RI-labeled humanized antibody

Abstract

The present invention provides 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 a method for using the radiopharmaceutical. [Solution] The complex is made of a chelating agent chelated with a radioactive nuclide, which is a metal nuclide that emits alpha rays or positrons, and a humanized antibody that specifically binds to mucin subtype 5AC, which is composed of a specific amino acid sequence.Since it has excellent specificity for MUC5AC and accumulation in tumors, it is extremely useful for the treatment and / or diagnosis of diseases in which MUC5AC is overexpressed, particularly cancer.

Description

Technical Field

[0001] The present invention relates to a complex of a chelating agent chelated with a radionuclide and a mucin subtype 5AC-specific humanized antibody, a radiopharmaceutical containing the same, and their uses.

Background Art

[0002] Mucin is the main component of mucus secreted from animal epithelial cells and is a glycoprotein containing a large amount of sugars with a molecular weight of 1 million to 10 million. Mucin includes secreted mucin produced by epithelial cells and membrane-bound mucin having a hydrophobic transmembrane site and existing in a state bound to the cell membrane. The core protein of mucin is collectively called MUC, and it is known that there are at least 20 types of genes encoding the core protein. Mucin subtype 5AC (MUC5AC), which is one of them, belongs to secreted mucin.

[0003] MUC5AC is expressed in the stomach and trachea in normal tissues, but its overexpression has been reported in pancreatic cancer, and its overexpression has also been reported in other cancers such as thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, endometrial cancer, and bile duct cancer. Regarding antibodies against MUC5AC, there are reports of mouse antibodies (Non-Patent Document 1) prepared using a pancreatic cancer mucin fraction purified from xenografts of the human pancreatic cancer cell line SW1990 as an antigen, chimeric antibodies (Patent Documents 1, 2, Non-Patent Documents 2, 3), and humanized antibodies (Patent Documents 3, 4) prepared based on them.

[0004] Antibodies are used as reagents, diagnostic agents for detecting target molecules, or pharmaceuticals for treating diseases by utilizing the specificity of the antibodies against the target molecules. For the purpose of further improving the detection performance and therapeutic effect, studies on antibodies conjugated with radionuclides or drugs have been advanced. In Non-Patent Document 1, radioimmunotherapy of pancreatic cancer model mice using a mouse antibody labeled with 131 I, a β-emitting radionuclide, has been reported. In Non-Patent Document 3, 111SPECT imaging of pancreatic cancer patients using chimeric antibodies labeled with In has been reported. Patent Documents 3 and 4 describe MUC5AC-specific humanized antibodies, 90 labeled with Y and 111 In, etc.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0007] An object of the present invention is to provide a radioisotope-labeled anti-MUC5AC humanized antibody having excellent specificity for mucin subtype 5AC (MUC5AC) and accumulation in tumors.

[0008] As a result of intensive studies in view of the above problems, the present inventors have succeeded in producing a complex of a chelating agent chelated with a radionuclide which is a metal nuclide and a humanized anti-MUC5AC antibody composed of a specific amino acid sequence, and have found that the complex is excellent in specificity for MUC5AC and accumulation in tumors, and have confirmed its effects and thus completed the present invention.

[0009] One aspect of the present invention provides a complex of a chelating agent chelated with a radionuclide and an antibody, wherein the radionuclide is a metal nuclide that emits α-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 an anti-MUC5AC humanized antibody labeled with a radionuclide, which is excellent in specificity for MUC5AC and accumulation in tumors, and uses thereof.

Brief Description of Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] The terms used in this specification can be used in the meaning commonly used in the technical field unless otherwise specified.

[0013] (1) Complex 1 The present invention provides a complex of a chelating agent chelated with a 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 α-rays, and the antibody is a humanized antibody that specifically binds to MUC5AC (hereinafter also referred to as the complex of the present invention).

[0014] (1-1) Radionuclide 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. Specifically, 212 Bi, 213 Bi, 227 Th or 225 Ac, etc. are preferably used, more preferably 227 Th or 225 Ac, and even more preferably 225 Ac (actinium-225). The metal nuclide that emits α-rays of the present invention can be produced by a known method using an accelerator such as a cyclotron or a linear accelerator. For example, 225 Ac can be produced by irradiating a 226 Ra target with protons through a (p,2n) nuclear reaction. The generated metal nuclide that emits α-rays can be purified by separating and purifying it from the target. For example, 225 Ac, 225 The target containing 225 Ac is dissolved with an acid or the like, and an alkali is added to the solution to precipitate a salt containing 225 Ac, and the purified

[0015] (1-2) Antibody The antibody contained in the complex 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 having the ability to specifically bind to MUC5AC, and preferably has stable physical properties and excellent tumor accumulation properties. The antibody may be used as its antigen-binding fragment, and such an embodiment is also included in the present invention. Specifically, it contains specific heavy-chain variable regions and light-chain variable regions described later, and can have appropriate heavy-chain constant regions and light-chain constant regions as desired. As used herein, the "antigen-binding fragment" means an antibody fragment composed of a part of the humanized antibody used in the present invention and having the ability to bind to MUC5AC. As long as it 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.

[0016] The following shows the preferred amino acid sequences as the heavy-chain variable regions of the humanized antibody used in the present invention. 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 in the sequence listing attached to this specification, respectively. The underlined sites are the CDR sites.

[0017]

Chemical formula

[0018] The following shows the preferred amino acid sequences as the light-chain variable regions of the humanized antibody in the present invention. 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 in the sequence listing attached to this specification, respectively. The underlined sites are the CDR sites.

[0019]

Chemical formula

[0020] In other words, the heavy chain variable region of the humanized antibody preferred in the present invention consists of the amino acid sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 4, and the light chain variable region consists of the amino acid sequence represented by any one of SEQ ID NO: 5 to SEQ ID NO: 8. That is, the humanized antibody used in the present invention is composed of a combination of the above-described four heavy chain variable regions (H01 to H04) and four light chain variable regions (L01 to L04).

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

[0022] The most preferred humanized antibody in 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 the humanized antibody is not limited to those defined by the amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 4, and also includes mutants that retain the function. That is, a mutated heavy chain variable region consisting of an amino acid sequence having 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 represented by SEQ ID NO: 1 to SEQ ID 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.

[0024] As used herein, the identity of amino acid sequences refers to the identity of the amino acid sequences between two target proteins, and is represented by the percentage of amino acid residues that match in the optimal alignment of amino acid sequences created using mathematical algorithms known in the art. The identity of amino acid sequences can be determined by visual inspection and mathematical calculations, 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, the identity of amino acid sequences herein can be determined using the phylogenetic analysis program ClustalW (http: / / clustalw.ddbj.nig.ac.jp / index.php?lang=ja) published on the website of DDBJ (DNA DataBank of Japan) under the initial settings (Version 2.1, Alignment type: slow, DNA Weight Matrix: Gonnet, GAP OPEN: 10, GAP EXTENSION: 0.1).

[0025] In addition, as the heavy chain variable region of the humanized antibody used in the present invention, among the amino acid sequences represented by SEQ ID NOs: 1 to 4, a mutated heavy chain variable region consisting of an amino acid sequence in which 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids are deleted, substituted, or added 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 region of the humanized antibody used in the present invention is not limited to the amino acid sequences represented by SEQ ID NO: 5 to SEQ ID NO: 8, and also includes mutants that retain the function. That is, a mutated light chain variable region consisting of an amino acid sequence having 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 represented by SEQ ID NO: 5 to SEQ ID NO: 8 is also used as the light 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 heavy chain variable region of the present invention.

[0027] In addition, as the light chain variable region of the humanized antibody used in the present invention, 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 represented by SEQ ID NO: 5 to SEQ ID NO: 8 is also used as the light 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 heavy chain variable region of the present invention.

[0028] The humanized antibody used in the present invention can be produced by a method generally practiced in the art or a method analogous thereto. Specifically, it can be carried out by the following procedure. Since the amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody used in the present invention are disclosed in SEQ ID NO: 1 to SEQ ID NO: 8, a nucleic acid encoding the antibody obtained based on the amino acid sequence information thereof is constructed and inserted into an appropriate expression vector. The expression vector may optionally contain, in addition to the nucleic acid encoding the humanized antibody used in the present invention, a Kozak sequence for increasing translation efficiency, a signal sequence for promoting the secretion of the humanized antibody used in the present invention into the medium when introduced into the host, and a promoter sequence. The vector that can be used in the present invention can be selected from those commonly used in the art, but pcDNA3.4, a plasmid vector, is preferred. The introduction of the expression vector into the host cell is not particularly limited, and methods conventionally used in the art for introducing genes into cells, such as the calcium phosphate method, electroporation method, lipofection method, and DEAE-dextran method, known to those skilled in the art, can be used. As carried out in the following examples, the introduction method using the lipofection method is particularly suitable. The host cells used for this purpose can also be those conventionally used in the art. Examples of such host cells include CHO cells, 293 cells, Escherichia coli, Pichia pastoris, and Sf9 cells. At present, kits for expression systems for expressing the target protein are also commercially available, and the ExpiCHO System (Thermo Fisher Scientific) used in the following examples is particularly preferred for the rapid and reliable expression of the target protein.

[0029] A nucleic acid encoding a humanized antibody used in the present invention is inserted into an expression vector, the nucleic acid is introduced into a host cell with the expression vector containing the nucleic acid, the host cell into which the nucleic acid has been introduced is cultured, and from the culture supernatant, the humanized antibody used in the present invention can be obtained by purification means such as chromatography. In this method, the humanized antibody used in the present invention is secreted into the culture supernatant by culturing the host cell. From the culture supernatant, the humanized antibody used in the present invention or its antigen-binding fragment can be obtained using purification means such as chromatography. As the means of chromatography, various means known in the art such as affinity chromatography, ion exchange chromatography, and size exclusion chromatography can be used. The affinity chromatography using a protein A column used in the following examples is particularly preferred.

[0030] Further, the above humanized antibody may be a polyclonal antibody or a monoclonal antibody.

[0031] (1-3) Chelating agent In the present invention, the chelating agent is not particularly limited as long as it has a site in its structure where a radionuclide coordinates. Preferably, it has a chelating moiety, which is a site where a radionuclide coordinates, and a substituent for enabling the complexation with an antibody. As the chelating moiety, for example, CB-TE2A (1,4,8,11-Tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), CDTA (Cyclohexane-trans-1,2-diamine tetra-acetic acid), CDTPA (4-cyano-4-[[(dodecylthio)thioxomethyl]thio]-Pentanoic acid), DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid), 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’-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N’-diacetic acid), H2bispa2 (6,6’-({9-hydroxy-1,5-bis(methoxycarbonyl)-2,4-di(pyridine-2-yl)-3,Examples include 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, etc., and preferably has a structure derived from the compound represented by the following formula (A).

[0032] [Chemical formula]

[0033] (In formula (A), R 11 , R 13 and R 14 are each independently a group selected from -(CH 2 ) p COOH, -(CH 2 ) p C 5 H 5 N, -(CH 2 ) p PO 3 H 2 -(CH 2 ) p CONH 2 or -(CHCOOH)(CH 2 ) p COOH, and R 12or R 15 One of them is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, the other is a substituent for complexing with the antibody, p is an integer of 0 or more and 3 or less, and R 12 is a substituent for complexing with the antibody, R 15 is a hydrogen atom, and when R 12 is not a substituent for complexing with the antibody, R 15 is a substituent for complexing with the antibody.)

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

[0035]

Chemical formula

[0036]

Chemical formula

[0037]

Chemical formula

[0038] The linking site between the chelate moiety and the substituent for enabling complexation with the antibody is preferably an amide bond or a thiourea bond, and from the viewpoint of stability, the amide bond is more preferable.

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

[0040] In the complex of the present invention, the chelating agent may be provided with at least 1 molecule or more per 1 molecule of the antibody, but preferably 1 molecule or more and 8 molecules or less are provided. However, from the viewpoint of maintaining the activity of the antibody itself (antigen recognition action, neutralizing action, complement activation action, and / or opsonin action), it is preferable that the chelating agent is site-specifically introduced into the Fc region (constant region) of the antibody. In the present invention, it is more preferable that the chelating agent is provided with 1 molecule or 2 molecules per 1 molecule of the antibody.

[0041] In the complex of the present invention, the chelating agent may be connected to the antibody via a linker. Examples of the linker include a substituted or unsubstituted alkyl group, a substituted or unsubstituted heteroalkyl group, a polyethylene glycol (PEG) group, a peptide, a sugar chain, a disulfide group, and combinations thereof. Preferably, the chelating agent modifies the antibody site-specifically via a linker, more preferably in the Fc region. In this case, the linker contains a peptide consisting of 13 to 17 amino acid residues represented by the following formula (i) (hereinafter also referred to as "antibody-modifying peptide"), and can be formed by a crosslinking reaction between the antibody-modifying peptide modified with a crosslinking agent and the antibody. In formula (i), it is described that the left side of the amino acid sequence on the paper represents the N-terminal side, and the right side of the amino acid sequence on the paper represents the C-terminal side. When the chelating agent is connected to the antibody via the antibody-modifying peptide as a linker, the position where the chelating agent and the antibody-modifying peptide are linked is not particularly limited, but can be directly or indirectly linked to, for example, the N-terminal or C-terminal of the antibody-modifying peptide, preferably the N-terminal. In addition, the C-terminal of the antibody-modifying peptide may be modified such as amidation for improving its stability.

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

[0043] Examples of the amino acid residue that can be included 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, methionine, etc. X may be an amino acid residue composed of the same type of amino acid, or may be an amino acid residue composed of different types of amino acids.

[0044] a, b, c, and d in formula (i) are not particularly limited as long as they are numbers within the above-described range. From the viewpoint of the binding stability between the peptide and the antibody, on the condition that a + b + c + d ≦ 14, 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.

[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 from each other. Examples of amino acids having a thiol group in the side chain include cysteine and homocysteine. Such amino acid residues are preferably bonded by a disulfide bond or a sulfide group is bonded via a linker represented by the following formula (4). In formula (4), the wavy line portion indicates the bonding portion with the sulfide group.

[0046]

Chemical formula

[0047] Instead of the above-described combination, Xaa1 and Xaa3 may be such that one of Xaa1 and Xaa3 is an amino acid residue derived from an amino acid having a thiol group in the side chain, and the other is an amino acid residue derived from an amino acid having a haloacetyl group in the side chain. These are bonded via a thioether bond. The haloacetyl group has its terminal substituted with a halogen such as iodine, and the halogen is eliminated by reaction with the thiol group in the other side chain 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 pamphlet, WO 2017 / 217347 pamphlet, and WO 2018 / 230257 pamphlet, and these can also be used.

[0049] Among these, it is preferable that the amino acid sequence of the antibody-modified peptide has any one of the following sequences (1) to (14), and it is more preferable that it 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 diaminopropionic acid, and (Xaa1) and (Xaa3) both represent homocysteine residues. In addition, in the following amino acid sequences (1) to (14), amino acids other than (Xaa1), (Xaa2) and (Xaa3) are represented by one-letter abbreviations.

[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] Method for producing the (1-4) complex The method for producing the complex of the present invention can be produced from two steps: a conjugation step of conjugating a chelating agent and an antibody, and a complex formation step of forming a complex between a radionuclide and a chelating agent. The conjugation step may be before or after the complex formation step.

[0052] In the conjugation step, various methods for chemically modifying antibodies are used. Specifically, the methods of (a) to (f) can be mentioned. (a) Amine coupling method (a method of modifying the amino group of the lysine residue of an antibody using a chelating agent or chelate having a carboxyl group activated by an N-hydroxysuccinimidyl (NHS) group) (b) A method of modifying the sulfhydryl (SH) group generated by partially reducing the disulfide bond (SS bond) between polypeptide chains at the hinge site of an antibody with a chelating agent or linker having a maleimide group reactive with the SH group (c) A method of modifying a chelating agent or linker having a maleimide group with respect to cysteine newly introduced into an antibody by amino acid mutation by genetic engineering (d) A method of modifying an azide group of azidolysine newly introduced into an antibody by amino acid mutation by genetic engineering with a chelating agent or linker having an alkyne (e.g., Dibensylciclooctene: DBCO) using a click reaction (e) A method of modifying glutamine introduced at a specific position of an antibody with a chelating agent or linker having a side chain of lysine using transglutaminase (f) A method of site-specifically modifying the Fc region of an antibody with a chelating agent or linker having the antibody modification peptide shown in (i) described above

[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 and more preferably in an ionic form from the viewpoint of enhancing the complex formation efficiency. In the complex formation step, the order of adding the radionuclide to the chelating agent is not limited as long as complex formation with the radionuclide is possible. For example, a solution in which a radioactive metal ion is dissolved in a solvent mainly composed of water can be used as the radionuclide. After complex formation, the obtained complex may be purified using a filtration filter, a membrane filter, a column filled with various fillers, chromatography, or the like.

[0054] In the method for producing the complex of the present invention, a 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 enabling complexation with an antibody. Then, in the conjugation step (B), a click reaction is carried out between a peptide-modified antibody having a site-specifically modified Fc region and the complexed chelating agent obtained in step (A) using an antibody-modified linker having an antibody-modifying peptide represented by (i) above and a second atomic group capable of click reaction to obtain the complex of the present invention. The following steps (A) and (B) will be described in detail.

[0055] As the combination of the first atomic group and the second atomic group capable of click reaction, an appropriate one is selected according to the type of click reaction. For example, combinations such as alkyne and azide, and combination of 1,2,4,5-tetrazine and alkene can be mentioned. These atomic groups only need to satisfy that the first atomic group has one of the above combinations of atomic groups, and the second atomic group has an atomic group different from the first atomic group among the above combinations of atomic groups. From the perspective of achieving both the stability of the chelating agent and the antibody and the improvement of their binding efficiency, it is preferable that the chelating linker is alkyne and the antibody-modifying linker is azide, or the chelating linker is 1,2,4,5-tetrazine and the antibody-modifying linker is alkene. Specific examples of click reactions by such combinations of atomic groups include Huisgen cycloaddition reaction, inverse electron demand Diels-Alder reaction, etc.

[0056] As specific examples of the combination of atomic groups capable of click reaction, as shown in the following formula, a combination of an atomic group containing dibenzylcyclooctyne (DBCO) as the alkyne of the first atomic group (formula (1a)) and an atomic group containing an azide group as the azide of the second atomic group (formula (2a)), or a combination of an atomic group containing 1,2,4,5-tetrazine as the first atomic group (formula (1b)) and an atomic group containing trans-cyclooctene (TCO) as the alkene of the second atomic group (formula (2b)) can be mentioned. Preferably, it is a combination of formula (1a) and formula (2a).

[0057]

Chemical formula

[0058] (In the formula, R 1 represents the linking site with the chelating agent, and R 2 represents the linking site with the antibody-modifying peptide in the antibody.)

[0059]

Chemical formula

[0060] (In the formula, R 3 and R 4 One of them represents a linking site with either a chelating agent or an antibody-modifying peptide in an antibody, and the other represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group. R 5 represents a linking site with either a chelating agent or an antibody-modifying peptide in an antibody according to R 3 or R 4 .)

[0061] When using an atomic group containing dibenzylcyclooctyne (DBCO) represented by the above formula (1a) as the alkyne of the first atomic group, various commercially available DBCO reagents can be mentioned. Specifically, for example, 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, DBCO-mPEG, etc. can be selected, but preferably Dibenzylcyclooctyne Maleimide is used.

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

[0063]

Chemical formula

[0064] In formula (iia), Ra, Rb, and Rc are independently -(CH 2) p COOH, -(CH 2 ) p C 5 H 5 N, -(CH 2 ) p PO 3 H 2 、-(CH 2 ) p CONH 2 Or, -(CHCOOH)(CH 2 ) p COOH, where p is an integer from 0 to 3, and either Rd or Re is the binding site (*) to B, and the other is a hydrogen atom or -(CH 2 ) p COOH, -(CH 2 ) p C 5 H 5 N, -(CH 2 )pPO 3 H 2 、-(CH 2 ) p CONH2 or, -(CHCOOH)(CH 2 ) p COOH, where p is an integer from 0 to 3. In formula (ii), B is represented by the following formula (iib).

[0065]

Chemical formula

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

[0067]

Chemical formula

[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 combine 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 combine together, or Rh and Ri may combine together to form a hydrocarbon ring. ** indicates the bonding site to B. In formula (iid), ** indicates the bonding site to B, and Rj represents a hydrogen atom, a methyl group, a phenyl group or a pyridyl group)

[0069] As the chelating agent used in step (A), in the above formula (iia), Ra to Rd are -(CH 2 ) p COOH, p is 1, and Re is the bonding site to B, a DOTA derivative; or Ra to Rc are -(CH 2 )pCOOH, p is 1, Rd is the bonding site (*) to B, and Re is a hydrogen atom, either a DO3A derivative or a DOTAGA derivative is more preferable.

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

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

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

[0073] The molar ratio of the chelating agent to the radionuclide is preferably such that the lower limit is 10 / 1 or more, more preferably 100 / 1 or more, and even more preferably 500 / 1 or more as the chelating moiety / radionuclide, and the upper limit is preferably 10000 / 1 or less, more preferably 8000 / 1 or less, and even more preferably 7000 / 1 or less. For example, a range of 100 / 1 or more and 7000 / 1 or less is preferable, and more preferably a range of 500 / 1 or more and 7000 / 1 or less.

[0074] The complex formation reaction is preferably carried out in a solvent. As the solvent, for example, water, physiological saline, or a buffer solution such as sodium acetate buffer solution, ammonium acetate buffer solution, phosphate buffer solution, phosphate buffered saline, tris(hydroxymethyl)aminomethane buffer solution (Tris buffer solution), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer solution (HEPES buffer solution), or tetramethylammonium acetate buffer solution can be used.

[0075] The liquid volume of the solvent is not particularly limited, but from the perspective of practicality in the manufacturing 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, still more preferably 10 mL or more, even more preferably 100 mL or more, and the upper limit is preferably 1000 or less, more preferably 100 mL or less, still more preferably 10 mL or less, even more preferably 1.0 mL or less. For example, it is in the range of 0.01 mL or more and 100 mL or less.

[0076] From the perspective of the yield of the target chelating agent, the concentration of the chelating agent in the reaction solution of the complex formation reaction is, independently of each other, at the start of step (A), the lower limit is preferably 0.001 μmol / L or more, more preferably 0.01 μmol / L or more, still more preferably 0.1 μmol / L or more, even more preferably 1 μmol / L or more, and the upper limit is preferably 1000 μmol / L or less, more preferably 100 μmol / L or less, still more preferably 10 μmol / L or less. For example, the range of 1 μmol / L or more and 100 μmol / L or less can be mentioned.

[0077] As the temperature of the complex formation reaction, for example, room temperature (25 °C) may be used, or it may be under heating conditions. However, from the perspective of achieving both suppression of the decomposition of the chelating agent and improvement of the complex formation efficiency, the lower limit is preferably 20 °C or more, more preferably 30 °C or more, still more preferably 35 °C or more, even more preferably 37 °C or more, particularly preferably 45 °C or more, and the upper limit is preferably 150 °C or less, more preferably 120 °C or less, still more preferably 100 °C or less, even more preferably 90 °C or less. For example, the range of 30 °C or more and 100 °C or less is preferred, and more preferably the range of 35 °C or more and 90 °C or less.

[0078] The reaction time, on the condition that it is the above-mentioned reaction temperature, preferably has a lower limit of 5 minutes or more, more preferably 10 minutes or more, still more preferably 20 minutes or more, even more preferably 30 minutes or more, particularly preferably 45 minutes or more, and an upper limit of preferably 180 minutes or less, more preferably 150 minutes or less, still more preferably 120 minutes or less, even more preferably 90 minutes or less, particularly preferably 60 minutes or less. For example, a range of 10 minutes or more and 150 minutes or less is preferable, and more preferably a range of 10 minutes or more and 60 minutes or less.

[0079] The antibody used in step (B) is a peptide-modified antibody in which the Fc region (constant region) of the humanized antibody detailed in the above item "(1-2) Antibody" is site-specifically modified using an antibody-modifying linker having the antibody-modifying peptide shown in the above-mentioned (i) and a second click-reactive atomic group.

[0080] The antibody-modifying peptide can be produced by using a combination of natural amino acids and amino acids regardless of natural or unnatural amino acids and subjecting them to peptide synthesis methods such as liquid phase synthesis method, solid phase synthesis method, automatic peptide synthesis method, gene recombination method, and phage display method. In peptide synthesis, if necessary, the functional groups of the amino acids used may be protected. These can be carried out, for example, according to the methods described in WO2017 / 217347 pamphlet and WO2018 / 230257 pamphlet.

[0081] The antibody-modifying linker may be one in which the antibody-modifying peptide is bound to a linker represented by the following formula (S1). *-((L 1 ) m -Z) k -L 2 -AG 2 ···(S1) (In the formula, * indicates the binding site to the N-terminus or C-terminus of the peptide, L 1 is a polyethylene glycol (PEG) linker part, m is an integer of 1 or more and 50 or less, Z is (L1 ) m and L 2 is a second linker part that binds to k is 0 or 1, L 2 is a second PEG linker part, AG 2 is a second atomic group.)

[0082] In the formula (S1), the structure of Z is such that (L 1 ) m and L 2 is not particularly limited as long as it is a linker structure that binds to each other, and for example, it can include an amino acid sequence composed of 1 or more and 5 or less amino acid residues. In this case, the amino acid sequence contained in Z preferably contains a cysteine residue, and is more preferably bound to L via a thioether group formed by the binding of the thiol group of the cysteine residue and a maleimide group. 2 and

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

[0084]

Chemical formula

[0085] One end of the structure of the PEG linker part may be modified by a structure derived from a commercially available PEGylation reagent or a structure derived from a reagent commonly used during PEGylation, and is not particularly limited. Examples include structures derived from diglycolic acid or its derivatives, maleimide or its derivatives.

[0086] The method for introducing the second atomic group into the antibody-modifying linker is as follows: After obtaining an antibody-modifying peptide having a desired amino acid sequence by the above-described method, the peptide is dissolved in a solution containing a solubilizing agent, a reducing agent, and, if necessary, an acid, and an organic solvent solution of an atomic group containing an azide group or trans-cyclooctene (TCO) as the second atomic group is added to the solution, followed by stirring at room temperature for introduction.

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

[0088] Also, when introducing an atomic group containing TCO as the second atomic group, a commercially available click chemistry reagent containing TCO can be used to directly introduce TCO to the N-terminus or C-terminus of the peptide or introduce an atomic group containing TCO via the linker structure described above according to a conventional method.

[0089] A method for obtaining a peptide-modified antibody by binding an antibody-modifying peptide and an antibody can be carried out, for example, using a cross-linking agent. A cross-linking agent is a chemical substance for linking an antibody-modifying peptide and an antibody by a covalent bond. Examples thereof include cross-linking agents preferably containing two or more succinimidyl groups such as disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), cross-linking agents composed of compounds preferably containing two or more imidic acid moieties such as dimethyl adipimidate or salts thereof, and those composed of compounds having a disulfide bond such as dimethyl 3,3'-dithiobispropionimidate and dithiobis(succinimidyl propionate) or salts thereof. By using such a cross-linking agent, a cross-linking reaction can occur between the amino acid residue of Xaa2 in the antibody-modifying peptide and the antibody. The cross-linking reaction in the antibody occurs site-specifically, for example, between the amino acid residue of Xaa2 and the Lys252 residue according to the Eu numbering in the humanized antibody of the present invention when the humanized antibody of the present invention is used as the antibody. These Lys residues are present in the Fc region of the humanized antibody of the present invention.

[0090] The method for binding an antibody-modifying peptide and an antibody can be carried out, for example, by dispersing the above-described antibody-modifying peptide, the antibody, the cross-linking agent, and optionally a catalyst in an appropriate buffer solution in an environment of 10°C or higher and 30°C or lower. The reaction time can be about 10 minutes or more and 2 hours. The molar ratio during the reaction between the peptide and the antibody, with antibody / peptide, has a lower limit that is preferably 1 / 5 or more, more preferably 1 / 3 or more, still more preferably 1 / 1.5 or more, and an upper limit that is preferably 20 / 1 or less, more preferably 10 / 1 or less, still more preferably 5 / 1 or less, even more preferably 1 / 1 or less, particularly preferably 1 / 1.7 or less. For example, a range of 1 / 5 or more and 20 / 1 or less is preferred, and more preferably 1 / 1.5 or more and 1 / 1.7 or less.

[0091] The peptide-modified antibody obtained through the above steps is a mixture containing, at an arbitrary ratio, an antibody (hereinafter referred to as "monovalent antibody") in which one molecule of the antibody-modifying peptide is bound to one molecule of the antibody, and an antibody (hereinafter referred to as "divalent antibody") in which two molecules of the antibody-modifying peptide are bound to one molecule of the antibody. However, this mixture may be directly used in the subsequent steps as it is, or after separating and purifying the unmodified antibody, the monovalent antibody, and the divalent antibody by methods such as a filtration filter, a membrane filter, a column filled with various fillers, and various chromatographies, only the antibody of any valence may be used in the subsequent steps. As a result of purification, if the unmodified antibody cannot be separated from the antibodies of other valences, the mixture containing them may be used in the subsequent steps. When separating and purifying the unmodified antibody, the monovalent antibody, and the divalent antibody, any of the above purification methods may be used, but it is preferable to use a column filled with various fillers, and more preferably to use a column filled with a filler suitable for separating and purifying proteins such as antibodies.

[0092] As a filler suitable for the separation and purification of proteins such as antibodies, there is no particular limitation as long as it is an immunoglobulin-binding protein that specifically binds to an antibody and is immobilized on a carrier composed of a water-insoluble substrate. Examples of immunoglobulin-binding proteins include protein A, protein G, protein L, and the like. These immunoglobulin-binding proteins may be genetically engineered recombinant types. Examples of recombinant immunoglobulin-binding proteins include genetically modified protein A, genetically modified protein G, or a fusion type of the domain of protein A and the domain of protein G. In the present invention, protein A is more preferable as a filler suitable for the separation and purification of at least monovalent antibodies and divalent antibodies, and genetically modified protein A is even more preferable. Here, protein A and protein G are protein molecules that can specifically bind to IgG, which is an antibody molecule, and are classified as protein A (Staphylococcus aureus) or protein G (Streptococcus genus) depending on the difference in the separated microorganisms. Genetically modified protein A is protein A in which at least one amino acid mutation has been introduced into the amino acid residues of any one of the IgG-binding domains (E, D, A, B, and C domains) of protein A. In the present invention, a genetically modified protein A in which a domain into which at least one amino acid mutation has been introduced is multimerized is preferable, a multimerized A, B, or C domain of protein A into which at least one amino acid mutation has been introduced is more preferable, and a multimerized product having a dimer or more and a pentamer or less is even more preferable. The amino acid mutation may be derived from any mutation such as substitution, deletion, insertion, etc. of the amino acid sequence or the base sequence encoding the amino acid in the gene transcription and translation process. As an example not particularly limited, there are genetically modified protein A and the like described in WO 2003 / 080655 pamphlet and WO 2011 / 118699 pamphlet.

[0093] Examples of the water-insoluble substrate to which the immunoglobulin-binding protein is immobilized include inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, organic carriers composed of polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran, and further composite carriers such as organic-organic and organic-inorganic carriers obtained by combining these.

[0094] Examples of the column packed with the above-described genetically modified protein A as a filler include, for example, KanCap (registered trademark) series (KANEKA KanCapA prepacked column) of Kaneka Corporation, HiTrap (registered trademark) series (HiTrap Mabselect, HiTrap Mabselect SuRe, HiTrap Mabselect Xtra) of GE Healthcare, HiScreen series (HiScreen Mabselect SuRe) of GE Healthcare, or TOYOPEARL (registered trademark) series (TOYOPEARL AF-rProtein A-650F) of Tosoh Corporation, etc., which are commercially available.

[0095] Taking the case of separating and purifying the peptide-modified antibody used in the click reaction in step (B) as an example, it will be described below. The peptide-modified antibody is subjected to an antibody modification step of obtaining a modified antibody by site-specifically modifying the Fc region of the antibody with a linker (antibody modification linker) having an antibody modification peptide, and an antibody purification step of purifying the modified antibody using the carrier to which the above-described immunoglobulin-binding protein is immobilized, and then used in the click reaction in step (B). Further, the antibody purification step further includes a retention step of retaining the modified antibody on the carrier, a washing step of washing the modified antibody not retained on the carrier, and an elution step of eluting the modified antibody retained on the carrier in the retention step. More specifically, in the antibody modification step, a modified antibody is obtained as a mixture containing an unmodified antibody that is not modified by an antibody modification linker, a monovalent antibody, and a bivalent antibody. In the antibody purification step, differences in the respective interactions of the unmodified antibody, the monovalent antibody, and the bivalent antibody with the immunoglobulin-binding protein are utilized to elute a first antibody composition containing a relatively large amount of the unmodified antibody and the monovalent antibody and a second antibody composition containing a relatively large amount of the bivalent antibody, respectively. That is, in the holding step and the washing step of the antibody purification step, a second antibody composition containing a relatively large amount of a peptide-modified antibody (bivalent antibody) with a low degree of interaction with the immunoglobulin-binding protein is eluted. In the elution step of the antibody purification step, a first antibody composition containing a relatively large amount of a peptide-modified antibody (unmodified antibody and monovalent antibody) with a high degree of interaction with the immunoglobulin-binding protein is eluted. Here, "containing a relatively large amount of the unmodified antibody and the monovalent antibody" means that the total amount of the unmodified antibody and the monovalent antibody contained in the first antibody composition is greater than the amount of the bivalent antibody contained in the antibody composition, preferably meaning that the total amount of the unmodified antibody and the monovalent antibody is 55% or more, 63% or more, 70% or more, 80% or more, or 90% or more with respect to the total amount (100%) of the unmodified antibody and the modified antibody contained in the antibody composition. "Containing a relatively large amount of the bivalent antibody" means that the amount of the bivalent antibody contained in the second antibody composition is greater than the amount of the monovalent antibody contained in the antibody composition, preferably meaning that the amount of the bivalent antibody is 55% or more, 63% or more, 70% or more, 80% or more, or 90% or more with respect to the total amount (100%) of the unmodified antibody and the modified antibody contained in the antibody composition.

[0096] In the retention step, a solution containing a mixture of unmodified antibody, monovalent antibody, and divalent antibody obtained in the antibody modification step is added to a column to retain the unmodified antibody and monovalent antibody retained by the carrier in the column and allow the divalent antibody not retained by the carrier to pass through. Here, the solution that has passed through in the retention step constitutes part of the second antibody composition. In order to easily retain the unmodified antibody and monovalent antibody on the column and to prevent their aggregation or denaturation, it is preferable to dilute the mixed solution of the peptide-modified antibody with an appropriate dilution solvent and add it to the column. The dilution solvent is not particularly limited as long as the peptide-modified antibody dissolves in it and is less likely to aggregate or denature in the solvent. Water, physiological saline, or buffer solutions such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate buffered saline, 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) buffer, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer, etc. can be used. It is preferable to use any of the above-mentioned buffer solutions, and it is more preferable to use sodium acetate buffer. When a buffer solution is used as the dilution solvent, the concentration of the buffer agent is 10 mmol / L or more, preferably 15 mmol / L or more, more preferably 20 mmol / L or more as the lower limit, and 1000 mmol / L or less, preferably 500 mmol / L or less, more preferably 100 mmol / L or less as the upper limit. Also, from the viewpoint of reducing the non-specific binding of the divalent antibody and antibody modification peptide to the column carrier, the elution solvent may contain additives such as sodium chloride and potassium chloride. The concentration of the additives contained in the elution solvent is not particularly limited, but for example, 0.15 mol / L can be used.

[0097] In the washing step, the modified antibody remaining in the column is eluted from the column using a washing solvent. Since the solution that has passed through the column in the above-mentioned retention step and the solution eluted from the column in the washing step contain relatively more divalent antibody, these can be combined and used as the second antibody composition. As the washing solvent, there is no particular limitation as long as it is a buffer solution in which the peptide-modified antibody dissolves, is less likely to aggregate or denature in the solvent, and has an appropriate pH buffering ability. Examples include sodium acetate buffer solution, ammonium acetate buffer solution, phosphate buffer solution, phosphate buffered saline, 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris) buffer solution, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer solution, and other buffer solutions. It is preferable to use any of the above-mentioned buffer solutions, and it is more preferable to use sodium acetate buffer solution. The concentration of the buffer used in the washing solvent is 20 mmol / L or more, preferably 30 mmol / L or more, as the lower limit, and 200 mmol / L or less, preferably 70 mmol / L or less, as the upper limit. Also, the pH of the washing solvent is 4.0 or more, preferably 4.5 or more, more preferably 4.8 or more, as the lower limit, and 7.4 or less, preferably 6.0 or less, more preferably 5.2 or less, as the upper limit. Further, from the viewpoint of reducing non-specific binding of the bivalent antibody and antibody-modifying peptide 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 for example, 0.15 mol / L can be used.

[0098] In the elution step, the modified antibody retained on the carrier is eluted from the column using an elution solvent. That is, a first antibody composition containing relatively more unmodified antibody and monovalent antibody is eluted from the column using an elution solvent. As the elution solvent, buffer solutions such as sodium acetate buffer solution, ammonium acetate buffer solution, and citrate buffer solution can be used. Also, from the viewpoint of reducing non-specific binding of the antibody-modifying 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 for example, 0.15 mol / L can be used. When the dissolution solvent contains a buffer, the concentration of the buffer is, as a lower limit, 20 mmol / L or more, preferably 30 mmol / L or more, and as an upper limit, 200 mmol / L or less, preferably 70 mmol / L or less. Further, the pH of the dissolution solvent is preferably 3.0 or more and 4.2 or less as a lower limit and an upper limit, respectively, from the viewpoints of weakening the interaction between the unmodified antibody and the monovalent antibody and the immunoglobulin-binding protein and preventing antibody denaturation and aggregation.

[0099] The first antibody composition or the second antibody composition obtained in the antibody purification step may be directly used 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 the click-reactive first atomic group of the chelating agent and the click-reactive second atomic group of the peptide-modified antibody. A linking group (a substituent enabling complexation with the antibody) that links the chelating agent and the antibody is formed by such a click reaction.

[0101] If the peptide-modified antibody and the complex obtained in step (A) are click-reactive, the order of addition thereof is not limited. 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 thereto for reaction, or one of the chelating agent and the antibody may be added to a dispersion in which the other is dispersed in a solvent for reaction. Alternatively, these may be added simultaneously to a reaction vessel containing a solvent for reaction.

[0102] As the solvent used in the click reaction of step (B), a solvent containing water can be used. For example, water, physiological saline, or buffers such as sodium acetate buffer, ammonium acetate buffer, phosphate buffer, phosphate buffered saline, Tris buffer, HEPES buffer, or tetramethylammonium acetate buffer can be used. When using a buffer, from the perspective of achieving both the stability of the complex and the antibody and their binding efficiency, the pH at 25°C is preferably 4.0 or higher and 10.0 or lower, more preferably 5.5 or higher and 8.5 or lower.

[0103] The volume of the reaction solution is not particularly limited. However, from the perspective of practicality in the manufacturing 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 still more preferably 1 mL or more. The upper limit is preferably 1000 mL or less, more preferably 100 mL or less, even more preferably 10 mL or less, and still more preferably 1 mL or less. For example, a range of 0.001 mL or more and 1000 mL or less is preferred, and a range of 0.1 mL or more and 10 mL or less is more preferred.

[0104] Also, the concentrations of the chelating agent and the antibody in the reaction solution are each independently, at the start of step (B), 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 still more preferably 1.0 μmol / L or more. As the upper limit, it is preferably 1000 μmol / L or less, 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 perspective of the yield of the target complex.

[0105] From the perspective of enhancing the reaction efficiency while preventing unintended denaturation of the antibody, in step (B), for the click reaction, the upper limit of the reaction temperature is preferably 50°C or lower, more preferably 40°C or lower. Also, the lower limit of the reaction temperature is not particularly limited as long as the reaction proceeds, but is preferably 15°C or higher. The reaction time of the click reaction is preferably 5 minutes or longer, more preferably 10 minutes or longer, preferably 24 hours or shorter, more preferably 20 hours or shorter, for example, in the range of 5 minutes to 24 hours, more preferably in the range of 10 minutes to 20 hours, on the condition that the above-mentioned reaction temperature is maintained.

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

[0107] The complex produced by steps (A) and (B) is one in which a specific site of a humanized antibody that specifically binds to MUC5AC (for example, a lysine residue in the Fc region of the antibody) is specifically modified by a chelating agent. This complex has one or two molecules of the chelating agent per molecule of the antibody. The chelating agent specifically modifies the Fc region of the antibody of the present invention via a linker. The linker is composed of a chelating linker connecting to the chelating agent, a first atomic group connecting to the linker, a second atomic group capable of undergoing a click reaction with the first atomic group, and an antibody-modifying linker connecting to the second atomic group (including the antibody-modifying peptide represented by the above formula (i)). Therefore, the linker has a chemical structure derived from the first atomic group and the second atomic group. As such a chemical structure, a triazole skeleton-containing structure represented by the following formula (10a) or (10b) or a pyridazine skeleton-containing structure represented by the following formula (10c) can be considered. Since formula (10a) and formula (10b) are in an isomeric relationship, they may be included in any ratio.

[0108]

Chemical formula

[0109] In Formula (10a) and Formula (10b), R 1A represents a binding site with a chelate linker, and R 2A represents a binding site with an antibody-modifying linker. In Formula (10c), one of R 3A and R 4A represents a hydrogen atom, a methyl group, a phenyl group, or a pyridyl group, and the other represents a binding site with a chelate linker, and R 5A represents a binding site with an antibody-modifying linker.

[0110] (1 - 5) Radiopharmaceutical The complex produced by the method described in (1 - 4) above can be used as it is or after purification to prepare a radiopharmaceutical containing the complex as an active ingredient. A radiopharmaceutical refers to a composition containing the complex of the present invention, that is, an anti-MUC5AC humanized antibody or a derivative thereof labeled with a radionuclide (a metal nuclide that emits α-rays), and is in a form suitable for administration into a subject's body. The radiopharmaceutical can be produced, for example, by dissolving the complex of the present invention produced by the above method in a solvent mainly composed of water and having substantially the same osmotic pressure as the living body. In this case, the radiopharmaceutical is preferably in the form of an aqueous solution and may contain other pharmaceutically acceptable components as necessary. The radiopharmaceutical is administered to a living body orally or parenterally such as intravenously, subcutaneously, intraperitoneally, or intramuscularly in an effective amount and is used for the treatment of diseases, the diagnosis of diseases, or the detection of lesions. Here, the administration subject is a human or an animal such as a mouse, rat, monkey, guinea pig, chimpanzee, sheep, goat, dog, cat, pig, cow, or horse, but is not particularly limited. Preferably, it is a human. A preferred target disease is cancer. The cancers treated and diagnosed by the present invention can include pancreatic cancer, thyroid cancer, liver cancer, colorectal cancer, gastric cancer, urothelial cancer, breast cancer, cervical cancer, ovarian cancer, or endometrial cancer, and particularly, application to pancreatic cancer is preferred.

[0111] An example of the cancer treated and diagnosed by the present invention can also include cholangiocarcinoma.

[0112] In addition, it has been reported by multiple studies that MUC5AC is an antigen carrier of CA19-9 (PLoS ONE (December 2011, Volume 6, Issue 12, e29180, p1-10)). Therefore, cancers to be treated in the present invention can include biliary tract cancer, endometrial cancer, lung cancer, or esophageal cancer that overexpress CA19-9, and can be treated efficiently.

[0113] The "effective amount" herein refers to an amount capable of obtaining a therapeutically effective effect in the administration subject. The effective amount to be administered to the subject varies depending on the type of the subject, the weight of the subject, the dosage form (tablet, injection, etc.) and route (oral administration, parenteral administration, etc.) of administration, and the severity of the disease (e.g., cancer). Physicians and veterinarians can determine an appropriate effective amount in consideration of these factors.

[0114] By selecting a radionuclide having a therapeutic effect, the complex of the present invention can be used for radionuclide internal therapy (RI internal therapy). RI internal therapy involves administering a radiopharmaceutical intravenously or orally, accumulating this radiopharmaceutical at the lesion site such as the primary cancer focus or metastatic focus, and destroying the cancer cells at the lesion site by the radiation emitted from the radiopharmaceutical. Therefore, the complex of the present invention can be preferably used for RI internal therapy of cancer. In this case, the dosage and dose of the said medicine are appropriately selected according to the effectiveness of the active ingredient, the form and route of administration, the progression stage of the disease (especially cancer), the body type, weight, and age of the patient, and the type and amount of the therapeutic agent for other diseases to be used in combination, but can usually be administered at 250 kBq / kg or less per time. A dosage of 80 kBq / kg or less per time can also exert an effect.

[0115] In addition, as another aspect of the present invention, in the above-described complex, only the radionuclide, a radionuclide that emits positrons or gamma rays from an alpha-ray emitting nuclide ( 68 Ga, 64 Cu, 86 Y, 89 Zr, 111Prepare a radiopharmaceutical having, as an active ingredient, a complex replaced with (In), and this may be used for the diagnosis of cancer in the above-described RI internal therapy for cancer. The radiopharmaceutical for diagnosing cancer of the present invention may be used for diagnosis before performing RI internal therapy for cancer, or may be used for diagnosis after performing RI internal therapy for cancer. By being used for diagnosis before performing RI internal therapy for cancer, it can be used for determining a treatment option as to whether to perform RI internal therapy for cancer using the complex of the present invention having a metal nuclide that emits α-rays. Further, by being used for diagnosis after performing RI internal therapy for cancer, it can be used for determining whether the RI internal therapy for cancer using the complex of the present invention having a metal nuclide that emits α-rays is effective and for optimizing a treatment plan such as increasing or decreasing the dose.

[0116] (2) Complex 2 As another embodiment, the present invention provides a complex of a chelating agent chelated with a radionuclide and an antibody, wherein the radionuclide is a metal nuclide that emits a positron, and the antibody is a humanized antibody that specifically binds to MUC5AC.

[0117] The same definition as the above “(1) Complex 1” applies except that the radionuclide in the chelating agent is a metal nuclide that emits a positron. The metal nuclide that emits a positron may be any nuclide that emits an electron (positron) having a positive charge in the decay process of a radioactive metal. Specifically, 68 Ga, 64 Cu, 86 Y, and 89 Zr, etc. are preferably used, and more preferably 89 Zr (zirconium-89). The antibody labeled with a positron-emitting nuclide can be suitably used for a PET (Positron Emission Tomography) examination. In addition, the complex 2 using a positron-emitting radionuclide as the radionuclide can also be used as a radiopharmaceutical for cancer diagnosis for the above-mentioned RI internal therapy using the complex 1 using an α-ray-emitting radionuclide as the radionuclide. In this case, the dosage of the pharmaceutical is not particularly limited as long as it is sufficient for imaging the lesions of the disease (especially cancer) in a PET examination. However, it is preferably appropriately selected according to the progression stage of the disease (especially cancer), the body type, weight, and age of the patient, and the type and amount of the therapeutic agent for other diseases used in combination.

[0118] According to the embodiments of the present invention described above, an anti-MUC5AC antibody labeled with a radionuclide, particularly an α-ray-emitting radionuclide, which is excellent in specificity for MUC5AC and accumulation in tumors, particularly a humanized antibody, is provided. In addition, according to the embodiments of the present invention, an RI-labeled anti-MUC5AC antibody for cancer diagnosis and / or cancer treatment for achieving seranostics is provided.

[0119] The above embodiments of the present invention include the following technical ideas. [1] A complex of a chelating agent chelated with a radionuclide and an antibody, wherein the radionuclide is a metal nuclide that emits α-rays, and the antibody is a humanized antibody that specifically binds to MUC5AC. [2] The antibody is (1) The amino acid sequence shown in SEQ ID NO: 1 (H01), (2) The amino acid sequence shown in SEQ ID NO: 2 (H02), (3) The amino acid sequence shown in SEQ ID NO: 3 (H03), or (4) The amino acid sequence shown in SEQ ID NO: 4 (H04) comprising a heavy chain variable region, and (5) The amino acid sequence shown in SEQ ID NO: 5 (L01), (6) The amino acid sequence shown in SEQ ID NO: 6 (L02), (7) The amino acid sequence shown in SEQ ID NO: 7 (L03), or (8) The amino acid sequence shown in SEQ ID NO: 8 (L04) comprising a light chain variable region, and The conjugate according to [1] above, which is a humanized antibody having [3] The antibody is (1) a heavy chain variable region consisting of the amino acid sequence (H01) shown in SEQ ID NO: 1, and (7) a light chain variable region consisting of the amino acid sequence (L03) shown in SEQ ID NO: 7, and The conjugate according to [2] above, which is a humanized antibody having [4] The conjugate according to any one of [1] to [3] above, wherein the metal nuclide that emits α-rays is actinium-225. [5] The conjugate according to any one of [1] to [4] above, which comprises 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 represented by the following formula (i). (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)···(i) (In the formula, Xa, Xb, Xc and Xd each represent a consecutive a number of Xs, a consecutive b number of Xs, a consecutive c number of Xs, and a consecutive d number of Xs, 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 or more and 5 or less, and satisfy a + b + c + d ≦ 14, Xaa1 and Xaa3 are each independently represent an amino acid residue derived from an amino acid having a thiol group in the side chain, and are bonded via a disulfide bond or a sulfide group is bonded via a linker, or one represents an amino acid residue derived from an amino acid having a thiol group in the side chain, the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, and 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 complex according to [7] above, wherein in the antibody-modifying peptide, Xaa2 is a lysine residue in the formula (i). [9] The complex according to [7] or [8] above, comprising 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 [1] to [9] above, wherein the chelating agent has a structure derived from a compound represented by the following formula (A) or a salt thereof.)

[0120] [Chemical formula]

[0121] (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 PO 3 H 2 、-(CH 2 ) p CONH 2 or a group consisting of -(CHCOOH)(CH 2 ) p COOH, one of R 12 or R 15 is a hydrogen atom, a carboxyl group, or a carboxyalkyl group having 2 or 3 carbon atoms, the other is a substituent for complexing with the antibody, p is an integer of 0 or more and 3 or less, and when R 12 is a substituent for complexing with the antibody, R 15 is a hydrogen atom, and when R 12 is not a substituent for complexing with the antibody, R 15is a substituent for complexing with the antibody.)

[11] The complex 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 bonding group formed by a click reaction.

[12] The linker has a chelate linker connecting the chelating agent and the bonding group formed by the click reaction, and an antibody modification linker connecting the antibody and the bonding group formed by the click reaction, and the bonding group formed by the click reaction has a triazole skeleton-containing structure represented by the following formula (10a) or a pyridazine skeleton-containing structure. The complex according to

[11] above.

[0122]

Chemical formula

[0123] (In the formula, R 1A represents a binding site with the chelate linker, and R 2A represents a binding site with the antibody modification linker.)

[13] A radiopharmaceutical containing 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 of 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 is 80 kBq / kg or less per administration.

[17] A radiopharmaceutical containing a complex of a chelating agent chelated with a radionuclide 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 complex of a chelating agent chelated with a radionuclide and an antibody, The radioactive nuclide is a metal nuclide that emits positrons, A complex, wherein the antibody is a humanized antibody that specifically binds to MUC5AC.

[19] The antibody is (1) an amino acid sequence (H01) represented by SEQ ID NO: 1, (2) an amino acid sequence (H02) represented by SEQ ID NO: 2, (3) an amino acid sequence (H03) represented by SEQ ID NO: 3, or (4) an amino acid sequence (H04) represented by SEQ ID NO: 4 comprising a heavy chain variable region, and (5) an amino acid sequence (L01) represented by SEQ ID NO: 5, (6) an amino acid sequence (L02) represented by SEQ ID NO: 6, (7) an amino acid sequence (L03) represented by SEQ ID NO: 7, or (8) an amino acid sequence (L04) represented by SEQ ID NO: 8 comprising a light chain variable region, and The complex according to

[18] above, which is a humanized antibody having

[20] The antibody is (1) a heavy chain variable region consisting of the amino acid sequence (H01) represented by SEQ ID NO: 1, and (7) a light chain variable region consisting of the amino acid sequence (L03) represented by SEQ ID NO: 7, and The complex according to

[19] above, which is a humanized antibody having

[21] The metal nuclide that emits positrons is zirconium-89, and the complex according to any one of

[18] to

[20] above.

[22] The complex according to any one of

[18] to

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

[23] The complex according to any one of

[18] to

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

[24] The complex according to

[23] above, wherein the linker contains an antibody-modifying peptide consisting of 13 to 17 amino acid residues represented by the following formula (i). (Xa)-Xaa1-(Xb)-Xaa2-(Xc)-Xaa3-(Xd)···(i) (In the formula, Xa, Xb, Xc and Xd respectively represent a consecutive a number of Xs, a consecutive b number of Xs, a consecutive c number of Xs, and a consecutive d number of Xs, 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 or more and 5 or less, and satisfy a + b + c + d ≦ 14, Xaa1 and Xaa3 each independently represent an amino acid residue derived from an amino acid having a thiol group in the side chain, and are 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 the side chain, the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, and 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 complex according to

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

[26] The complex according to

[24] or

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

[27] The complex according to any one of

[18] to

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

[0124]

Chemical formula

[0125] (In the 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 PO 3 H 2 、-(CH 2 ) p CONH 2 or, -(CHCOOH)(CH 2 ) p COOH, a group consisting of 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 complexing with the antibody, p is an integer of 0 or more and 3 or less, and when R 12 is a substituent for complexing with the antibody, R 15 is a hydrogen atom, and when R 12 is not a substituent for complexing with the antibody, R 15 is a substituent for complexing with the antibody.)

[28] A radiopharmaceutical containing, as an active ingredient, the complex according to any one of

[18] to

[27] above. Radiopharmaceutical.

[29] A method for producing the complex according to any one of [1] to

[12] and

[18] to

[27] above, comprising a complexing step of complexing a chelating agent chelated with a radionuclide and an anti-MUC5AC antibody to produce a complex of the chelating agent and the anti-MUC5AC antibody.

[30] The chelating agent is connected to a chelating linker, the anti-MUC5AC antibody is specifically modified at the Fc region by an antibody-modifying linker having an antibody-modifying peptide, and in the complexing step, a click reaction is performed to connect the chelating linker and the antibody-modifying linker. A method for producing the complex according to

[29] above.

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

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

[33] The method for producing a modified antibody according to

[32] above, wherein the immunoglobulin-binding protein is protein A or a genetically modified protein A.

[34] The method for producing a modified antibody according to

[32] or

[33] above, wherein the antibody-purifying step is performed using a column packed with the carrier.

[35] The antibody-purifying step includes a holding step of holding the modified antibody on the carrier, and an elution step of eluting the modified antibody held on the carrier. The method for producing a modified antibody according to any one of

[32] to

[34] above.

[36] In the antibody-modifying step, the modified antibody is obtained as a mixture containing an unmodified antibody in which the antibody-modifying linker is not modified, a monovalent antibody in which one molecule of the antibody-modifying linker is modified per one molecule of the antibody, and a divalent antibody in which two molecules of the antibody-modifying linker are modified per one molecule of the antibody. In the antibody purification step, a first antibody composition relatively rich in unmodified antibodies and monovalent antibodies and a second antibody composition relatively rich in bivalent antibodies are obtained by utilizing the differences in the respective interactions of the unmodified antibody, the monovalent antibody, and the bivalent antibody with respect to the immunoglobulin-binding protein, respectively. The method for producing a modified antibody according to

[35] above.

[37] A method for producing a complex, comprising a modified antibody production step of obtaining a modified antibody by performing the method for producing a modified antibody according to any one of

[32] to

[36] above, and a complexing step of complexing the modified antibody with a chelating agent chelated with a radionuclide to generate a complex of the chelating agent and the modified antibody.

[38] In the modified antibody production step, a first antibody composition having a higher total ratio of unmodified antibodies not modified with the antibody modification linker and monovalent antibodies modified with one molecule of the antibody modification linker per one molecule of the antibody than a bivalent antibody modified with two molecules of the antibody modification linker per one molecule of the antibody is obtained. The method for producing a complex according to

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

[39] In the modified antibody production step, a second antibody composition having a higher ratio of a bivalent antibody modified with two molecules of the antibody modification linker per one molecule of the antibody than the total of unmodified antibodies not modified with the antibody modification linker and monovalent antibodies modified with one molecule of the antibody modification linker per one molecule of the antibody is obtained. The method for producing a complex according to

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

[40] The chelating agent is connected to a chelating linker, and in the complexing step, by performing a click reaction, the chelating linker and the antibody modification linker are connected. The method for producing a complex according to any one of

[37] to

[39] above.

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

[12] and

[18] to

[27] .

[42] The kit according to

[41] , further comprising (1) a first click-reactive group and (2) a second click-reactive group.

[43] The kit according to

[41] , further comprising a radionuclide capable of chelating with the chelating agent.

[0126] According to the radiopharmaceutical of

[14] above, since it contains, as an active ingredient, a complex comprising a humanized antibody that specifically binds to MUC5AC and a metal nuclide that emits α-rays, by using it in the RI internal therapy for cancer, it can specifically accumulate in tumors expressing MUC5AC, irradiate α-rays specifically to tumor cells without affecting normal cells, and higher safety and therapeutic effects can be obtained. According to the radiopharmaceutical of

[28] above, since it contains, as an active ingredient, a complex comprising a humanized antibody that specifically binds to MUC5AC and a metal nuclide that emits a positron, it is suitable for PET examination, and by showing the same accumulation property as the radiopharmaceutical used in the RI internal therapy of

[14] above, it can be efficiently used as a diagnostic radiopharmaceutical for the RI internal therapy of cancer expressing MUC5AC. According to the method for producing the complex of

[29] above, since it includes a complexing step of complexing a chelating agent chelated with a radionuclide and an anti-MUC5AC antibody, it is possible to prevent the denaturation of the antibody without subjecting the anti-MUC5AC antibody to the chelating step, which is a more severe condition for the antibody, and efficiently obtain the complex. According to the method for producing the complex of

[30] above, since the click reaction is included in the complexing step, complexing can be carried out under extremely mild conditions of normal temperature in a buffer solution, and the complex can be efficiently obtained without denaturing the anti-MUC5AC antibody. According to the modified antibody of

[31] above, since the Fc region of the anti-MUC5AC antibody is specifically modified by an antibody-modifying linker, the modified antibody can be used for a click reaction with a chelating linker provided in a chelating agent chelated with a radionuclide without impairing the antigen-binding ability of the anti-MUC5AC antibody. According to the method for producing the modified antibody of

[32] above, an antibody-modifying step of obtaining a modified antibody by site-specifically modifying the Fc region of an anti-MUC5AC antibody with a linker having an antibody-modifying peptide, and an antibody-purifying step of purifying the antibody using a carrier to which an immunoglobulin-binding protein is immobilized are provided, so that the purity of the modified antibody can be further increased. According to the method for producing the complex of

[38] or

[39] above, since a complexing step is performed using an antibody composition in which the ratio of either a monovalent antibody or a bivalent antibody is higher than the ratio of the other, the number of chelating agents binding to the anti-MUC5AC antibody can be adjusted according to the purpose, and a complex of a desired valence can be obtained with higher purity. According to the kit of

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

[12] and

[18] to

[27] at the time of use, enabling efficient treatment or diagnosis without impairing both the half-life of the radionuclide and the antibody activity. According to the kit of

[42] above, since a chelating agent capable of chelating a radionuclide and a complex containing a click reaction group, and a complex of an antibody and a click reaction group are provided separately, a radionuclide is chelated to the chelating agent at a necessary timing, and these are subjected to a click reaction to prepare a complex described in any of [1] to

[12] and

[18] to

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

[0127] Hereinafter, the present invention will be described in detail by way of examples and the like, but the present invention is not limited thereto.

Examples

[0128] Production Example 1: Preparation of Anti-MUC5AC Humanized Antibody The amino acid sequences of various variable regions with an added signal sequence and the amino acid sequences of various constant regions were converted into nucleotide sequences while considering a codon usage suitable for expression in CHO cells. A Kozak sequence was added to the start codon site of the signal sequence, and a stop codon was added to the C-terminal side of the constant region. Furthermore, restriction enzyme sites were added upstream of the Kozak sequence and downstream of the stop codon so that they could be introduced into the gene introduction site for expression of a mammalian cell expression plasmid (pcDNA3.4). Each DNA fragment designed in this way was prepared by chemical synthesis. A DNA fragment containing a variable region that would become the target H chain and the target L chain and a DNA fragment containing a constant region were ligated using fusion PCR.

[0129] Each of the prepared antibody genes was restriction-treated and then purified. Similarly, a mammalian cell transient expression plasmid (pcDNA3.4) was also treated with the same restriction enzyme and then purified. The two fragments were mixed at an appropriate mixing ratio and ligated. The ligation reaction solution was mixed with E. coli DH5α competent cells and transformed. From the resulting transformants, colony PCR, single colony isolation, plasmid extraction from a small-scale culture solution, and nucleotide sequencing of the insert portion were performed, and a plasmid (E. coli clone) in which the full-length designed antibody gene was correctly inserted in the intended direction with the designed sequence was selected. Large-scale culture was performed on the selected E. coli clone, and plasmid extraction and purification including an endotoxin removal step were carried out. The absorbance of the purified plasmid was measured at 260 nm to calculate the concentration.

[0130] Transient expression in CHO cells was performed using the ExpiCHO System (Thermo Fisher Scientific). From each prepared plasmid for H chain expression and each plasmid for L chain expression, one type of H chain and one type of L chain were selected to form the desired combination, and transfection was carried out by the lipofection method, followed by culturing and feeding. Seven to thirteen days after transfection, the culture medium was collected. The centrifuged and filter - filtered culture supernatant was added to a Protein A column, and the antibody was purified by ordinary affinity column chromatography (adsorption, washing, elution with an acidic buffer, and neutralization of the eluate). The absorbance of the purified antibody at 280 nm was measured to calculate the concentration.

[0131] The following anti - MUC5AC humanized antibodies were prepared using the method described above. The antibody numbers assigned to the combinations of the heavy - chain variable region and the light - chain variable region are shown below. Antibody 1: H01L03 Antibody 2: H01L04 Antibody 3: H02L04 Antibody 4: H04L04 Here, H01, H02, and H04 are the 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 the 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), light - chain constant region 1 (SEQ ID NO: 26), and the combinations of the heavy - chain variable regions and light - chain variable regions of Antibodies 1 to 4 described above.

[0132] Production Example 2: Site-Specific Antibody Modification with Peptide Linker (1) Antibody modification step The antibody - modifying peptide was produced by the method described in WO2017 / 217347 pamphlet to obtain a peptide containing 17 amino acid residues represented by the following formula (P3). The amino acid sequence of this peptide is identical to the sequence in which Xaa2 of SEQ ID NO: 10 is a lysine residue, and the side - chain terminal amino group of the lysine residue is R 1It was modified with the structure shown by. Also, two cysteine residues form a disulfide bond with each other, and the N-terminus of the peptide is ethyl azide as a group containing an azide group, which is a second group, bonded via a linker structure having diglycolic acid and eight PEGs.

[0133] [Chemical formula]

[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] A mixed solution obtained by mixing this peptide and the anti-MUC5AC humanized antibody (antibody 1) prepared in Production Example 1 in a sodium acetate buffer (pH 6.0) was reacted at room temperature for 30 minutes to obtain a solution containing the peptide-modified antibody. This peptide-modified antibody has the Fc region of the antibody site-specifically modified by the above peptide.

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

[0137] Example 1: 225 Preparation of Ac-Labeled Anti-MUC5AC Humanized Antibody ( 225 Ac-Labeled Monovalent Antibody) 1 (1) Chelating agent synthesis step 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 0.1 mol / L sodium acetate buffer (pH 6.0) as a solvent to obtain a solution containing 1.7 mmol / L of the chelating moiety. 0.005 mL of this solution and 225 a solution containing Ac ions (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 300 MBq / mL, prepared from Oak Ridge National Laboratory, liquid volume: 0.005 mL) 1.5 MBq (calculated value obtained by decay calculation from the radioactivity at the assay date and time) were mixed, and the reaction solution was reacted under heating conditions to 225 obtain an Ac complex solution. The molar ratio of the chelating moiety to the radioactive metal ion is chelating moiety: 225 Ac ion = approximately 2000:1, the heating temperature of the reaction solution was 70 °C, and the heating time was 90 minutes.

[0138]

Chemical formula

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

[0140] (2) Labeling step The eluate of the monovalent antibody obtained in Production Example 2 and the 225 solution of the Ac complex obtained in the above step (1) were each added to a 0.09 mol / L sodium acetate buffer containing 0.02 mol / L (20 mM) ascorbic acid, and subjected to a click reaction at 37°C for 120 minutes to 225 obtain an Ac-labeled monovalent antibody. 225 The amount of the Ac complex and the amount of the 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. Furthermore, the solution of the Ac-labeled monovalent antibody obtained by reacting at 37°C for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096) and used for subsequent experiments. After purification, 225 the radiochemical purity of the Ac-labeled monovalent antibody (radioactivity 0.303 MBq calculated by decay from the radioactivity at the assay date and time) was 93%, and the radiochemical yield was 39%. Here, the radiochemical purity is based on the total radioactivity count of the thin layer plate when analyzed by thin layer chromatography 225 to the 225It is the ratio (%) of the radioactivity count of the peak corresponding to the Ac-labeled monovalent antibody, and the radiochemical yield is calculated from the radioactivity count at the start of the labeling step measured with a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC), multi-channel analyzer: M7-000 (manufactured by Seiko Epson Corporation), data processing: Spectrum Navigator: DS-P300 (manufactured by Seiko Epson Corporation) and Gamma Studio: DS-P600 (manufactured by Seiko Epson Corporation)) with respect to the radioactivity amount 225 It is the ratio (%) of the radioactivity amount calculated from the radioactivity count of the Ac-labeled monovalent antibody.

[0141] Example 2: 225 Preparation of Ac-Labeled Anti-MUC5AC Humanized Antibody ( 225 Ac-Labeled Monovalent Antibody) 2 (1) Chelating agent synthesis step 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 obtain a solution containing 1.7 mmol / L of the chelating moiety. 0.0025 mL of this solution, 225 1.08 MBq (calculated value obtained by decay calculation from the radioactivity amount at the assay date and time) of an Ac ion-containing solution (0.2 mol / L hydrochloric acid aqueous solution, radioactivity concentration 432 MBq / mL, manufactured by Oak Ridge National Laboratory, liquid volume: 0.0025 mL) and 0.0375 mL of 0.1 mol / L sodium acetate buffer (pH 6.0) were mixed to obtain a reaction solution, which was reacted under heating conditions to 225 obtain an Ac complex solution. The molar ratio of the chelating moiety to the radioactive metal ion is chelating moiety:225 The Ac ion = was about 2000:1, the heating temperature of the reaction solution was 70 °C, and the heating time was 90 minutes.

[0142]

Chemical formula

[0143] The obtained 225 The radiochemical purity of the Ac complex was measured by the following method. That is, 225 A part of the Ac complex solution was developed by thin-layer chromatography (manufactured by Agilent, model number: SGI0001, developing solvent: acetonitrile / water mixture (volume ratio 1:1)), and then measured with a radio γ-TLC analyzer (manufactured by raytest, MODEL GITA Star). The percentage of the radioactivity (counts) of the peak detected near the original line to the total detected radioactivity (counts) was 225 taken as the radiochemical purity (%) of the Ac complex. As a result, 225 the radiochemical purity of the Ac complex was 98%. The obtained 225 Ac complex solution was directly used in the following labeling step.

[0144] (2) Labeling step The eluate of the monovalent antibody obtained in Production Example 2 and the 225 solution of the Ac complex obtained in the above step (1) were subjected to a click reaction at 37 °C for 120 minutes to 225 obtain an Ac-labeled monovalent antibody. 225 The amounts of the Ac complex and the 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 about 1:1, respectively. Furthermore, the 225 solution of the Ac-labeled monovalent antibody obtained by reacting at 37 °C for 120 minutes was added with a 90 mmol / L sodium acetate buffer solution (pH 6.0) containing 20 mmol / L ascorbic acid and purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096) for subsequent experiments. After purification, the 225The radiochemical purity of the [Ac-labeled monovalent antibody (radioactivity calculated by attenuation from the radioactivity at the assay date and time: 0.231 MBq)] was 86%, and the radiochemical yield was 21%. Here, the radiochemical purity is the ratio (%) of the radioactivity count of the peak corresponding to the [Ac-labeled monovalent antibody] to the total radioactivity count of the thin layer plate when analyzed by thin layer chromatography, and the radiochemical yield is the ratio (%) of the radioactivity calculated from the radioactivity count at the start of the labeling step measured with a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC), multi-channel analyzer: M7-000 (manufactured by Seiko Epson Corporation), data processing: Spectrum Navigator: DS-P300 (manufactured by Seiko Epson Corporation) and Gamma Studio: DS-P600 (manufactured by Seiko Epson Corporation)) to the radioactivity calculated from the radioactivity count of the [Ac-labeled antibody]. 225 It is the ratio (%) of the radioactivity count of the peak corresponding to the [Ac-labeled monovalent antibody], and the radiochemical yield is the ratio (%) of the radioactivity calculated from the radioactivity count of the [Ac-labeled antibody] to the radioactivity calculated from the radioactivity count at the start of the labeling step measured with a gamma-ray spectrometer (Ge semiconductor detector: GMX10P4-70 (manufactured by ORTEC), multi-channel analyzer: M7-000 (manufactured by Seiko Epson Corporation), data processing: Spectrum Navigator: DS-P300 (manufactured by Seiko Epson Corporation) and Gamma Studio: DS-P600 (manufactured by Seiko Epson Corporation)). 225 It is the ratio (%) of the radioactivity calculated from the radioactivity count of the [Ac-labeled antibody].

[0145] Example 3: 225 Preparation of Ac-Labeled Anti-MUC5AC Humanized Antibody ( 225 Ac-Labeled Bivalent Antibody) In Example 2, in the same manner, except that the eluate of the divalent antibody obtained in Production Example 2 was used instead of the eluate of the monovalent antibody, 225 an [Ac-labeled divalent antibody] was obtained. The obtained 225 The radiochemical purity of the [Ac-labeled divalent antibody (radioactivity calculated by attenuation from the radioactivity at the assay date and time: 0.168 MBq)] was 99%, and the radiochemical yield was 20%.

[0146] Example 4: Screening of Humanized Antibody Using In-111 ( 111 In)-Labeled Antibody Example 4-1: 111 Preparation of In-Labeled Antibody In order to find an anti-MUC5AC antibody with high tumor accumulation ability and high maximum tolerated dose, various antibodies were 111 In-labeled, administered to tumor-bearing mice, subjected to SPECT-CT imaging, and the cumulative radioactivity and absorbed dose of the tumor and liver were calculated from the obtained images and compared. Antibodies were labeled with 4 types of anti-MUC5AC humanized antibodies prepared in Production Example 1 and 1 type of anti-MUC5AC chimeric antibody disclosed in Patent Document 1. 111 They were labeled with In. The amino acid sequences of the heavy chain variable region and the light chain variable region of the chimeric antibody disclosed in Patent Document 1 (SEQ ID NOs: 23 and 24, respectively) are as follows. 111 The In-labeling was performed on the complex of the labeling precursor having the structure represented by the following formula and various antibodies.

[0147]

Chemical formula

[0148]

Chemical formula

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

[0150] Various 111 The radiochemical yields, radiochemical purities, and radioactivities administered to animals of the In-labeled antibodies are shown in Table 1. The radiochemical yield referred to here is the 111 ratio of the radioactivity of the111 It refers to the radioactive energy of the In-labeled antibody. The radioactivity measurement was performed using a radioisotope dose calibrator (manufactured by CAPINTEC, model number: CRC-15R). The radiochemical purity refers to the ratio (%) of the radioactivity count of the peak corresponding to the In-labeled antibody to the total radioactivity count of the filter paper when analyzed by filter paper chromatography. 111 Filter paper chromatography was developed using filter paper (manufactured by ADVANTEC, model number: No. 590) and a developing solvent (0.01% EDTA, 50 mM citric acid-sodium citrate aqueous solution), and the detection of radioactivity count was performed 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 preparing tumor-bearing mice) 0.7×10 7 cells of the human pancreatic cancer cell line SW1990 were subcutaneously administered from the flanks to the back of Balb / c nude mice (male). When the tumor size was approximately 150 - 300 mm 3 14 days after transplantation of SW1990, various 111 In-labeled antibodies prepared in Example 4-1 were administered via the tail vein of the mice. Also, the tumor volume was calculated from the following formula. Tumor volume = (short diameter of the tumor 2 × long diameter of the tumor) / 2

[0154]

Table 2

[0155] (Evaluation method) SPECT-CT imaging (Small animal SPECT-CT device: FX-3000, manufactured by Trifoil) was performed under the conditions shown in the following table. 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 of the tumor and liver was performed at each time point. The count number per organ volume was corrected to %ID / g, and the physical half-life was 111 converted from 225 In to

[0156]

Table 3

[0157]

Table 4

[0158] (Results) 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 confirming the in-vivo distribution and excretion pathway after completion of SPECT-CT imaging 168 hours after administration is shown in Figure 3. Accumulation in tumors was highest when using the humanized antibody (H01L03), while it was lowest when using the chimeric antibody. Accumulation in the liver was highest when using the chimeric antibody, and was lower when using the humanized antibody compared to the chimeric antibody in all cases. Excretion was slowest and blood retention was highest when using the humanized antibody (H01L03). In all cases of using any antibody, accumulation in the liver and spleen was high among normal organs, followed by high accumulation in the lungs and kidneys.

[0159] When the liver threshold dose was set at 30 Gy, the maximum tolerated dose was a high value of 3.90 MBq when using the humanized antibody (H01L03), while it was a low value of 0.43 MBq when using the chimeric antibody. The maximum tolerated dose (MBq) was calculated by the threshold dose (Gy) / absorbed dose (Gy / MBq).

[0160] Example 4-3: In Vitro Autoradiography Various prepared in Example 4-1 111 Using the In-labeled antibodies, the image results of evaluating the binding and specificity of various antibodies to MUC5AC by in vitro autoradiography (ARG) are shown in Figure 4. Also, a graph of the numerical values of the radioactivity density (Bq / mm 2 ) in the region of interest (ROI) calculated by setting the ROI for the whole section is shown in Figure 5. From the frozen blocks of MUC5AC high-expression tumors (SW1990 transplanted tumor tissues) or MUC5AC low-expression tumors (MIAPaCa2 transplanted tumor tissues) frozen in liquid nitrogen, sections with a thickness of 10 μm were prepared using a cryostat (manufactured by Leica) and used for in vitro ARG. The sections stored at -80 °C until use were returned to room temperature, dried for more than 30 minutes, immersed in phosphate-buffered saline for 30 minutes, and then immersed in phosphate-buffered saline containing 1% bovine serum albumin for 30 minutes to hydrophilize the sections. The hydrophilized frozen sections were various 111The In-labeled antibody was immersed in phosphate-buffered saline containing 1% bovine serum albumin at 5 kBq / mL for 30 minutes each. Subsequently, the sections were washed by immersing them in each solution for 5 minutes in the order of phosphate-buffered saline containing 1% bovine serum albumin, phosphate-buffered saline, and phosphate-buffered saline. The washed sections were air-dried, exposed to an imaging plate (BAS-SR2040, manufactured by Fujifilm) for about 15 hours, and an autoradiogram was obtained using a fluorimage analyzer (Typhoon FLA 7000 IP, manufactured by GE Healthcare). The obtained autoradiogram was used with the analysis software "Image Quant TL" attached to the fluorimage analyzer to set a region of interest (ROI) for the entire section, and the radioactivity density (Bq / mm 2 ) in the ROI was calculated. 111 It was confirmed that all humanized antibodies labeled with In retained the binding ability and specificity for MUC5AC (see Figure 5, data of MUC5AC-highly expressing tumors). Compared with the chimeric antibody, it was confirmed that various humanized antibodies had less non-specific binding (see Figure 5, data of MUC5AC-low / non-expressing tumors). From the results of Examples 4-2 and 4-3, it became clear that the humanized antibody has higher specificity for MUC5AC compared to the chimeric antibody, and also has high accumulation in tumors and low accumulation in normal organs such as the liver, thus providing a more excellent delivery technology for radiolabeled antibodies.

[0161] The results of this example are summarized in the following table. In the absorbed dose of SPECT in the table, the tumor volume was calculated assuming 150 mm 3 . Also, the absorbed dose of the liver was calculated based on the average value of the mouse liver weights used in this example (1.15 ± 0.14 g, n = 19). The numerical values of the biodistribution are expressed as the mean ± standard deviation of n = 4 (however, for H02L04, n = 3).

[0162]

Table 5

[0163] Example 5: Evaluation of Ac-Labeled Monovalent Antibody Using Tumor-Bearing Mice 225 Example 6: High-Dose Evaluation of Ac-Labeled Monovalent Antibody Using Tumor-Bearing Mice Manufactured according to Example 1 225 The Ac-labeled monovalent antibody (H01L03) was used. The tumor-bearing mice were 225 divided into three groups according to the administered radioactivity of the Ac-labeled monovalent antibody, namely the 2.5 kBq administration group, the 5 kBq administration group, and the 10 kBq administration group, and compared with the group administered with the humanized antibody (H01L03) manufactured in Production Example 1 (antibody control group). Six mice were in each group. Four weeks after administration, the general condition was observed, and the body weight and tumor volume were measured. The tumor-bearing mice used for evaluation were prepared by the same procedure as in Example 4-2, and when the tumor size reached approximately 200 mm 3 10 days after transplantation of SW1990, they were used for the experiment. The animal information of 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 last day of the observation period when the tumor volume before administration was set to 1.0 is shown in the following table. 225 The Ac-labeled monovalent antibody showed a dose-dependent tumor growth inhibitory effect and significantly inhibited tumor growth at all doses statistically.

[0166]

Table 7

[0167] Autopsy was performed on the last day of the observation period, and the tumors were collected and weighed. The results of comparing the tumor weights are shown in the following table. 225 The Ac-labeled monovalent antibody showed a dose-dependent decrease in tumor weight, 225 and it was confirmed that the tumor weight was significantly lower statistically in all groups administered with the Ac-labeled monovalent antibody compared with the antibody control group.

[0168]

Table 8

[0169] The relative value of the change in body weight over time is shown in Fig. 7. In all groups, no weight loss of 10% or more was observed compared to before dosing. Therefore 225 It was shown that there was no or a sufficiently low possibility of an effect on the general condition by administration of the Ac-labeled monovalent antibody.

[0170] Autopsy was performed on the last day of the observation period, and the liver, kidneys, and spleen were collected and weighed. The results of comparing the weights of each organ are shown in the following table. 225 In the group administered with 2.5 kBq of the Ac-labeled monovalent antibody, it was confirmed that the weight of the liver was significantly lower statistically compared to the antibody control group, but since no dose-dependency was observed, it was considered an accidental result. From the fact that no statistically significant difference was observed in the kidneys and spleen and in the liver at other doses compared to the antibody control group, it was shown that there was no or a sufficiently low possibility of an effect on the liver, kidneys, and spleen.

[0171]

Table 9

[0172] Using the blood samples collected at the end of the observation period, nephrotoxicity (measuring plasma creatinine using a Creatinine Assay Kit (manufactured by Cayman Chemical Company)), hepatotoxicity (measuring alanine aminotransferase (ALT) in plasma using an ALT activity Kit (manufactured by Bio Vision)), and hematotoxicity (measuring the number of white blood cells and platelets using an automatic blood cell counter (model: thinka CB-1010, manufactured by Arkray)) were secondarily evaluated. For each measured value, Stat PreClinica (manufactured by Takumi Information Technology) was used to confirm the homoscedasticity of the measured values. When there was homoscedasticity, analysis was performed by the Dunnett method of parametric testing, and when there was no homoscedasticity, analysis was performed by the Steel method of non-parametric testing.

[0173] The results for hepatotoxicity and nephrotoxicity are shown in Fig. 8. 225In the group administered with the total dose of the Ac-labeled monovalent antibody, no statistically significant difference was observed at the 5% significance level compared with the antibody control group. The results regarding blood toxicity are shown in Fig. 9. At each time point of blood sampling, 225 In each dose group of the Ac-labeled monovalent antibody, no statistically significant difference was observed at the 5% significance level compared with the antibody control group.

[0174] According to this example, 225 The tumor growth inhibitory effect of the Ac-labeled monovalent antibody was confirmed. The inhibitory effect was dose-dependent, and a statistically significant inhibitory effect was shown at the 5% significance level at all doses (2.5, 5, 10 kBq). Also, 225 In addition to no more than 10% weight loss being observed compared with before administration of the Ac-labeled monovalent antibody, 225 It was suggested that the possibility of hepatotoxicity, nephrotoxicity, and blood toxicity caused by the Ac-labeled monovalent antibody was low. From these results, 225 It became clear that the Ac-labeled monovalent antibody is a very useful cancer therapeutic agent with high safety while having a very high antitumor effect.

[0175] Example 7: Evaluation of Ac-Labeled Monovalent Antibody and 225 Ac-Labeled Bivalent Antibody Using Tumor-Bearing Mice Except that the administered radiation energy was 25 kBq / animal (10 times the minimum dose in Example 5), the Ac-labeled monovalent antibody was evaluated in the same manner as in Example 5, 225 and the Ac-labeled monovalent antibody was evaluated ( 225 Ac-labeled monovalent antibody administration group). Also, a group (antibody control group) was established that administered a solution in which only the antibody (H01L03) produced in Production Example 1 was dissolved in a 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. Five animals were used in each group, and the general condition, body weight, and tumor volume were observed and measured for 4 weeks after administration. The animal information for each group is summarized below.

[0176]

Table 10

[0177] The results of examining the change in tumor volume over time are shown in Fig. 10. 225 In the group administered with the Ac-labeled monovalent antibody, tumor growth was statistically significantly suppressed, and 225 the tumor growth inhibitory effect by administration of the high-dose Ac-labeled monovalent antibody was confirmed.

[0178] At the end of the observation period, autopsy was performed, and the tumors were collected and weighed. The results of comparing the tumor weights are shown in the following table. 225 It was confirmed that the tumor weight in the group administered with the Ac-labeled monovalent antibody was statistically significantly lower than that in the antibody control group.

[0179]

Table 11

[0180] The results of examining the change in body weight over time are shown in Fig. 11. 225 In the group administered with the Ac-labeled monovalent antibody, a decrease in body weight was observed in the early stage of administration, but it did not fall below 0.9 as a relative ratio.

[0181] At the end of the observation period, autopsy was performed, and the liver, kidneys, and spleen were collected and weighed. The results of comparing the organ weights are shown in the following table. 225 It was confirmed that only the weight of the spleen in the group administered with the Ac-labeled monovalent antibody was statistically significantly lower than that in the antibody control group.

[0182]

Table 12

[0183] The results regarding hepatotoxicity and nephrotoxicity are shown in Fig. 12. Since no statistically significant difference was observed compared to the antibody control group, it was suggested that liver and kidney damage were not induced at this dose. The results regarding hematotoxicity are shown in Fig. 13. Since no statistically significant difference was observed compared to the antibody control group, it was suggested that hematotoxicity was not induced at this dose.

[0184] From these results as well, 225The Ac-labeled monovalent antibody was suggested to be a very useful anti-cancer drug with high safety while having a very high anti-tumor effect.

[0185] As a method for converting the mouse dose to the human dose, there is a method using the following calculation formula. Animal dose in mg / kg x (animal weight in kg / human weight in kg) x 0.33 When following this formula, administering 25 kBq / 20 g to mice in this example is equivalent to administering at 89.0 kBq / kg (human).

[0186] Example 8: Comparison of Pharmacokinetics Using In-111 ( 225 In)-Labeled Monovalent Antibody and 225 In-Labeled Bivalent Antibody Using tumor-bearing mice 225 The Ac-labeled monovalent antibody and 225 The performance of each of the Ac-labeled bivalent antibodies was evaluated. The Ac-labeled monovalent antibody produced according to Example 2 was administered to tumor-bearing mice prepared in the same manner as in Example 4-2 at a radiation dose of 5 kBq / mouse or 10 kBq / mouse ( 225 Ac-labeled monovalent antibody administration group). Also, the Ac-labeled bivalent antibody produced according to Example 3 was administered to tumor-bearing mice prepared in the same manner as in Example 4-2 at a radiation dose of 5 kBq / mouse or 10 kBq / mouse ( 225 Ac-labeled bivalent antibody administration group). Also, an antibody control group was set up in the same manner as in Examples 5 and 6. There were 6 animals in each group, and after administration, the general condition was observed, and the body weight and tumor volume were measured for 4 weeks. The animal information of each group is summarized below. 225 Ac-labeled bivalent antibody was administered to tumor-bearing mice prepared in the same manner as in Example 4-2 at a radiation dose of 5 kBq / mouse or 10 kBq / mouse ( 225 Ac-labeled bivalent antibody administration group). Also, an antibody control group was set up in the same manner as in Examples 5 and 6. There were 6 animals in each group, and after administration, the general condition was observed, and the body weight and tumor volume were measured for 4 weeks. The animal information of each group is summarized below.

[0187]

Table 13

[0188] The results of confirming the change in tumor volume over time are shown in Fig. 14. The following table shows the relative ratio of the tumor volume on the final day of the observation period to the tumor volume before administration, with the tumor volume before administration set to 1.0. 225In the group administered with the Ac-labeled monovalent antibody, tumor growth was significantly suppressed statistically, and a dose-dependent tumor growth inhibitory effect was confirmed. 225 In the group administered with the Ac-labeled bivalent antibody, a tendency to suppress tumor growth was shown.

[0189]

Table 14

[0190] On the last day of the observation period, euthanasia was performed by bleeding under isoflurane anesthesia, and tumors were collected and weighed. The results of comparing tumor weights are shown in the table below. 225 In the group administered with the Ac-labeled monovalent antibody, it was confirmed that the tumor weight was significantly lower statistically compared to the antibody control group, and a dose-dependent tumor growth inhibitory effect was confirmed. 225 In the group administered with the Ac-labeled bivalent antibody, it was also confirmed that the tumor weight was lower compared to the antibody control group, but no statistically significant difference was observed.

[0191]

Table 15

[0192] The results of confirming the change in body weight over time are shown in Fig. 15. The average value of the relative ratio did not fall below 0.9. Also, the relative ratio of each individual did not fall below 0.8.

[0193] On the last day of the observation period, euthanasia was performed by bleeding under isoflurane anesthesia, and autopsy was performed. As a result of the autopsy, no abnormal findings were observed. Also, the liver, kidneys, and spleen were collected and weighed. The results of comparing the weights of normal organs are shown in the table below. None of the 225 In the group administered with the Ac-labeled antibody, no statistically significant decrease in organ weight was observed compared to the antibody control group.

[0194]

Table 16

[0195] 225Ac-labeled monovalent antibody and 225 The results of the hepatotoxicity of the Ac-labeled divalent antibody are shown in Fig. 16. The results of the nephrotoxicity are shown in Fig. 17. Since no statistically significant increase was observed compared to the antibody control group, it was suggested that liver and kidney damage were not induced at this dose. The results of the white blood cell counts at 1 week and 4 weeks after administration for hematotoxicity are shown in Fig. 18. Also, the results of the platelet counts at 1 week and 4 weeks after administration are shown in Fig. 19. Regarding the white blood cell count, at 1 week after administration, a statistically significant difference was observed in the monovalent antibody administration groups (5 kBq and 10 kBq) and the divalent antibody administration group (10 kBq) compared to the antibody control group, but it had recovered at 4 weeks after administration. Regarding the divalent antibody administration group (5 kBq) that showed a statistically significant difference compared to the antibody control group at 4 weeks after administration, it was not a dose-dependent event. Regarding the platelet count, except for one individual, it did not fall below 500×10 9 cells / L, which is the lower limit of the normal range, at any time point. From these results, it was suggested that hematotoxicity was not induced at this dose.

[0196] Example 8-1: Preparation of Each 111 In-Labeled Antibody 111 Example 8-2: Biodistribution in Tumor-Bearing Mice Example 9: 111 Production of Ac-Labeled Anti-MUC5AC Humanized Antibody Using Ac-Labeled DOTAGA-DBCO To compare the pharmacokinetics of the monovalent antibody and the divalent antibody, the monovalent antibody and the divalent antibody were 111 labeled with In and administered to tumor-bearing mice, and a biodistribution experiment was performed 20, 68, and 188 hours after administration to compare the pharmacokinetics of the monovalent antibody and the divalent antibody.

[0197] (1) Preparation of each antibody introduced with a chelator The antibodies were prepared in the same manner as in Production Example 1 and Production Example 2 to obtain a monovalent antibody and a divalent antibody of a peptide-modified antibody of the humanized antibody H01L03. A 0.1 mol / L sodium acetate buffer solution (pH 6.0) containing 34 nmol of the chelate moiety (structural formula: L1-4) and a 0.1 mol / L arginine-containing 0.1 mol / L histidine buffer solution (pH 6.0) containing 34 nmol of the monovalent antibody or bivalent antibody were reacted at 37 °C for 120 minutes to obtain a chelator-introduced antibody. This was passed through a desalting column (model number: PD-10, manufactured by GE Healthcare), and the fraction containing the chelator-introduced antibody was collected. The collected fraction was 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) For each antibody 111 In labeling As a radioactive metal source 111 An In ion-containing solution (indium chloride ( 111 In) injection solution, manufactured by Nippon Medi-Physics Co., Ltd.) was used with a radioactivity of 91 - 92 MBq, and 0.05 mL of each chelator-introduced antibody was added and mixed well. It was confirmed with pH test paper (manufactured by Merck) that the pH was 4. This was reacted at 45 °C for 120 minutes. The reaction solution was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096), and in addition, 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 radioactivity administered to the animals is shown in Table 17. The radiochemical yield referred to here means the radioactivity of the In-labeled antibody relative to the radioactivity of the 111 In used. Radioactivity measurement was performed using a radioisotope dose calibrator (manufactured by CAPINTEC, model number: CRC-15R). The radiochemical purity is the ratio of the radioactivity of the thin-layer plate analyzed by thin-layer chromatography to the total radioactivity count of the thin-layer plate. 111 111 ​Refers to the percentage (%) of the radioactivity count of the peak corresponding to the In-labeled antibody. Thin layer chromatography (thin layer plate: manufactured by Agilent, model number: SGI0001) was developed using a developing solvent of a 100 mmol / L EDTA solution (pH 5.0) / acetonitrile mixture (volume ratio 1:1), and radioactivity counts were detected using a radio γ-TLC analyzer (manufactured by raytest, MODEL GITA Star).

[0200]

Table 17

[0201] Example 10: 0.5×10 human pancreatic cancer cell line SW1990 7 individuals were subcutaneously administered from the flanks to the back of Balb / c nude mice (male). When the tumor volume reached approximately 300 mm 3 before and after, the 111 In-labeled monovalent antibody and bivalent antibody prepared in Example 8-1 were administered via the tail vein.

[0202] (Evaluation method) Tumor-bearing mice were each 111 fed in a metabolic cage after administration of the In-labeled antibody, and feces and urine excreted by each time point (20, 68, and 188 hours after administration) were collected. At each time point, the tumor-bearing mice were euthanized by bleeding under isoflurane anesthesia. Tumors, blood, and normal organs (including the rest of the body) were collected and weighed. In addition to the weighed organs, the radioactivity of the excreted feces and urine was measured (γ-ray well scintillation measuring device: JDC-1712, manufactured by Hitachi Aloka Medical). The radioactivity accumulation rate (%ID) relative to the administered dose was calculated from the radioactivity (counts) of each organ (including excreted feces and urine), and the radioactivity accumulation amount (%ID / g) was calculated as the radioactivity accumulation rate per organ weight.

[0203] (Results) The results showing the change over time in the radioactivity accumulation amount in each organ are shown in Fig. 20. The results showing the change over time in the radioactivity accumulation rate of excreted feces and urine and the total radioactivity accumulation rate of these are shown in Fig. 21. The radioactivity accumulation amount in the blood showed higher values at all time points for the monovalent antibody compared to the divalent antibody. From this, it was confirmed that the monovalent antibody has higher blood retention compared to the divalent antibody. The radioactivity accumulation amount in the tumor showed higher values at all time points for the monovalent antibody compared to the divalent antibody. As a tendency of radioactivity accumulation in normal organs, it was confirmed that for the monovalent antibody, the spleen, liver, lung, and kidney were in descending order of high values. For the divalent antibody, significantly high values were shown in the order of the liver and spleen, and it was confirmed that they were high in the order of the testis, heart, kidney, and femur. It was confirmed that the radioactivity accumulation in each normal organ decreased over time. The excretion amount showed higher values at all time points for the divalent antibody compared to the monovalent antibody, and it was confirmed that the excretion rate was faster for the divalent antibody. For the monovalent antibody, the feces and urine excretion amounts were comparable. On the other hand, for the divalent antibody, urine excretion was higher than feces excretion, and it was confirmed that it was mainly excreted through the renal urinary system.

[0204] Production of Zr-Labeled Anti-MUC5AC Humanized Antibody (HPLC Purification) Using Zr-Labeled DOTAGA-DBCO 225 Example 11: Stability Evaluation of Each 225 Zr- and (1. Chelating agent synthesis step) 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 0.1 mol / L sodium acetate buffer (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, as a radioactive metal source 225A reaction solution was prepared by mixing 4.9 MBq (a calculated value obtained by decay calculation from the radioactivity at the assay date and time) of an 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), and the reaction was carried out under heating conditions to 225 obtain an Ac complex solution. The molar ratio of the chelating part to the radioactive metal ion was chelating part: 225 Ac ion = approximately 670:1, the heating temperature of the reaction solution was 70 °C, and the heating time was 30 minutes.

[0205]

Chemical formula

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

[0207] (2. Labeling step) A solution of the Ac complex obtained through the above (1. Chelating agent synthesis step) and a solution containing a peptide-modified antibody (monovalent antibody; H01L03) produced in the same manner as in Production Example 2, except that they were reacted at room temperature for 60 minutes, were mixed as they were without purification, and a click reaction was carried out at 37 °C for 2 hours to 225 obtain an Ac complex-labeled antibody. The amounts of the chelating part or the chelating part 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. 225 Furthermore, the solution of the Ac complex-labeled antibody obtained by reacting at 37 °C for 2 hours was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096). After purification, 225 225 225The radiochemical purity of the Ac-labeled monovalent antibody was 96%, and the radiochemical yield was 68%. 225 The method for measuring the radiochemical purity and radiochemical yield of the Ac-labeled monovalent antibody was carried out in the same manner as in Example 1.

[0208] Ac-Labeled Anti-MUC5AC Humanized Antibody in Human and Mouse Plasma 89 Example 12: Binding Affinity and Specificity of RI-Labeled Anti-MUC5AC Humanized Antibody to MUC5AC 89 Example 13: PET-CT Imaging of Zr-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Zr-Labeled DOTA-Bn-DBCO and DOTAGA-DBCO (1-1. Chelating agent synthesis step) In this example, the same chelating moiety as that represented by the above formula (L1-5) was used. This chelating moiety was dispersed in a DMSO solution to obtain a dispersion containing 2.0 mmol / L of the chelating moiety. 0.150 mL of this dispersion, as a radioactive metal source 89 134 MBq of a Zr ion-containing solution (0.1 mol / L hydrochloric acid aqueous solution, radioactivity concentration 1335 MBq / mL, prepared from Nihon Medi-Physics Co., Ltd., liquid volume 0.100 mL) and 0.050 mL of a 780 mmol / L acetic acid buffer solution containing 300 mmol / L of gentidilic acid were mixed to obtain a reaction solution, which was reacted under heating conditions to 89 obtain a Zr complex solution. The molar ratio of the chelating moiety to the radioactive metal ion was chelating moiety: 89 Zr ion = approximately 3333:1, the heating temperature of the reaction solution was 70 °C, and the heating time was 60 minutes.

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

[0210] (1-2.89 Zr complex purification process) The 89 Zr complex solution obtained in the above (1-1. Chelating agent synthesis process) was fractionated using high performance liquid chromatography (HPLC) to remove unreacted DOTAGA-DBCO. The obtained fractionated solution was concentrated to about 30 μL by solvent evaporation and used in the labeling process. 89 The method for measuring the radiochemical yield (HPLC recovery rate) in the process of removing unreacted substances from the Zr complex-labeled antibody was as follows. That is, the percentage of the radioactivity of the fractionated solution with respect to the radioactivity charged 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 absorption photometer (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) Liquid delivery of the 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 89 solution of the Zr complex obtained through the above respective processes and a 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 a Zr complex-labeled antibody. 89The amounts of the chelate part containing the Zr-labeled complex and the peptide-modified antibody (monovalent antibody) were 73 pmol and 50 nmol, respectively, and the molar ratios of the first atomic group (DBCO) to the second atomic group (azide) were each about 1:685. Furthermore, obtained by reacting at 37°C for 1.5 hours 89 The solution of the Zr complex-labeled antibody was purified using an ultrafiltration filter (manufactured by Merck, model number: UFC505096). After purification 89 The radiochemical purity of the Zr complex-labeled antibody was 95%, and the radiochemical yield was 50%. 89 The measurement methods for the radiochemical purity and radiochemical yield of the Zr complex-labeled antibody were as follows. That is, thin-layer chromatography (manufactured by Agilent, model number: SGI0001, developing solvent is a mixed solution of acetonitrile: 0.1 mmol / L EDTA solution (volume ratio 1:1)) was measured with a radio γ-TLC analyzer (manufactured by raytest, MODEL GITA Star PS), and the percentage of the radioactivity (counts) of the peak detected near the origin to the total radioactivity (counts) detected was defined as the radiochemical purity (%). Also, the percentage of the radioactivity recovered after ultrafiltration purification to the total radioactivity added at the start of the labeling process was defined as the radiochemical yield (%).

[0213] Example 14: 89 In Vivo Biodistribution Experiment of Zr-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Zr-Labeled DOTA-Bn-DBCO and DOTAGA-DBCO 225 Example 15: 89 Prepared using Zr-labeled DOTAGA-DBCO 89 The Zr-labeled anti-MUC5AC humanized antibody was produced according to Example 10. 89 Prepared using Zr-labeled DOTA-Bn-DBCO 89 The Zr-labeled anti-MUC5AC humanized antibody was produced according to Example 10, except that the chelate part shown in the above formula (L1-4) was used as the chelate part. 225 Prepared using Ac-labeled DOTA-Bn-DBCO 225 The Ac-labeled anti-MUC5AC humanized antibody was produced according to Example 2. 225 Prepared using Ac-labeled DOTAGA-DBCO225 The Ac-labeled anti-MUC5AC humanized antibody was produced according to Example 9. In addition, 0.1 M sodium acetate buffer (pH 6.0) was used as the solvent in all cases.

[0214] Various 89 Zr-labeled antibody and 225 The stability of the Ac-labeled antibody when mixed with human and mouse plasma and incubated at 37°C at each time point was evaluated by cellulose acetate membrane electrophoresis. In addition, 89 Zr-labeled antibody and 225 To evaluate the degradation products in plasma other than the Ac-labeled antibody, 89 Zr or 225 Assuming the detachment of each chelator of Ac and the cleavage of each linker site, each monomer was separately mixed with each plasma, and cellulose acetate membrane electrophoresis was performed. Using each plasma sample collected from each incubated time point, cellulose acetate membrane electrophoresis was performed, and after electrophoresis, the cellulose acetate membrane was exposed to an imaging plate. The exposed imaging plate was read by a scanner-type image analysis device (manufactured by GE Healthcare, model number: Typhoon-7000), and various 89 Zr-labeled antibody and 225 The radiochemical purity of the Ac-labeled antibody was quantitatively evaluated. The composition of the evaluation samples is shown in Table 19. The percentage of the radioactivity (counts) of the peak corresponding to each labeled antibody to the total detected radioactivity (counts) was defined as the radiochemical purity (%).

[0215] [Table 19]

[0216] [Table 20]

[0217] Regarding the RI-labeled anti-MUC5AC humanized antibody prepared using RI-labeled DOTA-Bn-DBCO, the radiochemical purity of the plasma samples sampled immediately after the incubation time for stability evaluation and measured by thin-layer chromatography was 94% in mouse plasma and 95% in human plasma. Finally, the radiochemical purity at the time of incubation for 378 hours was 70% or more in both cases. The graph of the results is shown in the upper part of Figure 22A. Used in this evaluation 225 The radiochemical purity of the Ac-labeled antibody was 77%, and dissociation was observed with the radiochemical purity of the plasma samples sampled immediately after the incubation time for stability evaluation and measured by thin-layer chromatography (mouse: 90%, human: 80%). It was considered that some solid components were fixed to the original line, and the obtained radiochemical purity was lower than the actual value. Finally, the radiochemical purity at the time of incubation for 168 hours was 60% or more in both cases. The graph of the results is shown in the upper part of Figure 22B.

[0218] Regarding the RI-labeled anti-MUC5AC humanized antibody prepared using RI-labeled DOTAGA-DBCO, used in this evaluation 89 The radiochemical purity of the Zr-labeled antibody was 94%, which was approximately the same as the radiochemical purity of the plasma samples measured immediately after the incubation time for stability evaluation (mouse: 99%, human: 99%). Finally, the radiochemical purity at the time of incubation for 336 hours was 85% or more in both cases. The graph of the results is shown in the lower part of Figure 22A. Used in this evaluation 225 The radiochemical purity of the Ac-labeled antibody was 100%, which was approximately the same as the radiochemical purity of the plasma samples measured immediately after the incubation time for stability evaluation (mouse: 97%, human: 100%). Finally, the radiochemical purity at the time of incubation for 336 hours was 80% or more in both cases. The graph of the results is shown in the lower part of Figure 22B.

[0219] Efficacy Evaluation of Ac-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Ac-Labeled DOTAGA-DBCO 225 Ac or 89The radiolabeled anti-MUC5AC humanized antibody prepared using Zr-labeled DOTAGA-DBCO was manufactured according to Example 9 and Example 10. Also, 89 The radiolabeled anti-MUC5AC humanized antibody prepared using Zr-labeled DOTA-Bn-DBCO was manufactured according to Example 11. Using these radiolabeled anti-MUC5AC humanized antibodies, the binding and specificity of each radiolabeled antibody to MUC5AC by in vitro ARG were evaluated. The specific operation was performed according to Example 4-3, except that the above-mentioned radiolabeled anti-MUC5AC humanized antibody was used as the test substance. 89 The image of the results of the Zr-labeled antibody is shown in Figure 23, 225 and the results of the Ac-labeled antibody are shown in Figure 24, respectively. Also, 89 When using the Zr-labeled antibody, ROIs were set for the entire sections of each MUC5AC-positive tumor section and negative tumor section, and the binding ratios to MUC5AC-positive tumors and negative tumors were calculated using the numerical values in the calculated ROIs. 225 When using the Ac-labeled antibody, multiple small ROIs were set within the tumor tissues of the MUC5AC-positive tumor section and negative tumor section, and the binding ratios to MUC5AC-positive tumors and negative tumors were calculated using the average value. As a result, 225 The radiolabeled anti-MUC5AC humanized antibody prepared using Ac-labeled DOTAGA-DBCO showed a binding ratio of 6.5 times. 89 The radiolabeled anti-MUC5AC humanized antibody prepared using Zr-labeled DOTAGA-DBCO showed a binding ratio of 138 times. 89 The radiolabeled anti-MUC5AC humanized antibody prepared using Zr-labeled DOTA-Bn-DBCO showed a binding ratio of 151 times. 89 Zr or 225 Ac-labeled DOTAGA-DBCO was used to prepare 89 Zr or 225 Ac-labeled anti-MUC5AC humanized antibody and 89 Zr-labeled DOTA-Bn-DBCO was used to prepare 89 It was confirmed that all of the Zr-labeled anti-MUC5AC humanized antibodies retained the binding and specificity to MUC5AC.

[0220] Example 16:89 PET-CT Imaging of Zr-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Zr-Labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 In Vivo Biodistribution Experiment of Zr-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Zr-Labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Using Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Zr-labeled antibodies were prepared according to Example 10, and each was administered to tumor-bearing mice, and evaluation using PET-CT imaging was performed. 0.7×10 of SW1990, a tumor cell line highly expressing MUC5AC derived from human pancreatic cancer, 7 cells were subcutaneously administered from the flank to the back of Balb / c nude mice (male). When the tumor volume after transplantation was approximately 150 - 300 mm 3 at that time, various 89 Zr-labeled anti-MUC5AC humanized antibodies were administered via the mouse tail vein. The tumor volume was calculated from the following formula. Tumor volume = (short diameter of tumor 2 × long diameter of tumor) / 2 The radiochemical purity and animal information of various 89 Zr-labeled anti-MUC5AC humanized antibodies administered were 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 shown in the following table. The time points for imaging PET and CT were 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. VOI analysis of the SUV (standardized uptake value) of the tumor, heart (blood), and liver was performed at each time point, and the transition of SUV was compared from the time activity curve.

[0223]

Table 22

[0224]

Table 23

[0225] Each 89 The results of PET-CT imaging 48 hours after administration of the 89 Zr-labeled antibody are shown in Fig. 25. The results of VOI analysis of the tumor, heart (blood), and liver at each time point are shown in Fig. 26. In addition, the results of the tumor-to-liver ratio at each time point are shown in Fig. 27. The maximum value of accumulation in the tumor was 2.8 or more in all cases as SUV, and the maximum value of the tumor-to-liver ratio 84 hours after administration was 89 prepared using 89 Zr-labeled DOTA-Bn-DBCO 89 3.6 for the 89 Zr-labeled antibody, and 89 prepared using 89 Zr-labeled DOTAGA-DBCO 89 3.4 for the 89 Zr-labeled antibody. No statistically significant difference was observed in each value. Various 89 It was confirmed that the accumulation of various 89 Zr-labeled antibodies in the heart (blood) decreased over time, and it was confirmed that they almost disappeared from the blood 252 hours after administration. In addition, no statistically significant difference was observed in the accumulation of various 89 Zr-labeled antibodies in the heart (blood) at each time point. Similarly, it was confirmed that the accumulation of various 89 Zr-labeled antibodies in the liver and muscle also decreased over time, and no statistically significant difference was observed in the accumulation of various 89 Zr-labeled antibodies in each tissue at each time point.

[0226] Example 14: 89 In Vivo Biodistribution Experiment of Zr-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Zr-Labeled DOTA-Bn-DBCO and DOTAGA-DBCO 89 Example 15: To confirm more detailed in vivo kinetics, 89 the 89 Zr-labeled antibodies prepared using 89 Zr-labeled DOTA-Bn-DBCO and DOTAGA-DBCO were administered to tumor-bearing mice, and biodistribution experiments were performed 20, 68, and 188 hours after administration. Note that the various 89 Zr-labeled antibodies were produced according to Example 10 in the same manner as in Example 13. 89

[0227] In the biodistribution experiment, various 89 Zr-labeled antibodies were administered to tumor-bearing mice prepared by the same method as in Example 4-2 via the tail vein. The radiochemical purity and animal information of each 89 Zr-labeled anti-MUC5AC humanized antibody are shown in Table 24. Approximately 5 MBq was administered to each group as the administered radioactivity.

[0228]

Table 24

[0229] The tumor-bearing mice were housed in a metabolic cage after administration of various 89 Zr-labeled antibodies, and feces and urine excreted by each time point (20, 68, and 188 hours after administration) were collected. At each time point, the tumor-bearing mice were euthanized by bleeding under isoflurane anesthesia. Tumors, blood, and normal organs (including the remaining whole body) were collected and weighed. In addition to the weighed organs, the radioactivity of the excreted feces and urine was measured (γ-ray well scintillation measurement device: JDC-1712, manufactured by Hitachi Aloka Medical, Ltd.). The radioactivity accumulation rate (%ID) with respect to the administered dose was calculated from the radioactivity (counts) of each organ (including excreted feces and urine), and the radioactivity accumulation amount (%ID / g) was calculated as the radioactivity accumulation rate per organ weight. The results showing the time-course changes in the radioactivity accumulation amounts of the tumor tissue and each organ are shown in FIGS. 28A to D. The radioactivity accumulation rate (%ID) with respect to the administered dose was calculated from the radioactivity accumulation amounts (counts) of the excreted feces and urine, and the results showing the time-course changes are shown in FIG. 29.

[0230] Regarding the radioactivity accumulation amount of the tumor, 89 the 89 Zr-labeled antibody prepared using 89 Zr-labeled DOTA-Bn-DBCO was the highest at 188 hours after administration, and 89 the 89It also showed a tendency to decrease similarly with the Zr-labeled antibody, and it can be judged that the blood clearance was comparable. Regarding excretion, the radioactivity accumulation rates in feces and urine at 188 hours after administration were 65% ID or higher. Regarding the radioactivity accumulation in normal tissues, both were highest at 20 hours after administration, and a tendency for the radioactivity accumulation to decrease was observed after 20 hours. The normal tissues with high radioactivity accumulation at 20 hours after administration were the liver, lung, and spleen in that order.

[0231] Efficacy Evaluation of Ac-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Ac-Labeled DOTAGA-DBCO 225 Example 16: 225 Efficacy Evaluation of Ac-Labeled Anti-MUC5AC Humanized Antibody Prepared Using Ac-Labeled DOTAGA-DBCO 225 Prepared using Ac-labeled DOTAGA-DBCO 225 The Ac-labeled antibody was administered to tumor-bearing mice, and a study was conducted to confirm the tumor growth inhibitory effect. In addition, 225 The Ac-labeled antibody was manufactured according to Example 9. The tumor-bearing mice were prepared in the same manner as in Example 4-2. 225 The Ac-labeled antibody was administered to tumor-bearing mice at a radioactive dose of 5 kBq / mouse or 10 kBq / mouse, 225 and the performance of the Ac-labeled antibody was evaluated. In addition, a group (antibody control group) was provided in which a solution containing only the anti-MUC5AC humanized antibody dissolved in 0.1 M sodium acetate buffer (pH 6.0) was administered. There were 6 mice in each group, and the general condition was observed, and the body weight and tumor volume were measured for 4 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 Fig. 30(A). 225 In the Ac-labeled antibody administration group, tumor growth was statistically significantly inhibited compared to the antibody control group at any administered radioactive dose, 225 and the tumor growth inhibitory effect by the administration of the Ac-labeled antibody was confirmed.

[0234] Autopsy was performed on the last day of the observation period, and tumors were collected and weighed. The results of comparing tumor weights are shown in Table 26. Various 225 It was confirmed that the tumor weight in the Ac-labeled antibody administration group was statistically significantly lower than that in the antibody control group.

[0235]

Table 26

[0236] The results of confirming the change in body weight over time are shown in (B) of Figure 30. Various 225 In the group administered with 10 kBq of the Ac-labeled antibody, a decrease in body weight was observed in the early stage of the observation period, but it did not fall below 0.9 as a relative ratio. In the later stage of the observation period, the body weight recovered to the level before administration.

[0237] Autopsy was performed on the last day of the observation period, and the liver, kidneys, and spleen were collected and weighed. None of the 225 In the Ac-labeled antibody administration group, no statistically significant decrease in tissue weight was observed compared to the antibody control group.

[0238]

Table 27

[0239] The results regarding hepatotoxicity and nephrotoxicity are shown in Figures 33 and 34, respectively. Since no statistically significant difference was observed compared to the antibody control group, it was suggested that this dosage did not induce liver and kidney damage. The results regarding hematotoxicity are shown in Figures 31 and 32. Since no statistically significant difference was observed compared to the antibody control group, it was suggested that this dosage did not induce hematotoxicity.

[0240] Example 16: 89 Zr Random-Labeled Anti-MUC5AC Humanized Antibody ( 89 Zr]Random-DFO-Anti-MUC5AC Humanized Antibody) Preparation A peptide-modified antibody (monovalent antibody; H01L03) (0.1 mg, 0.7 nmol) prepared in the same manner as in Production Example 2 and 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) (0.26 mg, 0.35 μmol) were mixed in 0.1 M sodium bicarbonate buffer and reacted at room temperature for 120 minutes. After completion of the reaction, the mixture was purified by ultrafiltration and the solvent was replaced with 50 mmol histidine buffer containing 100 mmol arginine (pH 6.1) (hereinafter referred to as RH buffer), thereby producing a humanized anti-MUC5AC antibody with DFO randomly bound to the amino group of the humanized anti-MUC5AC antibody (hereinafter referred to as "Random-DFO-humanized anti-MUC5AC antibody"). As a result of protein concentration measurement using NanoDrop 2000 (ThermoFisher), the protein concentration of the Random-DFO-humanized anti-MUC5AC antibody solution was 1.12 mg / mL. 89.6 μL (0.1 mg, 0.67 μmol) of the obtained Random-DFO-humanized anti-MUC5AC antibody solution was mixed with 10 μL (11.8 MBq) of 89ZrCl3 solution and 301.6 μL of RH buffer to proceed with the complex formation reaction. After complex formation, purification was performed by ultrafiltration to obtain 89 89Zr]Random-DFO-humanized anti-MUC5AC antibody. As a result of calculating the radiochemical purity in the same manner as in Example 10, the radiochemical purity was 97.3%. Further, as a result of calculating the radiochemical yield based on the radioactivity at the start of the reaction, it was 43.0%.

[0241] Example 17: 89 Zr]Random-DFO-Anti-MUC5AC Humanized Antibody PET-CT Imaging Obtained in Example 16 89The Zr]Random-DFO-humanized anti-MUC5AC antibody was diluted and adjusted with RH buffer to a concentration of 1.27 MBq / 22.5 μg protein / 100 μL / mouse, and administered to tumor-bearing mice prepared in the same manner as in Example 4-2 (n = 3). At 19, 42, 86, and 158 hours after administration, evaluation using PET-CT imaging was performed. As a result of calculating the tumor volume of the animals used in this evaluation in the same manner as in Example 4-2, the average value of the tumor volume was 60.0 ± 20.0 mm 3 It was. The PET imaging conditions and image reconstruction method were in accordance with Example 13. VOI analysis of the SUV of the tumor, heart (blood), and liver was performed at each time point, and the change in SUV was compared from the time activity curve.

[0242] The results of the PET-CT imaging are shown in Fig. 35. The results of the VOI analysis of the tumor, heart, and liver at each time point are shown in Fig. 36. From the results of the VOI analysis, accumulation over time was observed in the tumor, and at any time point, the average value of the SUV was 2.7 or more, and the maximum average value of the SUV was 5.1 at the 158-hour time point. It was confirmed that the SUV in organs other than the tumor (heart (blood), liver) decreased over time. The tumor-liver ratio of the SUV at 158 hours after administration was 3.7.

[0243] Example 18: 89 Zr]Random-DFO-Anti-MUC5AC Humanized Antibody Biodistribution Experiment In Example 17, after completion of the PET-CT imaging at the 158-hour time point, euthanasia by bleeding was performed under isoflurane anesthesia. Tumors, blood, and normal organs (including the rest of the body) were collected and weighed. Furthermore, the radioactivity of each organ was measured using a gamma-ray well scintillation measuring device (JDC-1712, Hitachi Aloka Medical Ltd.). The radioactivity accumulation rate (%ID) with respect to the administered dose was calculated from the radioactivity (count) of each organ (including excreted feces and urine), and the radioactivity accumulation amount (%ID / g) was calculated as the radioactivity accumulation rate per organ weight.

[0244] The results of the in - vivo distribution evaluation after PET - CT imaging are shown in Table 28. The radioactive distribution rate of the tumor at 158 hours after administration was 5.1 ± 2.3%ID, and the radioactive distribution rate per unit weight was 49.5 ± 5.2%ID. A high radioactive distribution in the liver was confirmed. The radioactive distribution rate in the liver was 5.1%ID, and the radioactive distribution rate per unit weight was 10.4%ID / g. The normal organs with high radioactive distribution rates excluding the tumor were the liver, kidney, and lung in that order.

[0245]

Table 28

[0246] As described above, the present invention has been described with reference to the embodiments. However, the present invention is not limited to the above - described embodiments. Various changes 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 content described in all publications, including the patents and patent application specifications mentioned herein, is incorporated herein by reference to the same extent as if each were expressly stated.

Industrial Applicability

[0247] Since the radiolabeled anti - MUC5AC humanized antibody of the present invention is excellent in specificity and tumor - accumulating property, it is extremely useful for the treatment and / or diagnosis of diseases in which MUC5AC is overexpressed, particularly cancer.

[0248] This application is based on Japanese Patent Application No. 2019 - 191562 (filing date: October 18, 2019), the content of which is incorporated herein in its entirety.

Claims

1. A conjugate of an antibody and a chelating agent having a chelated radionuclide, The radioactive nuclide is a metal nuclide that emits alpha rays or positrons, The antibody specifically binds to mucin subtype 5AC. (1) an amino acid sequence (H01) represented by 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 shown in 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 shown in SEQ ID NO: 8 (L04) A light chain variable region consisting of A conjugate which is a humanized antibody having the formula:

2. 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 1, which is a humanized antibody having the following structure:

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

4. The complex according to any one of claims 1 to 3, comprising 1 to 8 molecules of the chelating agent per one molecule of the antibody.

5. The conjugate according to any one of claims 1 to 4, wherein the chelating agent site-specifically modifies the Fc region of the antibody via a linker.

6. The complex according to claim 5, 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 respectively represent a consecutive Xs, b consecutive Xs, c consecutive Xs, and d consecutive Xs; 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 represents an integer of 1 to 5, and a+b+c+d≦14; Xaa1 and Xaa3 are each independently 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, the other represents an amino acid residue derived from an amino acid having a haloacetyl group in the side chain, 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 the crosslinker.

7. The complex according to any one of claims 1 to 6, wherein the chelating agent has a structure derived from a compound represented by the following formula (A) or a salt thereof: 【Chemistry 1】 (In formula (A), R １１ , R １３ and R １４ Each independently represents -(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, 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, R １２ is not a substituent for conjugation with the antibody, R １５ is a substituent for conjugation to the antibody.

8. A radiopharmaceutical comprising the complex according to any one of claims 1 to 7 as an active ingredient.

9. The radiopharmaceutical according to claim 8, wherein the radionuclide is a metal nuclide that emits alpha rays and is used for RI internal therapy of cancer.

10. The radiopharmaceutical according to claim 9, which is administered to a subject at a dose of 250 kBq / kg or less per administration in the RI internal therapy.

11. The radiopharmaceutical of claim 10, wherein the dosage is 80 kBq / kg or less per administration.

12. The radiopharmaceutical of claim 8, wherein the radionuclide is a positron-emitting metal nuclide and is used for the diagnosis of cancer.

13. A radiopharmaceutical comprising a complex of a chelating agent having a radionuclide chelated thereto and an antibody, 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 9 to 11.

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