Modified antibody, radioactive metal-labeled antibody, and radioactive drug

Abstract

An objective of the present invention is to provide a radiolabeled antibody and a radioactive drug containing the same that have an excellent tumor-to-blood ratio. A modified antibody expressed by formula (I), which is A–C–La–B–(Lb)p–D (where the definition of each symbol is as described in the description), a radioactive metal-labeled antibody in which said modified antibody is labeled with a radioactive metal, and a radioactive drug containing the radioactive metal-labeled antibody as an active ingredient have an excellent tumor-to-blood ratio.

Description

Modified antibodies, radioactive metal-labeled antibodies and radiopharmaceuticals

[0001] The present invention relates to modified antibodies, the modified antibodies labeled with a radioactive metal (radiometal-labeled antibodies), and radiopharmaceuticals containing the same.

[0002] Antibodies have traditionally been widely used in various research and development projects to detect target molecules, and they also play an extremely important role in industry as detection reagents and diagnostic agents. Furthermore, due to their high specificity for target molecules, antibodies have also attracted attention as pharmaceuticals for disease treatment. For example, Patent Document 1 describes an antibody that binds to a glycosylated HEG1 protein obtained from mesothelioma, and includes a mouse antibody (SKM9-2 antibody) as an example. Furthermore, a technique has been reported for site-specific chemical modification to add functionality to an antibody without affecting its specificity for a target molecule, in which an amino acid capable of binding to a crosslinking agent is introduced into a peptide that specifically or selectively binds to an antibody (hereinafter also referred to as an IgG-binding peptide), and the amino acid is modified with the crosslinking agent to prepare a site-specifically modified IgG-binding peptide, and the peptide is then used to modify the antibody (Patent Document 2).

[0003] Radiolabeled antibodies (hereinafter also referred to as "radiolabeled antibodies") are expected to be useful as therapeutic agents in internal radiotherapy and diagnostic agents in PET and the like. For example, ibritumomab tiuxetan is a combination of ibritumomab, an anti-CD20 antibody, and DTPA-modified tiuxetan, and the combination with yttrium-90 has been approved in Japan as a therapeutic agent for CD20-positive lymphoma. In addition, although not approved in Japan, １３１I-Tositumomab has been approved overseas as a therapeutic agent for CD20-positive lymphoma. Furthermore, trastuzumab is a HER2-specific antibody, and has been reported to be labeled with various radionuclides, such as indium-111, lutetium-177, copper-64, zirconium-89, astatine-211, and radioactive iodine. Clinical applications for noninvasive imaging and targeted radiotherapy of tumors overexpressing HER2 are being investigated. Patent Document 3 describes a technique for labeling SKM9-2 humanized antibodies site-specifically modified with a specific peptide with a radioisotope.

[0004] While the clinical application of radiolabeled antibodies is progressing, there are concerns about side effects due to the long blood retention of radiolabeled antibodies themselves. Therefore, there is a need to further improve the accumulation in target cells. For example, the present applicant has attempted to improve tumor accumulation in tumor cells by increasing intracellular retention (Patent Document 4).

[0005] International Publication No. 2017 / 141604 International Publication No. 2017 / 217347 International Publication No. 2023 / 277144 International Publication No. 2024 / 181576 (PCT / JP2024 / 7907)

[0006] The present inventors aimed to obtain a labeled antibody with an improved ratio of radioactivity accumulation in tumor to blood (hereinafter also referred to as the tumor-blood ratio). In light of this problem, they conducted extensive research and succeeded in producing a modified antibody (hereinafter also referred to as the "modified antibody of the present invention") with an improved tumor-blood ratio by site-specifically introducing into the antibody a chelating moiety incorporating a functional unit that improves intracellular retention. Accordingly, one aspect of the present invention is a modified antibody of the present invention, which is represented by the following formula (I): A-C-La-B-(Lb)p-D (I) [wherein, A is an antibody that exhibits binding to a cell surface antigen, wherein the antibody has the ability to be internalized within a cell; B is a chelating moiety capable of coordinating to a radioactive metal ion; C is a peptide that binds to a specific site in the Fc region of the antibody; D is a functional unit that improves intracellular retention; La is a linker a connecting C and B; Lb is a linker b connecting B and D; and p is 0 or 1]. The details of each symbol in formula (I) will be described later. Another aspect of the present invention is a modified antibody represented by formula (I) that is labeled with a radioactive metal (hereinafter also referred to as the "radiometal-labeled antibody of the present invention"). Yet another aspect of the present invention is a radiopharmaceutical comprising the radiometal-labeled antibody of the present invention.

[0007] 1 is a graph showing the results of an evaluation of radioactivity distribution in the body using a tumor-implanted model mouse. The vertical axis indicates the tumor-blood ratio.

[0034] FIG. 1 is a diagram showing the results of PET / CT imaging using a tumor-implanted model mouse.

[0035] The results are from imaging 72 hours after administration of a radioactive metal-labeled antibody. The heart, tumor, and liver are indicated by arrows.

[0036] FIG. 1 is a graph showing the results of a drug efficacy evaluation using a tumor-implanted model mouse. The vertical axis indicates tumor volume.

[0037] FIG. 1 is a diagram showing the results of PET / CT imaging using a tumor-implanted model mouse. The results are from imaging 120 hours after administration of a radioactive metal-labeled antibody. The heart, tumor, and liver are indicated by arrows.

[0038] FIG. 1 shows a structural diagram of one embodiment of a radioactive metal-labeled antibody of the present invention.

[0008] Terms used herein have the meanings commonly used in the art unless otherwise specified. In the following description, when "T to U[V]" (T and U are arbitrary numbers, and [V] is a unit) is written, it means "at least T[V] and at most U[V]" unless otherwise specified. Furthermore, when an asymmetric carbon atom is present in a structure, unless otherwise specified, each may independently be in the S-configuration or the R-configuration. When amino acids are represented in this specification, either the well-known three-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission or one-letter symbols may be used.

[0009] The modified antibody of the present invention comprises an antibody (particularly one that exhibits binding to a cell surface antigen and has the ability to be internalized within a cell), a peptide that binds to a specific site in the Fc region of the antibody, a chelating moiety (hereinafter also simply referred to as a "chelating moiety") capable of coordinating with a radioactive metal ion, a functional unit (hereinafter also simply referred to as a "functional unit") that has the function of improving intracellular retention, and a linker connecting them. Furthermore, the radiometal-labeled antibody of the present invention is obtained by labeling the modified antibody of the present invention with a radiometal. Furthermore, the radiopharmaceutical of the present invention comprises the radiometal-labeled antibody of the present invention.

[0010] In one embodiment, the modified antibody of the present invention has a functional unit and an antibody linked via a chelating moiety. The inclusion of a chelating moiety capable of coordinating to a radioactive metal ion makes it possible to label the modified antibody with a radioactive metal, thereby producing a radiometal-labeled antibody. The modified antibody of the present invention is preferably represented by the following formula (I): A-C-La-B-(Lb)p-D (I) [wherein A is an antibody that exhibits binding to a cell surface antigen, and the antibody has the ability to be internalized within a cell; B is a chelating moiety capable of coordinating to a radioactive metal ion; C is a peptide that binds to a specific site in the Fc region of the antibody; D is a functional unit that improves intracellular retention; La is a linker a connecting C and B; Lb is a linker b connecting B and D; and p is 0 or 1].

[0011] In formula (I), the chelating moiety represented by symbol B is bound to the antibody represented by symbol A via the peptide represented by symbol C, and the peptide represented by symbol C and the chelating moiety represented by symbol B are bound via a linker a represented by symbol La. The peptide represented by symbol C is a peptide that binds to a specific site in the Fc region of the antibody represented by symbol A, and the structure (C-La) consisting of the peptide represented by symbol C and the linker a represented by symbol La may be referred to as an "antibody binding unit" in the present invention. The chelating moiety represented by symbol B is bound to the functional unit represented by symbol D directly (p=0 in formula (I)) or via a linker b represented by symbol Lb (p=1 in formula (I)).

[0012] Matters applicable to each of the above-mentioned embodiments will be described below. When the modified antibody of the present invention is a radioactive metal-labeled antibody, from the viewpoint of sufficiently increasing the affinity between the modified antibody of the present invention and the radioactive metal ion and thereby suppressing the release of the radioactive metal ion from the radioactive metal-labeled antibody before it is taken up by a target cell, it is preferred that in formula (I), the chelating moiety B has a cyclic structure having two or more nitrogen atoms, with the nitrogen atoms being linked to each other via two or more adjacent carbon atoms, or that the chelating moiety B has a chain structure having two or more nitrogen atoms, with the nitrogen atoms being linked to each other via two or more adjacent carbon atoms.

[0013] In the formula (I), when B has a cyclic structure, the skeleton of the cyclic structure may be composed only of nitrogen atoms and carbon atoms, or may be composed of oxygen atoms in addition to nitrogen atoms and carbon atoms. The bonds between the carbon atoms in the cyclic structure may be linear, or may form a cyclic structure. Furthermore, in the formula (I), when B has a chain structure, the bonds between the carbon atoms in the chain structure may be interrupted by a nitrogen atom. The bonds between the carbon atoms in the chain structure may be linear, or may form a cyclic structure.

[0014] Furthermore, in formula (I), when B has a cyclic structure or a chain structure, B preferably has a nitrogen-bonding atomic group directly bonded to a nitrogen atom constituting the cyclic structure or the chain structure. Specific examples of the nitrogen-bonding atomic group preferably include an atomic group containing one or more of a carboxy group, a phosphate group, an amide group, a benzene ring, and a pyridine ring, and more preferably the atomic group is chain-like. Furthermore, in formula (I), when B has a cyclic structure or a chain structure, D is bonded to any site on B, and the antibody binding unit (C-La) is bonded to any site on B different from the site on B to which D is bonded. When D is bonded to the nitrogen-bonding atomic group via Lb (p=1) or without Lb (p=0), the antibody binding unit is preferably bonded to a site other than the nitrogen-bonding atomic group to which D is bonded via Lb or without Lb.

[0015] Specifically, in formula (I), the chelating moiety capable of coordinating to a radioactive metal ion, represented by the symbol B, preferably has a structure derived from a compound represented by any one of the following formulas (A1) to (A9), and more preferably has a structure derived from a compound represented by the following formula (A1). In other words, the modified antibody of the present invention is preferably a derivative of a compound represented by any one of the following formulas (A1) to (A9), and more preferably a derivative of a compound represented by the following formula (A1). These structures can be appropriately selected depending on the type of radioactive metal, which will be described later. Regardless of the structure of the chelating moiety, when the modified antibody of the present invention is made into a radioactive metal-labeled antibody, the affinity between the modified antibody of the present invention and the radioactive metal ion can be sufficiently increased, thereby preventing the radioactive metal ion from being released from the radioactive metal-labeled antibody before it is taken up by a target cell.

[0016] In the formula (I), the chelating moiety represented by the symbol B may be, for example, a structure derived from the following compounds, but is not limited to these. These compounds are commercially available or can be produced based on known literature.

[0017] <DOTA or its derivatives> 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid (DOTPA) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphate (DOTMP) Hydroxypropyltetraazacyclododecanetriacetic acid (HP-DO3A) (1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAMA) 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAA) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylene)phosphonic acid (DOTA-A-AMP) Tetraazacyclododecanedimethanephosphonic acid (DO2P) α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA)

[0018] <HOPO or its derivatives> N,N',N'',N'''-tetra(1,2-dihydro-1-hydroxy-2-oxopyridine-6-carbonyl)-1,5,10,14-tetraazatetradodecane (1,2-HOPO)

[0019] <TETA or PEPA or derivatives thereof> 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid (TETPA) 1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid (PEPA)

[0020] <Chain structure (Octapa, Neunpa, or derivatives thereof)> Ethylenediaminetetraacetic acid (EDTA) 6,6'-((ethane-1,2-diylbis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (H ４octapa) 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))dipicolinic acid (H ２ bispa2) 1,2-[{6-(carboxy)-pyridin-2-yl}-methylamino]ethane (H ２ dedpa) N,N″-bis(6-carboxy-2-pyridylmethyl)-diethylenetriamine-N,N′,N″-triacetic acid (H ５ decapa) N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane (H ６ phospa) 6,6'-(((((4-isothiocyanatophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (p-SCN-Bn-H ４ neunpa) 6,6'-(((((4-nitrophenethyl)azanediyl)bis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl))bis(methylene))dipicolinic acid (p-NO ２ -Bn-H ４ 6,6'-((azanediylbis(ethane-2,1-diyl))bis((carboxymethyl)azanediyl)bis(methylene))dipicolinic acid (H ５ neunpa)

[0021] <Macropa and its derivatives> 6-(1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-N,N'-dimethyl)picolinic acid (H ２ macropa)

[0022] <NOTA or its derivatives> 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid (NOTA)

[0023]

[0024] In formula (A1), R １１ , R１２ , R １３ and R １４ are each independently -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ４ N, -(CH ２ ) ｐ P.O. ３ H ２ , -(CH ２ ) ｐ CONH ２ , -(CHCOOH)(CH ２ ) ｐ COOH, and p represents an integer of 0 to 3. If desired, the group may have a substituent.

[0025] In formula (A2), R ２１ , R ２２ , R ２３ and R ２４ each independently represents a carboxy group or a carboxyalkyl group having 2 or 3 carbon atoms, which may have a substituent if desired.

[0026] In formula (A3), R ３１ , R ３２ , R ３３ and R ３４ each independently represents an atomic group having a hydrogen atom and 2 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom; R ３５ represents a hydrogen atom, a carboxy group, or a carboxyalkyl group having 2 or 3 carbon atoms, which may have a substituent if desired.

[0027] In formula (A4), R ４１ , R ４２ , R ４３ and R ４４ each independently represents an atomic group having a hydrogen atom and 2 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom; R ４５ represents a hydrogen atom, a carboxy group, or a carboxyalkyl group having 2 or 3 carbon atoms, which may have a substituent if desired.

[0028] In formula (A5), R ４８and R ４９ each independently represents an atomic group having a hydrogen atom and 2 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom, and which may optionally have a substituent.

[0029] 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 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom, and which may optionally have a substituent.

[0030] 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 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom; R ６７ represents a hydrogen atom, a carboxy group, or a carboxyalkyl group having 2 or 3 carbon atoms, which may have a substituent if desired.

[0031]

[0032] In formula (A8), R ７１ , R ７２ and R ７３ each independently represents an atomic group having a hydrogen atom and 2 to 10 carbon atoms, which may contain a nitrogen atom or an oxygen atom, and which may optionally have a substituent.

[0033] In formula (A9), R ８１ and R ８２ each independently represents an alkyl group having 1 to 5 carbon atoms, the terminal of which may be substituted with a pyridyl group substituted with one or more carboxy groups; R ８７ represents an oxygen atom (=O) of a hydroxyl group or a carbonyl group, and R ８３ and R ８４ each independently represents a substituted or unsubstituted pyridyl group, R ８５ and R ８６each independently represents —COO—Ra, where Ra represents an alkyl group having 1 to 5 carbon atoms, and may have a substituent if desired.

[0034] Specific examples of the structure represented by formula (A1) include structures represented by the following formulae (A1-1) to (A1-7).

[0035]

[0036]

[0037]

[0038] Specific examples of the structure represented by formula (A2) include structures represented by the following formulae (A2-1) and (A2-2).

[0039]

[0040] Specific examples of the structure represented by formula (A3) include structures represented by the following formulae (A3-1) to (A3-7).

[0041]

[0042]

[0043] Specific examples of the structure represented by formula (A4) include structures represented by the following formulae (A4-1) and (A4-2).

[0044]

[0045] Specific examples of the structure represented by formula (A5) include structures represented by the following formulae (A5-1) to (A5-3).

[0046]

[0047] A specific example of the structure represented by formula (A6) is a structure represented by the following formula (A6-1).

[0048]

[0049] Specific examples of the structure represented by formula (A7) include structures represented by the following formulae (A7-1) and (A7-2).

[0050]

[0051] Specific examples of the structure represented by formula (A8) include structures represented by the following formulae (A8-1) to (A8-3).

[0052]

[0053] Specific examples of the structure represented by formula (A9) include structures represented by the following formulae (A9-1) to (A9-4).

[0054]

[0055] The chelating moiety is preferably DOTA or a derivative thereof. In particular, from the viewpoint of sufficiently increasing the affinity between the compound of the present invention and a radioactive metal ion, it is preferable that the chelating moiety represented by symbol B in formula (I) above has a structure derived from DOTA or any of its derivatives (A1-2) to (A1-7) or a compound represented by formula (A) below.

[0056]

[0057] In formula (A), R １１ and R １４ are each independently -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ４ N, -(CH ２ ) ｐ P.O. ３ H ２ , -(CH ２ ) ｐ CONH ２ Or -(CHCOOH)(CH ２ ) ｐ When the modified antibody of the present invention is represented by formula (I), for example, R １２ is a substituent for binding to an antibody binding unit, and R １３ is a substituent for bonding to the functional unit (D), and R １５ is a hydrogen atom, and p may be an integer of 0 to 3. Preferably, R １２, R １３ It is sufficient that the amino acid is introduced so that the carboxyl group remains in R １２ Lysine is introduced into R １３ It is more preferable that glutamine is introduced into the functional unit R. １１ and / or R １４ may be substituted with a group represented by the formula:

[0058] The radioactive metal that is coordinated in an ionic state to the chelating moiety of the radioactive metal-labeled antibody or modified antibody of the present invention can be a metal nuclide that emits α-rays, β-rays, γ-rays, or a combination thereof. Examples of such radioactive metal nuclides include alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, and radioactive isotopes of metals other than these metals. Among these, the following radioactive metal nuclides are preferred from the viewpoint of commercial availability and improving complex formation ability: 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 Ru, 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 When the radioactive metal-labeled antibody of the present invention is used for the purpose of treating a disease, it is preferable to use an α-ray emitting nuclide or β-Ac as the radioactive metal in order to enhance the therapeutic effect. －It is preferable to use an α-ray emitting nuclide. The α-ray emitting nuclide may be any nuclide that emits α-rays in the decay process of a radioactive metal. ２１２ Bi, ２１３ Bi, ２２７ Th or ２２５ Ac and the like are preferably used, and more preferably ２２７ Th or ２２５ Ac, more preferably ２２５ Ac. β － The radioactive nuclides are generated by the β － Any nuclide that emits radiation is acceptable. In detail, ６０ Co, ５９ Fe, ６４ Cu, ６７ Cu, ８９ Sr, ９０ Y. ９９ｍ Tc, １０３ Ru, １５３ Sm, １６５ Dy, １６６ Ho, １７７ Lu, １８６ Re, １８８ Re, １９８ Au, ２０３ Hg, ２１２ Bi, ２１３ Bi or ２１２ Pb and the like are preferably used, and more preferably ６４ Cu, ６７ Cu, ８９ Sr, ９０ Y or １７７ When the radiolabeled compound is used for the purpose of diagnosing a disease or detecting a lesion, β Lu is used as the radioactive metal in order to improve diagnostic performance. ＋ It is preferable to use a β-ray emitting nuclide, an electron capture decay nuclide, or a gamma ray emitting nuclide. ＋ The radiation-emitting nuclide may be any nuclide that emits positrons during the decay process of a radioactive metal. ４４ Sc, ５８ Co, ６８ Ga, ６４ Cu or ８９ Zr and the like are preferably used, and more preferably ６４ Cu or ８９The electron capture decay nuclide may be any nuclide that emits Auger electrons or characteristic X-rays during the decay process of the radioactive metal. ５１ Cr, ５７ Co, ５８ Co, ６７ Ga, ６８ Ga, ６４ Cu, ８９ Zr, １１１ In, １８６ Re, ２０１ Tl or １９７ The gamma-ray emitting nuclide may be any nuclide that emits gamma rays through gamma decay, and examples of the nuclide that emits gamma rays through gamma decay include: ９９ｍ Tc, ６８ Ga or ２０１ When the radioactive metal that is coordinated in an ionic state to the radioactive metal complex is selected based on the ionic radius, the following radioactive metals are preferred as radioactive metals with an ionic radius of about 70 to 130 pm: ６７ Ga, ６８ Ga, Al １８ F. ６４ Cu, ６７ Cu, ８９ Zr, ９０ Y. ９９ｍ Tc, １０３ Ru, 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, ２２５ A complex is formed between these radioactive metal ions and the chelating moiety B present in the modified antibody of the present invention. The radioactive metal coordinated to the chelating moiety of the radioactive metal-labeled antibody of the present invention or the modified antibody of the present invention is preferably Al. １８ F. ６４ Cu, ６７ Cu, ６８ Ga, ８９ Zr,９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu and ２２５ Ac.

[0059] In formula (I), the functional unit represented by the symbol D is an atomic group that has the function of improving the intracellular retention of the radiometal when the modified antibody of the present invention is made into a radiometal-labeled antibody, and is preferably an atomic group containing carbon and hydrogen, and further containing nitrogen or oxygen. Examples of functional units suitable for use in the present invention include a structure that has a positive charge in an acidic environment and a structure containing a unit for increasing the mass of the radiometal-labeled antibody. When the functional unit has a structure that has a positive charge in an acidic environment, the structure generates a positive charge in intracellular lysosomes, making it difficult for the radiometal to be excreted outside the cell. Therefore, it is believed that the radiometal coordinated to the functional chelating linker will be more likely to be retained inside the cell. Examples of such atomic groups include a structure containing one or more primary, secondary, or tertiary amino groups, and preferably an atomic group containing a structure in which ethyleneimine is polymerized (hereinafter also referred to as a "PEI structure"). Furthermore, it is believed that when the functional unit has a unit for increasing the mass of the radiometal-labeled antibody, it becomes difficult for metabolites derived from the radiometal-labeled antibody of the present invention to be excreted outside the cell. As such an atomic group, for example, an atomic group having an atomic weight of 400 or more may be designed, and more preferably an atomic group having an atomic weight of 400 or more and 10,000 or less may be used. An even more preferred example is an atomic group containing a polymerized ethylene glycol structure (hereinafter also referred to as a "PEG structure"). Furthermore, when the functional unit has a structure capable of binding to a molecule present in a target cell, the mass of the radioactive metal-labeled antibody increases due to this binding, making it difficult for metabolites derived from the radioactive metal-labeled antibody to be excreted outside the cell. Therefore, an atomic group having a structure capable of binding to a molecule present in a target cell can be used as the functional unit.

[0060] When the functional unit has a PEI structure, the PEI structure is preferably a structure in which ethyleneimine is polymerized with a degree of polymerization of 1 to 7. Specific examples of the PEI structure contained in the functional unit include a structure represented by the following formula (D-1).

[0061]

[0062] In formula (D-1), X represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxy group, an amino group, or a bond. n represents the degree of polymerization of ethyleneimine, and preferably n is 1 to 7. * (asterisk) represents a bond.

[0063] In formula (D-1), examples of the alkyl group having 1 to 4 carbon atoms represented by X include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group.

[0064] In formula (D-1), n ​​is preferably an integer of 1 or more and 7 or less, more preferably 1 or more and 5 or less, and even more preferably 1 or more and 3 or less.

[0065] The functional unit represented by formula (D-1) is protonated in an acidic environment, such as in a lysosome, to form a (poly)cation having a high ionic charge density, thereby providing high intracellular retention of the radiometal coordinated to the functional chelating linker.

[0066] When the functional unit has a PEG structure, the PEG structure is preferably a structure in which ethylene glycol is polymerized with a degree of polymerization of 11 or more and 48 or less. Specific examples of the PEG structure contained in the functional unit include a structure represented by the following formula (D-2).

[0067]

[0068] In formula (D-2), X represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, a hydroxymethyl group, an aminomethyl group, or a bond. n represents the degree of polymerization of ethylene glycol, and is preferably 11 to 48. * (asterisk) represents a bond.

[0069] In formula (D-2), examples of the alkyl group having 1 to 5 carbon atoms represented by X include 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 isopentyl group, a sec-pentyl group, a tert-pentyl group, and a neopentyl group.

[0070] When a compound having a functional unit represented by formula (D-2) is used to prepare a radiometal-labeled antibody, the radiometal coordinated to the functional chelating linker has high intracellular retention due to the large mass of the functional unit. From this viewpoint, n in formula (D-2) is preferably an integer of 11 or more and 48 or less, more preferably 12 or more and 48 or less, and even more preferably 24 or more and 48 or less.

[0071] Next, another embodiment of the functional unit will be described. In this embodiment, the functional unit is an atomic group having a structure capable of binding to a molecule present in a target cell. For example, when the target cell is a cancer cell, it is preferable to select a protein that is overexpressed in the cancer cell as the "molecule present in the target cell." An example of such a protein is cathepsin B. Therefore, the functional unit of this embodiment preferably has a structure capable of binding to cathepsin B.

[0072] There are no limitations on the type of bond between the functional unit and the molecule present in the target cell, and it can be a non-covalent bond such as an ionic bond, hydrogen bond, dipole-dipole interaction, or dispersion force, as well as a covalent bond. In particular, from the viewpoint of increasing affinity and binding specificity for the molecule present in the target cell, it is preferable that the bond between the functional unit and the molecule present in the target cell includes a covalent bond. The covalent bond formed between the functional unit and the molecule present in the target cell may be one or more. Furthermore, the functional unit and the molecule present in the target cell may have one or more non-covalent bonds in addition to the covalent bond.

[0073] Although there are no particular limitations on the binding site of the functional unit in the molecule present in the target cell, when the molecule present in the target cell is an enzyme, it is preferable that the functional unit binds to the binding site between the enzyme and its substrate, i.e., the active site of the enzyme, because by mimicking the structure of the enzyme's substrate, it is possible to easily design a functional unit structure that has excellent affinity and binding specificity for the enzyme.

[0074] In summary, when an atomic group capable of binding to a molecule present in a target cell is used as a 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 D-3"). For example, when a functional unit forms a covalent bond with a thiol group present in the active site of cathepsin B, Structure D-3 preferably has an epoxy group. Furthermore, in consideration of affinity with cathepsin B and stability in the bound state with cathepsin B, Structure D-3 is preferably an epoxysuccinyl peptide. An epoxysuccinyl peptide refers to a peptide containing a structure formed by dehydration condensation of trans-epoxysuccinic acid with two identical or different amines. Specific preferred examples of Structure D-3 include structures represented by the following formulae (D-3-1) to (D-3-3). *-HN-(CH ２ ) ｒ -HN-Gly-Aa-Bb-Xd-Cc-Dd (D-3-1) *-HN-(CH ２ ) ｒ -HN-Gly-Aa-Bb-Xd-Cc (D-3-2) *-HN-(CH ２ ) ｒ -HN-Gly-Aa-Bb-Xd (D-3-3)

[0075] In formulas (D-3-1) to (D-3-3), r represents an integer of 1 or more and 30 or less, preferably 6. Aa represents one selected from the group consisting of Gly, Phe, Asp, Glu, and 1-aminoadipic acid, preferably Gly. Bb represents an aliphatic or aromatic hydrophobic amino acid. Cc represents an aliphatic or aromatic hydrophobic amino acid. Dd represents an aliphatic or aromatic hydrophobic amino acid, or an aliphatic or aromatic amine. Xd represents an epoxide derivative. * (asterisk) represents a bond. Gly, Aa, Bb, Xd, Cc, and Dd are bonded to each other via an amide bond.

[0076] In formulas (D-3-1) to (D-3-3), 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 formulas (i) to (iv), and more preferably represents Leu:

[0077]

[0078] In formulas (D-3-1) and (D-3-2), Cc preferably represents one selected from the group consisting of Leu, Ile, Phe, Tyr, Val, Trp, and Nle, and more preferably represents Leu.

[0079] In formula (D-3-1), Dd is Trp, Val, He, Phe, Tyr, Ala, Ser, Thr, Pro, -NH-(CH ２ ) ｎ -CH ３ (n represents an integer of 0 to 4), —NH—CH(CH ３ ) ２ , —NH—CH ２ -CH(CH ３ ) ２ and -NH-(CH ２ ) ｍ It is preferable that m represents one selected from the group consisting of Ph (m represents 1 or 2), and more preferable that m represents Pro.

[0080] In the formulae (D-3-1) to (D-3-3), the epoxide derivative represented by Xd is preferably a dicarboxylic acid having an epoxy group or an amino acid having an epoxy group, and more preferably trans-epoxysuccinic acid.

[0081] 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 a target cell, or may have two or more of them. For example, the functional unit may have one PEI structure and one PEG structure. Furthermore, the radiometal-labeled antibody of the present invention may have only one functional unit or may have two or more functional units.

[0082] 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):

[0083]

[0084] In formula (c), X １ and X ２ X each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxymethyl group, an aminomethyl group, or a bond. ａ and X ｂ Each independently represents O or NH. 1 represents 0 or 1. X ｃ represents a hydrogen atom when l is 0, and represents a CH ２ m and n each independently represent the degree of polymerization of ethyleneimine or ethylene glycol. * (asterisk) represents a bond.

[0085] In the modified antibody of the present invention represented by formula (I), the partial structure "-B-(Lb)p-D" preferably includes a structure represented by the following formula (d): The structure represented by the following formula (d) includes a chelating moiety and one or more functional units.

[0086]

[0087] In formula (d), o, p, and q each independently represent 0 or 1, and satisfy the relationship 1≦o+p+q≦3. Xd represents a structure represented by formula (c) or a structure in which a structure represented by formula (D-2) is connected via an amino group (NH). Xe represents a hydrogen atom when o is 0, and a CH ２ When p is 0, Xf represents a hydrogen atom, and when p is 1, Xf represents CH ２ When q is 0, Xg represents a hydrogen atom, and when q is 1, Xg represents a CH ２ represents a bond. * (asterisk) represents a bond. In the modified antibody of the present invention, the structure shown in formula (d) is bound to the antibody binding unit via the bond (*). Furthermore, in a radiometal-labeled antibody obtained by labeling the modified antibody of the present invention with a radiometal, the structure shown in formula (d) is bound directly or indirectly to the antibody via the bond (*). Here, "indirectly bound" means that a linker is present between the bond and the antibody.

[0088] The structure shown in formula (d) is preferably represented by the following formula (a) or (b).

[0089]

[0090] (In formulas (a) and (b), * (asterisk) represents a bond, n represents an integer of 1 to 7, and m represents an integer of 11 to 48.)

[0091] In formula (I), the peptide represented by the symbol C is a peptide that binds to a specific site in the Fc region of an antibody. Preferably, it is a peptide that binds to a specific site in the Fc region of IgG (an IgG-binding peptide). In the present invention, "IgG" refers to IgG of mammals, for example, primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pet animals such as dogs and cats, preferably human IgG (IgG1, IgG2, IgG3, or IgG4). The IgG in the first embodiment of the present invention is more preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.

[0092] In one embodiment, the IgG-binding peptide of the present invention has the following formula (1): (X) １－３ -Cys-(X) ２ -His-(Xaa1)-Gly-(Xaa2)-Leu-Val-Trp-Cys-(X) １－３ (1) (wherein each X is independently any amino acid residue other than cysteine, Cys is a cysteine ​​residue, His is a histidine residue, Xaa1 is a lysine residue, arginine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Gly is a glycine residue, Xaa2 is a glutamic acid residue, glutamine residue, or asparagine residue, Leu is a leucine residue, Val is a valine residue, and Trp is a tryptophan residue).

[0093] In the above formula, (X) at the N-terminus or C-terminus １－３ means that 1 to 3 consecutive amino acid residues X are independently any amino acid residue other than cysteine ​​(C or Cys), and the amino acid residues constituting the sequence may be the same or different, but preferably, all three residues are not the same. ２means that two consecutive amino acid residues X are independently any amino acid residue other than cysteine ​​(C or Cys), and the amino acid residues constituting the two consecutive amino acid residues may be the same or different, but preferably the two consecutive amino acid residues are not the same.

[0094] The two cysteine ​​residues of formula (1) can be, and usually are, disulfide bonded to form a cyclic peptide. Alternatively, in the peptide of formula (1), the sulfide groups in the two cysteine ​​residues are bonded to the following formula:

[0095]

[0096] The wavy line in the above formula represents the bond with the sulfide group. This group is more stable against reduction reactions and the like than a normal disulfide bond. Such peptides can be synthesized, for example, according to the methods described in WO 2016 / 186206 and WO 2017 / 217347.

[0097] Some specific examples of the IgG-binding peptide of formula (1) are listed below in 1) to 18), but it goes without saying that the invention is not limited to these. 1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 1), 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2), 3) RCAYH(Xaa1)GELVWCS (SEQ ID NO: 3), 4) GPRCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 4), 5) SPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 5), 6) GDDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 6), 7) GPSCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 7), 8) GPDCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 8), 9) GPDCAYH(Xaa1)GELVWCTHH (SEQ ID NO: 9), 10) GPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 10), 11) SPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 11), 12) SDDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 12), 13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 13), 14) G(Xaa3)DCAYH(Xaa1)GELVWCT(Xaa3)H (SEQ ID NO: 14), 15) DCTYH(Xaa1)GNLVWCT (SEQ ID NO: 15), 16) DCAYH(Xaa1)GNLVWCT (SEQ ID NO: 16), 17) DCTYH(Xaa1)GELVWCT (SEQ ID NO: 17), and 18) DCAWH(Xaa1)GELVWCT (SEQ ID NO: 18), (wherein Xaa1 is a lysine residue, an arginine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and Xaa3 is homocysteine, preferably, the homocysteines form a disulfide bond with each other).

[0098] Specific preferred examples of the IgG-binding peptide of formula (1) include: 1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 1), 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2), 13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 13), and 14) G(Xaa3)DCAYH(Xaa1)GELVWCT(Xaa3)H (SEQ ID NO: 14). Particularly preferred examples include 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2) (wherein Xaa1 is a lysine residue, arginine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and Xaa3 is homocysteine, and preferably, two cysteines and / or two homocysteines form a disulfide bond with each other).

[0099] The IgG-binding peptides used in the present invention can be produced by peptide synthesis methods such as liquid phase synthesis, solid phase synthesis, automated peptide synthesis, recombinant DNA technology, and phage display using a combination of amino acids, regardless of whether they are natural or unnatural. When synthesizing the peptide, functional groups of the amino acids used may be protected as necessary. This can be done, for example, according to the methods described in WO 2016 / 186206, WO 2017 / 217347, and WO 2018 / 230257.

[0100] When the IgG-binding peptide used in the present invention is represented by, for example, formula (1), Xaa1 is adjacent to a lysine residue in a specific region of IgG Fc. For example, when the IgG is trastuzumab, Xaa1 is adjacent to Lys248 or Lys246, preferably Lys248, according to EU numbering in human IgG Fc. Xaa1 of the IgG-binding peptide is modified with a crosslinking agent, and a crosslinking reaction is carried out between the IgG-binding peptide modified with the crosslinking agent and IgG, thereby forming a site-specific crosslinked structure between Xaa1 of the IgG-binding peptide and the lysine residue in IgG Fc. By modifying Xaa1 of the IgG-binding peptide with a crosslinking agent in this way and crosslinking it with IgG, various compounds can be introduced into IgG in a site-specific and convenient manner. Furthermore, because compounds can be introduced via the IgG-binding peptide, compounds with various structures can be introduced into IgG. Such a method has the advantage that the yield of the obtained product is high and, since it does not involve modification of the antibody itself, there is little possibility of reducing the function of the antibody.

[0101] Here, the "crosslinking agent" refers to a chemical substance that covalently links the IgG-binding peptide and the IgG Fc. Those skilled in the art can select an appropriate crosslinking agent. For example, crosslinkers containing preferably two or more succinimidyl groups such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate) and crosslinkers containing preferably two or more imidic acid moieties such as DMA (dimethyl adipimidate.2HCl, dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate.2HCl, dimethyl pimelimidate dihydrochloride), and DMS (dimethyl suberimidate.2HCl, dimethyl suberimidate dihydrochloride) and DTBP (dimethyl Examples of crosslinking agents include crosslinkers having an SS bond, such as 3,3'-dithiobispropionimidate.2HCl (dimethyl 3,3'-dithiobispropionimidate dihydrochloride) and DSP (dithiobis(succinimidyl propionate)). Preferred crosslinking agents are those having a succinimide group that can bond to the terminal amino group of the side chain of an amino acid residue that constitutes the IgG-binding peptide, and DSG and DSS are particularly preferred.

[0102] In the present invention, the IgG-binding peptide may be modified with other functional substances, such as antibodies (e.g., IgA or VHH), labeling substances, and / or other drugs. Linking of the IgG-binding peptide to other functional substances can be performed by methods known to those skilled in the art, such as the reaction of an azide group with dibenzocyclooctyne or the reaction of a maleimide group with a sulfhydryl group. When labeled with a labeling substance, the IgG-binding peptide forms a complex with IgG, enabling detection or quantification of IgG via the labeling substance. Labeling substances include, but are not limited to, fluorescent dyes, chemiluminescent dyes, biotin and fluorescent proteins such as GFP (green fluorescent protein), luminescent proteins, and enzymes such as peroxidase. Preferred examples of labeling substances include fluorescein derivatives such as fluorescein and FITC, rhodamine derivatives such as rhodamine and tetramethylrhodamine, and fluorescent dyes such as Texas Red. When the IgG-binding peptide is modified with another drug, examples of the drug include, but are not limited to, anticancer drugs such as auristatin, maytansine, doxorubicin, bleomycin, or derivatives thereof; and targeting agents such as drugs that bind to receptors on the blood-brain barrier to enable migration to the central nervous system, or drugs that bind to cancer cells or the like to enable migration of an antibody into the cells.

[0103] Binding of an IgG-binding peptide to a specific site in the Fc region of an antibody (hereinafter, for convenience, also referred to as "modifying an antibody with an IgG-binding peptide") is carried out by a cross-linking reaction with the IgG-binding peptide modified with the above-mentioned cross-linking agent. In one embodiment, the cross-linking reaction comprises a step of mixing the IgG-binding peptide modified with the above-mentioned cross-linking agent with the antibody. The conditions for this mixing step are not particularly limited, as long as they are conditions that allow a cross-linking reaction between the IgG-binding peptide and the antibody. For example, the reaction can be carried out by mixing the IgG-binding peptide and the antibody in an appropriate buffer at room temperature (e.g., about 15°C to 30°C). The mixing step may be carried out by adding an appropriate amount of a catalyst that promotes the cross-linking reaction, as needed.

[0104] As an example, a solvent containing at least water is added to dissolve the antibody. Examples of this solvent include, in addition to water, dimethyl sulfoxide, acetonitrile, saline, and buffers such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, Tris buffer, HEPES buffer, and tetramethylammonium acetate buffer. When using a buffer, from the viewpoint of antibody stability, the pH at 25°C is preferably 4.0 to 10.0, more preferably 5.5 to 8.5. At the start of the crosslinking reaction, the antibody concentration is preferably 1.0 μmol / L or more at the lower limit and 1000 μmol / L or less at the upper limit, more preferably 500 μmol / L or less.

[0105] Next, the IgG-binding peptide modified with a crosslinker and, if necessary, a catalyst are added, and the mixture is dispersed at a temperature of 10° C. to 30° C. The mixing ratio of the IgG-binding peptide to the antibody in the mixing step is not particularly limited. The molar ratio of the IgG-binding peptide to the antibody can be, for example, 1:1 to 20:1, preferably 2:1 to 20:1 or 5:1 to 10:1.

[0106] In a preferred embodiment, the IgG-binding peptide to antibody molar ratio in the mixing step is 0.5 to 2.2, preferably 0.8 to 1.8, which allows efficient production of monovalent modified antibodies (i.e., complexes containing one IgG-binding peptide per antibody).

[0107] The mixing time (reaction time) in the mixing step is not limited as long as a cross-linking reaction occurs between the IgG-binding peptide and the antibody, but can be, for example, 1 minute to 5 hours, preferably 10 minutes to 2 hours.

[0108] In this way, the IgG-binding peptide can bind to the Fc region of the antibody. The antibody has one binding region for the peptide per heavy chain. Therefore, one or two peptides can bind to an antibody having two heavy chains. In this separation or purification step, it is not necessary to separate the unmodified antibody (i.e., naked antibody), the monovalent modified antibody (i.e., one antibody modified with one IgG-binding peptide), and the bivalent modified antibody (i.e., one antibody modified with two IgG-binding peptides). Alternatively, only the unmodified antibody may be removed from the mixture to obtain a mixture of the monovalent modified antibody and the bivalent modified antibody, or the monovalent modified antibody and the bivalent modified antibody may be individually isolated, concentrated, or purified. Removal of unmodified antibodies from a mixture or separation of monovalent modified antibodies and divalent modified antibodies can be achieved by, for example, chromatography such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC. In a preferred embodiment, at least one of the unmodified antibodies, monovalent modified antibodies, and divalent modified antibodies may be separated by an IgG-BP column method (see WO 2021 / 080008) or affinity chromatography (e.g., a protein A column or a protein G column). An IgG-BP column is a column onto which an IgG-binding peptide is immobilized. The divalent modified antibodies cannot bind to the column because their binding sites are already occupied by the IgG-binding peptide, and only the monovalent modified antibodies exhibit affinity for the column. Therefore, monovalent modified antibodies and divalent modified antibodies can be easily separated using an IgG-BP column. Thus, in a preferred embodiment, a composition can be provided comprising an unmodified antibody and a monovalent modified antibody, wherein the molar ratio of the unmodified antibody to the monovalent modified antibody is 4 to 47:53 to 96, preferably 4 to 30:70 to 96, more preferably 4 to 20:80 to 96, and even more preferably 4 to 10:90 to 96.

[0109] In the present invention, the linker a (La) is not particularly limited as long as it can link the chelating moiety (B) and the IgG-binding peptide (C). Examples of the linker a used in the present invention include a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a polyethylene glycol (PEG) group, a peptide, a sugar chain, a disulfide group, an amide group, and combinations thereof. As used herein, linker a refers to a linker used to connect the IgG-binding peptide or antibody A modified with the IgG-binding peptide to the chelating moiety, and includes an antibody-modifying linker (La1) that modifies the IgG-binding peptide and a chelating linker (La2) that modifies the chelating moiety. The antibody-modifying linker (La1) is introduced to the N-terminal side of the IgG-binding peptide, and the chelating linker (La2) is introduced to the functional group of the chelating moiety.

[0110] The linker a used in the present invention may contain a binding site formed by a click reaction, and preferably corresponds to a binding site formed by binding the antibody-modified linker (La1) and the chelate linker (La2) by a click reaction.

[0111] In the present invention, the combination of click-reactive atomic groups is selected appropriately depending on the type of click reaction, and examples include a combination of an alkyne and an azide, or a combination of a 1,2,4,5-tetrazine and an alkene. These atomic groups may be any atomic group, as long as the antibody binding unit has one of the atomic groups and the antibody has an atomic group that forms a combination of antibody binding units. Specific examples of click reactions using such combinations of atomic groups include the Huisgen cycloaddition reaction and the inverse electron demand Diels-Alder reaction. From the perspective of simplifying the click reaction process, the click-reactive atomic group is preferably an atomic group that can be used in a metal catalyst-free click reaction.

[0112] Specific examples of combinations include, as shown in the following formulas, a combination of an atomic group containing dibenzocyclooctyne (DBCO) as the alkyne (formula (11a)) and an atomic group containing an azide group as the azide (formula (12a)), or a combination of an atomic group containing 1,2,4,5-tetrazine (formula (11b)) and an atomic group containing trans-cyclooctene (TCO) as the alkene (formula (12b)).

[0113]

[0114] In formula (11a), R １ represents a chelating moiety or a moiety obtained by removing the click-reactive atomic group from the IgG-binding peptide. ２ represents the chelating moiety or the portion obtained by removing the click-reactive atomic group from the IgG-binding peptide.

[0115]

[0116] In formula (11b), R ３ and R ４ In formula (12b), one of R represents a chelating moiety or a moiety obtained by removing a click-reactive atomic group from the IgG-binding peptide, and the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group. ５ represents the chelating moiety or the portion obtained by removing the click-reactive atomic group from the IgG-binding peptide.

[0117] When a click-reactive atomic group is introduced into a chelating linker, it can be introduced using various commercially available reagents. Specifically, when an atomic group containing dibenzocyclooctyne (DBCO) is introduced as a click-reactive atomic group, for example, DBCO-C6-Acid, DBCO-Amine, DBCO-Maleimide, DBCO-PEG acid, DBCO-PEG-NHS, etc. DBCO reagents such as 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, and DBCO-mPEG can be used. Alternatively, a modified antibody into which an azide group has been introduced can be prepared as a corresponding modified antibody. Examples of methods for introducing an azide group into an antibody include introducing the group into the N-terminus of the amino acid sequence or into the amino side chain of a lysine residue.

[0118] The binding site formed by the click reaction is preferably a triazole skeleton-containing structure represented by the following formula (10a) or (10b) or a pyridazine skeleton-containing structure represented by the following formula (10c). Formula (10a) and formula (10b) are isomers and may be contained in any ratio.

[0119]

[0120] In formula (10a) and formula (10b), R1A represents a linking site with the chelating moiety, and R ２Ａ represents a linking site with the IgG-binding peptide. ３Ａ and R ４Ａ one of which represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a linking site with the chelating moiety; R ５Ａindicates a linking site to the IgG-binding peptide. In formulas (10a), (10b), and (10c), the linking site to the IgG-binding peptide is such that the peptide is linked via an antibody-modifying linker (La1), and the linking site to the chelating moiety is such that the chelating moiety is linked via a chelating linker (La2).

[0121] In the present invention, the linker b (Lb) is not particularly limited as long as it can link the functional unit (D) and the chelating moiety (B), but it may not be present when D is directly linked to B (p=0 in formula (I)). When present (p=1 in formula (I)), examples of the linker b used in the present invention include a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a polyethylene glycol (PEG) group, a peptide, a sugar chain, a disulfide group, an amide group, and combinations thereof.

[0122] In formula (I), the bonding mode between the functional unit represented by symbol D and the chelating moiety represented by symbol B is not particularly limited, and the bonding modes shown below can be used, for example: (a) Bonding by conjugate addition reaction (b) Bonding by click reaction (c) Bonding by amine coupling These bonds can form a linker structure (Lb) between the functional unit (D) and the chelating moiety (B). Preferred examples of linker b (Lb) include structures represented by the following formula:

[0123]

[0124] In the formula, R １Ｂ indicates the linking site with the chelating moiety, and R ２Ｂ indicates the linking site with the functional unit. ３Ｂ and R ４Ｂ one of which represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a linking site with the chelating moiety; R ５Ｂ indicates the linking site with the functional unit. ６Ｂ indicates the linking site with the chelating moiety, and R ７Ｂ indicates the linking site with the functional unit. ８Ｂ indicates the linking site with the chelating moiety, and R ９Ｂindicates the linking site with the functional unit. The structures of formulas (i) to (iii) can be obtained by a click reaction in the same manner as the linker La. The structure of formula (iv) (oxime) is ７Ｂ and hydroxylamine having R ６Ｂ By reaction with a ketone having the formula (v), the structure of formula (v) is obtained ７Ｂ and hydrazine having R ６Ｂ By reaction with a carbonyl compound having the formula (vi), the structure of formula (vi) is formed ８Ｂ and maleimides having R ９Ｂ can be obtained by Michael addition of thiols having the formula:

[0125] The linker structure represented by Lb in formula (I) may have a structure represented by the following formula (P): This structure is derived from ethylene glycol, and in 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 even more preferably an integer of 2 or more and 5 or less.

[0126]

[0127] The linker structure represented by Lb may consist of one type of linker structure, or may consist of a single type of linker structure repeated or a combination of multiple types of linker structures bonded in a linear or branched chain. In formula (I), Lb is preferably absent (i.e., p=0). In such a case, the amino group of the functional unit (D) and the carboxyl group of the chelating moiety (B) are connected by an amide bond.

[0128] By reacting the modified antibody of the present invention with a radioactive metal ion, a radiometal-labeled antibody in which a functional unit that improves intracellular retention is site-specifically conjugated to the antibody can be obtained. The radiometal-labeled antibody of the present invention can be prepared by first reacting the radioactive metal ion with an IgG-binding peptide (also referred to as an intracellular retention unit-containing linker) to which the intracellular retention unit is bound via a chelating moiety, followed by reaction with the antibody, or by first reacting the antibody with the intracellular retention unit-containing linker, followed by reaction with the radioactive metal ion. The radioactive metal ion can preferably be one of the radioactive metals listed above.

[0129] The antibody used in the present invention is a polyclonal antibody or a monoclonal antibody. The immunoglobulin class of the antibody can be any of IgG, IgA, IgM, IgD, or IgE, without any particular limitation. Of these, IgG is preferred because it is the major class in the secondary immune response, is present in the most amount in blood, and has high antigen recognition specificity. Mammalian IgG is also preferred, and specific examples of mammalian IgG include primates such as humans and chimpanzees; laboratory animals such as rats, mice, and rabbits; livestock animals such as pigs, cows, horses, sheep, and goats; and pet animals such as dogs and cats. Chimeric, humanized, or human monoclonal antibodies are even more preferred. The subclasses of these antibodies are also not particularly limited. For example, when human IgG is used, it may be at least one of IgG1, IgG2, IgG3, and IgG4. The antibody may be a full-length antibody or an antibody fragment (e.g., F(ab')2, Fab', Fab, Fv, single-chain antibody), with full-length antibodies being preferred. The antibody may be directed against any antigen, such as a protein, a sugar chain, a nucleic acid, or a low-molecular-weight compound. Preferably, the antibody is directed against a cell surface antigen, more preferably a tumor cell surface antigen. The antibody has a cellular internalization function. An antibody must not only accumulate in target cells but also be efficiently taken up into the target cells (antibody internalization function). The antibody internalization function is derived from both the membrane surface protein to which the antibody binds and the antibody itself. The antibody internalization function can be measured by known methods. For example, a method is known in which an antibody (or secondary antibody) is labeled with RI, a fluorescent dye, or the like and the amount of uptake (RI, fluorescence intensity) is measured, and a method is known in which cell death is measured using a toxin such as saporin (e.g., a saporin-labeled anti-mouse IgG antibody). However, a method using an antibody labeled with saporin toxin is superior in terms of sensitivity and simplicity.

[0130] For example, an antibody that recognizes an antigen that is specifically expressed in tumor cells can be used.Specifically, for example, PD-L1, GD2, PDGFRα (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, phosphatidylinositol phosphate (PHSP), Dyserine (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, CPAAs, PSMA, ICAM-1, MUC1, MUC5AC, EGFR, EGFRvIII, KIR2DL1, KIR3DL2, NKG2A, tenascin-C, IGF (insulin-like growth factor receptor 1 (IGF)), factor), CTLA-4, mesothelin, c-Met, Ang2, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, Notch1, Notch2, Notch3, TLR-2, C SF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7-H3, gpA33, Frizzled7 receptor, RSPO, LIV-1, SLITRK6, Nectin-4, MET, Tissue Factor, IL-8, P-cadherin, CEA, GITR, FGFR2, FGFR3, FGFR4, CXCR4, LAG-3, Fucosyl It is preferable to use an antibody whose antigen is GM1, IGF-1, CSF-1R, OX40, BCMA, PTK7, EFNA4, FAP, DR5, Ly6E, CA6, CAIX, CA72-4 (TAG-72), LAMP1, EPHA2, α2-PI, ROR1, TBA, TIM-3, PIGF, Axl, MICA / B, Thomsen-Friedenreich, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, immune suppressor protein, NaPi2b, Lewis blood group antigen, A34, DLK-1, or α9 integrin.

[0131] Examples of antibodies that use HER2 as an antigen include trastuzumab and pertuzumab. Examples of antibodies that use EGFR as an antigen include cetuximab and panitumumab. Examples of antibodies that use sialylated HEG1 as an antigen include antibodies that bind to glycosylated HEG1 proteins obtained from mesothelioma, antibodies that bind to HEG1 proteins on the cell membrane of mesothelioma, and antibodies that bind to peptides expressed in mesothelioma cell lines (e.g., peptides having the amino acid sequence SKSPSLVSLPT). More specifically, examples include the SKM9-2 antibody described in WO 2017 / 141604, or humanized antibodies thereof. Examples of SKM9-2 antibodies include, for example, mouse anti-sialylated HEG1 monoclonal antibodies commercially available from Nichirei Biosciences. Examples of humanized SKM9-2 antibodies include humanized antibodies that can bind to human HEG1 proteins expressed in mesothelioma cells. The humanized SKM9-2 antibody preferably includes the antibody described in WO2023 / 277113.

[0132] Specifically, the humanized SKM9-2 antibody has a heavy chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 26 shown in Table 1, and more preferably has the heavy chain variable region having the amino acid sequence of SEQ ID NO: 24. The "reference numbers" shown below are the SEQ ID NOs of WO2023 / 277113.

[0133]

[0134] Specifically, the humanized SKM9-2 antibody has a light chain variable region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 39 shown in Table 2, and more preferably has the amino acid sequence of SEQ ID NO: 36 in the light chain variable region.

[0135]

[0136] The radioactive metal-labeled antibody of the present invention can be used as a radiopharmaceutical containing the labeled antibody as an active ingredient. Radiopharmaceuticals containing the radioactive metal-labeled antibody of the present invention as an active ingredient can be formulated using known pharmaceutical methods. For example, the radiopharmaceutical of the present invention may contain a pharmaceutically acceptable excipient. The excipient can be an excipient that can be appropriately administered to provide an effective amount of the radioactive metal-labeled antibody of the present invention, which is the active ingredient, to a subject. In some embodiments, the radiopharmaceutical of the present invention can be made into an injection, and the injection excipient can be a sterile aqueous solution, for example, physiological saline or a pharmaceutically acceptable buffer, and an isotonic solution containing auxiliary agents such as stabilizers and solubilizers. When in the form of an injection, the radiopharmaceutical of the present invention can be administered parenterally (e.g., intravenously or intrathoracically) and used for disease treatment, disease diagnosis, or lesion detection. Radiopharmaceuticals using antibodies that recognize antigens specifically expressed in tumor cells can be suitably used for cancer diagnosis or internal cancer radiotherapy by selecting the radiometal.

[0137] In each of the above-described embodiments, examples of substituents that can substitute each atomic group and each chemical structure of the compound include halogen atoms, saturated or unsaturated alkyl groups, hydroxy groups, formyl groups, carboxy groups, acyl groups, amino groups, nitro groups, ester groups, isothiocyanate groups, thioxy groups, cyano groups, amide groups, imide groups, phosphate groups, phenyl groups, benzyl groups, pyridyl groups, naphthyl groups, etc. These substituents may be used alone or in combination of two or more types of substituents.

[0138] Although the present invention has been described above based on its preferred embodiments, the present invention is not limited to the above-described embodiments. For example, in the above-described embodiments, compounds having one each of a chelating moiety, a functional unit, and an antibody-binding unit have been described. However, as long as the present invention is achieved, the compounds of the present invention may have multiple functional units and / or multiple antibody-binding units.

[0139] The above-described embodiments of the present invention encompass the following technical concepts. [1] A modified antibody represented by the following formula (I): A-C-La-B-(Lb)p-D (I) [wherein A is an antibody that exhibits binding to a cell surface antigen, wherein the antibody has a cellular internalization function; B is a chelating moiety capable of coordinating to a radioactive metal ion; C is a peptide that binds to a specific site in the Fc region of the antibody; D is a functional unit that improves intracellular retention; La is a linker a connecting C and B; Lb is a linker b connecting B and D; and p is 0 or 1]. [2] The modified antibody according to [1], wherein the antibody (A) is an antibody that exhibits binding to a tumor cell surface antigen. [3] The modified antibody according to [1] or [2], wherein the antibody (A) is an SKM9-2 antibody. [4] The modified antibody according to any of [1] to [3], wherein the chelating moiety (B) is DOTA or a derivative thereof. [5] The radioactive metal is Al １８ F. ６４ Cu, ６７ Cu, ６８ Ga, ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu and ２２５ [6] The modified antibody according to any one of [1] to [4], wherein the peptide (C) is one selected from the group consisting of peptides represented by the following formula (1): １－３ -Cys-(X) ２ -His-(Xaa1)-Gly-(Xaa2)-Leu-Val-Trp-Cys-(X) １－３(1) The modified antibody according to any one of [1] to [5], comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by the formula: [wherein each X independently represents any amino acid residue other than cysteine, Cys represents a cysteine ​​residue, His represents a histidine residue, Xaa1 represents a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or a diaminopropionic acid, Gly represents a glycine residue, Xaa2 represents a glutamic acid residue, a glutamine residue, or an asparagine residue, Leu represents a leucine residue, Val represents a valine residue, and Trp represents a tryptophan residue].[7] The modified antibody according to any one of [1] to [6], wherein the amino acid sequence of the peptide (C) is any one of those listed in 1) to 18) below: 1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 1), 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2), 3) RCAYH(Xaa1)GELVWCS (SEQ ID NO: 3), 4) GPRCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 4), 5) SPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 5), 6) GDDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 6), 7) GPSCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 7), 8) GPDCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 8), 9) GPDCAYH(Xaa1)GELVWCTHH (SEQ ID NO: 9), 10) GPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 10), 11) SPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 11), 12) SDDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 12), 13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 13), 14) G(Xaa3)DCAYH(Xaa1)GELVWCT(Xaa3)H (SEQ ID NO: 14), 15) DCTYH(Xaa1)GNLVWCT (SEQ ID NO: 15), 16) DCAYH(Xaa1)GNLVWCT (SEQ ID NO: 16), 17) DCTYH(Xaa1)GELVWCT (SEQ ID NO: 17), and 18) DCAWH(Xaa1)GELVWCT (SEQ ID NO: 18) [wherein Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and Xaa3 is homocysteine, preferably wherein the homocysteines form a disulfide bond with each other]. [8] The modified antibody according to any of [1] to [7], wherein the functional unit (D) has a structure in which ethyleneimine or ethylene glycol is polymerized, or a structure capable of binding to cathepsin B. [9] The modified antibody according to any of [1] to [8], wherein the functional unit (D) has a structure in which ethyleneimine is polymerized with a degree of polymerization of 1 or more and 7 or less.

[10] The modified antibody according to any one of [1] to [8], wherein the functional unit (D) has a structure in which ethylene glycol is polymerized with a degree of polymerization of 11 to 48.

[11] The modified antibody according to any one of [1] to

[10] , wherein the linker a (La) comprises a structure represented by the following formula (10a), formula (10b), or formula (10c):

[0140]

[0141] [In formula (10a) and formula (10b), R １Ａ represents the linking site with the chelating moiety (B), and R ２Ａ represents a linking site with the peptide (C), and in formula (10c), R ３Ａ and R ４Ａ one of which represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a linking site with the chelating moiety (B), and R ５Ａ indicates a linking site to the peptide.

[12] The modified antibody according to any one of [1] to

[11] , wherein the linker b (Lb) has a structure represented by the following formula (L):

[0142]

[0143] [wherein k is an integer of 2 to 10]. [12'] The modified antibody according to any one of [1] to

[11] , wherein the linker b (Lb) is not present.

[13] The modified antibody according to any one of [1] to

[11] , wherein in formula (I), the partial structure -B-(Lb)p-D is represented by the following formula (a) or (b):

[0144]

[0145] (In formulas (a) and (b), * (asterisk) represents a bond, n represents an integer of 1 to 7, and m represents an integer of 11 to 48.)

[14] In formula (I), the antibody (A) is an SKM9-2 antibody, and the partial structure -B-(Lb)p-D is represented by the following formula (b):

[0146]

[0147] (wherein * (asterisk) is a bond, and m is an integer of 11 to 48), and the linker a (La) contains a structure represented by the following formula (10a) or formula (10b):

[0148]

[0149] [In formula (10a) and formula (10b), R １Ａ represents the linking site with the chelating moiety (B), and R ２Ａ indicates a linking site to the peptide (C), wherein the peptide (C) has the amino acid sequence set forth in SEQ ID NO: 2.

[15] A radioactive metal-labeled antibody in which the modified antibody set forth in any one of [1] to

[14] is labeled with a radioactive metal.

[16] The radioactive metal is Al １８ F. ６４ Cu, ６７ Cu, ６８ Ga, ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu and ２２５

[17] A radioactive metal-labeled antibody according to

[16] , wherein the radioactive metal is one selected from Ac.

[17] A radioactive pharmaceutical comprising the radioactive metal-labeled antibody according to

[16] as an active ingredient.

[18] The radioactive pharmaceutical according to

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

[19] The modified antibody according to [1] or [2], wherein the antibody (A) is an antibody against EGFR as an antigen.

[20] A radioactive metal-labeled antibody, wherein the modified antibody according to

[19] is labeled with a radioactive metal.

[21] The radioactive metal is ８９ Zr or ２２５

[22] The radiometal-labeled antibody according to any one of

[15] ,

[16] , and

[20] , wherein the partial structure -B-(Lb)p-D in formula (I) comprises a structure represented by formula (d):

[0150]

[0151] In formula (d), o, p, and q each independently represent 0 or 1, and satisfy the relationship 1≦o+p+q≦3. Xd represents a structure represented by formula (c) or a structure in which a structure represented by formula (D-2) is connected via an amino group (NH). Xe represents a hydrogen atom when o is 0, and a CH ２ When p is 0, Xf represents a hydrogen atom, and when p is 1, Xf represents CH ２ When q is 0, Xg represents a hydrogen atom, and when q is 1, Xg represents a CH ２ * (asterisk) represents a bond.

[0152]

[0153] In formula (c), X １ and X ２ X each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxymethyl group, an aminomethyl group, or a bond. ａ and X ｂ Each independently represents O or NH. 1 represents 0 or 1. X ｃ represents a hydrogen atom when l is 0, and represents a CH ２ m and n each independently represent the degree of polymerization of ethyleneimine or ethylene glycol. * (asterisk) represents a bond.

[0154]

[0155] In formula (D-2), X represents a hydrogen atom, an alkyl group having from 1 to 5 carbon atoms, an aminomethyl group, a hydroxymethyl group, or a bond. n represents the degree of polymerization of ethylene glycol, and is preferably from 11 to 48. * (asterisk) represents a bond.

[23] The radiometal-labeled modified antibody according to any one of

[15] ,

[16] , and

[20] to

[22] , wherein the peptide (C) is bound to a lysine residue in the Fc region of the antibody (A), and the N-terminus of the amino acid sequence constituting the peptide (C) is bound to a linker (La).

[24] The radiometal-labeled modified antibody according to any one of

[15] ,

[16] and

[20] to

[23] , wherein the amino acid sequence of the peptide (C) is the following 2): 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2) [wherein Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid].

[25] The radiometal-labeled modified antibody according to

[24] , wherein the two cysteine ​​residues in 2) are disulfide-bonded to form a cyclic peptide.

[26] The radiometal-labeled modified antibody according to

[24] or

[25] , wherein Xaa1 in 2) is modified with a crosslinker, and the crosslinker is preferably DSG (disuccinimidyl glutarate) or DSS (disuccinimidyl suberate).

[27] The radiometal-labeled modified antibody according to any one of

[15] ,

[16] , and

[20] to

[26] , wherein the functional unit (D) has a structure in which ethylene glycol is polymerized with a degree of polymerization of 11 to 48.

[28] The radiometal-labeled modified antibody according to any one of

[15] ,

[16] , and

[20] to

[27] , wherein in formula (I), p = 0, and preferably B and D are connected via an amide bond.

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

[15] ,

[16] , and

[20] to

[28] , wherein in formula (I), La includes an antibody-modifying linker (La1) that modifies the IgG-binding peptide and a chelating linker (La2) that modifies the chelating moiety, and preferably the antibody-modifying linker (La1) is introduced to the N-terminus of the IgG-binding peptide (C), and the chelating linker (La2) is introduced to a functional group of the chelating moiety (B).

[30] The radioactive metal-labeled modified antibody according to

[29] , wherein the antibody-modifying linker (La1) and the chelating linker (La2) are bonded by a click reaction.

[31] The radioactive metal-labeled modified antibody according to

[29] or

[30] , wherein the binding site between the antibody-modifying linker (La1) and the chelating linker (La2) comprises the structure shown in formula (10a) or (10b) above.

[32] The radioactive metal-labeled modified antibody according to any of

[29] to

[31] , wherein the antibody-modifying linker (La1) comprises a polyethylene glycol (PEG) group.

[33] The radioactive metal-labeled modified antibody according to any of

[15] ,

[16] , and

[20] to

[32] , wherein in formula (I), the partial structure -B-(Lb)p-D is represented by formula (b) above.

[34] The radioactive metal-labeled modified antibody according to any of

[15] ,

[16] , and

[20] to

[33] , wherein the modified antibody represented by formula (I) has a structure represented by the following formula:

[0156]

[0157]

[35] The radiometal-labeled modified antibody according to

[34] , wherein the antibody (A) is an SKM9-2 antibody or an antibody against EGFR as an antigen.

[36] The radiometal-labeled modified antibody according to

[34] or

[35] , wherein the lysine residue of the antibody (A) to which the peptide (C) is bound is Lys248 or Lys246 in human IgG Fc according to EU numbering.

[37] A radioactive pharmaceutical comprising the radiometal-labeled antibody according to any of

[20] to

[36] as an active ingredient.

[38] The radioactive pharmaceutical according to

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

[39] A compound comprising the structure of the following formula (d), wherein a group containing dibenzocyclooctyne (DBCO) is attached to the position indicated by an asterisk in formula (d):

[0158]

[0159] In formula (d), o, p, and q each independently represent 0 or 1, and satisfy the relationship 1≦o+p+q≦3. Xd represents a structure represented by formula (c) or a structure in which a structure represented by formula (D-2) is connected via an amino group (NH). Xe represents a hydrogen atom when o is 0, and a CH ２When p is 0, Xf represents a hydrogen atom, and when p is 1, Xf represents CH ２ When q is 0, Xg represents a hydrogen atom, and when q is 1, Xg represents a CH ２ The asterisk (*) represents a bond.

[40] The compound according to

[39] , which is represented by the following formula:

[0160]

[0161] The present invention will be described in more detail below using examples, but the scope of the present invention is not limited thereto.

[0162] In the following examples, unless otherwise specified, all commercially available reagents were used as they were. For mass spectrometry (MS), an LCMS 2020 from Shimadzu Corporation (Kyoto, Japan) was used, and for high-resolution mass spectrometry (HRMS), an LCMS-IT-TOF was used. For reversed-phase HPLC, a Cosmosil C from Nacalai Tesque, Inc. (Tokyo, Japan) was used. １８ Column (5C １８ The analysis was carried out on a Shimadzu system equipped with a LC-20AD pump equipped with an SPD-20A UV detector, detection wavelength: 220 or 254 nm.

[0163] DBCO-DOTA-PEG48 (Example 1-1) Synthesis Scheme

[0164]

[0165] Synthesis of DBCO-DOTA-PEG48 (Example 1-1) (Synthesis of Compound 1) Compound 1 was synthesized according to a previous report (J. Med. Chem., 2023, 66, 12812-12827).

[0166] (Synthesis of Compound 2) Compound 1 (33 mg, 36 μmol) was dissolved in anhydrous N,N-dimethylformamide (DMF, 330 μL), and 1-((1-(cyano-2-ethoxy-2-oxo-ethylideneaminooxy)dimethylaminomorpholino))uronium hexafluorophosphate (COMU, 96 mg, 0.22 mmol) and N,N-diisopropylethylamine (DIPEA, 39 μL, 0.22 mmol) were added at 0° C., followed by stirring at 0° C. for 10 minutes. Thereafter, m-dPEG48-amine (60 mg, 28 μmol) was added, followed by stirring at room temperature overnight. The solution was washed with Cosmosil C １８ Column (5C １８ -AR-II, 10 mm ID x 250 mm) and mobile phase [H ２ The resulting mixture was purified by reverse-phase HPLC using a solvent mixture of 0.05% CO₂ / acetonitrile (MeCN) / trifluoroacetic acid (TFA) = 80 / 20 / 0.1 (0 min) to 40 / 60 / 0.1 (40 min) (flow rate 4 mL / min) to give 28 mg of compound 2 (33%). MS (ESI): m / z calculated for C １４４ H ２７６ N ６ O ５９ ２＋ , 1517.4 [M+2H] ２＋ ;found, 1517.2.

[0167] (Synthesis of Compound 3) A mixed solution of TFA / triisopropylsilane / ultrapure water = 95 / 4 / 1 (2 mL) was added to Compound 2 (20 mg, 6.4 μmol) and stirred at room temperature for 12 hours. Thereafter, the solvent was distilled off under an argon gas stream, and the resulting residue was dissolved in MeCN. After the solvent was distilled off under reduced pressure, the resulting residue was dissolved in Cosmosil C １８ Column (5C １８ -AR-II, 10 mm ID x 250 mm) and mobile phase [H ２The residue was purified by reverse-phase HPLC using a solvent mixture of 0.05% CO₂ / MeCN / TFA = 85 / 15 / 0.1 (0 min) to 55 / 45 / 0.1 (30 min) (flow rate 4 mL / min) to give 7.7 mg of compound 3 (45%). MS (ESI): m / z calculated for C １２０ H ２４０ N ６ O ５７ ４＋ ,669.7[M+4H] ４＋ ;found, 669.7.

[0168] (Synthesis of Compound 4: DBCO-DOTA-PEG48 (Example 1-1)) Compound 3 (7.7 mg, 2.9 μmol) was dissolved in anhydrous DMF (100 μL), and DBCO-NHS ester (2.3 mg, 5.7 μmol) and triethylamine (3.2 μL, 23 μmol) were added, followed by stirring at room temperature for 20 hours. The solution was purified by Cosmosil C １８ Column (5C １８ -AR-II, 10 mm ID x 250 mm) and mobile phase [H ２ The residue was purified by reverse-phase HPLC using a solvent mixture of 0.05% CO₂ / MeCN / TFA = 80 / 20 / 0.1 (0 min) to 50 / 50 / 0.1 (30 min) (flow rate 4 mL / min) to give 2.9 mg of compound 4 (35%). MS (ESI): m / z calculated for C １３９ H ２５３ N ７ O ５９ ４＋ ,741.4[M+4H] ４＋ ;found, 741.4.

[0169] Synthesis of DBCO-DOTA (Comparative Example 1-1) (Synthesis of Compound 5) DBCO-DOTA represented by the following formula was produced based on the method described in Bernhard et al. DOTAGA-Anhydride: A Valuable Building Block for the Preparation of DOTA-Like Chelating Agents Chem. Eur. J. 2012, 18, 7834-7841.

[0170]

[0171] Maleimide-DOTA-PEG48 (Comparative Example 1-2) Synthesis Scheme

[0172]

[0173] Synthesis of maleimide-DOTA-PEG48 (Comparative Example 1-2) (Synthesis of Compound 6) Compound 6 was synthesized according to a previous report (J. Chem. Soc., Chem. Commun., 1995, 185-186).

[0174] (Synthesis of Compound 7) Compound 7 was synthesized according to a previous report (Org. Biomol. Chem., 2013, 11, 1294-1305).

[0175] (Synthesis of Compound 8) Compound 8 was synthesized according to a previous report (Bioorg. Med. Chem. Lett., 2000, 10, 2133-2135).

[0176] (Synthesis of Compound 9) Compound 6 (1400 mg, 3.5 mmol) was dissolved in acetonitrile (25 mL), and compound 7 (1678 mg, 4.2 mmol) and potassium carbonate (579 mg, 4.2 mmol) were added thereto, followed by stirring at 50°C for 24 hours. Thereafter, filtration was performed, the filtrate was collected, and the solvent was distilled off. The residue was dissolved in acetonitrile (30 mL), and compound 8 (2496 mg, 7.0 mmol) and potassium carbonate (966 mg, 7.0 mmol) were added thereto, followed by stirring at 55°C for 15 hours. Thereafter, filtration was performed, the filtrate was collected, and the solvent was distilled off. The residue was purified by amine silica gel chromatography using a mobile phase (hexane / ethyl acetate = 1 / 1), to obtain 1274 mg of compound 9 (yield 37%). When MS (electrospray ionization) was performed on the obtained compound 9, the measured m / z was 996.5 (theoretical m / z was 996.6268 (C ５４ H ８６ N ５ O １２ ＋ , [M+H] ＋ )).

[0177] (Synthesis of Compound 10) Compound 9 (1274 mg, 1.28 mmol) was dissolved in methanol (25 mL), 10% palladium-carbon (350 mg) was added, and the mixture was vigorously stirred at room temperature for 48 hours under a hydrogen gas atmosphere. The mixture was then filtered using Hyflo Super Cel, and the solvent was distilled off from the filtrate. A portion of the residue (100 mg, 0.13 mmol) was dissolved in anhydrous DMF (700 μL), and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (160 mg, 0.52 mmol) and triethylamine (52 mg, 0.52 mmol) were added, followed by stirring at room temperature for 71 hours. The solution was purified by COSMOSILC １８ Column (5C １８ The mixture was purified by reversed-phase HPLC using a column separator (-AR-II, 10 mm ID x 250 mm) and a mobile phase (development conditions: 0.1% aqueous trifluoroacetic acid solution / 0.1% acetonitrile trifluoroacetic acid solution = 9 / 1 (0 min) - 3 / 7 (30 min), flow rate: 4 mL / min), and further purified by COSMOSILC １８ Column (5C １８ The resulting mixture was purified by reversed-phase HPLC using a column separator (4.6 mm ID x 150 mm) and a mobile phase (development conditions: 0.1% aqueous trifluoroacetic acid solution / 0.1% trifluoroacetic acid acetonitrile solution = 7 / 3 (0 min) to 4 / 6 (30 min), flow rate 1 mL / min) to obtain 19.6 mg of compound 10 (yield 16%). MS (electrospray ionization) of the obtained compound 10 revealed that the measured m / z was 965.4 (theoretical m / z was 965.6170 (C ４９ H ８５ N ６ O １３ ＋ , [M+H] ＋ )).

[0178] (Synthesis of Compound 11) Compound 10 (17 mg, 18 μmol) was dissolved in anhydrous DMF (300 μL), and COMU (7.7 mg, 36 μmol) and DIPEA (2.3 mg, 36 μmol) were added at 0°C, followed by stirring at 0°C for 15 minutes. Then, m-dPEG48-amine (39 mg, 18 μmol) was added, followed by stirring at room temperature for 136 hours. The solution was subjected to a COSMOSILC １８ Column (5C１８ The resulting mixture was purified by reversed-phase HPLC using a column separator (10 mm ID x 250 mm) and a mobile phase (development conditions: 0.1% aqueous trifluoroacetic acid solution / 0.1% trifluoroacetic acid acetonitrile solution = 9 / 1 (0 min) to 3 / 7 (30 min), flow rate 4 mL / min) to obtain 10 mg of compound 11 (yield 18%). When MS (electrospray ionization) was performed on the obtained compound 11, the measured m / z was 1546.7 (theoretical m / z was 1546.9588 (C １４６ H ２８１ N ７ O ６０ ２＋ , [M+2H] ２＋ )).

[0179] (Synthesis of Compound 12: Maleimide-DOTA-PEG48 (Comparative Example 1-2)) Compound 11 (5.0 mg, 1.6 μmol) was dissolved in a mixed solution of trifluoroacetic acid (1.6 mL) and dichloromethane (0.4 mL), and the solution was stirred at room temperature for 14 hours. Thereafter, the solvent was distilled off, and the residue was purified by COSMOSILC. １８ Column (5C １８ The resulting mixture was purified by reversed-phase HPLC using a column separator (4.6 mm ID x 150 mm) and a mobile phase (development conditions: 0.1% aqueous trifluoroacetic acid solution / 0.1% trifluoroacetic acid acetonitrile solution = 3 / 1 (0 min) to 11 / 9 (20 min), flow rate 1 mL / min) to obtain 2.0 mg of compound 12 (yield 43%). HRMS (electrospray ionization) of the obtained compound 12 revealed that the actual m / z was 1434.8340 (theoretical m / z was 1434.8336 (C １３０ H ２４９ N ７ O ６０ ２＋ , [M+2H] ２＋ )).

[0180] ・ ８９ Synthesis of Zr-labeled antibody drug (Example 2-1 and Comparative Example 2-1) ８９ The structure of the Zr-labeled antibody is shown in Figure 5. ８９Zr is chelated to the DOTA moiety. (Synthesis of Peptide-Modified SKM9-2 Antibody) A peptide containing 17 amino acid residues represented by the following formula (P3) was obtained using the method described in WO 2017 / 217347. The amino acid sequence of this peptide was identical to the sequence in SEQ ID NO: 2 described in paragraph

[0038] of the specification of the same document, in which Xaa1 is a lysine residue, and the terminal amino group of the side chain of the lysine residue was modified with the structure represented by R1. In addition, two cysteine ​​residues were disulfide-bonded to each other, and the N-terminus of the peptide was bound to ethyl azide as an atomic group containing an azide group via a linker structure having diglycolic acid and eight PEGs.

[0181]

[0182] (In formula (P3), Gly represents glycine, Pro represents proline, Asp represents aspartic acid, Cys represents cysteine, Ala represents alanine, Tyr represents tyrosine, His represents histidine, Lys represents lysine, Glu represents glutamic acid, Leu represents leucine, Val represents valine, Trp represents tryptophan, The represents threonine, and Phe represents phenylalanine.)

[0183] In this example and comparative example, the humanized SKM9-2 antibody described in Production Example 3 of WO 2023 / 277144 was used as the antibody. The above-mentioned peptide DSG (disuccinimidyl glutarate) was reacted with the humanized SKM9-2 antibody in accordance with the method described in WO 2017 / 217347. A solution containing the DSG-modified peptide and a solution containing the SKM9-2 antibody were mixed in 0.02 mol / L acetic acid / sodium acetate buffer (pH 6.0) and reacted at room temperature for 60 minutes to obtain a solution containing the peptide-modified antibody. This peptide-modified antibody had its Fc region site-specifically modified with the above-mentioned peptide. The solution was then passed through an IgG-BP column to obtain a first antibody composition containing a relatively large amount of monovalent antibodies. The concentration of the total antibody in the collected fractions was adjusted to 22 mg / mL with 0.02 mol / L histidine buffer (pH 6.0) containing 6 w / v % trehalose. The resulting monovalent peptide-modified humanized SKM9-2 antibody solution was subjected to the labeling step described below.

[0184] (1. Complex Formation Step) ( ８９ Synthesis of Zr-labeled DBCO-DOTA-PEG48 Compound 4 (Example 1-1) was dispersed in DMSO as a solvent to prepare a dispersion containing 3.375 mmol / L of a chelating agent. 0.008 mL of this dispersion, 0.082 mL of 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0), and 0.12 mL of 75 mmol / L gentisic acid solution (dissolved in 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0)) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ８９ A Zr complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ８９ The Zr ion ratio was about 218:1, and the reaction solution was heated to 70°C for 60 minutes. ８９The radiochemical purity of the Zr complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent was a mixture of acetonitrile and water (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected around Rf 0.2-0.5 relative to the total detected radioactivity (counts) was calculated. ８９ The radiochemical purity (%) of the Zr complex was determined. As a result, the radiochemical purity was 79.37%. ８９ The Zr complex solution was used directly in the labeling step.

[0185] ( ８９ Synthesis of Zr-labeled DBCO-DOTA) Compound 5 (Comparative Example 1-1) was dispersed in 156 mmol / L acetic acid-sodium acetate buffer solution (pH 5.5) as a solvent to prepare a dispersion containing 0.3 mmol / L of a chelating agent. 0.06 mL of this dispersion and 0.04 mL of a 150 mmol / L gentisic acid solution (dissolved in 156 mmol / L acetic acid-sodium acetate buffer solution (pH 5.5)) were added to a radioactive metal source (dissolved in 0.04 mL of 100 mmol / L hydrochloric acid solution) from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ８９ A Zr complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ８９ The Zr ion ratio was about 68:1, and the reaction solution was heated to 70°C for 60 minutes. ８９ The radiochemical purity of the Zr complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent was a mixture of acetonitrile and water (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the solvent front relative to the total detected radioactivity (counts) was calculated as ８９ The radiochemical purity (%) of the Zr complex was determined. As a result, the radiochemical purity was 96.73%. ８９ The Zr complex solution was used directly in the labeling step.

[0186] (2. Labeling step) The unpurified cellulose obtained through the above step (1) ８９ Zr complex (８９ Zr-labeled DBCO-DOTA-PEG48 and ８９ A solution containing the monovalent peptide-modified humanized SKM9-2 antibody was added to a solution of Zr-labeled DBCO-DOTA, and a click reaction was carried out at 37°C for 2 hours. ８９ A Zr-labeled antibody was obtained. The molar ratio of DBCO groups to azide groups during the reaction was approximately 1:1. ８９ The labeling rate of the Zr-labeled antibody was measured using a thin layer chromatography (Agilent, model number: SGI0001, developing solvent was a mixture of acetonitrile and 0.1 mol / L EDTA solution (pH 5.0) (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star), and the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts) detected was taken as the labeling rate (%). ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 (Example 2-1) was 78.60%, ８９ The Zr-labeled DBCO-DOTA-SKM9-2 (Comparative Example 2-1) was 78.16%. ８９ The Zr-labeled antibody solution was purified using an ultrafiltration filter (Merck, model number: UFC803024). ８９ The radiochemical purity and radiochemical yield of the Zr-labeled antibody were measured as follows: ８９ The radiochemical purity (%) of the Zr-labeled antibody was determined by measuring it. The radiochemical yield (%) was calculated as the percentage of the radioactivity recovered after purification relative to the total radioactivity added at the start of the labeling process. ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 was 68.54%; ８９ The radiochemical purity of Zr-labeled DBCO-DOTA-SKM9-2 was 70.30%. ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 was 89.41%; ８９ The Zr-labeled DBCO-DOTA-SKM9-2 was 96.42%.

[0187] ・ １１１Synthesis of In-Labeled Antibody Drug (Comparative Example 3-2) (Introduction of Thiol Groups into Humanized SKM9-2 Antibody) In this comparative example, the humanized SKM9-2 antibody described in Production Example 3 of WO 2023 / 277144 was used. A solution of humanized SKM9-2 antibody (891 μg) in 0.1 mol / L borate buffer solution (pH 8.0, 72.1 μL) containing 2 mmol / L EDTA was prepared. Furthermore, 2-iminothiolane hydrochloride was dissolved in 0.1 mol / L borate buffer solution (pH 8.0) containing 2 mmol / L EDTA to prepare a 2-iminothiolane hydrochloride solution with a concentration of 0.7 mmol / L. Next, the 2-iminothiolane hydrochloride solution was added to the humanized SKM9-2 solution. The amount of the 2-iminothiolane hydrochloride solution added was such that the amount of 2-iminothiolane hydrochloride was 2 molar equivalents relative to SKM9-2. The reaction solution thus obtained was incubated at room temperature in a light-shielded environment for 1 hour. The reaction solution was purified using a ZebaSpin Desalting Column (40K MWCO, 0.5 mL) to obtain humanized SKM9-2 (hereinafter also referred to as "SKM9-2-SH") into which a thiol group had been introduced. The purified SKM9-2-SH was immediately used in the labeling reaction described below.

[0188] ( １１１ Synthesis of In-labeled maleimide-DOTA-PEG48 (First-stage labeling) Compound 12 (1.0 mg / mL DMSO solution, 3 μL) was dissolved in 0.1 mol / L acetate buffer (pH 5.5, 200 μL) and [ １１１ In]InCl ３ After adding the solution (150 μL, 8.3 MBq), the reaction mixture was heated at 90° C. for 5 minutes. １８ Column (5C １８ -AR-II, 4.6 mm ID x 150 mm) and mobile phase (MeCN / H ２ The product was purified by reverse-phase HPLC using O / TFA [10 / 90 / 0.1 (0 min) to 30 / 70 / 0.1 (20 min)], flow rate 1.0 mL / min), and the solvent from the fractions containing the target compound was evaporated under a stream of nitrogen gas.

[0189] (Second-stage labeling) 0.1 mol / L phosphate buffer (pH 7, 500 μL) was added to the residue obtained in the first-stage labeling, and then 0.1 mol / L phosphate buffer solution (pH 7, 100 μL) of SKM9-2-SH (0.4 mg) was added, followed by incubation at room temperature in a light-shielded environment for 7.5 hours. The reaction solution was then purified and concentrated using Amicon Ultra-4 (collected fraction 50 kDa) to obtain the target product. １１１ The In-labeled antibody was obtained. The radiochemical purity was measured as follows: TSKgel G3000SW ＸＬ The radiochemical purity (%) was determined as the percentage of the area value of the radiolabeled compound relative to the area values ​​of all detected peaks. The radiochemical yield was measured as follows: １１１ The radioactivity of the In-labeled chelating linker after purification １１１ The radioactivity of the In-labeled antibody was calculated as a percentage. As a result, the radiochemical yield was 74.4%.

[0190] Formulation process (Example 4-1 and Comparative Examples 4-1 and 4-2) Produced according to Example 2-1 and Comparative Example 2-1 ８９ The Zr-labeled antibody was diluted with 0.02 mol / L histidine buffer (pH 6.0) containing 6 w / v% trehalose and 0.02 mol / L histidine buffer (pH 6.0) containing 0.3 w / v% polysorbate 20 and 6 w / v% trehalose to give 0.02 mol / L histidine buffer (pH 6.0) containing 0.03 w / v% polysorbate 20 and 6 w / v% trehalose. １１１ The In-labeled antibody was diluted in saline.

[0191] ・ ８９ Evaluation of antigen binding activity and internalization of Zr-labeled antibodies to live cells (Example 5-1) Using cultured cells, Zr-labeled antibodies were produced as described in Example 2-1 and formulated as described in Example 4-1. ８９The antigen binding activity of the Zr-labeled antibody was confirmed. HEG1-positive human malignant pleural mesothelioma cells, ACC-MESO-4 (Cancer Sci 2006; 97: 387-394) (purchased from the Cell Engineering Division of the RIKEN BioResource Research Center (hereinafter also referred to as "RIKEN BRC")), and HEG1-negative lung cancer cells, A549 (purchased from the European Collection of Authenticated Cell Cultures), were cultured in RPMI 1640 medium (Gibco) (containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco)) or Ham's medium. The cells were suspended in F-12K medium (Gibco) (containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco)), and plated onto a 6-well plate (3.8 × 10 ５ Cells / well or 4.0 x 10 ５ The cells were incubated in a 1000-well (1000-well) culture medium at 37°C under 5% carbon dioxide and atmospheric pressure for 24 hours. ８９Zr-labeled antibody (final added radioactivity: 1.1 kBq / well or 2.5 kBq / well) was added. 2.0 mL of medium containing 0.3 μg / well of antibody was added, and the plate was incubated for 24 or 48 hours at 37°C, 5% carbon dioxide, and atmospheric pressure. Each well was then washed twice with 1.0 mL of phosphate-buffered saline, and the removed medium and the phosphate-buffered saline used for washing were collected as the unreacted fraction. The phosphate-buffered saline was prepared by adding 1 L of ultrapure water (Millipore) to two packets of Dulbecco's phosphate-buffered saline powder (Ca- and Mg-free) (Fujifilm Wako Pure Chemical Industries, Ltd.). Then, 1 mL of 0.1 mol / L glycine-HCl buffer (pH 2.2) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the plate was left to stand on ice for 15 minutes. After collecting the 0.1 mol / L glycine-hydrochloric acid buffer (pH 2.2) as a cell surface fraction, a mixture of 0.05 mL of 2N aqueous sodium hydroxide solution (Nacalai Tesque) and ultrapure water (Millipore) was added to make a total volume of 1.0 mL, and the cells were lysed at room temperature. 0.9 mL of the mixture was collected as an internalized fraction, and the radioactivity in each fraction was measured using a gamma counter (model: 2470 WIZARD ２ The absorbance at 562 nm of the solution remaining in the wells was measured using a Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) with a plate reader (SpectraMax i3x, manufactured by Molecular Devices) to calculate the protein concentration. The ratio of the radioactivity of each fraction to the total radioactivity of all fractions was corrected for the protein amount (Uptake ratio (% added amount / mg)). ８９ The results of the antigen-binding activity of the Zr-labeled antibody are shown in Table 3. The radioactivity of the antibody bound to the cell surface (cell surface fraction) and the antibody internalized after binding (internalized fraction) was higher in HEG1-positive cells than in HEG1-negative cells, and the radioactivity of the cell surface fraction and internalized fraction increased up to 48 hours after addition of the agent.

[0192]

[0193] ・ ８９Evaluation of Stability of Zr-Labeled Antibody in Human Plasma (Example 6-1 and Comparative Example 6-1) Zr-labeled antibodies were produced according to the description in Example 2-1 or Comparative Example 2-1, and formulated according to the description in Example 4-1 or Comparative Example 4-1. ８９ The Zr-labeled antibody was stored in human plasma (pooled heparin Na, Cosmo Bio) at 37°C for 2 weeks, and the stability in the plasma was evaluated at each time point (day 0, day 1, day 6, and day 14). The human plasma was incubated at 37°C. ８９ Zr-labeled antibody was added to a final concentration of 60 kBq / mL. At each evaluation time point, immunoprecipitation was performed using Dynabeads® Protein G (Thermo Fisher Scientific). After washing with phosphate buffered saline containing 0.1% Tween 20 (BioRad) (hereinafter referred to as 0.1% Tween 20-containing PBS), the antibodies bound to the beads were recovered. The 0.1% Tween 20-containing PBS used for washing was recovered, and the radioactivity of the recovered wash solution and the radioactivity of the recovered antibodies were measured using an Autowell gamma counter (2480 WIZARD). ２ The measurement was performed using a gamma counter (PerkinElmer). The results of the evaluation of stability in human plasma were calculated by calculating the ratio of the radioactivity of the recovered antibody to the total radioactivity of the 0.1% Tween 20-containing PBS used to wash the beads and the recovered antibody at each time point, and then calculating the ratio to the average value at day 0 to calculate a numerical value (%). This value represents the radioactivity in human plasma. ８９ This can be considered as the stability of the Zr-labeled antibody. ８９ The Zr-labeled antibody was also shown to be 80% or more stable up to 14 days after the start of treatment.

[0194] Preparation of tumor-implanted mouse model HEG1-positive ACC-MESO-4 cells (purchased from RIKEN BRC) (5.0 × 10 ６ The cells (100 cells / mouse) were suspended and implanted subcutaneously in the flank of 5-6 week old male SHO mice under isoflurane anesthesia. The mice were kept for 10 weeks, and the tumor volume reached 250 mm３ Mice that reached this age were used in the experiments.

[0195] Evaluation of Internal Radioactivity Distribution Using Tumor-Injected Model Mice (Example 7-1 and Comparative Examples 7-1 and 7-2) Manufactured according to the description of Example 2-1 or Comparative Example 2-1, and formulated according to the description of Example 4-1 or Comparative Example 4-1. ８９ Zr-labeled antibody was prepared at 100 kBq / animal (n=3) or as described in Comparative Example 3-2 and formulated as described in Comparative Example 4-2. １１１ Tumor-implanted mouse models were administered 148 kBq / mouse (n=4) of In-labeled antibody (100 μL) via the tail vein, and the mice were euthanized 72 hours after administration. Blood and each organ were collected, and the organ weight and radioactivity were measured. The ratio of the amount of radioactivity to the amount of administered radioactivity (% ID) divided by the blood weight or organ weight (g) (% ID / g) was calculated as an index of internal radioactivity distribution. The results are shown in Table 4. In addition, the ratio of internal radioactivity distribution between tumor and blood (tumor-blood ratio) was calculated, and a parametric Dunnett multiple comparison test was performed using Stat Preclinica (manufactured by Takumi Information Technology Co., Ltd.) to conduct a significance test. The results are shown in Figure 1. As shown in Figure 1, with regard to the tumor-blood ratio, ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 (Example 2-1) ８９ Zr-labeled DBCO-DOTA-SKM9-2 (Comparative Example 2-1) and １１１ This was significantly higher than that of the In-labeled antibody (Comparative Example 3-2).

[0196]

[0197] PET / CT imaging using tumor-implanted model mice (Example 8-1 and Comparative Example 8-1) Manufactured according to the description of Example 2-1 or Comparative Example 2-1, and formulated according to the description of Example 4-1 or Comparative Example 4-1. ８９Zr-labeled antibody was administered (100 μL) via the tail vein of tumor-implanted mouse models at approximately 1 MBq / animal, and imaging was performed using small animal PET (Positron Emission Tomography) / CT (Computed Tomography) at 24, 48, 72, 120, and 168 hours after administration. PET imaging conditions were an acquisition time of 1200 seconds and an energy window of 357.7-664.3 KeV. Image reconstruction was performed using the Maximum Likelihood-Expectation Maximization method (Iterations: 12), incorporating safety (scatter correction, random correction, and attenuation correction), and PET images were obtained. The obtained PET quantitative values ​​were converted into the ratio of the radioactivity concentration at each site (Standard Uptake Value: SUV) using image analysis software PMOD (manufactured by PMOD), assuming that the administered radioactivity is uniformly distributed and not excreted, with the radioactivity concentration being set at 1. The imaging results 72 hours after administration are shown in Figure 2. The gradation bar in Figure 2 reflects the SUV, and the heart, tumor, and liver are indicated by arrows. As shown in Figure 2, ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 (Example 2-1) ８９ The tumor was visualized more clearly than with Zr-labeled DBCO-DOTA-SKM9-2 (Comparative Example 2-1). For quantitative analysis, regions of interest (Volume of Interest) were set on the tumor, heart (reflecting the blood radioactivity concentration), and muscle on the SUV-converted PET image, and the average SUV of the tumor at each time point (SUV ｍｅａｎ ) and cardiac and muscle SUVs ｍｅａｎ Obtain tumor SUVs ｍｅａｎ SUV of the heart or muscle ｍｅａｎ The tumor-organ ratio was calculated by dividing the value by 1. The results are shown in Tables 5 and 6. As shown in Tables 4 to 6, the tumor radioactivity uptake and the tumor-to-heart or tumor-to-muscle ratio were significantly higher at all time points except for the tumor-to-muscle ratio at 24 hours. ８９ Zr-labeled DBCO-DOTA-PEG48-SKM9-2 (Example 2-1) ８９It was higher than that of Zr-labeled DBCO-DOTA-SKM9-2 (Comparative Example 2-1).

[0198]

[0199]

[0200] ・ ２２５ Synthesis of Ac-labeled antibody drugs (SKM9-2 antibody and IgG4 antibody) (Example 9-1 and Comparative Examples 9-1 and 9-1-1) ２２５ The structure of the Ac-labeled antibody is shown in Figure 5. ２２５ Ac is chelated to the DOTA moiety. (Synthesis of Peptide-Modified SKM9-2 Antibody) A monovalent peptide-modified humanized SKM9-2 antibody solution was prepared using the methods described in Example 2-1 and Comparative Example 2-1. (Synthesis of Peptide-Modified IgG4 Antibody) The peptide DSG (disuccinimidyl glutarate) used in the synthesis of the peptide-modified SKM9-2 antibody was reacted with an IgG4 antibody (manufactured by Bio X Cell) according to the method described in WO 2017 / 217347. A solution containing the DSG-modified peptide and a solution containing the IgG4 antibody were mixed in a 0.02 mol / L histidine buffer (pH 6.0) and reacted at room temperature for 60 minutes to obtain a solution containing the peptide-modified antibody. This peptide-modified antibody had the Fc region of the antibody site-specifically modified with the above peptide. Next, the solution was passed through an IgG-BP column to obtain a first antibody composition containing a relatively large amount of monovalent antibodies. The obtained monovalent peptide-modified IgG4 antibody solution was subjected to the labeling step described below. (1. Complex Formation Step) ( ２２５ Synthesis of Ac-labeled DBCO-DOTA-PEG48 Compound 4 (Example 1-1) was dispersed in DMSO as a solvent to prepare a dispersion containing 3.375 mmol / L of a chelating agent. 0.004 mL of this dispersion, 0.041 mL of 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0), and 0.06 mL of 75 mmol / L gentisic acid solution (pH 5.0) (dissolved in 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0) and pH adjusted with 1 mol / L sodium hydroxide solution) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ２２５An Ac complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ２２５ The reaction mixture was heated to 70°C for 30 minutes. ２２５ The radiochemical purity of the Ac complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent: acetonitrile:water mixture (volume ratio 1:1)) after radioactive equilibration using a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the solvent front relative to the total detected radioactivity (counts) was calculated as ２２５ The radiochemical purity (%) of the Ac complex was determined. As a result, the radiochemical purity was 95.94%. ２２５ The Ac complex solution was used as is in the labeling step. ２２５ Synthesis of Ac-labeled DBCO-DOTA) Compound 5 (Comparative Example 1-1) was dispersed in 100 mmol / L acetic acid-sodium acetate buffer solution (pH 5.0) as a solvent to prepare a dispersion containing 0.3 mmol / L of a chelating agent. 0.045 mL of this dispersion and 0.06 mL of 75 mmol / L gentisic acid solution (pH 5.0) (dissolved in 100 mmol / L acetic acid-sodium acetate buffer solution (pH 5.0) and pH adjusted with 1 mol / L sodium hydroxide solution) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ２２５ An Ac complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ２２５ The reaction mixture was heated to 70°C for 30 minutes. ２２５ The radiochemical purity of the Ac complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent: acetonitrile:water mixture (volume ratio 1:1)) after radioactive equilibration using a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the solvent front relative to the total detected radioactivity (counts) was calculated as ２２５ The radiochemical purity (%) of the Ac complex was determined. As a result, the radiochemical purity was 99.39 to 100.00%.２２５ The Ac complex solution was used as is in the labeling step.

[0201] (2. Labeling step (SKM9-2 antibody)) The unpurified SKM9-2 antibody obtained through the above (1. Complex formation step) ２２５ Ac complex ( ２２５ Ac-labeled DBCO-DOTA-PEG48 and ２２５ A solution containing monovalent peptide-modified SKM9-2 antibody was added to a solution of Ac-labeled DBCO-DOTA, and a click reaction was carried out at 37°C for 2 hours. ２２５ An Ac-labeled antibody was obtained. The molar ratio of DBCO groups to azide groups during the reaction was approximately 1:1. ２２５ The labeling rate of the Ac-labeled antibody was determined by measuring the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts) detected using thin layer chromatography (Agilent, model number: SGI0001, developing solvent: a mixture of acetonitrile and 0.05 mol / L EDTA solution (pH 5.0) (volume ratio 1:1)) after radioactive equilibration using a radio-γ-TLC analyzer (Raytest, Model GITA Star). The labeling rate (%) was calculated as the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts). ２２５ Ac-labeled DBCO-DOTA-PEG48-SKM9-2 was 82.06%; ２２５ The Ac-labeled DBCO-DOTA-SKM9-2 was 89.56%. ２２５ The Ac-labeled antibody solution was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC803024). ２２５ The radiochemical purity and radiochemical yield of the Ac-labeled antibody were measured as follows: ２２５ The radiochemical purity (%) of the Ac-labeled antibody was determined by measuring the antibody. The radiochemical yield (%) was calculated as the percentage of the radioactivity recovered after purification relative to the total radioactivity added at the start of the labeling process. ２２５ Ac-labeled DBCO-DOTA-PEG48-SKM9-2 was 72.39%; ２２５ The radiochemical purity of Ac-labeled DBCO-DOTA-SKM9-2 was 80.90%. ２２５Ac-labeled DBCO-DOTA-PEG48-SKM9-2 was 97.68%; ２２５ The yield of Ac-labeled DBCO-DOTA-SKM9-2 was 97.80%.

[0202] (3. Labeling Step (IgG4 Antibody)) The unpurified IgG4 antibody obtained through the above step (1) ２２５ Ac complex ( ２２５ A solution containing monovalent peptide-modified IgG4 antibody was added to a solution of Ac-labeled DBCO-DOTA, and a click reaction was carried out at 37°C for 2 hours. ２２５ An Ac-labeled antibody was obtained. The molar ratio of DBCO groups to azide groups during the reaction was approximately 1:1. ２２５ The labeling rate of the Ac-labeled antibody was determined by measuring with a radio-γ-TLC analyzer (manufactured by raytest, Model GITA Star) after radio equilibration using thin layer chromatography (manufactured by Agilent, Model SGI0001, developing solvent was a mixture of acetonitrile and 0.1 mol / L EDTA solution (pH 5.0) (volume ratio 1:1)), and the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total detected radioactivity (counts) was taken as the labeling rate (%). As a result, the labeling rate was 84.54%. Next, the obtained ２２５ The Ac-labeled antibody solution was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC803024). ２２５ The radiochemical purity and radiochemical yield of the Ac-labeled antibody were measured as follows: ２２５ The radiochemical purity (%) of the Ac-labeled antibody was determined by measuring the antibody. The radiochemical yield (%) was determined as the percentage of the radioactivity recovered after purification relative to the total radioactivity added at the start of the labeling process. ２２５ The radiochemical yield of Ac-labeled DBCO-DOTA-IgG4 was 73.10%, and the radiochemical purity was 99.22%.

[0203] Formulation process (Example 10-1 and Comparative Examples 10-1 and 10-1-1) Produced according to Example 9-1 and Comparative Examples 9-1 and 9-1-1 ２２５The Ac-labeled antibody was diluted with 0.02 mol / L histidine buffer (pH 6.0) containing 6 w / v% trehalose and 0.02 mol / L histidine buffer (pH 6.0) containing 0.3 w / v% polysorbate 20 and 6 w / v% trehalose to give 0.02 mol / L histidine buffer (pH 6.0) containing 0.03 w / v% polysorbate 20 and 6 w / v% trehalose.

[0204] Preparation of tumor-transplanted mouse model for drug efficacy evaluation HEG1-positive ACC-MESO-4 cells (Cancer Sci 2006; 97: 387-394) (purchased from the Cell Engineering Division of the RIKEN BioResource Research Center (hereinafter also referred to as "RIKEN BRC")) (5.0 × 10 ６ The cells (1000 cells / mouse) were suspended and implanted subcutaneously in the flank of 7-8 week old male SHO mice under isoflurane anesthesia. The mice were kept for 3 weeks, and the tumor volume was 35.0-99.9 mm. ３ The mice were allowed to grow until they reached a size suitable for tumor diameter measurement, and were randomly divided into groups based on the shape of the individual mice. The average tumor volume and average body weight of each group are shown in Table 6. The tumor volume was calculated according to the following formula: Tumor volume (mm ３ ) = (tumor long diameter × (tumor short diameter) ２ ) x 1 / 2

[0205]

[0206] - Evaluation of efficacy using tumor-implanted model mice (Example 11-1 and Comparative Examples 11-1 and 11-1-1) Manufactured according to the description of Example 9-1, Comparative Example 9-1 or Comparative Example 9-1-1, and formulated according to the description of Example 10-1, Comparative Example 10-1 or Comparative Example 10-1-1. ２２５The Ac-labeled antibody was administered (100 μL) at a dose of approximately 300 kBq / kg via the tail vein of tumor-implanted model mice, and the antitumor effects were compared. A vehicle group was set up as a control group, to which 0.02 mol / L histidine buffer (pH 6.0) containing 0.03 w / v% polysorbate 20 and 6 w / v% trehalose was administered. Each group consisted of 5 to 6 mice, and their general condition was observed, and their body weight and tumor volume were measured over time up to 53 days after administration. The progression of tumor volume in each group of mice is shown in Figure 3. ２２５ The increase in tumor volume was also slower in the Ac-labeled antibody administration group compared to the vehicle group, confirming the antitumor effect. ２２５ The tumor volume in the Ac-labeled DBCO-DOTA-PEG48-SKM9-2 (Example 9-1) administration group was the smallest, ２２５ Ac-labeled DBCO-DOTA-SKM9-2 (Comparative Example 9-1) administration group, ２２５ A significant difference in antitumor effect was observed compared to the Ac-labeled DBCO-DOTA-IgG4 (Comparative Example 9-1-1) administration group and the vehicle group (P<0.05). A parametric Dunnett multiple comparison test was performed using the statistical analysis software Stat Preclinica to test for significance. Furthermore, there was no significant change in the general condition of either group, and no signs of toxicity, such as significant weight loss, were observed.

[0207] ・ ８９Synthesis of Zr-Labeled Antibody Drug (Panitumumab (Pmab)) (Example 12-1-2 and Comparative Examples 12-1-2 and 12-2-2) (Synthesis of Peptide-Modified Pmab) In accordance with the method described in WO 2017 / 217347, the peptide DSG (disuccinimidyl glutarate) used in the synthesis of the peptide-modified SKM9-2 antibody was reacted with Pmab (Vectibix (registered trademark), manufactured by Amgen). A solution containing the DSG-modified peptide and a solution containing -Pmab were mixed in 0.02 mol / L acetic acid / sodium acetate buffer (pH 6.0) and reacted at room temperature for 60 minutes to obtain a solution containing the peptide-modified antibody. This peptide-modified antibody had the Fc region of the antibody site-specifically modified with the above-mentioned peptide. Next, the solution was passed through an IgG-BP column to obtain a first antibody composition containing a relatively large amount of monovalent antibodies. The concentration of the total antibody in the collected fractions was adjusted to 22 mg / mL with 50 mmol / L sodium acetate buffer (pH 5.8) containing 0.1 mol / L sodium chloride. The resulting monovalent peptide-modified Pmab solution was subjected to the labeling step described below.

[0208] (Introduction of Thiol Groups into Pmab) A solution of Pmab (10.0 mg) in 0.1 mol / L borate buffer (pH 8.0, 200 μL) containing 2 mmol / L EDTA was prepared. Also, 2-iminothiolane hydrochloride was dissolved in 0.1 mol / L borate buffer (pH 8.0) containing 2 mmol / L EDTA to prepare a 2-iminothiolane hydrochloride solution with a 2-iminothiolane hydrochloride concentration of 0.7 mmol / L. Next, the 2-iminothiolane hydrochloride solution was added to the Pmab solution. The amount of the 2-iminothiolane hydrochloride solution added was such that the amount of 2-iminothiolane hydrochloride relative to the Pmab was 2 molar equivalents. The reaction solution thus obtained was incubated at room temperature in a light-shielded environment for 1 hour. The reaction solution was purified using a ZebaSpin Desalting Column (40K MWCO, 0.5 mL) to obtain Pmab having a thiol group introduced therein (hereinafter also referred to as "Pmab-SH"). The purified Pmab-SH was immediately subjected to the labeling step described below.

[0209] (1. Complex Formation Step) ( ８９Synthesis of Zr-labeled DBCO-DOTA-PEG48 Compound 4 (Example 1-1) was dispersed in DMSO as a solvent to prepare a dispersion containing 3.375 mmol / L of a chelating agent. 0.004 mL of this dispersion, 0.041 mL of 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0), and 0.06 mL of 75 mmol / L gentisic acid solution (dissolved in 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0)) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ８９ A Zr complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ８９ The Zr ion ratio was approximately 182:1, and the reaction solution was heated to 70°C for 60 minutes. ８９ The radiochemical purity of the Zr complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent was a mixture of acetonitrile and water (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected around Rf 0.2-0.5 relative to the total detected radioactivity (counts) was calculated. ８９ The radiochemical purity (%) of the Zr complex was determined. As a result, the radiochemical purity was 37.56%. ８９ The Zr complex solution was used directly in the labeling step.

[0210] ( ８９ Synthesis of Zr-labeled DBCO-DOTA) Compound 5 (Comparative Example 1-1) was dispersed in 100 mmol / L acetic acid-sodium acetate buffer solution (pH 5.0) as a solvent to prepare a dispersion containing 0.3 mmol / L of a chelating agent. 0.045 mL of this dispersion and 0.06 mL of 75 mmol / L gentisic acid solution (dissolved in 100 mmol / L acetic acid-sodium acetate buffer solution (pH 5.0)) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ８９ A Zr complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ８９ The Zr ion ratio was approximately 179:1, and the reaction solution was heated to 70°C for 60 minutes. ８９The radiochemical purity of the Zr complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent was a mixture of acetonitrile and water (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the solvent front relative to the total detected radioactivity (counts) was calculated as ８９ The radiochemical purity (%) of the Zr complex was determined. As a result, the radiochemical purity was 49.75%. ８９ The Zr complex solution was used directly in the labeling step.

[0211] ( ８９ Synthesis of Zr-labeled maleimide-DOTA-PEG48) Compound 6 was dispersed in DMSO as a solvent to prepare a dispersion containing 1.74 mmol / L of a chelating agent. 0.0078 mL of this dispersion, 0.0372 mL of 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0), and 0.06 mL of 75 mmol / L gentisic acid solution (dissolved in 100 mmol / L acetic acid-sodium acetate buffer (pH 5.0)) were added to the radioactive metal source from which the solvent had been distilled off, and the mixture was allowed to react under heating conditions. ８９ A Zr complex solution was obtained. The molar ratio of the chelating agent to the radioactive metal ions was as follows: ８９ The ratio of Zr ions to total ions was approximately 150:1, and the reaction solution was heated to 70°C for 60 minutes. ８９ The radiochemical purity of the Zr complex was measured by thin layer chromatography (Agilent, Model No. SGI0001, developing solvent was a mixture of acetonitrile and water (volume ratio 1:1)) with a radio-γ-TLC analyzer (Raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected around Rf 0.2-0.5 relative to the total detected radioactivity (counts) was calculated. ８９ The radiochemical purity (%) of the Zr complex was determined. As a result, the radiochemical purity was 52.80%. ８９ The Zr complex solution was used directly in the labeling step.

[0212] (2. Labeling process) ( ８９Zr-labeled DBCO-DOTA-PEG48-Pmab (Example 12-1-2) and ８９ Synthesis of Zr-labeled DBCO-DOTA-Pmab (Comparative Example 12-1-2) The unpurified DBCO-DOTA-Pmab obtained through the above (1. Complex formation step) ８９ Zr complex ( ８９ Zr-labeled DBCO-DOTA-PEG48 and ８９ A solution containing the monovalent peptide-modified Pmab was added to a solution of Zr-labeled DBCO-DOTA, and a click reaction was carried out at 37°C for 2 hours. ８９ A Zr-labeled antibody was obtained. The molar ratio of DBCO groups to azide groups during the reaction was approximately 1:1. ８９ The labeling rate of the Zr-labeled antibody was measured using a radio-γ-TLC analyzer (manufactured by Raytest, Model GITA Star) on thin layer chromatography (manufactured by Agilent, Model SGI0001, developing solvent was a mixture of acetonitrile and 0.05 mol / L EDTA solution (pH 5.0) (volume ratio 1:1)), and the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts) detected was taken as the labeling rate (%). ８９ Zr-labeled DBCO-DOTA-PEG48-Pmab was 40.44%; ８９ The Zr-labeled DBCO-DOTA-Pmab was 54.12%. ８９ The Zr-labeled antibody solution was purified using an ultrafiltration filter (Merck, model number: UFC803024). ８９ The radiochemical purity and radiochemical yield of the Zr-labeled antibody were measured as follows: ８９ The radiochemical purity (%) of the Zr-labeled antibody was determined by measuring it. The radiochemical yield (%) was calculated as the percentage of the radioactivity recovered after purification relative to the total radioactivity added at the start of the labeling process. ８９ Zr-labeled DBCO-DOTA-PEG48-Pmab was 39.98%; ８９ The radiochemical purity of Zr-labeled DBCO-DOTA-Pmab was 43.06%. ８９ Zr-labeled DBCO-DOTA-PEG48-Pmab was 78.38%;８９ The Zr-labeled DBCO-DOTA-Pmab was 83.21%.

[0213] ( ８９ Synthesis of Zr-labeled maleimide-DOTA-PEG48-Pmab (Comparative Example 12-2-2) ８９ Zr complex ( ８９ A solution containing -Pmab-SH was added to a solution of Zr-labeled maleimide-DOTA-PEG48, and the mixture was allowed to react overnight at 37°C. ８９ A Zr-labeled antibody was obtained. The molar ratio of maleimide to Pmab-SH during the reaction was approximately 1:2. ８９ The labeling rate of the Zr-labeled antibody was measured using a radio-γ-TLC analyzer (manufactured by raytest, Model GITA Star) on thin layer chromatography (manufactured by Agilent, Model SGI0001, developing solvent was a mixture of acetonitrile and 0.05 mol / L EDTA solution (pH 5.0) (volume ratio 1:1)), and the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total detected radioactivity (counts) was taken as the labeling rate (%). As a result, the labeling rate was 47.21%. Next, ８９ The Zr-labeled antibody solution was purified using an ultrafiltration filter (Merck, model number: UFC803024). ８９ The radiochemical purity and radiochemical yield of the Zr-labeled antibody were measured as follows: ８９ The radiochemical purity (%) of the Zr-labeled antibody was determined by measuring the antibody. The radiochemical yield (%) was calculated as the percentage of radioactivity recovered after purification relative to the total radioactivity added at the start of the labeling process. The radiochemical yield was 22.03%, and the radiochemical purity was 91.24%.

[0214] Formulation process (Example 13-1-2 and Comparative Examples 13-1-2 and 13-2-2) Produced according to Example 12-1-2 and Comparative Examples 12-1-2 and 12-2-2 ８９ The Zr-labeled antibody was diluted with 50 mmol / L sodium acetate buffer (pH 5.8) containing 0.1 mol / L sodium chloride.

[0215] ・ ８９ Evaluation of antigen binding activity and internalization of Zr-labeled antibodies to live cells (Example 14-1-2) Using cultured cells, Zr-labeled antibodies were produced as described in Example 12-1-2 and formulated as described in Example 13-1-2. ８９ The antigen binding activity of Zr-labeled DBCO-DOTA-PEG48-Pmab was confirmed. EGFR-positive human epidermoid carcinoma cells, A431 (purchased from the European Collection of Authenticated Cell Culture), were suspended in Dulbecco's Modified Eagle Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). The cells were plated onto a 24-well plate (1.5 × 10 ５ The cells were incubated in a 1000-well (1000-well) culture medium at 37°C under 5% carbon dioxide and atmospheric pressure for 24 hours. ８９ Zr-labeled antibody (final added radioactivity: 2.0 kBq / well, final added antibody amount: 0.1 μg / well; hereinafter also referred to as the experimental group) or an excess amount of panitumumab was added. ８９1.1 mL of medium containing Zr-labeled antibody (final added radioactivity: 2.0 kBq / well, final added antibody amount: 158.4 μg / well; hereafter referred to as the competitive inhibition group) was added and incubated for 24 hours at 37°C, 5% carbon dioxide, and atmospheric pressure. Each well was then washed twice with 1.0 mL of phosphate-buffered saline (PBS). The removed medium and the PBS used for washing were collected as the unreacted fraction. PBS was prepared by adding 1 L of ultrapure water (Millipore) to two packets of Dulbecco's phosphate-buffered saline powder (Ca- and Mg-free) (Fujifilm Wako Pure Chemical Industries, Ltd.). Then, 1 mL of 0.1 mol / L glycine-HCl buffer (pH 2.2) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added and the wells were left to stand on ice for 15 minutes. After collecting the 0.1 mol / L glycine-HCl buffer (pH 2.2) as a cell surface fraction, a mixture of 0.05 mL of 2N aqueous sodium hydroxide solution (Nacalai Tesque) and ultrapure water (Millipore) was added to make a total volume of 1.0 mL, and the cells were lysed at room temperature. 0.9 mL of the mixture was collected as an internalized fraction, and the radioactivity in each fraction was measured using a gamma counter (model: 2470 WIZARD). ２ The protein concentration was calculated by measuring the absorbance at 562 nm of the solution remaining in the wells using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and a plate reader (SpectraMax i3x, Molecular Devices). The protein concentration was calculated by correcting the ratio of the radioactivity of each fraction to the total radioactivity of all fractions by the amount of protein (Uptake ratio (% added amount / mg)). The results of antigen binding activity are shown in Table 7. The antibody bound to the cell surface (cell surface fraction) and the antibody internalized after binding (internalized fraction) were higher in the experimental group than in the competitive inhibition group.

[0216]

[0217] ・ ８９Evaluation of Stability of Zr-Labeled Antibody in Human Plasma (Example 15-1-2 and Comparative Example 15-1-2) Zr-Labeled Antibody was produced according to the description in Example 12-1-2 or Comparative Example 12-1-2, and formulated according to the description in Example 13-1-2 or Comparative Example 13-1-2. ８９ The Zr-labeled antibody was stored in human plasma (pooled heparin Na, manufactured by Cosmo Bio) at 37°C for 15 days, and the stability in the plasma was evaluated at each time point (1 day, 7 days, and 15 days). The human plasma was incubated at 37°C. ８９ Zr-labeled antibody was added to a final concentration of 1 MBq / mL. At each evaluation time point, immunoprecipitation was performed using Dynabeads (registered trademark) Protein G (manufactured by Thermo Fisher Scientific). After washing with PBS containing 0.1% Tween 20 (manufactured by BioRad) (hereinafter referred to as 0.1% Tween 20-containing PBS), the antibodies bound to the beads were recovered. The 0.1% Tween 20-containing PBS used for washing was recovered, and the radioactivity of the recovered wash solution and the radioactivity of the recovered antibody were measured using an Autowell gamma counter (2480 WIZARD ２ Measurements were made using a gamma counter (PerkinElmer). At each time point, the ratio of the radioactivity of the recovered antibody to the total of the radioactivity of the 0.1% Tween 20-containing PBS used to wash the beads and the radioactivity of the recovered antibody was calculated, and the percentage of this ratio relative to the average value of the ratio at day 0 measured by thin-layer chromatography was calculated. This value was the ratio of the radioactivity of the recovered antibody to the total radioactivity of the recovered antibody in human plasma. ８９ This can be considered the stability of the Zr-labeled antibody, and after 15 days ８９ Zr-labeled DBCO-DOTA-PEG48-Pmab (Example 12-1-2) was 85.7%, ８９ Zr-labeled DBCO-DOTA-Pmab (Comparative Example 12-1-2) remained stable at 84.7%. The measurement results at all time points are shown in Table 8. There was almost no change after 1 day (proportion of unchanged substance: almost 100%).

[0218]

[0219] Preparation of tumor-implanted mouse model for PET / CT imaging EGFR-positive HCT116 cells (purchased from American Type Culture Collection) (3.0 × 10 cells) were inoculated into a mixture (1:1, 100 μL) of McCoy's 5A (Gibco) and high-concentration Matrigel basement membrane matrix (Corning Life Sciences). ６ The cells (1000 cells / mouse) were suspended and implanted subcutaneously into the flank of 5-week-old male BALB / c nu / nu mice under isoflurane anesthesia. The mice were kept for 12 days, and tumor volumes were determined to be 417.9-1122.7 mm. ３ Mice were used in the experiment.

[0220] PET / CT imaging using tumor-implanted model mice (Example 16-1-2 and Comparative Examples 16-1-2 and 16-2-2) Produced according to the description of Example 12-1-2, Comparative Example 12-1-2, or Comparative Example 12-2-2, and formulated according to the description of Example 13-1-2, Comparative Example 13-1-2, or Comparative Example 13-2-2. ８９Approximately 2-3 MBq / animal of Zr-labeled panitumumab antibody was administered (100 μL) via the tail vein of tumor-implanted model mice, and imaging was performed using small animal PET (Positron Emission Tomography) / CT (Computed Tomography) 24, 48, 72, 120, and 168 hours after administration. The PET imaging conditions were an acquisition time of 600 seconds and an energy window of 357.7-664.3 KeV. Image reconstruction was performed using the Maximum Likelihood-Expectation Maximization method (iterations: 12) incorporating accuracy (scatter correction, random correction, and attenuation correction), and PET images were obtained. The quantitative PET values ​​obtained were converted into the ratio of radioactivity concentration at each site (Standard Uptake Value: SUV) using image analysis software PMOD (manufactured by PMOD), assuming that the administered radioactivity is uniformly distributed and not excreted, with the radioactivity concentration set to 1. The imaging results 120 hours after administration are shown in Figure 4. The gradient bar in Figure 4 reflects the SUV, and the heart, tumor, and liver are indicated by arrows. For quantitative analysis, regions of interest (Volume of Interest) were set on the tumor, heart (reflecting blood radioactivity concentration), and muscle on the SUV-converted PET image, and the average SUV of the tumor at each time point (SUV ｍｅａｎ ) and cardiac and muscle SUVs ｍｅａｎ Obtain tumor SUVs ｍｅａｎ SUV of the heart or muscle ｍｅａｎ The tumor-organ ratio was calculated by dividing the value by 1. The results are shown in Tables 9 to 11. As shown in Tables 9 to 11, the tumor radioactivity uptake and the ratio of tumor to heart or muscle were significantly higher at all time points. ８９ Zr-labeled DBCO-DOTA-PEG48-Pmab (Example 12-1-2) ８９ Zr-labeled DBCO-DOTA-Pmab (Comparative Example 12-1-2) and ８９ It was higher than that of Zr-labeled DOTA-PEG48-Pmab (Comparative Example 12-2-2).

[0221]

[0222]

[0223]

[0224] This application is based on patent application No. 2024-153439 filed in Japan (filing date: September 5, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A modified antibody represented by the following formula (I): A-C-La-B-(Lb)p-D (I) [wherein, A is an antibody that exhibits binding to a cell surface antigen, wherein the antibody has a cellular internalization function; B is a chelating moiety that can coordinate to a radioactive metal ion; C is a peptide that binds to a specific site in the Fc region of an antibody; D is a functional unit that improves intracellular retention; La is a linker a connecting C and B; Lb is a linker b connecting B and D; and p is 0 or 1].

2. The modified antibody according to claim 1, wherein the antibody (A) is an antibody that exhibits binding to a tumor cell surface antigen.

3. The modified antibody of claim 1, wherein the antibody (A) is an SKM9-2 antibody.

4. The modified antibody of claim 1, wherein the chelating moiety (B) is DOTA or a derivative thereof.

5. The radioactive metal is Al １８ F. ６４ Cu, ６７ Cu, ６８ Ga, ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu and ２２５ The modified antibody of claim 1, wherein the modified antibody is one selected from the group consisting of:

6. The peptide (C) is represented by the following formula (1) (X): １－３ -Cys-(X) ２ -His-(Xaa1)-Gly-(Xaa2)-Leu-Val-Trp-Cys-(X) １－３ (1) The modified antibody according to claim 1, comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by the formula: [wherein each X is independently any amino acid residue other than cysteine, Cys is a cysteine ​​residue, His is a histidine residue, Xaa1 is a lysine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, Gly is a glycine residue, Xaa2 is a glutamic acid residue, glutamine residue, or asparagine residue, Leu is a leucine residue, Val is a valine residue, and Trp is a tryptophan residue].

7. The modified antibody according to claim 1, wherein the amino acid sequence of peptide (C) is any one of those listed in 1) to 18) below: 1) DCAYH(Xaa1)GELVWCT (SEQ ID NO: 1), 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2), 3) RCAYH(Xaa1)GELVWCS (SEQ ID NO: 3), 4) GPRCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 4), 5) SPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 5), 6) GDDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 6), 7) GPSCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 7), 8) GPDCAYH(Xaa1)GELVWCSFH (SEQ ID NO: 8). 9) GPDCAYH(Xaa1)GELVWCTHH (SEQ ID NO: 9), 10) GPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 10), 11) SPDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 11), 12) SDDCAYH(Xaa1)GELVWCTFY (SEQ ID NO: 12), 13) RGNCAYH(Xaa1)GQLVWCTYH (SEQ ID NO: 13), 14) G(Xaa3)DCAYH(Xaa1)GELVWCT(Xaa3)H (SEQ ID NO: 14), 15) DCTYH(Xaa1)GNLVWCT (SEQ ID NO: 15), 16) DCAYH(Xaa1)GNLVWCT (SEQ ID NO: 16), 17) DCTYH(Xaa1)GELVWCT (SEQ ID NO: 17), and 18) DCAWH(Xaa1)GELVWCT (SEQ ID NO: 18) (wherein Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and Xaa3 is homocysteine, preferably wherein the homocysteines form a disulfide bond with each other).

8. The modified antibody according to claim 1, wherein the functional unit (D) has a structure in which ethyleneimine or ethylene glycol is polymerized, or a structure capable of binding to cathepsin B.

9. The modified antibody according to claim 1, wherein the functional unit (D) has a structure in which ethyleneimine is polymerized with a degree of polymerization of 1 or more and 7 or less.

10. The modified antibody described in claim 1, wherein the functional unit (D) has a structure in which ethylene glycol is polymerized with a degree of polymerization of 11 or more and 48 or less.

11. The modified antibody of claim 1, wherein the linker a (La) comprises a structure shown in formula (10a), formula (10b), or formula (10c): [In formula (10a) and formula (10b), R １Ａ represents the linking site with the chelating moiety (B), and R ２Ａ represents a linking site with the peptide (C), and in formula (10c), R ３Ａ and R ４Ａ one of which represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a linking site with the chelating moiety (B), and R ５Ａ indicates the linking site to the peptide].

12. The modified antibody of claim 1, wherein the linker b (Lb) is absent.

13. The modified antibody of claim 1, wherein in formula (I), the partial structure -B-(Lb)p-D is represented by the following formula (a) or (b): (In formulas (a) and (b), * (asterisk) represents a bond, n represents an integer of 1 to 7, and m represents an integer of 11 to 48).

14. In formula (I), the antibody (A) is an SKM9-2 antibody, and the partial structure -B-(Lb)p-D is represented by the following formula (b): (wherein * (asterisk) is a bond, and m is an integer of 11 to 48), and the linker a (La) contains a structure represented by the following formula (10a) or formula (10b): [In formula (10a) and formula (10b), R １Ａ represents the linking site with the chelating moiety (B), and R ２Ａ indicates the linking site to said peptide (C), and peptide (C) has the amino acid sequence shown in SEQ ID NO:

2. The modified antibody according to claim 1 .

15. A radioactive metal-labeled antibody in which the modified antibody of claim 1 is labeled with a radioactive metal.

16. The radioactive metal is Al １８ F. ６４ Cu, ６７ Cu, ６８ Ga, ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu and ２２５ The radioactive metal-labeled antibody of claim 15, wherein the radioactive metal-labeled antibody is one selected from the group consisting of IgG, ...

17. A radiopharmaceutical containing the radioactive metal-labeled antibody according to claim 16 as an active ingredient.

18. The radiopharmaceutical according to claim 17, which is used for cancer diagnosis or internal radiotherapy of cancer.

19. A compound having a structure represented by the following formula (d), in which a group containing dibenzocyclooctyne (DBCO) is connected to the position indicated by an asterisk in formula (d): In formula (d), o, p, and q each independently represent 0 or 1 and satisfy 1≦o+p+q≦3, and Xd represents a structure represented by the following formula (c) or a structure in which a structure represented by formula (D-2) is connected via an amino group (NH): In formula (c), X １ and X ２ each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxymethyl group, an aminomethyl group, or a bond; ａ and X ｂ each independently represents O or NH, Xe represents a hydrogen atom when o is 0, and represents CH when o is 1. ２ Xf represents a hydrogen atom when p is 0, and represents CH when p is 1. ２ Xg represents a hydrogen atom when q is 0, and represents CH when q is 1. ２ Represents. In formula (D-2), X represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, an aminomethyl group, a hydroxymethyl group, or a bond; n represents 11 to 48; and * (asterisk) represents a bond.

Images