Method for producing radioactive metal-labeled antibodies

Abstract

To provide a method for producing a radioactive metal-labeled antibody.SOLUTION: A method for producing a radioactive metal-labeled antibody of the present invention comprises a step of generating a radioactive metal-labeled antibody by causing a radioactive metal complex to undergo a click reaction with an antibody site-specifically modified by a peptide. The click reaction is performed between a first atomic group included in the radioactive metal complex and a second atomic group directly or indirectly linked to the peptide. The second atomic group is an atomic group containing an azide or an atomic group containing trans-cyclooctene.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing radioactive metal-labeled antibodies. [Background technology]

[0002] Antibodies are used as reagents for detecting target molecules, diagnostic agents, or pharmaceuticals for treating diseases, utilizing their specificity for target molecules. To further improve detection performance and therapeutic efficacy, research is underway on antibodies conjugated with radionuclides or drugs.

[0003] Patent Document 1 describes a peptide comprising an amino acid sequence of 13 to 17 amino acid residues that can bind to a human antibody. The same document also describes that the peptide can be modified with labeling substances such as complexes containing radioisotopes or drugs such as anticancer agents, and that it can form a complex with an antibody.

[0004] Patent Document 2 describes a radioactive metal. 111 A complex of a peptide modified with a DTPA complex of In and an antibody is described. It is also described that this antibody complex specifically binds to a target molecule expressed in tumors. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2016 / 186206 Pamphlet [Patent Document 2] International Publication No. 2017 / 217347 Brochure [Patent Document 3] International Publication No. 2019 / 125982 brochure [Overview of the project]

[0006] Incidentally, when producing antibodies bound to radioactive metal complexes, depending on the combination of ligand and radioactive metal used, it may be necessary to heat the reaction system to increase the efficiency of complex formation. However, under these conditions, the antibody is denatured by heat, making it impossible to obtain the desired antibody. Patent documents 1 and 2 do not address any reaction conditions that can solve this problem.

[0007] Therefore, the object of the present invention is to provide a method for producing radiometal-labeled antibodies that exhibits excellent labeling efficiency of radiometals to antibodies, even under mild reaction conditions.

[0008] The present invention comprises a step of generating a radiometal-labeled antibody by causing a click reaction between a radiometal complex and an antibody site-specifically modified with a peptide. The click reaction takes place between a first atomic group of the radioactive metal complex and a second atomic group that is directly or indirectly linked to the peptide. The present invention provides a method for producing a radioactive metal-labeled antibody, wherein the second atomic group is an atomic group containing an azide group or an atomic group containing trans-cyclooctene.

[0009] Furthermore, the present invention relates to a radioactive metal-labeled antibody that is site-specifically modified with a peptide, The present invention provides a radiometal-labeled antibody in which a radioactive metal complex is directly or indirectly linked to a peptide, and a triazole skeleton-containing structure represented by the following formula (10a) is present between the peptide and the radioactive metal complex, or a pyridazine skeleton-containing structure is present between the peptide and the radioactive metal complex.

[0010] [ka]

[0011] (In the formula, R 1A R indicates the bonding site with the modified or chelated portion. 2A (This indicates the binding site with the peptide.)

[0012] The present invention also provides a chelate linker having a chelate portion that coordinates to a radioactive metal and a modification portion comprising a click-reactive first atomic group, wherein the first atomic group is an atomic group represented by either formula (1a) or (1b) below.

[0013] [ka]

[0014] (In formula (1a), R1 represents a bonding site with the modified or chelated moiety, and in formula (1b), one of R3 and R4 represents a bonding site with the modified or chelated moiety, while the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group.)

[0015] Furthermore, the present invention relates to a peptide-modified antibody that is site-specifically modified by a peptide comprising an amino acid sequence consisting of 13 to 17 amino acid residues, represented by the following formula (i). The present invention provides a peptide-modified antibody having a click-reactable second atomic group at the N-terminus or C-terminus of the peptide, wherein the second atomic group is represented by either formula (2a) or (2b) below. (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) (In the formula, Xa, Xb, Xc, and Xd represent a consecutive a number of X's, a consecutive b number of X's, a consecutive c number of X's, and a consecutive d number of X's, respectively.) X is an amino acid residue that does not have either a thiol group or a haloacetyl group in its side chain. a, b, c, and d are each independent integers between 1 and 5, and satisfy a+b+c+d≦14. Xaa1 and Xaa3 operate independently of each other. Represents an amino acid residue derived from an amino acid having a thiol group in its side chain, and is linked via a disulfide bond or the sulfide group is linked via a linker. Or, One side represents an amino acid residue derived from an amino acid having a thiol group in its side chain, and the other side represents an amino acid residue derived from an amino acid having a haloacetyl group in its side chain, and they are linked via a thioether bond. Xaa2 is a lysine residue, arginine residue, cysteine ​​residue, aspartic acid residue, glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid.

[0016] [ka]

[0017] (In formula (2a), R2 represents the binding site to the N-terminus or C-terminus of the peptide, and in formula (2b), R5 represents the binding site to the N-terminus or C-terminus of the peptide.)

[0018] According to the present invention, even under mild reaction conditions, the labeling efficiency of radioactive metals to antibodies is excellent. [Modes for carrying out the invention]

[0019] The method for producing the radioactive metal-labeled antibody of the present invention will be described below based on a preferred embodiment.

[0020] The present invention provides a manufacturing method comprising a step (labeling step) of generating a radioactive metal-labeled antibody by causing a click reaction between a radioactive metal complex and an antibody site-specifically modified with a peptide (hereinafter also simply referred to as a "peptide-modified antibody"). Details of the radioactive metal, its complex, and the peptide will be described later.

[0021] The radioactive metal complex and the peptide-modified antibody each possess click-reaction-capable atomic groups, which react with each other to bind the radioactive metal complex and the peptide-modified antibody to one another. In other words, the click reaction in this process takes place between the first atomic group of the radioactive metal complex and the second atomic group that is directly or indirectly linked to the peptide in the peptide-modified antibody.

[0022] "Directly or indirectly" refers to whether or not a linker structure, as described below, exists between the second atomic group and the peptide. "Directly" means that there is no linker structure, and more specifically, that the second atomic group is bound to the N-terminus or C-terminus of the peptide without a linker structure. "Indirectly" means that there is a linker structure, and more specifically, that the second atomic group is bound to the N-terminus or C-terminus of the peptide via a linker structure. In either the "directly" or "indirectly" mode, it is preferable that the second atomic group is bound to the N-terminus of the peptide.

[0023] The linker structure described above is represented by the following equation (S1). **-((L1) m -Z) k -L2-AG2···(S1) (In the formula, ** indicates the binding site to the N-terminus or C-terminus of the peptide.) L1 is the polyethylene glycol (PEG) linker portion. m is an integer between 1 and 50, Z is (L1) m This is the second linker section that connects L2, k is either 0 or 1. L2 is the second PEG linker section, AG2 is the second atomic group.

[0024] In the above equation (S1), the structure of Z is (L1) m The linker structure that connects Z and L2 is not particularly limited, but for example, it may include an amino acid sequence consisting of 1 to 5 amino acid residues. In this case, the amino acid sequence contained in Z preferably contains a cysteine ​​residue, and more preferably it is bound to L2 via a thioether group formed by the bonding of the thiol group and maleimide group of the cysteine ​​residue.

[0025] In the present invention, the PEG linker portion constituting L2 preferably has the structure shown in the following formula (P1). In formula (P1), n ​​is an integer, preferably 1 to 50, more preferably 1 to 20, and even more preferably 2 to 10.

[0026] [ka]

[0027] One end of the PEG linker structure may be modified by a structure derived from a commercially available PEGylation reagent or a reagent commonly used in PEGylation, and is not particularly limited, but examples include structures derived from diglycolic acid or its derivatives, maleimide or its derivatives.

[0028] In this process, the combination of atomic groups that can undergo a click reaction is selected appropriately depending on the type of click reaction. Examples include a combination of alkyne and azide, or a combination of 1,2,4,5-tetrazine and alkene. These atomic groups only need to have one of the aforementioned atomic groups as the first atomic group and an atomic group that is a combination of the first atomic group as the second atomic group. From the viewpoint of achieving both the stability of the radioactive metal complex and the peptide-modified antibody and the improvement of their binding efficiency, it is preferable that the first atomic group is an alkyne and the second atomic group is an azide, or that the first atomic group is 1,2,4,5-tetrazine and the second atomic group is an alkene. Specific examples of click reactions using such atomic group combinations include the Huisgen cycloaddition reaction or the reverse electron-demanded Diels-Alder reaction.

[0029] Specific examples of click-reaction-compatible atomic group combinations include, as shown in the following formulas, a combination of an atomic group containing dibenzylcyclooctyne (DBCO) as the alkyne in the first atomic group (formula (1a)) and an atomic group containing an azide group as the azide in the second atomic group (formula (2a)), or a combination of an atomic group containing 1,2,4,5-tetrazine as the first atomic group (formula (1b)) and an atomic group containing trans-cyclooctene (TCO) as the alkene in the second atomic group (formula (2b)).

[0030] [ka]

[0031] (In formula (1a), R1 indicates a binding site to the modified or chelated portion, and in formula (2a), R2 indicates a binding site to the peptide in the peptide-modified antibody.)

[0032] [ka]

[0033] (In formula (1b), one of R3 and R4 represents a bonding site to another structure, and the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group; in formula (2b), R5 represents a bonding site to another structure.)

[0034] In this process, the order of addition of the radioactive metal complex and the peptide-modified antibody does not matter as long as they can react via a click reaction. For example, one of the radioactive metal complex and the peptide-modified antibody may be added to a reaction vessel containing a solvent, followed by the other, or one of the radioactive metal complex and the peptide-modified antibody may be dispersed in a solvent and then the other may be added to the dispersion and reacted. Alternatively, they may be added simultaneously to a reaction vessel containing a solvent and reacted.

[0035] As the solvent used in this process, a solvent containing water can be used. For example, water, physiological saline, or buffers such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered physiological saline, tris-hydroxymethylaminomethane buffer (hereinafter simply referred to as "Tris buffer"), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (hereinafter simply referred to as "HEPES buffer"), or tetramethylammonium acetate buffer can be used. When using a buffer, from the viewpoint of achieving both the stability of the complex and the antibody and their binding efficiency, the lower limit of the pH at 25°C is preferably 3.5 or higher, more preferably 4.0 or higher, even more preferably 4.5 or higher, even more preferably 5.0 or higher, and particularly preferably 5.5 or higher. The upper limit is preferably 10.0 or lower, more preferably 9.5 or lower, even more preferably 9.0 or lower, even more preferably 8.5 or lower, and particularly preferably 8.0 or lower. The preferred range is 4.0 to 10.0, and the even more preferred range is 5.5 to 8.5.

[0036] From the viewpoint of preventing unintended denaturation of peptide-modified antibodies while increasing the binding efficiency between the radioactive metal complex and the peptide-modified antibody, the reaction temperature for the click reaction in this step is preferably 120°C or lower, more preferably 90°C or lower, even more preferably 50°C or lower, and even more preferably 40°C or lower. The lower limit of the reaction temperature is not particularly limited as long as it is a temperature at which a click reaction can occur, but it is preferably 10°C or higher, more preferably 15°C or higher, even more preferably 20°C or higher, even more preferably 30°C or higher, and especially preferably 35°C or higher. The reaction time for the click reaction, provided that the above reaction temperature is met, is preferably 5 minutes or more, more preferably 10 minutes or more, even more preferably 20 minutes or more, even more preferably 30 minutes or more, and especially preferably 60 minutes or more. The upper limit is preferably 36 hours or less, more preferably 24 hours or less, even more preferably 20 hours or less, and especially preferably 15 hours or less. The preferred range is 5 minutes to 24 hours, and the even more preferred range is 10 minutes to 20 hours.

[0037] The volume of the reaction solution is not particularly limited, but from the viewpoint of practicality in the manufacturing process, at the start of this process, the lower limit is preferably 0.01 mL or more, more preferably 0.1 mL or more, and even more preferably 1 mL or more, and the upper limit is preferably 1000 mL or less, more preferably 100 mL or less, and even more preferably 10 mL or less, for example, 0.1 mL or more and 10 mL or less is preferred. Furthermore, the concentrations of the radioactive metal complex and the peptide-modified antibody in the reaction solution, independently at the start of this process, are preferably 0.01 μmol / L or more, more preferably 0.1 μmol / L or more, and even more preferably 1 μmol / L or more, and the upper limit is preferably 10000 μmol / L or less, more preferably 1000 μmol / L or less, for example, 1 μmol / L or more and 100 μmol / L or less is preferred from the viewpoint of the yield of the target radioactive metal-labeled antibody.

[0038] The obtained radioactive metal-labeled antibody may be used as is, or it may be purified using a filtration filter, membrane filter, column packed with various packing materials, chromatography, etc.

[0039] According to the manufacturing method of the present invention having the steps described above, a radioactive metal complex and a peptide-modified antibody can be bound under mild reaction conditions to obtain a radioactive metal-labeled antibody in high yield. Furthermore, since the obtained labeled antibody can be specifically labeled at a site that does not inhibit the antigen specificity of the antibody, the antigen specificity inherent in the antibody itself is maintained. In particular, the above steps allow the binding reaction between the complex and the antibody to proceed sufficiently and quickly without the need for heat treatment to promote the binding reaction between the radioactive metal complex and the peptide-modified antibody. Therefore, even when using radioactive metals with short half-lives such as positron-emitting nuclides, labeled antibodies with high radiochemical purity and radiochemical yield can be obtained in a short time.

[0040] The present invention's manufacturing method, from the viewpoint of allowing the use of any radioactive metal to be labeled without particular limitation depending on the application and purpose of the labeled antibody, preferably includes a step of reacting a ligand with a radioactive metal to form a radioactive metal complex (complex formation step) before the labeling step. The ligand used in this step preferably has a first atomic group. Furthermore, from the viewpoint of increasing the efficiency of complex formation, the radioactive metal in this step is preferably used in the form of an ionizable radioactive metal compound, and more preferably in the form of a radioactive metal ion (hereinafter, these forms are collectively referred to as "radioactive metal source").

[0041] Furthermore, from the viewpoint of increasing the efficiency of radioactive metal complex formation without depending on the combination of ligand and radioactive metal, it is preferable to heat the ligand and radioactive metal during the complex formation process. In this regard, depending on the combination of ligand and radioactive metal, complex formation may not proceed well under non-heating conditions. However, by performing the formation of the complex to which the radioactive metal ion is coordinated under heating conditions, complex formation can be efficiently carried out without depending on the combination of ligand and radioactive metal.

[0042] In the complex formation step, the order of addition of the ligand and the radioactive metal source is not important, as long as complex formation with the radioactive metal ion is possible. For example, one of the ligand and the radioactive metal source may be added to a reaction vessel containing a solvent, followed by the other, or one of the ligand and the radioactive metal source may be dispersed in a solvent and then the other may be added to the dispersion and reacted. Alternatively, both may be added simultaneously to a reaction vessel containing a solvent and reacted.

[0043] The reaction conditions in the complex formation step can be, for example, the following conditions. The solvent used in this step can be, for example, water, physiological saline, or buffers such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered physiological saline, Tris buffer, HEPES buffer, or tetramethylammonium acetate buffer, or water-soluble organic solvents such as alcohols with 1 to 5 carbon atoms, acetonitrile, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and acetone, or mixed solvents thereof. The reaction temperature can be, for example, room temperature (25°C), or it can be under heated conditions, but from the viewpoint of suppressing ligand decomposition and improving the efficiency of complex formation, the upper limit is preferably 120°C or lower, more preferably 90°C or lower, even more preferably 50°C or lower, and even more preferably 40°C or lower. The lower limit is not particularly limited as long as it is a temperature at which a click reaction can occur, but it is preferably 0°C or higher, more preferably 10°C or higher, even more preferably 15°C or higher, even more preferably 20°C or higher, even more preferably 30°C or higher, and especially preferably 35°C or higher. Preferably, the mixture is heated to 30°C or higher and 100°C or lower, more preferably 37°C or higher and 90°C or lower. The reaction time, provided the reaction temperature is as described above, is preferably 5 minutes or more at the lower limit, more preferably 10 minutes or more, even more preferably 20 minutes or more, even more preferably 30 minutes or more, and particularly preferably 60 minutes or more. The upper limit is preferably 300 minutes or less, more preferably 150 minutes or less, even more preferably 120 minutes or less, particularly preferably 60 minutes or less, preferably 10 minutes or more and 150 minutes or less, and even more preferably 30 minutes or more and 60 minutes or less.

[0044] In the complex formation process, the radioactive metal source can be, for example, a solution in which radioactive metal ions are dispersed or dissolved in a solvent mainly composed of water.

[0045] The amount of reaction solution in this process is not particularly limited, but from the viewpoint of practicality in the manufacturing process, at the start of this process, the lower limit is preferably 0.01 mL or more, more preferably 0.1 mL or more, and even more preferably 1 mL or more, and the upper limit is preferably 1000 mL or less, more preferably 100 mL or less, for example, 0.01 mL or more and 100 mL or less is preferred. Furthermore, the concentrations of the ligand and radioactive metal ions in the reaction solution, independently at the start of this process, are preferably 0.01 μmol / L or more, more preferably 0.1 μmol / L or more, and even more preferably 1 μmol / L or more, and the upper limit is preferably 10000 μmol / L or less, more preferably 1000 μmol / L or less, for example, 1 μmol / L or more and 100 μmol / L or less is preferred from the viewpoint of yielding the desired radioactive metal complex. The molar ratio of ligand to radioactive metal ion varies depending on the type of ligand and radioactive metal ion used, but the lower limit of ligand / radioactive metal ion is preferably 10 / 1 or more, more preferably 100 / 1 or more, even more preferably 200 / 1 or more, 300 / 1 or more, and even more preferably 500 / 1 or more. The upper limit is preferably 10000 / 1 or less, more preferably 9000 / 1 or less, even more preferably 8000 / 1 or less, and even more preferably 7000 / 1 or less. Preferably, it is in the range of 200 / 1 to 10000 / 1, and particularly preferably 500 / 1 to 7000 / 1.

[0046] The resulting radioactive metal complex may be used as is, or it may be purified using a filtration filter, membrane filter, column packed with various packing materials, chromatography, etc. In particular, this process is advantageous because even when using low-energy radiation that is difficult to detect or radioactive metal nuclides that emit alpha rays, complex formation proceeds well and the yield of the product is high, so the complex containing the radioactive metal nuclide can be used in subsequent processes in an unpurified state.

[0047] From the viewpoint of achieving both improved reaction efficiency of the reaction between the ligand and the radioactive metal ion and improved reaction efficiency of the click reaction between the resulting complex and the peptide-modified antibody, it is preferable that the ligand has a chelate portion, which is the site to which the radioactive metal ion coordinates, and a modified portion bound to the first atomic group.

[0048] As shown in the following formula (3a), the modification portion (indicated as Rm in formula (3a)) is bonded to the chelate portion (indicated as Ch in formula (3a)) and to the first atomic group (indicated as AG in formula (3a)) described above. In formula (3a), Rm is a linear or branched chain, substituted or unsubstituted, and an atomic group with a total of 10 to 50 carbon atoms.

[0049] [ka]

[0050] The modification portion Rm is not particularly restricted in its binding mode to the chelate portion Ch, as long as it enables complex formation between the ligand and the radioactive metal ion. However, from the viewpoint of efficiently forming a complex between the ligand and the radioactive metal ion, it is preferable that the modification portion Rm and the chelate portion Ch are bound by either a thiourea bond or an amide bond. Furthermore, from the viewpoint of increasing the labeling efficiency of the radioactive metal complex and the antibody, it is also preferable that the modification portion Rm is bound to R1 and R3 or R4 represented by formulas (1a) and (1b) above in the first atomic group.

[0051] From the viewpoint of further improving the labeling efficiency of the radioactive metal complex and the antibody, it is preferable that the modified portion has a structure shown in formula (P2) attached to the structure represented by Rm in formula (3a). This structure is derived from ethylene glycol, and in formula (P2), r is preferably an integer between 2 and 50, and more preferably an integer between 2 and 30.

[0052] [ka]

[0053] Furthermore, the chelate portion is not particularly limited as long as it has a site in its structure to which a radioactive metal can coordinate, but examples include CB-TE2A (1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), CDTA (Cyclohexane-trans-1,2-diamine tetra-acetic acid), CDTPA (4-cyano-4-[[(dodecylthio)thioxomethyl]thio]-Pentanoic acid), DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTMA ((1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid acid), DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTA-GA(α-(2-Carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic DOTMP(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid), DOTMP((1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrakis(methylene) DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamidomethylenephosphonic acid)), D02P(Tetraazacyclododecane dimethanephosphonic acid), Deferoxamine (DFO), DTPA(Glycine, N,N-bis[2-[bis(carboxymethyl)amino]ethyl]-), DTPA-BMA(5,8-Bis(carboxymethyl)-11-[2-(methylamino)-2-oxoethyl]-3-oxo-2,5,8,11-tetraazatridecan-13-oic acid)、EDTA(2,2’,2’’,2’’’-(ethane-1,2-diylbis(azanetriyl))tetraacetic acid)、NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid)、NOTP(1,4,7-Triazacyclononane-1,4,7-triyltris(methylenephosphonic acid)、TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid)、TETA(1,4,8,11-Tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid)、TTHA(3,6,9,12-Tetrakis(carboxymethyl)-3,6,9,12-tetraazatetradecanedioic acid)、HEHA(1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid)、1,2-HOPO(N,N’,N’’,N’’’-tetra(1,2-dihydro-1-hydroxy-2-oxopyridine-6-carbonyl)-1,5,10,14-tetraazatetradecane)、PEPA(1,4,7,10,13-pentaazacyclopentadecane-N,N’,N’’,N’’’,N’’’’-penta-acetic acid)、H4octapa(N,N’-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N’-diacetic acid)、H2bispa2(6,6’-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridine-2-yl)-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl}bis(-methylene))dipicolinic acid), H2dedpa (1,2 - [{6 - (carboxy) - pyridin - 2 - yl}-methylamino]ethane), H2macropa (6 - (1,4,10,13 - tetraoxa - 7,16 - diazacyclooctadecan - N,N’ - methyl)picolinic acid), H5decapa (N,N’’ - bis(6 - carboxy - 2 - pyridylmethyl)-diethylenetriamine - N,N’,N’’ - triacetic acid), H6phospa (N,N’ - (methylenephosphonate)-N,N’ - [6 - (methoxycarbonyl)pyridin - 2 - yl]-methyl - 1,2 - diaminoethane), HP - D03A (Hydroxypropyltetraazacyclododecanetriacetic acid) or porphyrin or any one of the following formulas (A) to (K) is preferable. These structures can be appropriately selected according to the type of radioactive metal described later. Even with any chelate part, the effects of the present invention can be sufficiently achieved.,

[0054]

Chemical formula

[0055]

Chemical formula

[0056] In formula (A), R 11 , R 13 and R 14 are each independently a group consisting of -(CH2) p COOH, -(CH2) p C5H5N, -(CH2) p PO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p COOH, and R 12 or R15 One of the members is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a group bonded to the above-mentioned modification, where p is an integer between 0 and 3.

[0057] In formula (B), R 21 , R 22 , R 23 and R 24 Each is independently a carboxyl group or a carboxyalkyl group having 2 or 3 carbon atoms, but R 21 , R 22 , R 23 or R 24 One of the groups is bonded to the above-mentioned modification portion.

[0058] In formula (C), R 31 , R 32 , R 33 and R 34 Each of these is a group consisting of an atomic group having a hydrogen atom and 2 to 10 carbon atoms, and which may also contain a nitrogen atom or an oxygen atom, R 35 This is a group that is bonded to the above-mentioned modified portion.

[0059] In formula (D), R 41 or R 42 One of the groups is a group having a hydrogen atom and 5 to 20 carbon atoms, and containing one or more atoms selected from nitrogen, oxygen, and sulfur atoms, while the other is a group bonded to the above-mentioned modification portion.

[0060] In formula (E), R 51 , R 52 , R 53 , R 54 and R 55 Each of these is independently a carboxyl group or a carboxyalkyl group having 2 or 3 carbon atoms, however, R 51 , R 52 , R 53 , R 54 or R 55 One of the groups is the group that is bonded to the above-mentioned modification portion.

[0061] In formula (F), R 61 , R 62 , R 63 , R 64 , R 65 and R 66 Each is independently a carboxyl group or a carboxyalkyl group having 2 or 3 carbon atoms, and R 67 This is a group that is bonded to the above-mentioned modified portion.

[0062] In formula (G), R 71 and R 72 is -O(CH2CH2O) n CH3 (where n is an integer between 1 and 5), and R 73 , R 75 , R 76 and R 78 Each of these is an alkyl group having 1 to 5 carbon atoms, and R 74 or R 77 The group is one in which either side is a hydroxyalkyl group having 1 to 5 carbon atoms, and the other side is bonded to the above-mentioned modified portion.

[0063] In formula (H), R 81 and R 82 Each of these is independently an alkyl group having 1 to 5 carbon atoms, and the terminus of the alkyl group may be substituted with a pyridyl group substituted with one or more carboxyl groups, R 87 R is -CHOH or -C=O, but 81、 R 82 or R 87 One of the groups is bonded to the above-mentioned modification portion, R 83 and R 84 R is a pyridinyl group which may be substituted, 85 and R 86 Each of these is independently -COORa, and Ra is an alkyl group having 1 to 5 carbon atoms.

[0064] In formula (I), R 91 , R 92 , R 93 and R 94 Each of these is independently -OCH2COOH, but R91 , R 92 , R 93 or R 94 Any one of the groups is a group bonded to the above-mentioned modification portion, R 95 , R 96 , R 97 and R 98 Each of these is an alkyl group having between 1 and 6 carbon atoms.

[0065] In formula (J), R 101 , R 102 and R 103 Each of these is independently a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, or in formula (J), R 101 , R 102 and R 103 At least one of these groups is a group bonded to the modified portion, and the other group is a carboxyl group or a carboxyalkyl group having 2 or 3 carbon atoms.

[0066] In the above formulas (A) to (J), "group bonded to the modification" refers to a structure in which the modification is bonded to a structure derived from a carboxyl group, an amino group, an N-hydroxysuccinimide ester (NHS) group, a 2,6-dioxotetrahydro-2H-pyranyl group, an isocyanate group, or an isothiocyanate group.

[0067] Specific structures represented by formula (A) include structures derived from the compounds represented by the following formulas (A-1) to (A-12).

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] Specific structures represented by formulas (B) and (C) include structures derived from the compounds represented by the following formulas (B-1) to (B-2) and (C-1) to (C-5).

[0072] [ka]

[0073] [ka]

[0074] Specific structures represented by formulas (D) and (E) include structures derived from the compounds represented by the following formulas (D-1) to (D-3) and (E-1).

[0075] [ka]

[0076] Specific structures represented by formulas (F) and (G) include structures derived from the compounds represented by the following formulas (F-1) to (F-2) and (G-1).

[0077] [ka]

[0078] Specific structures represented by formulas (H) and (I) include structures derived from the compounds represented by the following formulas (H-1) to (H-4) and (I-1).

[0079] [ka]

[0080] [ka]

[0081] Specific structures represented by formula (J) include structures derived from the compounds represented by the following formulas (J-1) to (J-5).

[0082] [ka]

[0083] As mentioned above, the bonding site between the chelate and modified portions is preferably an amide bond or a thiourea bond, but an amide bond is more preferable from the viewpoint of increasing the yield.

[0084] Amide bonds are formed, for example, by the reaction of a carboxyl group of the compounds shown in formulas (B-1) to (B-2), (G-1), (H-1) to (H-4), (I-1), (J-1) to (J-3) above, an N-hydroxysuccinimide (NHS) group of formulas (A-10) or (A-11) above, or a 2,6-dioxotetrahydro-2H-pyranyl group of formula (A-12) above with a primary amine, or by the reaction of the amino group shown on the far right of the diagram of the compound shown in formula (K) with a reagent having a hydroxyl group, a carboxyl group, or an NHS group. Here, when reacting with a reagent having a hydroxyl group, the hydroxyl group is converted to a carboxyl group before use. Thiourea bonds are formed by the reaction of an isothiocyanate group of the compounds shown in formulas (A-2), (A-3), (D-2), or (F-2) above with a primary amine or a maleimide group.

[0085] The modified portion has a desired primary atomic group bonded to it and can be formed by selecting from various commercially available reagents containing a primary amine or commercially available reagents capable of forming amide bonds or thiourea bonds. As such reagents, when using Dibenzylcyclooctyne (DBCO) represented by the above formula (1a) as the first atomic group, DBCO-amine, DBCO-maleimide, DBCO-PEG-NHS ester, DBCO-PEG-alcohol, DBCO-PEG-amine, DBCO-PEG-maleimide, etc. can be selected, but preferably DBCO-amine, DBCO-maleimide, DBCO-PEG-amine, DBCO-PEG-maleimide can be selected.

[0086] The radioactive metal complex is not particularly limited as long as it is formed by reacting a ligand having a structure appropriately selected from the above-described first atomic group, chelate moiety, and modification moiety with a radioactive metal. However, the radioactive metal complex is preferably formed by reacting a ligand having a structure represented by the following formula (ii) with a radioactive metal. A-B-C ···(ii) In formula (ii), A is represented by the following formula (iia).

[0087]

Chemical formula

[0088] In formula (iia), Ra, Rb, and Rc are independently -(CH2) p COOH, -(CH2) p C5H5N, -(CH2) p PO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p COOH, and p is an integer of 0 or more and 3 or less. Either Rd or Re is the binding site (*) with B, and the other is a hydrogen atom or -(CH2) p COOH, -(CH​​​​​​​​​In formula (ii), B is represented by the following formula (iib).

[0089] [Chemical formula]

[0090] In formula (iib), La and Lb are, independently, linking linkers having 1 to 50 carbon atoms containing at least an amide bond or a thiourea bond, t is an integer of 0 or more and 30 or less, s is 0 or 1, * is a bonding site with A, and ** is a bonding site with C.

[0091] In formula (ii), C is either an alkyne derivative represented by the following formula (iic) or a tetrazine derivative represented by the following formula (iid).

[0092] t[Chemical formula]

[0093] In formula (iic), X is CHRk—** or N—**, Y is CHRk or C═O, Rk is independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and when X is CHRk—** and Y is CHRk, the Rk moieties may together form a cycloalkyl group. Rf, Rg, Rh, and Ri are independently a hydrogen atom, a halogen atom, or an alkyl group having 1 to 5 carbon atoms, and Rf and Rg may together or Rh and Ri may together form a hydrocarbon ring. ** indicates a bonding site with B. In formula (iid), ** indicates a bonding site with B, and Rj represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group.

[0094] [[ID=​​​Either a DO3A derivative or a DOTAGA derivative is more preferable, where the compound is COOH, p is 1, Rd is the binding site to B (*), and Re is a hydrogen atom.

[0095] In formula (ii), if A is the above DOTA derivative, then B is a linker with 1 to 50 carbon atoms containing a thiourea bond, s is 0 or 1, and if s is 1, t is an integer between 0 and 30, Lb is a linker with 1 to 50 carbon atoms containing an amide bond or a thiourea bond, and C is an alkyne derivative represented by formula (iic), where X is N-**, Y is CHRk, Rk is a hydrogen atom, and Rf and Rg together form a benzene ring. A DOTA-PEGt-DBCO derivative is more preferable, in which Rh and Ri together form a benzene ring, and ** is the bonding site with B; or a DOTA-PEGt-Tz derivative is more preferable, in which La is a linker having 1 to 50 carbon atoms and containing a thiourea bond, s is 0 or 1, and if s is 1, t is an integer between 0 and 30, Lb is a linker having 1 to 50 carbon atoms and containing an amide bond or a thiourea bond, and C is a tetrazine derivative represented by formula (iid).

[0096] In formula (ii), if A is the above DO3A derivative, B is a bond linker having 1 to 50 carbon atoms, La is an amide bond or a thiourea bond, s is 0 or 1, and if s is 1, t is an integer from 0 to 30, Lb is a bond linker having 1 to 50 carbon atoms, C is an alkyne derivative represented by formula (iic), in formula (iic), where X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg together form a benzene ring, Rh and Ri together form a benzene ring, and ** is a bonding site with B, and a DO3A-PEGt-DBCO derivative is even more preferred.

[0097] In formula (ii), if A is the DOTAGA derivative described above, B is a bond linker having 1 to 50 carbon atoms, La being an amide bond or a thiourea bond, s being 0 or 1, and if s is 1, t being an integer between 0 and 30, Lb being a bond linker having 1 to 50 carbon atoms, C being an alkyne derivative represented by formula (iic), where X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg together form a benzene ring, Rh and Ri together form a benzene ring, and ** is a bonding site with B, and a DOTAGA-PEGt-DBCO derivative is even more preferred.

[0098] The following describes matters that are applicable to all of the embodiments described above. The peptide-modified antibody used in the present invention is one in which a specific site of the antibody is specifically modified by a peptide, preferably the Fc region (constant region) of the antibody is specifically modified, and particularly preferably a lysine residue in the Fc region of the antibody is site-specifically modified. In other words, in the present invention, the step of reacting a peptide with an antibody to obtain a peptide-modified antibody (antibody modification step) may be provided before the labeling step.

[0099] The antibody used in this invention may be any polypeptide containing an Fc region, and may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred. Monoclonal antibodies also include genetically modified antibodies such as antibody variants (chimeric antibodies, humanized antibodies, etc.).

[0100] The peptide used in the present invention may be a chain peptide or a cyclic peptide, as long as it site-specifically modifies the Fc region of the antibody, but a cyclic peptide is preferred. Furthermore, it is preferable that the peptide contains an amino acid sequence consisting of 13 to 17 amino acid residues, represented by the following formula (i). In the following description of the amino acid sequence, the left side of the amino acid sequence on the page represents the N-terminus, and the right side of the amino acid sequence on the page represents the C-terminus. The binding position of the second atomic group linked to the peptide is not particularly limited as long as it is possible to perform a click reaction with the first atomic group, but from the viewpoint of improving reactivity, it is preferable that the second atomic group is located at the N-terminus or C-terminus of the peptide, and more preferably at the N-terminus of the peptide.

[0101] (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) In the above equation (i), Xa, Xb, Xc, and Xd represent a consecutive a number of X's, a consecutive b number of X's, a consecutive c number of X's, and a consecutive d number of X's, respectively. X is an amino acid residue that does not have either a thiol group or a haloacetyl group in its side chain. a, b, c, and d are each independent integers between 1 and 5, and satisfy a+b+c+d≦14. Xaa1 and Xaa3 operate independently of each other. Represents an amino acid residue derived from an amino acid having a thiol group in its side chain, and is linked via a disulfide bond, or the sulfide group is linked via a linker, or One side represents an amino acid residue derived from an amino acid having a thiol group in its side chain, and the other side represents an amino acid residue derived from an amino acid having a haloacetyl group in its side chain, and they are linked via a thioether bond. Xaa2 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 is preferably modified with a crosslinking agent.

[0102] Examples of amino acid residues that can be included in X in formula (i) above include those derived from amino acids such as glycine, alanine, phenylalanine, proline, asparagine, aspartic acid, glutamic acid, arginine, histidine, serine, threonine, tyrosine, and methionine. X may consist of amino acid residues of the same type, or it may consist of amino acid residues of different types.

[0103] In formula (i), a, b, c, and d are not particularly limited as long as they are within the range described above. However, from the viewpoint of the binding stability between the peptide and the antibody, a+b+c+d≦14 is the condition, where a is preferably an integer between 1 and 3, b is preferably an integer between 1 and 3, c is preferably an integer between 3 and 5, and d is preferably an integer between 1 and 3.

[0104] Xaa1 and Xaa3 are amino acid residues derived from amino acids having a thiol group in their side chains, and these amino acids may be the same or different. Examples of amino acids having a thiol group in their side chains include cysteine ​​and homocysteine. It is preferable that such amino acid residues are linked by disulfide bonds or that a sulfide group is linked via the structure shown in formula (4) below. In formula (4), the dashed line portion indicates the portion linked to the sulfide group.

[0105] [ka]

[0106] Xaa1 and Xaa3 may be different from the above combination; one of Xaa1 and Xaa3 may be an amino acid residue derived from an amino acid having a thiol group in its side chain, and the other may be an amino acid residue derived from an amino acid having a haloacetyl group in its side chain. These are linked via a thioether bond. The haloacetyl group has a halogen such as iodine at its terminus, and the halogen is removed by reaction with the thiol group in the other side chain, forming a thioether bond.

[0107] Specific amino acid sequences of the peptide represented by formula (i) can be found in, for example, the peptides described in International Publication No. 2016 / 186206, International Publication No. 2017 / 217347, and International Publication No. 2018 / 230257, and these can also be used.

[0108] Of these, it is preferable that the peptide has one of the following amino acid sequences (1) to (14), and more preferably sequence (1), (2), (13), or (14). Having such an amino acid sequence can increase the binding affinity between the peptide and the antibody (e.g., human IgG). In the following amino acid sequences (1) to (14), (Xaa2) represents a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and is preferably modified with a crosslinking agent, and (Xaa1) and (Xaa3) both represent homocysteine ​​residues. In addition, in the following amino acid sequences (1) to (14), amino acids other than (Xaa1), (Xaa2), and (Xaa3) are denoted by single-letter abbreviations.

[0109] (1) DCAYH(Xaa2)GELVWCT (2)GPDCAYH(Xaa2)GELVWCTFH (3) RCAYH(Xaa2)GELVWCS (4)GPRCAYH(Xaa2)GELVWCSFH (5) SPDCAYH(Xaa2)GELVWCTFH (6) GDDCAYH(Xaa2)GELVWCTFH (7)GPSCAYH(Xaa2)GELVWCTFH (8)GPDCAYH(Xaa2)GELVWCSFH (9)GPDCAYH(Xaa2)GELVWCTHH (10)GPDCAYH(Xaa2)GELVWCTFY (11)SPDCAYH(Xaa2)GELVWCTFY (12)SDDCAYH(Xaa2)GELVWCTFY (13)RGNCAYH(Xaa2)GQLVWCTYH (14)G(Xaa1)DCAYH(Xaa2)GELVWCT(Xaa3)H

[0110] The peptides used in the present invention can be produced by using a combination of amino acids, including both natural and unnatural amino acids, and subjecting them to peptide synthesis methods such as liquid-phase synthesis, solid-phase synthesis, automated peptide synthesis, genetic recombination, or phage display. During peptide synthesis, the functional groups of the amino acids used may be protected as needed. These methods can be carried out, for example, in accordance with the methods described in International Publication No. 2017 / 217347 and International Publication No. 2018 / 230257.

[0111] The present invention provides a method for introducing a second atomic group into a peptide, which involves first obtaining a peptide having a desired amino acid sequence by the method described above, then dissolving the peptide in a solution containing a solubilizer, a reducing agent, and, if necessary, an acid, and adding an organic solvent solution of an atomic group containing an azide group or TCO as the second atomic group to the solution, and stirring at room temperature to introduce the second atomic group.

[0112] When introducing an atomic group containing an azide group as the second atomic group, the azide group can be directly introduced to the N-terminus or C-terminus of the peptide using a commercially available azide group introduction reagent in accordance with a conventional method, or the atomic group containing an azide group can be introduced via the linker structure described above. Examples of azide group introduction reagents that can be used include silyl azides, phosphate azides, alkylammonium azides, inorganic azides, sulfonyl azides, or PEG azides.

[0113] Furthermore, when introducing a group containing TCO as the second group, TCO can be directly introduced to the N-terminus or C-terminus of the peptide using a commercially available click chemistry reagent containing TCO, following a conventional method, or the group containing TCO can be introduced via the linker structure described above.

[0114] A method for obtaining peptide-modified antibodies by linking peptides and antibodies can be carried out, for example, using a crosslinking agent. A crosslinking agent is a chemical substance used to covalently link peptides and antibodies. Examples include crosslinking agents that preferably contain two or more succinimidyl groups, such as disuccinimidyl glutarate (DSG) and disuccinimidyl severate (DSS); crosslinking agents consisting of compounds or salts thereof that preferably contain two or more imido acid portions, such as dimethyl adipimidoate; and compounds or salts thereof that have disulfide bonds, such as dimethyl 3,3'-dithiobispropionimidoate and dithiobissuccinimidylpropionic acid. By using such a crosslinking agent, a crosslinking reaction can be brought about between the Xaa2 amino acid residue in the peptide and the antibody. In antibodies, when human IgG (e.g., trastuzumab) is used as the antibody, the site of cross-linking occurs via a site-specific cross-linking structure between the amino acid residue Xaa2 and at least one of the Lys246 and Lys248 residues, which follow the Eu numbering in trastuzumab. These Lys residues are located in the Fc region of human IgG, and even with antibodies other than trastuzumab, those skilled in the art can align the amino acid sequence of the antibody and identify the corresponding Lys residues.

[0115] The method for conjugating peptides and antibodies can be carried out by dispersing the aforementioned peptide, antibody, crosslinking agent, and, if necessary, a catalyst in a suitable buffer solution at an environment between 10°C and 30°C. The reaction time can be between 10 minutes and 2 hours. The molar ratio of peptide to antibody during the reaction can be in the range of 1:1 to 20:1 as peptide:antibody.

[0116] The peptide-modified antibody obtained through the above process only needs to have at least one peptide molecule bound to one antibody molecule (preferably an antibody of immunoglobulin class IgG). However, from the viewpoint of maintaining the activity of the antibody itself (antigen recognition, neutralization, complement activation, and opsonization), it is preferable that the peptide is site-specifically bound to the Fc region (constant region) of the antibody. In the present invention, it is more preferable that the peptide-modified antibody has one or two peptide molecules bound to one antibody molecule.

[0117] The peptide-modified antibody described above is a mixture containing, in any proportion, antibodies in which one peptide molecule is bound to one antibody molecule (hereinafter referred to as "monovalent antibody") and antibodies in which two peptide molecules are bound to one antibody molecule (hereinafter referred to as "bivalent antibody"). This mixture may be used as is in subsequent processes, or the unmodified antibody, monovalent antibody, and bivalent antibody may be separated and purified using methods such as filtration filters, membrane filters, columns packed with various packing materials, and various types of chromatography, and only the antibody of one of the valencies may be used in subsequent processes. If the unmodified antibody cannot be separated from the antibody of the other valency as a result of purification, the mixture containing them may be used in subsequent processes. When separating and purifying unmodified antibodies, monovalent antibodies, and bivalent antibodies, any of the above purification methods may be used. However, it is preferable to use a column packed with various packing materials, and more preferably to use a column packed with a packing material suitable for separating and purifying proteins such as antibodies.

[0118] The radioactive metal coordinated in the radioactive metal complex can be a metal nuclide that emits alpha rays, beta rays, or gamma rays, or a combination thereof. Examples of such radioactive metal nuclides include alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, or radioactive isotopes of metals other than these. Of these, from the viewpoint of being commercially usable and improving complex formation, the radioactive metal nuclide can be: 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 The or 225 Ac is preferred. These radioactive metals can be manufactured according to conventional methods, and are preferably obtained as a solution containing the radioactive metals in an ionized form.

[0119] When using radioactive metal-labeled antibodies for the treatment of diseases, from the perspective of enhancing therapeutic efficacy, alpha-emitting radionuclides or beta-emitting radionuclides should be used as the radioactive metal. - It is preferable to use alpha-emitting radionuclides. Alpha-emitting radionuclides can be any radionuclides that emit alpha particles during the decay process of radioactive metals; in detail, 212 Bi, 213 Bi, 227 The or 225 Ac and the like are preferably used, more 227 The or 225 It is Ac, and more preferably Ac. 225 It is Ac. - Radioactive radionuclides undergo the decay process of radioactive metals, including the β-emitting process. - Any radionuclide that emits radiation will suffice; in more detail, 60 Co, 59 Fe, 64 Cu, 67 Cu, 89 Sr, 90 Y, 99m Tc, 103Ru, 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, more 64 Cu, 67 Cu, 89 Sr, 90 Y or 177 Lu is used.

[0120] Furthermore, when using radioactive metal-labeled antibodies for the purpose of diagnosing diseases or detecting lesions, from the perspective of improving diagnostic performance, β is used as the radioactive metal. + It is preferable to use a radioactive emission nuclide, an electron capture decay nuclide, or a gamma-ray emitting nuclide. + A radioactive emission nuclide can be any nuclide that emits positrons during the decay process of radioactive metals. 44 Sc, 58 Co, 68 Ga, 64 Cu or 89 Zr and the like are preferably used, more 64 Cu or 89 It is Zr. An electron capture decay nuclide can be any nuclide that emits Auger electrons or characteristic X-rays during the decay process of a radioactive metal. 51 Cr, 57 Co, 58 Co, 67 Ga, 68 Ga, 64 Cu, 89 Zr, 111 In, 186 Re, 201 Tl or 197 Hg and the like are preferably used. The gamma-ray emitting nuclide can be any nuclide that emits gamma rays through gamma decay. Examples of nuclides that emit gamma rays through gamma decay include: 99m Tc, 68 Ga or 201 Tl is preferably used.

[0121] For example, when selecting a radioactive metal coordinated in an ionic state to a radioactive metal complex based on its ionic radius, a radioactive metal with an ionic radius of approximately 70-130 pm would be selected. 67 Ga, 68 Ga, 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 Examples include Ac, which can preferably form radioactive metal complexes with ligands having a chelate portion of the structure shown in formulas (A) to (K) above.

[0122] For example, when using radioactive metal-labeled antibodies for the purpose of treating a disease, the radioactive metal is 225 Ac, 177 Lu or 90 When Y is used, it is preferable to use a ligand having a chelate portion of the structure shown in any of the above formulas (A) or (D) to (I), more preferably a ligand having a chelate portion of the structure shown in any of the above formulas (A), (D), or (F), and even more preferably a ligand having a chelate portion of the structure shown in the above formula (A). Furthermore, when radioactive metal-labeled antibodies are used for the purpose of diagnosing diseases or detecting lesions, the radioactive metal is 89 Zr or 111 When using In, it is preferable to use a ligand having a chelate portion of the structure represented by any of the above formulas (A), (C), or (K) as the ligand, and it is even more preferable to use a ligand having a chelate portion of the structure represented by the above formula (A).

[0123] In the present invention, the immunoglobulin class of the antibody used can be any of IgG, IgA, IgM, IgD, or IgE without particular limitation. Of these, IgG is preferred because it is the main class in the secondary response of the immune response, is the most abundant in the blood, and has high recognition specificity for antigens. Mammalian IgG is preferred, and specific mammals include primates such as humans and chimpanzees; experimental animals such as rats, mice, and rabbits; domestic animals such as pigs, cattle, horses, sheep, and goats; and companion animals such as dogs and cats. Human IgG or rabbit IgG is even more preferred, and human IgG is even more preferred. Furthermore, there are no particular limitations on the subclass of these antibodies. For example, when using human IgG, it may be at least one of IgG1, IgG2, IgG3, and IgG4, and IgG1, IgG3, or IgG4 is preferred.

[0124] The radioactive metal-labeled antibody obtained through the above-described manufacturing method is one in which a specific site of the antibody is specifically modified by a peptide. The radioactive metal complex is directly or indirectly linked to the peptide, and there is a binding site between the peptide and the radioactive metal complex, preferably formed by a click reaction. The binding site is preferably a chemical structure derived from a first atomic group of the radioactive metal complex and a second atomic group linked to the peptide. For example, such a chemical structure may be one in which a structure containing a substituted triazole skeleton is formed at the binding site, or a structure containing a substituted pyridazine skeleton is formed. For example, a structure containing the substituted skeleton may be one in which a structure containing at least one of a substituent, an aliphatic ring, and an aromatic ring is bound to the triazole skeleton or pyridazine skeleton, and which has a binding site to the modification site or chelate site and a binding site to the peptide.

[0125] Regarding the specific structure of the bonding site, for example, if the first and second atomic groups are a combination of an atomic group containing DBCO and an atomic group containing an azide group, depending on the reaction reagent used, a structure containing the triazole skeleton shown in formula (10a) or formula (10b) below is formed, and since these are isomers, they may be present in any proportion. Also, if the first and second atomic groups are a combination of an atomic group containing 1,2,4,5-tetrazine and an atomic group containing TCO, depending on the reaction reagent used, a structure containing the pyridazine skeleton shown in formula (10c) below is formed. In formulas (10a) and (10b), R 1A R indicates the bonding site with the modified or chelated portion. 2A The symbol indicates the binding site with the peptide. In formula (10c), R 3A and R 4A One of them represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a bonding site with the modification or chelate part, R 5A This indicates the binding site with the peptide.

[0126] [ka]

[0127] Radiopharmaceutical compositions can be prepared using radiometal-labeled antibodies as they are, or after purification, as an active ingredient. A radiopharmaceutical composition refers to a composition containing radiometal-labeled antibodies or their derivatives, in a form suitable for administration into the body. A radiopharmaceutical composition can be prepared, for example, by dissolving radiometal-labeled antibodies produced by the method described above in a solvent mainly composed of water and approximately isotonic with the body. In this case, the radiopharmaceutical composition is preferably in the form of an aqueous solution, and may contain other pharmaceutically acceptable components as needed. Radiopharmaceutical compositions are administered to the body orally, or parenterally, such as intravenously, subcutaneously, intraperitoneally, or intramuscularly, and are used for the treatment of diseases, the diagnosis of diseases, or the detection of lesions.

[0128] Substituents that can be substituted into the above formulas (A) to (J), as well as the triazole skeleton-containing structure and the pyridazine skeleton-containing structure, include, for example, halogen atoms, saturated or unsaturated alkyl groups, hydroxyl groups, aldehyde groups, carboxyl 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, and pyridyl groups. These substituents may be substituted individually or in combination of two or more types. [Examples]

[0129] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. In the following table, the "-" column indicates that the invention was not implemented.

[0130] [Examples 1-4] (1-1. Complex Formation Process) The structures of the ligands used in this example are shown in the following formulas (L1-1) to (L1-3). DO3A-DBCO, represented by formula (L1-1), was synthesized according to the method described in Liang Y, Jiang X, Yuan R, Zhou Y, Ji C, Yang L et al. Metabolism-Based Click-Mediated Platform for Specific Imaging and Quantification of Cell Surface Sialic Acids. Anal Chem. Jan 3; 89(1): 538-543. (2017). DOTA-DBCO, represented by formula (L1-2), was synthesized according to the method described in Wang H, Wang R, Cai K, He H, Liu Y, Yen J et al. Selective in vivo metabolic cell-labeling-mediated cancer targeting. Nat Chem Biol. Apr; 13(4): 415-424. (2017). Furthermore, the DO3A-PEG4-DBCO represented by formula (L1-3) was a commercially available product from Iris Biotech GmbH. These ligands were dispersed in 0.1 mol / L sodium acetate buffer (pH 6.0) as a solvent to obtain a dispersion containing 1.7 mmol / L of ligand. 0.0025 mL of this dispersion was used, along with a radioactive metal source. 225 A reaction solution containing Ac ions (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 160 MBq / mL, prepared from Oak Ridge National Laboratory, volume 0.0025 mL) and 0.4 MBq (calculated value calculated from the radioactivity amount at the time of calibration) were mixed and reacted under heating conditions. 225 An Ac complex solution was obtained. The molar ratio of ligand to radioactive metal ion was: ligand: 225 The Ac ion ratio was 3000:1, the reaction solution was heated to 70°C for 90 minutes, and the heating time was 90 minutes.

[0131] [ka]

[0132] obtained 225 The radiochemical purity of the Ac complex was measured by the following method: 225 A portion of the Ac complex solution was developed using thin-layer chromatography (Agilent, model number: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured using a radio-γ-TLC analyzer (raytest, MODEL GITA Star). The percentage of the radioactivity (count) of the peak detected near the origin relative to the total detected radioactivity (count) was calculated. 225 The radiochemical purity (%) of the Ac complex was determined. 225 The radiochemical purity of the Ac complex was 89-99%. 225 The Ac complex solution was used directly in the labeling step.

[0133] (1-2. Antibody modification step) Separately, the peptide was prepared by the method described in International Publication No. 2017 / 217347 to obtain a peptide containing 17 amino acid residues represented by the following formula (P3). The amino acid sequence of this peptide was identical to the sequence in Sequence ID No. (2) where Xaa2 is a lysine residue, and the amino group at the side chain terminal of the lysine residue was modified in a structure indicated by R1. Furthermore, two cysteine ​​residues were disulfide-bonded to each other, and the N-terminus of the peptide was bonded to an ethyl azide group, which is the second atomic group containing an azide group, via a linker structure having diglycolic acid and eight PEGs.

[0134] [ka]

[0135] (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, Glu represents glutamic acid, Leu represents leucine, Val represents valine, Trp represents tryptophan, and Phe represents phenylalanine.)

[0136] This peptide was mixed with a human IgG antibody (rituximab; Roche, or trastuzumab; Roche, Inc.) in sodium acetate buffer (pH 6.0), and the mixture was reacted at room temperature for 30 minutes to obtain a solution containing the peptide-modified antibody. This peptide-modified antibody is one in which the Fc region of the antibody has been site-specifically modified by the above-mentioned peptide.

[0137] (2. Separation process of peptide-modified antibodies) The peptide-modified antibody described above was diluted with 1 mol / L sodium acetate buffer (pH 6.0) and added to a Protein A column (GE Healthcare, HiTrap MabSelect SuRe). A 0.05 mol / L sodium acetate buffer (pH 5.7) containing 0.15 mol / L sodium chloride was passed through the column, and the solution containing the bivalent antibody was collected. The concentration of the collected fraction was adjusted so that the concentration of the bivalent antibody was 15 mg / mL. Subsequently, a 0.05 mol / L sodium acetate buffer (pH 3.5) containing 0.15 mol / L sodium chloride was passed through the column, and the solution containing the monovalent antibody was collected. The concentration of the collected fraction was adjusted so that the concentrations of the unmodified antibody and monovalent antibody were between 17 and 40 mg / mL.

[0138] (3. Labeling process) The results obtained through the above-mentioned processes 225 The Ac complex solution and the peptide-modified antibody (monovalent antibody) were each added unpurified to a 0.09 mol / L sodium acetate buffer containing 0.02 mol / L ascorbic acid, and a click reaction was carried out at 37°C for 120 minutes to produce the results of Examples 1 to 4. 225 Ac-conjugate labeled antibody was obtained. 225 The amounts of Ac complex and peptide-modified antibody (monovalent antibody) were 43 μmol and 46 μmol, respectively (Example 1), or 100 μmol each (Examples 2 to 4), and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:1. 225 The reaction rates (%) of Ac-conjugated antibodies are shown in Table 1 below. Here, the reaction rate (%) is the percentage of the labeling rate (%) in the complex formation process. 225This refers to the radiochemical purity (%) of the Ac complex-labeled antibody, and the labeling rate (%) is the percentage relative to the initial radioactivity. 225 This represents the radioactivity (%) of the Ac complex. Furthermore, the reaction was carried out at 37°C for 2 hours. 225 The solution of Ac complex-labeled antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096). 225 The radiochemical purity (RCP) and radiochemical yield (RCY) of the Ac complex-labeled antibody are shown in Table 1 below.

[0139] 225 The radiochemical purity and radiochemical yield of Ac complex-labeled antibodies were measured as follows: Thin-layer chromatography (Agilent, model number: SGI0001, developing solvent: acetonitrile:0.1 mmol / L EDTA solution mixture (volume ratio 1:1)) was measured using a radio-γ-TLC analyzer (raytest, MODEL GITA Star), and the radiochemical purity (%) was defined as the percentage of the radioactivity (count) of the peak detected near the origin relative to the total detected radioactivity (count). Furthermore, the radiochemical yield (%) was defined as the percentage of the radioactivity recovered after ultrafiltration purification (calculated from the count measured by the gamma-ray spectrometer, as described above) relative to the total radioactivity added at the start of the labeling process (calculated from the count measured by a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (ORTEC), multi-channel analyzer: M7-000 (Seiko Easy & G), data processing: Spectrum Navigator: DS-P300 (Seiko Easy & G) and Gamma Studio: DS-P600 (Seiko Easy & G))).

[0140] [Example 5] (1-1. Complex Formation Process) In this example, the procedure was the same as in Example 1, except that a ligand with the structure shown in the following formula (L2) was used. 225An Ac complex solution was obtained. DOTA-PEG7-Tz represented by formula (L2) was synthesized according to the method described in Poty S, Membreno R, Glaser JM, Ragupathi A, Scholz WW, Zeglis BM et al. The inverse electron-demand Diels-Alder reaction as a new methodology for the synthesis of 225Ac-labelled radioimmunoconjugates. Chem Commun (Camb). Mar 8;54(21):2599-2602. (2018).

[0141] [Chemical formula]

[0142] (1-2. Antibody modification step) The peptide used in this example was a peptide consisting of 17 amino acid residues represented by the following formula (P4). The amino acid sequence of this peptide was the same as the sequence in which Xaa2 of SEQ ID NO: (2) was a lysine residue, and the side-chain terminal amino group of the lysine residue was modified with the structure represented by R2. In addition, the thiol groups of the two cysteine residues were linked by the linker represented by the above formula (4), and the N-terminus of the peptide had a linker structure having, in this order from the N-terminal side, five PEGs, a cysteine residue substituted with the structure represented by R1 containing TCO whose side-chain thiol group was the second atomic group, two glutamic acid residues, and an acetyl group.

[0143] [Chemical formula]

[0144] (In formula (P4), Gly represents glycine, Pro represents proline, Asp represents aspartic acid, Cys represents cysteine, Ala represents alanine, Tyr represents tyrosine, His represents histidine, Glu represents glutamic acid, Leu represents leucine, Val represents valine, Trp represents tryptophan, and Phe represents phenylalanine.)

[0145] This peptide was mixed with a human IgG antibody (trastuzumab; manufactured by Roche) in sodium acetate buffer (pH 6), and the mixture was reacted at room temperature for 30 minutes to obtain a solution containing the peptide-modified antibody.

[0146] (2. Labeling process) The click response was triggered using the same procedure as in Example 1. 225 Ac-conjugate labeled antibody was obtained. 225 The amount of Ac complex and the amount of peptide-modified antibody were both 100 μmol, and the molar ratio of the first atomic group (1,2,4,5-tetrazine) to the second atomic group (TCO) was 1:1. 225 The reaction rates (%) of Ac-conjugated antibodies are shown in Table 1 below. Here, the reaction rate (%) is the percentage of the labeling rate (%) in the complex formation process. 225 This refers to the radiochemical purity (%) of the Ac complex-labeled antibody, and the labeling rate (%) is the percentage relative to the initial radioactivity. 225 This represents the radioactivity (%) of the Ac complex. Also, after purification using an ultrafiltration filter, similar to Example 1. 225 The radiochemical purity (RCP) and radiochemical yield (RCY) of the Ac complex-labeled antibody are shown in Table 1 below.

[0147] [Table 1]

[0148] As shown in Table 1, in the example where the complex was formed and then reacted with the antibody, the labeling reaction proceeded under mild reaction conditions without excessive heating of the antibody, and the labeling efficiency of the radioactive metal to the antibody was excellent.

[0149] [Examples 6-7] 225 Tmab and Rmab production using Ac-labeled DOTAGA-DBCO (1. Complex formation process) The structure of the ligand used in this example is shown in the following formulas (L1-4). DOTAGA-DBCO represented by formula (L1-4) was prepared 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. These ligands were dispersed in 0.1 mol / L sodium acetate buffer (pH 6.0) as a solvent to obtain a dispersion containing 1.7 mmol / L of ligand. 0.005 mL of this dispersion and a radioactive metal source were used. 225 A reaction solution prepared by mixing Ac ion-containing solution (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 320-340 MBq / mL, prepared from Oak Ridge National Laboratory, volume 0.005 mL) with 1.6-1.7 MBq (calculated value based on decay calculation from the radioactivity amount on the day of the test) was reacted under heating conditions. 225 An Ac complex solution was obtained. The molar ratio of ligand to radioactive metal ion was: ligand: 225 The Ac ion ratio was approximately 2500:1, the reaction solution was heated to 70°C for 30 minutes, and the heating time was 30 minutes.

[0150] [ka]

[0151] obtained 225 The radiochemical purity of the Ac complex was measured in the same manner as in Example 1, and the result was 90%. 225 The Ac complex solution was used directly in the labeling step.

[0152] (2. Labeling process) The unrefined product obtained through the above process (1) 225The Ac complex solution and the solution containing the peptide-modified antibody (monovalent antibody) obtained in the same manner as in Example 1 were each added to 0.1 mol / L arginine-containing 0.1 mol / L histidine buffer pH 6.0, and a click reaction was carried out at 37°C for 120 minutes. 225 Ac-conjugate labeled antibody was obtained. 225 The amounts of Ac complex and peptide-modified antibody (monovalent antibody) were 85 nmol and 100 nmol, respectively (Examples 6 and 7), and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:1.2. 225 The reaction rates (%) of Ac-conjugated antibodies are shown in Table 2 below. Here, the reaction rate (%) is the percentage of the labeling rate (%) in the complex formation step. 225 This refers to the radiochemical purity (%) of the Ac complex-labeled antibody, and the labeling rate (%) is the percentage relative to the initial radioactivity. 225 This represents the radioactivity (%) of the Ac complex. Furthermore, the reaction was carried out at 37°C for 2 hours. 225 The solution of Ac complex-labeled antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096). 225 The radiochemical purity (RCP) and radiochemical yield (RCY) of the Ac complex-labeled antibody are shown in Table 2 below.

[0153] 225 The radiochemical purity and radiochemical yield of the Ac-labeled monovalent antibody were measured in the same manner as in Example 1.

[0154] [Table 2]

[0155] [Example 8] 89 Tmab fabrication using Zr-labeled DOTAGA-DBCO (1. Complex formation process) The structure of the ligand used in this embodiment is the same as that of formula (L1-4) described above. This ligand was dispersed in DMSO as a solvent to obtain a dispersion containing 0.33 mmol / L of ligand. 0.030 mL of this dispersion and a radioactive metal source were used. 89A reaction solution obtained by mixing 60 MBq of a Zr ion-containing solution (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 181 MBq / mL, prepared from Nippon Medi-Physics Co., Ltd., liquid volume 0.33 mL) was reacted under heating conditions to 89 obtain a Zr complex solution. The molar ratio of the ligand to the radioactive metal ion was ligand: 89 Zr ion = approximately 250:1, the heating temperature of the reaction solution was 70 °C, and the heating time was 60 minutes.

[0156] The obtained 89 radiochemical purity of the Zr complex was measured by the following method. That is, 89 A part of the Zr complex solution was developed by thin-layer chromatography (manufactured by Agilent, model number: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured with a radio γ-TLC analyzer (manufactured by raytest, MODEL GITA Star PS). The percentage of the radioactivity (counts) of the peak detected near the origin to the total detected radioactivity (counts) was 89 taken as the radiochemical purity (%) of the Zr complex. As a result, 89 the radiochemical purity of the Zr complex was 90%. The obtained 89 Zr complex solution was used as it was in the labeling step. <0,

[0157] (2. Labeling step) The solution of the unpurified Zr complex obtained through the above step (1) and the solution containing the peptide-modified antibody (monovalent antibody) obtained in the same manner as in Example 1 were each added to a 0.1 mol / L arginine-containing 0.1 mol / L histidine buffer solution at pH 6.0 as they were, and subjected to a click reaction at 37 °C for 120 minutes to obtain the 89 Zr complex-labeled antibody of Example 8. 89 The amount of the Zr complex and the amount of the peptide-modified antibody (monovalent antibody) were each 100 nmol, and the molar ratio of the first atomic group (DBCO ) to the second atomic group (azide) was approximately 1:1 each. The reaction rate (%) of the 89 Zr complex-labeled antibody in the unpurified example is shown in Table 3 below. Here, the reaction rate (%) is the ratio to the labeling rate (%) in the complex formation step 89 ​89 This refers to the radiochemical purity (%) of the Zr-complex labeled antibody, and the labeling rate (%) is the percentage relative to the initial radioactivity. 89 This represents the radioactivity (%) of the Zr complex. Furthermore, the reaction was carried out at 37°C for 2 hours. 89 The solution of Zr-labeled antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096). 89 The radiochemical purity (RCP) and radiochemical yield (RCY) of the Zr-labeled antibody are shown in Table 3 below.

[0158] 89 The radiochemical purity and radiochemical yield of the Zr complex-labeled antibody were measured in the same manner as in Example 8.

[0159] [Table 3]

[0160] [Example 9] 89 Tmab fabrication using Zr-labeled DOTA-DBCO (1-1. Complex Formation Process) The structure of the ligand used in this embodiment is the same as that of formula (L1-2) described above. This ligand was dispersed in DMSO as a solvent to obtain a dispersion containing 1 mmol / L of ligand. 0.1 mL of this dispersion and a radioactive metal source were used. 89 A reaction solution containing Zr ions (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 450 MBq / mL, prepared from Okayama University, volume 0.1 mL) was mixed with 45 MBq of Zr ions and reacted under heating conditions. 89 A Zr complex solution was obtained. The molar ratio of ligand to radioactive metal ion was: ligand: 89 The Zr ion ratio was approximately 10,000:1, the reaction solution was heated to 70°C for 60 minutes, and the heating time was 60 minutes.

[0161] obtained 89 The radiochemical purity of the Zr complex was determined in accordance with Example 1. As a result, 89 The radiochemical purity of the Zr complex was 99%.

[0162] (1-2. Removal of unreacted materials) obtained 89 The Zr complex solution was parsected using high-performance liquid chromatography (HPLC), and unreacted DOTA-DBCO was removed. The HPLC conditions were as follows: Detector: UV absorbance spectrophotometer (measurement wavelength: 254 nm) / scintillation detector; Column: XBridge C18 3.5 μm, 4.6 × 100 nm; Waters; Mobile phase A: 10 mmol / L histidine buffer pH 6.5; Mobile phase B: Acetonitrile for liquid chromatography; Mobile phase delivery: The concentration gradient was controlled by changing the mixing ratio of mobile phase A and mobile phase B as follows (A:B = 90:10 (0 min) → 50:50 (40 min) (vol% / vol%)); Flow rate: 0.5 mL / min; Retention time of parsecting peak: approximately 32 minutes. The obtained parsected solution was diluted to approximately 20 μL and used in the labeling step. 89 The radiochemical yield (HPLC recovery rate) in the unreacted material removal step of the Zr-labeled antibody is shown in Table 4 below. The radiochemical yield (HPLC recovery rate) was measured by taking the percentage of the radioactivity of the preparative solution relative to the amount of radioactivity added at the start of the process, and this percentage was defined as the HPLC recovery rate (%) in the unreacted material removal step.

[0163] (2. Labeling process) The results obtained through the above-mentioned processes 89 A solution of the Zr complex and a solution containing the peptide-modified antibody (monovalent antibody) obtained in the same manner as in Example 1 were each added to 0.1 mol / L arginine-containing 0.1 mol / L histidine buffer pH 6.0, and a click reaction was carried out at 37°C for 120 minutes. 89 Zr-conjugated antibody was obtained. (Examples of the unpurified state) 89 The labeling reaction rate (%) of Zr-conjugated antibodies is shown in Table 4 below. Here, the labeling reaction rate (%) is the percentage of the radioactivity of the HPLC fraction. 89 This represents the radioactivity (%) of the Zr complex. Furthermore, the reaction was carried out at 37°C for 2 hours. 89 The solution of Zr-labeled antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096). 89The radiochemical purity (RCP) and radiochemical yield (RCY) of the Zr-labeled antibody are shown in Table 4 below.

[0164] 89 The radiochemical purity and radiochemical yield of the Zr complex-labeled antibody were measured in accordance with Example 1.

[0165] [Table 4]

[0166] Although the present invention has been described above with reference to embodiments, the present invention is not limited to the above embodiments. Various modifications to the configuration and details of the present invention can be made that will be understood by those skilled in the art within the scope of the present invention. All content contained herein, including patents and patent application specifications, is incorporated herein to the same extent as if it were explicitly stated, by its sole reference. [Industrial applicability]

[0167] According to the present invention, high labeling efficiency of radioactive metals to antibodies is possible even under mild reaction conditions.

[0168] This application is based on Japanese Patent Application No. 2019-191561 (filing date: October 18, 2019), the contents of which are fully incorporated herein.

Claims

1. A method for producing a radiometal-labeled antibody, comprising the step of causing a click reaction between a radiometal complex and a peptide-modified antibody that has been site-specifically modified with a peptide to produce a radiometal-labeled antibody, The radioactive metal complex is a complex of a radioactive metal with DOTAGA-DBCO represented by the following formula (L1-4), A method for producing a radioactive metal-labeled antibody, wherein the peptide-modified antibody is an antibody modified with a peptide represented by the following formula (P3). 【Chemistry 1】 【Chemistry 2】

2. A method for producing a radiometal-labeled antibody according to claim 1, further comprising the step of reacting DOTAGA-DBCO with a radiometallic metal to form the radiometal complex.

3. A method for producing a radioactive metal-labeled antibody according to claim 2, comprising heating and reacting the DOTAGA-DBCO with the radioactive metal.

4. where the radioactive metal is 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 Ac, the method for producing a radioactive metal-labeled antibody according to any one of claims 1 to 3.

5. The method for producing a radiometal-labeled antibody according to claim 4, wherein the radioactive metal is an alpha-emitting radionuclide.

6. A method for producing a radioactive metal-labeled antibody according to any one of claims 1 to 5, wherein the click reaction is performed at a temperature of 50°C or lower.

7. A method for producing a radioactive metal-labeled antibody according to any one of claims 1 to 6, wherein the peptide-modified antibody is site-specifically modified with one or two peptide molecules for one molecule of antibody.

8. A method for producing a radioactive metal-labeled antibody according to any one of claims 1 to 7, wherein the antibody is trastuzumab or rituximab.

9. A method for producing a radiometal-labeled antibody according to any one of claims 1 to 8, wherein the succinimidyl group in the structure of the peptide represented by formula (P3) reacts with a lysine residue present in the Fc region of the IgG antibody, thereby site-specifically modifying the IgG antibody with the peptide linked to the radiometal complex.

10. A radiometal-labeled antibody site-specifically modified with a peptide linked to a radiometal complex, The radioactive metal complex is a complex of a radioactive metal with DOTAGA-DBCO represented by the following formula (L1-4), The peptide is a peptide represented by the following formula (P3): Radioactive metal-labeled antibody. 【Transformation 3】 【Chemistry 4】

11. The aforementioned radioactive metal is 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 The or 225 The radioactive metal-labeled antibody according to claim 10, wherein the antibody is Ac.

12. The radioactive metal-labeled antibody according to claim 11, wherein the radioactive metal is an alpha-emitting radionuclide.

13. The radioactive metal-labeled antibody according to any one of claims 10 to 12, wherein the linkage between the radioactive metal complex and the peptide comprises a triazole skeleton shown in formula (10a) or formula (10b) below, formed by the reaction of a dibenzocyclooctinyl group in the structure represented by formula (L1-4) and an azide group in the structure represented by formula (P3). 【Transformation 5】 (In formulas (10a) and (10b), R 1A represents the linking site with the radioactive metal complex, and R 2A represents the linking site with the peptide.)

14. The radioactive metal-labeled antibody according to any one of claims 10 to 13, wherein the succinimidyl group in the structure of the peptide represented by formula (P3) reacts with a lysine residue present in the Fc region of the IgG antibody, thereby site-specifically modifying the IgG antibody with the peptide linked to the radioactive metal complex.

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