl of 0.05 M EDTA was added and the resulting mixture was incubated at 30°C for 10 minutes. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-0.5 mL centrifuge filter. The isolated solution was washed with phosphate-buffered saline 7 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#N)], having a molecular weight of 1074, was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it.

[0355] (Example 5-104. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -( A-4 and/or A-5)]p-Fab ⁵ ))

(i) Synthesis of conjugate no. 104

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (1 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (40 μl). 0.1 M borate buffer (40 μl) was added to the resulting solution and the pH was adjusted to 10.2 using 0.1 M aqueous sodium carbonate solution.

[0356] The entire pre-prepared solution was added to 5.2 mg/ml Fab ⁵ borate buffer (240 μl) and the resulting mixture was incubated at 30°C for 2 hours. The mixture was isolated using an Amicon Ultra-15 ml centrifuge filter and washed with phosphate buffered saline.

Buffer exchange was carried out twice using 3 ml of HEPES-NaOH buffer (pH=4.5) and concentration was carried out to obtain 100 μl of a solution with pH=4.52 and then the solution was incubated at 45°C for 30 minutes.

The solution was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 mL centrifuge filter. The isolated solution was washed with phosphate-buffered saline 3 times, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-4 and/or A-5)], having a molecular weight of 1092, was associated with one Fab ⁵ having a molecular weight of 47.5 kDa, a conjugate in which two such molecules were associated with it, and a conjugate , in which three such molecules were associated with it.

[0357] (Example 5-107. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#S)]p-Fab ⁵ ))

(i) Synthesis of conjugate no. 107

[N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ ,N ⁴ , N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl] -L-isoleucine amidato(3-)]gadolinium (0.75 mg) synthesized in Example 2-18 (vii) was dissolved in DMSO (40 μl), 0.1 M borate buffer (40 μl) was added and then the pH was adjusted to 8.2 using 0.1 M aqueous sodium carbonate and 0.25 M acetate buffer.

2 mg/ml 2-IT solution (7.5 μl) prepared using 0.1 M borate buffer was added to 5.2 mg/ml Fab ⁵ borate buffer (240 μl) and the resulting mixture was incubated at 37°C in for 40 minutes. Excess 2-IT was washed with phosphate buffered saline using an Amicon Ultra-0.5 ml centrifuge filter and concentrated, then the pre-prepared solution was added and the resulting mixture was incubated at 30°C for 2 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate.

MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -Z ₁ (#S)], having a molecular weight of 1175, was associated with one Fab ⁵ having a molecular weight of 47.5 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were bound to it.

(Example 5-112. Synthesis of ([Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-3 )]p-Fab ⁵ ))

(i) Synthesis of conjugate no. 112

[0358] [N-({4-[(2-{4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecan-1-yl-κ ⁴ N ¹ , N ⁴ ,N ⁷ ,N ¹⁰ }acetamido-co)methyl]phenyl}carbamoyl)-L-methionyl-N ¹ -[2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl]-L-isoleucine amidato(3- )]gadolinium (0.46 mg) synthesized in Example 2-68 (x) was dissolved in DMSO (155 μl).

30 µl of 0.1 M aqueous sodium carbonate solution and the pre-prepared solution were added to the Fab ⁵ solution (240 µl) prepared to 5.2 mg/ml using borate buffer and glycerol solution to adjust the pH to 8.8 and the resulting mixture incubated at 37°C for 2 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [Gd/DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-3)], having a molecular weight of 1187, was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it.

[0359] (Example 5-113. Synthesis of ([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 ))

(i) Synthesis of conjugate no. 113

{N-[2-({4,7,10-tris[(carboxy-co)methyl]-1,4,7,10-tetraazacyclododecane-1-y-κ ⁴ N ¹ ,N ⁴ ,N ⁷ ,N ¹⁰ }acetyl-co)-1,2,3,4-tetrahydroisoquinoline-5-carbonyl]-L-methionylglycyl-N ⁶ -[(4-isothiocyanatophenyl)carbamothioyl]-L-lysinato(3-)}gadolinium (0, 22 mg) synthesized in Example 2-69 (vii) was dissolved in DMSO (72 μl).

30 µl of 0.1 M aqueous sodium carbonate solution and pre-prepared solution was added to the Fab ⁵ solution (240 µl) prepared to 5.2 mg/ml using borate buffer and glycerol solution to adjust the pH to 9.3 and the resulting mixture incubated at 37°C for 2 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate. MS analysis confirmed that the conjugate was a mixture of a conjugate in which 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, was associated with one Fab ⁵ having a molecular weight of 47.5 kDa, and the conjugate, in in which two such molecules were associated with it.

The chemical structural formulas of the conjugates obtained in the Preparation Examples described above are shown in Tables 37 and 38.

[0360]

Table 37

[0361]

Table 38

[0362] The compounds of Preparation Example No. C1 and C2 in Table 39 were synthesized using the same methods as in the above Preparation Examples or methods known to those skilled in the art, and used these compounds or used the compounds synthesized in Example 2 , to obtain conjugates with Fab⁵, shown in Tables 40 and 41 below.

[0363]

Table 39

[0364]

Table 40

[0365]

Table 41

[0366] MS analyzes of the compounds of the Examples shown in Tables 40 and 41 are shown in the table below.

[0367]

Table 42 Pr.-No. MS 102 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1278 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it . 013 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1115 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it . 105 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1190 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 106 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1380 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 108 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 542 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, a conjugate in which two such molecules were associated with it, and a conjugate in which three such molecules were associated with it. 109 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1216 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it . 110 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1201 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it . 111 MS analysis confirmed that the conjugate was a mixture of a conjugate in which one small molecule having a molecular weight of 1391 was associated with one Fab ⁵ having a molecular mass of 47.5 kDa, and a conjugate in which two such molecules were associated with it .

[0368] The Compound of Example in Table 43 was prepared using the same method as the Preparation Examples described above, or a method known to those skilled in the art.

Table 43

[0369] MS analysis of the compound of the Example shown in Table 43 is shown in Table 44 below.

[0370]

Table 44 Pr.-No. MS 114 MS analysis confirmed that the conjugate was a mixture of conjugate in which one small molecule having a molecular weight of 1059 was associated with one Fab ⁵ having a molecular weight of 47.5 kDa.

In addition, the conjugates in Tables 45-50 can be prepared using the same methods as in the Preparation Examples described above, or methods known to those skilled in the art.

[0371]

Table 45

[0372]

Table 46

[0373]

Table 47

[0374]

Table 48

[0375]

Table 49

[0376]

Table 50

[0377] Example 6-1: Evaluation of Anti-Human MUC1 Antibody Fragment Fab ⁵ Conjugate Binding Activity

Each anti-human MUC1 antibody fragment Fab ⁵ conjugate prepared by the method of Example 5 was subjected to an ELISA assay to evaluate its binding activity to human cancer-specific MUC1. In this test, 20X PBS/Tween 20 Buffer (Thermo Fisher Scientific Inc., 28352) diluted 20 times with distilled water was used as the wash liquid. In addition, regarding the bovine serum albumin (BSA) used in this test, 30% bovine serum albumin solution (Sigma-Aldrich, Inc., A9576-50ML) was added in a suitable proportion for use. Human cancer-specific MUC1 peptide (PTL 1) at 0.5 μmol/L was added to a white 384 Nunc MaxiSorp plate (Nunc, 460372) at 30 μL per well and the plate was incubated overnight at 4°C for immobilization. MUC1 peptide was removed by reverse centrifugation and then blocked by adding PBS/Tween 20 buffer containing 5.0% BSA. The blocking solution was then removed by reverse centrifugation, a solution of each anti-human MUC1 antibody fragment Fab ⁵ conjugate (Product No. 104, 108 or 112) described above at about 10,000 ng/ml was diluted in 14 steps by 3-fold dilution using PBS/Tween 20 buffer containing 1.0% BSA was added at 30 μl per well and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and horseradish peroxidase-labeled goat anti-human Igκ antibody (Southern Biotechnology Associates, Inc.) was diluted 10,000-fold using PBS/Tween 20 buffer containing 5.0% BSA added at 30 μl per well. and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and ECL Prime Western Blotting Detection reagent (GE Healthcare, RPN2232) was added as substrate at 30 μl per well. The substrate was incubated at room temperature for 15 minutes and then its signal value was measured using an Envision counter (PerkinElmer, Inc.).

The test for each antibody was performed in duplicate and the EC ₅₀ value was calculated using a 4-parameter logistic curve model. The EC ₅₀ value (nM) for each of the 3 experiments for P10-2 is shown in Table 51. Additionally, the EC ₅₀ value of each conjugate tested in duplicate is shown in Table 52.

[0378]

Table 51 P10-2 (nM) 0.06 0.06 0.14

[0379]

Table 52 Pr.-No. EC50 (nM) 104 0.08 112 0.15 108 0.07

[0380] Example 6-2: Evaluation of Anti-Human MUC1 Antibody Fragment Fab ⁵ Conjugate Binding Activity

Each anti-human MUC1 antibody fragment Fab ⁵ conjugate prepared by the method of Example 5 was subjected to an ELISA assay to evaluate its binding activity to human cancer-specific MUC1. In this test, 20X PBS/Tween 20 Buffer (Thermo Fisher Scientific Inc., 28352) diluted 20 times with distilled water was used as the wash liquid. In addition, regarding the bovine serum albumin (BSA) used in this test, 30% bovine serum albumin solution (Sigma-Aldrich, Inc., A9576-50ML) was added in a suitable proportion for use. Human cancer-specific MUC1 peptide (PTL 1) at 0.5 μmol/L was added to a white 384 Nunc MaxiSorp plate (Nunc, 460372) at 30 μL per well and the plate was incubated overnight at 4°C for immobilization. MUC1 peptide was removed by reverse centrifugation and then blocked by adding PBS/Tween 20 buffer containing 5.0% BSA. The blocking solution was then removed by reverse centrifugation, and a solution of each anti-human MUC1 antibody fragment Fab ⁵ conjugate (Product No. 101-113) described above at about 10,000 ng/ml was diluted in 14 steps by 3-fold dilution using PBS/ Tween 20 buffer containing 1.0% BSA and 30 μl were added per well and incubated at room temperature for 60 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and horseradish peroxidase-labeled goat anti-human Igκ antibody (Southern Biotechnology Associates, Inc.) was diluted 10,000-fold using PBS/Tween 20 buffer containing 5.0% BSA added at 30 μl per well. and incubated at room temperature for 30 minutes. The plate was washed 3 times with PBS/Tween 20 buffer and ECL Prime Western Blotting Detection reagent (GE Healthcare, RPN2232) was added as substrate at 30 μl per well. The substrate was incubated at room temperature for 15 minutes and then its signal value was measured using an Envision counter (PerkinElmer, Inc.).

The test for each antibody was performed in duplicate and the EC ₅₀ value was calculated using a 4-parameter logistic curve model. The EC ₅₀ value (nM) for each of the 3 experiments for P10-2 is shown in Table 53. Additionally, the EC ₅₀ value of each conjugate tested in duplicate is shown in Table 54.

[0381]

Table 53 R 10-02(nM) 0.06 0.06 0.14

[0382]

Table 54 Pr.-No. EC50 (nM) 101 0.08 102 0.07 103 0.08 105 0.11 106 0.10 107 0.09 108 0.07 109 0.08 110 0.19 111 0.16 113 0.19

[0383] Example 7: Test to Assess Renal Accumulation of Gd-Labeled Conjugates (Normal Mice)

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to mice (BALB/c or BALB/c nu/nu) such that the protein weight was 0.02 mg ( 100 µl) per animal. After approximately 24 hours, the mice were sacrificed and the kidneys were removed and the amount of Gd in the kidneys was measured using ICP-MS. This test was performed on 3 cases in each group, the average of the 3 cases was calculated, and the amount of Gd contained per kidney after administration was shown as a percentage (% dose/tissue) of the total amount of Gd administered. The same test was carried out using PR-No. 108 obtained in Preparation Example 5, which did not contain a peptide linker, as a control conjugate. The average value for 2 experiments for Pr.-No. 108 is shown in Tables 55.

(Results)

The results are shown in Table 56. Based on the amount of Gd contained in the kidneys using the control conjugate, the percentage reduction in the amount of Gd contained in the kidneys was determined for each of the conjugates containing the peptide linker. As shown in Table 56, the accumulation of the labeling moiety in the kidneys was 91.52-93.03% points lower in the conjugates containing the peptide linker compared to the control conjugate.

[0384]

Table 55
Residuals in kidneys (normal mice) Ave.-No. 108 Average value(% dose/tissue) 33.00

[0385]

Table 56
Residuals in kidneys (normal mice) Pr.-No. % dose/tissue Reduction percentage (%) 104 2.30 93.03 112 2.80 91.52

[0386] Example 8: Test to Assess Renal Accumulation of Gd-Labeled Conjugates (Tumor-Bearing Mice)

(Test method)

A Gd-labeled conjugate, such as the peptide linker-containing conjugate prepared in one of the Examples described above, was administered via the tail vein to tumor-bearing mice such that the protein mass was 0.02 mg (100 μl) per animal. The tumor-bearing mice used in this Example were mice (BALB/c nu/nu) that were in a tumor-bearing state as a result of transplantation of the human breast cancer cell line MDA-MB-468 (ATCC (registered trademark ); HTB-132) at 5.0 × 10 ⁶ cells/mouse subcutaneously in the dorsal region before sample injection. 24 hours after sample administration, mice were sacrificed, kidneys and tumor were removed, and the amount of Gd in the kidney and tumor was measured using ICP-MS. This test was performed on 3 cases in each group and the average of the 3 cases was calculated. The amount of Gd contained per kidney after administration was shown as a percentage of the amount of Gd administered (% dose/tissue), and the amount of Gd contained per g of tumor tissue was shown as a percentage of the total amount of Gd administered (% dose/g). The results are shown in Tables 59 and 60. Pr.-No. in the tables represents the number of the conjugate in Example 5. Moreover, the same test was carried out using Pr.-No. 108 obtained in Preparation Example 5, which did not contain a peptide linker, as a control conjugate. Average values for 2 experiments for Pr.-No. 108 are shown in Tables 57 and 58.

(Results)

As shown in Tables 57-60, the accumulation of the labeling moiety in the kidney was 88.08-90.87% points lower in the conjugates containing the peptide linker compared to the control conjugate.

[0387]

Table 57
Residual amount in kidneys Residual amount in kidneys
(% dose/tissue) Average value 34.67

[0388]

Table 58
Accumulation in tumor Amount accumulated in tumor
(% dose/g) Average value 0.92

[0389]

Table 59
Residual amounts in kidneys (tumor-bearing mice) Pr.-No. % dose/tissue Reduction percentage (%) 104 3.17 90.87 112 4.13 88.08

[0390]

Table 60
Amount accumulated in tumor (tumor-bearing mice) Pr.-No. % dose/g 104 1.04 112 1.27

[0391] Example 9: Preparation of Anti-Human MUC1 Antibody Fragment Fab ⁵ Conjugate

(Example 9-115: Synthesis of [Gd/DOTA]p-Fab ⁵ )

[0392] [Chemical formula 124]

[0393] 1 M 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) when cooled with ice to bring the pH to 5.5-6. To the resulting solution (239 μL), sodium 1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate (2.3 mg) dissolved in water (117 μL) was added under ice-cooling. Then, an aqueous solution of EDC HCl (8.3 μL, 25 mg/mL) was added, and the resulting mixture was stirred under ice-cooling for 30 minutes to obtain an N-hydroxysulfosuccinimidyl DOTA solution. Before adding Fab ^5, 0.2 M aqueous disodium hydrogen phosphate (pH 9) (40 μl) was added to adjust the pH to 7.

The resulting N-hydroxysulfosuccinimidyl DOTA solution (300 μl) was added to a 0.1 M aqueous solution of disodium hydrogen phosphate (585 μl) with 20.8 mg/ml Fab ⁵ (90 μl) and the resulting mixture was incubated at 4°C for 23 hours. Excess linker was washed with 10 mM phosphate buffer using an Amicon Ultra-15 ml centrifuge filter twice, washed with 0.3 M ammonium acetate buffer, finally concentrated and then filtered through a membrane filter to obtain the conjugate Pr.-No. 115. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one molecule of [DOTA], having a molecular weight of 387, was associated with one Fab ⁵ having a molecular weight of 47.5 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it.

(Example 9-116. Synthesis of [DOTA-NH-CH ₂ -(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH ₂ ) ₂ -(A-3)]p -Fab ⁵ )

[0394] [Chemical formula 125]

[0395] N-{[4-({2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetamido}methyl)phenyl]carbamoyl}-L- Methionyl-N ¹ -(2-{[(4-isothiocyanatophenyl)carbamothioyl]amino}ethyl)-L-isoleucinamide tetrakis(trifluoroacetate) (1 mg), synthesized in Example 2-68 (ix), was dissolved in DMSO (336 μl ).

30 µl of the pre-prepared solution was added to the 5.2 mg/ml Fab solution⁵V phosphate-buffered saline (240 μl), the pH was adjusted to about 8.0 using 0.1 M aqueous sodium carbonate solution and the resulting mixture was incubated at 30°C for 2 hours. The mixture was purified using a PD-10 column and the resulting solution was isolated using an Amicon Ultra-15 ml centrifuge filter. The isolated solution was washed twice with phosphate-buffered saline, finally concentrated and then filtered through a membrane filter to obtain the conjugate Pr.-No. 116. The same procedure was carried out again and the resulting conjugate Pr.-No. 116 were mixed. MS analysis confirmed that the conjugate was a mixture of a conjugate in which one [DOTA-NH-CH₂-(1,4-Ph)-NH-C(=O)-Met-Ile-NH-(CH₂)₂-(A-3)], having a molecular weight of 1032, was associated with one Fab⁵, having a molecular weight of 47.5 kDa, a conjugate in which two such molecules were associated with it, a conjugate in which three such molecules were associated with it, and a conjugate in which four such molecules were associated with it.

[0396] Example 10: Pharmacokinetics Study of Metal Complex Compounds in Common Marmosets

Example of preparation of 1 ⁶⁴ Cu-labeled protein

139 μL of 0.1 mol/L sodium acetate buffer (pH 6.5) was added to 10 μL (19.8 mBq, FUJIFILM Toyama Chemical Co., Ltd.) of 0.05 mol/L solution [⁶⁴Cu]CuCl₂in hydrochloric acid and mixed. 51 μl of the protein conjugate prepared in Example 9-115 was then added and the mixture was incubated at room temperature for 60 minutes to react. The reaction liquid was added to an ultrafiltration membrane (Amicon Ultra 10K, Millipore), and then 50 mmol/L sodium acetate buffer was added to carry out ultrafiltration purification to obtain a solution⁶⁴Cu-protein conjugate (A) of interest. 140 μl of protein conjugate and 145 μl of PBS solution were mixed in the resulting solution and the resulting mixture was filtered using a filter syringe (Millex-GV 0.22 μm, Millipore) to obtain a PBS solution containing⁶⁴Cu-protein conjugate (A) (11.5 mBq, 19.17 mBq/mg).

An example of obtaining 2 ⁶⁴ Cu-labeled protein

285 μL of 0.1 mol/L sodium acetate buffer (pH 6.5) was added to 20 μL (37.1 mBq, FUJIFILM Toyama Chemical Co., Ltd.) of 0.05 mol/L solution [⁶⁴Cu]CuCl₂in hydrochloric acid and mixed. Then, 94.7 μl of the protein conjugate obtained in Example 9-116 was added, and the mixture was incubated at room temperature for 60 minutes to react. The reaction liquid was added to an ultrafiltration membrane (Amicon Ultra 10K, Millipore), and then 50 mmol/L sodium acetate buffer was added to carry out ultrafiltration purification to obtain a solution⁶⁴Cu-protein conjugate (B) of interest. 260 μl of protein conjugate and 377 μl of PBS solution were mixed in the resulting solution and the resulting mixture was filtered using a filter syringe (Millex-GV 0.22 μm, Millipore) to obtain a PBS solution containing⁶⁴Cu-protein conjugate (B) (31.5 mBq, 26.27 mBq/mg).

PET/CT imaging

A common marmoset (2 years old) was anesthetized with isoflurane and 175-185 μl of PBS containing solution conjugate⁶⁴Cu protein (A) or (B) was injected through the tail vein. After administration, under anesthesia, images were acquired using a PET/CT imaging device (PET: Clairvivo PET, manufactured by Shimadzu Corporation, CT: Aquilion, manufactured by TOSHIBA).

Fig. 1 is a PET/CT image obtained approximately 3 hours after administration of a PBS solution containing conjugate⁶⁴Cu protein (A). In addition, FIG. 2 is a PET/CT image obtained approximately 3 hours after administration of a PBS solution containing conjugate⁶⁴Cu protein (B). Fig. 3 represents SUV (standardized accumulation level). SUV is the value obtained by dividing the radioactivity per g of tissue by the administered radioactivity per g of body weight, i.e. value represented by SUV=radioactivity per g tissue/radioactivity administered per g body weight. The radioactivity value is the attenuation-corrected value. It was observed that the accumulation in the kidneys was lower in Fig. 2 than in Fig. 1.

Industrial applicability

[0397] The present invention includes a conjugate having excellent binding activity to human CEACAM5 and which is expected to be useful for the diagnosis and/or treatment of cancer involving human CEACAM5.

The present invention further includes a conjugate having excellent binding activity to MUC1 and which is expected to be useful for diagnosing and/or treating cancers involving MUC1.

In addition, the conjugate consisting of 3arm DOTA, a specific spacer, a specific peptide linker and a biomolecule, which is the conjugate of the present invention, is degraded in the kidneys and excreted, and is thus expected to be useful for diagnosis and/or treatment disease associated with a biomolecule.

Free text list of sequences

[0398] At number <223> in the following sequence listing is a description of a typical "Artificial Sequence". Specifically, the nucleotide sequences represented by SEQ ID NO: 1 and 3 in the sequence listing are the nucleotide sequence of the heavy chain fragment and the nucleotide sequence of the light chain of PB009-01, respectively, and the amino acid sequences represented by SEQ ID NO: 2 and 4 are is the amino acid sequence of the heavy chain fragment and the amino acid sequence of the light chain encoded by SEQ ID NO: 1 and 3, respectively. In addition, the nucleotide sequences represented by SEQ ID NO: 5 and 9 in the sequence listing represent the nucleotide sequence of the heavy chain fragment and the nucleotide sequence of the light chain of P10-1, respectively, and the amino acid sequences represented by SEQ ID NO: 6 and 10 represent is the amino acid sequence of the heavy chain fragment and the amino acid sequence of the light chain encoded by SEQ ID NO: 5 and 9, respectively. the nucleotide sequences represented by SEQ ID NO: 7 and 9 in the sequence listing are the nucleotide sequence of the heavy chain fragment and the nucleotide sequence of the light chain of P10-2, respectively, and the amino acid sequences represented by SEQ ID NO: 8 and 10 are the amino acid sequence heavy chain fragment and the light chain amino acid sequence encoded by SEQ ID NOs: 7 and 9, respectively. SEQ ID NO: 11 and 15 are the heavy chain variable region and the light chain variable region of P10-1, respectively, and the amino acid sequences represented by SEQ ID NO: 12 and 16 are the amino acid sequences of the heavy chain variable region and the light chain variable region , encoded by SEQ ID NO: 11 and 15, respectively. SEQ ID NO: 13 and 15 are the heavy chain variable region and the light chain variable region of P10-2, respectively, and the amino acid sequences represented by SEQ ID NO: 14 and 16 are the amino acid sequences of the heavy chain variable region and the light chain variable region , encoded by SEQ ID NO: 13 and 15, respectively.

--->

LIST OF SEQUENCES

<110> Astellas Pharma Inc.

<120> A conjugate comprising a ligand, a spacer, a peptide linker and

biomolecule

<130> A20002A00

<150> JP 2019-000530

<151> 2019-01-07

<150>JP 2019-206560

<151> 2019-11-14

<160> 19

<170> Patent In version 3.5

<210> 1

<211> 678

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding heavy chain Fab PB009-01

<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> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<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 light chain 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> Protein

<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 Fab P10-1 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> Protein

<213> Artificial sequence

<220>

<223> Fab P10-1 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 Fab P10-2 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> Protein

<213> Artificial sequence

<220>

<223> Fab P10-2 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 the light chain of an antibody

<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> Protein

<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 Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Gly Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 11

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding the heavy chain variable region of Fab P10-1

<400> 11

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 ctca

354

<210> 12

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Fab P10-1 heavy chain variable region

<400> 12

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

115

<210> 13

<211> 354

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding the heavy chain variable region of Fab P10-2

<400> 13

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 ctca

354

<210> 14

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain variable region Fab P10-2

<400> 14

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

115

<210> 15

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<223> DNA encoding the variable region of an antibody light chain

<400> 15

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 atcaagcgt

339

<210> 16

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain variable region

<400> 16

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Gly Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 17

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Heavy chain signal sequence

<400> 17

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser

<210> 18

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Light chain signal sequence

<400> 18

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys

20

<210> 19

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Extracellular domain tandem repeat sequence

MUC1

<400> 19

His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr

1 5 10 15

Ala Pro Pro Ala

20

<---

Claims (102)

1. A conjugate which has binding activity to human MUC1 and is represented by the following formula (Ic): , where DOTA ¹ represents 3arm DOTA, which is selected from the following: U: bond or -NH(CH ₂ ) ₂ O(CH ₂ ) ₂ O(CH ₂ ) ₂ OCH ₂ C(=O)-, Q: -C(=O)-, -NH-C(=O)- or -NH-C(=S)-, R ^1a , R ^1b : the same or different from each other, H or C _1-6 alkyl, with the proviso that R ^1a and R ^1b together can form C _1-6 alkylene; p represents a natural number from 1 to 25 and is bonded to an adjacent carbon atom through p the number of amino groups or thiol groups in the Biomolecule; R ¹ : H, halogen, C _1-6 alkyl or halogen C _1-6 alkyl, R ² : C _1-6 alkyl or halogen C _1-6 alkyl, L ³ : communication, L ⁴ : -NH-(CH ₂ ) ₂ - or -NHCH(C(=O)OH)(CH ₂ ) ₄ -, V ¹ : group represented by any of the following formulas (A-1)-(A-5): [Chemical formula 127] or the formula -L ³ -L ⁴ -V ¹ - together forms the following formula: [Chemical formula 128] The biomolecule is a Fab fragment of an anti-human MUC1 antibody selected from the group consisting of: (a) an anti-human MUC1 antibody Fab fragment containing a heavy chain fragment including a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14 and a light chain including a light chain variable region, consisting of the amino acid sequence represented by SEQ ID NO: 16, and (b) an anti-human MUC1 antibody Fab fragment selected from the group consisting of an anti-human MUC1 antibody Fab fragment containing a heavy chain fragment comprising a heavy chain variable region that consists of the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO: 14, and wherein glutamine, amino acid 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 represented by SEQ ID NO: 16, and the fragment is connected to V ¹ through p the number of amino groups or thiol groups in the fragment, dotted line Q is bonded to either of the two carbon atoms on the ring. 2. The conjugate according to claim 1, where the conjugate is represented by the following formula (Id): [Chemical formula 135] . 3. Conjugate according to any one of paragraphs. 1, 2, where V ¹ in formula (Id) is any of the following formulas (A-3)-(A-5): [Chemical formula 136] . 4. Conjugate according to any one of paragraphs. 1-3, where 3arm DOTA represents . 5. Conjugate according to any one of paragraphs. 1-4, where the conjugate is selected from the group consisting of compounds represented by the following formulas: [Chemical formula 137] , [Chemical formula 138] , [Chemical formula 139] , [Chemical formula 140] , [Chemical formula 141] , Biomolecule1 is the Biomolecule disclosed in claim 1. 6. Conjugate according to any one of paragraphs. 1-5, where Biomolecule is A Fab fragment of an anti-human MUC1 antibody selected from the group consisting of: (a) a Fab fragment of an anti-human MUC1 antibody comprising a heavy chain fragment consisting of the amino acid sequence set forth in SEQ ID NO: 8 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10; And (b) an anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and light chain consisting of the amino acid sequence set forth in SEQ ID NO: 10. 7. The conjugate of claim 6, wherein the Biomolecule is a Fab fragment of an anti-human MUC1 antibody comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8, and in which glutamine, amino acid 1 in SEQ ID NO: : 8, modified to pyroglutamic acid, and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 10. 8. A conjugate that can bind to human MUC1 and is represented by the following formula: [Chemical formula 144] , where Fab ⁵ represents the following Fab antibody fragment: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain, consisting of the amino acid sequence presented in SEQ ID NO: 10; p represents a natural number from 1 to 25; And Fab ⁵ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ⁵ . 9. A conjugate that can bind to human MUC1 and is represented by the following formula: [Chemical formula 145] , where Fab ⁵ represents the following Fab antibody fragment: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain, consisting of the amino acid sequence presented in SEQ ID NO: 10; p represents a natural number from 1 to 25; And Fab ⁵ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ⁵ . 10. A conjugate that can bind to human MUC1 and is represented by the following formula: [Chemical formula 146] , where Fab ⁵ represents the following Fab antibody fragment: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain, consisting of the amino acid sequence presented in SEQ ID NO: 10; p represents a natural number from 1 to 25; And Fab ⁵ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ⁵ . 11. A conjugate that can bind to human MUC1 and is represented by the following formula: [Chemical formula 147] , where Fab ⁵ represents the following Fab antibody fragment: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain, consisting of the amino acid sequence presented in SEQ ID NO: 10; p represents a natural number from 1 to 25; And Fab ⁵ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ⁵ . 12. A conjugate that can bind to human MUC1 and is represented by the following formula: [Chemical formula 148] , where Fab ⁵ represents the following Fab antibody fragment: An anti-human MUC1 antibody Fab fragment comprising a heavy chain fragment that consists of the amino acid sequence set forth in SEQ ID NO: 8 and in which glutamine, amino acid 1 in SEQ ID NO: 8, is modified to pyroglutamic acid, and a light chain, consisting of the amino acid sequence presented in SEQ ID NO: 10; p represents a natural number from 1 to 25; And Fab ⁵ is bonded to an adjacent carbon atom through p number of amino groups or thiol groups in Fab ⁵ . 13. Conjugate according to any one of paragraphs. 1-12, where p represents a natural number from 1 to 4. 14. A metal chelate complex of a conjugate having binding activity to human MUC1, wherein the conjugate is a conjugate as disclosed in any one of claims. 1-13, and the metal is coordinated with 3arm DOTA. 15. Chelate complex according to claim 14, where the metal is a radioisotope of a metal. 16. Chelate complex according to claim 15, where the metal is ⁸⁹ Zr. 17. Chelate complex according to claim 14, where the metal is a paramagnetic metal ion. 18. Chelate complex according to claim 17, where the metal is Gd ³⁺ . 19. Chelate complex according to any one of paragraphs. 14-16, where the chelate complex is used as a radiopharmaceutical for PET. 20. Chelate complex according to claim 15, where the conjugate is a conjugate according to any one of claims. 8-12. 21. A diagnostic composition for diagnosing cancer expressing human MUC1, comprising an effective amount of one or more chelate complexes according to any one of paragraphs. 14-19 and a pharmaceutically acceptable carrier. 22. The diagnostic composition according to claim 21, wherein the diagnostic composition is used as a drug for early diagnosis or a drug for determining the stage of a disease. 23. Diagnostic composition according to any one of paragraphs. 21 or 22, 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. 24. A pharmaceutical composition for the treatment of cancer expressing human MUC1, comprising an effective amount of one or more chelate complexes according to any one of paragraphs. 14-18 and a pharmaceutically acceptable carrier. 25. The pharmaceutical composition according to claim 24, 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. 26. Use of a chelate complex according to any one of paragraphs. 14-18 to prepare a composition for diagnosing cancer expressing human MUC1. 27. Use of a chelate complex according to any one of paragraphs. 14-18 for the preparation of a pharmaceutical composition for the treatment of cancer expressing human MUC1. 28. Chelate complex according to any one of paragraphs. 14-18, wherein the chelate complex is used to diagnose cancers expressing human MUC1. 29. Chelate complex according to any one of paragraphs. 14-18, wherein the chelate complex is used to treat cancer expressing human MUC1. 30. A method for diagnosing cancer expressing human MUC1, comprising administering to a subject an effective amount of a chelate complex according to any one of claims. 14-18. 31. A method for treating cancer expressing human MUC1, comprising administering to a subject a therapeutically effective amount of a chelate complex according to any one of claims. 14-18.