Ri-labeled humanized antibody

Abstract

To provide a radionuclide-labeled anti-MUC5AC humanized antibody having superior specificity for mucin subtype 5AC (MUC5AC) and superior accumulation in tumors.SOLUTION: A radionuclide-labeled anti-MUC5AC humanized antibody is a conjugate of a chelating agent chelated with a radionuclide and an antibody (the radionuclide being a metal nuclide that emits α particles or positrons, and the antibody being a humanized antibody that specifically binds to MUC5AC), and is superior in specificity for MUC5AC and accumulation in tumors. Therefore, it is extremely useful for the treatment and / or diagnosis of diseases in which MUC5AC is overexpressed, particularly cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a conjugate of a chelating agent having a radionuclide chelated thereto and a mucin subtype 5AC-specific humanized antibody, a radiopharmaceutical containing the same, and uses thereof. [Background technology]

[0002] Mucins are the main component of mucus secreted by animal epithelial cells and are glycoproteins containing large amounts of sugars with a molecular weight of 1 to 10 million. Mucins are classified into secretory mucins produced by epithelial cells and membrane-bound mucins that have hydrophobic transmembrane domains and exist bound to the cell membrane. The core proteins of mucins are collectively called MUCs, and at least 20 genes encoding these core proteins are known. One of these, mucin subtype 5AC (MUC5AC), belongs to the secretory mucin family.

[0003] MUC5AC is expressed in normal tissues such as the stomach and trachea, but its overexpression has been reported in pancreatic cancer, as well as in thyroid cancer, liver cancer, colon cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, and bile duct cancer. Antibodies against MUC5AC have been reported, including a mouse antibody produced using a pancreatic cancer mucin fraction purified from xenografts of the human pancreatic cancer cell line SW1990 as an antigen (Non-Patent Document 1), as well as chimeric antibodies (Patent Documents 1 and 2, Non-Patent Documents 2 and 3), and humanized antibodies (Patent Documents 3 and 4).

[0004] Antibodies are used as reagents for detecting target molecules, diagnostic agents, or pharmaceuticals for treating diseases, taking advantage of the specificity of antibodies for target molecules. With the aim of further improving detection performance and therapeutic effects, studies are underway on antibodies bound to radionuclides or drugs. In Non-Patent Document 1, a β-ray emitting nuclide, 131 Radioimmunotherapy of pancreatic cancer model mice using mouse antibodies labeled with I has been reported. 111SPECT imaging of pancreatic cancer patients using an In-labeled chimeric antibody has been reported. Patent documents 3 and 4 describe MUC5AC-specific humanized antibodies, 90 Y and 111 Labeled with In etc. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 7-203974 [Patent Document 2] Japanese Patent Application Publication No. 11-5749 [Patent Document 3] International Publication No. 2013 / 157102 [Patent Document 4] International Publication No. 2013 / 157105 [Non-patent literature]

[0006] [Non-Patent Document 1] Japanese Journal of Cancer Research, 87, 977-984, 1996 [Non-patent document 2] Japanese Journal of Clinical Medicine, Vol. 64, Special Issue 1, 2006, 274-278 [Non-patent document 3] Japanese Journal of Cancer Research, 90, 1179-1186, 1999 Summary of the Invention

[0007] An object of the present invention is to provide a radionuclide-labeled humanized anti-MUC5AC antibody that has excellent specificity for mucin subtype 5AC (MUC5AC) and excellent tumor accumulation.

[0008] The present inventors have conducted extensive research in light of the above-mentioned problems, and have found that a chelating agent containing a radionuclide, which is a metal nuclide, and an anti-MUC5AC humanized antibody consisting of a specific amino acid sequence can be used. The present inventors have succeeded in producing a complex of the above, found that the complex has excellent specificity for MUC5AC and accumulation in tumors, and confirmed its effects, thereby completing the present invention.

[0009] One aspect of the present invention provides a conjugate of a chelating agent to which a radionuclide has been chelated and an antibody, wherein the radionuclide is a metal nuclide that emits alpha rays or positrons, and the antibody is a humanized antibody that specifically binds to MUC5AC.

[0010] According to the present invention, there can be provided a radionuclide-labeled humanized anti-MUC5AC antibody that has excellent specificity for MUC5AC and tumor accumulation, and uses of the antibody. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the results of SPECT-CT imaging using each In-labeled antibody. SPECT images of each In-labeled antibody are shown. [Figure 2] FIG. 2 is a graph showing the results of VOI (volume of interest, three-dimensional ROI) analysis of the tumor and liver at each time point from SPECT images of each 111In-labeled antibody. [Figure 3] FIG. 3 is a graph showing the results of examining the distribution and excretion route in the body 168 hours after administration of each 111In-labeled antibody. [Figure 4] FIG. 4 is an image showing the results of comparing the in vitro ARG-binding ability of each 111In-labeled antibody. [Figure 5] FIG. 5 is a graph showing the results of quantifying and comparing the in vitro ARG-binding ability of each 111In-labeled antibody. [Figure 6] FIG. 6 is a graph showing the change in tumor volume over time in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody. [Figure 7]FIG. 7 is a graph showing the change in body weight over time in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody, expressed as a relative value with the weight before administration set at 1.0. [Figure 8] FIG. 8 is a graph showing the results of examining hepatotoxicity and nephrotoxicity in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody. [Figure 9] FIG. 9 is a graph showing the results of examining hematotoxicity (white blood cell count, platelet count) in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody. [Figure 10] FIG. 10 is a graph showing the change in tumor volume over time in tumor-bearing mice after administration of a high dose of 225Ac-labeled monovalent antibody. [Figure 11] FIG. 11 is a graph showing the change in body weight over time in cancer-bearing mice after administration of a high dose of 225Ac-labeled monovalent antibody, expressed as a relative value with the weight before administration set at 1.0. [Figure 12] FIG. 12 is a graph showing the results of examining hepatotoxicity and nephrotoxicity in cancer-bearing mice after administration of a high dose of 225Ac-labeled antibody. [Figure 13] FIG. 13 is a graph showing the results of examining hematotoxicity (white blood cell count, platelet count) in cancer-bearing mice after administration of a high dose of 225Ac-labeled antibody. [Figure 14] FIG. 14 is a graph showing the change in tumor volume over time in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody. [Figure 15] FIG. 15 is a graph showing the change in body weight over time in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody, expressed as a relative value with the weight before administration set at 1.0. [Figure 16] FIG. 16 is a graph showing the results of examining hepatotoxicity in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody. [Figure 17] FIG. 17 is a graph showing the results of examining nephrotoxicity in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody. [Figure 18]FIG. 18 is a graph showing the results of examining hematotoxicity (white blood cell count) in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody. [Figure 19] FIG. 19 is a graph showing the results of examining hematotoxicity (platelet count) in cancer-bearing mice after administration of 225Ac-labeled monovalent antibody or 225Ac-labeled bivalent antibody. [Figure 20] FIG. 20 is a graph showing the results of radioactivity accumulation per unit weight (% ID / g) in each organ in the body 20, 68, and 188 hours after administration of 111In-labeled monovalent or bivalent antibody. [Figure 21] Figure 21 is a graph showing the time course of radioactivity accumulation rates in the tumor, blood, excreted urine, excreted feces, and the total of excreted urine and excreted feces in the body 20, 68, and 188 hours after administration of 111In-labeled monovalent or bivalent antibody. [Figure 22A] Figure 22A shows the stability of Zr-labeled anti-MUC5AC humanized antibodies in human (-■-, ...■...) and mouse (-●-, ...●...) plasma. Various Zr-labeled antibodies were mixed with human and mouse plasma and incubated at 37°C. The graph shows the time course of radiochemical purity at each time point. The upper panel shows the results for Zr-labeled anti-MUC5AC humanized antibodies produced using Zr-labeled DOTA-Bn-DBCO (24, 48, 168, and 378 hours after incubation), and the lower panel shows the results for Zr-labeled anti-MUC5AC humanized antibodies produced using Zr-labeled DOTAGA-DBCO (24, 168, and 336 hours after incubation). [Figure 22B]Figure 22B shows the stability of 225Ac-labeled anti-MUC5AC humanized antibodies in human (-■-, ...■...) and mouse (-●-, ...●...) plasma. Various 225Ac-labeled antibodies were mixed with human and mouse plasma and incubated at 37°C. The graph shows the time course of radiochemical purity at each time point. The upper panel shows the results for the 225Ac-labeled anti-MUC5AC humanized antibody produced using 225Ac-labeled DOTA-Bn-DBCO (21, 115, 140, and 168 hours after incubation), and the lower panel shows the results for the 225Ac-labeled anti-MUC5AC humanized antibody produced using 225Ac-labeled DOTAGA-DBCO (48, 168, and 336 hours after incubation). [Figure 23] FIG. 23 is an image showing the results of comparing the in vitro ARG-binding ability of each 89Zr-labeled antibody. [Figure 24] FIG. 24 is an image showing the results of comparing the in vitro ARG-binding ability of 225Ac-labeled antibodies. [Figure 25] FIG. 25 shows images showing the results of PET-CT imaging performed 48 hours after administration of each 89Zr-labeled antibody. [Figure 26] Figure 26 is a graph showing the results of VOI (volume of interest, three-dimensional ROI) analysis of the tumor (top panel), heart (middle panel), and liver (bottom panel) at each time point (12, 48, 84, 168, and 252 hours) from SPECT images of each 89Zr-labeled antibody prepared using 89Zr-labeled DOTA-Bn-DBCO (-■-) and 89Zr-labeled DOTAGA-DBCO (...●...). [Figure 27] Figure 27 is a graph showing the results of calculating the tumor-liver ratio from the accumulation in the tumor and liver at each time point (12, 48, 84, 168, and 252 hours) after administration of each 89Zr-labeled antibody prepared using 89Zr-labeled DOTA-Bn-DBCO (-●-) and 89Zr-labeled DOTAGA-DBCO (...●...). [Figure 28A]Figure 28A is a graph showing the radioactivity accumulation per unit weight (%ID / g) in each organ during biodistribution at 20, 68, and 188 hours after administration of Zr-labeled antibodies prepared using Zr-labeled DOTA-Bn-DBCO (-■-) and Zr-labeled DOTAGA-DBCO (...●...). The upper and lower panels show the results for tumor and blood, respectively. [Figure 28B] Figure 28B is a graph showing the radioactivity accumulation per unit weight (%ID / g) in each organ during biodistribution at 20, 68, and 188 hours after administration of Zr-labeled antibodies prepared using Zr-labeled DOTA-Bn-DBCO (-■-) and Zr-labeled DOTAGA-DBCO (...●...). The upper and lower panels show the results for the liver and kidney, respectively. [Figure 28C] Figure 28C is a graph showing the radioactivity accumulation per unit weight (%ID / g) in each organ during biodistribution at 20, 68, and 188 hours after administration of Zr-labeled antibodies prepared using Zr-labeled DOTA-Bn-DBCO (-■-) and Zr-labeled DOTAGA-DBCO (...●...). The upper and lower panels show the results for the heart and lung, respectively. [Figure 28D] Figure 28D is a graph showing the radioactivity accumulation per unit weight (%ID / g) in each organ during biodistribution at 20, 68, and 188 hours after administration of Zr-labeled antibodies prepared using Zr-labeled DOTA-Bn-DBCO (-■-) and Zr-labeled DOTAGA-DBCO (...●...). The results for the spleen and pancreas are shown, respectively. [Figure 29] Figure 29 is a graph showing the time course of the radioactivity accumulation rate (%ID) in excreted feces (-□-, ....O...) and urine (-■-, ...●...) 20, 68, and 188 hours after administration of each 89Zr-labeled antibody produced using 89Zr-labeled DOTA-Bn-DBCO (-■-, -□-) and 89Zr-labeled DOTAGA-DBCO (...●..., ....O...). [Figure 30]Figure 30 is a graph showing the changes in tumor volume (A) and body weight (B) over time after administration of 225Ac-labeled antibodies produced using 225Ac-labeled DOTAGA-DBCO to cancer-bearing mice at a radioactivity dose of 5 kBq / mouse or 10 kBq / mouse. [Figure 31] FIG. 31 is a graph showing the results of examining hematotoxicity (white blood cell count) in cancer-bearing mice after administration of a 225Ac-labeled antibody prepared using 225Ac-labeled DOTAGA-DBCO. [Figure 32] FIG. 32 is a graph showing the results of examining hematotoxicity (platelet count) in cancer-bearing mice after administration of a 225Ac-labeled antibody prepared using 225Ac-labeled DOTAGA-DBCO. [Figure 33] FIG. 33 is a graph showing the results of examining hepatotoxicity (ALT, AST) in cancer-bearing mice after administration of 225Ac-labeled antibodies prepared using 225Ac-labeled DOTAGA-DBCO. [Figure 34] FIG. 34 is a graph showing the results of examining nephrotoxicity (BUN) in cancer-bearing mice after administration of a 225Ac-labeled antibody prepared using 225Ac-labeled DOTAGA-DBCO. [Figure 35] FIG. 35 shows the results of time-course PET-CT imaging using [ 89 Zr]Random-DFO-anti-MUC5AC humanized antibody. [Figure 36] Figure 36 is a graph showing the results of VOI (volume of interest, three-dimensional ROI) analysis of the tumor (-●-), heart (blood) (...■...), and liver (...△...) at each time point from PET images of [89Zr]Random-DFO-anti-MUC5AC humanized antibody. DETAILED DESCRIPTION OF THE INVENTION

[0012] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art.

[0013] (1) Complex 1 The present invention provides a conjugate (hereinafter also referred to as the conjugate of the present invention) of a chelating agent having a chelated radionuclide (hereinafter also referred to as the chelating agent of the present invention) and an antibody, wherein the radionuclide is a metal nuclide that emits alpha rays, and the antibody is a humanized antibody that specifically binds to MUC5AC.

[0014] (1-1) Radionuclides The radionuclide contained in the complex of the present invention is a metal nuclide that emits α-rays. The metal nuclide may be any nuclide that emits α-rays during the decay process of the radioactive metal, and more specifically, 212 Bi, 213 Bi, 227 Th or 225 Ac and the like are preferably used, and more preferably 227 Th or 225 Ac, and more preferably 225 Ac (actinium-225). The metal nuclides that emit alpha rays in the present invention are generated using accelerators such as cyclotrons and linear accelerators. It can be produced by known methods, for example 225 Ac is measured using a cyclotron. 226 It can be produced by the (p,2n) nuclear reaction by irradiating a Ra target with protons. The produced alpha-emitting metal nuclide can be purified by separating it from the target, for example. 225 Ac is 225 By dissolving the target containing Ac in acid, etc. and adding alkali to the solution, 225 The salt containing Ac is precipitated, and the salt is separated and purified to obtain a purified product. 225 The α-ray emitting nuclide purified in this way can be chemically treated as needed to obtain a chemical form suitable for RI labeling, and can then be used for RI labeling.

[0015] (1-2) Antibody The antibody contained in the conjugate of the present invention is a humanized antibody that specifically binds to MUC5AC (hereinafter also referred to as the humanized antibody used in the present invention). The antibody is not particularly limited as long as it is a humanized antibody that has the ability to specifically bind to MUC5AC, and it is preferable that it has stable physical properties and excellent tumor accumulation. The antibody may be used as its antigen-binding fragment, and such embodiments are also encompassed by the present invention. Specifically, it contains the specific heavy chain variable region and light chain variable region described below, and can optionally have appropriate heavy chain constant region and light chain constant region. As used herein, the term "antigen-binding fragment" refers to an antibody fragment consisting of a portion of the humanized antibody used in the present invention, which has the ability to bind to MUC5AC. The number of amino acids contained in the polypeptide constituting the antigen-binding fragment is not particularly limited, as long as it has the ability to bind to MUC5AC.

[0016] Preferred amino acid sequences for the heavy chain variable region of the humanized antibody used in the present invention are shown below. Heavy chain variable region 1 (H01), heavy chain variable region 2 (H02), heavy chain variable region 3 (H03), and heavy chain variable region 4 (H04) correspond to SEQ ID NOS: 1 to 4, respectively, in the sequence listing attached hereto. The underlined regions are CDR regions.

[0017] [ka]

[0018] Preferred amino acid sequences for the light chain variable region of the humanized antibody of the present invention are shown below. Light chain variable region 1 (L01), light chain variable region 2 (L02), light chain variable region 3 (L03), and light chain variable region 4 (L04) correspond to SEQ ID NOS: 5 to 8, respectively, in the sequence listing attached hereto. The underlined regions are CDR regions.

[0019] [ka]

[0020] In other words, the heavy chain variable region of a preferred humanized antibody of the present invention consists of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 4, and the light chain variable region consists of the amino acid sequence shown in any one of SEQ ID NOs: 5 to 8. That is, the humanized antibody used in the present invention consists of a combination of the four heavy chain variable regions (H01 to H04) and four light chain variable regions (L01 to L04) described above.

[0021] In the present invention, a preferred humanized antibody is one in which the heavy chain variable region is H01, H03, or H04 and the light chain variable region is any one of L01 to L04.

[0022] The most preferred humanized antibody of the present invention is an antibody in which the heavy chain variable region is H01 and the light chain variable region is L03.

[0023] In the present invention, the heavy chain variable region of a humanized antibody is not limited to those defined by the amino acid sequences set forth in SEQ ID NOs: 1 to 4, and also includes mutants that retain function. That is, mutated heavy chain variable regions consisting of amino acid sequences that share 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more sequence identity with the amino acid sequences set forth in SEQ ID NOs: 1 to 4 can also be used as the heavy chain variable region of a humanized antibody used in the present invention, as long as they retain the ability to bind to MUC5AC when combined with the light chain variable region of the present invention.

[0024] As used herein, amino acid sequence identity refers to the identity of the amino acid sequences between two proteins of interest, and is expressed as the percentage (%) of identical amino acid residues in an optimal alignment of amino acid sequences generated using mathematical algorithms known in the art. Amino acid sequence identity can be determined by visual inspection and mathematical calculation, and can be calculated using homology search programs (e.g., BLAST, FASTA), sequence alignment programs (e.g., ClustalW), or genetic information processing software (e.g., GENETYX [registered trademark]) well known to those skilled in the art. Specifically, amino acid sequence identity herein was measured using the phylogenetic analysis program ClustalW (http: / / clustalw.ddbj.nig.ac.jp / index.php?lang=ja) available on the DDBJ (DNA DataBank of Japan) website under the default conditions (Version 2.1, Alignment type: slow, DNA Weight Matrix: Gonnet, GAP OPEN: 10, GAP EXTENSION: 0.1). You can ask for it.

[0025] In addition, the heavy chain variable region of the humanized antibody used in the present invention includes SEQ ID NO: 1 to SEQ ID NO: A mutated heavy chain variable region consisting of an amino acid sequence in which 10 or less, preferably 8 or less, more preferably 5 or less, and most preferably 3 or less amino acids are deleted, substituted, or added in the amino acid sequence shown in No. 4 can also be used as the heavy chain variable region of the humanized antibody used in the present invention, as long as it has the ability to bind to MUC5AC when combined with the light chain variable region of the present invention.

[0026] The light chain variable regions of the humanized antibodies used in the present invention are not limited to the amino acid sequences set forth in SEQ ID NOs: 5 to 8, and also include mutants that retain their function. That is, mutated light chain variable regions consisting of amino acid sequences that share 90% or more, preferably 95% or more, more preferably 98% or more, and most preferably 99% or more sequence identity with the amino acid sequences set forth in SEQ ID NOs: 5 to 8 can also be used as the light chain variable regions of the humanized antibodies used in the present invention, as long as they retain the ability to bind to MUC5AC when combined with the heavy chain variable regions of the present invention.

[0027] In addition, the light chain variable region of the humanized antibody used in the present invention may be a mutated light chain variable region consisting of an amino acid sequence in which 10 or less, preferably 8 or less, more preferably 5 or less, and most preferably 3 or less amino acids are deleted, substituted, or added in the amino acid sequences shown in SEQ ID NO:5 to SEQ ID NO:8, as long as it has the ability to bind to MUC5AC when combined with the heavy chain variable region of the present invention.

[0028] The humanized antibody used in the present invention can be produced by a method commonly used in the art or a method based thereon. Specifically, it can be produced by the following procedure. The amino acid sequences of the heavy and light chain variable regions of the humanized antibodies used in the present invention are disclosed in SEQ ID NOs: 1 to 8. Based on these amino acid sequence information, nucleic acids encoding the antibodies are constructed and inserted into an appropriate expression vector. In addition to the nucleic acid encoding the humanized antibodies used in the present invention, the expression vector may optionally contain a Kozak sequence to increase translation efficiency, a signal sequence that promotes secretion of the humanized antibodies used in the present invention into the culture medium when introduced into a host, and a promoter sequence. Vectors that can be used in the present invention can be selected from those commonly used in the art, but the plasmid vector pcDNA3.4 is preferred. The introduction of expression vectors into host cells is not particularly limited. Methods for introducing genes into cells can be conventionally used in the art, such as the calcium phosphate method, electroporation, lipofection, and DEAE-dextran method, which are well known to those skilled in the art. As demonstrated in the Examples below, lipofection is particularly preferred. Host cells used for this purpose can also be conventionally used in the art. Examples of such host cells include CHO cells, 293 cells, Escherichia coli, Pichia yeast, Sf9 cells, etc. Expression system kits for expressing target proteins are now commercially available, and the ExpiCHO System (Thermo Fisher Scientific) used in the following examples is a rapid This is particularly preferred for rapid and reliable expression of the target protein.

[0029] The nucleic acid encoding the humanized antibody used in the present invention is inserted into an expression vector, the nucleic acid is introduced into host cells using the expression vector containing the nucleic acid, the host cells into which the nucleic acid has been introduced are cultured, and the humanized antibody used in the present invention can be obtained from the culture supernatant by a purification method such as chromatography. In this method, the humanized antibody used in the present invention is secreted into the culture supernatant by culturing the host cells. The humanized antibody or antigen-binding fragment thereof used in the present invention can be obtained from the culture supernatant by a purification method such as chromatography. Chromatography methods known in the art include affinity chromatography, ion exchange chromatography, and size exclusion chromatography. Affinity chromatography using a Protein A column, as used in the Examples below, is particularly preferred.

[0030] The humanized antibody may be a polyclonal or monoclonal antibody.

[0031] (1-3) Chelating agents In the present invention, the chelating agent is not particularly limited as long as it has a site in its structure where a radionuclide is coordinated, but preferably has a chelating moiety, which is the site where a radionuclide is coordinated, and a substituent that enables conjugation with an antibody. (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)、DOTMA((1R,4R,7R,10R)-α,α’,α”,α’”-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic 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 acid)、DOTP(((1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(methylene))tetraphosphonic acid)、DOTMP(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid))、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 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), porphyrin, However, it is preferable that the compound has a structure derived from the compound represented by the following formula (A):

[0032] [ka]

[0033] (In formula (A), R 11 , R 13 and R 14 are each independently -(CH2) p COOH, -(CH2) p C5H5N, -(CH2) p PO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2)p is a group consisting of COOH, and R 12 or R 15 one of which is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a substituent for conjugating with the antibody, p is an integer of 0 to 3, and R 12 is a substituent for conjugation with the antibody, R 15 is a hydrogen atom, and R 12 is not a substituent for conjugation with the antibody, R 15 is a substituent for conjugation to the antibody.

[0034] Specific examples of the structure represented by formula (A) include structures derived from compounds represented by the following formulae (A-1) to (A-12).

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] The linking site between the chelating moiety and the substituent that enables conjugation with an antibody is preferably an amide bond or a thiourea bond, with the amide bond being more preferred from the viewpoint of stability.

[0039] An amide bond is formed, for example, by the reaction of the N-hydroxysuccinimide ester (NHS) group of the above formula (A-10) or (A-11) or the 2,6-dioxotetrahydro-2H-pyranyl group of the above formula (A-12) with a primary amine. A thiourea bond is formed by the reaction of the isothiocyanate group of the compounds represented by the above formula (A-2) or (A-3) with a primary amine or a maleimide group.

[0040] In the conjugate of the present invention, at least one molecule of the chelating agent is provided per antibody molecule. However, from the viewpoint of maintaining the activity of the antibody itself (antigen recognition activity, neutralization activity, complement activation activity and / or opsonization activity), it is preferable that the chelating agent is site-specifically introduced into the Fc region (constant region) of the antibody, and in the present invention, one or two chelating agent molecules are provided per antibody molecule. It is more preferable to have

[0041] In the conjugate of the present invention, the chelating agent may be connected to the antibody via a linker, such as 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, or any of these. Combinations are also possible. Preferably, the chelating agent site-specifically modifies the antibody via a linker, more preferably the Fc region. In this case, the linker can be one that contains a peptide consisting of 13 to 17 amino acid residues (hereinafter also referred to as "antibody-modifying peptide") represented by the following formula (i), and is formed by a crosslinking reaction between the antibody-modifying peptide modified with a crosslinking agent and the antibody. In formula (i), the left side of the amino acid sequence on the paper represents the N-terminus, and the right side of the amino acid sequence on the paper represents the C-terminus. When the chelating agent is connected to the antibody via the antibody-modifying peptide as a linker, the position at which the chelating agent and the antibody-modifying peptide are linked is not particularly limited, and they can be linked, for example, directly or indirectly to the N-terminus or C-terminus of the antibody-modifying peptide, preferably the N-terminus. In addition, the antibody-modifying peptide can be The C-terminus of the amino acid sequence may be modified by amidation or the like to improve its stability.

[0042] (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) In formula (i), Xa, Xb, Xc, and Xd represent a consecutive Xs, b consecutive Xs, c consecutive Xs, and d consecutive Xs, respectively; X is an amino acid residue having neither a thiol group nor a haloacetyl group in the side chain, a, b, c, and d are each independently an integer of 1 to 5, and satisfy a+b+c+d≦14. Xaa1 and Xaa3 each independently represent represents an amino acid residue derived from an amino acid having a thiol group in the side chain, or one represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, and Xaa1 and Xaa3 are linked together; Xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or a diaminopropionic acid residue, and is modified with a cross-linking agent.

[0043] Examples of amino acid residues that can be contained in X in the above formula (i) 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 be amino acid residues consisting of the same type of amino acid, or may be amino acid residues consisting of different types of amino acids.

[0044] There are no particular limitations on a, b, c, and d in formula (i) as long as they are numbers within the above-mentioned ranges. However, from the viewpoint of binding stability between the peptide and the antibody, a is preferably an integer of 1 or more and 3 or less, b is preferably an integer of 1 or more and 3 or less, c is preferably an integer of 3 or more and 5 or less, and d is preferably an integer of 1 or more and 3 or less, provided that a+b+c+d≦14.

[0045] Xaa1 and Xaa3 are amino acid residues derived from amino acids having a thiol group in the side chain, and the amino acids may be the same or different. Examples of amino acids having a thiol group in the side chain include cysteine and homocysteine. Such amino acid residues are preferably bonded via a disulfide bond, or the sulfide group is bonded via a linker shown in formula (4) below. In formula (4), the wavy line indicates the bond to the sulfide group.

[0046] [ka]

[0047] Alternatively, 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 bonded via a thioether bond. The haloacetyl group has a terminal substituted with a halogen such as iodine, and upon reaction with the thiol group in the other side chain, the halogen is eliminated to form a thioether bond.

[0048] Specific amino acid sequences of the antibody-modifying peptide represented by formula (i) include, for example, the peptides described in WO 2016 / 186206, WO 2017 / 217347, and WO 2018 / 230257, and these can also be used.

[0049] Among these, the antibody-modifying peptide preferably has any one of the following sequences (1) to (14) as its amino acid sequence, and more preferably has the following sequence (1), (2), (13), or (14). 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 a diaminopropionic acid residue, and (Xaa1) and (Xaa3) both represent homocysteine residues. In the following amino acid sequences (1) to (14), amino acids other than (Xaa1), (Xaa2), and (Xaa3) are abbreviated with one-letter codes.

[0050] (1) DCAYH(Xaa2)GELVWCT (SEQ ID NO: 9) (2) GPDCAYH(Xaa2)GELVWCTFH (SEQ ID NO: 10) (3) RCAYH(Xaa2)GELVWCS (SEQ ID NO: 11) (4) GPRCAYH(Xaa2)GELVWCSFH (SEQ ID NO: 12) (5) SPDCAYH(Xaa2)GELVWCTFH (SEQ ID NO: 13) (6) GDDCAYH(Xaa2)GELVWCTFH (SEQ ID NO: 14) (7) GPSCAYH(Xaa2)GELVWCTFH (SEQ ID NO: 15) (8) GPDCAYH(Xaa2)GELVWCSFH (SEQ ID NO: 16) (9) GPDCAYH(Xaa2)GELVWCTHH (SEQ ID NO: 17) (10) GPDCAYH(Xaa2)GELVWCTFY (SEQ ID NO: 18) (11) SPDCAYH(Xaa2)GELVWCTFY (SEQ ID NO: 19) (12) SDDCAYH(Xaa2)GELVWCTFY (SEQ ID NO: 20) (13) RGNCAYH(Xaa2)GQLVWCTYH (SEQ ID NO: 21) (14) G(Xaa1)DCAYH(Xaa2)GELVWCT(Xaa3)H (SEQ ID NO: 22)

[0051] (1-4) Method for producing the composite The method for producing the conjugate of the present invention comprises two steps: a conjugation step in which a chelating agent is conjugated to an antibody, and a complex formation step in which a complex between a radionuclide and a chelating agent is formed. The conjugation step may be performed before or after the complex formation step.

[0052] In the conjugation step, various methods for chemically modifying antibodies are used. The methods are (a) to (f). (a) Amine coupling method (Carbohydrate activated with N-hydroxysuccinimide (NHS) group) A method for modifying the amino group of an antibody lysine residue using a chelating agent or chelate having an xyl group. (b) A method of modifying sulfhydryl (SH) groups generated by partially reducing disulfide bonds (SS bonds) between polypeptide chains in the hinge region of an antibody with a chelating agent or linker having a maleimide group that is reactive to SH groups. (c) A method of modifying a cysteine newly introduced into an antibody by amino acid mutation through genetic engineering with a chelating agent or linker having a maleimide group. (d) The azide group of the azide-modified lysine, which has been newly introduced into the antibody by amino acid mutation through genetic engineering, is converted to an alkyne (e.g., dibensylcyclooctene: DBCO) using the click reaction. Methods for modifying chelators or linkers with (e) A method using transglutaminase to modify glutamine introduced at a specific position of an antibody with a chelating agent or linker having a lysine side chain. (f) A chelating agent or linker having the antibody-modifying peptide shown in (i) above is added to an antibody. Method for site-specific modification of the Fc region of the body - Patents.com

[0053] In the complex formation step, a radionuclide is chelated (complexed) with a chelating agent. The radionuclide used here is preferably used in an ionizable form, more preferably in an ionic form, from the viewpoint of increasing the efficiency of complex formation. In the complex formation step, the order of adding the radionuclide to the chelating agent is not important, as long as a complex can be formed with the radionuclide. For example, a solution in which radioactive metal ions are dissolved in a solvent mainly composed of water can be used as the radionuclide. After the complex formation, the resulting complex may be purified using a filter, a membrane filter, a column filled with various packing materials, chromatography, or the like.

[0054] In the method for producing the complex of the present invention, the conjugation step is preferably carried out after the complex formation step. In a more preferred embodiment, in the complex formation step (A), a complex is formed between a radionuclide and a chelating agent having a first atomic group capable of Click reaction as a substituent for enabling conjugation with an antibody. Then, in the conjugation step (B), the above-mentioned (i) Using an antibody-modifying peptide and an antibody-modifying linker having a second atomic group capable of Click reaction, a Click reaction is carried out between the peptide-modified antibody whose Fc region has been site-specifically modified and the complexed chelating agent obtained in step (A), thereby obtaining the conjugate of the present invention. Steps (A) and (B) will be described in detail below.

[0055] The combination of the first and second atomic groups capable of Click reaction 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 selected such that the first atomic group has one of the combinations of atomic groups listed above, and the second atomic group has an atomic group that is different from the first atomic group. From the viewpoint of achieving both stability of the chelating agent and the antibody and improved binding efficiency, it is preferable that the chelating linker be an alkyne and the antibody-modifying linker be an azide, or that the chelating linker be a 1,2,4,5-tetrazine and the antibody-modifying linker be an alkene. Specific examples of Click reactions using such combinations of atomic groups include the Huisgen cycloaddition reaction and the inverse electron demand Diels-Alder reaction.

[0056] A specific example of a combination of atomic groups capable of a click reaction is a combination of an atomic group (formula (1a)) containing dibenzylcyclooctyne (DBCO) as the alkyne of the first atomic group and an atomic group (formula (2a)) containing an azide group as the azide of the second atomic group, as shown in the following formula: Alternatively, a combination of an atomic group (formula (1b)) in which the first atomic group contains 1,2,4,5-tetrazine and an atomic group (formula (2b)) in which the second atomic group contains trans-cyclooctene (TCO) as an alkene can be mentioned. A combination of formula (1a) and formula (2a) is preferred.

[0057] [ka]

[0058] (In the formula, R1 represents a linking site with a chelating agent, and R2 represents a linking site with an antibody-modifying peptide in the antibody.)

[0059] [ka]

[0060] (In the formula, one of R3 and R4 represents a linking site to either a chelating agent or an antibody-modifying peptide in the antibody, the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and R5 represents a linking site to either a chelating agent or an antibody-modifying peptide in the antibody, depending on R3 or R4.)

[0061] As the alkyne of the first atomic group, dibenzylcyclooctyne ( When an atomic group containing DBCO is used, various commercially available DBCO reagents can be used. Specific examples include DBCO-C6-Acid, Dibenzylcyclooctyne-Amine, Dibenzylcyclooctyne Maleimide, DBCO-PEG acid, DBCO-PEG-NHS ester, DBCO-PEG-Alcohol, DBCO-PEG-amine, DBCO-PEG-NH-Boc, Carboxyrhodamine-PEG-DBCO, Sulforhodamine-PEG-DBCO, TAMRA-PEG-DBCO, DBCO-PEG-Biotin, DBCO-PEG-DBCO, DBCO-PEG-Maleimide, TCO-PEG-DBCO, and DBCO-mPEG. Dibenzylcyclooctyne Maleimide is preferred.

[0062] In step (A), a chelating agent having a structure represented by the following formula (ii) is more preferably used. ABC (ii) In formula (ii), A is a chelating moiety represented by the following formula (iia):

[0063] [ka]

[0064] In formula (iia), Ra, Rb and Rc are independently —(CH2) p COOH, -(CH2) p C5H5N, -(CH2) pPO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p A group consisting of COOH, p is an integer of 0 to 3, one of Rd and Re is a bonding site (*) with B, and the other is a hydrogen atom or -(CH2) p COOH, -(CH2) p C5H5N, -(CH2)pPO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p It is a group consisting of COOH, and p is an integer of 0 or more and 3 or less. In formula (ii), B is represented by the following formula (iib).

[0065] [ka]

[0066] In formula (iib), La and Lb are independently a linker having 1 to 50 carbon atoms and containing at least an amide bond or a thiourea bond; t is an integer of 0 to 30; s is 0 or 1; * is a bonding site with A; and ** is a bonding site with C. In formula (ii), C is either an alkyne derivative represented by the following formula (iic) or a tetrazine derivative represented by formula (iid).

[0067] [ka]

[0068] 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, when X is CHRk-** and Y is CHRk, the Rk moieties may be joined together to 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, Rf and Rg may be joined together or Rh and Ri may be joined together to form a hydrocarbon ring, ** represents a bonding site with B, and In iid), ** indicates the bonding site with B, and Rj indicates a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group.

[0069] The chelating agent used in step (A) is a compound represented by the formula (iia) in which Ra to Rd are each -(CH2) p More preferred are DOTA derivatives in which R a to R c are —(CH 2 ) pCOOH, p is 1, R d is a bonding site (*) with B, and Re is a hydrogen atom; or DO 3 A derivatives or DOTAGA derivatives in which R a to R c are —(CH 2 ) pCOOH, p is 1, R d is a bonding site (*) with B, and Re is a hydrogen atom.

[0070] In formula (ii), when A is the DOTA derivative, B is a linker having 1 to 50 carbon atoms, in which La contains a thiourea bond, and s is 0 or 1. When s is 1, a DOTA-PEGt-DBCO derivative in which La is a linker having 1 to 50 carbon atoms and containing an amide bond or a thiourea bond, t is an integer of 0 to 30, Lb is a linker having 1 to 50 carbon atoms and containing an amide bond or a thiourea bond, C is an alkyne derivative represented by formula (iic), in which X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg are joined together to form a benzene ring, Rh and Ri are joined together to form a benzene ring, and ** is a bonding site with B; or B is a DOTA-PEGt-DBCO derivative in which La is a linker having 1 to 50 carbon atoms and containing a thiourea bond, s is 0 or 1, and when s is 1, t is an integer of 0 to 30 Further preferred is a DOTA-PEGt-Tz derivative in which Lb is a linker containing an amide bond or a thiourea bond and having 1 to 50 carbon atoms, and C is a tetrazine derivative represented by formula (iid).

[0071] In formula (ii), when A is the above-mentioned DO3A derivative, B is even more preferably a DO3A-PEGt-DBCO derivative in which La is a linker having 1 to 50 carbon atoms and containing an amide bond, s is 0 or 1, and when s is 1, t is an integer of 0 to 30, and Lb is a linker having 1 to 50 carbon atoms and containing an amide bond; and C is an alkyne derivative represented by formula (iic), in which X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg are joined together to form a benzene ring, Rh and Ri are joined together to form a benzene ring, and ** is a bonding site with B.

[0072] In formula (ii), when A is the DOTAGA derivative, B is even more preferably a DOTAGA-PEGt-DBCO derivative in which La is a linker having 1 to 50 carbon atoms and containing an amide bond or a thiourea bond, s is 0 or 1, and when s is 1, t is an integer of 0 to 30, and Lb is a linker having 1 to 50 carbon atoms and containing an amide bond or a thiourea bond, and C is an alkyne derivative represented by formula (iic), in which X is N-**, Y is CHRk, Rk is a hydrogen atom, Rf and Rg are joined together to form a benzene ring, Rh and Ri are joined together to form a benzene ring, and ** is a bonding site with B.

[0073] The molar ratio of the chelating agent to the radionuclide, in terms of chelating portion / radionuclide, is preferably 10 / 1 or more in lower limit, more preferably 100 / 1 or more, and even more preferably 500 / 1 or more in upper limit, and is preferably 10,000 / 1 or less, more preferably 8,000 / 1 or less, and even more preferably 7,000 / 1 or less in upper limit, for example, preferably in the range of 100 / 1 or more and 7,000 / 1 or less, more preferably 500 / 1 or more and 7,000 / 1 or less.

[0074] The complex-forming reaction is preferably carried out in a solvent, such as water, saline, sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, trishydroxymethylaminomethane buffer (Tris buffer), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES buffer), or For example, a buffer solution such as tetramethylammonium acetate buffer can be used.

[0075] The volume of the solvent is not particularly limited, but from the viewpoint of practicality in the production process, at the start of step (A), the lower limit is 0.01 mL or more, preferably 0.1 mL or more, more preferably 1.0 mL or more, even more preferably 10 mL or more, and even more preferably 100 mL or more, and the upper limit is preferably 1000 mL or less, more preferably 100 mL or less, even more preferably 10 mL or less, and even more preferably 1.0 mL or less, for example, in the range of 0.01 mL or more and 100 mL or less.

[0076] From the viewpoint of the yield of the desired chelating agent, the concentration of the chelating agent in the reaction solution for the complex formation reaction at the start of step (A) is, independently, preferably 0.001 μmol / L or more in lower limit, more preferably 0.01 μmol / L or more, even more preferably 0.1 μmol / L or more, and more preferably 1 μmol / L or more in upper limit, and is preferably 1000 μmol / L or less, more preferably 100 μmol / L or less, and even more preferably 10 μmol / L or less in upper limit, for example, in the range of 1 μmol / L or more and 100 μmol / L or less.

[0077] The temperature for the complex formation reaction may be, for example, room temperature (25°C) or may be under heated conditions. From the viewpoint of simultaneously suppressing decomposition of the chelating agent and improving the efficiency of complex formation, the lower limit is preferably 20°C or higher, more preferably 30°C or higher, even more preferably 35°C or higher, still more preferably 37°C or higher, and particularly preferably 45°C or higher, and the upper limit is preferably 150°C or lower, more preferably 120°C or lower, even more preferably 100°C or lower, and still more preferably 90°C or lower. For example, a range of 30°C or higher and 100°C or lower is preferred, and a range of 35°C or higher and 90°C or lower is more preferred.

[0078] Provided that the reaction temperature is as described above, the lower limit of the reaction time is preferably 5 minutes or more, more preferably 10 minutes or more, even more preferably 20 minutes or more, still more preferably 30 minutes or more, and particularly preferably 45 minutes or more, and the upper limit is preferably 180 minutes or less, more preferably 150 minutes or less, even more preferably 120 minutes or less, still more preferably 90 minutes or less, and particularly preferably 60 minutes or less, for example, a range of 10 minutes or more and 150 minutes or less is preferred, and a range of 10 minutes or more and 60 minutes or less is more preferred.

[0079] The antibody used in step (B) is a compound obtained by combining the antibody-modified peptide shown in (i) above with a click. This is a peptide-modified antibody in which the Fc region (constant region) of the humanized antibody described in detail in the section "(1-2) Antibodies" above has been site-specifically modified using an antibody-modifying linker having a reactive second atomic group.

[0080] Antibody-modified peptides 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 peptides, functional groups of the amino acids used may be protected as needed. This can be done, for example, according to the methods described in International Publication Nos. 2017 / 217347 and 2018 / 230257.

[0081] The antibody-modifying linker may be an antibody-modifying peptide bound to a linker represented by the following formula (S1). *-((L1) m -Z) k -L2-AG2···(S1) (wherein * represents a binding site to the N-terminus or C-terminus of the peptide, L1 is a polyethylene glycol (PEG) linker moiety; m is an integer between 1 and 50, Z is (L1) m and a second linker portion connecting L2 and L3, k is 0 or 1; L2 is a second PEG linker moiety; AG2 is the second atomic group.)

[0082] In the formula (S1), the structure of Z is (L1): m and L2. The linker structure is not particularly limited as long as it bonds L2 to Z, and may include, for example, an amino acid sequence consisting of 1 to 5 amino acid residues. In this case, the amino acid sequence included in Z preferably includes a cysteine residue, and more preferably is bonded to L2 via a thioether group formed by bonding a thiol group of the cysteine residue with a maleimide group.

[0083] In the present invention, the PEG linker moiety constituting L2 preferably has a structure shown in the following formula (P2): In formula (P2), n is an integer, preferably 1 to 50, more preferably 1 to 20, even more preferably 2 to 10, and even more preferably 2 to 6.

[0084] [ka]

[0085] One end of the PEG linker structure may be modified with a structure derived from a commercially available PEGylation reagent or a structure derived from a reagent commonly used for PEGylation, and examples thereof include, but are not limited to, a structure derived from diglycolic acid or a derivative thereof, or maleimide or a derivative thereof.

[0086] A method for introducing the second atomic group into the antibody-modifying linker includes obtaining an antibody-modifying peptide having the desired amino acid sequence by the above-mentioned method, dissolving the peptide in a solution containing a solubilizing agent, a reducing agent, and, if necessary, an acid, and adding to the solution an organic solvent solution of an atomic group containing an azide group or trans-cyclooctene (TCO) as the second atomic group, and stirring the mixture at room temperature.

[0087] When an atomic group containing an azide group is to be introduced as the second atomic group, the azide group can be introduced directly into the N-terminus or C-terminus of the peptide according to a standard method using a commercially available azide group-introducing reagent, or the atomic group containing an azide group can be introduced via the above-mentioned linker structure. Examples of the azide group-introducing reagent that can be used include silyl azide, phosphoric acid azide, alkylammonium azide, inorganic azide, sulfonyl azide, and PEG azide.

[0088] Furthermore, when an atomic group containing TCO is introduced as the second atomic group, TCO can be introduced directly to the N-terminus or C-terminus of the peptide using a commercially available click chemistry reagent containing TCO according to a standard method, or the atomic group containing TCO can be introduced via the above-mentioned linker structure.

[0089] The method of binding an antibody-modified peptide to an antibody to obtain a peptide-modified antibody can be carried out using, for example, a crosslinking agent. A crosslinking agent is a chemical substance that covalently links an antibody-modified peptide to an antibody, and examples thereof include succinimidyl groups such as disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS). Examples of suitable cross-linking agents include cross-linkers containing two or more imidic acid moieties, cross-linkers consisting of compounds containing preferably two or more imidic acid moieties, such as dimethyl adipimidate, or salts thereof, and cross-linkers consisting of compounds having a disulfide bond, such as dimethyl 3,3'-dithiobispropionimidate and dithiobissuccinimidyl propionic acid, or salts thereof. The use of such cross-linking agents enables a cross-linking reaction to occur between the Xaa2 amino acid residue in the antibody-modifying peptide and the antibody. For example, when a humanized antibody of the present invention is used as the antibody, the cross-linking reaction in the antibody occurs site-specifically between the Xaa2 amino acid residue and the Lys252 residue (according to the EU numbering system) in the humanized antibody of the present invention. These Lys residues are present in the Fc region of the humanized antibody of the present invention.

[0090] The antibody-modifying peptide can be bound to an antibody, for example, by dispersing the above-mentioned antibody-modifying peptide, antibody, crosslinker, and, if necessary, catalyst in an appropriate buffer solution at a temperature of 10°C to 30°C. The reaction time can be approximately 10 minutes to 2 hours. The molar ratio of antibody / peptide during the reaction between the peptide and antibody is preferably 1 / 5 or more, more preferably 1 / 3 or more, and even more preferably 1 / 1.5 or more, and preferably 20 / 1 or less, more preferably 10 / 1 or less, even more preferably 5 / 1 or less, even more preferably 1 / 1 or less, and particularly preferably 1 / 1.7 or less, for example, preferably in the range of 1 / 5 to 20 / 1, and more preferably 1 / 1.5 to 1 / 1.7.

[0091] The peptide-modified antibody obtained through the above steps is a mixture containing an arbitrary ratio of antibodies in which one antibody molecule is bound to one antibody-modifying peptide molecule (hereinafter referred to as "monovalent antibodies") and antibodies in which two antibody-modifying peptide molecules are bound to one antibody molecule (hereinafter referred to as "bivalent antibodies"), and this may be subjected to subsequent steps as is, or the unmodified antibody, monovalent antibody, and bivalent antibody may be separated and purified using a filtration filter, membrane filter, columns filled with various packing materials, various types of chromatography, or other methods, and only antibodies of one valency may be subjected to subsequent steps. If separation of unmodified antibodies from antibodies of other valencies is not possible as a result of purification, the mixture containing these may be subjected to subsequent steps. When separating and purifying an unmodified antibody, a monovalent antibody, and a divalent antibody, any of the purification methods described above may be used. However, it is preferable to use a column packed with various packing materials, and it is more preferable to use a column packed with a packing material suitable for separating and purifying proteins such as antibodies.

[0092] Packings suitable for separating and purifying proteins such as antibodies are not particularly limited, as long as they are made of a carrier comprising a water-insoluble base material and an immunoglobulin-binding protein that specifically binds to antibodies is immobilized thereon. Examples of immunoglobulin-binding proteins include protein A, protein G, and protein L. These immunoglobulin-binding proteins may be genetically engineered recombinant proteins, and examples of recombinant immunoglobulin-binding proteins include genetically modified protein A, genetically modified protein G, and fusions of protein A domains with protein G domains. In the present invention, protein A is more preferred as a packing suitable for separating and purifying at least monovalent and bivalent antibodies, and genetically modified protein A is even more preferred. Here, protein A and protein G are protein molecules that can specifically bind to IgG, an antibody molecule, and are classified as protein A (Staphylococcus aureus) or protein G (Streptococcus genus) depending on the microorganism from which they are isolated. Genetically modified Protein A is Protein A in which at least one amino acid mutation has been introduced into an amino acid residue in any of the IgG-binding domains (E, D, A, B, and C domains) of Protein A. In the present invention, genetically modified Protein A in which a domain in which at least one amino acid mutation has been introduced is multimerized is preferred, and the A, B, or C domain in which at least one amino acid mutation has been introduced of Protein A is preferred. A main multimer is more preferred, and a multimer of dimer to pentamer is even more preferred. The amino acid mutation may be derived from any mutation, such as substitution, deletion, or insertion, of the amino acid sequence or the nucleotide sequence encoding the amino acid, during the gene transcription / translation process. Non-limiting examples include genetically modified protein A described in WO 2003 / 080655 and WO 2011 / 118699.

[0093] Examples of water-insoluble substrates onto which immunoglobulin-binding proteins can be immobilized include inorganic supports such as glass beads and silica gel; organic supports made of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; and polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran; and composite supports such as organic-organic and organic-inorganic composites obtained by combining these.

[0094] Examples of columns packed with the above-mentioned genetically modified protein A as a packing material include KanCap (registered trademark) series (KANEKA KanCapA prepacked column) from Kaneka Corporation, GE GE Healthcare's HiTrap® series (HiTrap Mabselect, HiTrap Mabselect SuRe, HiTrap Mabselect Xtra), GE Healthcare's HiScreen series (HiScreen Mabselect SuRe), or the TOYOPEARL (registered trademark) series (TOYOPEARL AF-rProtein A-650F) from Tosoh Corporation.

[0095] The following will explain the separation and purification of a peptide-modified antibody used in the click reaction in step (B) as an example. The peptide-modified antibody is subjected to a click reaction in step (B) through an antibody modification step in which the Fc region of the antibody is site-specifically modified with a linker (antibody-modifying linker) comprising the antibody-modifying peptide to obtain the modified antibody, and an antibody purification step in which the modified antibody is purified using a carrier to which the above-mentioned immunoglobulin-binding protein has been immobilized. The antibody purification step further includes a retention step in which the modified antibody retained on the carrier is retained on the carrier, a washing step in which the modified antibody not retained on the carrier is washed, and an elution step in which the modified antibody retained on the carrier in the retention step is eluted. More specifically, in the antibody modification step, modified antibodies are obtained as a mixture containing unmodified antibodies (those not modified with antibody-modifying linkers), monovalent antibodies, and divalent antibodies, and in the antibody purification step, differences in the interactions of unmodified antibodies, monovalent antibodies, and divalent antibodies with immunoglobulin-binding proteins are utilized to elute a first antibody composition relatively rich in unmodified antibodies and monovalent antibodies, and a second antibody composition relatively rich in divalent antibodies. That is, in the retention step and washing step of the antibody purification step, a second antibody composition relatively rich in peptide-modified antibodies (divalent antibodies) that interact to a low degree with immunoglobulin-binding proteins is eluted, and in the elution step of the antibody purification step, a first antibody composition relatively rich in peptide-modified antibodies (unmodified antibodies and monovalent antibodies) that interact to a high degree with immunoglobulin-binding proteins is eluted. Here, "containing a relatively large amount of unmodified antibodies and monovalent antibodies" means that the total amount of unmodified antibodies and monovalent antibodies contained in the first antibody composition is greater than the bivalent antibodies contained in the antibody composition, and preferably means that the total amount of unmodified antibodies and monovalent antibodies is 55% or more, 63% or more, 70% or more, 80% or more, or 90% or more of the total amount (100%) of unmodified antibodies and modified antibodies contained in the antibody composition; and "containing a relatively large amount of bivalent antibodies" means that the amount of bivalent antibodies contained in the second antibody composition is greater than the monovalent antibodies contained in the antibody composition, and preferably means that the amount of bivalent antibodies is 55% or more, 63% or more, 70% or more, 80% or more, or 90% or more of the total amount (100%) of unmodified antibodies and modified antibodies contained in the antibody composition.

[0096] In the retention step, a solution containing the mixture of unmodified, monovalent, and bivalent antibodies obtained in the antibody modification step is applied to a column, allowing the unmodified and monovalent antibodies retained on the support to be retained on the column, while allowing the bivalent antibodies not retained on the support to pass through. Here, the solution passed through in the retention step constitutes part of the second antibody composition. To facilitate retention of the unmodified and monovalent antibodies on the column and to prevent their aggregation or denaturation, it is preferable to dilute the mixed solution of peptide-modified antibodies with an appropriate diluent solvent before applying it to the column. The diluent solvent is not particularly limited as long as it dissolves the peptide-modified antibodies and is unlikely to aggregate or denature in the solvent. Examples of the diluent include water, saline, and buffers such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) buffer, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer. Use of any of the above buffers is preferred, with sodium acetate buffer being more preferred. When a buffer solution is used as the dilution solvent, the buffer concentration is 10 mmol / L or more, preferably 15 mmol / L or more, and more preferably 20 mmol / L or more, and 1000 mmol / L or less, preferably 500 mmol / L or less, and more preferably 100 mmol / L or less. Furthermore, to reduce nonspecific binding of bivalent antibodies or antibody-modified peptides to the column carrier, the elution solvent may contain an additive such as sodium chloride or potassium chloride. The concentration of the additive contained in the elution solvent is not particularly limited, but can be, for example, 0.15 mol / L.

[0097] In the washing step, the modified antibody remaining in the column is eluted from the column using a washing solvent. The solution that passed through the column in the above-mentioned retention step and the solution that was eluted from the column in the washing step contain a relatively large amount of bivalent antibody, and therefore, these can be combined and used as the second antibody composition. The washing solvent is not particularly limited as long as it dissolves the peptide-modified antibody, is resistant to aggregation or denaturation in the solvent, and has appropriate pH buffering capacity. Examples of suitable buffers include sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) buffer, and 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) buffer. The use of any of the above buffers is preferred, with sodium acetate buffer being more preferred. The concentration of the buffer used in the washing solvent is 20 mmol / L or more, preferably 30 mmol / L or more, with an upper limit of 200 mmol / L or less, preferably 70 mmol / L or less. The pH of the washing solvent is 4.0 or more, preferably 4.5 or more, more preferably 4.8 or more, and 7.4 or less, preferably 6.0 or less, more preferably 5.2 or less. Furthermore, in order to reduce nonspecific binding of the bivalent antibody or antibody-modified peptide to the column carrier, the elution solvent may contain an additive such as sodium chloride or potassium chloride. The concentration of the additive contained in the elution solvent is not particularly limited, but for example, 0.15 mol / L can be used.

[0098] In the elution step, the modified antibodies retained on the carrier are eluted from the column using an elution solvent, i.e., the first antibody composition containing relatively large amounts of unmodified antibodies and monovalent antibodies is eluted from the column using an elution solvent. Examples of elution solvents that can be used include buffers such as sodium acetate buffer, ammonium acetate buffer, and citrate buffer. Furthermore, to reduce nonspecific binding of the antibody-modified linker, unmodified antibody, and modified antibody to the column carrier, the elution solvent may contain additives such as sodium chloride and potassium chloride. The concentration of the additive contained in the elution solvent is not particularly limited, but a concentration of 0.15 mol / L, for example, can be used. When the elution solvent contains a buffer, the concentration of the buffer is 20 mmol / L or more, preferably 30 mmol / L or more, and 200 mmol / L or less, preferably 70 mmol / L or less, as a lower limit. The pH of the elution solvent is adjusted to the pH of the unmodified antibody and the monovalent antibody. In order to weaken the interaction with the immunoglobulin-binding protein and to prevent denaturation and aggregation of the antibody, the lower limit of the pH is preferably 3.0 or more and the upper limit is preferably 4.2 or less.

[0099] The first antibody composition or the second antibody composition obtained in the antibody purification step may be used directly in the click reaction in the subsequent step (B), or may be used in the click reaction in step (B) after adjusting the protein concentration of the peptide-modified antibody contained therein.

[0100] The click reaction in step (B) is carried out between a first click-reactive atomic group of the chelating agent and a second click-reactive atomic group of the peptide-modified antibody, forming a linking group (a substituent that enables conjugation with the antibody) that links the chelating agent to the antibody.

[0101] As long as the peptide-modified antibody and the complex obtained in step (A) can undergo a click reaction, the order of addition is not important. For example, one of the complex and the peptide-modified antibody may be added to a reaction vessel containing a solvent, and then the other may be added to allow the reaction to occur; one of the chelating agent and the antibody may be dispersed in a solvent and the other may be added to the dispersion, and then the reaction may occur. Alternatively, they may be added simultaneously to a reaction vessel containing a solvent and allowed to react.

[0102] The solvent used in the click reaction of step (B) may be a water-containing solvent, such as water, saline, or a buffer solution such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate-buffered saline, Tris buffer, HEPES buffer, or tetramethylammonium acetate buffer. When a buffer solution is used, the pH at 25°C is preferably 4.0 to 10.0, more preferably 5.5 to 8.5, from the viewpoint of achieving both stability of the complex and antibody and their binding efficiency.

[0103] The volume of the reaction solution is not particularly limited, but from the viewpoint of practicality in the production process, at the start of step (B), the lower limit is preferably 0.001 mL or more, more preferably 0.01 mL or more, even 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, even more preferably 10 mL or less, and even more preferably 1 mL or less, for example, preferably in the range of 0.001 mL or more and 1000 mL or less, and more preferably in the range of 0.1 mL or more and 10 mL or less.

[0104] Furthermore, the concentrations of the chelating agent and antibody in the reaction solution at the start of step (B) are each independently preferably 0.001 μmol / L or more as the lower limit, more preferably 0.01 μmol / L or more, even more preferably 0.1 μmol / L or more, and even more preferably 1.0 μmol / L or more, and preferably 1000 μmol / L or less as the upper limit, and more preferably 100 μmol / L or less, for example, a range of 0.1 μmol / L or more and 1000 μmol / L or less is preferred, and a range of 1 μmol / L or more and 100 μmol / L or less is more preferred from the viewpoint of the yield of the desired conjugate.

[0105] From the viewpoint of increasing the reaction efficiency while preventing unintended denaturation of the antibody, the upper limit of the reaction temperature for the click reaction in step (B) is preferably 50°C or lower, more preferably 40°C or lower. The lower limit of the reaction temperature is not particularly limited as long as the reaction proceeds at that temperature, but is preferably 15°C or higher. The reaction time for the click reaction, provided that the reaction temperature is as described above, is preferably 5 minutes or longer, more preferably 10 minutes or longer, and is preferably 24 hours or shorter, more preferably 20 hours or shorter; for example, a range of 5 minutes to 24 hours is preferred, more preferably 10 minutes to 20 hours.

[0106] The resulting complex may be used as it is, or may be purified using a filter, a membrane filter, a column filled with various packing materials, chromatography, or the like.

[0107] The conjugate produced by steps (A) and (B) is a humanized antibody that specifically binds to MUC5AC, with a specific site (e.g., a lysine residue in the Fc region of the antibody) specifically modified with a chelating agent. This conjugate contains one or two molecules of the chelating agent per antibody molecule. The chelating agent site-specifically modifies the Fc region of the antibody of the present invention via a linker. The linker is composed of a chelating linker connected to the chelating agent, a first atomic group connected to the linker, a second atomic group capable of Click reaction with the first atomic group, and an antibody-modifying linker (including the antibody-modifying peptide represented by formula (i) above) connected to the second atomic group. Therefore, the linker has a chemical structure derived from the first atomic group and the second atomic group. Examples of such chemical structures include a triazole skeleton-containing structure represented by formula (10a) or (10b) below or a pyridazine skeleton-containing structure represented by formula (10c) below. Formula (10a) and formula (10b) are isomers and may be present in any ratio.

[0108] [ka]

[0109] In formula (10a) and formula (10b), R 1A indicates the binding site with the chelating linker, and R 2A represents the binding site with the antibody-modifying linker. 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 bonding site with the chelating linker; R 5A indicates the binding site with the antibody-modified linker.

[0110] (1-5) Radioactive drugs The conjugate produced by the method shown in (1-4) above can be used as is or after purification to prepare a radiopharmaceutical containing the conjugate as an active ingredient. A radiopharmaceutical refers to a composition containing the conjugate of the present invention, i.e., an anti-MUC5AC humanized antibody or derivative thereof labeled with a radionuclide (a metal nuclide that emits α-particles), in a form suitable for administration to a subject's living body. A radiopharmaceutical can be produced, for example, by dissolving the conjugate of the present invention produced by the above method in a solvent that is primarily water and is approximately isotonic with the living body. In this case, the radiopharmaceutical is preferably in the form of an aqueous solution, and may contain other pharmaceutically acceptable ingredients as needed. An effective amount of a radiopharmaceutical is administered to a living body orally or parenterally, such as intravenously, subcutaneously, intraperitoneally, or intramuscularly, for use in the treatment, diagnosis, or detection of disease. The subjects of administration here include, but are not limited to, humans, or animals such as mice, rats, monkeys, guinea pigs, chimpanzees, sheep, goats, dogs, cats, pigs, cows, and horses, with humans being preferred. A preferred target disease is cancer. The cancer to be treated and diagnosed by the present invention is pancreatic cancer. Examples of cancers that can be treated include thyroid cancer, liver cancer, colon cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, and endometrial cancer, and application to pancreatic cancer is particularly preferred.

[0111] An example of a cancer that can be treated or diagnosed by the present invention is bile duct cancer.

[0112] In addition, MUC5AC has been reported to be an antigen carrier for CA19-9 (PLOS ONE (December 2011, Volume 6, Issue 12, e 29180, pp. 1-10). Therefore, cancers that can be treated by the present invention include biliary tract cancer, uterine cancer, lung cancer, and esophageal cancer, which overexpress CA19-9, and these cancers can be treated effectively.

[0113] Here, the term "effective amount" refers to an amount that can achieve a therapeutically effective effect in a subject to which it is administered. The effective amount to be administered to a subject varies depending on the type of subject, the subject's weight, the dosage form (tablet, injection, etc.) and route (oral administration, parenteral administration, etc.) of administration, and the severity of the disease (e.g., cancer). A physician or veterinarian can determine an appropriate effective amount taking these factors into consideration.

[0114] By selecting a radionuclide with therapeutic efficacy, the conjugate of the present invention can be used in radionuclide internal therapy (RI internal therapy). RI internal therapy involves administering a radiopharmaceutical intravenously or orally, allowing the radiopharmaceutical to accumulate at a lesion site, such as a primary cancer tumor or a metastatic tumor, and then destroying cancer cells at the lesion site via radiation emitted from the radiopharmaceutical. Therefore, the conjugate of the present invention is preferably used in RI internal cancer therapy. In this case, the dosage of the pharmaceutical is appropriately selected depending on the efficacy of the active ingredient, the form and route of administration, the stage of the disease (especially cancer), the patient's body type, weight, and age, and the type and amount of other therapeutic drugs used in combination. Typically, the dosage is 250 kBq / kg or less per administration. Effectiveness can also be achieved with a single dose of 80 kBq / kg or less.

[0115] In another embodiment of the present invention, in the above-mentioned complex, only the radioactive nuclide is used, and the radioactive nuclide that emits positrons or gamma rays from an α-ray emitting nuclide ( 68 Ga, 64 Cu, 86 Y, 89 Zr, 111In addition, a radiopharmaceutical containing as an active ingredient a complex in which α-rays are substituted with α-rays (In) may be prepared and used for the diagnosis of cancer in the aforementioned RI internal therapy for cancer. The cancer diagnostic radiopharmaceutical of the present invention may be used for diagnosis before or after RI internal therapy for cancer. When used for diagnosis before RI internal therapy for cancer, it can be used to determine the treatment selection of whether to perform RI internal therapy for cancer using the complex of the present invention equipped with an α-ray-emitting metal nuclide. Furthermore, when used for diagnosis after RI internal therapy for cancer, it can be used to determine whether RI internal therapy for cancer using the complex of the present invention equipped with an α-ray-emitting metal nuclide is effective or to optimize the treatment plan, such as increasing or decreasing the dosage.

[0116] (2) Complex 2 In another embodiment, the present invention provides a conjugate of an antibody and a chelating agent to which a radionuclide is chelated, wherein the radionuclide is a positron-emitting metal nuclide and the antibody is a humanized antibody that specifically binds to MUC5AC.

[0117] The same definition as in "(1) Complex 1" above applies, except that the radionuclide in the chelating agent is a metal nuclide that emits positrons. The metal nuclide that emits positrons may be any nuclide that emits positively charged electrons (positrons) during the decay process of radioactive metals. 68 Ga, 64 Cu, 86 Y and 89 Zr and the like are preferably used, and more preferably 89 Zr (zirconium-89). Antibodies labeled with positron-emitting nuclides are used in PET (Positron Emission Tomography) studies. It can be suitably used for the following. Furthermore, complex 2 using a positron-emitting nuclide as the radionuclide can also be used as a cancer diagnostic radiopharmaceutical for RI internal therapy using the above complex 1 using an α-ray-emitting nuclide as the radionuclide. In this case, the dosage of the pharmaceutical is not particularly limited as long as it is an amount necessary and sufficient for imaging disease (particularly cancer) lesions in PET examinations, but it is preferable to select an appropriate dosage depending on the stage of progression of the disease (particularly cancer), the patient's body type, weight, and age, and the type and amount of therapeutic drugs for other diseases used in combination.

[0118] According to the above-described embodiments of the present invention, there are provided anti-MUC5AC antibodies, particularly humanized antibodies, labeled with radionuclides, particularly α-ray-emitting nuclides, which have excellent specificity for MUC5AC and excellent accumulation in tumors. Furthermore, according to an embodiment of the present invention, there is provided an RI-labeled anti-MUC5AC antibody for cancer diagnosis and / or cancer treatment to achieve theranostics.

[0119] The above-described embodiments of the present invention encompass the following technical ideas. [1] A conjugate of an antibody and a chelating agent to which a radionuclide has been chelated, wherein the radionuclide is a metal nuclide that emits alpha rays, and the antibody is a humanized antibody that specifically binds to MUC5AC. [2] The antibody, (1) the amino acid sequence (H01) shown in SEQ ID NO: 1; (2) the amino acid sequence (H02) shown in SEQ ID NO: 2; (3) the amino acid sequence (H03) shown in SEQ ID NO: 3, or (4) Amino acid sequence (H04) shown in SEQ ID NO: 4 a heavy chain variable region consisting of (5) the amino acid sequence (L01) shown in SEQ ID NO: 5; (6) Amino acid sequence (L02) shown in SEQ ID NO: 6; (7) the amino acid sequence (L03) shown in SEQ ID NO: 7, or (8) Amino acid sequence (L04) shown in SEQ ID NO: 8 a light chain variable region consisting of The conjugate according to [1] above, which is a humanized antibody having the following structure: [3] The antibody, (1) a heavy chain variable region consisting of the amino acid sequence (H01) shown in SEQ ID NO: 1; (7) a light chain variable region consisting of the amino acid sequence (L03) shown in SEQ ID NO: 7; The conjugate according to [2] above, which is a humanized antibody having the following structure: [4] The complex according to any one of the above [1] to [3], wherein the metal nuclide that emits α-rays is actinium-225. [5] The complex according to any one of [1] to [4] above, comprising 1 to 8 molecules of the chelating agent per molecule of the antibody. [6] The conjugate according to any one of [1] to [5] above, wherein the chelating agent site-specifically modifies the Fc region of the antibody via a linker. [7] The conjugate according to [6] above, wherein the linker comprises an antibody-modifying peptide consisting of 13 to 17 amino acid residues and represented by the following formula (i): (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) (In the formula, Xa, Xb, Xc, and Xd represent a number of consecutive Xs, b number of consecutive Xs, c number of consecutive Xs, and d number of consecutive Xs, respectively; X is an amino acid residue having neither a thiol group nor a haloacetyl group in the side chain, a, b, c, and d are each independently an integer of 1 to 5, and satisfy a+b+c+d≦14; Xaa1 and Xaa3 each independently represent It represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and is a disulfide or the sulfide group is attached via a linker; or one represents an amino acid residue derived from an amino acid having a thiol group in a side chain, and the other represents an amino acid residue derived from an amino acid having a haloacetyl group in a side chain, and they are bonded 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. [8] The conjugate according to [7] above, wherein in the antibody-modifying peptide, Xaa2 in formula (i) is a lysine residue. [9] The conjugate according to [7] or [8] above, wherein the antibody-modifying peptide comprises an antibody-modifying peptide consisting of the amino acid sequence represented by SEQ ID NO: 10 (wherein Xaa2 is a lysine residue).

[10] The complex according to any one of the above [1] to [9], wherein the chelating agent has a structure derived from a compound represented by the following formula (A) or a salt thereof:

[0120] [ka]

[0121] (In formula (A), R 11 , R 13 and R 14 are each independently -(CH2) p COOH, -(CH2) p C5H5N, -(CH2) p PO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p COOH, R 12 or R 15 one of which is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a substituent for conjugating with the antibody, p is an integer of 0 to 3, and R 12 is a substituent for conjugation with the antibody, R 15 is a hydrogen atom, and R 12 is not a substituent for conjugation with the antibody, R 15 is a substituent for conjugation to the antibody.

[11] The conjugate according to any one of [6] to

[10] above, wherein the chelating agent site-specifically modifies the Fc region of the antibody via a linker, and the linker has a binding group formed by a click reaction.

[12] The conjugate according to

[11] above, wherein the linker comprises a chelating linker connecting the chelating agent and the binding group formed by the click reaction, and an antibody-modifying linker connecting the antibody and the binding group formed by the click reaction, and the binding group formed by the click reaction has a triazole skeleton-containing structure or a pyridazine skeleton-containing structure represented by the following formula (10a):

[0122] [ka]

[0123] (In the formula, R 1A represents the binding site with the chelating linker, and R 2A indicates the binding site with the antibody-modified linker.

[13] A radiopharmaceutical comprising the complex according to any one of [1] to

[12] above as an active ingredient.

[14] The radiopharmaceutical according to

[13] above, which is used for RI internal therapy for cancer.

[15] The radiopharmaceutical according to

[14] above, which is administered to a subject at a dose of 250 kBq / kg or less per administration in the RI internal therapy.

[16] The radiopharmaceutical according to

[15] above, wherein the dose per administration is 80 kBq / kg or less.

[17] A radiopharmaceutical comprising a conjugate of a chelating agent to which a radionuclide is chelated and an antibody, wherein the antibody is a humanized antibody that specifically binds to MUC5AC. A radiopharmaceutical for cancer diagnosis in RI internal therapy using the radiopharmaceutical according to any one of

[14] to

[16] above.

[18] A conjugate of an antibody and a chelating agent to which a radionuclide is chelated, the radionuclide is a positron-emitting metal nuclide, A conjugate wherein the antibody is a humanized antibody that specifically binds to MUC5AC.

[19] The antibody, (1) the amino acid sequence (H01) shown in SEQ ID NO: 1; (2) the amino acid sequence (H02) shown in SEQ ID NO: 2; (3) the amino acid sequence (H03) shown in SEQ ID NO: 3, or (4) Amino acid sequence (H04) shown in SEQ ID NO: 4 a heavy chain variable region consisting of (5) the amino acid sequence (L01) shown in SEQ ID NO: 5; (6) Amino acid sequence (L02) shown in SEQ ID NO: 6; (7) the amino acid sequence (L03) shown in SEQ ID NO: 7, or (8) Amino acid sequence (L04) shown in SEQ ID NO: 8 a light chain variable region consisting of The conjugate according to

[18] above, which is a humanized antibody having the formula:

[20] The antibody, (1) a heavy chain variable region consisting of the amino acid sequence (H01) shown in SEQ ID NO: 1; (7) a light chain variable region consisting of the amino acid sequence (L03) shown in SEQ ID NO: 7; The conjugate according to

[19] above, which is a humanized antibody having the formula:

[21] The complex according to any one of the above

[18] to

[20] , wherein the positron-emitting metal nuclide is zirconium-89.

[22] The complex according to any one of

[18] to

[21] above, comprising 1 to 8 molecules of the chelating agent per molecule of the antibody.

[23] The conjugate according to any one of

[18] to

[21] above, wherein the chelating agent site-specifically modifies the Fc region of the antibody via a linker.

[24] The conjugate according to

[23] above, wherein the linker comprises an antibody-modifying peptide consisting of 13 to 17 amino acid residues and represented by the following formula (i): (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) (In the formula, Xa, Xb, Xc, and Xd represent a number of consecutive Xs, b number of consecutive Xs, c number of consecutive Xs, and d number of consecutive Xs, respectively; X is an amino acid residue having neither a thiol group nor a haloacetyl group in the side chain, a, b, c, and d are each independently an integer of 1 to 5, and satisfy a+b+c+d≦14; Xaa1 and Xaa3 each independently represent represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and is bonded via a disulfide bond or the sulfide group is bonded via a linker, or one represents an amino acid residue derived from an amino acid having a thiol group in a side chain, and the other represents an amino acid residue derived from an amino acid having a haloacetyl group in a side chain, and they are bonded 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.

[25] The conjugate according to

[24] above, wherein in the antibody-modifying peptide, Xaa2 in formula (i) is a lysine residue.

[26] The conjugate according to

[24] or

[25] above, wherein the antibody-modifying peptide comprises an antibody-modifying peptide consisting of the amino acid sequence represented by SEQ ID NO: 10 (wherein Xaa2 is a lysine residue).

[27] The complex according to any one of the above

[18] to

[26] , wherein the chelating agent has a structure derived from a compound represented by the following formula (A):

[0124] [ka]

[0125] (In formula (A), R 11 , R 13 and R 14 are each independently -(CH2) p COOH, -(CH2) p C5H5N, -(CH2) pPO3H2, -(CH2) p CONH2 or -(CHCOOH)(CH2) p COOH, R 12 or R 15 one of which is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a substituent for conjugating with the antibody, p is an integer of 0 to 3, and R 12 is a substituent for conjugation with the antibody, R 15 is a hydrogen atom, and R 12 is not a substituent for conjugation with the antibody, R 15 is a substituent for conjugation to the antibody.

[28] A radiopharmaceutical comprising the complex according to any one of

[18] to

[27] above as an active ingredient.

[29] A method for producing a conjugate according to any one of [1] to

[12] and

[18] to

[27] above, comprising a conjugation step of conjugating a chelating agent having a radioactive nuclide chelated thereto with an anti-MUC5AC antibody to produce a conjugate of the chelating agent and the anti-MUC5AC antibody.

[30] The chelating agent is connected to a chelating linker, and the anti-MUC5AC antibody has an Fc region specifically modified with an antibody-modifying linker comprising an antibody-modifying peptide. and in the conjugation step, a click reaction is carried out to connect the chelate linker and the antibody-modified linker.

[31] A modified antibody in which the Fc region of the antibody is specifically modified with an antibody-modifying linker comprising an antibody-modifying peptide, wherein the antibody is an anti-MUC5AC antibody, and the antibody-modifying linker has an atomic group for connecting via a click reaction to a chelating linker comprising a chelating agent to which a radionuclide has been chelated.

[32] A method for producing a modified antibody in which the Fc region of the antibody is specifically modified with an antibody-modifying linker comprising an antibody-modifying peptide, comprising: an antibody modification step of site-specifically modifying the Fc region of the antibody with a linker comprising an antibody-modifying peptide to obtain a modified antibody; an antibody purification step of purifying the antibody using a carrier on which an immunoglobulin-binding protein is immobilized; Including, A method for producing a modified antibody, wherein the antibody is an anti-MUC5AC antibody.

[33] The immunoglobulin-binding protein is protein A or a genetically modified protein. The method for producing the modified antibody according to

[32] above, wherein the modified antibody is tein A.

[34] The method for producing a modified antibody described in

[32] or

[33] above, wherein the antibody purification step is carried out using a column packed with the carrier.

[35] The antibody purification step includes a retention step of retaining the modified antibody on the carrier. a step of eluting the modified antibody retained on the carrier.

[36] In the antibody modification step, a mixture containing an unmodified antibody that is not modified with the antibody-modifying linker, a monovalent antibody in which one antibody molecule is modified with one antibody-modifying linker molecule, and a bivalent antibody in which one antibody molecule is modified with two antibody-modifying linker molecules. to obtain the modified antibody, The method for producing the modified antibody described in

[35] above, comprising the step of obtaining a first antibody composition containing a relatively large amount of unmodified antibody and monovalent antibody, and a second antibody composition containing a relatively large amount of bivalent antibody, respectively, by utilizing differences in the interactions of the unmodified antibody, the monovalent antibody, and the bivalent antibody with the immunoglobulin-binding protein in the antibody purification step.

[37] A method for producing a conjugate, comprising: a modified antibody production step of obtaining a modified antibody by carrying out the method for producing a modified antibody described in any one of

[32] to

[36] above; and a conjugation step of conjugating the modified antibody with a chelating agent having a radioactive nuclide chelated thereto to produce a conjugate of the chelating agent and the modified antibody.

[38] In the modified antibody production process, the antibody-modified linker is obtaining a first antibody composition in which the total ratio of unmodified antibodies not modified with the antibody-modifying linker and monovalent antibodies modified with one antibody-modifying linker molecule per antibody molecule is higher than that of bivalent antibodies modified with two antibody molecules, The method for producing a complex according to

[37] above, wherein in the complexing step, a complex is formed between the chelating agent and the monovalent antibody.

[39] In the modified antibody production process, an unmodified antibody to which the antibody-modifying linker is not modified and a monovalent antibody to which one antibody molecule is modified with one antibody-modifying linker molecule are a bivalent antibody modified with two molecules of the antibody-modifying linker per one molecule of the antibody, obtaining a second antibody composition having a higher proportion of the antibody; The method for producing a complex according to

[37] above, wherein in the complexing step, a complex is formed between the chelating agent and the bivalent antibody.

[40] The method for producing a conjugate according to any one of

[37] to

[39] above, wherein the chelating agent is connected to a chelating linker, and the chelating linker and the antibody-modified linker are connected by carrying out a click reaction in the conjugation step.

[41] A kit for producing a conjugate between a radionuclide-chelated chelating agent and an antibody. The kit comprises (1) a chelating agent capable of chelating a radionuclide and (2) an anti-MUC5AC antibody, wherein the complex is a complex according to any one of [1] to

[12] and

[18] to

[27] above.

[42] The kit according to

[41] , further comprising (1) a first atomic group capable of Click reaction and (2) a second atomic group capable of Click reaction.

[43] The kit according to

[41] , further comprising a radionuclide capable of being chelated to a chelating agent.

[0126] The radiopharmaceutical described above in

[14] contains as its active ingredient a complex comprising a humanized antibody that specifically binds to MUC5AC and a metal nuclide that emits alpha rays. When used in RI internal cancer therapy, the drug accumulates specifically in tumors that express MUC5AC, allowing alpha rays to be irradiated specifically to tumor cells without affecting normal cells, resulting in greater safety and therapeutic efficacy. The radiopharmaceutical described in

[28] above contains as its active ingredient a complex comprising a humanized antibody that specifically binds to MUC5AC and a positron-emitting metal nuclide, making it suitable for PET examinations. Furthermore, it exhibits similar accumulation to the radiopharmaceutical used in RI internal therapy described in

[14] above, and can therefore be efficiently used as a diagnostic radiopharmaceutical for RI internal therapy of cancers that express MUC5AC. The method for producing the conjugate described above in

[29] includes a conjugation step in which a chelating agent containing a chelated radionuclide is conjugated to an anti-MUC5AC antibody. This prevents denaturation of the antibody and allows the conjugate to be obtained efficiently without subjecting the anti-MUC5AC antibody to a chelation step, which is a harsher condition for the antibody. According to the conjugate production method

[30] , the conjugation step involves a click reaction, which allows conjugation under extremely mild conditions, i.e., in a buffer solution at room temperature, and the conjugate can be obtained efficiently without denaturing the anti-MUC5AC antibody. According to the modified antibody of

[31] above, the Fc region of the anti-MUC5AC antibody is specifically modified with the antibody-modifying linker, and therefore the modified antibody can be used in a click reaction with a chelating linker provided in a chelating agent to which a radionuclide has been chelated, without impairing the antigen-binding ability of the anti-MUC5AC antibody. The method for producing the modified antibody described above in

[32] includes an antibody modification step in which the Fc region of an anti-MUC5AC antibody is site-specifically modified with a linker comprising an antibody-modifying peptide to obtain the modified antibody, and an antibody purification step in which the antibody is purified using a carrier on which an immunoglobulin-binding protein is immobilized, thereby further increasing the purity of the modified antibody. According to the conjugate production methods

[38] or

[39] above, the conjugation step is carried out using an antibody composition in which the proportion of either monovalent antibodies or bivalent antibodies is greater than the proportion of the other. This makes it possible to adjust the number of chelating agents bound to the anti-MUC5AC antibody according to the purpose, and to obtain a conjugate of the desired valency with higher purity. According to the kit

[41] above, a complex of an antibody and a chelating agent capable of chelating a radionuclide can be reacted with the radionuclide at the required timing to prepare a complex described in any of [1] to

[12] and

[18] to

[27] immediately before use, thereby enabling efficient treatment or diagnosis without impairing both the half-life of the radionuclide and the activity of the antibody. The kit of

[42] above separately comprises a conjugate comprising a chelating agent capable of chelating a radionuclide and a click reaction atomic group, and a conjugate comprising an antibody and a click reaction atomic group. Therefore, the radionuclide can be chelated to the chelating agent at the required timing, and then the conjugate can be subjected to a click reaction to prepare the conjugate described in any of [1] to

[12] and

[18] to

[27] immediately before use, enabling efficient treatment or diagnosis without impairing both the half-life of the radionuclide and the activity of the antibody.

[0127] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. [Example]

[0128] Production Example 1: Preparation of anti-MUC5AC humanized antibody The amino acid sequences of the various variable regions with added signal sequences and the various constant regions were converted to base sequences, taking into consideration the codon usage appropriate for expression in CHO cells. A Kozak sequence was added to the start codon of the signal sequence, and a stop codon was added to the C-terminus of the constant region. Furthermore, restriction enzyme sites were added upstream of the Kozak sequence and downstream of the stop codon to enable insertion into the expression gene introduction site of a mammalian cell expression plasmid (pcDNA3.4). Each DNA fragment designed in this way was produced by chemical synthesis. DNA fragments containing the variable regions of the desired H chain and L chain were ligated to DNA fragments containing the constant regions using fusion PCR.

[0129] The various antibody genes constructed were restriction digested and then purified. Similarly, a mammalian cell transient expression plasmid (pcDNA3.4) was digested with the same restriction enzymes and then purified. Both fragments were mixed at the appropriate ratio and ligated. The ligation reaction mixture was then mixed with E. coli DH5α competent cells for transformation. From the resulting transformants, colony PCR, single colony isolation, plasmid extraction from small-scale cultures, and base sequencing of the insert were performed to select plasmids (E. coli clones) in which the designed full-length antibody gene was correctly inserted in the intended direction with the designed sequence. The selected E. coli clones were cultured on a large scale, and plasmid extraction and purification, including an endotoxin removal step, were performed. The absorbance of the purified plasmid at 260 nm was measured to calculate the concentration.

[0130] CHO cells were cultured using the ExpiCHO System (Thermo Fisher Scientific). Transient expression was performed using the plasmids. One H chain and one L chain were selected from the prepared H chain expression plasmids and L chain expression plasmids to create the desired combination, and transfected using the lipofection method, followed by culturing and feeding. Seven to 13 days after transfection, the culture medium was collected. The culture supernatant was centrifuged and filtered, and then loaded onto a Protein A column. The antibody was purified by standard affinity column chromatography (adsorption followed by washing, elution with an acidic buffer, and neutralization of the eluate). The absorbance of the purified antibody at 280 nm was measured, and the concentration was calculated.

[0131] The following anti-MUC5AC humanized antibodies were produced using the method described above. The antibody numbers assigned to the combinations of heavy and light chain variable regions are shown below. Antibody 1:H01L03 Antibody 2:H01L04 Antibody 3:H02L04 Antibody 4:H04L04 Here, H01, H02, and H04 are heavy chain variable regions shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4, respectively, and L03 and L04 are light chain variable regions shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The antibodies used in the following examples consist of heavy chain constant region 1 (SEQ ID NO: 25) and light chain constant region 1 (SEQ ID NO: 26), as well as a combination of the heavy chain variable region and light chain variable region of antibody 1 to antibody 4 described above.

[0132] Preparation Example 2: Site-specific antibody modification with a peptide linker (1) Antibody modification step The antibody-modified peptide was produced by the method described in WO 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 SEQ ID NO: 10 in which Xaa2 was a lysine residue, and the terminal amino group of the side chain of the lysine residue was modified with the structure represented by R1. The cysteine residues form disulfide bonds with each other, and the N-terminus of the peptide is linked to ethyl azide as the second atomic group containing an azide group via a linker structure containing diglycolic acid and eight PEGs.

[0133] [ka]

[0134] (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.)

[0135] This peptide was mixed with the anti-MUC5AC humanized antibody (antibody 1) prepared in Preparation Example 1 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 has an Fc region site-specifically modified with the above peptide.

[0136] (2) Peptide-modified antibody separation process This peptide-modified antibody was diluted with 1 mol / L sodium acetate buffer (pH 6.0) and loaded onto a Protein A column (GE Healthcare, HiTrap MabSelect SuRe). 0.15 mol / L sodium chloride-containing 0.05 mol / L sodium acetate buffer (pH 5.7) was applied. The peptide-modified antibody modified with two peptide molecules (hereinafter also referred to as "bivalent antibody") was recovered, and the concentration of the bivalent antibody in the recovered fraction was adjusted to 15 mg / mL. Subsequently, 0.15 mol / L sodium chloride-containing 0.05 mol / L sodium acetate buffer (pH 3.5) was applied to the Protein A column. The peptide-modified antibody modified with one peptide molecule (hereinafter also referred to as "monovalent antibody") was recovered, and the concentration of the monovalent antibody in the recovered fraction was adjusted to 15 mg / mL.

[0137] Example 1: 225 Ac-labeled anti-MUC5AC humanized antibody ( 225 Preparation of Ac-labeled monovalent antibody 1 (1) Chelating agent synthesis process The structure of the chelating moiety (manufactured by Iris Biotech GmbH) used in this example is shown in the following formula (L1-3). This chelating moiety was dissolved in a 0.1 mol / L sodium acetate buffer solution (pH 6.0) as a solvent to prepare a solution containing 1.7 mmol / L of the chelating moiety. 0.005 mL of this solution and 1.7 mmol / L of ... 225 A reaction solution containing Ac ions (0.2 mol / L hydrochloric acid solution, radioactivity concentration 300 MBq / mL, prepared by Oak Ridge National Laboratory, liquid volume: 0.005 mL) was mixed with 1.5 MBq (calculated value calculated from the radioactivity at the time of testing) and reacted under heating conditions. 225 The molar ratio of the chelate moiety to the radioactive metal ion was Part: 225 Ac ion = approximately 2000:1, the heating temperature of the reaction solution was 70°C, and the heating time The duration was 90 minutes.

[0138] [ka]

[0139] obtained 225 The radiochemical purity of the Ac complex was determined by the following method: 225 A portion of the Ac complex solution was developed by thin layer chromatography (Agilent, model number: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured with a radio-γ-TLC analyzer (raytest, model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the origin relative to the total detected radioactivity (counts) was calculated as follows: 225 The radiochemical purity of the Ac complex was calculated as follows (%). 225The radiochemical purity of the Ac complex was 86%. 225 The Ac complex solution was used as is in the next labeling step.

[0140] (2) Labeling process The eluate of the monovalent antibody obtained in Production Example 2 and the eluate of the monovalent antibody obtained in the above step (1) 225 The Ac complex solution was added to a 0.09 mol / L sodium acetate buffer solution containing 0.02 mol / L (20 mM) ascorbic acid, and the click reaction was carried out at 37°C for 120 minutes. 225 Ac-labeled monovalent antibodies were obtained. 225 The amounts of Ac complex and peptide-modified antibody were 44 μmol and 46 μmol, respectively, and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:1, respectively. Furthermore, the resulting mixture was reacted at 37°C for 2 hours. 225 The solution of Ac-labeled monovalent antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096) and subjected to the subsequent experiments. 225 The radiochemical purity of the Ac-labeled monovalent antibody (radioactivity amount 0.303 MBq calculated by decay from the radioactivity amount at the time of assay) was 93%, and the radiochemical yield was 39%. Here, radiochemical purity is the ratio of the total radioactivity counts in the thin-layer plate when analyzed by thin-layer chromatography. 225 The radiochemical yield is the percentage (%) of radioactive counts of the peak corresponding to Ac-labeled monovalent antibody. The radiochemical yield was measured using a γ-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (ORTEC) and multi-channel analyzer: M7-000 (Seiko Easy-An)). Data processing: Spectrum Navigator: DS-P300 (Seiko Easy and Measured using a Gamma Studio: DS-P600 (Seiko E&G) and a Gamma Studio: DS-P600 (Seiko E&G) The radioactivity calculated from the radioactivity count at the start of the labeling process was 225 The percentage of radioactivity calculated from the radioactivity count of Ac-labeled monovalent antibody.

[0141] Example 2: 225 Ac-labeled anti-MUC5AC humanized antibody ( 225 Preparation of Ac-labeled monovalent antibody 2 (1) Chelating agent synthesis process The structure of the chelating moiety used in this example is shown in the following formula (L1-4). DOTA-Bn-DBCO shown in formula (L1-4) was produced 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). This chelating moiety was dissolved in 0.1 mol / L sodium acetate buffer (pH 6.0) as a solvent to prepare a solution containing 1.7 mmol / L of the chelating moiety. 0.0025 mL of this solution and 1.7 mmol / L of benzophenone-3 as a radioactive metal source were mixed. 225 Ac ion-containing solution (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 43 A reaction mixture of 1.08 MBq (calculated value calculated from the radioactivity at the time of assay) of 2 MBq / mL (Oak Ridge National Laboratory, liquid volume: 0.0025 mL) and 0.0375 mL of 0.1 mol / L sodium acetate buffer (pH 6.0) was reacted under heating conditions. 225 An Ac complex solution was obtained. The molar ratio of the chelating moiety to the radioactive metal ion was: 225 Ac ion = approximately 2000:1, and the reaction solution The heating temperature was 70°C and the heating time was 90 minutes.

[0142] [ka]

[0143] obtained 225 The radiochemical purity of the Ac complex was determined by the following method: 225A portion of the Ac complex solution was developed by thin layer chromatography (Agilent, Model: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured with a radio-γ-TLC analyzer (raytest, Model GITA Star). The percentage of the radioactivity (counts) of the peak detected near the origin to the total detected radioactivity (counts) was calculated as follows: 225 The radiochemical purity of the Ac complex was calculated as follows (%). 225 The radiochemical purity of the Ac complex was 98%. 225 The Ac complex solution is This was then used in the next labeling step.

[0144] (2) Labeling process The eluate of the monovalent antibody obtained in Production Example 2 and the eluate of the monovalent antibody obtained in the above step (1) 225 The solution of Ac complex was subjected to a click reaction at 37°C for 120 minutes. 225 Ac-labeled monovalent antibodies were obtained. 225 The amounts of Ac complex and peptide-modified antibody were 44 μmol and 46 μmol, respectively, and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:1, respectively. Furthermore, the resulting mixture was reacted at 37°C for 120 minutes. 225 The Ac-labeled monovalent antibody solution was added to a 90 mmol / L sodium acetate buffer solution (pH 6.0) containing 20 mmol / L ascorbic acid, and purified using an ultrafiltration filter (Merck, model number: UFC505096) for subsequent experiments. 225 The radiochemical purity of the Ac-labeled monovalent antibody (radioactivity calculated by decay from the radioactivity at the time of assay: 0.231 MBq) was 86%, and the radiochemical yield was 21%. Here, radiochemical purity is the ratio of the total radioactivity counts in the thin-layer plate when analyzed by thin-layer chromatography. 225 The radiochemical yield is the percentage (%) of radioactivity counts of the peak corresponding to Ac-labeled monovalent antibody. The radiochemical yield was calculated using a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC), multi-channel analyzer: M7-000 (manufactured by Seiko)). Easy & G), Data processing: Spectrum Navigator: DS-P300 (Seiko Easy & G) and Gamma Studio: DS-P600 (Seiko Easy & G) The radioactivity calculated from the radioactivity count at the start of the labeling process measured by a 225 The percentage of radioactivity calculated from the radioactivity count of Ac-labeled antibody.

[0145] Example 3: 225 Ac-labeled anti-MUC5AC humanized antibody ( 225 Preparation of Ac-labeled bivalent antibody The procedure was repeated in Example 2, except that the eluate of the bivalent antibody obtained in Production Example 2 was used instead of the eluate of the monovalent antibody. 225 The Ac-labeled bivalent antibody was obtained. 225 The radiochemical purity of the Ac-labeled bivalent antibody (radioactivity calculated by decay from the radioactivity on the date of assay: 0.168 MBq) was 99%, and the radiochemical yield was 20%.

[0146] Example 4: In-111( 111 Screening of humanized antibodies using In)-labeled antibodies Example 4-1: 111 Preparation of In-labeled antibodies In order to find an anti-MUC5AC antibody with high tumor accumulation and a high maximum tolerated dose, various antibodies were tested. 111 The tumor-bearing mice were administered In-labeled drugs and SPECT-CT images were taken. The cumulative radioactivity and absorbed dose in the tumor and liver were calculated from the images obtained and compared. The antibodies used were four types of anti-MUC5AC humanized antibodies prepared in Production Example 1 and one type of anti-MUC5AC chimeric antibody disclosed in Patent Document 1. 111 In labeled. The amino acid sequences of the heavy chain variable region and light chain variable region of the chimeric antibody disclosed in Patent Document 1 (SEQ ID NOs: 23 and 24, respectively) are as follows: 111In labeling was performed on complexes of various antibodies with a label precursor having the structure shown in the following formula:

[0147] [ka]

[0148] [ka]

[0149] The label precursor has DOTA as a chelating moiety linked to the N-terminus of an antibody-modifying peptide (a peptide containing 17 amino acid residues having the same sequence as the sequence in SEQ ID NO: 10 in which Xaa2 is a lysine residue) via eight polyethylene glycol groups, and also has the N-hydroxysuccinimide ester groups in the structure linked to the chelating moiety of various antibodies, according to EU numbering. It had a structure linked to the 252nd lysine residue. 450 μg of this labeled precursor was dissolved in 100 mmol / L of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (pH 5.5) as a solvent. 111 In ion-containing solution (indium chloride ( 111 Injection solution (manufactured by Nippon Medi-Physics Co., Ltd.) was mixed with 10 MBq of radioactivity, and the labeling reaction was carried out at 45°C for 120 minutes.

[0150] Various 111 The radiochemical yield, radiochemical purity, and amount of radioactivity administered to animals for the In-labeled antibody are shown in Table 1. 111 In response to the amount of radioactivity 111 This refers to the amount of radioactivity in the In-labeled antibody. Radioactivity was measured using a radioisotope dose calibrator (manufactured by CAPINTEC, model number: CRC-15R). Radiochemical purity is the ratio of the total radioactivity counts on the filter paper analyzed by filter chromatography. 111The radioactivity counts of the peak corresponding to the In-labeled antibody are expressed as a percentage (%). Filter paper chromatography was performed using filter paper manufactured by ADVANTEC, model number: No. 590, and developing solvent: 0.01% EDTA, 50 mM citric acid-sodium citrate aqueous solution. The activity counts were detected using a radio γ-TLC analyzer (manufactured by raytest, Model GITA Star).

[0151] [Table 1]

[0152] Example 4-2: Biodistribution in tumor-bearing mice

[0153] (Method for producing tumor-bearing mice) Human pancreatic cancer cell line SW1990 was used at 0.7 × 10 7 The dose was administered subcutaneously from the flank to the back of a Balb / c nude mouse (male). 14 days after transplantation of SW1990, the tumor size was approximately 150-300 mm 3 At this point, various types of 111 In-labeled antibody was administered to the mice via the tail vein. The tumor volume was calculated using the following formula. Tumor volume = (minor axis of tumor 2 × longest diameter of tumor) / 2

[0154] [Table 2]

[0155] (Evaluation method) SPECT-CT imaging (SPECT-CT device for small animals: FX-3000, T The imaging was performed under the conditions in the table below. The imaging time points were 1, 6, 24, 48, 72, and 168 hours after administration. Image reconstruction was performed using the OSEM method for SPECT and the FBP method for CT. VOI (volume of interest, 3D ROI) analysis was performed on the tumor and liver at each time point. The counts per organ volume were corrected to %ID / g, and the physical half-life was calculated. 111 From In 225 The time activity curve was obtained by converting the radioactivity to Ac, correcting for differences in physical half-life, and taking biological half-life into account. The cumulative radioactivity was calculated from the integral of this time activity curve, and the absorbed dose was calculated using a spherical model (OLINDA / EXM ver2.0), which was compared for each antibody. However, for H01L03, the time activity Since no decrease in the curve was observed, only the physical half-life was taken into account, without considering the biological half-life.

[0156] [Table 3]

[0157] [Table 4]

[0158] (result) The results of SPECT-CT imaging are shown in Figure 1. The results of VOI analysis of the tumor and liver at each time point are shown in Figure 2. A graph showing the results of examining the distribution and excretion pathway in the body after SPECT-CT imaging 168 hours after administration is shown in Figure 3. Tumor accumulation was highest when humanized antibody (H01L03) was used, while it was lowest when chimeric antibody was used. Liver accumulation was highest when chimeric antibody was used. When the humanized antibody was used, the excretion rate was lower than that of the chimeric antibody. When the humanized antibody (H01L03) was used, excretion was slowest and blood retention was high. When both antibodies were used, accumulation in the liver and spleen was high among normal organs, followed by the lungs and kidneys.

[0159] When the liver threshold dose was set at 30 Gy, the maximum tolerated dose (MTD) was high (3.90 MBq) when the humanized antibody (H01L03) was used, but low (0.43 MBq) when the chimeric antibody was used. The MTD (MTD) was calculated as threshold dose (Gy) / absorbed dose (Gy / MBq).

[0160] Example 4-3: In vitro autoradiography Various preparations prepared in Example 4-1 111 Figure 4 shows the image results of in vitro autoradiography (ARG) using In-labeled antibodies to evaluate the binding and specificity of various antibodies to MUC5AC. The radioactivity density (Bq / mm ) in the region of interest (ROI) calculated by setting the ROI over the entire section is also shown. 2 ) is shown in Figure 5. MUC5AC-highly expressing tumors (SW1990 transplanted tumor tissue) or MUC5AC-lowly expressing tumors (MIAPaCa2 transplanted tumor tissue) were frozen in liquid nitrogen, and 10 μm-thick sections were prepared from the frozen blocks using a cryostat (Leica) and used for in vitro ARG. The sections were stored at -80°C until use, and then brought to room temperature and dried for at least 30 minutes. The sections were then immersed in phosphate-buffered saline for 30 minutes, and then in phosphate-buffered saline containing 1% by volume of bovine serum albumin for 30 minutes to hydrophilize the sections. Hydrophilized frozen sections were 111The sections were immersed in phosphate-buffered saline containing 1% bovine serum albumin (volume %) containing 5 kBq / mL of In-labeled antibody for 30 minutes. The sections were then washed by immersing them in phosphate-buffered saline containing 1% bovine serum albumin (volume %), phosphate-buffered saline, and phosphate-buffered saline, in that order, for 5 minutes each. After washing, the sections were air-dried and exposed to light on an imaging plate (BAS-SR2040, Fujifilm) for approximately 15 hours. An autoradiogram was then obtained using a fluoroimage analyzer (Typhoon FLA 7000 IP, GE Healthcare). Regions of interest (ROIs) were set across the entire section, and the radioactivity density (Bq / mm) within the ROI was analyzed using the Image Quant TL analysis software provided with the fluoroimage analyzer. 2 ) was calculated. 111 We confirmed that all In-labeled humanized antibodies retained their binding and specificity to MUC5AC (see Figure 5, data for MUC5AC-high expressing tumors). Compared to the chimeric antibody, we confirmed that the various humanized antibodies exhibited less nonspecific binding (see Figure 5, data for MUC5AC-low / non-expressing tumors). The results of Examples 4-2 and 4-3 demonstrated that humanized antibodies have higher specificity for MUC5AC than chimeric antibodies, and also have high accumulation in tumors and low accumulation in normal organs such as the liver, thereby providing a superior delivery technology for RI-labeled antibodies.

[0161] The results of this example are summarized in the table below. In the table, the tumor volume was 150 mm at the SPECT absorbed dose. 3 The absorbed dose in the liver was calculated based on the average liver weight of the mice used in this example (1.15±0.14 g, n=19). The values for biodistribution are expressed as the mean ± standard deviation of n=4 (except for H02L04, n=3).

[0162] [Table 5]

[0163] Example 5: Tumor-bearing mice were used 225 Evaluation of Ac-labeled monovalent antibodies Produced according to Example 1 225 Ac-labeled monovalent antibody (H01L03) was used. 225 The mice were divided into three groups based on the radioactivity dose of Ac-labeled monovalent antibody administered: a 2.5 kBq administration group, a 5 kBq administration group, and a 10 kBq administration group. These groups were compared with a group administered with the humanized antibody (H01L03) produced in Production Example 1 (antibody control group). There were six mice in each group, and for four weeks after administration, observation of the general condition, weight, and tumor volume were measured. The tumor-bearing mice used for evaluation were prepared using the same procedure as in Example 4-2, and 10 days after transplantation of SW1990, tumors had grown to approximately 200 mm 3 The information on the animals in each group is summarized below.

[0164] [Table 6]

[0165] The change in tumor volume over time is shown in FIG. 6, and the relative ratio of the tumor volume on the final day of the observation period, assuming the tumor volume before administration to be 1.0, is shown in the table below. 225 The Ac-labeled monovalent antibody exhibited a dose-dependent tumor growth inhibitory effect, and statistically significant tumor growth inhibition was observed at all doses.

[0166] [Table 7]

[0167] On the final day of the observation period, autopsies were performed, and tumors were collected and weighed. The results of a comparison of tumor weights are shown in the table below. 225 The Ac-labeled monovalent antibody reduced tumor weight in a dose-dependent manner. 22 5 It was observed that tumor weights were statistically significantly lower in all groups administered with Ac-labeled monovalent antibodies compared to the antibody control group.

[0168] [Table 8]

[0169] The relative changes in body weight over time are shown in Figure 7. In all groups, no weight loss of 10% or more was observed compared to before administration. 225 It was shown that administration of Ac-labeled monovalent antibodies may have no or a sufficiently low effect on the overall condition.

[0170] On the final day of the observation period, autopsies were performed, and the liver, kidneys, and spleen were collected and weighed. The results of comparing the weights of each organ are shown in the table below. 225 Although liver weight was statistically significantly lower in the group administered 2.5 kBq of Ac-labeled monovalent antibody compared to the antibody control group, this was considered an incidental result because no dose-dependency was observed. No statistically significant differences were observed in the kidneys, spleen, or liver at other doses compared to the antibody control group, suggesting that there may be no or minimal effects on the liver, kidneys, and spleen.

[0171] [Table 9]

[0172] Blood samples collected at the end of the observation period were used to evaluate nephrotoxicity (plasma creatinine was measured using the Creatinine Assay Kit (Cayman Chemical Company)), hepatotoxicity (plasma alanine aminotransferase (ALT) was measured using the ALT activity Kit (BioVision)), and hematotoxicity (white blood cell and platelet counts were measured using an automated hematology analyzer (model: thinka CB-1010, Arkray)). Equality of variance was confirmed for each measurement using Stat PreClinica (Takumi Information Technology). If homogeneity of variance was confirmed, analysis was performed using the Dunnett method (parametric test). If homogeneity of variance was not confirmed, analysis was performed using the Steel method (nonparametric test).

[0173] The results for hepatotoxicity and nephrotoxicity are shown in FIG. 225 In all groups administered with Ac-labeled monovalent antibodies, no statistically significant difference was observed compared to the antibody control group at a significance level of 5%. The results of hematotoxicity are shown in Figure 9. 225 No statistically significant difference was observed at the 5% significance level between each dose group of Ac-labeled monovalent antibody and the antibody control group.

[0174] According to this embodiment, 225 The tumor growth inhibitory effect of Ac-labeled monovalent antibody was confirmed. The inhibitory effect was dose-dependent, and all doses (2.5, 5, 10 kBq) showed statistically significant inhibitory effects at the 5% significance level. 225 Compared with before administration of Ac-labeled monovalent antibody In addition, no weight loss of more than 10% was observed. 225 These results suggest that Ac-labeled monovalent antibodies are unlikely to be hepatotoxic, nephrotoxic, or hematotoxic. 225 It has been revealed that Ac-labeled monovalent antibodies have a very high antitumor effect while being highly safe, making them a very useful cancer treatment drug.

[0175] Example 6: Tumor-bearing mice were used 225 High-dose evaluation of Ac-labeled monovalent antibodies The same procedure as in Example 5 was carried out except that the radioactivity dose administered was 25 kBq / animal (10 times the minimum dose in Example 5). 225 Ac-labeled monovalent antibodies were evaluated ( 225 Ac-labeled monovalent antibody administration group). In addition, a group (antibody control group) was set up to receive a solution in which only the antibody (H01L03) produced in Preparation Example 1 was dissolved in 0.1 M acetate buffer containing 20 mM ascorbic acid. The antibody used in this example was prepared in the same manner as in Example 5. The tumor-bearing mice used for evaluation were prepared in the same manner as in Example 4-2. There were five mice in each group, and for four weeks after administration, observation of the general condition, and measurements of body weight and tumor volume were carried out. Information on the animals in each group is summarized below.

[0176] [Table 10]

[0177] The results of examining the change in tumor volume over time are shown in FIG. 225 The Ac-labeled monovalent antibody administration group showed statistically significant suppression of tumor growth, and the high dose 225 The tumor growth suppression effect of administration of Ac-labeled monovalent antibody was confirmed.

[0178] On the final day of the observation period, autopsies were performed, and tumors were collected and weighed. The results of a comparison of tumor weights are shown in the table below. 225 The tumor weight was statistically significantly lower in the Ac-labeled monovalent antibody-administered group compared to the antibody control group.

[0179] [Table 11]

[0180] The results of the changes in body weight over time are shown in FIG. 225 In the Ac-labeled monovalent antibody-administered group, weight loss was observed early after administration, but the relative weight loss did not fall below 0.9.

[0181] On the final day of the observation period, autopsies were performed, and the liver, kidneys, and spleen were collected and weighed. The results of a comparison of organ weights are shown in the table below. 225 Only the spleen weight was statistically significantly lower in the Ac-labeled monovalent antibody-administered group compared with the antibody control group.

[0182] [Table 12]

[0183] The results for hepatotoxicity and nephrotoxicity are shown in Figure 12. No statistically significant difference was observed compared to the antibody control group, suggesting that this dose did not induce liver or kidney damage. The results for hematotoxicity are shown in Figure 13. No statistically significant difference was observed compared to the antibody control group, suggesting that this dose did not induce hematotoxicity.

[0184] From these results, 225 The Ac-labeled monovalent antibody has a very high antitumor effect while being highly safe, suggesting that it is a very useful cancer treatment drug.

[0185] The following formula can be used to convert the mouse dose into the human dose. Animal dose in mg / kg x (animal weight in kg / human weight in kg) x 0.33 According to this formula, the administration of 25 kBq / 20 g to mice in this example corresponds to the administration of 89.0 kBq / kg (human).

[0186] Example 7: Tumor-bearing mice were used 225 Ac-labeled monovalent antibody and 225 Evaluation of Ac-labeled bivalent antibodies Using tumor-bearing mice 225 Ac-labeled monovalent antibody and 225 The performance of each Ac-labeled bivalent antibody was evaluated. 225 The Ac-labeled monovalent antibody was administered to tumor-bearing mice prepared in the same manner as in Example 4-2 at a radioactivity dose of 5 kBq / mouse or 10 kBq / mouse. 225 Ac-labeled monovalent antibody administration group). 225 The Ac-labeled bivalent antibody was administered to tumor-bearing mice prepared in the same manner as in Example 4-2 at a radioactivity dose of 5 kBq / mouse or 10 kBq / mouse. 225 Ac-labeled bivalent antibody administration group). An antibody control group was also set up as in Examples 5 and 6. Each group consisted of six animals, and for four weeks after administration, observation of the general condition, body weight, and tumor volume were measured. Information on the animals in each group is summarized below.

[0187] [Table 13]

[0188] The results of examining the change in tumor volume over time are shown in Figure 14. The relative ratio of the tumor volume on the final day of the observation period to the tumor volume before administration, which was set at 1.0, is shown in the table below. 225 The Ac-labeled monovalent antibody administration group showed statistically significant suppression of tumor growth, and a dose-dependent tumor growth suppression effect was confirmed. did. 225 The Ac-labeled bivalent antibody administration group showed a tendency to suppress tumor growth.

[0189] [Table 14]

[0190] On the final day of the observation period, the mice were euthanized by exsanguination under isoflurane anesthesia, and the tumors were collected and weighed. The results of a comparison of tumor weights are shown in the table below. 225 The tumor weight was statistically significantly lower in the Ac-labeled monovalent antibody-administered group compared to the antibody control group, confirming a dose-dependent tumor growth inhibitory effect. 225 The Ac-labeled bivalent antibody-treated group also showed lower tumor weights than the antibody control group, but the difference was not statistically significant.

[0191] [Table 15]

[0192] The results of examining changes in body weight over time are shown in Figure 15. The average relative ratio did not fall below 0.9, and the relative ratio for each individual did not fall below 0.8.

[0193] On the final day of the observation period, the animals were euthanized by exsanguination under isoflurane anesthesia, and autopsies were performed. No abnormal findings were found in the autopsies. The liver, kidneys, and spleen were also collected and weighed. The results of a comparison of normal organ weights are shown in the table below. 225 No statistically significant decrease in organ weight was observed in the Ac-labeled antibody-administered group compared with the antibody control group.

[0194] [Table 16]

[0195] 225 Ac-labeled monovalent antibody and 225 The results of hepatotoxicity of Ac-labeled bivalent antibodies are shown in Figure 16. The results of nephrotoxicity are shown in Figure 17. A statistically significant increase was observed compared to the antibody control group. No significant changes were observed, suggesting that this dose does not induce liver or kidney damage. Regarding hematotoxicity, the results of the white blood cell count one week and four weeks after administration are shown in Figure 18. The results of the platelet count one week and four weeks after administration are shown in Figure 19. Regarding the white blood cell count, Between the antibody control group and the monovalent antibody administration group (5kBq and 10kBq) and the bivalent antibody administration group A statistically significant difference was observed in the bivalent antibody group (10 kBq), but this recovered 4 weeks after administration. The bivalent antibody group (5 kBq), which showed a statistically significant difference compared to the antibody control group 4 weeks after administration, was not a dose-dependent event. With regard to platelet counts, except for one animal, the lower limit of the normal range was 500 × 10 at all time points. 9 The cytotoxicity rate did not fall below 100 cells / L. These results suggest that this dose does not induce hematotoxicity.

[0196] Example 8: In-111( 111 In) labeled monovalent antibody and 111 Comparison of pharmacokinetics using In-labeled bivalent antibodies Example 8-1: Each 111 Preparation of In-labeled antibodies To compare the pharmacokinetics of monovalent and bivalent antibodies, we compared monovalent and bivalent antibodies. 111 The antibodies were labeled with In and administered to tumor-bearing mice. Biodistribution experiments were performed 20, 68, and 188 hours after administration to compare the pharmacokinetics of monovalent and bivalent antibodies.

[0197] (1) Preparation of chelator-conjugated antibodies The antibodies were produced in the same manner as in Production Examples 1 and 2, and monovalent and bivalent peptide-modified antibodies of the humanized antibody H01L03 were obtained. A 0.1 mol / L sodium acetate buffer solution (pH 6.0) containing 34 nmol of the chelating moiety (structural formula: L1-4) was reacted with a 0.1 mol / L histidine buffer solution (pH 6.0) containing 0.1 mol / L arginine and containing 34 nmol of either a monovalent antibody or a bivalent antibody at 37°C for 120 minutes to obtain a chelator-introduced antibody. The solution was passed through a desalting column (model number: PD-10, manufactured by GE Healthcare) and fractions containing chelator-conjugated antibodies were collected. The collected fractions were further purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096). The concentration of the purified monovalent antibody was 7.13 mg / mL, and the concentration of the bivalent antibody was 5.07 mg / mL.

[0198] (2) Each antibody 111 In sign As a source of radioactive metals 111 In ion-containing solution (indium chloride ( 111 Injection solution (manufactured by Nippon Medi-Physics Co., Ltd.) with a radioactivity of 91-92 MBq was used, and 0.05 mL of each chelator-introduced antibody was added and mixed well. The pH was confirmed to be 4 using pH test paper (manufactured by Merck). This was reacted at 45°C for 120 minutes. The reaction solution was purified using an ultrafiltration filter (Merck, model number: UFC505096), and the solvent was replaced with a 90 mmol / L sodium acetate buffer solution containing 20 mmol / L ascorbic acid.

[0199] each 111 The radiochemical yield of the In-labeled antibody was 55% for the monovalent antibody and 59% for the bivalent antibody. The radiochemical purity was 97% for the monovalent antibody and 98% for the bivalent antibody. The amount of radioactivity administered to the animals is shown in Table 17. The radiochemical yield here refers to the amount of radioactivity used. 111 In response to the amount of radioactivity 111 This refers to the amount of radioactivity in In-labeled antibodies. Radioactivity was measured using a radioisotope dose calibrator (manufactured by CAPINTEC, model number: CRC-15R). Radiochemical purity is the ratio of the total radioactivity counts in a thin-layer plate analyzed by thin-layer chromatography. 111This refers to the percentage (%) of radioactivity counts of the peak corresponding to In-labeled antibody. Thin-layer chromatography (thin-layer plate: Agilent, model number: SGI0001) was performed using a developing solvent of 100 mmol / L EDTA solution (pH 5.0) / acetonitrile (volume ratio 1:1). Radioactivity counts were detected using a radio-γ-TLC analyzer (Raytest, Model GITA Star).

[0200] [Table 17]

[0201] Example 8-2: Biodistribution in tumor-bearing mice Human pancreatic cancer cell line SW1990 was used at 0.5 × 10 7 The dose was administered subcutaneously to the flank or back of a Balb / c nude mouse (male). 3 Before and after At this point, the solution prepared in Example 8-1 111 In-labeled monovalent and bivalent antibodies were administered via the tail vein.

[0202] (Evaluation method) Tumor-bearing mice were 111 After administration of In-labeled antibodies, mice were housed in metabolic cages, and feces and urine excreted up to each time point (20, 68, and 188 hours after administration) were collected. At each time point, tumor-bearing mice were euthanized by exsanguination under isoflurane anesthesia. Tumors, blood, and normal organs (including the remaining body) were collected and weighed. Radioactivity in the weighted organs, as well as excreted feces and urine, was measured (γ-ray well scintillation counter: JDC-1712, Hitachi Aloka Medical). The radioactivity accumulation rate (%ID) relative to the administered dose was calculated from the radioactivity counts in each organ (including excreted feces and urine), and the radioactivity accumulation rate per organ weight (%ID / g) was calculated.

[0203] (result) The results showing the time-dependent changes in the amount of radioactivity accumulated in each organ are shown in Figure 20. The results showing the time-dependent changes in the radioactivity accumulation rates in excreted feces and excreted urine, and the combined radioactivity accumulation rate of these, are shown in Figure 21. The amount of radioactivity accumulated in the blood was higher for monovalent antibodies than for bivalent antibodies at all time points. This confirmed that monovalent antibodies have a longer blood retention than bivalent antibodies. The amount of radioactivity accumulated in tumors was higher for monovalent antibodies than bivalent antibodies at all time points. Regarding the tendency of radioactivity accumulation in normal organs, the monovalent antibodies showed the highest levels in the spleen, liver, lungs, and kidneys, in that order. For bivalent antibodies, the liver and spleen showed significantly higher levels, followed by the testis, heart, kidney, and femur, in that order. Radioactivity accumulation in each normal organ was confirmed to decrease over time. The amount of excretion was higher for bivalent antibodies than for monovalent antibodies at all time points, and the excretion rate was faster for bivalent antibodies. For monovalent antibodies, fecal and urinary excretion levels were similar. On the other hand, urinary excretion was higher than fecal excretion for bivalent antibodies, confirming that they were mainly excreted via the renal and urinary system.

[0204] Example 9: 225 Ac-labeled DOTAGA-DBCO 225 Preparation of Ac-labeled anti-MUC5AC humanized antibody (1. Chelating Agent Synthesis Process) The structure of the chelating moiety (DOTAGA-DBCO) used in this example is shown in the following formula (L1-5): DOTAGA-DBCO shown in formula (L1-5) was produced according to 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. This chelating moiety was dispersed in a 0.1 mol / L sodium acetate buffer solution (pH 6.0) as a solvent to obtain a dispersion containing 1.7 mmol / L of the chelating moiety. 0.004 mL of this dispersion and 0.004 mL of a radioactive metal source were added. 225A reaction mixture of 4.9 MBq (calculated value calculated from the radioactivity at the time of assay) of Ac ion-containing solution (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 1225 MBq / mL, prepared from Oak Ridge National Laboratory, liquid volume 0.004 mL) and 0.06 mL of 0.1 mol / L sodium acetate buffer (pH 6.0) was reacted under heating conditions. 225 An Ac complex solution was obtained. The molar ratio of the chelating moiety to the radioactive metal ion was: 225 The ratio of Ac ion to total ion was approximately 670:1, and the reaction solution was heated to 70°C for 30 minutes.

[0205] [ka]

[0206] obtained 225 The radiochemical purity of the Ac complex was measured in the same manner as in Example 1. 225 The radiochemical purity of the Ac complex was 85%. 225 The Ac complex solution was used directly in the labeling step.

[0207] (2. Labeling process) The compound obtained through the above (1. Chelating agent synthesis process) 225 The Ac complex solution was mixed with a solution containing a peptide-modified antibody (monovalent antibody; H01L03) prepared in the same manner as in Preparation Example 2, except that the reaction was carried out at room temperature for 60 minutes, without further purification. The mixture was then subjected to a click reaction at 37°C for 2 hours. 225 The Ac complex-labeled antibody was obtained. 225 The amounts of the chelating moiety containing the Ac-labeled complex and the peptide-modified antibody (monovalent antibody) were 68 nmol and 80 nmol, respectively, and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:1.2, respectively. Furthermore, the resulting mixture was reacted at 37°C for 2 hours. 225 The solution of the Ac complex-labeled antibody was purified using an ultrafiltration filter (Merck, model number: UFC505096). 225The radiochemical purity of the Ac-labeled monovalent antibody was 96%, and the radiochemical yield was 68%. 225 The radiochemical purity and radiochemical yield of the Ac-labeled monovalent antibody were measured in the same manner as in Example 1.

[0208] Example 10: 89 Zr-labeled DOTAGA-DBCO 89 Production of Zr-labeled anti-MUC5AC humanized antibody (HPLC purification) (1-1. Chelating agent synthesis process) In this example, the same chelating moiety as that shown in formula (L1-5) above was used. The chelating moiety was dispersed in a DMSO solution to prepare a dispersion containing 2.0 mmol / L of the chelating moiety. 0.150 mL of this dispersion and 0.150 mL of DMSO were used as a radioactive metal source. 89 A reaction mixture of 134 MBq of a Zr ion-containing solution (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 1335 MBq / mL, prepared by Nippon Medi-Physics Co., Ltd., liquid volume 0.100 mL) and 0.050 mL of a 780 mmol / L acetate buffer solution containing 300 mmol / L gentisic acid was reacted under heating conditions. 89 A Zr complex solution was obtained. The molar ratio of the chelate moiety to the radioactive metal ion was: 89 The Zr ion ratio was about 3333:1, and the reaction solution was heated to 70°C for 60 minutes.

[0209] obtained 89 The radiochemical purity of the Zr complex was determined by the following method: 89 A portion of the Zr complex solution was developed by thin layer chromatography (Agilent, model number: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured with a radio-γ-TLC analyzer (raytest, MODEL GITA Star PS). The percentage of the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts) detected was calculated as follows: 89 The radiochemical purity (%) of the Zr complex was calculated. 89 The radiochemical purity of the Zr complex was 98%.

[0210] (1-2. 89 (Zr complex purification process) The Zr complex solution obtained in the above (1-1. Chelating agent synthesis process) 89 was fractionated using high performance liquid chromatography (HPLC) to remove unreacted DOTAGA-DBCO. The obtained fractionated solution was concentrated to about 30 μL by evaporation of the solvent and used in the labeling process. 89 The method for measuring the radiochemical yield (HPLC recovery rate) in the process of removing unreacted substances of the Zr complex-labeled antibody was as follows. That is, the percentage of the radioactivity of the fractionated solution with respect to the charged radioactivity at the start of this process was defined as the HPLC recovery rate (%) in the process of removing unreacted substances. The HPLC conditions were as follows, and the fraction with a retention time of around 27 minutes was fractionated. <HPLC conditions> Detector: Ultraviolet absorptiometer (measurement wavelengths: 220 nm, 254 nm) / Scintillation detector Column: XBridge C18 3.5 μm, 4.6 x 100 mm, manufactured by Waters Flow rate: 0.5 mL per minute Area measurement range: 45 minutes after sample injection Mobile phase A: 10 mmol / L histidine buffer solution pH 6.5 Mobile phase B: Acetonitrile for liquid chromatography Mobile phase C: Acetonitrile / water mixture (1:1) Delivery of mobile phase: The concentration gradient control was performed by changing the mixing ratio of mobile phase A, mobile phase B, and mobile phase C as follows.

[0211] [Table 18]

[0212] (2. Labeling process) The solution of the Zr complex obtained through the above respective processes 89 and the solution containing the peptide-modified antibody (monovalent antibody; H01L03) produced in the same manner as in Production Example 2 were mixed and subjected to a click reaction at 37°C for 1.5 hours to 89 obtain the Zr complex-labeled antibody.​​89 The amounts of the chelating moiety containing the Zr-labeled complex and the peptide-modified antibody (monovalent antibody) were 73 pmol and 50 nmol, respectively, and the molar ratio of the first atomic group (DBCO) to the second atomic group (azide) was approximately 1:685. Further, the reaction was continued at 37°C for 1.5 hours. 89 The Zr complex-labeled antibody solution was purified using an ultrafiltration filter (Merck, model number: UFC505096). 89 The radiochemical purity of the Zr complex-labeled antibody was 95%, and the radiochemical yield was 50%. 89 The radiochemical purity and radiochemical yield of Zr complex-labeled antibodies were measured as follows. Specifically, thin-layer chromatography (Agilent, Model No. SGI0001, developing solvent: acetonitrile:0.1 mmol / L EDTA solution (volume ratio 1:1)) was performed using a radio-γ-TLC analyzer (raytest, Model GITA Star PS). The radiochemical purity (%) was calculated as the percentage of the radioactivity (counts) of the peak detected near the origin relative to the total radioactivity (counts). The radiochemical yield (%) was calculated as the percentage of the radioactivity recovered after ultrafiltration purification relative to the total radioactivity added at the start of the labeling process.

[0213] Example 11: Each 89 Zr and 225 Stability evaluation of Ac-labeled anti-MUC5AC humanized antibody in human and mouse plasma 89 Prepared using Zr-labeled DOTAGA-DBCO 89 The Zr-labeled anti-MUC5AC humanized antibody was produced in accordance with Example 10. 89 Prepared using Zr-labeled DOTA-Bn-DBCO 89 The Zr-labeled anti-MUC5AC humanized antibody was produced in accordance with Example 10, except that the chelating moiety shown in the above formula (L1-4) was used as the chelating moiety. 225 Prepared using Ac-labeled DOTA-Bn-DBCO 225 Ac-labeled anti-MUC5AC humanized antibody was produced in accordance with Example 2. 225Prepared using Ac-labeled DOTAGA-DBCO 225 Ac-labeled anti-MUC5AC humanized antibody was produced in accordance with Example 9. In both cases, 0.1 M sodium acetate buffer (pH 6.0) was used as the solvent.

[0214] Various 89 Zr-labeled antibodies and 225 The Ac-labeled antibody was mixed with human and mouse plasma and incubated at 37°C. The stability at each time point was evaluated by cellulose acetate membrane electrophoresis. 89 Zr-labeled antibodies and 225 To evaluate degradation products in plasma other than Ac-labeled antibodies, 89 Zr or 225 Assuming the separation of Ac from each chelator and the cleavage of each linker site, each unit was mixed with each plasma separately to measure cellulose acetate. Cellulose acetate membrane electrophoresis was performed using each plasma sample collected at each incubation time point, and after electrophoresis, the cellulose acetate membrane was exposed to an imaging plate. The exposed imaging plate was read using a scanner-type image analyzer (GE Healthcare, model number: Typhoon-7000), and various images were analyzed using imaging analysis software (GE Healthcare, software name: ImageQuant). 89 Zr-labeled antibodies and 225 The radiochemical purity of the Ac-labeled antibodies was quantitatively evaluated. The composition of the evaluation samples is shown in Table 19. The radiochemical purity (%) was calculated as the percentage of the radioactivity (counts) of the peak corresponding to each labeled antibody relative to the total detected radioactivity (counts).

[0215] [Table 19]

[0216] [Table 20]

[0217] For the radioisotope-labeled anti-MUC5AC humanized antibody produced using radioisotope-labeled DOTA-Bn-DBCO, the radiochemical purity of plasma samples sampled immediately after the incubation period for stability evaluation and measured by thin-layer chromatography was 94% in mouse plasma and 95% in human plasma. The final radiochemical purity at the 378-hour incubation point was 70% or higher in both cases. The results are shown in the upper panel of Figure 22A. Used for this evaluation 225 The radiochemical purity of the Ac-labeled antibody was 77%, and the radiochemical purity and dissociation of plasma samples sampled immediately after the incubation period for stability evaluation and measured by thin-layer chromatography were confirmed (mouse: 90%, human: 80%). This was thought to be due to some solid components adhering to the antibody, causing the calculated radiochemical purity to be lower than the actual value. Ultimately, the radiochemical purity at the 168-hour incubation time was 60% or higher in all cases. The graph of the results is shown in the upper panel of Figure 22B.

[0218] The RI-labeled anti-MUC5AC humanized antibody produced using RI-labeled DOTAGA-DBCO was subjected to this evaluation. 89 The radiochemical purity of the Zr-labeled antibody was 94%, roughly equivalent to the radiochemical purity of the plasma samples measured immediately after the incubation period for stability evaluation (mouse: 99%, human: 99%). The final radiochemical purity after 336 hours of incubation was 85% or higher in both cases. The results are shown in the lower panel of Figure 22A. Used for this evaluation 225 The radiochemical purity of the Ac-labeled antibody was 100%, which was roughly equivalent to the radiochemical purity of the plasma sample measured immediately after the incubation period for stability evaluation (mouse: 97%, human: 100%). The final radiochemical purity after 336 hours of incubation was 80% or higher in both cases. The results are shown in the lower panel of Figure 22B.

[0219] Example 12: Binding and specificity of RI-labeled anti-MUC5AC humanized antibodies to MUC5AC 225 Ac or 89The RI-labeled anti-MUC5AC humanized antibody prepared using Zr-labeled DOTAGA-DBCO was produced in accordance with Examples 9 and 10. 89 RI-labeled anti-MUC5AC humanized antibodies were prepared using Zr-labeled DOTA-Bn-DBCO in accordance with Example 11. These RI-labeled anti-MUC5AC humanized antibodies were used to evaluate the binding affinity and specificity of each RI-labeled antibody to MUC5AC in vitro using ARG. Specific procedures were performed in accordance with Example 4-3, except that the RI-labeled anti-MUC5AC humanized antibodies were used as test substances. 89 The image of the Zr-labeled antibody is shown in Figure 23. 225 The results for the Ac-labeled antibody are shown in Figure 24. 89 When Zr-labeled antibodies were used, ROIs were set over the entire MUC5AC-positive and -negative tumor sections, and the binding ratio to MUC5AC-positive and -negative tumors was calculated using the values in the calculated ROIs. 225 When Ac-labeled antibodies were used, multiple small ROIs were set within the tumor tissue of MUC5AC-positive and -negative tumor sections, and the binding ratio to MUC5AC-positive tumors and -negative tumors was calculated using the average value. 225 The RI-labeled anti-MUC5AC humanized antibody produced using Ac-labeled DOTAGA-DBCO showed a 6.5-fold binding ratio. 89 The RI-labeled anti-MUC5AC humanized antibody produced using Zr-labeled DOTAGA-DBCO showed a 138-fold binding ratio. 89 The RI-labeled anti-MUC5AC humanized antibody produced using Zr-labeled DOTA-Bn-DBCO showed a 151-fold binding ratio. 89 Zr or 225 Prepared using Ac-labeled DOTAGA-DBCO 89 Zr or 225 Ac-labeled anti-MUC5AC humanized antibody and 89 Prepared using Zr-labeled DOTA-Bn-DBCO 89 It was confirmed that all Zr-labeled anti-MUC5AC humanized antibodies retained their binding and specificity to MUC5AC.

[0220] Example 13:89 Prepared using Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 PET-CT imaging of Zr-labeled anti-MUC5AC humanized antibody 89 Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Zr-labeled antibodies were produced according to Example 10, and each was administered to tumor-bearing mice, followed by evaluation using PET-CT imaging. SW1990, a human pancreatic cancer-derived tumor cell line with high MUC5AC expression, was cultured at 0.7 × 10 7 The tumor volume after transplantation was approximately 150-300 mm. 3 At this point, various 89 Zr-labeled anti-MUC5AC humanized antibody was administered via the tail vein of mice. Tumor volume was calculated using the following formula. Tumor volume = (minor axis of tumor 2 × longest diameter of tumor) / 2 Various administered 89 The radiochemical purity and animal information of the Zr-labeled anti-MUC5AC humanized antibody are shown in Table 21.

[0221] [Table 21]

[0222] PET-CT imaging (small animal PET-CT device: Si78, manufactured by Bruker) ) was performed under the conditions in the table below. PET and CT were taken at 12, 48, 84, 168, and 252 hours after administration. Image reconstruction was performed using the MLEM method for PET and the FBP method for CT. SUV (standardized uptake value) of the tumor, heart (blood), and liver at each time point VOI analysis was performed and the SUV trends were compared using the time activity curve.

[0223] [Table 22]

[0224] [Table 23]

[0225] each 89 The results of PET-CT imaging performed 48 hours after administration of the Zr-labeled antibody are shown in Figure 25. The results of VOI analysis of the tumor, heart (blood), and liver at each time point are shown in Figure 26. In addition, the tumor-liver ratio results at each time point are shown in Figure 27. The maximum tumor accumulation was 2.8 or more in SUV in all cases, and the maximum tumor-liver ratio 84 hours after administration was 89 Prepared using Zr-labeled DOTA-Bn-DBCO 89 Zr mark The antibody titer was 3.6, 89 Prepared using Zr-labeled DOTAGA-DBCO 89 The Zr-labeled antibody showed a mean value of 3.4. No statistically significant differences were observed between the values. Various 89 It was confirmed that the accumulation of Zr-labeled antibody in the heart (blood) decreased over time, and that it had almost completely disappeared from the blood 252 hours after administration. 89 No statistically significant difference was observed in the accumulation of Zr-labeled antibodies in the heart (blood) at each time point. 89 It was confirmed that the accumulation of Zr-labeled antibodies in the liver and muscle also decreased over time. 89 No statistically significant differences were observed in the accumulation of Zr-labeled antibodies in each tissue at each time point.

[0226] Example 14: 89 Prepared using Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Biodistribution experiment of Zr-labeled anti-MUC5AC humanized antibody To confirm more detailed pharmacokinetics, 89 Prepared using Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Zr-labeled antibodies were administered to tumor-bearing mice, and biodistribution experiments were performed 20, 68, and 188 hours after administration. 89The Zr-labeled antibody was produced in the same manner as in Example 13, as described in Example 10.

[0227] Biodistribution experiments were carried out in tumor-bearing mice prepared in the same manner as in Example 4-2. 89 Zr-labeled antibodies were administered via the tail vein. 89 The radiochemical purity of the Zr-labeled anti-MUC5AC humanized antibody and animal information are shown in Table 24. The administered radioactivity was approximately 5 MBq for each group.

[0228] [Table 24]

[0229] Various tumor-bearing mice 89 After administration of Zr-labeled antibodies, mice were housed in metabolic cages, and feces and urine excreted up to each time point (20, 68, and 188 hours after administration) were collected. At each time point, tumor-bearing mice were euthanized by exsanguination under isoflurane anesthesia. Tumors, blood, and normal organs (including the remaining body) were collected and weighed. Radioactivity levels in the weighted organs, as well as excreted feces and urine, were measured (using a gamma-ray scintillation counter: JDC-1712, Hitachi Aloka Medical). The radioactivity accumulation rate (%ID) relative to the administered dose was calculated from the radioactivity counts in each organ (including excreted feces and urine), and the radioactivity accumulation rate (%ID / g) was calculated as the radioactivity accumulation rate per organ weight. The time course of radioactivity accumulation in tumor tissue and each organ is shown in Figures 28A–D. The radioactivity accumulation rate (%ID) relative to the administered dose was calculated from the amount of radioactivity accumulated (counts) in excreted feces and urine, and the results showing the time course are shown in FIG.

[0230] Regarding the amount of radioactivity accumulated in the tumor, 89 Prepared using Zr-labeled DOTA-Bn-DBCO 89 For Zr-labeled antibodies, the highest level was 188 hours after administration. 89 Prepared using Zr-labeled DOTAGA-DBCO 89 The Zr-labeled antibody showed the highest level 68 hours after administration. The amount of radioactivity accumulated was over 20% ID / g. 89 A similar tendency for radioactivity to decrease was observed with the Zr-labeled antibody, and it can be determined that blood clearance was at the same level. Regarding excretion, the radioactivity accumulation rate in feces and urine was 65%ID or more 188 hours after administration. Regarding radioactivity accumulation in normal tissues, the highest level was 20 hours after administration in both cases, and radioactivity accumulation tended to decrease after 20 hours. The normal tissues with the highest radioactivity accumulation 20 hours after administration were the liver, lungs, and spleen, in that order.

[0231] Example 15: 225 Prepared using Ac-labeled DOTAGA-DBCO 225 Efficacy evaluation of Ac-labeled anti-MUC5AC humanized antibody 225 Prepared using Ac-labeled DOTAGA-DBCO 225 The Ac-labeled antibody was administered to tumor-bearing mice to confirm its tumor growth suppression effect. 225 The Ac-labeled antibody was produced in accordance with Example 9. The tumor-bearing mice were produced in the same manner as in Example 4-2. 225 Ac-labeled antibodies were administered to tumor-bearing mice at a radioactivity dose of 5 kBq / mouse or 10 kBq / mouse. 225 The performance of the Ac-labeled antibody was evaluated. Additionally, a group (antibody control group) was administered a solution containing only the anti-MUC5AC humanized antibody dissolved in 0.1 M sodium acetate buffer (pH 6.0). Six mice were included in each group, and general observations, body weights, and tumor volumes were measured for four weeks after administration. The results are shown in Table 25.

[0232] [Table 25]

[0233] The results of confirming the change in tumor volume over time are shown in Figure 30(A). 225 The Ac-labeled antibody administration group showed statistically significant tumor growth inhibition at all doses of radioactivity compared to the antibody control group. 225 The tumor growth suppression effect of administration of Ac-labeled antibody was confirmed.

[0234] On the final day of the observation period, autopsies were performed, and tumor samples were collected and weighed. The results of a comparison of tumor weights are shown in Table 26. 225 The tumor weight was statistically significantly lower in the Ac-labeled antibody-administered group compared with the antibody control group.

[0235] [Table 26]

[0236] The results of the changes in body weight over time are shown in Figure 30 (B). 225 In the Ac-labeled antibody group administered 10 kBq, weight loss was observed in the early observation period, but the relative ratio did not fall below 0.9. In the later observation period, weight returned to pre-administration levels.

[0237] On the final day of the observation period, an autopsy was performed, and the liver, kidneys, and spleen were collected and weighed. Out of place 225 In the Ac-labeled antibody-administered group, no statistically significant decrease in tissue weight was observed compared with the antibody control group.

[0238] [Table 27]

[0239] The results for hepatotoxicity and nephrotoxicity are shown in Figures 33 and 34, respectively. No statistically significant difference was observed compared to the antibody control group, suggesting that this dose did not induce liver or kidney damage. The results for hematotoxicity are shown in Figures 31 and 32. No statistically significant difference was observed compared to the antibody control group, suggesting that this dose did not induce hematotoxicity.

[0240] Example 16: 89 Zr randomly labeled anti-MUC5AC humanized antibody ([ 89 Zr]Random-DFO-Anti-M Creation of UC5AC humanized antibody 0.1 mg (0.7 nmol) of the peptide-modified antibody (monovalent antibody; H01L03) prepared in the same manner as in Preparation Example 2 and 0.26 mg (0.35 μmol) of 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptaeicosine] thiourea (p-SCN-Bn-DFO, Macrocyclics) were mixed in 0.1 M sodium bicarbonate buffer. After the reaction was completed, the product was purified by ultrafiltration, and the solvent was replaced with 50 mmol histidine buffer (pH 6.1) containing 100 mmol arginine (hereinafter referred to as RH buffer). Anti-MUC5AC humanized antibody in which DFO was randomly bound to the amino groups of the MUC5AC humanized antibody. A Random-DFO-anti-MUC5AC humanized antibody (hereinafter referred to as "Random-DFO-anti-MUC5AC humanized antibody") was produced. Protein concentration measurement using a NanoDrop2000 (ThermoFisher) revealed that the protein concentration of the Random-DFO-anti-MUC5AC humanized antibody solution was 1.12 mg / mL. The resulting Random-DFO-anti-MUC5AC humanized antibody solution (89.6 μL, 0.1 mg, 0.67 μmol) was mixed with 10 μL (11.8 MBq) of ZrCl3 solution and 301.6 μL of RH buffer to allow the complex formation reaction to proceed. After complex formation, the antibody was purified by ultrafiltration to obtain the desired antibody. 89 [Zr] Random-DFO-anti-MUC5AC humanized antibody was obtained. The radiochemical purity was calculated to be 97.3%. The radiochemical yield was calculated based on the radioactivity at the start of the reaction to be 43.0%.

[0241] Example 17: 89 Zr]Random-DFO-PET-CT imaging of anti-MUC5AC humanized antibody The compound obtained in Example 16 89 Zr] Random-DFO-anti-MUC5AC humanized antibody 1.27MBq / 22.5μg The solution was diluted and adjusted with RH buffer to a concentration of protein / 100 μL / mouse, and the same procedure as in Example 4-2 was repeated. The compound was administered to tumor-bearing mice (n=3) prepared in the same manner, and evaluation was performed using PET-CT imaging at 19, 42, 86, and 158 hours after administration. The tumor volume of the animals subjected to this evaluation was calculated in the same manner as in Example 4-2, and the average tumor volume was 60.0±20.0 mm 3 The PET imaging conditions and image reconstruction method were the same as in Example 13. VOI analysis of SUV for the tumor, heart (blood), and liver at each time point was performed, and the transitions in SUV were compared using time activity curves.

[0242] The results of PET-CT imaging are shown in Figure 35. The results of VOI analysis of the tumor, heart, and liver at each time point are shown in Figure 36. The results of VOI analysis showed accumulation in the tumor over time, with the average SUV value being 2.7 or higher at all time points, and the maximum average SUV value being 5.1 at 158 hours. It was confirmed that the SUV values in organs other than the tumor (heart (blood), liver) decreased over time. SU at 158 hours after administration The tumor-liver ratio of V was 3.7.

[0243] Example 18: 89 Zr]Random-DFO-anti-MUC5AC humanized antibody biodistribution experiment In Example 17, after PET-CT imaging at 158 hours, blood was exsanguinated under isoflurane anesthesia. Euthanasia was performed by IV sterilization. Tumors, blood, and normal organs (including the remaining body) were collected and weighed. Furthermore, the amount of radioactivity in each organ was measured using a gamma-ray well scintillation counter (JDC-1712, Hitachi Aloka Medical). The radioactivity accumulation rate (%ID) relative to the administered dose was calculated from the amount of radioactivity (counts) in each organ (including excreted feces and urine), and the radioactivity accumulation rate per organ weight (%ID / g) was calculated.

[0244] The results of biodistribution evaluation after PET-CT imaging are shown in Table 28. At 158 hours after administration, the radioactivity distribution rate in the tumor was 5.1±2.3%ID, and the radioactivity distribution rate per unit weight was 49.5±5.2%ID. High radioactivity distribution in the liver was confirmed, with the radioactivity distribution rate in the liver being 5.1%ID and the radioactivity distribution rate per unit weight being 10.4%ID / g. Normal organs with high radioactivity distribution rates, excluding the tumor, were the liver, kidneys, and lungs, in that order.

[0245] [Table 28]

[0246] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention. The contents of all publications, including patents and patent applications, mentioned herein are hereby incorporated by reference to the same extent as if fully set forth. [Industrial Applicability]

[0247] The RI-labeled humanized anti-MUC5AC antibody of the present invention has excellent specificity and tumor accumulation, and is therefore extremely useful in treating and / or diagnosing diseases in which MUC5AC is overexpressed, particularly cancer.

[0248] This application is based on patent application No. 2019-191562 filed in Japan (filing date: October 18, 2019), the contents of which are incorporated in their entirety herein.

Claims

1. A conjugate of an antibody and a chelating agent to which a radionuclide is chelated, the radioactive nuclide is a metal nuclide that emits alpha rays or positrons, A conjugate wherein the antibody is a humanized antibody that specifically binds to mucin subtype 5AC.

2. The antibody (1) the amino acid sequence (H01) shown in SEQ ID NO: 1; (2) the amino acid sequence (H02) shown in SEQ ID NO: 2; (3) the amino acid sequence (H03) shown in SEQ ID NO: 3, or (4) Amino acid sequence represented by SEQ ID NO: 4 (H04) a heavy chain variable region consisting of (5) the amino acid sequence (L01) shown in SEQ ID NO: 5; (6) the amino acid sequence (L02) shown in SEQ ID NO: 6; (7) The amino acid sequence (L03) shown in SEQ ID NO: 7, or (8) Amino acid sequence (L04) shown in SEQ ID NO: 8 a light chain variable region consisting of The conjugate of claim 1, which is a humanized antibody having the following structure:

3. The antibody (1) a heavy chain variable region consisting of the amino acid sequence (H01) shown in SEQ ID NO: 1; (7) a light chain variable region consisting of the amino acid sequence (L03) shown in SEQ ID NO: 7; The conjugate of claim 2, which is a humanized antibody having the following structure:

4. 4. The complex according to claim 1, wherein the metal nuclide that emits α rays is actinium-225 and the metal nuclide that emits positrons is Zr-89.

5. The complex according to any one of claims 1 to 4, comprising 1 to 8 molecules of the chelating agent per antibody molecule.

6. The conjugate according to any one of claims 1 to 5, wherein the chelating agent site-specifically modifies the Fc region of the antibody via a linker.

7. The complex according to claim 6, wherein the linker comprises a peptide consisting of 13 to 17 amino acid residues represented by the following formula (i), and is formed by a crosslinking reaction between the peptide modified with a crosslinking agent and the antibody: (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)...(i) (In the formula, Xa, Xb, Xc, and Xd represent a consecutive Xs, b consecutive Xs, c consecutive Xs, and d consecutive Xs, respectively; X is an amino acid residue having neither a thiol group nor a haloacetyl group in the side chain, a, b, c, and d each independently represent an integer of 1 to 5, and satisfy a+b+c+d≦14; Xaa1 and Xaa3 each independently represent represents an amino acid residue derived from an amino acid having a thiol group in the side chain, or one represents an amino acid residue derived from an amino acid having a thiol group in the side chain, and the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, and Xaa1 and Xaa3 are linked together; Xaa2 is a lysine residue, an arginine residue, a cysteine residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or a diaminopropionic acid, and is modified with the crosslinker.

8. The complex according to any one of claims 1 to 7, wherein the chelating agent has a structure derived from a compound represented by the following formula (A) or a salt thereof: 【Chemical 1】 (In formula (A), R １１ , R １３ and R １４ are each independently -(CH ２ ) ｐ COOH, -(CH ２ ) ｐ C ５ H ５ N, -(CH ２ ) ｐ P.O. ３ H ２ , -(CH ２ ) ｐ CONH ２ Or -(CHCOOH)(CH ２ ) ｐ COOH, and R １２ or R １５ one of which is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, and the other is a substituent for conjugating with the antibody, p is an integer of 0 to 3, and R １２ is a substituent for conjugating with the antibody, R １５ is a hydrogen atom, and R １２ is not a substituent for conjugation with the antibody, R １５ is a substituent for conjugation with the antibody.

9. A radiopharmaceutical comprising the complex according to any one of claims 1 to 8 as an active ingredient.

10. The radiopharmaceutical according to claim 9, wherein the radionuclide is a metal nuclide that emits α-rays and is used for RI internal therapy of cancer.

11. The radiopharmaceutical according to claim 10, which is administered to a subject at a dose of 250 kBq / kg or less per administration in the RI internal therapy.

12. The radiopharmaceutical of claim 11, wherein the dose is 80 kBq / kg or less per administration.

13. The radiopharmaceutical of claim 9, wherein the radionuclide is a positron-emitting metal nuclide and is used for cancer diagnosis.

14. A radiopharmaceutical comprising a complex of an antibody and a chelating agent to which a radionuclide is chelated, The antibody is a humanized antibody that specifically binds to mucin subtype 5AC. A radiopharmaceutical for cancer diagnosis in RI internal therapy using the radiopharmaceutical according to any one of claims 10 to 12.

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