Radiometal-labeled antibodies, radiopharmaceuticals and compounds

Abstract

The invention relates to a radioactive metal labeled antibody obtained by labeling an antibody with a radioactive metal. A functional chelate linker is coupled to the antibody, and the functional chelate linker is provided with: a chelate part capable of coordinating with a radioactive metal ion; and a functional unit having a function of improving intracellular retention. It is preferable that the functional unit has a structure obtained by polymerizing ethyleneimine or ethylene glycol, or a structure capable of binding to cathepsin B. The functional unit preferably has a structure obtained by polymerizing ethyleneimine at a degree of polymerization of 1-7 (inclusive).

Description

TECHNICAL FIELD

[0001] The present application relates to radiometal labeled antibodies, radiopharmaceuticals and compounds. BACKGROUND

[0002] Radiolabeled antibodies (hereinafter, also referred to as "radiolabeled antibodies") are expected to function as therapeutic drugs in internal radiotherapy or diagnostic drugs in PET or the like. For example, Ibritumomab tiuxetan is a substance obtained by combining tiuxetan modified with DTPA on Ibritumomab which is an anti-CD20 antibody specific antibody, to which yttrium-90 is additionally attached, and has been approved in Japan as a therapeutic drug for CD20-positive lymphoma. In addition, Ibritumomab tiuxetan has been approved as a therapeutic drug for CD20-positive lymphoma in overseas. 131 Ibritumomab tiuxetan has not been approved in Japan, but has been approved as a therapeutic drug for CD20-positive lymphoma in overseas.

[0003] In addition, trastuzumab is a HER2 specific antibody, and it has been reported that various radionuclides such as indium 111, lutetium 177, copper 64, zirconium 89, astatine 211, and radioactive iodine are labeled, and clinical application thereof in non-invasive imaging and targeted radiotherapy of tumors overexpressing HER2 is being studied.

[0004] As a problem of radiolabeled antibodies, it is known that iodinated tyrosine rapidly diffuses from cells due to endocytosis and lysosomal decomposition. In order to address this problem, Non-Patent Literature 1 describes that iodinated tyrosine labeled with radioactive iodine is combined with DOTA in view of the fact that In-111 DOTA lysine accumulates in target cells, thereby improving intracellular retention of trastuzumab labeled with radioactive iodine. In addition, Non-Patent Literature 2 describes that intracellular retention of trastuzumab labeled with radioactive iodine can be improved by introducing a guanidino group having high basicity into trastuzumab.

[0005] In addition, Non-Patent Literature 3 describes that the retention of a peptide targeting a gastrin-releasing peptide receptor (GRPR) in tumor cells can be improved by introducing a structure capable of binding to cathepsin B into the peptide.

[0006] Furthermore, Non-Patent Literature 4 describes that a peptide having an epoxy succinyl group has excellent affinity for the active site of cathepsin B.

[0007] PRIOR ART DOCUMENTS

[0008] NON-PATENT LITERATURE

[0009] Non-patent literature 1: C. Andrew Boswell et al., J. Med. Chem. 2013, 56, 9418-9426

[0010] Non-patent literature 2: Satish K. Chitneni et al., Molecules, 2019, 24, 3907

[0011] Non-patent literature 3: Wenting Zhang et al., J. Nucl. Med., 2020, 61, 443-450

[0012] Non-patent literature 4: N. Schaschke et al., FEBS Lett., 2000, 482, 91-96 SUMMARY

[0013] As described in Non-patent literatures 1 and 2, it has been conventionally considered that a radio metal ion-labeled antibody (hereinafter, also referred to as "radio metal-labeled antibody") is retained in a cell. However, among antibodies, there are antibodies that are rapidly discharged from a cell to the outside of a cell. In the case of a radio metal-labeled antibody, it is also desired to further improve the efficacy by improving the accumulation of radioactivity in a target cell.

[0014] In addition, although it is described in Non-patent literature 3 that the retention of a peptide targeting GRPR in a tumor cell is improved by introducing a structure capable of binding to cathepsin B as described above, this document does not study the introduction of a structure capable of binding to cathepsin B into a radio metal-labeled antibody.

[0015] Therefore, an object of the present application is to provide a radio metal-labeled antibody capable of improving the intracellular accumulation of radioactivity in a target cell.

[0016] The present application provides a radio metal-labeled antibody, which is a radio metal-labeled antibody obtained by labeling an antibody with a radio metal,

[0017] wherein a functional chelating linker having:

[0018] a chelating portion capable of coordinating with a radio metal ion; and

[0019] a functional unit having a function of improving intracellular retention is coupled to the aforementioned antibody.

[0020] In addition, the present application provides a compound having:

[0021] a chelating portion capable of coordinating with a radio metal ion;

[0022] a functional unit having a function of improving intracellular retention; and

[0023] An antibody binding site comprising the binding portion to an antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] [ Figure 1 ] Figure 1 It is a graph showing the radioactivity percentage of the internalized fraction and the culture medium fraction in Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-3, and their ratio.

[0025] [ Figure 2 ] Figure 2 It is a graph showing the radioactivity percentages of the dissociated fraction and the metabolized fraction in Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-3, and their ratios.

[0026] [ Figure 3 ] Figure 3 It is a graph showing the area under the curve (AUC) in blood and tumor in Examples 5-1 to 5-3 and Comparative Examples 5-1 to 5-3 and their ratios.

[0027] [ Figure 4 ] Figure 4 For Example 7-1, [ 111 SPECT / CT images of In]In-TMDPEI2.

[0028] [ Figure 5 ] Figure 5 The graphs are the area under the curve (AUC) of blood and tumor and their ratios in Examples 5-4 to 5-6.

[0029] [ Figure 6 ] Figure 6 The autoradiographic images of the gels after polyacrylamide electrophoresis in Example 4-7 and Comparative Example 4-4 are shown.

[0030] [ Figure 7 ] Figure 7 It is a graph showing the radioactivity percentage of the internalized fraction and the metabolized fraction in Example 5-7 and Comparative Example 5-4.

[0031] [ Figure 8 ] Figure 8 This is an autoradiograph of the gel after polyacrylamide electrophoresis in Example 6-7.

[0032] [ Figure 9 ] Figure 9 for Figure 8 The autoradiogram shown is an enlarged view of the molecular weight range of 100 kDa to 250 kDa.

[0033] [ Figure 10 ] Figure 10A graph showing the tumor / blood ratio of the accumulation of radioactivity, and the area under the curve (AUC) in the blood and tumor in Examples 6 to 7 and Comparative Examples 4 to 4. DETAILED DESCRIPTION

[0034] Hereinafter, the radio-metal labeled antibody and the compound of the present application will be described based on preferred embodiments. In the following description, in the case where "T ~ U [V]" (T and U are arbitrary numbers, and [V] is a unit) is described, unless otherwise specified, it means "T [V] or more and U [V] or less". In addition, in the case where a chiral carbon atom exists in the structure, unless otherwise specified, it can be independently either S configuration or R configuration.

[0035] The radio-metal labeled antibody of the present application is a radio-metal labeled antibody obtained by labeling an antibody with a radio-metal, wherein a functional chelating linker having a chelating portion capable of coordinating with a radio-metal ion and a functional unit having a function of improving intracellular retention is coupled to the antibody.

[0036] In addition, the compound of the present application is a functional chelating linker before being coupled to an antibody, and its chemical structure is roughly classified as having: a chelating portion capable of coordinating with a radio-metal ion; a functional unit having a function of improving intracellular retention; and an antibody binding unit including a binding portion to an antibody.

[0037] The compound of the present application can also be a precursor compound for labeling with a radio-isotope such as a radio-metal ion, that is, a precursor for radio-labeling. The description related to a radio-metal ion is described later.

[0038] In one embodiment, in the compound of the present application, the functional unit having a function of improving intracellular retention and the antibody binding unit including a binding portion to an antibody are bound via the chelating portion. In other words, the chelating portion is interposed between the antibody binding unit and the functional unit. Thus, when the compound of this embodiment is made into a radio-metal labeled antibody, it becomes a radio-metal labeled antibody in which the chelating portion is interposed between the antibody and the functional unit.

[0039] Such a compound is preferably represented by the following general formula (I).

[0040] [Chemical Formula 01]

[0041]

[0042] In formula (I), A represents a chelating portion capable of coordinating with a radio-metal ion, B represents a functional unit having a function of improving intracellular retention, and C represents an antibody binding unit including a binding portion to an antibody.

[0043] L a represents a linker structure.

[0044] L b represents the same or different linker structure. a

[0045] m and n each independently represent 0 (zero) or 1.

[0046] In the case where n represents 1, L a is bound to any site of A.

[0047] In the case where n represents 0, B is bound to any site of A.

[0048] In the case where m represents 1, L b is bound to a site of A different from the site to which B or L a is bound.

[0049] In the case where m represents 0, C is bound to A at a site different from the site to which B or L a is bound.

[0050] In General Formula (I), A and B, and A and C can each independently be bound indirectly with a linker structure or directly without a linker structure. In General Formula (I), in the case where A and B, and A and C each have a linker structure, the linker structures can each be the same or different. One or two or more B is directly or indirectly bound to A.

[0051] Details of the linker structure are described later.

[0052] In General Formula (I), the chelating portion represented by symbol A preferably has a first amino acid residue and a second amino acid residue, and the chelating portion is bound to the antibody binding unit represented by symbol C via the first amino acid residue, and the chelating portion is bound to the functional unit represented by symbol B via the second amino acid residue.

[0053] Preferably, the first amino acid residue is a lysine residue, and the second amino acid residue is a glutamine residue.

[0054] When the compound of the present application has this configuration, it has advantages in that both ease of synthesis and stability of the chemical structure can be achieved.

[0055] Next, another embodiment of the compound of the present application will be described. In the compound of the present application in another embodiment, the antibody binding unit including the binding portion to the antibody is bound to the chelating portion via the functional unit having a function of improving intracellular retention. In other words, the functional unit is interposed between the antibody binding unit and the chelating portion. ​

[0056] Such a compound is preferably represented by the following general formula (II).

[0057] [Chemical Formula 02]

[0058]

[0059] In formula (II), A represents a chelating portion capable of coordinating with a radioactive metal ion, B represents a functional unit having a function of improving intracellular retention, and C represents an antibody binding unit including a binding portion to an antibody.

[0060] L a represents a linker structure.

[0061] L b represents a linker structure identical with or different from L a .

[0062] m and n each independently represent 0 (zero) or 1.

[0063] In the case where n represents 1, L a is bound to an arbitrary site of B.

[0064] In the case where n represents 0, A is bound to an arbitrary site of B.

[0065] In the case where m represents 1, L b is bound to a site different from the site of B to which A or L a is bound.

[0066] In the case where m represents 0, C is bound to A at a site different from the site of B to which A or L a is bound.

[0067] In general formula (II), A and B, and B and C can each independently be indirectly bound with a linker structure or directly bound without a linker structure. In formula (II), in the case where A and B, and B and C each have a linker structure, the linker structures can each be identical or different.

[0068] From the viewpoint of easiness of manufacture and economy, the compound of the present application is particularly preferably represented by general formula (I), i.e., the functional unit and the antibody binding unit are bound via the chelating portion.

[0069] Hereinafter, matters applicable to each of the above-described embodiments will be described.

[0070] The following description is appropriately applied to the description relating to general formula (I) and general formula (II) unless otherwise specified, and can also be adopted in combination as appropriate.

[0071] When the compound of the present application is made into a radiometal-labeled antibody, from the viewpoint of sufficiently increasing the affinity of the compound of the present application for a radiometal ion, thereby suppressing the dissociation of the radiometal ion from the radiometal-labeled antibody before being taken up by a target cell, in the aforementioned formulae (I) and (II), preferably: A has a cyclic structure, and the cyclic structure has 2 or more nitrogen atoms each linked via 2 or more adjacent carbon atoms; or A has a chain structure, the chain structure has 2 or more nitrogen atoms each linked via 2 or more adjacent carbon atoms.

[0072] In the aforementioned formulae (I) and (II), in the case where A has a cyclic structure, the skeleton of the cyclic structure can be composed of only nitrogen atoms and carbon atoms, or can be composed of oxygen atoms in addition to nitrogen atoms and carbon atoms. The bonds between the carbon atoms in the cyclic structure can be chain-like, or can form a cyclic structure.

[0073] In addition, in the aforementioned formulae (I) and (II), in the case where A has a chain structure, the bonds between the carbon atoms in the chain structure can be interrupted by a nitrogen atom. The bonds between the carbon atoms in the chain structure can be chain-like, or can form a cyclic structure.

[0074] In addition, in the aforementioned formulae (I) and (II), in the case where A has a cyclic structure or a chain structure, A preferably has a nitrogen-binding atom group directly bound to a nitrogen atom constituting the cyclic structure or the chain structure. As a specific example of the nitrogen-binding atom group, an atom group containing one or more of a carboxyl group, a phosphoric acid group, an amide group, a benzene ring, and a pyridine ring can be preferably selected, and further preferably the atom group is chain-like.

[0075] Further, in the aforementioned formulae (I) and (II), in the case where A has a cyclic structure or a chain structure, provided that "B is bound to any site of A, and C is bound to any site of A different from the binding site of B", when B is bound to the aforementioned nitrogen-binding atom group via L a or not via L a C is preferably bound to a site other than the "nitrogen-binding atom group to which B is bound via L or not via L".

[0076] Specifically, in aforementioned formula (I) and (II), the chelating part that can coordinate with radioactive metal represented as symbol A, preferably has the structure of the compound represented by any one of the following formulas (A1) to (A9), more preferably has the structure of the compound represented by the following formula (A1). That is, the compound of the present invention is preferably a derivative of the compound represented by any one of the following formulas (A1) to (A9), more preferably a derivative of the compound represented by the following formula (A1). These structures can be appropriately selected according to the type of radioactive metal described later. No matter what kind of structure the chelating part has, when the compound of the present invention is made into radioactive metal labeled antibody, the affinity of the compound of the present invention and radioactive metal ions can be fully improved, therefore radioactive metal ions can be suppressed from being freed from the radioactive metal labeled antibody before being taken up by target cells.

[0077] In the aforementioned formulae (I) and (II), the chelating moiety represented by symbol A may be, for example, structures derived from the following compounds, but is not limited thereto.

[0078] <DOTA or its derivatives>

[0079] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)

[0080] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA)

[0081] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid (DOTMP)

[0082] Hydroxypropyl tetraazacyclododecane triacetic acid (HP-DO3A)

[0083] (1R,4R,7R,10R)-α,α',α",α'"-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAMA)

[0084] 1,4,7,10-Tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAA)

[0085] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylene)phosphonic acid (DOTA-A-AMP)

[0086] Tetraazacyclododecanedimethylphosphonic acid (DO2P)

[0087] α-(2-Carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA)

[0088] <HOPO or its derivatives>

[0089] N,N',N",N'"-Tetrakis(1,2-dihydro-1-hydroxy-2-oxopyridine-6-carbonyl)-1,5,10,14-tetraazatetradecane (1,2-HOPO)

[0090] <TETA or PEPA or their derivatives>

[0091] 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA)

[0092] 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETPA)

[0093] 1,4,7,10,13-Pentaazacyclopentadecane-N,N',N",N'",N""-pentaacetic acid (PEPA)

[0094] <Chain structure (Octapa, Neunpa, or their derivatives)>

[0095] Ethylenediaminetetraacetic acid (EDTA)

[0096] 6,6'-((Ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(methylene))picolinic acid (H4octapa)

[0097] 6,6'-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridin-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(methylene))picolinic acid (H2bispa2)

[0098] 1,2-[{6-(Carboxy)-pyridin-2-yl}-methylamino]ethane (H2dedpa)

[0099] N,N"-bis(6-carboxy-2-pyridylmethyl)-diethylenetriamine-N,N',N"-triacetic acid (H5decapa)

[0100] N,N'-(Methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane (H6phospa)

[0101] 6,6'-(((((4-Isothiocyanatophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))picolinic acid (p-SCN-Bn-H4neunpa)

[0102] • 6,6'-(((4-nitrophenethyl)azanediyl)bis(ethane-2, 1 -diyl))bis((carboxymethyl)azanediyl)bis(methylene))pyridinedicarboxylic acid (p-NO2-Bn-H4neunpa)

[0103] • 6,6'-(((azanediylbis(ethane-2, 1 -diyl))bis((carboxymethyl)azanediyl)bis(methylene))pyridinedicarboxylic acid (H5neunpa)

[0104] <Macropa and derivatives thereof>

[0105] • 6-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-N,N'-dimethyl)picolinic acid (H2macropa)

[0106] <NOTA or derivatives thereof>

[0107] • 2-[4,7-bis(carboxymethyl)-1,4,7-triazacyclononan-1 -yl]acetic acid (NOTA)

[0108] [Chemical Formula 03]

[0109]

[0110] In Formula (A1), R 11 , R 12 , R 13 , and R 14 each independently represent any one of a group consisting of - (CH2) p COOH, -(CH2) p C5H4N, -(CH2) p PO3H2, -(CH2) p CONH2, -(CHCOOH)(CH2) p COOH, and p represents an integer of 0 or more and 3 or less.

[0111] In Formula (A2), R 21 , R 22 , R 23 , and R 24 each independently represent a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms.

[0112] In Formula (A3), R 31 , R 32 , R 33 , and R 34 each independently represent an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom, and R35 represents a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms.

[0113] In formula (A4), R 41 , R 42 , R 43 , and R 44 each independently represents an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom. 45 represents a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms.

[0114] In formula (A5), R 48 , and R 49 each independently represents an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom.

[0115] In formula (A6), R 51 , R 52 , R 53 , R 54 , and R 55 each independently represents an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom.

[0116] In formula (A7), R 61 , R 62 , R 63 , R 64 , R 65 , and R 66 each independently represents an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom. 67 represents a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms.

[0117] [Chemical Formula 04]

[0118]

[0119] In formula (A8), R 71 , R 72 , and R 73 each independently represents an atomic group having a hydrogen atom and 2 or more and 10 or less carbon atoms, and can contain a nitrogen atom or an oxygen atom.

[0120] In formula (A9), R 81 , and R 82 each independently represents an alkyl group having 1 or more and 5 or less carbon atoms, and the terminal of the alkyl group can be substituted with a pyridyl group substituted with 1 or more carboxyl groups. 87 represents a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms.83 and R 84 represents a substituted or unsubstituted pyridyl group, R 85 and R 86 each independently represents -COO-R a , R a represents an alkyl group having 1 to 5 carbon atoms.

[0121] As a specific structure represented by formula (A1), for example, structures represented by the following formulae (A1-1) to (A1-7) can be given.

[0122] [Chemical Formula 05]

[0123]

[0124] [Chemical Formula 06]

[0125]

[0126] [Chemical Formula 07]

[0127]

[0128] As a specific structure represented by formula (A2), for example, structures represented by the following formulae (A2-1) to (A2-2) can be given.

[0129] [Chemical Formula 08]

[0130]

[0131] As a specific structure represented by formula (A3), for example, structures represented by the following formulae (A3-1) to (A3-7) can be given.

[0132] [Chemical Formula 09]

[0133]

[0134] [Chemical Formula 10]

[0135]

[0136] As a specific structure represented by formula (A4), structures represented by the following formulae (A4-1) to (A4-2) can be given.

[0137] [Chemical Formula 11]

[0138]

[0139] As a specific structure represented by formula (A5), for example, structures represented by the following formulae (A5-1) to (A5-3) can be given.

[0140] [Chemical formula 12]

[0141]

[0142] As a specific structure represented by Formula (A6), for example, a structure represented by Formula (A6-1) below can be given.

[0143] [Chemical formula 13]

[0144]

[0145] As a specific structure represented by Formula (A7), for example, structures represented by Formulas (A7-1) to (A7-2) below can be given.

[0146] [Chemical formula 14]

[0147]

[0148] As a specific structure represented by Formula (A8), for example, structures represented by Formulas (A8-1) to (A8-3) below can be given.

[0149] [Chemical formula 15]

[0150]

[0151] As a specific structure represented by Formula (A9), for example, structures represented by Formulas (A9-1) to (A9-4) below can be given.

[0152] [Chemical formula 16]

[0153]

[0154] The chelating portion is preferably DOTA or a derivative thereof, and particularly, from the viewpoint of sufficiently improving the affinity of the compound of the present application to a radioactive metal ion, the chelating portion represented by symbol A in the aforementioned Formulas (I) and (II) is preferably a structure of a compound represented by any one of DOTA or a derivative thereof (A1-2 to A1-7) or the following Formula (A).

[0155] [Chemical formula 17]

[0156]

[0157] In Formula (A), R 11 and R 14 are each independently -(CH2) p COOH, -(CH2) p C5H4N, -(CH2) p PO3H2, -(CH2) pCONH2or -(CHCOOH)(CH2) p COOH group.

[0158] In the case where the compound of the present application is represented by the aforementioned formula (I), for example, R 12 R is a substituent for binding to the antibody-binding unit, 13 R is a substituent for binding to the functional unit (B), 15 R is a hydrogen atom, and p is an integer of 0 or more and 3 or less. Preferably, an amino acid is introduced into R 12 R is a hydrogen atom, and p is an integer of 0 or more and 3 or less. Preferably, an amino acid is introduced into R 13 R is a hydrogen atom, and p is an integer of 0 or more and 3 or less. Preferably, an amino acid is introduced into R 12 R is a hydrogen atom, and p is an integer of 0 or more and 3 or less. Preferably, an amino acid is introduced into R 13 R is a hydrogen atom, and p is an integer of 0 or more and 3 or less. Preferably, an amino acid is introduced into R

[0159] In the case where the compound of the present application is represented by formula (II), R 12 or R 15 one of R is a hydrogen atom, a carboxyl group, or a carboxylalkyl group having 2 or 3 carbon atoms, the other is a substituent for binding to the functional unit (B), p is an integer of 0 or more and 3 or less, and R 12 R is a hydrogen atom, and R 15 R is a hydrogen atom, and R 12 R is a hydrogen atom, and R 15 R is a hydrogen atom, and R

[0160] As the radioactive metal coordinated in the ionic state on the chelate portion of the radiometallabeled antibody of the present application or the compound of the present application, a metal nuclide emitting α-rays, β-rays, or γ-rays or a combination thereof can be used. As the nuclide of such a radioactive metal, for example, radioactive isotopes of alkali metals, alkaline earth metals, lanthanoid elements, actinoid elements, transition metals, or metals other than these metals can be mentioned.

[0161] Among them, from the viewpoint of commercial availability and achievement of improvement in complex-forming property, as the nuclide of the radioactive metal, it is preferable to use 44 Sc, 51 Cr, 57 Co, 58 Co, 60 Co, 59 Fe, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 89 Sr, 89 Zr, 90 Y,99m Tc, 103 Such as 111 In, 153 Sm, 165 Dy, 166 Ho, 177 Lu, 186 Re、 188 Re、 198 Au, 201 Tl, 197 Hg, 203 Hg, 212 Bi, 213 Bi, 212 Pb, 227 Th or 225 Ac.

[0162] When the radioactive metal-labeled antibody of the present invention is used for the purpose of treating a disease, it is preferable to use a nuclide emitting α rays or a nuclide emitting β rays from the viewpoint of improving the therapeutic effect. - The nuclide emitting α rays is a radioactive metal. The nuclide emitting α rays may be a nuclide that emits α rays during the decay process of the radioactive metal. Specifically, it is preferable to use 212 Bi, 213 Bi, 227 Th or 225 Ac, etc., more preferably 227 Th or 225 Ac, more preferably 225 Ac.

[0163] Release β - The nuclides of radiation are those that release beta ray during the decay of radioactive metals. - The nuclides of radiation can be used. Specifically, it is preferable to use 60 Co、 59 Fe, 64 Cu, 67 Cu, 89 Sr. 90 Y. 99m Tc, 103 Such as 153 Sm, 165 Dy, 166 Ho, 177 Lu, 186 Re、 188 Re、 198 Au, 203 Hg, 212 Bi, 213 Bior 212 Pb, etc., more preferably 64 Cu, 67 Cu,89 Sr, 90 Y or 177 Lu.

[0164] In addition, in the case where a radiolabeled compound is used for the purpose of diagnosis of a disease or detection of a lesion, from the viewpoint of improving the diagnostic performance, as the radioactive metal, a nuclide which emits β + rays, an electron capture decay nuclide, or a nuclide which emits γ rays is preferably used. The nuclide which emits β + rays is a nuclide which emits a positron in the course of decay of the radioactive metal, and 44 Sc, 58 Co, 68 Ga, 64 Cu or 89 Zr, and more preferably 64 Cu or 89 Zr.

[0165] The electron capture decay nuclide is a nuclide which emits Auger electrons or characteristic X-rays in the course of decay of the radioactive metal, and 51 Cr, 57 Co, 58 Co, 67 Ga, 68 Ga, 64 Cu, 89 Zr, 111 In, 186 Re, 201 Tl or 197 Hg, and the like.

[0166] The nuclide which emits γ rays is a nuclide which emits γ rays by γ decay, and as the nuclide which emits γ rays by γ decay, a nuclide which emits γ rays by γ decay is preferably used. 99m Tc, 68 Ga or 201 Tl.

[0167] In the case where the radioactive metal which coordinates in the state of an ion in the radioactive metal complex is selected on the basis of the ionic radius, as the radioactive metal whose ionic radius is 70 to 130 pm, there can be mentioned 67 Ga, 68 Ga, 64 Cu, 67 Cu, 89 Zr, 90 Y, 99m Tc, 103 Ru, 111 In, 153 Sm, 165 Dy, 166 Ho, 177Lu, 186 Re、 188 Re、 198 Au、 201 Tl、 197 Hg、 203 Hg、 212 Bi、 213 Bi、 212 Pb、 225 Ac, etc. They are preferably capable of forming a complex of the compound of the present application with a radioactive metal ion having a structure represented by the aforementioned formula (A).

[0168] As the radioactive metal coordinated on the chelating portion of the radioactive metal-labeled antibody of the present application or the compound of the present application, one or more kinds selected from the group consisting of Al 18 F、 64 Cu、 67 Cu、 68 Ga、 89 Zr、 90 Y、 99m Tc、 111 In、 177 Lu and 225 Ac are preferred.

[0169] In formulae (I) and (II), the functional unit represented by the symbol B is a functional group having a function of improving the intracellular retention of a radioactive metal when the compound of the present application is made into a radioactive metal-labeled antibody, and is preferably an atomic group containing carbon and hydrogen, and further containing nitrogen or oxygen. As the functional unit suitable for use in the present application, for example, a structure having a positive charge in an acidic environment, and a structure having a unit for increasing the mass of the radioactive metal-labeled antibody can be cited.

[0170] In the case where the functional unit has a structure having a positive charge in an acidic environment, the structure generates a positive charge in the lysosome in the cell, and is not easily discharged to the outside of the cell. Therefore, it is considered that the radioactive metal coordinated with the functional chelating linker is easily retained in the cell. As an example of the atomic group, a structure having one or more primary, secondary or tertiary amino groups can be cited, and preferably an atomic group containing a structure polymerized from ethyleneimine (hereinafter, also referred to as "PEI structure") can be cited.

[0171] Further, it is considered that by providing the functional unit with a unit for increasing the mass of the radiometal-labeled antibody, metabolites from the radiometal-labeled antibody of the present application are less likely to be excreted outside the cell. As such a group, a group composed of atoms having an atomic weight of 400 or more can be designed, and more preferably a group composed of atoms having an atomic weight of 400 or more and 10,000 or less can be used. As a further preferred example, a group containing a structure polymerized from ethylene glycol (hereinafter also referred to as "PEG structure") can be given.

[0172] Further, in the case where the functional unit has a structure capable of binding to a molecule present in the target cell, by this binding, the mass of the radiometal-labeled antibody increases, and thus metabolites from the radiometal-labeled antibody are less likely to be excreted outside the cell. Therefore, as the functional unit, a group having a structure capable of binding to a molecule present in the target cell can be used.

[0173] In the case where the functional unit has a PEI structure, the PEI structure is preferably a structure polymerized from ethylene imine at a polymerization degree of 1 or more and 7 or less.

[0174] As a specific structure of the functional unit containing a PEI structure, a structure represented by the following formula (1) can be given.

[0175] [Chemical Formula 18]

[0176]

[0177] In formula (1), X represents a hydrogen atom, an alkyl group having 1 or more and 4 or less carbon atoms, a hydroxyl group, an amino group, or a bond.

[0178] n represents the polymerization degree of ethylene imine.

[0179] * (asterisk) represents a bond.

[0180] In formula (1), as the alkyl group having 1 or more and 4 or less carbon atoms represented by X, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, and the like can be given.

[0181] In formula (1), n is preferably an integer of 1 or more and 7 or less, more preferably an integer of 1 or more and 5 or less, and further preferably an integer of 1 or more and 3 or less.

[0182] The functional unit represented by formula (1) is protonated in an acidic environment such as in a lysosome, and becomes a (poly) cation having a high ionic charge density. Therefore, the radiometal coordinated with the functional chelate linker has a high intracellular retention property.

[0183] In the case where the functional unit has a PEG structure, the PEG structure is preferably a structure formed by polymerization of ethylene glycol with a polymerization degree of 11 or more and 48 or less.

[0184] As a specific structure of the functional unit including a PEG structure, a structure represented by the following formula (2) can be given.

[0185] [Chemical Formula 19]

[0186]

[0187] In formula (2), X represents a hydrogen atom, an alkyl group having 1 or more and 5 or less carbon atoms, a hydroxyl group, an amino group, a hydroxymethyl group, or a bond.

[0188] n represents a polymerization degree of ethylene glycol.

[0189] * (asterisk) represents a bond.

[0190] In formula (2), as the alkyl group having 1 or more and 5 or less carbon atoms represented by X, a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a pentyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an isoamyl group, a sec-amyl group, a tert-amyl group, a neopentyl group, and the like can be given.

[0191] In the case where a compound having a functional unit represented by formula (2) is made into a radiometal-labeled antibody, the radiometal coordinated with the functional chelating linker has high intracellular retention due to the large mass of the functional unit.

[0192] From this viewpoint, in formula (2), n is preferably an integer of 11 or more and 48 or less, more preferably an integer of 12 or more and 48 or less, and further preferably an integer of 24 or more and 48 or less.

[0193] Next, another embodiment of the functional unit will be described. In the present embodiment, the functional unit is a group having a structure capable of binding to a molecule present in a target cell. For example, in the case where the target cell is a cancer cell, as the aforementioned "molecule present in a target cell", a protein overexpressed in a cancer cell is preferably selected. As the protein, for example, cathepsin B can be given. Therefore, the functional unit of the present embodiment preferably has a structure capable of binding to cathepsin B.

[0194] The functional unit is not limited in the form of binding to the molecule present in the target cell, and can be an ionic bond, a hydrogen bond, a dipole-dipole interaction, a dispersion force, and the like, a non-covalent bond, and a covalent bond. Among them, from the viewpoint of improving the affinity and the binding specificity to the molecule present in the target cell, the binding of the functional unit to the molecule present in the target cell preferably includes a covalent bond. The covalent bond formed between the functional unit and the molecule present in the target cell can be one or a plurality. In addition, the functional unit and the molecule present in the target cell can have one or two or more non-covalent bonds in addition to the covalent bond.

[0195] The binding site of the molecule present in the target cell to the functional unit is not particularly limited, and in the case where the molecule present in the target cell is an enzyme, the functional unit is preferably bound to the binding site of the enzyme to its substrate, that is, the active site of the enzyme. The reason for this is that by mimicking the structure of the substrate of the enzyme, it is possible to easily design the structure of the functional unit having excellent affinity and binding specificity to the enzyme.

[0196] As described above, when an atomic group capable of binding to the molecule present in the target cell is used as the functional unit, the functional unit preferably has a structure capable of forming a covalent bond with Cathepsin B, and more preferably has a structure capable of forming a covalent bond with the active site of Cathepsin B (hereinafter, also referred to as "Structure A").

[0197] For example, in the case where a sulfhydryl group present on the active site of Cathepsin B forms a covalent bond with the functional unit, Structure A preferably has an epoxy group.

[0198] Furthermore, if the affinity to Cathepsin B and the stability of the binding state to Cathepsin B are taken into consideration, Structure A is preferably an epoxy succinyl peptide. The epoxy succinyl peptide refers to a peptide including a structure obtained by dehydration condensation of trans-epoxysuccinic acid and two amines of the same kind or different kinds.

[0199] As a suitable specific example of Structure A, the structures represented by the following formulas (X-1) to (X-3) can be given.

[0200] *-HN-(CH2) r -HN-Gly-Aa-Bb-X-Cc-Dd(X-1)

[0201] *-HN-(CH2) r -HN-Gly-Aa-Bb-X-Cc(X-2)

[0202] *-HN-(CH2) r -HN-Gly-Aa-Bb-X(X-3)

[0203] In formulae (X-1) to (X-3), X represents an epoxide derivative.

[0204] r represents an integer of 1 or more and 30 or less, preferably 6.

[0205] Aa represents one selected from the group consisting of Gly, Phe, Asp, Glu and 1-aminoadipic acid, preferably Gly.

[0206] Bb represents an aliphatic or aromatic hydrophobic amino acid.

[0207] Cc represents an aliphatic or aromatic hydrophobic amino acid.

[0208] Dd represents an aliphatic or aromatic hydrophobic amino acid, or an aliphatic or aromatic amine.

[0209] X represents an epoxide derivative.

[0210] * represents a bond.

[0211] Gly, Aa, Bb, X, Cc and Dd are bonded to each other via amide bonds.

[0212] In formulae (X-1) to (X-3), Bb preferably represents one selected from the group consisting of Leu, Ile, Phe, Tyr, Val, 3-(2-naphthyl)alanine, 3-cyclohexylalanine, 3-(4-bromophenyl)alanine and amino acids represented by the following formulae (X-4) to (X-7), more preferably Leu.

[0213] [Chemical Formula 20]

[0214]

[0215] In formulae (X-1) and (X-2), Cc preferably represents one selected from the group consisting of Leu, Ile, Phe, Tyr, Val, Trp and Nle, more preferably Leu.

[0216] In formula (X-1), Dd preferably represents one selected from the group consisting of Trp, Val, Ile, Phe, Tyr, Ala, Ser, Thr, Pro, -NH-(CH2) n -CH3 (n represents an integer of 0 or more and 4 or less), -NH-CH(CH3)2, -NH-CH2-CH(CH3)2 and -NH-(CH2) m Ph (m represents 1 or 2), more preferably Pro.

[0217] In formulae (X-1) to (X-3), the epoxide derivative represented by X is preferably a dicarboxylic acid having an epoxy group or an amino acid having an epoxy group, and more preferably trans-epoxysuccinic acid.

[0218] The functional unit may have only one of the above-mentioned PEI structure, PEG structure and / or structure capable of binding to a molecule present in the target cell, or may have two or more. For example, the functional unit may have one PEI structure and one PEG structure.

[0219] Furthermore, the radioactive metal-labeled antibody of the present invention may have only one functional unit or may have two or more functional units.

[0220] When the functional unit has a PEI structure and / or a PEG structure, the functional unit preferably has a structure represented by the following formula (c).

[0221] [Chemical Formula 21]

[0222]

[0223] In formula (c), X1 and X2 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxyl group, an amino group, or a linking bond.

[0224] X a and X b Each independently represents O or NH.

[0225] l represents 0 or 1.

[0226] When l is 0, Xc represents a hydrogen atom, and when l is 1, Xc represents CH2.

[0227] m and n each independently represent the degree of polymerization of ethyleneimine or ethylene glycol.

[0228] * indicates a connection key.

[0229] The compound of the present invention (functional chelate linker) preferably comprises a structure represented by the following formula (d): The structure represented by the following formula (d) comprises a chelating moiety and one or more functional units.

[0230] [Chemical Formula 22]

[0231]

[0232] In formula (d), o, p, and q each independently represent 0 or 1, and satisfy 1≤o+p+q≤3.

[0233] Xd represents the structure represented by the above formula (c).

[0234] Xe represents a hydrogen atom when o is 0, and Xe represents CH2when o is 1.

[0235] Xf represents a hydrogen atom when p is 0, and Xf represents CH2when p is 1.

[0236] Xg represents a hydrogen atom when q is 0, and Xg represents CH2when q is 1.

[0237] (*) is a connecting bond.

[0238] In the compound of the present application, the structure represented by formula (d) is directly or indirectly bound to the antibody binding unit via the connecting bond (*).

[0239] In addition, in the radiometal labeled antibody comprising the compound of the present application and the antibody, the structure represented by formula (d) is directly or indirectly bound to the antibody via the connecting bond (*). The "indirectly bound" referred to here means that a linker is present between the connecting bond and the antibody.

[0240] In formulae (I) and (II), the antibody binding unit represented by the symbol C is an antibody binding unit comprising a binding portion to an antibody. The form of binding of the antibody binding unit to the antibody is not particularly limited, and can be set to the following binding forms, for example.

[0241] (a) Binding of antibody to antibody binding unit based on conjugate addition reaction

[0242] (b) Binding of antibody to antibody binding unit based on click reaction

[0243] (c) Binding of antibody to antibody binding unit based on amine coupling

[0244] These will be described in turn below.

[0245] <Binding based on conjugate addition reaction>

[0246] By a conjugate addition reaction with the antibody binding unit from the thiol group of the side chain of the amino acid constituting the antibody, it is possible to bind the antibody to the compound of the present application. For this purpose, the antibody binding unit preferably has a Michael acceptor as the binding portion to the antibody. As this Michael acceptor, from the viewpoint of the balance between stability and reactivity to the conjugate addition reaction, and the viewpoint that it can be introduced to the antibody binding unit inexpensively, it is particularly preferable to use a maleimide group. In other words, the binding portion to the antibody is particularly preferably a maleimide group.

[0247] In addition, instead of the thiol group from the side chain of the amino acid constituting the antibody, after introducing a thiol group to the surface of the antibody by appropriate chemical modification, the antibody-binding unit can be bound to the antibody by conjugate addition reaction of the thiol group with a Michael acceptor. As a method of introducing a thiol group to the antibody, for example, a method of treating the antibody with 2-iminothiolane hydrochloride can be given.

[0248] As the antibody-binding unit having a maleimide group, for example, one selected from the atomic groups represented by the following formulae (3) to (8) can be used.

[0249] [Chemical Formula 23]

[0250]

[0251] As for the atomic groups represented by formulae (3) to (8), since BMPS, GMBS, MBS, SMCC, EMCS, or SMPH, which are commercially available reagents, can be easily introduced into the compound of the present application, they can be particularly preferably used as the antibody-binding unit.

[0252] <Combination based on click reaction>

[0253] In the case where the antibody is bound to the antibody-binding unit by the click reaction, the antibody-binding unit preferably uses a substance containing an atomic group that can undergo the click reaction as a binding portion to the antibody. By reacting this antibody-binding unit with an antibody (hereinafter, also referred to as "modified antibody") into which another atomic group that can undergo the click reaction is introduced, the antibody can be bound to the antibody-binding unit.

[0254] In the present application, as a combination of atomic groups that can undergo the click reaction, an appropriate combination is selected depending on the kind of the click reaction, for example, a combination of alkyne and azide, a combination of 1,2,4,5-tetrazine and olefin, and the like can be given. As for these atomic groups, the antibody-binding unit has one of the aforementioned atomic groups, and the antibody has an atomic group that becomes a combination with the antibody-binding unit.

[0255] From the viewpoint of achieving the simplicity of the click reaction process, the atomic group that can undergo the click reaction is preferably an atomic group that can be used in the click reaction without a metal catalyst.

[0256] As a specific example of the combination of the antibody binding unit and the modified antibody, the following can be given: a combination of a moiety containing dibenzocyclooctyne (DBCO) as the alkyne (Formula (11a)) and a moiety containing an azido group as the azide (Formula (12a)); or, a combination of a moiety containing 1,2,4,5-tetrazine (Formula (11b)) and a moiety containing trans-cyclooctene (TCO) as the olefin (Formula (12b)).

[0257] [Chemical Formula 24]

[0258]

[0259] In Formula (11a), R1represents a portion after removal of the moiety capable of click reaction from the antibody binding unit or the modified antibody.

[0260] In Formula (12a), R2represents a portion after removal of the moiety capable of click reaction from the antibody binding unit or the modified antibody.

[0261] [Chemical Formula 25]

[0262]

[0263] In Formula (11b), one of R3and R4represents a portion after removal of the moiety capable of click reaction from the antibody binding unit or the modified antibody, and the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group.

[0264] In Formula (12b), R5represents a portion after removal of the moiety capable of click reaction from the antibody binding unit or the modified antibody.

[0265] In the case where an atom group that can undergo a click reaction is introduced to the antibody-binding unit, various reagents available on the market can be used for the introduction. Specifically, in the case where an atom group containing dibenzocyclooctyne (DBCO) is introduced as an atom group that can undergo a click reaction, for example, DBCO-C6-Acid, DBCO-Amine, DBCOMaleimide, DBCO-PEG acid, DBCO-PEG-NHS ester, DBCO-PEG-Alcohol, DBCO-PEG-amine, DBCO-PEG-NH-Boc, Carboxyrhodamine-PEG-DBCO, Sulforhodamine-PEG-DBCO, TAMRA-PEG-DBCO, DBCO-PEG-Biotin, DBCO-PEG-DBCO, DBCO-PEG-Maleimide, TCO-PEG-DBCO, DBCO-mPEG, and the like DBCO reagents can be used. In addition, as a modified antibody corresponding thereto, a modified antibody into which an azido group is introduced is prepared. As a method of introducing an azido group into an antibody, for example, a method of introducing into the N terminus of an amino acid sequence, the amino side chain of a lysine residue, and the like can be given.

[0266] <Based on amine coupling>

[0267] In the case where the antibody-binding unit is bound to the antibody by amine coupling, the antibody-binding unit preferably uses a substance having a functional group capable of reacting with a primary amine as a binding portion to the antibody. By using such an antibody-binding unit, a bond is formed by reacting with the binding portion to the antibody, which is present on the N terminus of a peptide chain constituting the antibody, or the side chain of a lysine residue possessed by the peptide chain, or the like, and an amino group.

[0268] As the binding portion to the antibody having a functional group capable of reacting with a primary amine, an atom group having a structure of isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide (NHS) ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, carbonate, aryl halide, imido ester, carbodiimide, anhydride, and fluoroester, and the like can be given.

[0269] L of formula (I) and (II) a or L b The linker structure represented by the formula (P) can have a structure represented by the following formula (P). The structure is a structure from ethylene glycol, and in the formula (P), k is preferably an integer of 2 or more and 10 or less, more preferably an integer of 2 or more and 8 or less, and further preferably an integer of 2 or more and 5 or less.

[0270] [Chemical Formula 26]

[0271]

[0272] L a L b The linker structure represented by L

[0273] By reacting the compound of the present application (functional chelate linker), a radioactive metal ion, and an antibody, a radioactive metal-labeled antibody in which the functional chelate linker is coupled to the antibody can be obtained. The compound of the present application can be reacted with the antibody first and then with the radioactive metal ion, or can be reacted with the radioactive metal ion first and then with the antibody. Alternatively, the compound of the present application, the radioactive metal ion, and the antibody can be reacted simultaneously.

[0274] As the aforementioned radioactive metal ion, one of the radioactive metals listed above can be preferably used.

[0275] The antibody used in the present application is a polyclonal antibody or a monoclonal antibody. As for the class of immunoglobulin of the antibody, any of IgG, IgA, IgM, IgD, or IgE can be used without particular limitation, among which IgG is the main class in the secondary response of the immune response, is present most in the blood, and has high recognition specificity for antigens, and thus IgG can be preferably used from these aspects. Further preferably, it is a mammalian IgG, and as the mammal, specifically, primates such as humans and chimpanzees; experimental animals such as rats, mice, and rabbits; livestock animals such as pigs, cattle, horses, sheep, and goats; and pets such as dogs and cats can be mentioned, and further preferably, it is a chimeric antibody, a humanized antibody, or a human antibody of a monoclonal antibody. In addition, as for the subclass of these antibodies, there is no particular limitation, and for example, in the case of using human IgG, it can be at least one of IgGl, IgG2, IgG3, and IgG4. The antibody is a full-length antibody or an antibody fragment (for example, F(ab')2, Fab', Fab, Fv, single-chain antibody), and preferably a full-length antibody.

[0276] The antibody is an antibody against any antigen. As such an antigen, for example, proteins, sugar chains, nucleic acids, low-molecular compounds can be mentioned, and preferably, it can be an antibody against a protein. As the protein, for example, cell membrane receptors, cell membrane proteins other than cell membrane receptors, ligands, soluble receptors, and more specifically, it can be a disease-targeting protein.

[0277] For example, an antibody that recognizes an antigen specifically expressed in tumor cells can be used. Specifically, for example, an antibody using as an antigen PD-L1, GD2, PDGFRa (platelet-derived growth factor receptor), CD3, CD16A, CD19, CD20, CD22, CD25, CD27, CD30, CD32B, CD33, CD37, CD38, CD39, CD40, CD70, CD73, CD74, CD79b, CD100 (SEMA4D), CD105 (endoglin), CD123, CD137 (4-1BB), CD138, CD157 (Bst1), CD166, CD200, HER2, HER3, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGF, VEGFR-2, VEGF-A, HGF, gpNMB, DEC-205, folate receptor, sialylated HEG1, HEG1, Trop2, CEACAM5, S1P, IGF-1R, DLL4, TNT-1 / B, CPAA, PSMA, ICAM-1, MUC1, MUC5AC, EGFR, EGFRvIII, KIR2DL1, KIR3DL2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, mesothelin, c-Met, Ang2, ENPD3, folate receptor a, TEM-1, GM2, glypican-3, macrophage inhibitory factor, Notch1, Notch2, Notch3, TLR-2, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, gpA33, Frizzled7 receptor, DLL4, RSP0, LIV-1, SLITRK6, Nectin-4, MET, tissue factor, IL-8, P-cadherin, CEA, GITR, TAM (tumor-associated macrophage), CD44, CD44v6, CD44v7 / 8, CD44s, CD44v3, CD44v10, CD44v8 / 10, CD44v2, CD44v5, CD44v6, CD44v7, CD44v8, CD44v9, CD44v10, CD44v11, CD44v12, CD44v13, CD44v14, CD44v15, CD44v16, CD44v17, CD44v18, CD44v19, CD44v20, CD44v21, CD44v22, CD44v23, CD44v24, CD44v25, CD44v26, CD44v27, CD44v28, CD44v29, CD44v30, CD44v31, CD44v32, CD44v33, CD44v34, CD44v35, CD44v36, CD44v37, CD44v38, CD44v39, CD44v40, CD44v41, CD44v42, CD44v43, CD44v44, CD44v45, CD44v46, CD44v47, CD44v48, CD44v49, CD44v50, CD44v51, CD44v52, CD44v53, CD44v54, CD44v55, CD44v56, CD44v57, CD44v58, CD44v59, CD44v60, CD44v61, CD44v62, CD44v63, CD44v64, CD44v65, CD44v66, CD44v67, CD44v68, CD44v69, CD44v70, CD44v71, CD44v72, CD44v73, CD44v74, CD44v75, CD44v76, CD44v77, CD44v78, CD44v79, CD44v80, CD44v81, CD44v82, CD44v83, CD44v84, CD44v85, CD44v86, CD44v87, CD44v88, CD44v89, CD44v90, CD44v91, CD44v92, CD44v93, CD44v94, CD44v95, CD44v96, CD44v97, CD44v98, CD44v99, CD44v100, CD44v101, CD44v102, CD44v103, CD44v104, CD44v105, CD44v106, CD44v107, CD44v108, CD44v109, CD44v110, CD44v111, CD44v112, CD44v113, CD44v114, CD44v115, CD44v116, CD44v117, CD44v118, CD44v119, CD44v120, CD44v121, CD44v122, CD44v123, CD44v124, CD44v125, CD44v126, CD44v127, CD44v128, CD44v129, CD44v130, CD44v131, CD44v132, CD44v133, CD44v134, CD44v135, CD44v136, CD44v137, CD44v138, CD44v139, CD44v140, CD44v141, CD44v142, CD44v143, CD44v144, CD44v145, CD44v146, CD44v147, CD44v148, CD44v149, CD44v150, CD44v151, CD44v152, CD44v153, CD44v154, CD44v155, CD44v156, CD44v157, CD44v158, CD44v159, CD44v160, CD44v161, CD44v162, CD44v163, CD44v164, CD44v165, CD44v166, CD44v167, CD44v168, CD44v169, CD44v170, CD44v171, CD44v172, CD44v173, CD44v174, CD44v175, CD44v176, CD44v177, CD44v178, CD44v179, CD44v180, CD44v181, CD44v182, CD44v183, CD44v184, CD44v185, CD44v186, CD44v187, CD44v188, CD44v189, CD44v190, CD44v191, CD44v192, CD44v193, CD44v194, CD44v195, CD44v196, CD44v197, CD44v198, CD44v199, CD44v200, CD44v201, CD44v202, CD44v203, CD44v204, CD44v205, CD44v206, CD44v207, CD44v208, CD44v209, CD44v210, CD44v211, CD44v212, CD44v213, CD44v214, CD44v215, CD44v216, CD44v217, CD44v218, CD44v219, CD44v220, CD44v221, CD44v222, CD44v223, CD44v224, CD44v225, CD44v226, CD44v227, CD44v228, CD44v229, CD44v230, CD44v231, CD44v232, CD44v233, CD44v234, CD44v235, CD44v236, CD44v237, CD44v238, CD44v239, CD44v240, CD44v241, CD44v242, CD44v243, CD44v244, CD44v245, CD44v246, CD44v247, CD44v248, CD44v249, CD44v250, CD44v251, CD44v252, CD44v253, CD44v254, CD44v255, CD44v256, CD44v257, CD44v258, CD44v259, CD44v260, CD44v261, CD44v262, CD44v263, CD44v264, CD44v265, CD44v266, CD44v267, CD44v268, CD44v269, CD44v270, CD44v271, CD44v272, CD44v273, CD44v274, CD44v275, CD44v276, CD44v277, CD44v278, CD44v279, CD44v280, CD44v281, CD44v282, CD44v283, CD44v284, CD44v285, CD44v286, CD44v287, CD44v288, CD44v289, CD44v290, CD44v291, CD44v292, CD44v293, CD44v294, CD44v295, CD44v296, CD44v297, CD44v298, CD44v299, CD44v300, CD44v301, CD44v302, CD44v303, CD44v304, CD44v305, CD44v306, CD44v307, CD44v308, CD44v309, CD44v310, CD44v311, CD44v312, CD44v313, CD44v314, CDDLL4, Ang2, FGFR2, FGFR3, FGFR4, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R, OX40, BCMA, ErbB3, PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CAIX, CA72-4 (TAG-72) CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, alpha2-PI, A33, GDF15, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CLEC12A, Lgr3, Transferrin receptor, TGF beta, IL-17, 5T4, RTK, immunosuppressive protein, NaPi2b, Lewis blood group B antigen, A34, Lysil-Oxidase, DLK-1, or integrin alpha 9.

[0278] As an example of an antibody against HER2, trastuzumab, pertuzumab can be given.

[0279] In addition, as an antibody against sialylated HEG1, an antibody that binds to HEG1 protein having a sugar chain modification obtained from mesothelioma, an antibody that binds to HEG1 protein on the cell membrane of mesothelioma, and an antibody that binds to a peptide expressed by a mesothelioma cell line (for example, a peptide having the amino acid sequence of SKSPSLVSLPT) can be given. More specifically, the SKM9-2 antibody described in WO2017 / 141604, or a humanized antibody thereof can be given. In the case of the SKM9-2 antibody, for example, Nichirei Biosciences sells a mouse-derived anti-sialylated HEG1 monoclonal antibody. In addition, as a humanized SKM9-2 antibody, a humanized antibody that binds to human HEG1 protein expressed in mesothelioma cells can be given. As a humanized SKM9-2 antibody, an antibody having the heavy chain variable region and the light chain variable region shown in Table 1 described in WO2023 / 277113 (hereinafter, also referred to as "antibody A") can be given. Note that the "reference number" shown below refers to the sequence number in WO2023 / 277113.

[0280] [Table 1]

[0281] Table 1: Heavy chain variable region and light chain variable region of humanized SKM9-2 antibody (antibody A)

[0282] Table 1: Heavy chain variable region and light chain variable region of humanized SKM9-2 antibody (antibody A)

[0283] Here, the heavy chain CDR1 of antibody A is preferably the amino acid sequence in which X in SEQ ID NO: 1 is T (Thr) (SEQ ID NO: 7, reference number 104). In addition, the heavy chain CDR2 of antibody A is preferably the amino acid sequence in which X in SEQ ID NO: 2 is A (Ala) (SEQ ID NO: 8, reference number 105).

[0284] Here, the light chain CDR1 of antibody A is preferably the amino acid sequence in which both X at the 9th and 12th positions in SEQ ID NO: 4 are Q (Gin) (SEQ ID NO: 9, reference number 63), and more preferably the amino acid sequence in which X at the 10th position is N (Asn) and X at the 11th position is G (Gly) in SEQ ID NO: 4 (SEQ ID NO: 10, reference number 149). In addition, the light chain CDR2 of antibody A is preferably the amino acid sequence in which X at the 2nd position is R (Arg) and X at the 8th position is S (Ser) in SEQ ID NO: 5 (SEQ ID NO: 11, reference number 150). In addition, the light chain CDR3 of antibody A is preferably the amino acid sequence in which X in SEQ ID NO: 6 is I (Ile) (SEQ ID NO: 12, reference number 151).

[0285] The heavy chain variable region of antibody A can have the framework regions 1 to 4 shown in Table 2.

[0286] [Table 2]

[0287] Table 2: Framework regions of the heavy chain variable region of humanized SKM9-2 antibody (antibody A)

[0288]

[0289] In antibody A, the light chain variable region can have the framework regions 1 to 4 shown in Table 3.

[0290] [Table 3]

[0291] Table 3: Framework regions of the light chain variable region of humanized SKM9-2 antibody (antibody A)

[0292]

[0293] Antibody A more preferably has the heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 28 shown in Table 4, and further preferably has the amino acid sequence of SEQ ID NO: 18 in the heavy chain variable region.

[0294] [Table 4]

[0295] Table 4: Amino acid sequences of the heavy chain variable region of humanized SKM9-2 antibody (antibody A)

[0296]

[0297] The antibody A more preferably has an amino acid sequence selected from the group consisting of SEQ ID Nos. 29 to 41 shown in Table 5 in the light chain variable region, and further preferably has the amino acid sequence of SEQ ID No. 30 in the light chain variable region.

[0298] [Table 5]

[0299] Table 5: Amino acid sequences of light chain variable regions of humanized SKM9-2 antibody (antibody A)

[0300]

[0301] The radiometal labeled antibody of the present application can be used as a radiopharmaceutical containing the labeled antibody as an active ingredient. The radiopharmaceutical containing the radiometal labeled antibody of the present application as an active ingredient can be formulated using known formulation techniques. For example, the radiopharmaceutical of the present application can contain a pharmaceutically acceptable excipient. The excipient can be an excipient that can be appropriately administered for administering an effective amount of the radiometal labeled antibody of the present application as an active ingredient to a subject. In some aspects, the radiopharmaceutical of the present application can be formulated as an injection, and the excipient for injection can be a sterile aqueous solution, for example, an isotonic solution containing physiological saline or a pharmaceutically acceptable buffer, a stabilizer, a solubilizer, and the like. When the radiopharmaceutical of the present application is in the form of an injection, it can be administered parenterally (for example, intravenously or intrathoracically) for the treatment of a disease, the diagnosis of a disease, or the detection of a lesion, and the like. This radiopharmaceutical using an antibody that recognizes an antigen specifically expressed in a tumor cell can be suitably used for the diagnosis of cancer or the internal radiotherapy of cancer by selection of the radiometal.

[0302] In each of the above-described embodiments, as a substituent that can substitute each chemical structure of each atomic group and compound, for example, a halogen atom, a saturated or unsaturated alkyl group, a hydroxyl group, a formyl group, a carboxyl group, an acyl group, an amino group, a nitro group, an ester group, an isothiocyanate group, a sulfoxy group, a cyano group, an amide group, an imide group, a phosphoric acid group, a phenyl group, a benzyl group, a pyridyl group, a naphthyl group, and the like can be exemplified. These substituents can be a single group or a group combined from two or more substituents.

[0303] The above-described embodiments of the present application have been described based on preferred embodiments, but the present application is not limited to the above-described embodiments. For example, in each of the above-described embodiments, a compound each having one chelating portion, one functional unit, and one antibody-binding unit has been described, but the compound of the present application can have a plurality of at least one of the functional unit and the antibody-binding unit as long as the present application can be achieved.

[0304] The above-described embodiments of the present application include the following technical ideas.

[0305] [1] A radiometal-labeled antibody, which is a radiometal-labeled antibody obtained by radiometal labeling of an antibody,

[0306] wherein a functional chelating linker having the following is coupled to the aforementioned antibody:

[0307] a chelating portion capable of coordinating with a radiometal ion; and

[0308] a functional unit having a function of improving intracellular retention.

[0309] [2] The radiometal-labeled antibody according to [1], wherein the aforementioned functional unit has a structure polymerized from ethyleneimine or ethylene glycol.

[0310] [3] The radiometal-labeled antibody according to [2], wherein the aforementioned functional unit has a structure polymerized from ethyleneimine at a polymerization degree of 1 or more and 7 or less.

[0311] [4] The radiometal-labeled antibody according to [2], wherein the aforementioned functional unit has a structure polymerized from ethylene glycol at a polymerization degree of 11 or more and 48 or less.

[0312] [5] The radiometal-labeled antibody according to any one of [1] to [4], wherein the aforementioned chelating portion is interposed between the aforementioned antibody and the aforementioned functional unit.

[0313] [6] The radiometal-labeled antibody according to [5], wherein the aforementioned functional chelating linker further has an antibody-binding unit having a binding portion to the aforementioned antibody,

[0314] the aforementioned chelating portion has a first amino acid residue and a second amino acid residue,

[0315] the aforementioned chelating portion binds to the aforementioned antibody-binding unit via the aforementioned first amino acid residue, and the aforementioned chelating portion binds to the aforementioned functional unit via the aforementioned second amino acid residue.

[0316] [7] The radiometal-labeled antibody according to any one of [1] to [6], wherein the aforementioned chelating portion is DOTA or a derivative thereof.

[0317] [8] The radiometal-labeled antibody according to any one of [1] to [7], wherein the aforementioned radiometal is selected from the group consisting of Al 18 F, 64 Cu, 67 Cu, 68 Ga, 89 Zr, 90 Y, 99m Tc, 111 In, 177 Lu, and225 One of the Ac.

[0318] [9] The radioactive metal-labeled antibody according to any one of [1] to [8], wherein the functional chelating linker is introduced into the thiol group of the antibody.

[0319]

[10] The radioactive metal-labeled antibody according to any one of [1] to [9], wherein the antibody is an antibody that recognizes an antigen specifically expressed in tumor cells.

[0320]

[11] The radioactive metal-labeled antibody according to any one of [1] to

[10] , wherein the chelating portion capable of coordinating with the radioactive metal ion has a structure represented by the aforementioned formula (A),

[0321] The functional unit has a structure formed by polymerizing ethyleneimine or a structure formed by polymerizing ethylene glycol with a degree of polymerization of 11 or more and 48 or less.

[0322]

[12] The radioactive metal-labeled antibody according to any one of [1] to

[11] , wherein the functional chelating linker comprises a structure represented by the following formula (a) or (b).

[0323] [Chemical Formula 27]

[0324]

[0325] [Chemical Formula 28]

[0326]

[0327] In formulae (a) and (b), * (asterisk) represents a connecting bond, n represents 1 to 7, and m represents 11 to 48.

[0328]

[13] The radioactive metal-labeled antibody according to any one of [1] to

[12] , wherein the functional unit has a structure represented by the aforementioned formula (c).

[0329]

[14] The radioactive metal-labeled antibody according to any one of [1] to

[13] , wherein the functional chelating linker comprises a structure represented by the aforementioned formula (d).

[0330]

[15] A radiopharmaceutical comprising the radioactive metal-labeled antibody according to any one of [1] to

[14] as an active ingredient.

[0331]

[16] The radiopharmaceutical according to

[15] , which is used for diagnosis of cancer or internal radiotherapy of cancer.

[0332]

[17] A compound having in its structure:

[0333] a chelating moiety capable of coordinating with a radioactive metal ion;

[0334] a functional unit having a function of improving intracellular retention;

[0335] an antibody binding unit comprising a binding moiety to an antibody.

[0336]

[18] The compound according to

[17] , wherein the aforementioned chelating moiety is interposed between the aforementioned antibody binding unit and the aforementioned functional unit.

[0337]

[19] The compound according to

[17] or

[18] , wherein the aforementioned functional unit has a structure polymerized from ethyleneimine at a polymerization degree of 1 or more and 7 or less.

[0338]

[20] The compound according to

[17] or

[18] , wherein the aforementioned functional unit has a structure polymerized from ethylene glycol at a polymerization degree of 11 or more and 48 or less.

[0339]

[21] The compound according to any one of

[17] to

[20] , wherein the binding moiety to the aforementioned antibody is a maleimide group.

[0340]

[22] The compound according to any one of

[17] to

[21] , wherein the chelating moiety capable of coordinating with a radioactive metal ion has a structure represented by the aforementioned formula (A),

[0341] the aforementioned functional unit has a structure polymerized from ethyleneimine or a structure polymerized from ethylene glycol at a polymerization degree of 11 or more and 48 or less.

[0342]

[23] The compound according to any one of

[17] to

[22] , comprising a structure represented by the aforementioned formula (a) or (b).

[0343]

[24] The compound according to any one of

[17] to

[23] , wherein the aforementioned functional unit has a structure represented by the aforementioned formula (c).

[0344]

[25] The compound according to any one of

[17] to

[24] , comprising a structure represented by the aforementioned formula (d).

[0345]

[26] The radioactive metal-labeled antibody according to any one of [1] and [5] to

[10] , wherein the aforementioned functional unit has a structure capable of forming a covalent bond with an active site of cathepsin B.

[0346]

[27] The radioactive metal-labeled antibody according to

[26] , wherein the aforementioned structure capable of forming a covalent bond with an active site of cathepsin B is an epoxy succinyl peptide.

[0347]

[28] The compound according to

[17] or

[18] , wherein the aforementioned functional unit has a structure comprising an epoxy succinyl peptide.

[0348]

[29] The radiometal labeled antibody according to any one of [1] to

[14] , wherein the aforementioned antibody is trastuzumab or pertuzumab.

[0349]

[30] The radiometal labeled antibody according to any one of [1] to

[14] , wherein the aforementioned antibody is an antibody capable of binding to HEG1 protein having a sugar chain modification derived from mesothelioma, preferably a humanized antibody thereof, more preferably the aforementioned antibody A.

[0350]

[31] The radiometal labeled antibody according to any one of [1] to

[14] ,

[29] and

[30] , wherein the aforementioned functional chelating linker is represented by the aforementioned general formula (I).

[0351]

[32] The radiometal labeled antibody according to any one of

[17] to

[25] and

[28] , wherein the aforementioned functional chelating linker is represented by the aforementioned general formula (II).

[0352] Examples

[0353] Hereinafter, the present application will be described in more detail by way of examples. However, the scope of the present application is not limited to these examples.

[0354] Reagents were used as commercially available materials, and were used as they were, unless otherwise specified.

[0355] In the case of performing mass spectrometry (MS) analysis, LCMS2020 manufactured by Shimadzu Corporation was used, and in the case of performing high resolution mass spectrometry (HRMS) analysis, LCMS-IT-TOF manufactured by Shimadzu Corporation was used.

[0356] In terms of 1 H and 13 In terms of nuclear magnetic resonance (NMR) spectra of1H and13C, tetramethylsilane (0 ppm) was used as an internal standard substance, and recording was performed using JNM-ECS400 system or JNM-ECA500 system manufactured by JEOL Ltd. In terms of coupling of peaks, singlet was written as s, triplet as t, and multiplet as m.

[0357] Reverse phase (RP)-HPLC was performed using a column equipped with COSMOSIL C 18 18 column (5C 18- AR-II, 4.6 mm I.D. x 150 mm or 10 mm I.D. x 250 mm) or COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) of Shimadzu system (LC-20AT pump equipped with SPD-20A UV detector, measuring wavelength: 220 or 254 nm) was measured, and size exclusion (SEC)-HPLC was measured using Shimadzu system (LC-20A pump equipped with SPD-20A UV detector, measuring wavelength: 254 nm) with TSKgel G3000SW XL - AR-II, 4.6 mm I.D. x 150 mm or 10 mm I.D. x 250 mm) or COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) of Shimadzu system (LC-20AT pump equipped with SPD-20A UV detector, measuring wavelength: 220 or 254 nm) was measured, and size exclusion (SEC)-HPLC was measured using Shimadzu system (LC-20A pump equipped with SPD-20A UV detector, measuring wavelength: 254 nm) with TSKgel G3000SW

[0358] The concentration of the antibody was calculated using the UV absorption at 280 nm measured by NanoVue PLUS of Central Scientific Commerce, Inc.

[0359] Autoradiography was measured using Amersham Typhoon scanner of Global Life Science Technologies Japan Co., Ltd.

[0360] [Chemical Formula 29]

[0361]

[0362] • Synthesis of compound 8 and its In complex 10 (Example 1-1) and synthesis of compound 9 and its In complex 11 (Example 1-2)

[0363] < Synthesis of compound 1>

[0364] Compound 1 was synthesized according to the reported method (Org. Biomol. Chem., 2014, 12, 9389).

[0365] < Synthesis of compound 1.1>

[0366] Compound 1 (613 mg, 0.67 mmol) was dissolved in methanol (12 mL), and 0.2 equivalents of NaOH aqueous solution (80 mg, 2.0 mmol, 10 mL) were added and stirred at room temperature for 20 hours. A 5% aqueous HCl solution was added to adjust the pH to 3, and then the methanol was removed using a rotary evaporator. Ethyl acetate was added to the remaining aqueous solution, the organic layer was recovered, and the recovered organic layer was dried with sodium sulfate and filtered. After the solvent of the filtrate was distilled off, 376 mg of the residue was obtained as compound 1.1 (yield 62%) without purification. HRMS (electrospray ionization method) of the obtained compound 1.1 was performed, and the measured m / z was 906.5799 (the theoretical m / z was 906.5798 (C 47 H 80 N5O 12 + 、[M+H] + )).

[0367] <Synthesis of Compounds 2 and 3>

[0368] Compounds 2 and 3 were synthesized according to previous reports (J. Am. Chem. Soc., 2008, 130, 14, 4618-4627).

[0369] <Synthesis of Compound 4>

[0370] Compound 1.1 (376 mg, 0.41 mmol) was dissolved in anhydrous DMF (1 mL), and 1-((1-(cyano-2-ethoxy-2-oxo-ethyleneaminooxy)dimethylaminomorpholino))uronium hexafluorophosphate (COMU) (351 mg, 0.82 mmol) was added at 0°C, and stirred at 0°C for 15 minutes. Then, compound 2 (249 mg, 0.82 mmol) was added, and stirred at room temperature for 13 hours. COSMOSIL C was used. 18 Column (5C 18 -AR-II, 10mmI.D.×250mm) and mobile phase (development conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) to 1 / 9 (40 min), flow rate 4mL / min), the solution was purified by reverse phase HPLC to obtain 272mg of compound 4 (yield 56%). HRMS (electrospray ionization method) of the obtained compound 4 was performed, and the measured m / z was 596.3962 (theoretical m / z was 596.3962 (C 61 H 108 N8O 15 2+ 、[M+2H] 2+ )).

[0371] < Synthesis of Compound 5 >

[0372] Compound 1.1 (392 mg, 0.43 mmol) was dissolved in dry DMF (800 μL), COMU (184 mg, 0.43 mmol) was added at 0°C and stirred for 15 minutes at 0°C. Then, compound 3 (635 mg, 0.43 mmol) was added and stirred for 11 hours at room temperature. The solution was purified by silica gel chromatography using a mobile phase (chloroform / methanol = 9 / 1) to give 293 mg of compound 5 (yield 46%). HRMS (electrospray ionization) of the obtained compound 5 was performed and the result gave m / z of 739.4905 (m / z of 739.4908 (C 75 H 134 N 10 O 19 2+ , [M+2H] 2+ ).

[0373] < Synthesis of Compound 6 >

[0374] Compound 4 (262 mg, 0.22 mmol) was dissolved in methanol (20 mL), 10% palladium carbon (100 mg) was added and stirred vigorously for 13 hours at room temperature under a hydrogen atmosphere. Then, filtration was performed using celite and the solvent of the filtrate was distilled off. The residue (159 mg, 0.15 mmol) was dissolved in dry DMF (800 μL), 6-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS) (56 mg, 0.18 mmol) and triethylamine (23 mg, 0.23 mmol) were added and stirred for 12 hours at room temperature. The solution was purified by reverse phase HPLC using a COSMOSIL CH 18 AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 1 / 9 (40 minutes), flow rate 4 mL / min) to give 21 mg of compound 6 (yield 11%). HRMS (electrospray ionization) of the obtained compound 6 was performed and the result gave m / z of 1250.8219 (m / z of 1250.8222 (C 18 H 63 N9O 112 16 + , [M+H] + ).

[0375] < Synthesis of Compound 7 >

[0376] ​Compound 5 (293 mg, 0.20 mmol) was dissolved in methanol (20 mL), 10% palladium carbon (100 mg) was added, and stirred vigorously under a hydrogen atmosphere at room temperature for 16 hours. Then, filtration was performed using celite, and the solvent of the filtrate was distilled off. A portion of the residue (50 mg, 37 μmol) was dissolved in anhydrous DMF (550 μL), 6-maleimidocaproic acid N-hydroxysuccinimidyl ester (46 mg, 0.15 mmol) and triethylamine (15 mg, 0.15 mmol) were added, and stirred at room temperature for 21 hours. The solution was purified by reverse phase HPLC using a COSMOSIL C 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 1 / 9 (40 min), flow rate 4 mL / min) to give 24 mg of compound 7 (yield 42%). HRMS (electrospray ionization) of the obtained compound 7 was performed, and the result of m / z of the found value was 1537.0113 (m / z of the theoretical value was 1537.0115 (C 77 H 138 N 11 O 20 + , [M+H] + ).

[0377] Synthesis of Compound 8 (Maleimide-DOTAGL-PEI2 (MDPEI2))

[0378] Compound 6 (21 mg, 17 μmol) was dissolved in a mixed solution of trifluoroacetic acid (2 mL) and dichloromethane (1 mL), and stirred at room temperature for 12 hours. Then, the solvent was distilled off, and the residue was purified by reverse phase HPLC using a COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 1.9 mg of compound 8 (yield 14%). HRMS (electrospray ionization) of the obtained compound 8 was performed, and the result of m / z of the found value was 826.4669 (m / z of the theoretical value was 826.4669 (C 37 H 64 N9O 12 + , [M+H] + ).

[0379] <Synthesis of Maleimide-DOTAGL-PEI4 (MDPEI4) (Compound 9)>

[0380] Compound 7 (12 mg, 8.0 μmol) was dissolved in a mixed solution of trifluoroacetic acid (800 μL) and dichloromethane (200 μL), and stirred at room temperature for 14 hours. The solvent was then distilled off and COSMOSIL C was used to 18 Column (5C 18 -AR-II, 4.6mmI.D.×150mm) and mobile phase (development conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) to 7 / 3 (40 min), flow rate 1 mL / min), the residue was purified by reverse phase HPLC to obtain 0.49 mg of compound 9 (yield 6.9%). HRMS (electrospray ionization method) of the obtained compound 9 showed that the measured m / z was 912.5515 (theoretical m / z was 912.5513 (C 41 H 74 N 11 O 12 + 、[M+H] + )).

[0381] <Maleimide-[ nat In]In-DOTAGL-PEI2([ nat Synthesis of In]In-MDPEI2) (Compound 10)

[0382] In this synthesis, indium(III) ( nat In) indium(III) chloride ( nat InCl3). Compound 8 (1.5 mg, 1.8 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 200 μL), indium (III) chloride (3.9 mg, 18 μmol) was added, and heated at 90°C for 30 minutes. The reaction solution was then centrifuged to remove the insoluble matter. The supernatant was purified by COSMOSIL C 18 Column (5C 18-AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) using reverse phase HPLC, and further using COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.01% trifluoroacetic acid aqueous solution / 0.01% trifluoroacetic acid acetonitrile solution = 10 / 0 (0 min) - 7 / 3 (30 min), flow rate 1 mL / min) using reverse phase HPLC, to give 0.45 mg of compound 10 (yield 27%). HRMS (electrospray ionization method) was performed on the obtained compound 10, and as a result, m / z of the found value was 938.3473 (m / z of the theoretical value was 938.3473 (C 37 H 61 InN9O 12 + , [M+2H] + )).

[0383] nat In] In-DOTAGL-PEI4([ nat In] In-MDPEI4) (compound 11)

[0384] Compound 9 (0.68 mg, 0.74 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 120 μL), and indium (III) chloride (1.6 mg, 7.4 μmol) was added, and heated at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. Using COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 8 / 2 (20 min), flow rate 1 mL / min) using reverse phase HPLC, to give 0.07 mg of compound 11 (yield 9.2%). HRMS (electrospray ionization method) was performed on the obtained compound 11, and as a result, m / z of the found value was 512.7197 (m / z of the theoretical value was 512.7195 (C 41 H 72 InN 11 O 12 2+ , [M+3H] 2+ )).

[0385] [Chemical Formula 30] ​

[0386]

[0387] • Synthesis of compound 14 and its In complex 15 (Comparative Example 1-1)

[0388] < Synthesis of compound 12 >

[0389] Compound 12 was synthesized according to the reported procedure (Angew. Chem. Int. Ed. Engl., 2017, 56, 9825-9828).

[0390] < Synthesis of compound 13 >

[0391] Compound 12 (784 mg, 0.94 mmol) was dissolved in methanol (15 mL), 10% palladium on carbon (150 mg) was added, and stirred vigorously under hydrogen atmosphere at room temperature for 19 hours. Then, filtration was performed using celite, and the solvent of the filtrate was distilled off. A portion of the residue (100 mg, 0.14 mmol) was dissolved in anhydrous DMF (400 μL), 6-maleimidocaproic acid N-hydroxysuccinimidyl ester (88 mg, 0.28 mmol) and triethylamine (58 mg, 0.57 mmol) were added, and stirred at room temperature for 13 hours. The solution was purified by reverse phase HPLC using COSMOSIL CH 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 4 mL / min), to obtain 20 mg of compound 13 (yield 15%). HRMS (electrospray ionization method) of the obtained compound 13 was performed, and the result showed m / z of 893.5959 (m / z of 893.5958 (C 46 H 81 N6O 11 + , [M+H] + ). In addition, the following shows NMR-based identification data of the obtained compound 13. 1 HNMR (400 MHz, CDCl3) δ 6.65 (s, 2H), 3.85-2.57 (m, 27H), 2.16 (t, 2H), 1.57-1.25 (m, 48H).

[0392] < Synthesis of maleimide-DOTAGA (MD) (compound 14) >

[0393] Compound 13 (20 mg, 22 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (0.4 mL) and stirred at room temperature for 14 hours. Then, the solvent was distilled off and the residue was purified by reverse phase HPLC using a COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 8.3 mg of compound 14 (yield 57%). HRMS (electrospray ionization) of the obtained compound 14 was performed and the result showed m / z of 669.3454 (m / z of 669.3454 (C 30 H 49 N6O 11 + , [M+H] + ).

[0394] < Maleimide-[ nat In]In-DOTAGA([ nat In]In-MD) (compound 15)

[0395] Compound 14 (1.0 mg, 1.5 μmol) was dissolved in an acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL) and indium (III) chloride (3.3 mg, 15 μmol) was added, followed by heating at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matters. The supernatant was purified by reverse phase HPLC using a COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 0.41 mg of compound 15 (yield 35%). HRMS (electrospray ionization) of the obtained compound 15 was performed and the result showed m / z of 781.2258 (m / z of 781.2258 (C 30 H 46 InN6O 11 + , [M+2H] + ).

[0396] [Chemical Formula 31]

[0397]

[0398] • Synthesis of compound 20 and its In complex 22 (comparative example 1-2) and synthesis of compound 21 and its In complex 23 (comparative example 1-3)

[0399] < Synthesis of compound 16 >

[0400] Compound 1.1 (376 mg, 0.41 mmol) was dissolved in dry DMF (700 μL), and 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl) (44 mg, 0.23 mmol), 1-hydroxy-7-azabenzotriazole (HOAt) (57 mg, 0.42 mmol), and N, N- diisopropylethylamine (DIPEA) (54 mg, 0.42 mmol) were added at 0°C, and stirred for 15 minutes at 0°C. Then, 2-(2-aminoethoxy)ethanol (33 mg, 0.31 mmol) was added, and stirred for 11 hours at room temperature. The solution was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minutes) - 1 / 9 (40 minutes), flow rate 4 mL / min) to give 113 mg of compound 16 (yield 54%). HRMS (electrospray ionization) of the obtained compound 16 was performed, and the result showed m / z of 993.6483 (m / z of 993.6483 (C 51 H 89 N6O 13 + [M+H] + ).

[0401] < Synthesis of compound 17 >

[0402] Compound 1.1 (376 mg, 0.41 mmol) was dissolved in dry DMF (700 μL), and 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl) (44 mg, 0.23 mmol), 1-hydroxy-7-azabenzotriazole (HOAt) (57 mg, 0.42 mmol), and N, N- diisopropylethylamine (DIPEA) (54 mg, 0.42 mmol) were added at 0°C, and stirred for 15 minutes at 0°C. Then, 2-(2-aminoethoxy)ethanol (33 mg, 0.31 mmol) was added, and stirred for 11 hours at room temperature. The solution was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18-AR-II, 10 mm I.D. x 250 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 1 / 9 (40 min), flow rate 4 mL / min), the solution was purified by reverse phase HPLC to obtain 61 mg of compound 17 (yield 67%). HRMS (electrospray ionization) of the obtained compound 17 was performed, and as a result, m / z of the found value was 1081.7009 (m / z of the theoretical value was 1081.7007 (C 55 H 97 N6O 15 + , [M+H] + )).

[0403] < Synthesis of compound 18 >

[0404] Compound 16 (113 mg, 0.11 mmol) was dissolved in methanol (15 mL), 10% palladium carbon (100 mg) was added, and it was stirred vigorously under a hydrogen gas atmosphere at room temperature for 20 hours. Then, filtration was performed using celite, and the solvent of the filtrate was distilled off. A part of the residue (50 mg, 58 μmol) was dissolved in anhydrous DMF (500 μL), 6-maleimidocaproic acid N-hydroxysuccinimide ester (18 mg, 58 μmol) and triethylamine (6.5 mg, 64 μmol) were added, and it was stirred at room temperature for 17 hours. The solution was purified by reverse phase HPLC using COSMOSIL C 18 18-P (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 4 mL / min), the solution was purified by reverse phase HPLC to obtain 24 mg of compound 18 (yield 39%). HRMS (electrospray ionization) of the obtained compound 18 was performed, and as a result, m / z of the found value was 1052.6857 (m / z of the theoretical value was 1052.6854 (C 53 H 94 N7O 14 + , [M+H] + )).

[0405] < Synthesis of compound 19 >

[0406] Compound 17 (34 mg, 31 μmol) was dissolved in methanol (10 mL), 10% palladium carbon (11 mg) was added, and the mixture was stirred vigorously under a hydrogen atmosphere at room temperature for 13 hours. Then, the mixture was filtered using celite, and the solvent of the filtrate was distilled off. The residue was dissolved in dry DMF (600 μL), 6-maleimidocaproic acid N-hydroxysuccinimide ester (38 mg, 0.12 mmol) and triethylamine (13 mg, 0.12 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The solution was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 1 / 9 (40 min), flow rate 4 mL / min) to give 14 mg of compound 19 (yield 40%). HRMS (electrospray ionization) of the obtained compound 19 was performed, and the result gave m / z of 570.8724 (m / z of 570.8724 (C 57 H 103 N7O 16 2+ , [M+2H] 2+ ).

[0407] Synthesis of Compound 20 (Maleimide-DOTAGL-PEG2 (MDPEG2))

[0408] Compound 18 (24 mg, 23 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (0.4 mL), and the mixture was stirred at room temperature for 14 hours. Then, the solvent was distilled off, and the residue was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 9.4 mg of compound 20 (yield 50%). HRMS (electrospray ionization) of the obtained compound 20 was performed, and the result gave m / z of 828.4350 (m / z of 828.4350 (C 37 H 62 N7O 14 + , [M+H] + ).

[0409] Synthesis of Maleimide-DOTAGL-PEG4 (MDPEG4) (Compound 21)

[0410] Compound 19 (11 mg, 10 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1280 μL) and dichloromethane (320 μL) and stirred at room temperature for 14 hours. Then, the solvent was distilled off and the residue was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 2.6 mg of Compound 19 (yield 28%). HRMS (electrospray ionization) of the obtained Compound 21 was performed and the result gave m / z of 916.4874 (m / z of 916.4874 (C 41 H 70 N7O 16 + , [M+H] + ).

[0411] Synthesis of Maleimide-[ nat In]In-DOTAGL-PEG2([ nat In]In-MDPEG2) (Compound 22)

[0412] Compound 20 (1.0 mg, 1.2 μmol) was dissolved in an acetic acid buffer (1.0 mol / L, pH 5.1, 200 μL) and indium (III) chloride (2.7 mg, 12 μmol) was added and heated at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. The supernatant was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 7 / 3 (20 min), flow rate 1 mL / min) to give 0.24 mg of Compound 22 (yield 21%). HRMS (electrospray ionization) of the obtained Compound 22 was performed and the result gave m / z of 940.3161 (m / z of 940.3154 (C 37 H 59 InN7O 14 + , [M+2H] + ).

[0413] <Maleimide-[ nat In]In-DOTAGL-PEG4([ nat Synthesis of In]In-MDPEG4) (Compound 23)

[0414] Compound 21 (2.3 mg, 2.5 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL), indium (III) chloride (5.6 mg, 25 μmol) was added, and heated at 90°C for 30 minutes. The reaction solution was then centrifuged to remove insoluble matter. 18 Column (5C 18 -AR-II, 4.6mmI.D.×150mm) and mobile phase (development conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) to 7 / 3 (20 minutes), flow rate 1mL / min), the supernatant was purified by reverse phase HPLC to obtain 0.69mg of compound 23 (yield 30%). HRMS (electrospray ionization method) of the obtained compound 23 was performed, and the measured m / z was 514.6876 (theoretical m / z was 514.6875 (C 41 H 68 InN7O 16 + 、[M+3H] 2+ )).

[0415] [Chemical Formula 32]

[0416]

[0417] Synthesis of Compound 30 and its In complex 31 (Examples 1-3)

[0418] <Synthesis of Compound 24>

[0419] Compound 24 was synthesized according to previous reports (J. Chem. Soc., Chem. Commun., 1995, 185-186).

[0420] <Synthesis of Compound 25>

[0421] Compound 25 was synthesized according to previous reports (Org. Biomol. Chem., 2013, 11, 1294-1305).

[0422] <Synthesis of Compound 26>

[0423] Compound 26 was synthesized according to the reported procedure (Bioorg. Med. Chem. Lett., 2000, 10, 2133-2135).

[0424] <SYNTHESIS OF COMPOUND 27>

[0425] Compound 24 (1400 mg, 3.5 mmol) was dissolved in acetonitrile (25 mL), and compound 25 (1678 mg, 4.2 mmol) and potassium carbonate (579 mg, 4.2 mmol) were added, and stirred at 50°C for 24 hours. Then, filtration was performed, and the filtrate was recovered, and the solvent was distilled off. The residue was dissolved in acetonitrile (30 mL), and compound 26 (2496 mg, 7.0 mmol) and potassium carbonate (966 mg, 7.0 mmol) were added, and stirred at 55°C for 15 hours. Then, filtration was performed, and the filtrate was recovered, and the solvent was distilled off. The residue was purified by an amine-based silica gel chromatography using a mobile phase (hexane / ethyl acetate = 1 / 1) to obtain 1274 mg of compound 27 (yield 37%). MS (electrospray ionization) of the obtained compound 27 was performed, and the result showed m / z of 996.5 (m / z of 996.6268 (C 54 H 86 N5O 12 + , [M+H] + ).

[0426] <SYNTHESIS OF COMPOUND 28>

[0427] Compound 27 (1274 mg, 1.28 mmol) was dissolved in methanol (25 mL), and 10% palladium carbon (350 mg) was added, and stirred vigorously under a hydrogen gas atmosphere at room temperature for 48 hours. Then, filtration was performed using celite, and the solvent of the filtrate was distilled off. A part of the residue (100 mg, 0.13 mmol) was dissolved in anhydrous DMF (700 μL), and 6-maleimidocaproic acid N-hydroxysuccinimidyl ester (160 mg, 0.52 mmol) and triethylamine (52 mg, 0.52 mmol) were added, and stirred at room temperature for 71 hours. Purification was performed by reverse phase HPLC using COSMOSIL CH 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 3 / 7 (30 minutes), flow rate 4 mL / min) to obtain 20 mg of compound 28 (yield 20%). MS (electrospray ionization) of the obtained compound 28 was performed, and the result showed m / z of 1000.5 (m / z of 1000.6268 (C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 3 / 7 (30 minutes), flow rate 4 mL / min) to obtain 20 mg of compound 28 (yield 20%). MS (electrospray ionization) of the obtained compound 28 was performed, and the result showed m / z of 1000.5 (m / z of 1000.6268 (C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 3 / 7 (30 minutes), flow rate 4 mL / min) to obtain 20 mg of compound 28 (yield 20%). MS (electrospray ionization) of the obtained compound 28 was performed, and the result showed m / z of 1000.5 (m / z of 1000.6268 (C 18-AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 7 / 3 (0 min) - 4 / 6 (30 min), flow rate 1 mL / min), purification was performed by reverse phase HPLC to obtain 19.6 mg of compound 28 (yield 16%). MS (electrospray ionization method) was performed on the obtained compound 28, and as a result, m / z of the observed value was 965.4 (m / z of the theoretical value was 965.6170 (C 49 H 85 N6O 13 + , [M+H] + )).

[0428] < Synthesis of compound 29 >

[0429] Compound 28 (17 mg, 18 μmol) was dissolved in dry DMF (300 μL), COMU (7.7 mg, 36 μmol) and DIPEA (2.3 mg, 36 μmol) were added at 0°C, and stirred at 0°C for 15 minutes. Then, m-dPEG48-amine (39 mg, 18 μmol) was added, and stirred at room temperature for 136 hours. Purification was performed by reverse phase HPLC on the solution using COSMOSIL C 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 4 mL / min), purification was performed by reverse phase HPLC on the solution to obtain 10 mg of compound 29 (yield 18%). MS (electrospray ionization method) was performed on the obtained compound 29, and as a result, m / z of the observed value was 1546.7 (m / z of the theoretical value was 1546.9588 (C 146 H 281 N7O 60 2+ , [M+2H] 2+ )).

[0430] < Synthesis of maleimide-DOTAGL-PEG48 (MDPEG48) (compound 30) >

[0431] Compound 29 (5.0 mg, 1.6 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (0.4 mL), and stirred at room temperature for 14 hours. Then, the solvent was distilled off, and purification was performed by reverse phase HPLC on the solution using COSMOSIL C 18 column (5C 18-AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 3 / 1 (0 min) - 11 / 9 (20 min), flow rate 1 mL / min), the residue was purified by reverse phase HPLC to obtain 2.0 mg of compound 30 (yield 43%). HRMS (electrospray ionization) of the obtained compound 30 was performed, and the result of m / z of the found value was 1434.8340 (m / z of the theoretical value was 1434.8336 (C 130 H 249 N7O 60 2+ , [M+2H] 2+ ).

[0432] < Maleimide-[ nat In]In-DOTAGL-PEG48([ nat In]In-MDPEG48) (compound 31)

[0433] Compound 30 (0.20 mg, 52 nmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL), and indium (III) chloride (0.11 mg, 0.52 μmol) was added, and heated at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. The supernatant was purified by reverse phase HPLC using COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 3 / 1 (0 min) - 11 / 9 (20 min), flow rate 1 mL / min), the residue was purified by reverse phase HPLC to obtain 2.0 mg of compound 30 (yield 43%). HRMS (electrospray ionization) of the obtained compound 30 was performed, and the result of m / z of the found value was 1434.8340 (m / z of the theoretical value was 1434.8336 (C 130 H 246 InN7O 60 2+ , [M+3H] 2+ ).

[0434] Synthesis of radiolabeled antibody agents (Examples 2-1 to 2-3 and Comparative Examples 2-1 to 2-3)

[0435] < Introduction of thiol group into trastuzumab>

[0436] Herceptin (3.0 mg) (Roche) was dissolved in 0.1 mol / L boric acid buffer (pH 8.0, 4 mL) containing 2 mmol / L EDTA, and purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa) manufactured by Millipore. 2-iminothiolane hydrochloride was dissolved in 0.1 mol / L boric acid buffer (pH 8.0) containing 2 mmol / L EDTA to prepare a 7 mmol / L solution, and after the trastuzumab in the concentrated solution was added as 2 molar equivalents, it was incubated for 1 hour in a light-shielded environment. The reaction solution was purified using a Zeba desalting centrifugal column (40K MWCO, 0.5 mL) to obtain thiol-introduced trastuzumab (trastuzumab-SH). The purified trastuzumab-SH was promptly used for the labeling reaction.

[0437] The number of introduced thiols was determined using the Ellman method. 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman reagent) (3.3 mg) was dissolved in 0.1 mol / L boric acid buffer (pH 8.0, 833 μL) containing 2 mmol / L EDTA to prepare an Ellman reagent solution. Separately, 0.1 mol / L boric acid buffer (pH 8.0) containing 2 mmol / L EDTA was prepared as a control solution not containing an antibody. The Ellman reagent solution (4 μL) was added to a trastuzumab-SH solution (20 μL) of a known antibody concentration or a control solution (20 μL), and incubated at room temperature for 20 minutes. After the incubation, the absorbance of the antibody solution at 412 nm was measured with the control solution as a reference. From the antibody concentration before the addition of the Ellman reagent and the solution amounts before and after the addition of the Ellman reagent, the antibody concentration at the time of the measurement of the absorbance was calculated. The molar absorption coefficient of the product of the reaction of the Ellman reagent with a thiol, 5-nitro-2-thiobenzoic acid, at 412 nm was set to 14.15 mmol / L cm -1 cm -1 , and the thiol concentration was calculated. The thiol concentration was divided by the antibody concentration, and thus the number of introduced thiols per antibody was calculated. The number of introduced thiols per antibody was 1.32 ± 0.16.

[0438] <Labeling reaction>

[0439] <Labeling in the first stage>

[0440] Compound 8, 9, 14, 20, 21, and 30 (1.0 mg / mL DMSO solution, 3-8 μL) were each dissolved in 0.1 mol / L acetic acid buffer (pH 5.1, 350 μL), and 111In] InCl3 solution (500-600 μL, 35-37 MBq) was added and heated at 90°C for 6 minutes. 18 Column (5C 18 -AR-II, 4.6 mm I.D. × 150 mm) and a mobile phase (MeCN / H2O / TFA [10 / 90 / 0.1 (0 min) ~ 30 / 70 / 0.1 (20 min)], flow rate 1.0 mL / min), the reaction solution was purified by reverse-phase HPLC, and the solvent containing the components of the target compound was distilled off under a nitrogen flow.

[0441] In this specification, compounds 8, 9, 14, 20, 21 and 30 are 111 In 3+ The radiolabeled compounds obtained by coordination are respectively called [ 111 In]In-MDPEI2, [ 111 In]In-MDPEI4, [ 111 In]In-MD, [ 111 In]In-MDPEG2, [ 111 In]In-MDPEG4, and [ 111 In]In-MDPEG48.

[0442] <Stage 2 Marking>

[0443] To the residue obtained in the first stage of labeling, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, followed by trastuzumab-SH (0.6 mg), and incubated at room temperature for 4-6 hours under a light-shielding environment. Then, using Amicon Ultra-4 (recovery fraction 50 kDa), the reaction solution was purified and concentrated to obtain the target radioactive metal-labeled antibody (hereinafter also referred to as "radioactive antibody agent"). Radiochemical purity (RCP) was determined as follows. By using TSKgel G3000SW XL The analysis was performed by size exclusion HPLC using a column (5 μm, 7.8 mm I.D. × 300 mm) and a mobile phase (0.1 mol / L phosphate buffer (pH 6.8), flow rate 0.5 mL / min). The percentage of the area value of the radiolabeled compound relative to the area value of all detected peaks was recorded as RCP (%).

[0444] In this specification, [ 111 In]In-MDPEI2, [ 111 In]In-MDPEI4, [ 111 In]In-MD, [ 111In-In-MDPEG2, 111 In-In-MDPEG4, and 111 In-In-MDPEG48, respectively. The radiochemical yields (RCY) of each labeling stage in the two-stage labeling are calculated in the form of the percentage of radioactivity after purification, relative to the radioactivity of the radioisotope added at each stage. 111 In-In-TMDPEI2, 111 In-In-TMDPEI4, 111 In-In-TMD, 111 In-In-TMDPEG2, 111 In-In-TMDPEG4, and 111 In-In-TMDPEG48. The radiochemical yields (RCY) of each labeling stage in the two-stage labeling are calculated in the form of the percentage of radioactivity after purification, relative to the radioactivity of the radioisotope added at each stage. 111 In the form of the percentage of radioactivity of the In-labeled antibody agent. In addition, the total radiochemical yield is calculated in the form of the percentage of radioactivity of the In-labeled antibody agent. 111 In the form of the percentage of radioactivity of the In-labeled antibody agent. In addition, the total radiochemical yield is calculated in the form of the percentage of radioactivity of the In-labeled antibody agent. 111 In the form of the percentage of radioactivity of the In-labeled antibody agent. In addition, the total radiochemical yield is calculated in the form of the percentage of radioactivity of the In-labeled antibody agent.

[0445] Example 2-1 (In-In-TMDPEI2) 111 The RCY of the first stage of In-In-TMDPEI2 was 59.1%, the RCY of the second stage was 73.5%, and the total radiochemical yield was 37.8%.

[0446] Example 2-2 (In-In-TMDPEI4) 111 The RCY of the first stage of In-In-TMDPEI4 was 65.1%, the RCY of the second stage was 49.3%, and the total radiochemical yield was 26.7%.

[0447] Example 2-3 (In-In-TMDPEG48) 111 The RCY of the first stage of In-In-TMDPEG48 was 76.1%, the RCY of the second stage was 65.7%, and the total radiochemical yield was 46.7%.

[0448] Comparative Example 2-1 (In-In-TMD) 111 The RCY of the first stage of In-In-TMD was 80.4%, the RCY of the second stage was 25.8%, and the total radiochemical yield was 20%.

[0449] Comparative Example 2-2 (In-In-TMDPEG2) 111 The RCY of the first stage of In-In-TMDPEG2 was 75.8%, the RCY of the second stage was 31.4%, and the total radiochemical yield was 23.8%.

[0450] Comparative Example 2-3 (In-In-TMDPEG4) 111The RCY of the first stage and the second stage of the PEG-1-[In]In-TMDPEG4) were 69.6%, 40.9%, and the total radiochemical yield was 28.5%.

[0451] Each antibody agent is synthesized through a two-stage labeling reaction. 111 The radiochemical purity of the In-labeled antibody preparations exceeded 95%.

[0452] All animal experiments were conducted in accordance with the guidelines of the Animal Experimentation Committee of Kyoto University. Female BALB / c-nu / nu mice (5 weeks old) were purchased from Shimizu Laboratory Materials Co., Ltd. The animals were housed under a 12-hour / 12-hour day / night cycle and had free access to feed and water.

[0453] HER2-positive ovarian cancer cell line SK-OV-3 was purchased from American Type Culture Collection, Inc. The cells were cultured in RPMI-1640 (manufactured by Nacalai Tesque, Inc.) containing antibiotics (penicillin and streptomycin) (100 U / mL) and 10% by mass heat-inactivated fetal bovine serum (FBS) at 37°C and 5% by volume CO2.

[0454] <Evaluation of Immunoreactivity> (Examples 3-1 to 3-3 and Comparative Examples 3-1 to 3-3)

[0455] SK-OV-3 cells (0.63, 1.25, 2.5, 5.0×10 6 cells) were suspended in culture medium (300 μL). 111 In-labeled antibody agent (0.74kBq) was incubated while mixing inverted for 1 hour. Non-specific binding was evaluated by adding an excess of trastuzumab (50μg). After incubation, the cell suspension was centrifuged at 860×g for 3 minutes, and the supernatant was removed. The cell pellet was resuspended with phosphate-buffered saline (PBS) containing 1% BSA by mass, and centrifuged again at 860×g for 3 minutes, and the supernatant was removed. After repeating the washing with PBS, the radioactivity of the cell pellet was measured using a Gamma Counter 2480WIZARD2 manufactured by PerkinElmer Co., Ltd.

[0456] The immunoreactive fraction of each radiolabeled antibody agent was calculated as reported (J. Immunol. Methods, 1984, 72, 77-89). In detail, for each antibody agent, a scatter plot was prepared with the reciprocal of the cell concentration (mL / million) on the abscissa and the reciprocal of the specific binding rate (total applied / specific binding) on the ordinate, and the reciprocal of the intercept of the approximate straight line was calculated to give the immunoreactive fraction. The results were as follows.

[0457] Example 3-1 (In-In-TMDPEI2) 111 Example 3-2 (In-In-TMDPEI4) 111 Example 3-3 (In-In-TMDPEG48) 111

[0458] Comparative Example 3-1 (In-In-TMD) 111 Comparative Example 3-2 (In-In-TMDPEG2) 111 Comparative Example 3-3 (In-In-TMDPEG4) 111

[0459] Each agent showed good HER2 binding, and no decrease in the targeting binding of the antibody was observed after the two-stage labeling reaction.

[0460] <Intracellular internalization experiment> (Examples 4-1 to 4-3 and Comparative Examples 4-1 to 4-3)

[0461] A suspension of SK-OV-3 cells (1 mL) was prepared. 111 The In-labeled antibody agent (148 kBq / 10 6 cells) was added to the suspension and incubated at 4°C for 1 hour. After the incubation, the cells were washed with PBS containing 1 mass% BSA according to the method described in the <Evaluation of immunoreactivity>. The cell pellet was resuspended with fresh medium to become 1.0 x 10 6 ​​The cells were adjusted and dispensed in a cell-specific manner and incubated at 37°C in a 5% CO2 atmosphere for 0, 1, 2, 4, or 24 hours. Then, centrifugal separation was performed at 860 x g for 3 minutes, and the supernatant was recovered as a medium fraction. Also, the cell pellets were washed twice with PBS containing 1% by mass of BSA, and the total supernatant was combined and recovered as a medium fraction. For the purpose of separating the radioactive component bound to the cell surface and the radioactive component present inside the cell, the cell pellets were resuspended with a 0.2 mol / L sodium citrate buffer (300 μL) at pH 2. After incubation at 4°C for 3 minutes, centrifugal separation was performed at 860 x g for 3 minutes, and the supernatant was recovered as a cell surface fraction. Also, the cell pellets were washed again with the sodium citrate buffer, and the supernatant was combined and recovered as a cell surface fraction, and the remaining cell pellets were recovered as an intracellular fraction. For each fraction, the radioactivity was measured using a gamma counter.

[0462] For the medium fraction after incubation for 24 hours, size-based separation was performed using Amicon Ultra-4 (recovery fraction 50 kDa) to separate the radioactive component from the dissociated radioactive antibody agent from the cell surface and the radioactive component from the cell metabolite. The concentrate was recovered as a dissociation fraction, and the radioactivity was measured using a gamma counter. In addition, the radioactivity remaining in the Amicon was the radioactivity of the metabolite fraction. For the significance test in the intracellularization experiment, one-way analysis of variance and Bonferroni's post-hoc test were combined at a 95% confidence interval.

[0463] The radioactivity percentages of each fraction are shown in Tables 6 to 11. In Tables 6 to 11, "N.D." indicates that data were not obtained. In addition, for the radioactivity percentages of the medium fraction (medium) and the intracellularization fraction (internalization), Figure 1 The results are summarized in Table 12. Note that for each radioactive antibody agent, the proportion of the radioactivity of the recovered fraction to the total radioactivity bound to the cell was recorded as the radioactivity percentage. Regarding the intracellularization fraction, TMDPEI2 / 4 and TMDPEG48 showed higher values than TMD and TMDPEG2 / 4 after incubation for 24 hours, indicating high intracellular retention. Regarding the medium fraction, TMDPEI2 / 4 showed a value about twice as low as TMD and TMDPEG2 / 4, and TMDPEG48 showed a slightly low value. In addition, regarding the ratio obtained by dividing the radioactivity of the intracellularization fraction by the radioactivity of the medium fraction, TMDPEI2 / 4, TMDPEG48, TMDPEG2 / 4, and TMD were in that order from the agent with the high ratio after incubation for 24 hours.

[0464] Regarding the radioactivity percentages of the dissociation fraction (dissociation) and the metabolite fraction (degradation),Figure 2 The results were summarized. No significant difference was observed among the antibody drugs with respect to the amount of radioantibody drug dissociated from the cells. With respect to the radioactivity from the metabolized radioantibody drug, TMDPEI2 / 4 and TMDPEG48 showed lower values compared to TMD and TMDPEG2 / 4. In addition, with respect to the ratio of the internalization rate to the proportion of metabolites outflowing to the outside of the cells 24 hours after incubation (internalization / degradation), it was TMDPEI2 / 4, TMDPEG48, TMDPEG2 / 4, and TMD in order from the drug with the high ratio.

[0465] [Table 6]

[0466]

[0467] [Table 7]

[0468]

[0469] [Table 8]

[0470]

[0471] [Table 9]

[0472]

[0473] [Table 10]

[0474]

[0475] [Table 11]

[0476]

[0477] Preparation of tumor xenograft model mice

[0478] SK-OV-3 cells (1.0 x 10 6 or 4.0 x 10 6 cells / mouse) were suspended in a mixture of RPMI-1640 and Matrigel (1:1, 100 μL) manufactured by Corning Life Sciences, and transplanted subcutaneously into the right flank of female BALB / c-nu / nu mice (5 weeks old) under isoflurane anesthesia. The mice were reared for 3 to 5 weeks, and mice in which the tumor diameter reached 0.5 cm were used for the in vivo radioactivity distribution experiment, and mice in which the tumor diameter reached 0.8 cm were used for the SPECT / CT imaging described later.

[0479] In vivo radioactivity distribution experiment using tumor xenograft model mice (Examples 5-1 to 5-3 and Comparative Examples 5-1 to 5-3)

[0480] The solution containing 111 In-labeled antibody agents (315-333 kBq, 18 μg) in physiological saline solution (100 μL) were administered from the tail vein to SK-OV-3 tumor-transplanted model mice, and the mice were euthanized at 4, 24, 48, 72, 96, or 192 hours after the administration (n = 4 each). The blood and each organ were recovered, and the weight and radioactivity of the organ were measured. The value obtained by dividing the percentage of radioactivity (%ID) relative to the administered radioactivity by the weight of the blood or the weight of the organ (g) (%ID / g) was calculated as an index indicating the in vivo radioactivity distribution. The results thereof are shown in Tables 12 to 17 for each of the radioactive antibody agents administered to the mice.

[0481] In addition, based on the results of the in vivo radioactivity distribution experiment without decay correction, the area under the curve (AUC) of the blood and the tumor was calculated using the GraphPad Prism software. For the significance test of the in vivo radioactivity distribution experiment, a post-hoc test combining one-way analysis of variance and the Bonferroni method was performed at a 95% confidence interval. The results thereof are shown in Figure 3 .

[0482] [Table 12]

[0483]

[0484] [Table 13]

[0485]

[0486] [Table 14]

[0487]

[0488] [Table 15]

[0489]

[0490] [Table 16]

[0491]

[0492] [Table 17]

[0493]

[0494] As shown in Tables 12 to 17, regarding the radioactivity uptake of the tumor, all 111 The In-labeled antibody reached the maximum value at approximately 72 hours after the administration. 111 In] In-TMDPEI2, 111 In] In-TMDPEI4, and 111The tumor accumulation of In]In-TMDPEG48 at 72 hours after administration was 33.5±11.3, 41.3±13.1, and 62.9±20.6% ID / g, which was significantly different from that of [ 111 In]In-TMDPEG2, [ 111 In]In-TMDPEG4, and [ 111 The content of In]In-TMD was higher than that of In-TMD (26.0±4.6, 23.9±2.7, and 24.7±4.2% ID / g).

[0495] like Figure 3 As shown, regarding the AUC in tumors, [ 111 In]In-TMDPEG48 showed the highest value, compared with [ 111 In]In-TMD and [ 111 Compared with In]In-TMDPEG2 / 4, [ 111 In]In-TMDPEI2 / 4 also showed high values.

[0496] <In vivo inhibition experiment using tumor transplantation model mice> (Example 6-1)

[0497] will contain [ 111 SK-OV-3 tumor-transplanted mice were administered a physiological saline solution (100 μL) of In]In-TMDPEI2 (148 kBq) and trastuzumab (2.0 mg / mouse) via the tail vein. Mice were euthanized 72 hours after administration (n=4). Blood and organs were recovered, and organ weights and radioactivity were measured. The significance of the in vivo inhibition experiment was tested using a Student's t-test with a 95% confidence interval. The results are shown in Table 18.

[0498] As shown in Table 18, by co-administration of trastuzumab, [ 111 The tumor accumulation of In]In-TMDPEI2 was significantly reduced (14.8±1.0% ID / g).

[0499] [Table 18]

[0500]

[0501] <SPECT / CT imaging> (Example 7-1)

[0502] will contain [ 111Inl-TMDPEI2(0.7 MBq, 50 μg) in a physiological saline solution (100 μL) was administered to SK-OV-3 tumor-implanted model mice from the tail vein. After 72 hours from the administration, SPECT / CT was performed using a FX3300 pre-clinical imaging system manufactured by Gamma Medica-Ideas Inc. The SPECT image was taken using a pinhole collimator with a diameter of 1.0 mm and a focal length of 75 mm under the conditions of isoflurane anesthesia, a rotation radius of 35 mm, a projection time of 100 seconds, and a number of projections of 32. After the SPECT, CT was performed (tube voltage: 60 kV, tube current: 270 μA). With respect to the projection data of the SPECT, image reconstruction was performed based on the three-dimensional ordered subset expectation maximization (3D-OSEM) method. The resulting SPECT / CT image was analyzed using PMOD software (Version 3.6, PMOD Technologies).

[0503] The results of the SPECT / CT imaging are shown in Figure 4 . 111 Inl-TMDPEI2was able to clearly delineate the tumor, although it partially showed background accumulation from the blood.

[0504] [Chemical Formula 33]

[0505]

[0506] • Synthesis of Compound 33 and its In complex 34 (Examples 1-4)

[0507] <Synthesis of Compound 32>

[0508] Compound 28 (11 mg, 11 μmol) was dissolved in anhydrous DMF (200 μL), COMU (20 mg, 46 μmol) and DIPEA (5.9 mg, 46 μmol) were added at 0°C, and stirring was performed at 0°C for 15 minutes. Then, m-dPEG24-amine (25 mg, 23 μmol) was added, and stirring was performed at room temperature for 14 hours. COSMOSIL C 18 18S column (5C 18- AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 1 mL / min), the solution was purified by reverse phase HPLC to give 12 mg of compound 32 (yield 50%). MS (electrospray ionization method) was performed on the obtained compound 32, and as a result, m / z of the found value was 1018.6 (m / z of the theoretical value was 1018.6442 (C 98 H 185 N7O 36 2+ , [M+2H] 2+ ).

[0509] Synthesis of Maleimide-DOTAGL-PEG24 (MDPEG24) (Compound 33)

[0510] Compound 32 (5.8 mg, 2.9 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.9 mL), thioanisole (60 μL), triisopropylsilane (20 μL), and water (20 μL), and stirred at room temperature for 56 hours. Then, the solvent was distilled off, and the residue was purified by reverse phase HPLC using COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.01% trifluoroacetic acid aqueous solution / 0.01% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 2 (30 min), flow rate 1 mL / min) to give 0.47 mg of compound 33 (yield 9.1%). MS (electrospray ionization method) was performed on the obtained compound 33, and as a result, m / z of the found value was 906.5 (m / z of the theoretical value was 906.0173 (C 82 H 153 N7O 36 2+ , [M+2H] 2+ ).

[0511] Synthesis of Maleimide-[ nat In]In-DOTAGL-PEG24([ nat In]In-MDPEG24) (Compound 34)

[0512] Compound 33 (0.47 mg, 0.26 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL), and indium (III) chloride (0.57 mg, 2.6 μmol) was added, and heated at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. To the supernatant, COSMOSIL C18 Column (5C 18 purification using reverse phase HPLC with a COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.01% trifluoroacetic acid aqueous solution / 0.01% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 2 (30 min), flow rate 1 mL / min), thereby obtaining 0.13 mg of compound 34 (yield 26%). MS (electrospray ionization method) was performed on the obtained compound 34, as a result, m / z of the found value was 647.6 (m / z of the theoretical value was 647.3163 (C 82 H 154 InN8O 36 2+ , [M+3H+NH4] 3+ ))).

[0513] [Chemical Formula 34]

[0514]

[0515] • Synthesis of compound 38 and its In complex 39 (Examples 1-5)

[0516] < Synthesis of compound 35 >

[0517] Compound 2 (306 mg, 1.01 mmol) was added thereto, and stirred at room temperature for 12 hours. To the reaction solution was added water and ethyl acetate, and subjected to liquid separation, and then the organic layer was washed with a saturated aqueous sodium chloride solution, and the recovered organic layer was dried over sodium sulfate, and subjected to filtration. The filtrate was distilled off under reduced pressure, and the residue was purified by silica gel chromatography using a mobile phase (chloroform / methanol = 7 / 3), to obtain 188 mg of compound 35 (yield 35%). MS (electrospray ionization method) was performed on the obtained compound 35, as a result, m / z of the found value was 641.2 (m / z of the theoretical value was 641.3182 (C 33 H 45 N4O9 + , [M+H] +).

[0518] < Synthesis of Compound 36 >

[0519] Compound 35 (9.0 mg, 14 μmol) was dissolved in dry DMF (240 μL), COMU (12 mg, 28 μmol) and DIPEA (3.6 mg, 28 μmol) were added at 0°C, and stirring was performed at 0°C for 15 minutes. Then, m-dPEG48-amine (30 mg, 14 μmol) was added, and stirring was performed at room temperature for 11 hours. Pipedine (60 μL) was added to the reaction solution, and stirring was performed at room temperature for 4 hours, after which the solvent was distilled off under reduced pressure. The residue was purified using a COSMOSIL CAPTO 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minutes) - 3 / 7 (30 minutes), flow rate 4 mL / min) by reverse phase HPLC to obtain 20 mg of Compound 36 (yield 55%). MS (electrospray ionization method) was performed on the obtained Compound 36, and the result showed m / z of 849.2 (m / z of the theoretical value was 849.5193 (C 115 H 232 N5O 54 3+ , [M+3H] 3+ ).

[0520] [Chemical Formula 35]

[0521]

[0522] < Synthesis of Compound 37 >

[0523] Compound 28 (15 mg, 15 μmol) was dissolved in dry DMF (220 μL), COMU (33 mg, 78 μmol) and DIPEA (10 mg, 78 μmol) were added at 0°C, and stirring was performed at 0°C for 15 minutes. Then, Compound 36 (20 mg, 7.8 μmol) was added, and stirring was performed at room temperature for 17 hours. The residue was purified using a COSMOSIL CAPTO 18 column (5C 18-AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 1 mL / min), the solution was purified by reverse phase HPLC to give 11 mg of compound 37 (yield 41%). MS (electrospray ionization) was performed on the obtained compound 37, and as a result, m / z of the found value was 874.0 (m / z of the theoretical value was 874.0411 (C 164 H 315 N 11 O 66 4+ , [M+4H] 4+ )).

[0524] < Synthesis of Maleimide-DOTAGL-PEI2-PEG48 (MDPEI2-PEG48) (Compound 38) >

[0525] Compound 37 (11 mg, 3.2 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (0.4 mL), and stirred at room temperature for 14 hours. Then, the solvent was distilled off, and the residue was purified by reverse phase HPLC using COSMOSIL C 18 18S column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 4 / 1 (0 min) - 3 / 2 (20 min), flow rate 1 mL / min), and then the same column was used again, and purification was performed under the elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 3 / 1 (0 min) - 13 / 7 (20 min), flow rate 1 mL / min to give 2.3 mg of compound 38 (yield 23%). MS (electrospray ionization) was performed on the obtained compound 38, and as a result, m / z of the found value was 776.4 (m / z of the theoretical value was 776.4655 (C 138 H 273 N 13 O 62 4+ , [M+2H+2NH4] 4+ )).

[0526] < Synthesis of Maleimide-[ nat In]In-DOTAGL-PEI2-PEG48([ nat In]In-MDPEI2-PEG48) (Compound 39) >

[0527] Compound 38 (0.5 mg, 0.16 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL), and indium (III) chloride (3.6 mg, 16 μmol) was added, followed by heating at 90°C for 30 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. The supernatant was purified by reverse phase HPLC using a COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (elution conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 4 / 1 (0 minutes) - 1 / 1 (30 minutes), flow rate 1 mL / min), to give 0.14 mg of compound 39 (yield 27%). MS (electrospray ionization method) was performed on the obtained compound 39, and the result showed m / z of 804.3 (m / z of the theoretical value was 804.4356 (C 138 H 270 InN 13 O 62 4+ , [M+3H+2NH4] 4+ ))).

[0528] [Chemical Formula 36]

[0529]

[0530] • Synthesis of compound 44 and its In complex 45 (Examples 1-6)

[0531] < Synthesis of compound 40 >

[0532] Compound 40 was synthesized according to the reported method (Angew. Chem. Int. Ed. Engl., 2017, 56, 9825-9828).

[0533] < Synthesis of compound 41 >

[0534] Compound 40 (285 mg, 0.58 mmol) was dissolved in acetonitrile (20 mL), and compound 26 (1858 mg, 5.2 mmol) and potassium carbonate (722 mg, 5.2 mmol) were added, and stirred at 50°C for 33 hours. Then, compound 26 (600 mg, 1.7 mmol) and potassium carbonate (240 mg, 1.7 mmol) were added to the reaction solution, and further stirred at 50°C for 10 hours. The reaction solution was filtered, and the filtrate was recovered, and the solvent was distilled off under reduced pressure. The residue was purified by an amine-based silica gel chromatography using a mobile phase (hexane / ethyl acetate = 3 / 7) to obtain 351 mg of compound 41 (yield 46%). MS (electrospray ionization) was performed on the obtained compound 41, and the result showed m / z of 1320.8 (m / z of 1320.7630 (C 74 H 106 N5O 16 + , [M+H] + ).

[0535] < Synthesis of Compound 42 >

[0536] Compound 41 (350 mg, 0.27 mmol) was dissolved in methanol (30 mL), and 10% palladium-carbon (140 mg) was added, and stirred vigorously under a hydrogen gas atmosphere at room temperature for 43 hours. Then, filtration was performed using diatomaceous earth, and the solvent of the filtrate was distilled off under reduced pressure. The residue was dissolved in anhydrous DMF (1.0 mL), and 6-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS) (323 mg, 1.1 mmol) and triethylamine (106 mg, 1.1 mmol) were added, and stirred at room temperature for 9 hours. The solution was purified by reverse phase HPLC using a SMOSIL C 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate 4 mL / min) to obtain 114 mg of compound 42 (yield 39%). MS (electrospray ionization) was performed on the obtained compound 42, and the result showed m / z of 1109.6 (m / z of 1109.6592 (C 55 H 93 N6O 17 + , [M+H] + ).

[0537] < Synthesis of Compound 43 >

[0538] Compound 42 (3.1 mg, 2.8 μmol) was dissolved in dry DMF (200 μL), COMU (12 mg, 28 μmol) and DIPEA (3.6 mg, 28 μmol) were added at 00C and stirred for 15 min at 00C. Then, m-dPEG48-amine (30 mg, 14 μmol) was added. Stirred for 24 h at room temperature. The solution was purified by reverse phase HPLC using COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 4 / 1 (0 min) - 1 / 1 (30 min), flow rate 1 mL / min) to give 0.38 mg of compound 44 (yield 66%). MS (electrospray ionization method) was performed on the obtained compound 44, and as a result, the found value of m / z was 808.3 (theoretical value of m / z was 808.3719 (C 346 H 686 N9O 158 9+ ,

[0539] [M+9H] 9+ ))).

[0540] [Formula 37]

[0541]

[0542] Synthesis of Maleimide-DOTA-PEG48-3 (MDPEG48-3) (Compound 44)

[0543] Compound 43 (0.59 mg, 79 nmol) was dissolved in trifluoroacetic acid (2.0 mL) and stirred for 84 h at room temperature. Then, the solvent was distilled off and the residue was purified by reverse phase HPLC using COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and mobile phase (elution condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 4 / 1 (0 min) - 1 / 1 (30 min), flow rate 1 mL / min) to give 0.38 mg of compound 44 (yield 66%). MS (electrospray ionization method) was performed on the obtained compound 44, and as a result, the found value of m / z was 808.3 (theoretical value of m / z was 808.3719 (C 330 H 654 N9O 158 9+ , [M+9H] 9+ )).

[0544] <Maleimide-[ nat In]In-DOTA-PEG48-3([ nat Synthesis of In]In-MDPEG48-3) (Compound 45)

[0545] Compound 44 (0.46 mg, 63 nmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.1, 100 μL), indium (III) chloride (1.4 mg, 6.3 μmol) was added, and heated at 90°C for 30 minutes. The reaction solution was then centrifuged to remove insoluble matter. 18 Column (5C 18 -AR-II, 4.6mmI.D.×150mm) and mobile phase (development conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 4 / 1 (0 min) to 1 / 1 (30 min), flow rate 1 mL / min), the supernatant was purified by reverse phase HPLC to obtain 0.05 mg of compound 45 (yield 11%). MS (electrospray ionization method) of the obtained compound 45 was performed, and the measured m / z was 671.7 (theoretical m / z was 671.7493 (C 330 H 653 InN9O 158 11+ 、[M+12H] 11+ )).

[0546] Synthesis of radioactive antibody drugs (Examples 2-4 to 2-6)

[0547] <Labeling reaction>

[0548] <Stage 1 Marking>

[0549] Compound 33 (1.0 mg / mL DMSO solution, 4 μL) was dissolved in 0.1 mol / L acetate buffer (pH 5.8, 450 μL) and [ 111 In] InCl3 solution (400 μL, 32.3 MBq) was added and heated at 90°C for 5 minutes. COSMOSIL C 18 Column (5C 18 -AR-II, 4.6 mm I.D. × 150 mm) and a mobile phase (H2O / MeCN / TFA [80 / 20 / 0.1 (0 min) to 50 / 50 / 0.1 (30 min)], flow rate 1.0 mL / min), the reaction solution was purified by reverse-phase HPLC, and the solvent containing the component of the target compound was distilled off under a stream of argon.

[0550] Compound 38 (1.0 mg / mL DMSO solution, 15 μL) was dissolved in 0.1 mol / L acetate buffer (pH 5.5, 500 μL) and [ 111 In] InCl3 solution (450 μL, 31.5 MBq) was added and heated at 90°C for 6 minutes. COSMOSIL C 18 Column (5C 18 -AR-II, 4.6 mm I.D. × 150 mm) and a mobile phase (H2O / MeCN / TFA [75 / 25 / 0.1 (0 min) ~ 55 / 45 / 0.1 (20 min)], flow rate 1.0 mL / min), the reaction solution was purified by reverse-phase HPLC, and the solvent containing the component of the target compound was distilled off under a stream of argon.

[0551] Compound 44 (2.0 mg / mL DMSO solution, 55 μL) was dissolved in 0.1 mol / L acetate buffer (pH 5.8, 300 μL) and [ 111 In] InCl3 solution (400 μL, 32.4 MBq) was added and heated at 90°C for 7 minutes. COSMOSIL C 18 Column (5C 18 -AR-II, 4.6 mm I.D. × 150 mm) and a mobile phase (H2O / MeCN / TFA [80 / 20 / 0.1 (0 min) to 50 / 50 / 0.1 (30 min)], flow rate 1.0 mL / min), the reaction solution was purified by reverse-phase HPLC, and the solvent containing the component of the target compound was distilled off under a stream of argon.

[0552] In this manual, 111 In 3+ The radiolabeled compounds obtained by coordinating to compounds 33, 38 and 44 are respectively called [ 111 In]In-MDPEG24, [ 111 In]In-MDPEI2-PEG48 and [ 111 In]In-MDPEG48-3.

[0553] <Stage 2 Marking>

[0554] [ 111 In]In-MDPEG24 and [ 111In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. XL column (5 μm, 7.8 mm I.D. x 300 mm) and a mobile phase (0.1 mol / L phosphate buffer (pH 6.8), flow rate 0.5 mL / min) was analyzed by size exclusion HPLC, and the percentage of the area value of the radiolabeled compound relative to the area value of all peaks detected was taken as RCP (%). XL column (5 μm, 7.8 mm I.D. x 300 mm) and a mobile phase (0.1 mol / L phosphate buffer (pH 6.8), flow rate 0.5 mL / min) was analyzed by size exclusion HPLC, and the percentage of the area value of the radiolabeled compound relative to the area value of all peaks detected was taken as RCP (%).

[0555] In this specification, the radiopharmaceuticals obtained by introducing 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111 In]In-MDPEI2-PEG48, 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added, and then trastuzumab-SH (0.6 mg) was added, followed by incubation for 9 to 10 hours in a dark room at room temperature. Thereafter, the reaction solution was purified and concentrated using Amicon Ultra-4 (recovery fraction 50 kDa), to obtain a target radiopharmaceutical. In addition, the radiopharmaceutical obtained in the first stage of labeling was used as a starting material for the second stage of labeling. 111In the form of percentage of radioactivity of the labeling agent. In addition, the total radiochemical yield (Total RCY) was calculated in terms of the radioactivity of the [In]InCl3 solution added at the time of labeling in the first stage 111 In the form of percentage of radioactivity of the labeling agent. In addition, the total radiochemical yield (Total RCY) was calculated in terms of the radioactivity of the [In]InCl3 solution added at the time of labeling in the first stage 111 In the form of percentage of radioactivity of the labeling agent. In addition, the total radiochemical yield (Total RCY) was calculated in terms of the radioactivity of the [In]InCl3 solution added at the time of labeling in the first stage

[0556] Example 2-4 ([ 111 The RCY of [In]In-TMDPEG24) in the first stage was 61.4%, the RCY in the second stage was 67.4%, and the total radiochemical yield was 23.6%.

[0557] Example 2-5 ([ 111 The RCY of [In]In-TMDPEI2-PEG48) in the first stage was 24.6%, the RCY in the second stage was 33.6%, and the total radiochemical yield was 5.6%.

[0558] Example 2-6 ([ 111 The RCY of [In]In-TMDPEG48-3) in the first stage was 44.4%, the RCY in the second stage was 21.5%, and the total radiochemical yield was 8.3%, and each antibody agent was synthesized by undergoing the two-stage labeling reaction. 111 The radiochemical purity of each of the [In]In-labeled antibody agents exceeded 95%.

[0559] Evaluation of immunoreactivity (Examples 3-4 to 3-6)

[0560] Using the same method as in Examples 3-1 to 3-3, the immunoreactive fraction of each of the radiolabeled antibody agents was calculated. The results were as follows.

[0561] Example 3-4 ([ 111 The result of [In]In-TMDPEG24) was 0.98 ± 0.09, Example 3-5 ([ 111 The result of [In]In-TMDPEI2-PEG48) was 0.84 ± 0.01, and Example 3-6 ([ 111 The result of [In]In-TMDPEG48-3) was 0.86 ± 0.03. Each of the agents showed good HER2 binding, and no decrease in the targeting binding of the antibody was observed after undergoing the two-stage labeling reaction.

[0562] Cellular internalization experiment (Examples 4-4 to 4-6)

[0563] Using the same method as in Examples 4-1 to 4-3, a cellular internalization experiment was performed.

[0564] The radioactivity of the dissociation fraction and the metabolic fraction after 24 hours was measured by the same method as in Example 4, and the results are shown in Tables 19 to 21 as the radioactivity percentage for each radioantibody agent with respect to the medium fraction and the internalization fraction. Note that the proportion of the radioactivity of the recovered fraction with respect to the total radioactivity bound to the cells is expressed as the radioactivity percentage for each radioantibody agent. With respect to the internalization fraction, TMDPEG24, TMDPEI2-PEG48, and TMDPEG48-3 all showed high values after 24 hours of incubation, indicating high intracellular retention. With respect to the medium fraction, each radioantibody agent showed equivalent values. In addition, no significant difference was observed in the ratio of the radioactivity of the internalization fraction to that of the medium fraction among the radioantibody agents, indicating that all the radioantibody agents had a strong cell retention tendency.

[0565] With respect to the radioactivity percentage of the dissociation fraction and the metabolic fraction, a tendency was observed that the amount of the radioantibody agent dissociated from the cells increased as the molecular weight increased. On the other hand, a tendency was observed that the radioactivity from the metabolized radioantibody agent decreased as the molecular weight increased. In addition, a tendency was observed that the larger the molecular weight of the radioantibody agent, the higher the ratio of the internalization rate after 24 hours of incubation to the proportion of the metabolite that flowed out to the outside of the cell showed. The above results indicate that the increase in the molecular weight improves the radioactivity retention, on the other hand, promotes the dissociation of the radioantibody agent from the cell surface, and the combination of the functional unit having a positive charge and the functional unit having a high molecular weight, the combination of a plurality of functional units having a high molecular weight do not show a synergistic effect.

[0566] [Table 19]

[0567]

[0568] [Table 20]

[0569]

[0570] [Table 21]

[0571]

[0572] <In vivo radioactivity distribution experiment using tumor transplantation model mice> (Examples 5-4 to 5-6)

[0573] Model mice were prepared by the same method as in Examples 5-1 to 5-3, and an in vivo radioactivity distribution experiment was performed. The radioactivity of 111 In]In-TMDPEG24 (148 kBq, 18 μg), [ 111In]In-MDPEI2-PEG48 (111 kBq, 18 μg), and [ 111 In] In-TMDPEG48-3 (148 kBq, 18 μg). The results are shown in Tables 22 to 24 for each radioactive antibody agent administered to mice.

[0574] In addition, based on the results of the in vivo radioactivity distribution experiment without attenuation correction, the area under the curve (AUC) of blood and tumor was calculated using GraphPadPrism software. The results are shown in Figure 5 .

[0575] [Table 22]

[0576]

[0577] [Table 23]

[0578]

[0579] [Table 24]

[0580]

[0581] As shown in Tables 22 to 24, regarding the radioactivity uptake of tumors, all 111 In-labeled antibodies all reached their maximum values ​​in the late period of administration. 111 In]In-TMDPEG24 was 34.3±7.4%ID / g 192 hours after administration, [ 111 In]In-MDPEI2-PEG48 was 23.3±3.3%ID / g 96 hours after administration, and [ 111 In]In-TMDPEG48-3 was 32.3±7.1%ID / g 72 hours after administration. In addition, regarding the radioactivity in the blood, 24 hours after administration, [ 111 In]In-TMDPEG24 was 21.1±0.3% ID / g, and [ 111 In]In-TMDPEG48-3 was 24.4±2.5%ID / g, in contrast, [ 111 In]In-TMDPEI2-PEG48 is 16.2±3.1% ID / g.

[0582] like Figure 5 As shown, all radioantibody agents showed similar AUC in tumors compared to [ 111 In]In-TMD equivalent value. Regarding AUC in blood, [ 111In]In-TMDPEI2-PEG48 showed slightly lower values ​​compared to the other two RICs. In terms of tumor-to-blood ratio, none of the RICs showed a value higher than [ 111 In]In-TMDPEG48 values, from high RIC onwards, are [ 111 In]In-TMDPEG24, [ 111 In]In-TMDPEI2-PEG48, [ 111 In]In-TMDPEG48-3.

[0583] [Chemical Formula 38]

[0584]

[0585] Synthesis of Compound 49 and its In complex 50 (Examples 1-7)

[0586] <Synthesis of Compound 46>

[0587] Compound 27 (1.25 g, 1.26 mmol) was dissolved in methanol (12 mL), 10% palladium carbon (624 mg) was added, and the mixture was vigorously stirred at room temperature for 20 hours under a hydrogen atmosphere. Then, the mixture was filtered using celite, and the solvent in the filtrate was distilled off. COSMOSIL C 18 Column (5C 18 -AR-II, 10mmI.D.×250mm) and mobile phase (development conditions 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) to 3 / 7 (30 minutes), flow rate 4mL / min), the residue was purified by reverse phase HPLC to obtain 200mg of compound 46 (yield 21%). HRMS (electrospray ionization method) of the obtained compound 46 showed that the measured m / z was 772.5426 (theoretical m / z was 772.5431 (C 39 H 74 N5O 10 + 、[M+H] + )) In addition, the identification data of the obtained compound 46 based on NMR are shown below. 1 HNMR (400MHz, CD3OD) δ4.53-2.55(m, 26H), 1.86-1.25(m, 44H).

[0588] <Synthesis of Compound 47>

[0589] Compound 46 (200 mg, 0.26 mmol) was dissolved in dry DMF (1 mL), 4-azidobutyric acid succinimidyl ester (88 mg, 0.39 mmol) and triethylamine (52 mg, 0.52 mmol) were added, and stirred at room temperature for 7 hours. After distilling off the solvent, the residue was purified by reverse phase HPLC using a COSMOSIL CH 18 column (5C 18 -AR-II, 10 mm I.D. x 250 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 7 / 3 (0 min) - 1 / 9 (30 min), flow rate 4 mL / min) to give 84 mg of compound 47 (yield 37%). HRMS (electrospray ionization) of the obtained compound 47 gave found m / z 883.5862 (theoretical m / z 883.5863 (C 43 H 79 N8O 11 + , [M+H] + ). In addition, the following shows NMR-based identification data of the obtained compound 47. 1 HNMR (400 MHz, DMSO-d6) δ 4.63-2.40 (m, 28H), 2.16-2.07 (m, 2H), 1.82-1.27 (m, 46H).

[0590] < Synthesis of Compound 48 >

[0591] Compound 48 was synthesized according to the reported method (FEBS Lett., 2000, 482, 91-96).

[0592] < Synthesis of Azido-DOTA Cathepsin B-substrate (ADCS) (Compound 49) >

[0593] Compound 47 (6.9 mg, 9.0 μmol) was dissolved in dry DMF (150 μL), COMU (7.6 mg, 27 μmol) and DIPEA (3.5 mg, 27 μmol) were added at 0°C and stirred for 20 minutes at 0°C. Then, compound 48 (6.5 mg, 9.0 μmol) was added and stirred for 24 hours at room temperature. The solvent was distilled off, the residue was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (400 μL) and stirred for 24 hours at room temperature. Then, the solvent was distilled off and the residue was purified by reverse phase HPLC using a COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.01% trifluoroacetic acid aqueous solution / 0.01% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 1 / 9 (40 minutes), flow rate 1 mL / min) to give 0.50 mg of compound 49 (yield 4.2%). HRMS (electrospray ionization) of the obtained compound 49 was performed and the result gave m / z of 1308.7157 (m / z of 1308.7158 (C 58 H 98 N 15 O 19 + , [M+H] + ).

[0594] nat In]In-DOTA- Cathepsin B-substrate([ nat In]In-ADCS) (compound 50)

[0595] Compound 49 (0.20 mg, 0.15 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.2, 150 μL), indium (III) chloride (0.34 mg, 1.5 μmol) was added and heated for 20 minutes at 90°C. Then, the reaction solution was centrifuged to remove insoluble matter. The supernatant was purified by reverse phase HPLC using a COSMOSIL PBr column (3PBr, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.01% trifluoroacetic acid aqueous solution / 0.01% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 1 / 9 (40 minutes), flow rate 1 mL / min) to give 0.20 mg of compound 50 (yield 94%). HRMS (electrospray ionization) of the obtained compound 50 was performed and the result gave m / z of 1420.5953 (m / z of 1420.5962 (C 58 H 98 InN 15 O 19 ​+ , [M+2H] + )).

[0596] [Chemical Formula 39]

[0597]

[0598] • Synthesis of compound 51 and its In complex 52 (Comparative Examples 1-4)

[0599] < Synthesis of azido-DOTA (AD) (compound 51) >

[0600] Compound 47 (3.6 mg, 4.0 μmol) was dissolved in trifluoroacetic acid (1 mL) and stirred at room temperature for 71 hours. Then, the solvent was distilled off, and the residue was purified by reverse phase HPLC using a COSMOSIL C 18 column 5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) - 1 / 9 (40 min), flow rate 1 mL / min) to give 0.44 mg of compound 51 (yield 16%). HRMS (electrospray ionization) of the obtained compound 51 was performed, and the result showed m / z of 659.3358 (m / z of 659.3359 (C 27 H 47 N8O 11 + , [M+H] + ). In addition, the following shows NMR-based identification data of the obtained compound 51. 1 HNMR (500 MHz, CD3CN) δ 7.93 (s, 1H), 3.3 (t, J = 13.5 Hz, 2H), 2.90-2.39 (m, 16H), 2.22-2.15 (m, 2H), 2.08-2.05 (m, 2H), 1.94-1.62 (m, 16H), 1.52-1.39 (m, 2H).

[0601] < Synthesis of azido-[ nat In] In-DOTA([ nat In] In-AD) (compound 52) >

[0602] Compound 51 (0.40 mg, 0.61 μmol) was dissolved in acetic acid buffer (1.0 mol / L, pH 5.2, 200 μL), and indium (III) chloride (1.3 mg, 6.1 μmol) was added, and the mixture was heated at 90°C for 20 minutes. Then, the reaction solution was centrifuged to remove insoluble matter. The supernatant was purified using COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (developing condition 0.1% trifluoroacetic acid aqueous solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 minute) - 1 / 9 (40 minutes), and the supernatant was purified by reverse phase HPLC to give 0.26 mg of compound 52 (55%). HRMS (electrospray ionization) of the obtained compound 52 was performed, and the found value of m / z was 771.2162 (theoretical value of m / z was 771.2163 (C 27 H 44 InN8O 11 + , [M+2H] + )).

[0603] • Synthesis of radiolabeled antibody agents (Examples 2-7 and Comparative Examples 2-4)

[0604] < Introduction of a dibenzocyclooctyne (DBCO) group to trastuzumab >

[0605] Herceptin (4.0 mg, Roche) was dissolved in PBS (4 mL), and purified and concentrated using a Millipore Amicon Ultra-4 (recovery fraction 30 kDa). To the concentrated trastuzumab in PBS solution (1.0 mg, 50.2 μL, 6.8 nmol) was added a DMSO solution of DBCO-PEG4-NHS ester (6.76 μL, 67.6 nmol) and PBS (78.2 μL), and incubated for 1 hour in the dark. The reaction solution was purified using a Zeba desalting spin column (40K MWCO, 0.5 mL), and the obtained filtrate was further purified using an Amicon Ultra-4 (recovery fraction 50 kDa), and further purified using a Zeba desalting spin column (40K MWCO, 0.5 mL), to give trastuzumab-DBCO having a DBCO group introduced thereto. The purified trastuzumab-DBCO was promptly used for a labeling reaction.

[0606] The number of DBCO groups introduced per antibody molecule was calculated according to existing reports (Bioconjug. Chem., 2016, 27, 217-225). The absorbance of the trastuzumab-DBCO solution at 280 nm and 309 nm was measured, and the molar absorption coefficient of trastuzumab at 280 nm was set to 218 mmol / L. -1 cm -1 , the molar absorption coefficient of DBCO at 309 nm is set to 12 mmol / L - 1 cm -1 The concentrations of trastuzumab and DBCO were calculated. The number of DBCO groups introduced per antibody molecule was calculated by dividing the DBCO concentration by the antibody concentration. The number of DBCO groups introduced per antibody molecule was 4.3 ± 0.3.

[0607] <Labeling reaction>

[0608] <Stage 1 Marking>

[0609] Compounds 49 and 51 (1.0 mg / mL DMSO solution, 1-2 μL) were each dissolved in 0.1 mol / L acetate buffer (pH 5.9, 80 μL), and [ 111 In] InCl3 solution (70 μL, 5.0 MBq) was added and heated at 90°C for 20 minutes. COSMOSIL C 18 Column (5C 18 -AR-II, 4.6mmI.D.×150mm) and mobile phase (development conditions of the reaction solution of compound 49: H2O / MeCN / TFA [80 / 20 / 0.1 (0 minute) ~ 50 / 50 / 0.1 (30 minutes)], development conditions of the reaction solution of compound 51: H2O / MeCN / TFA [92 / 8 / 0.1], flow rate 1.0mL / min), the reaction solution was purified by reverse phase HPLC, and the solvent containing the components of the target compound was distilled off under a flow of argon.

[0610] In this manual, 111 In 3+ The radiolabeled compounds obtained by coordinating to compounds 49 and 51 are respectively called [ 111 In]In-ADCS and [ 111 In]In-AD.

[0611] <Stage 2 Marking>

[0612] After adding 0.1 mol / L phosphate buffer (pH 6.7, 300 μL) to the residue obtained in the first stage of labeling, trastuzumab-DBCO (0.6 mg) was added and incubated for 12 hours under a light-shielded environment at room temperature. Then, the reaction solution was purified and concentrated using an Amicon Ultra-4 (recovery fraction 50 kDa) to obtain the target radioactive antibody agent. The radiochemical purity (RCP) was determined as follows. By using TSKgel G3000SW XL The analysis was performed by size exclusion HPLC using a column (5 μm, 7.8 mm I.D. × 300 mm) and a mobile phase (0.1 mol / L phosphate buffer (pH 6.8), flow rate 0.5 mL / min). The percentage of the area value of the radiolabeled compound relative to the area value of all detected peaks was recorded as RCP (%).

[0613] In this specification, [ 111 In]In-ADCS and [ 111 In]In-AD, the RICs are respectively called [ 111 In]In-TADCS and [ 111 In] In-TAD. The radiochemical yield (RCY) of the labeling stage in the two-stage labeling is expressed as the radioactivity of the purified 111 The radioactivity of the In-labeled form was calculated as a percentage. In addition, the total radiochemical yield was expressed as [ 111 In] InCl3 solution radioactivity, the obtained 111 The radioactivity of the In-labeled antibody drug was calculated as a percentage.

[0614] Example 2-7 ([ 111 The RCY of the first stage and the second stage of the 1st stage of the 2nd ...2nd stage of the 1st stage of the 2nd stage of the 1st stage of the 2nd stage of the 2nd stage of the 1st stage of the

[0615] Comparative Example 2-8 ([ 111 The RCY of the first stage and the second stage of the 1st stage of the 2nd stage of the 1st stage of the 2nd stage of the 2nd stage of the 2nd stage of the 3rd stage of the 1st stage of the 2nd stage of the 3rd ...3rd stage of the

[0616] Each antibody agent is synthesized through a two-stage labeling reaction. 111 The radiochemical purity of the In-labeled antibody preparations exceeded 5%.

[0617] <Evaluation of stability in plasma> (Examples 3-7)

[0618] To mouse plasma (200 μL) was added 111 In]In-ADCS in PBS solution (740 kBq, 50 μL) was incubated at 37°C for 0, 24 or 96 hours. Then, MeCN (250 μL) was added, and the suspension was centrifuged at 1500 x g for 5 minutes, and the supernatant was filtered using a Cosmonice Filter S (0.45 μm, 4 mm). The solvent of the filtrate was distilled off under an argon stream, and the radiochemical purity of the residue was analyzed by reverse phase HPLC. Reverse phase HPLC was performed using a COSMOSIL C 18 column (5C 18 -AR-II, 4.6 mm I.D. x 150 mm) and a mobile phase (H2O / MeCN / TFA = 80 / 20 / 0.1 (0 minutes) - 50 / 50 / 0.1 (30 minutes), flow rate 1.0 mL / min).

[0619] The radiochemical purity of [In]In-ADCS was determined at each time point, and the results were 99.0 ± 0.7% at the 0-hour time point, 99.9 ± 0.2% at the 24-hour time point, and 97.5 ± 0.6% at the 96-hour time point. This radiochemical purity can be considered as 111 the stability of [In]In-ADCS. That is, [In]In-ADCS maintained a radiochemical purity of 95% or more for 96 hours, indicating high stability in mouse plasma. 111 111

[0620] <Cathepsin B binding experiment> (Examples 4-7 and Comparative Examples 4-4)

[0621] The cathepsin B binding experiment was performed substantially according to the existing report (J. Nucl. Med., 2020, 61, 443-450).

[0622] ​​As the buffer used in the experiment, an aqueous solution containing sodium acetate at a final concentration of 50 mmol / L and ethylenediaminetetraacetic acid at 1.0 mmol / L, namely storage buffer (pH 6.0), and an aqueous solution containing trishydroxyaminomethane at a final concentration of 5.0 mmol / L, magnesium chloride at 5.0 mmol / L, and dithiothreitol at 2.0 mmol / L, namely binding assay buffer (pH 5.5) were prepared. A storage buffer solution of cathepsin B from human liver (600 nM, 10 μL) was added to a storage buffer (10 μL) containing CA-074 (2 mM), a representative inhibitor of cathepsin B, or not containing CA-074, which was pre-incubated at room temperature for 30 minutes. Then, [ 111 In]In-ADCS or [ 111 In]In-AD binding assay buffer solution (111 kBq, 30 μL). 111 In]In-ADCS or [ 111 The storage buffer (20 μL) was added to the binding assay buffer solution of In]In-AD (111 kBq, 30 μL), but cathepsin B was not added. After incubation at 37°C for 2 hours, Laemmli sample buffer (2X) (50 μL) was added and incubated at room temperature for another 10 minutes. Each reaction solution (15 μL) and Precision Plus Protein TM DualXtra standard (15 μL) was injected into the polyacrylamide gel and subjected to polyacrylamide electrophoresis (SDS-PAGE) at 40 mA for 80 minutes. After the electrophoresis, the gel was removed and [ 111 In] InCl3 solution (1-2 kBq, 0.5 μL). The removed gel was placed in contact with an imaging plate under a light-shielding environment for 20 minutes, and then autoradiography was performed using an Amersham Typhoon scanner.

[0623] The results of the autoradiography are shown in Figure 6 . Figure 6 In [ 111 In the group incubated with In-ADCS, only the group without CA-074 addition observed a band of RI at around 30 kDa. In the group with CA-074 addition, a band was observed at around 10 kDa, which is consistent with the [ 111In]In-ADCS. However, bands were detected at the same position also in the group to which Cathepsin B was not added, and thus it was considered that these bands were derived from unbound[ 111 In]In-ADCS. As for[ 111 In]In-AD, bands were confirmed at a position of 2 kDa or less in the group to which CA-074 was not added and the group to which CA-074 was added. Bands were observed at the same position also in the group to which Cathepsin B was not added, and thus it was considered that these bands were derived from unbound[ 111 In]In-ADCS.

[0624] <Intracellular uptake experiment> (Examples 5-7 and Comparative Examples 5-4)

[0625] Using the same method as in Examples 4-1 to 4-3, an intracellular uptake experiment was performed. As for the medium fraction after incubation for 24 and 48 hours, a separation based on molecular size was performed using Amicon Ultra-4 (recovery fraction 50 kDa) to separate radioactivity from the radioactivity of the metabolic product from the cells. The concentrate was recovered as a dissociation fraction, and the radioactivity was measured using a gamma counter. In addition, the radioactivity remaining in the Amicon was the radioactivity of the metabolic fraction. As for the significance test in the intracellular uptake experiment, a one-way analysis of variance and a post-hoc test of the Bonferroni method were combined to perform a 95% confidence interval. The percentage of radioactivity of each fraction is shown in Table 25 and Table 26. In Table 25 and Table 26, "N.D." indicates that data was not obtained. In addition, as for the percentage of radioactivity of the intracellular uptake fraction (internalization) and the metabolic fraction (degradation), Figure 7 The results are summarized in Table 25 and Table 26. As for the intracellular uptake fraction, the values after incubation for 24 and 48 hours were 111 In]In-TADCS and[ 111 In]In-TAD, showed significantly high values, indicating that the intracellular retention of radioactivity was high. As for the metabolic fraction, the values after incubation for 24 and 48 hours were 111 In]In-TADCS and[ 111 In]In-TAD, showed low values, and in particular after incubation for 48 hours, the difference was significant.

[0626] [Table 25]

[0627]

[0628] [Table 26]

[0629]

[0630] <Intracellular Cathepsin B binding experiment> (Examples 6-7)

[0631] The intracellular Cathepsin B binding experiment was performed substantially according to the previously reported method (J. Nucl. Med., 2020, 61, 443-450). To a suspension of SK-OV-3 cells (1.0 x 10 7 cells / 1 mL) was added PBS (10 μL) containing CA-074Me (1 mM) as a Cathepsin B inhibitor or PBS (10 μL) not containing CA-074Me. Further, [In]In-TADS (740 kBq, 100 μg) was added, and incubation was performed at 37°C for 24 hours. After the incubation, the cell suspension was centrifuged at 860 x g for 3 minutes, and the supernatant was removed. The cell pellet was resuspended with 0.2 M sodium citrate buffer (500 μL) at pH 2. After incubation at 4°C for 3 minutes, centrifugation was performed, and the supernatant containing the radiolabeled antibody agent dissociated from the cell surface was removed. Acid washing was performed again. To the cell pellet was added a solution (1 μL) in which a Protease Inhibitor Cocktail was dissolved in 2 mL of distilled water, and a solution (100 μL) in which RIPA buffer was mixed, and sonication was performed for 1 minute. The suspension was centrifuged at 12000 x g for 5 minutes, and the supernatant was recovered. To the supernatant (40 μL) was added Laemmli sample buffer (2X) (40 μL), and incubation was performed at 85°C for 2 minutes. Each solution (15 μL) and Precision Plus Protein 111 Dual Xtra Standard (15 μL) were injected into a polyacrylamide gel, and SDS-PAGE was performed at 20 mA for 80 minutes. After the electrophoresis, the gel was removed, and a solution (0.1-0.2 kBq, 0.5 μL) of [In]InCl3was added to each band of the molecular weight marker. After the removed gel was contacted with an imaging plate in a light-shielded environment for 7 days, autoradiography was performed using an Amersham Typhoon scanner. TM TM Dual Xtra Standard (15 μL) were injected into a polyacrylamide gel, and SDS-PAGE was performed at 20 mA for 80 minutes. After the electrophoresis, the gel was removed, and a solution (0.1-0.2 kBq, 0.5 μL) of [In]InCl3was added to each band of the molecular weight marker. After the removed gel was contacted with an imaging plate in a light-shielded environment for 7 days, autoradiography was performed using an Amersham Typhoon scanner. 111

[0632] The results of the autoradiography are shown in Figure 8 ​​In the group without the addition of CA-074Me, a band from the radioactivity was confirmed at a position of a molecular weight of 30 kDa, whereas in the group with the addition of CA-074Me, almost no band was confirmed. In addition, in both the group without the addition of CA-074Me and the group with the addition of CA-074Me, radioactivity was detected at a position of a molecular weight of 200 to 250 kDa. Focusing on only the band at the position of 200 to 250 kDa, the resulting results are shown in FIG. 6, with the contrast adjusted. Figure 9 In the group without the addition of CA-074Me, a band from the radioactivity was confirmed at a position of a molecular weight of 30 kDa, whereas in the group with the addition of CA-074Me, almost no band was confirmed. In addition, in both the group without the addition of CA-074Me and the group with the addition of CA-074Me, radioactivity was detected at a position of a molecular weight of 200 to 250 kDa. Focusing on only the band at the position of 200 to 250 kDa, the resulting results are shown in FIG. 6, with the contrast adjusted. 111 In]In-TADCS and cathepsin B.

[0633]

[0634] Model mice were produced in the same manner as in Examples 5-1 to 5-3, and an in vivo radioactivity distribution experiment was performed. 111 In]In-TADCS and cathepsin B. 111 In]In-TAD was administered at 130 kBq, 15 μg of radioactivity. The results are shown in Table 27 and Table 28, respectively, for each radiopharmaceutical administered to the mice. In addition, based on the results of the in vivo radioactivity distribution experiment without decay correction, the area under the curve (AUC) of the blood and the tumor was calculated using GraphPad Prism software.

[0635] [Table 27]

[0636]

[0637] [Table 28]

[0638]

[0639] [ 111 In]In-TADCS and cathepsin B. 111 In]In-TAD exhibited high blood retention and showed similar accumulation tendencies in the tumor and other organs. Regarding tumor accumulation, 111 In]In-TADCS showed a maximum value at 72 hours after administration, 111 In]In-TAD showed a maximum value at 192 hours after administration. The tumor / blood ratio of radioactivity accumulation and the AUC values in the tumor and the blood are shown in Table 29. Figure 10 .

[0640] ​• Synthesis of radiolabeled antibody drugs (Examples 2-8 and 2-9 and Comparative Example 2-5)

[0641] < Introduction of thiol group to humanized SKM9-2 antibody>

[0642] In the present example and comparative example, as the antibody, the humanized SKM9-2 antibody described in Production Example 3 of International Publication No. 2023 / 277144 was used.

[0643] A solution of humanized SKM9-2 antibody (891 μg) in 0.1 mol / L boric acid buffer containing 2 mmol / L EDTA (pH 8.0, 72.1 μL) was prepared. In addition, a 2-iminothiolane hydrochloride solution was prepared by dissolving 2-iminothiolane hydrochloride in 0.1 mol / L boric acid buffer containing 2 mmol / L EDTA (pH 8.0) so that the concentration of 2-iminothiolane hydrochloride was 0.7 mmol / L. Next, the aforementioned 2-iminothiolane hydrochloride solution was added to the aforementioned solution of humanized SKM9-2. The amount of the aforementioned 2-iminothiolane hydrochloride solution to be added was set to an amount in which 2 mol equivalents of 2-iminothiolane hydrochloride with respect to SKM9-2. The reaction solution obtained in this manner was incubated at room temperature in the dark for 1 hour. The reaction solution was purified using a Zeba desalting spin column (40K MWCO, 0.5 mL) to obtain humanized SKM9-2 into which a thiol group was introduced (hereinafter, also referred to as "SKM9-2-SH"). The purified SKM9-2-SH was promptly used in the labeling reaction described below.

[0644] < Labeling reaction>

[0645] To 0.1 mol / L phosphate buffer solutions (pH 7, 500 μL) of [In]In-MDPEI2, 111 In]In-MDPEI2, 111 In]In-MD, and 111 In]In-MDPEG48 prepared in the same manner as in Example 2-1, Comparative Example 2-1, and Example 2-3, respectively, 0.1 mol / L phosphate buffer solutions (pH 7, 100 μL) of SKM9-2-SH (0.4 to 0.5 mg) were added, and incubated at room temperature in the dark for 7.5 to 10 hours. Then, the reaction solutions were purified and concentrated using Amicon Ultra-4 (nominal fraction 50 kDa) to obtain the target radiolabeled antibody drugs. The RCP was determined as follows. By using TSKgel SW3000 XLThe RICs were analyzed by size exclusion HPLC with a column (5 μm, 7.8 mm I.D. x 300 mm) and a mobile phase (0.1 mol / L phosphate buffer (pH 7), flow rate 0.5 mL / min), and the percentage of the area value of the radiolabeled compound relative to the area value of all peaks detected was recorded as RCP (%).

[0646] In the present specification, the RICs obtained by introducing [ 111 In]In-MDPEI2, 111 In]In-MD, and [ 111 In]In-MDPEG48into SKM9-2-SH are referred to as [ 111 In]In-SMDPEI2, 111 In]In-SMD, and [ 111 In]In-SMDPEG48, respectively. The RCY of the labeling reaction was calculated as a percentage relative to the amount of added 111 In]In-SMDPEG48. The radioactivity of the purified RICs was calculated as a percentage relative to the radioactivity of the labeled chelating linker. 111 In]In-SMDPEG48. The radioactivity of the purified RICs was calculated as a percentage relative to the radioactivity of the labeled chelating linker.

[0647] Example 2-8 (RCY of [ 111 In]In-SMDPEI2) was 70.6%, Example 2-9 (RCY of [ 111 In]In-SMDPEG48) was 74.4%, and Comparative Example 2-5 (RCY of [ 111 In]In-SMD) was 60.4%, and the RCP of each of the synthesized RICs exceeded 95%.

[0648] Sialylated HEGl-positive malignant pleural mesothelioma cells, ACC-MESO-4 cells (Cancer Sci 2006; 97: 387-394), were provided by the National Institute for Basic Biology. The cells were cultured in RPMI-1640 containing antibiotics (penicillin and streptomycin) (100 U / mL) and 10 mass% heat-inactivated fetal bovine serum (FBS) (manufactured by Nacalai Tesque, Inc.) at 37°C in a 5% CO2environment.

[0649] Preparation of tumor transplantation model mice

[0650] ACC-MESO-4 cells (2.0 x 10 6 cells / mouse) were suspended in RPMI-1640 (100 μL) and transplanted subcutaneously into the back of male SHO mice (5 weeks old) under isoflurane anesthesia. The mice were reared for 4 to 5 weeks, and mice in which the tumor diameter reached 0.5 cm were used in the following experiments.

[0651] <In vivo radioactivity distribution experiment using tumor transplantation model mice> (Examples 5-8 and 5-9 and Comparative Example 5-5)

[0652] will contain [ 111 In]In-SMDPEI2, [ 111 In]In-SMD, and 111 In] In-SMDPEG48 (148kBq, 20μg) in a physiological saline solution (100μL) was administered to ACC-MESO-4 tumor transplanted mice via the tail vein. The mice were euthanized 4, 24, 48, 72, 96, or 192 hours after administration (n=3-4 each). Blood and organs were recovered, and each weight and radioactivity were measured. As an indicator of the distribution of radioactivity in the body, the percentage (%ID) of radioactivity relative to the administered radioactivity was divided by the blood weight or organ weight (g) to obtain the value (%ID / g). The results are shown in Tables 29 to 31 according to each RIC administered to the mice.

[0653] In addition, the AUCs for blood and tumors were calculated using GraphPad Prism software based on the average of the %ID / g values ​​at each time point without attenuation correction. The results are shown in Table 32.

[0654] [Table 29]

[0655]

[0656] [Table 30]

[0657]

[0658] [Table 31]

[0659]

[0660] [Table 32]

[0661]

[0662] As shown in Tables 29 to 31, regarding the radioactivity uptake of tumors, all 111 In-labeled antibody reached its maximum value in the late administration period. 111 In]In-SMDPEI2 was 25.7±2.2%ID / g 96 hours after administration, [ 111 In]In-SMD was 17.0±1.9%ID / g 96 hours after administration, and [ 111 In]In-SMDPEG48 was 32.3±8.6%ID / g 192 hours after administration.111 In]In-SMDPEI2 and [ 111 The tumor accumulation of In]In-SMDPEG48 at 96 hours after administration was 25.7±2.2%ID / g and 28.5±5.8%ID / g, respectively. 111 Compared with In-SMD, 111 In]In-SMDPEI2 and [ 111 Excellent tumor accumulation was observed for In]In-SMDPEG48.

[0663] As shown in Table 32, regarding the AUC in tumors, [ 111 In]In-SMDPEG48 showed the highest value, [ 111 In]In-SMDPEI2 also showed 111 In]In-SMD compared to the higher value. Regarding the AUC in blood, compared with [ 111 Compared with In-SMD, 111 In]In-SMDPEI2 and [ 111 In]In-SMDPEG48 showed a higher value. In terms of the tumor / blood ratio of AUC, the order from the highest RIC was [ 111 In]In-SMDPEG48, [ 111 In]In-SMDPEI2, and [ 111 In]In-SMD, the same tendency was observed as when each chelating linker was applied to trastuzumab.

[0664] <In vivo inhibition experiment using tumor transplantation model mice> (Examples 6-9)

[0665] will contain [ 111 In]In-SMDPEG48 (148 kBq) and humanized SKM9-2 antibody (2.0 mg / mouse) in physiological saline (100 μL) were administered via the tail vein to ACC-MESO-4 tumor-transplanted mice. Mice were euthanized 72 hours after administration (n=4). Blood and organs were collected, and their weights and radioactivity were measured. The significance of the in vivo inhibition experiment was tested using a Student's t-test with a 95% confidence interval. The results are shown in Table 33.

[0666] [Table 33]

[0667]

[0668] As shown in Table 33, by co-administration of humanized SKM9-2 antibody, [ 111In]In-SMDPEG48 significantly reduced tumor accumulation (4.2 ± 1.9 %ID / g). A significant reduction in blood radioactivity (6.8 ± 0.6 %ID / g) was also observed, and a significant reduction in tumor / blood ratio (0.62 ± 0.25) was observed, showing that 111 Targeting specificity of In-SMDPEG48 in vivo.

[0669] According to the present application, a radioactive metal-labeled antibody capable of improving intracellular accumulation of radioactivity to target cells is provided.

[0670] This application is based on Japanese Patent Application No. 2023-032317 (filed on March 2, 2023) and Japanese Patent Application No. 2023-203279 (filed on November 30, 2023) in Japan, the contents of which are incorporated herein in their entirety.

Claims

1. Radioactive metal-labeled antibodies, which are antibodies labeled with radioactive metals. in, The following functional chelating linker is coupled to the antibody, wherein the functional chelating linker has: a chelating moiety capable of coordinating with a radioactive metal ion; and A functional unit having the function of improving intracellular retention.

2. The radioactive metal-labeled antibody according to claim 1, wherein The functional unit has a structure formed by polymerization of ethyleneimine or ethylene glycol, or a structure capable of binding to cathepsin B.

3. The radioactive metal-labeled antibody according to claim 2, wherein The functional unit has a structure in which ethyleneimine is polymerized at a polymerization degree of 1 to 7.

4. The radioactive metal-labeled antibody according to claim 2, wherein The functional unit has a structure in which ethylene glycol is polymerized at a degree of polymerization of 11 or more and 48 or less.

5. The radioactive metal-labeled antibody according to claim 2, wherein The functional unit has a structure represented by the following formula (c), [Chemical formula 01] In formula (c), X1 and X2 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxyl group, an amino group, or a linking bond. X a and X b Each independently represents O or NH, l represents 0 or 1, When l is 0, Xc represents a hydrogen atom; when l is 1, Xc represents CH2. m and n each independently represent the degree of polymerization of ethyleneimine or ethylene glycol, * indicates a connection key.

6. The radioactive metal-labeled antibody according to claim 5, wherein The functional chelate linker comprises a structure represented by the following formula (d): [Chemical formula 02] In formula (d), o, p, and q each independently represent 0 or 1, and satisfy 1≤o+p+q≤3, Xd represents the structure represented by the formula (c), When o is 0, Xe represents a hydrogen atom, and when o is 1, Xe represents CH2. When p is 0, Xf represents a hydrogen atom, and when p is 1, Xf represents CH2. When q is 0, Xg represents a hydrogen atom, and when q is 1, Xg represents CH2. * is the connection key.

7. The radioactive metal-labeled antibody according to claim 2, wherein The structure capable of binding to cathepsin B is an epoxysuccinyl peptide.

8. The radioactive metal-labeled antibody according to claim 1 or 2, wherein The chelating portion is located between the antibody and the functional unit.

9. The radioactive metal-labeled antibody according to claim 8, wherein The functional chelate linker further comprises an antibody binding unit having a binding portion to the antibody, The chelating portion has a first amino acid residue and a second amino acid residue, The chelating portion binds to the antibody-binding unit via the first amino acid residue, and the chelating portion binds to the functional unit via the second amino acid residue.

10. The radioactive metal-labeled antibody according to claim 1 or 2, wherein The chelating part is DOTA or a derivative thereof.

11. The radioactive metal-labeled antibody according to claim 1 or 2, wherein The radioactive metal is selected from Al 18 F. 64 Cu, 67 Cu, 68 Ga, 89 Zr, 90 Y. 99m Tc, 111 In, 177 Lu and 225 One of the Ac.

12. The radioactive metal-labeled antibody according to claim 1 or 2, wherein The functional chelating linker is introduced into the sulfhydryl group of the antibody.

13. The radioactive metal-labeled antibody according to claim 1 or 2, wherein The antibody is an antibody that recognizes antigens specifically expressed in tumor cells.

14. A radiopharmaceutical comprising the radioactive metal-labeled antibody according to claim 1 or 2 as an active ingredient.

15. The radiopharmaceutical according to claim 14, which is used for diagnosis of cancer or internal radiation therapy of cancer.

16. A compound having in its structure: a chelating portion capable of coordinating with radioactive metal ions; A functional unit having a function of improving intracellular retention; and An antibody binding unit comprising a binding portion to an antibody.

17. The compound according to claim 16, wherein The chelating portion is located between the antibody binding unit and the functional unit.

18. The compound according to claim 16 or 17, wherein The binding portion to the antibody is a maleimide group.

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